44147253
40 Years of Research Put p53 in Translation
Since its discovery in 1979, p53 has shown multiple facets. Initially the tumor suppressor p53 protein was considered as a stress sensor able to maintain the genome integrity by regulating transcription of genes involved in cell cycle arrest, apoptosis and DNA repair. However, it rapidly came into light that p53 regulates gene expression to control a wider range of biological processes allowing rapid cell adaptation to environmental context. Among them, those related to cancer have been extensively documented. In addition to its role as transcription factor, scattered studies reported that p53 regulates miRNA processing, modulates protein activity by direct interaction or exhibits RNA-binding activity, thus suggesting a role of p53 in regulating several layers of gene expression not restricted to transcription. After 40 years of research, it appears more and more clearly that p53 is strongly implicated in translational regulation as well as in the control of the production and activity of the translational machinery. Translation control of specific mRNAs could provide yet unsuspected capabilities to this well-known guardian of the genome.
38710686
Characterization of a 54K Dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells
SV40 infection or transformation of murine cells stimulated the production of a 54K dalton protein that was specifically immunoprecipitated, along with SV40 large T and small t antigens, with sera from mice or hamsters bearing SV40-induced tumors. The same SV40 anti-T sera immunoprecipitated a 54K dalton protein from two different, uninfected murine embryonal carcinoma cell lines. These 54K proteins from SV40-transformed mouse cells and the uninfected embryonal carcinomas cells had identical partial peptide maps which were completely different from the partial peptide map of SV40 large T antigen. An Ad2+ND4-transformed hamster cell line also expressed a 54K protein that was specifically immunoprecipitated by SV40 T sera. The partial peptide maps of the mouse and hamster 54K protein were different, showing the host cell species specificity of these proteins. The 54K hamster protein was also unrelated to the Ad2+ND4 SV40 T antigen. Analogous proteins immunoprecipitated by SV40 T sera, ranging in molecular weight from 44K to 60K, were detected in human and monkey SV40-infected or -transformed cells. A wide variety of sera from hamsters and mice bearing SV40-induced tumors immunoprecipitated the 54K protein of SV40-transformed cells and murine embryonal carcinoma cells. Antibody produced by somatic cell hybrids between a B cell and a myeloma cell (hybridoma) against SV40 large T antigen also immunoprecipitated the 54K protein in virus-infected and -transformed cells, but did not do so in the embryonal carcinoma cell lines. We conclude that SV40 infection or transformation of mouse cells stimulates the synthesis or enhances the stability of a 54K protein. This protein appears to be associated with SV40 T antigen in SV40-infected and -transformed cells, and is co-immunoprecipitated by hybridomas sera to SV40 large T antigen. The 54K protein either shares antigenic determinants with SV40 T antigen or is itself immunogenic when in association with SV40 large T antigen. The protein varies with host cell species, and analogous proteins were observed in hamster, monkey and human cells. The role of this protein in transformation is unclear at present.
43026891
The first 30 years of p53: growing ever more complex
Thirty years ago p53 was discovered as a cellular partner of simian virus 40 large T-antigen, the oncoprotein of this tumour virus. The first decade of p53 research saw the cloning of p53 DNA and the realization that p53 is not an oncogene but a tumour suppressor that is very frequently mutated in human cancer. In the second decade of research, the function of p53 was uncovered: it is a transcription factor induced by stress, which can promote cell cycle arrest, apoptosis and senescence. In the third decade after its discovery new functions of this protein were revealed, including the regulation of metabolic pathways and cytokines that are required for embryo implantation. The fourth decade of research may see new p53-based drugs to treat cancer. What is next is anybody's guess.
30651700
p53 isoforms - A conspiracy to kidnap p53 tumor suppressor activity?
Abstract.For 25 years, the p53 tumor suppressor protein was considered the only protein expressed by the TP53 gene. However, in several studies the existence of p53 alternative transcripts in mouse and human cells has been documented, while their expression patterns and functions remained a mystery. Since 2002, several groups have identified and described the existence of up to 10 p53 isoforms and have demonstrated their roles in modulation of p53 suppressive activity. It is now clear that the patterns of p53 expression are much more complex than previously recognized and that these isoforms have the potential to act either synergistically or antagonistically, depending on their structure and mechanism of production. This review focuses on the different ways to produce p53 isoforms, on their specific properties, on their effect on p53 suppressive activity as well as on their implication in a new potential mechanism involved in p53 deregulation in cancer.
38127304
Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas.
Previous studies have demonstrated that allelic deletions of the short arm of chromosome 17 occur in over 75% of colorectal carcinomas. Twenty chromosome 17p markers were used to localize the common region of deletion in these tumors to a region contained within bands 17p12 to 17p13.3. This region contains the gene for the transformation-associated protein p53. Southern and Northern blot hybridization experiments provided no evidence for gross alterations of the p53 gene or surrounding sequences. As a more rigorous test of the possibility that p53 was a target of the deletions, the p53 coding regions from two tumors were analyzed; these two tumors, like most colorectal carcinomas, had allelic deletions of chromosome 17p and expressed considerable amounts of p53 messenger RNA from the remaining allele. The remaining p53 allele was mutated in both tumors, with an alanine substituted for valine at codon 143 of one tumor and a histidine substituted for arginine at codon 175 of the second tumor. Both mutations occurred in a highly conserved region of the p53 gene that was previously found to be mutated in murine p53 oncogenes. The data suggest that p53 gene mutations may be involved in colorectal neoplasia, perhaps through inactivation of a tumor suppressor function of the wild-type p53 gene.
25943294
TP53 Variations in Human Cancers: New Lessons from the IARC TP53 Database and Genomics Data
TP53 gene mutations are one of the most frequent somatic events in cancer. The IARC TP53 Database (http://p53.iarc.fr) is a popular resource that compiles occurrence and phenotype data on TP53 germline and somatic variations linked to human cancer. The deluge of data coming from cancer genomic studies generates new data on TP53 variations and attracts a growing number of database users for the interpretation of TP53 variants. Here, we present the current contents and functionalities of the IARC TP53 Database and perform a systematic analysis of TP53 somatic mutation data extracted from this database and from genomic data repositories. This analysis showed that IARC has more TP53 somatic mutation data than genomic repositories (29,000 vs. 4,000). However, the more complete screening achieved by genomic studies highlighted some overlooked facts about TP53 mutations, such as the presence of a significant number of mutations occurring outside the DNA‐binding domain in specific cancer types. We also provide an update on TP53 inherited variants including the ones that should be considered as neutral frequent variations. We thus provide an update of current knowledge on TP53 variations in human cancer as well as inform users on the efficient use of the IARC TP53 Database.
4348340
Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours
Mutations in the p53 tumour-suppressor gene are the most frequently observed genetic lesions in human cancers. To investigate the role of the p53 gene in mammalian development and tumori-genesis, a null mutation was introduced into the gene by homologous recombination in murine embryonic stem cells. Mice homozygous for the null allele appear normal but are prone to the spontaneous development of a variety of neoplasms by 6 months of age. These observations indicate that a normal p53 gene is dispensable for embryonic development, that its absence predisposes the animal to neoplastic disease, and that an oncogenic mutant form of p53 is not obligatory for the genesis of many types of tumours.
34609995
Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms.
Familial cancer syndromes have helped to define the role of tumor suppressor genes in the development of cancer. The dominantly inherited Li-Fraumeni syndrome (LFS) is of particular interest because of the diversity of childhood and adult tumors that occur in affected individuals. The rarity and high mortality of LFS precluded formal linkage analysis. The alternative approach was to select the most plausible candidate gene. The tumor suppressor gene, p53, was studied because of previous indications that this gene is inactivated in the sporadic (nonfamilial) forms of most cancers that are associated with LFS. Germ line p53 mutations have been detected in all five LFS families analyzed. These mutations do not produce amounts of mutant p53 protein expected to exert a trans-dominant loss of function effect on wild-type p53 protein. The frequency of germ line p53 mutations can now be examined in additional families with LFS, and in other cancer patients and families with clinical features that might be attributed to the mutation.
4234370
Germ-line transmission of a mutated p53 gene in a cancer-prone family with Li–Fraumeni syndrome
TUMOUR suppressor genes, whose usual function seems to be controlling normal cell proliferation1,2, have been implicated in many inherited and sporadic forms of malignancies (for reviews, see refs 3 and 4). Much evidence supports the concept of tumour formation by loss-of-function mutations in suppressor genes, as predicted by the two-hit model of Knudson5 and DeMars6. The suppressor gene, p53, is affected in such a manner by numerous mutations, which occur in a variety of human tumours7-10. These mutations usually represent the loss of one allele and the substitution of a single base in the other. We have now analysed the p53 gene in a family affected by Li–Fraumeni syndrome11, a rare autosomal dominant syndrome characterized by the occurrence of diverse mesenchymal and epithelial neoplasms at multiple sites. In some instances the neoplasms seem to be related to exposure to carcinogens, including ionizing radiation. The Li–Fraumeni family that we studied had noncancerous skin fibroblasts (NSF) with an unusual radiation-resistant phenotype12–15. DNA derived from the NSF cells of four family members, spanning two generations, had the same point mutation in codon 245 (GGC→GAC) of the p53 gene. This mutation leads to substitution of aspartic acid for glycine in one of the regions identified9 as a frequent target of point mutations in p53. The NSF cell lines with the mutation also retained the normal p53 allele. This inherited p53 mutation may predispose the members of this family to increased susceptibility to cancer.
1433663
Distinct p53 Transcriptional Programs Dictate Acute DNA-Damage Responses and Tumor Suppression
The molecular basis for p53-mediated tumor suppression remains unclear. Here, to elucidate mechanisms of p53 tumor suppression, we use knockin mice expressing an allelic series of p53 transcriptional activation mutants. Microarray analysis reveals that one mutant, p53(25,26), is severely compromised for transactivation of most p53 target genes, and, moreover, p53(25,26) cannot induce G(1)-arrest or apoptosis in response to acute DNA damage. Surprisingly, p53(25,26) retains robust activity in senescence and tumor suppression, indicating that efficient transactivation of the majority of known p53 targets is dispensable for these pathways. In contrast, the transactivation-dead p53(25,26,53,54) mutant cannot induce senescence or inhibit tumorigenesis, like p53 nullizygosity. Thus, p53 transactivation is essential for tumor suppression but, intriguingly, in association with a small set of novel p53 target genes. Together, our studies distinguish the p53 transcriptional programs involved in acute DNA-damage responses and tumor suppression-a critical goal for designing therapeutics that block p53-dependent side effects of chemotherapy without compromising p53 tumor suppression.
36892185
p53 efficiently suppresses tumor development in the complete absence of its cell-cycle inhibitory and proapoptotic effectors p21, Puma, and Noxa.
Activation of apoptosis through transcriptional induction of Puma and Noxa has long been considered to constitute the critical (if not sole) process by which p53 suppresses tumor development, although G1/S boundary cell-cycle arrest via induction of the CDK inhibitor p21 has also been thought to contribute. Recent analyses of mice bearing mutations that impair p53-mediated induction of select target genes have indicated that activation of apoptosis and G1/S cell-cycle arrest may, in fact, be dispensable for p53-mediated tumor suppression. However, the expression of Puma, Noxa, and p21 was not abrogated in these mutants, only reduced; therefore, the possibility that the reduced levels of these critical effectors of p53-mediated apoptosis and G1/S-cell-cycle arrest sufficed to prevent tumorigenesis could not be excluded. To resolve this important issue, we have generated mice deficient for p21, Puma, and Noxa (p21-/-puma-/-noxa-/- mice). Cells from these mice were deficient in their ability to undergo p53-mediated apoptosis, G1/S cell-cycle arrest, and senescence. Nonetheless, these animals remained tumor free until at least 500 days, in contrast to p53-deficient mice, which had all succumbed to lymphoma or sarcoma by 250 days. Interestingly, DNA lesions induced by γ-irradiation persisted longer in p53-deficient cells compared to wild-type or p21-/-puma-/-noxa-/- cells, and the former failed to transcriptionally activate several p53 target genes implicated in DNA repair. These results demonstrate beyond a doubt that the induction of apoptosis, cell-cycle arrest, and possibly senescence is dispensable for p53-mediated suppression of spontaneous tumor development and indicate that coordination of genomic stability and possibly other processes, such as metabolic adaptation, may instead be critical.
1377010
Tumor Suppression in the Absence of p53-Mediated Cell-Cycle Arrest, Apoptosis, and Senescence
Cell-cycle arrest, apoptosis, and senescence are widely accepted as the major mechanisms by which p53 inhibits tumor formation. Nevertheless, it remains unclear whether they are the rate-limiting steps in tumor suppression. Here, we have generated mice bearing lysine to arginine mutations at one (p53(K117R)) or three (p53(3KR); K117R+K161R+K162R) of p53 acetylation sites. Although p53(K117R/K117R) cells are competent for p53-mediated cell-cycle arrest and senescence, but not apoptosis, all three of these processes are ablated in p53(3KR/3KR) cells. Surprisingly, unlike p53 null mice, which rapidly succumb to spontaneous thymic lymphomas, early-onset tumor formation does not occur in either p53(K117R/K117R) or p53(3KR/3KR) animals. Notably, p53(3KR) retains the ability to regulate energy metabolism and reactive oxygen species production. These findings underscore the crucial role of acetylation in differentially modulating p53 responses and suggest that unconventional activities of p53, such as metabolic regulation and antioxidant function, are critical for suppression of early-onset spontaneous tumorigenesis.
208186769
Deconstructing networks of p53-mediated tumor suppression in vivo.
The transcription factor p53 is a vital tumor suppressor. Upon activation by diverse stresses including oncogene activation, DNA damage, hypoxia and nutrient deprivation, p53 activates a panoply of target genes and orchestrates numerous downstream responses that suppress tumorigenesis. Although early studies of p53 suggested that its ability to induce cell cycle arrest, senescence and apoptosis programs accounted for its tumor-suppressor activity, more recent studies have challenged this notion. Moreover, p53 regulates a suite of additional processes, such as metabolism, stem cell function, invasion and metastasis. The processes p53 coordinately regulates to enact tumor suppression, and how such regulation occurs, thus remain elusive. In this review, we will summarize our current knowledge of p53-mediated tumor-suppressive mechanisms gleaned from in vivo studies in mouse models.
9008238
Influenza NS1 interacts with p53 and alters its binding to p53‐responsive genes, in a promoter‐dependent manner
p53 physically interacts with NS1 by anti bait coimmunoprecipitation (View interaction)
24564588
Modulation of p53β and p53γ expression by regulating the alternative splicing of TP53 gene modifies cellular response
In addition to the tumor suppressor p53 protein, also termed p53α, the TP53 gene produces p53β and p53γ through alternative splicing of exons 9β and 9γ located within TP53 intron 9. Here we report that both TG003, a specific inhibitor of Cdc2-like kinases (Clk) that regulates the alternative splicing pre-mRNA pathway, and knockdown of SFRS1 increase expression of endogenous p53β and p53γ at mRNA and protein levels. Development of a TP53 intron 9 minigene shows that TG003 treatment and knockdown of SFRS1 promote inclusion of TP53 exons 9β/9γ. In a series of 85 primary breast tumors, a significant association was observed between expression of SFRS1 and α variant, supporting our experimental data. Using siRNA specifically targeting exons 9β/9γ, we demonstrate that cell growth can be driven by modulating p53β and p53γ expression in an opposite manner, depending on the cellular context. In MCF7 cells, p53β and p53γ promote apoptosis, thus inhibiting cell growth. By transient transfection, we show that p53β enhanced p53α transcriptional activity on the p21 and Bax promoters, while p53γ increased p53α transcriptional activity on the Bax promoter only. Moreover, p53β and p53γ co-immunoprecipitate with p53α only in the presence of p53-responsive promoter. Interestingly, although p53β and p53γ promote apoptosis in MCF7 cells, p53β and p53γ maintain cell growth in response to TG003 in a p53α-dependent manner. The dual activities of p53β and p53γ isoforms observed in non-treated and TG003-treated cells may result from the impact of TG003 on both expression and activities of p53 isoforms. Overall, our data suggest that p53β and p53γ regulate cellular response to modulation of alternative splicing pre-mRNA pathway by a small drug inhibitor. The development of novel drugs targeting alternative splicing process could be used as a novel therapeutic approach in human cancers.
23064539
Polysome-profiling in small tissue samples
Abstract Polysome-profiling is commonly used to study translatomes and applies laborious extraction of efficiently translated mRNA (associated with >3 ribosomes) from a large volume across many fractions. This property makes polysome-profiling inconvenient for larger experimental designs or samples with low RNA amounts. To address this, we optimized a non-linear sucrose gradient which reproducibly enriches for efficiently translated mRNA in only one or two fractions, thereby reducing sample handling 5–10-fold. The technique generates polysome-associated RNA with a quality reflecting the starting material and, when coupled with smart-seq2 single-cell RNA sequencing, translatomes in small tissues from biobanks can be obtained. Translatomes acquired using optimized non-linear gradients resemble those obtained with the standard approach employing linear gradients. Polysome-profiling using optimized non-linear gradients in serum starved HCT-116 cells with or without p53 showed that p53 status associates with changes in mRNA abundance and translational efficiency leading to changes in protein levels. Moreover, p53 status also induced translational buffering whereby changes in mRNA levels are buffered at the level of mRNA translation. Thus, here we present a polysome-profiling technique applicable to large study designs, primary cells and frozen tissue samples such as those collected in biobanks.
206235557
Identification of a core TP53 transcriptional program with highly distributed tumor suppressive activity.
The tumor suppressor TP53 is the most frequently mutated gene product in human cancer. Close to half of all solid tumors carry inactivating mutations in the TP53 gene, while in the remaining cases, TP53 activity is abrogated by other oncogenic events, such as hyperactivation of its endogenous repressors MDM2 or MDM4. Despite identification of hundreds of genes regulated by this transcription factor, it remains unclear which direct target genes and downstream pathways are essential for the tumor suppressive function of TP53. We set out to address this problem by generating multiple genomic data sets for three different cancer cell lines, allowing the identification of distinct sets of TP53-regulated genes, from early transcriptional targets through to late targets controlled at the translational level. We found that although TP53 elicits vastly divergent signaling cascades across cell lines, it directly activates a core transcriptional program of ∼100 genes with diverse biological functions, regardless of cell type or cellular response to TP53 activation. This core program is associated with high-occupancy TP53 enhancers, high levels of paused RNA polymerases, and accessible chromatin. Interestingly, two different shRNA screens failed to identify a single TP53 target gene required for the anti-proliferative effects of TP53 during pharmacological activation in vitro. Furthermore, bioinformatics analysis of thousands of cancer genomes revealed that none of these core target genes are frequently inactivated in tumors expressing wild-type TP53. These results support the hypothesis that TP53 activates a genetically robust transcriptional program with highly distributed tumor suppressive functions acting in diverse cellular contexts.
2138602
p53-directed translational control can shape and expand the universe of p53 target genes
The increasing number of genome-wide transcriptome analyses focusing on p53-induced cellular responses in many cellular contexts keeps adding to the already numerous p53-regulated transcriptional networks. To investigate post-transcriptional controls as an additional dimension of p53-directed gene expression responses, we performed a translatome analysis through polysomal profiling on MCF7 cells upon 16 hours of doxorubicin or nutlin-3a treatment. The comparison between the transcriptome and the translatome revealed a considerable level of uncoupling, characterized by genes whose transcription variations did not correlate with translation variations. Interestingly, uncoupled genes were associated with apoptosis, DNA and RNA metabolism and cell cycle functions, suggesting that post-transcriptional control can modulate classical p53-regulated responses. Furthermore, even for well-established p53 targets that were differentially expressed both at the transcriptional and translational levels, quantitative differences between the transcriptome, subpolysomal and polysomal RNAs were evident. As we searched mechanisms underlying gene expression uncoupling, we identified the p53-dependent modulation of six RNA-binding proteins, where hnRNPD (AUF1) and CPEB4 are direct p53 transcriptional targets, whereas SRSF1, DDX17, YBX1 and TARDBP are indirect targets (genes modulated preferentially in the subpolysomal or polysomal mRNA level) modulated at the translational level in a p53-dependent manner. In particular, YBX1 translation appeared to be reduced by p53 via two different mechanisms, one related to mTOR inhibition and the other to miR-34a expression. Overall, we established p53 as a master regulator of translational control and identified new p53-regulated genes affecting translation that can contribute to p53-dependent cellular responses.
1770881
p53 induces transcriptional and translational programs to suppress cell proliferation and growth
BackgroundCell growth and proliferation are tightly connected to ensure that appropriately sized daughter cells are generated following mitosis. Energy stress blocks cell growth and proliferation, a critical response for survival under extreme conditions. Excessive oncogenic stress leads to p53 activation and the induction of senescence, an irreversible state of cell-cycle arrest and a critical component in the suppression of tumorigenesis. Nutrient-sensing and mitogenic cues converge on a major signaling node, which regulates the activity of the mTOR kinase. Although transcriptional responses to energy and oncogenic stresses have been examined by many gene-expression experiments, a global exploration of the modulation of mRNA translation in response to these conditions is lacking.ResultsWe combine RNA sequencing and ribosomal profiling analyses to systematically delineate modes of transcriptional and translational regulation induced in response to conditions of limited energy, oncogenic stress and cellular transformation. We detect a key role for mTOR and p53 in these distinct physiological states, and provide the first genome-wide demonstration that p53 activation results in mTOR inhibition and a consequent global repression of protein translation. We confirm the role of the direct p53 target genes Sestrin1 and Sestrin2 in this response, as part of the broad modulation of gene expression induced by p53 activation.ConclusionsWe delineate a bimodal tumor-suppressive regulatory program activated by p53, in which cell-cycle arrest is imposed mainly at the transcriptional level, whereas cell growth inhibition is enforced by global repression of the translation machinery.
198084
Targeting IRES-Mediated p53 Synthesis for Cancer Diagnosis and Therapeutics
While translational regulation of p53 by the internal ribosome entry site (IRES) at its 5′-untranslated region following DNA damage has been widely accepted, the detailed mechanism underlying the translational control of p53 by its IRES sequence is still poorly understood. In this review, we will focus on the latest progress in identifying novel regulatory proteins of the p53 IRES and in uncovering the functional connection between defective IRES-mediated p53 translation and tumorigenesis. We will also discuss how these findings may lead to a better understanding of the process of oncogenesis and open up new avenues for cancer diagnosis and therapeutics.
15624598
Ribosomal Proteins Control or Bypass p53 during Nucleolar Stress
The nucleolus is the site of ribosome biogenesis, a complex process that requires the coordinate activity of all three RNA polymerases and hundreds of non-ribosomal factors that participate in the maturation of ribosomal RNA (rRNA) and assembly of small and large subunits. Nevertheless, emerging studies have highlighted the fundamental role of the nucleolus in sensing a variety of cellular stress stimuli that target ribosome biogenesis. This condition is known as nucleolar stress and triggers several response pathways to maintain cell homeostasis, either p53-dependent or p53-independent. The mouse double minute (MDM2)-p53 stress signaling pathways are activated by multiple signals and are among the most important regulators of cellular homeostasis. In this review, we will focus on the role of ribosomal proteins in p53-dependent and p53-independent response to nucleolar stress considering novel identified regulators of these pathways. We describe, in particular, the role of ribosomal protein uL3 (rpL3) in p53-independent nucleolar stress signaling pathways.
27700160
p53 and ribosome biogenesis stress: The essentials
Cell proliferation and cell growth are two tightly linked processes, as the proliferation program cannot be executed without proper accumulation of cell mass, otherwise endangering the fate of the two daughter cells. It is therefore not surprising that ribosome biogenesis, a key element in cell growth, is regulated by many cell cycle regulators. This regulation is exerted transcriptionally and post‐transcriptionally, in conjunction with numerous intrinsic and extrinsic signals. Those signals eventually converge at the nucleolus, the cellular compartment that is not only responsible for executing the ribosome biogenesis program, but also serves as a regulatory hub, responsible for integrating and transmitting multiple stress signals to the omnipotent cell fate gatekeeper, p53. In this review we discuss when, how and why p53 is activated upon ribosomal biogenesis stress, and how perturbation of this critical regulatory interplay may impact human disease.
15375229
The role of the 5' terminal region of p53 mRNA in the p53 gene expression.
The p53 tumour suppressor protein is one of the major factors responsible for cell cycle regulation and protection against cancer development. This is why it is often referred to as "the guardian of the genome". On the other hand, mutations in the p53 gene are connected with more than 50% of tumours of various types. The thirty-six years of extensive research on the p53 gene and its protein products have shown how sophisticated the p53-based cell system control is. An additional level of complexity of the p53 research is connected with at least twelve p53 isoforms which have been identified in the cell. Importantly, disturbance of the p53 isoforms' expression seems to play a key role in tumorigenesis, cell differentiation and cell response to pathogenic bacteria, and RNA and DNA viruses. Expression of various p53 isoforms results from the usage of different transcription promoters, alternative splicing events and translation initiation from alternative AUG codons. The importance of the 5'-terminal regions of different p53 mRNA transcripts in the multi-level regulation of the p53 gene has recently been documented. In this review we focus on the structural features of these regions and their specific role in the p53 translation initiation process.
5995660
Biological functions of p53 isoforms through evolution: lessons from animal and cellular models
The TP53 tumour-suppressor gene is expressed as several protein isoforms generated by different mechanisms, including use of alternative promoters, splicing sites and translational initiation sites, that are conserved through evolution and within the TP53 homologues, TP63 and TP73. Although first described in the eighties, the importance of p53 isoforms in regulating the suppressive functions of p53 has only become evident in the last 10 years, by analogy with observations that p63 and p73 isoforms appeared indispensable to fully understand the biological functions of TP63 and TP73. This review summarizes recent advances in the field of ‘p53 isoforms’, including new data on p63 and p73 isoforms. Details of the alternative mechanisms that produce p53 isoforms and cis- and trans-regulators identified are provided. The main focus is on their biological functions (apoptosis, cell cycle, aging and so on) in cellular and animal models, including mouse, zebrafish and Drosophila. Finally, the deregulation of p53 isoform expression in human cancers is reviewed. Based on these latest results, several developments are expected in the future: the identification of drugs modulating p53 isoform expression; the generation of animal models and the evaluation of the use of p53 isoform as biomarkers in human cancers.
24727790
Synergistic Rescue of Nonsense Mutant Tumor Suppressor p53 by Combination Treatment with Aminoglycosides and Mdm2 Inhibitors
The tumor suppressor gene TP53 is inactivated by mutation in a large fraction of human tumors. Around 10% of TP53 mutations are nonsense mutations that lead to premature termination of translation and expression of truncated unstable and non-functional p53 protein. Aminoglycosides G418 (geneticin) and gentamicin have been shown to induce translational readthrough and expression of full-length p53. However, aminoglycosides have severe side effects that limit their clinical use. Here, we show that combination treatment with a proteasome inhibitor or compounds that disrupt p53-Mdm2 binding can synergistically enhance levels of full-length p53 upon aminoglycoside-induced readthrough of R213X nonsense mutant p53. Full-length p53 expressed upon combination treatment is functionally active as assessed by upregulation of p53 target genes, suppression of cell growth, and induction of cell death. Thus, our results demonstrate that combination treatment with aminoglycosides and compounds that inhibit p53 degradation is synergistic and can provide significantly improved efficacy of readthrough when compared with aminoglycosides alone. This may have implications for future cancer therapy based on reactivation of nonsense mutant TP53.
4703117
Gentamicin B1 is a minor gentamicin component with major nonsense mutation suppression activity
Significance The small number of patients for each of the >5,000 rare genetic diseases restricts allocation of resources for developing disease-specific therapeutics. However, for all these diseases about 10% of patients share a common mutation type, nonsense mutations. They introduce a premature termination codon (PTC) that forms truncated proteins. Pharmaceutical gentamicin, a mixture of several related aminoglycosides, is an antibiotic frequently used in humans that shows weak and variable PTC readthrough activity. Using a variety of in vitro and in vivo assays we report that the major gentamicin components lack PTC readthrough activity but that a minor component, gentamicin B1, is responsible for most of the PTC readthrough activity of this drug and has potential to treat patients with nonsense mutations. Nonsense mutations underlie about 10% of rare genetic disease cases. They introduce a premature termination codon (PTC) and prevent the formation of full-length protein. Pharmaceutical gentamicin, a mixture of several related aminoglycosides, is a frequently used antibiotic in humans that can induce PTC readthrough and suppress nonsense mutations at high concentrations. However, testing of gentamicin in clinical trials has shown that safe doses of this drug produce weak and variable readthrough activity that is insufficient for use as therapy. In this study we show that the major components of pharmaceutical gentamicin lack PTC readthrough activity but the minor component gentamicin B1 (B1) is a potent readthrough inducer. Molecular dynamics simulations reveal the importance of ring I of B1 in establishing a ribosome configuration that permits pairing of a near-cognate complex at a PTC. B1 induced readthrough at all three nonsense codons in cultured cancer cells with TP53 (tumor protein p53) mutations, in cells from patients with nonsense mutations in the TPP1 (tripeptidyl peptidase 1), DMD (dystrophin), SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1), and COL7A1 (collagen type VII alpha 1 chain) genes, and in an in vivo tumor xenograft model. The B1 content of pharmaceutical gentamicin is highly variable and major gentamicins suppress the PTC readthrough activity of B1. Purified B1 provides a consistent and effective source of PTC readthrough activity to study the potential of nonsense suppression for treatment of rare genetic disorders.
205895792
Characterization of new-generation aminoglycoside promoting premature termination codon readthrough in cancer cells
ABSTRACT Nonsense mutations, generating premature termination codons (PTCs), account for 10% to 30% of the mutations in tumor suppressor genes. Nonsense translational suppression, induced by small molecules including gentamicin and G418, has been suggested as a potential therapy to counteract the deleterious effects of nonsense mutations in several genetic diseases and cancers. We describe here that NB124, a synthetic aminoglycoside derivative recently developed especially for PTC suppression, strongly induces apoptosis in human tumor cells by promoting high level of PTC readthrough. Using a reporter system, we showed that NB124 suppressed several of the PTCs encountered in tumor suppressor genes, such as the p53 and APC genes. We also showed that NB124 counteracted p53 mRNA degradation by nonsense-mediated decay (NMD). Both PTC suppression and mRNA stabilization contributed to the production of a full-length p53 protein capable of activating p53-dependent genes, thereby specifically promoting high levels of apoptosis. This new-generation aminoglycoside thus outperforms the only clinically available readthrough inducer (gentamicin). These results have important implications for the development of personalised treatments of PTC-dependent diseases and for the development of new drugs modifying translation fidelity.
25085442
p53, a translational regulator: contribution to its tumour-suppressor activity
A growing body of evidences indicate that deregulation of translation contributes to tumourigenesis. In tumours, alterations of translational control of specific mRNAs encoding oncogenes or tumour suppressors have been extensively reported. Moreover, restricting the rate of protein synthesis has been shown to delays tumourigenesis in C-Myc overexpressing or PTEN deleted mice models. Finally, the specific inhibition of RNA polymerase I (RNA pol I) has been shown to kill cancer cells without affecting normal cells. It thus emerges that a tight coordination between the rate of global protein synthesis and a defined translational program is required to prevent tumour development. In this review, we expose the evidences supporting that p53 acts as a translational regulator. In addition, this review discusses the notion that the ability to maintain both a selective translational program and a low level of protein synthesis could directly contribute to the p53 tumour-suppressor activity.
4562944
The mechanism of eukaryotic translation initiation and principles of its regulation
Protein synthesis is principally regulated at the initiation stage (rather than during elongation or termination), allowing rapid, reversible and spatial control of gene expression. Progress over recent years in determining the structures and activities of initiation factors, and in mapping their interactions in ribosomal initiation complexes, have advanced our understanding of the complex translation initiation process. These developments have provided a solid foundation for studying the regulation of translation initiation by mechanisms that include the modulation of initiation factor activity (which affects almost all scanning-dependent initiation) and through sequence-specific RNA-binding proteins and microRNAs (which affect individual mRNAs).
10840093
Regulation of Translation Initiation in Eukaryotes: Mechanisms and Biological Targets
Translational control in eukaryotic cells is critical for gene regulation during nutrient deprivation and stress, development and differentiation, nervous system function, aging, and disease. We describe recent advances in our understanding of the molecular structures and biochemical functions of the translation initiation machinery and summarize key strategies that mediate general or gene-specific translational control, particularly in mammalian systems.
27107846
Regulation of mRNA translation by signaling pathways.
mRNA translation is the most energy consuming process in the cell. In addition, it plays a pivotal role in the control of gene expression and is therefore tightly regulated. In response to various extracellular stimuli and intracellular cues, signaling pathways induce quantitative and qualitative changes in mRNA translation by modulating the phosphorylation status and thus the activity of components of the translational machinery. In this work we focus on the phosphoinositide 3-kinase (PI3K)/AKT and the mitogen-activated protein kinase (MAPK) pathways, as they are strongly implicated in the regulation of translation in homeostasis, whereas their malfunction has been linked to aberrant translation in human diseases, including cancer.
4360955
Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function
The cloning is described of two related human complementary DNAs encoding polypeptides that interact specifically with the translation initiation factor elF-4E, which binds to the messenger RNA 5'-cap structure. Interaction of these proteins with elF-4E inhibits translation but treatment of cells with insulin causes one of them to become hyperphosphorylated and dissociate from elF-4E, thereby relieving the translational inhibition. The action of this new regulator of protein synthesis is therefore modulated by insulin, which acts to stimulate the overall rate of translation and promote cell growth.
33457819
Cap-dependent translation initiation in eukaryotes is regulated by a molecular mimic of eIF4G.
eIF4G uses a conserved Tyr-X-X-X-X-Leu-phi segment (where X is variable and phi is hydrophobic) to recognize eIF4E during cap-dependent translation initiation in eukaryotes. High-resolution X-ray crystallography and complementary biophysical methods have revealed that this eIF4E recognition motif undergoes a disorder-to-order transition, adopting an L-shaped, extended chain/alpha-helical conformation when it interacts with a phylogenetically invariant portion of the convex surface of eIF4E. Inhibitors of translation initiation known as eIF4E-binding proteins (4E-BPs) contain similar eIF4E recognition motifs. These molecules are molecular mimics of eIF4G, which act by occupying the same binding site on the convex dorsum of eIF4E and blocking assembly of the translation machinery. The implications of our results for translation initiation are discussed in detail, and a molecular mechanism for relief of translation inhibition following phosphorylation of the 4E-BPs is proposed.
206671728
Revisiting the Nucleolus: From Marker to Dynamic Integrator of Cancer Signaling
Deregulations in ribosome biogenesis contribute to tumorigenesis and could be targeted to treat cancer. Key signaling pathways (such as phosphoinositide 3-kinase, Myc, and RAS) act as sensors of energy, stress, and nutrient availability and integrate these inputs to directly control ribosome production and gene expression at the translational level. This activity is normally directly coupled to cell growth, division, and survival. However, it remains poorly understood the extent to which changes in ribosome number and nucleolar integrity downstream of these key signaling pathways contribute to their oncogenic activity. Emerging studies provide interesting insight into how deregulations in RNA polymerase I activity may lead to tumorigenesis and suggest that new drugs targeting ribosomal DNA transcription may hold great promise for the treatment of cancer.
28475165
An encore for ribosome biogenesis in the control of cell proliferation
Although the importance of cell growth for cell-cycle progression has been recognized for thirty years, the molecular basis of this relationship is poorly understood. However, researchers have begun to tease apart these two processes in model systems. This commentary focuses on one potential mechanism by which ribosome biogenesis antagonizes cell-cycle progression until the cell has grown to an adequate size.
23906871
MYC as a regulator of ribosome biogenesis and protein synthesis
MYC regulates the transcription of thousands of genes required to coordinate a range of cellular processes, including those essential for proliferation, growth, differentiation, apoptosis and self-renewal. Recently, MYC has also been shown to serve as a direct regulator of ribosome biogenesis. MYC coordinates protein synthesis through the transcriptional control of RNA and protein components of ribosomes, and of gene products required for the processing of ribosomal RNA, the nuclear export of ribosomal subunits and the initiation of mRNA translation. We discuss how the modulation of ribosome biogenesis by MYC may be essential to its physiological functions as well as its pathological role in tumorigenesis.
13491541
Translational control in cancer etiology.
The link between perturbations in translational control and cancer etiology is becoming a primary focus in cancer research. It has now been established that genetic alterations in several components of the translational apparatus underlie spontaneous cancers as well as an entire class of inherited syndromes known as "ribosomopathies" associated with increased cancer susceptibility. These discoveries have illuminated the importance of deregulations in translational control to very specific cellular processes that contribute to cancer etiology. In addition, a growing body of evidence supports the view that deregulation of translational control is a common mechanism by which diverse oncogenic pathways promote cellular transformation and tumor development. Indeed, activation of these key oncogenic pathways induces rapid and dramatic translational reprogramming both by increasing overall protein synthesis and by modulating specific mRNA networks. These translational changes promote cellular transformation, impacting almost every phase of tumor development. This paradigm represents a new frontier in the multihit model of cancer formation and offers significant promise for innovative cancer therapies. Current research, in conjunction with cutting edge technologies, will further enable us to explore novel mechanisms of translational control, functionally identify translationally controlled mRNA groups, and unravel their impact on cellular transformation and tumorigenesis.
6438075
Translational control in cancer
Remarkable progress has been made in defining a new understanding of the role of mRNA translation and protein synthesis in human cancer. Translational control is a crucial component of cancer development and progression, directing both global control of protein synthesis and selective translation of specific mRNAs that promote tumour cell survival, angiogenesis, transformation, invasion and metastasis. Translational control of cancer is multifaceted, involving alterations in translation factor levels and activities unique to different types of cancers, disease stages and the tumour microenvironment. Several clinical efforts are underway to target specific components of the translation apparatus or unique mRNA translation elements for cancer therapeutics.
12611406
Nucleolus, ribosomes, and cancer.
The complex aspects linking the nucleolus and ribosome biogenesis to cancer are reviewed here. The available evidence indicates that the morphological and functional changes in the nucleolus, widely observed in cancer tissues, are a consequence of both the increased demand for ribosome biogenesis, which characterizes proliferating cells, and the changes in the mechanisms controlling cell proliferation. In fact, the loss or functional changes in the two major tumor suppressor proteins pRB and p53 cause an up-regulation of ribosome biogenesis in cancer tissues. In this context, the association in human carcinomas of nucleolar hypertrophy with bad prognoses is worthy of note. Further, an increasing amount of data coming from studies on both hepatitis virus-induced chronic liver diseases and a subset of rare inherited disorders, including X-linked dyskeratosis congenita, suggests an active role of the nucleolus in tumorigenesis. Both an up-regulation of ribosome production and changes in the ribosome structure might causally contribute to neoplastic transformation, by affecting the balance of protein translation, thus altering the synthesis of proteins that play an important role in the genesis of cancer.
18987782
Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency
The Myc oncogene regulates the expression of several components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, RNA polymerase III and ribosomal DNA. Whether and how increasing the cellular protein synthesis capacity affects the multistep process leading to cancer remains to be addressed. Here we use ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in Eμ-Myc/+ transgenic mice to normal levels, and show that the oncogenic potential of Myc in this context is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc-overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a new mechanism that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation used to regulate the expression of selective messenger RNAs. We show that an aberrant increase in cap-dependent translation downstream of Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site (IRES)-dependent translation that is required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic-specific expression of the endogenous IRES-dependent form of Cdk11 (also known as Cdc2l and PITSLRE), which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in Eμ-Myc/+ mice. When accurate translational control is re-established in Eμ-Myc/+ mice, genome instability is suppressed. Our findings demonstrate how perturbations in translational control provide a highly specific outcome for gene expression, genome stability and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post-genomic level.
5351000
A subset of platinum-containing chemotherapeutic agents kills cells by inducing ribosome biogenesis stress
Cisplatin and its platinum analogs, carboplatin and oxaliplatin, are some of the most widely used cancer chemotherapeutics. Although cisplatin and carboplatin are used primarily in germ cell, breast and lung malignancies, oxaliplatin is instead used almost exclusively to treat colorectal and other gastrointestinal cancers. Here we utilize a unique, multi-platform genetic approach to study the mechanism of action of these clinically established platinum anti-cancer agents, as well as more recently developed cisplatin analogs. We show that oxaliplatin, unlike cisplatin and carboplatin, does not kill cells through the DNA-damage response. Rather, oxaliplatin kills cells by inducing ribosome biogenesis stress. This difference in drug mechanism explains the distinct clinical implementation of oxaliplatin relative to cisplatin, and it might enable mechanistically informed selection of distinct platinum drugs for distinct malignancies. These data highlight the functional diversity of core components of front-line cancer therapy and the potential benefits of applying a mechanism-based rationale to the use of our current arsenal of anti-cancer drugs.
2447856
Inhibition of RNA polymerase I as a therapeutic strategy to promote cancer-specific activation of p53.
Increased transcription of ribosomal RNA genes (rDNA) by RNA Polymerase I is a common feature of human cancer, but whether it is required for the malignant phenotype remains unclear. We show that rDNA transcription can be therapeutically targeted with the small molecule CX-5461 to selectively kill B-lymphoma cells in vivo while maintaining a viable wild-type B cell population. The therapeutic effect is a consequence of nucleolar disruption and activation of p53-dependent apoptotic signaling. Human leukemia and lymphoma cell lines also show high sensitivity to inhibition of rDNA transcription that is dependent on p53 mutational status. These results identify selective inhibition of rDNA transcription as a therapeutic strategy for the cancer specific activation of p53 and treatment of hematologic malignancies.
1090039
A targeting modality for destruction of RNA polymerase I that possesses anticancer activity.
We define the activity and mechanisms of action of a small molecule lead compound for cancer targeting. We show that the compound, BMH-21, has wide and potent antitumorigenic activity across NCI60 cancer cell lines and represses tumor growth in vivo. BMH-21 binds GC-rich sequences, which are present at a high frequency in ribosomal DNA genes, and potently and rapidly represses RNA polymerase I (Pol I) transcription. Strikingly, we find that BMH-21 causes proteasome-dependent destruction of RPA194, the large catalytic subunit protein of Pol I holocomplex, and this correlates with cancer cell killing. Our results show that Pol I activity is under proteasome-mediated control, which reveals an unexpected therapeutic opportunity.
25753284
Translation Control by p53
The translation of mRNAs plays a critical role in the regulation of gene expression and therefore, in the regulation of cell proliferation, differentiation and apoptosis. Unrestricted initiation of translation causes malignant transformation and plays a key role in the maintenance and progression of cancers. Translation initiation is regulated by the ternary complex and the eukaryotic initiation factor 4F (eIF4F) complex. The p53 tumor suppressor protein is the most well studied mammalian transcription factor that mediates a variety of anti-proliferative processes. Post-transcriptional mechanisms of gene expression in general and those of translation in particular play a major role in shaping the protein composition of the cell. The p53 protein regulates transcription and controls eIF4F, the ternary complex and the synthesis of ribosomal components, including the down-regulation of rRNA genes. In summary, the induction of p53 regulates protein synthesis and translational control to inhibit cell growth.
20693285
New frontiers in translational control of the cancer genome
The past several years have seen dramatic leaps in our understanding of how gene expression is rewired at the translation level during tumorigenesis to support the transformed phenotype. This work has been driven by an explosion in technological advances and is revealing previously unimagined regulatory mechanisms that dictate functional expression of the cancer genome. In this Review we discuss emerging trends and exciting new discoveries that reveal how this translational circuitry contributes to specific aspects of tumorigenesis and cancer cell function, with a particular focus on recent insights into the role of translational control in the adaptive response to oncogenic stress conditions.
206645997
Translational control by 5′-untranslated regions of eukaryotic mRNAs
The eukaryotic 5′ untranslated region (UTR) is critical for ribosome recruitment to the messenger RNA (mRNA) and start codon choice and plays a major role in the control of translation efficiency and shaping the cellular proteome. The ribosomal initiation complex is assembled on the mRNA via a cap-dependent or cap-independent mechanism. We describe various mechanisms controlling ribosome scanning and initiation codon selection by 5′ upstream open reading frames, translation initiation factors, and primary and secondary structures of the 5′UTR, including particular sequence motifs. We also discuss translational control via phosphorylation of eukaryotic initiation factor 2, which is implicated in learning and memory, neurodegenerative diseases, and cancer.
11502334
Derepression of the Her-2 uORF is mediated by a novel post-transcriptional control mechanism in cancer cells.
Transcripts harboring 5' upstream open reading frames (uORFs) are often found in genes controlling cell growth including receptors, oncogenes, or growth factors. uORFs can modulate translation or RNA stability and mediate inefficient translation of these potent proteins under normal conditions. In dysregulated cancer cells, where the gene product, for example Her-2 receptor, is overexpressed, post-transcriptional processes must exist that serve to override the inhibitory effects of the uORFs. The 5' untranslated region (UTR) of Her-2 mRNA contains a short uORF that represses translation of the downstream coding region. We demonstrate that in Her-2 overexpressing breast cancer cells, the 3' UTR of the Her-2 mRNA can override translational inhibition mediated by the Her-2 uORF. Within this 3' UTR, a translational derepression element (TDE) that binds to a 38-kDa protein was identified. These results define a novel biological mechanism in which translational control of genes harboring a 5' uORF can be modulated by elements in their 3' UTRs.
30782788
The Functions of MicroRNAs: mRNA Decay and Translational Repression.
MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs, which regulate complementary mRNAs by inducing translational repression and mRNA decay. Although this dual repression system seems to operate in both animals and plants, genetic and biochemical studies suggest that the mechanism underlying the miRNA-mediated silencing is different in the two kingdoms. Here, we review the recent progress in our understanding of how miRNAs mediate translational repression and mRNA decay, and discuss the contributions of the two silencing modes to the overall silencing effect in both kingdoms.
5844855
RNA‐binding proteins and post‐transcriptional gene regulation
RNAs in cells are associated with RNA‐binding proteins (RBPs) to form ribonucleoprotein (RNP) complexes. The RBPs influence the structure and interactions of the RNAs and play critical roles in their biogenesis, stability, function, transport and cellular localization. Eukaryotic cells encode a large number of RBPs (thousands in vertebrates), each of which has unique RNA‐binding activity and protein–protein interaction characteristics. The remarkable diversity of RBPs, which appears to have increased during evolution in parallel to the increase in the number of introns, allows eukaryotic cells to utilize them in an enormous array of combinations giving rise to a unique RNP for each RNA. In this short review, we focus on the RBPs that interact with pre‐mRNAs and mRNAs and discuss their roles in the regulation of post‐transcriptional gene expression.
21341113
Cap-independent regulation of gene expression in apoptosis.
Expression of the proteome is tightly regulated at the level of protein synthesis. Translational control is a critical homeostatic mechanism that allows the cell to rapidly change its phenotype in the face of an intra- and extra-cellular environment in constant flux. It is becoming increasingly clear that when it comes to protein translation during cell stress, all mRNAs are not treated equally. The translation of the majority of mRNAs is compromised during cell stresses that induce programmed cell death such as hypoxia, or DNA damage. However, cellular messages harbouring Internal Ribosome Entry Site elements (IRES) within their 5' untranslated regions are insensitive to stress-induced repression of global translation. Instead, these IRES-containing mRNAs use a poorly understood alternative mechanism of translation that allows continued expression of proteins that are required for the cell to recover from a transient stress or to proceed down the path toward apoptotic death. This review will highlight recent literature that suggests why global translation rates are impaired during stress and apoptosis and how these conditions mediate a switch in the mechanism by which pertinent proteins are synthesized. In addition, recent advances towards our understanding of the physiological role and mechanism of IRES-mediated translation in the context of cell stress-induced apoptosis and human disease will be examined.
10493830
The role of IRES trans-acting factors in regulating translation initiation.
The majority of mRNAs in eukaryotic cells are translated via a method that is dependent upon the recognition of, and binding to, the methylguanosine cap at the 5' end of the mRNA, by a set of protein factors termed eIFs (eukaryotic initiation factors). However, many of the eIFs involved in this process are modified and become less active under a number of pathophysiological stress conditions, including amino acid starvation, heat shock, hypoxia and apoptosis. During these conditions, the continued synthesis of proteins essential to recovery from stress or maintenance of a cellular programme is mediated via an alternative form of translation initiation termed IRES (internal ribosome entry site)-mediated translation. This relies on the mRNA containing a complex cis-acting structural element in its 5'-UTR (untranslated region) that is able to recruit the ribosome independently of the cap, and is often dependent upon additional factors termed ITAFs (IRES trans-acting factors). A limited number of ITAFs have been identified to date, particularly for cellular IRESs, and it is not yet fully understood how they exert their control and which cellular pathways are involved in their regulation.
42556303
Cis-regulatory RNA elements that regulate specialized ribosome activity
Recent evidence has shown that the ribosome itself can play a highly regulatory role in the specialized translation of specific subpools of mRNAs, in particular at the level of ribosomal proteins (RP). However, the mechanism(s) by which this selection takes place has remained poorly understood. In our recent study, we discovered a combination of unique RNA elements in the 5′UTRs of mRNAs that allows for such control by the ribosome. These mRNAs contain a Translation Inhibitory Element (TIE) that inhibits general cap-dependent translation, and an Internal Ribosome Entry Site (IRES) that relies on a specific RP for activation. The unique combination of an inhibitor of general translation and an activator of specialized translation is key to ribosome-mediated control of gene expression. Here we discuss how these RNA regulatory elements provide a new level of control to protein expression and their implications for gene expression, organismal development and evolution.
22441056
Specialized ribosomes: a new frontier in gene regulation and organismal biology
Historically, the ribosome has been viewed as a complex ribozyme with constitutive rather than intrinsic regulatory capacity in mRNA translation. However, emerging studies reveal that ribosome activity may be highly regulated. Heterogeneity in ribosome composition resulting from differential expression and post-translational modifications of ribosomal proteins, ribosomal RNA (rRNA) diversity and the activity of ribosome-associated factors may generate 'specialized ribosomes' that have a substantial impact on how the genomic template is translated into functional proteins. Moreover, constitutive components of the ribosome may also exert more specialized activities by virtue of their interactions with specific mRNA regulatory elements such as internal ribosome entry sites (IRESs) or upstream open reading frames (uORFs). Here we discuss the hypothesis that intrinsic regulation by the ribosome acts to selectively translate subsets of mRNAs harbouring unique cis-regulatory elements, thereby introducing an additional level of regulation in gene expression and the life of an organism.
3943564
Heterogeneous Ribosomes Preferentially Translate Distinct Subpools of mRNAs Genome-wide.
Emerging studies have linked the ribosome to more selective control of gene regulation. However, an outstanding question is whether ribosome heterogeneity at the level of core ribosomal proteins (RPs) exists and enables ribosomes to preferentially translate specific mRNAs genome-wide. Here, we measured the absolute abundance of RPs in translating ribosomes and profiled transcripts that are enriched or depleted from select subsets of ribosomes within embryonic stem cells. We find that heterogeneity in RP composition endows ribosomes with differential selectivity for translating subpools of transcripts, including those controlling metabolism, cell cycle, and development. As an example, mRNAs enriched in binding to RPL10A/uL1-containing ribosomes are shown to require RPL10A/uL1 for their efficient translation. Within several of these transcripts, this level of regulation is mediated, at least in part, by internal ribosome entry sites. Together, these results reveal a critical functional link between ribosome heterogeneity and the post-transcriptional circuitry of gene expression.
11814882
Reprogramming of tRNA modifications controls the oxidative stress response by codon-biased translation of proteins
Selective translation of survival proteins is an important facet of the cellular stress response. We recently demonstrated that this translational control involves a stress-specific reprogramming of modified ribonucleosides in tRNA. Here we report the discovery of a step-wise translational control mechanism responsible for survival following oxidative stress. In yeast exposed to hydrogen peroxide, there is a Trm4 methyltransferase-dependent increase in the proportion of tRNALEU(CAA) containing m5C at the wobble position, which causes selective translation of mRNA from genes enriched in the TTG codon. Of these genes, oxidative stress increases protein expression from the TTG-enriched ribosomal protein gene RPL22A, but not its unenriched paralog. Loss of either TRM4 or RPL22A confers hypersensitivity to oxidative stress. Proteomic analysis reveals that oxidative stress causes a significant translational bias toward proteins coded by TTG-enriched genes. These results point to stress-induced reprogramming of tRNA modifications and consequential reprogramming of ribosomes in translational control of cell survival.
6196135
Modulated Expression of Specific tRNAs Drives Gene Expression and Cancer Progression
Transfer RNAs (tRNAs) are primarily viewed as static contributors to gene expression. By developing a high-throughput tRNA profiling method, we find that specific tRNAs are upregulated in human breast cancer cells as they gain metastatic activity. Through loss-of-function, gain-of-function, and clinical-association studies, we implicate tRNAGluUUC and tRNAArgCCG as promoters of breast cancer metastasis. Upregulation of these tRNAs enhances stability and ribosome occupancy of transcripts enriched for their cognate codons. Specifically, tRNAGluUUC promotes metastatic progression by directly enhancing EXOSC2 expression and enhancing GRIPAP1-constituting an "inducible" pathway driven by a tRNA. The cellular proteomic shift toward a pro-metastatic state mirrors global tRNA shifts, allowing for cell-state and cell-type transgene expression optimization through codon content quantification. TRNA modulation represents a mechanism by which cells achieve altered expression of specific transcripts and proteins. TRNAs are thus dynamic regulators of gene expression and the tRNA codon landscape can causally and specifically impact disease progression.
6571664
Translatome profiling: methods for genome-scale analysis of mRNA translation.
During the past decade, there has been a rapidly increased appreciation of the role of translation as a key regulatory node in gene expression. Thereby, the development of methods to infer the translatome, which refers to the entirety of mRNAs associated with ribosomes for protein synthesis, has facilitated the discovery of new principles and mechanisms of translation and expanded our view of the underlying logic of protein synthesis. Here, we review the three main methodologies for translatome analysis, and we highlight some of the recent discoveries made using each technique. We first discuss polysomal profiling, a classical technique that involves the separation of mRNAs depending on the number of bound ribosomes using a sucrose gradient, and which has been combined with global analysis tools such as DNA microarrays or high-throughput RNA sequencing to identify the RNAs in polysomal fractions. We then introduce ribosomal profiling, a recently established technique that enables the mapping of ribosomes along mRNAs at near-nucleotide resolution on a global scale. We finally refer to ribosome affinity purification techniques that are based on the cell-type-specific expression of tagged ribosomal proteins, allowing the capture of translatomes from specialized cells in organisms. We discuss the advantages and disadvantages of these three main techniques in the pursuit of defining the translatome, and we speculate about future developments.
12129665
Identification of eukaryotic mRNAs that are translated at reduced cap binding complex eIF4F concentrations using a cDNA microarray.
Although most eukaryotic mRNAs need a functional cap binding complex eIF4F for efficient 5' end- dependent scanning to initiate translation, picornaviral, hepatitis C viral, and a few cellular RNAs have been shown to be translated by internal ribosome entry, a mechanism that can operate in the presence of low levels of functional eIF4F. To identify cellular mRNAs that can be translated when eIF4F is depleted or in low abundance and that, therefore, may contain internal ribosome entry sites, mRNAs that remained associated with polysomes were isolated from human cells after infection with poliovirus and were identified by using a cDNA microarray. Approximately 200 of the 7000 mRNAs analyzed remained associated with polysomes under these conditions. Among the gene products encoded by these polysome-associated mRNAs were immediate-early transcription factors, kinases, and phosphatases of the mitogen-activated protein kinase pathways and several protooncogenes, including c-myc and Pim-1. In addition, the mRNA encoding Cyr61, a secreted factor that can promote angiogenesis and tumor growth, was selectively mobilized into polysomes when eIF4F concentrations were reduced, although its overall abundance changed only slightly. Subsequent tests confirmed the presence of internal ribosome entry sites in the 5' noncoding regions of both Cyr61 and Pim-1 mRNAs. Overall, this study suggests that diverse mRNAs whose gene products have been implicated in a variety of stress responses, including inflammation, angiogenesis, and the response to serum, can use translational initiation mechanisms that require little or no intact cap binding protein complex eIF4F.
17911153
Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling
Techniques for systematically monitoring protein translation have lagged far behind methods for measuring messenger RNA (mRNA) levels. Here, we present a ribosome-profiling strategy that is based on the deep sequencing of ribosome-protected mRNA fragments and enables genome-wide investigation of translation with subcodon resolution. We used this technique to monitor translation in budding yeast under both rich and starvation conditions. These studies defined the protein sequences being translated and found extensive translational control in both determining absolute protein abundance and responding to environmental stress. We also observed distinct phases during translation that involve a large decrease in ribosome density going from early to late peptide elongation as well as widespread regulated initiation at non–adenine-uracil-guanine (AUG) codons. Ribosome profiling is readily adaptable to other organisms, making high-precision investigation of protein translation experimentally accessible.
21974419
Translational reprogramming in cellular stress response
Cell survival in changing environments requires appropriate regulation of gene expression, including translational control. Multiple stress signaling pathways converge on several key translation factors, such as eIF4F and eIF2, and rapidly modulate messenger RNA (mRNA) translation at both the initiation and the elongation stages. Repression of global protein synthesis is often accompanied with selective translation of mRNAs encoding proteins that are vital for cell survival and stress recovery. The past decade has seen significant progress in our understanding of translational reprogramming in part due to the development of technologies that allow the dissection of the interplay between mRNA elements and corresponding binding proteins. Recent genome‐wide studies using ribosome profiling have revealed unprecedented proteome complexity and flexibility through alternative translation, raising intriguing questions about stress‐induced translational reprogramming. Many surprises emerged from these studies, including wide‐spread alternative translation initiation, ribosome pausing during elongation, and reversible modification of mRNAs. Elucidation of the regulatory mechanisms underlying translational reprogramming will ultimately lead to the development of novel therapeutic strategies for human diseases.
30886079
p53 RNA interactions: new clues in an old mystery.
The p53 tumor suppressor protein is typically considered to be a sequence-specific DNA-binding transcription factor. However, reports over the last 15 years have described RNA binding by p53 in a variety of contexts, suggesting the possibility of new p53 functions. It is clear that p53-RNA interactions are mediated by a nucleic acid-binding domain of p53 independent of the sequence-specific core domain responsible for DNA recognition. Reports disagree on several aspects of the putative RNA interaction, including sequence specificity and biological relevance. Here we review the history and recent advances in the study of p53-RNA interactions. We argue that p53-RNA interactions are sequence nonspecific and depend on incomplete post-translational modification of the p53 C-terminal domain when the protein is expressed in heterologous systems. It is unknown what fraction of p53 protein exists in a state competent for RNA binding in vivo. Thus, potential physiological roles of p53-RNA interactions remain mysterious.
20472842
The tumor suppressor p53 is bound to RNA by a stable covalent linkage.
We have previously shown that the carboxyl-terminal tryptic peptide of the tumor suppressor p53 coeluted from reverse-phase high-performance liquid chromatography (HPLC) with ribonucleotides, suggesting the possible linkage of RNA to p53. In this report, we establish that p53 is covalently linked to RNA, using biochemical criteria at the levels of both tryptic peptide and intact protein: the electrophoretic properties of a tryptic peptide containing phosphorylated Ser-389 and the HPLC chromatographic properties of p53 depend on the linked RNA, p53, purified through urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, copurifies with RNA, and Ser-389 liberates ribonucleotides upon RNase or alkali treatment. Wild-type and mutant p53s from both simian virus 40 (SV40)-transformed and SV40-nontransformed cells are RNA linked, indicating that RNA linkage may be a general property of p53. The RNA is labeled in vivo with 3H-uridine and in vitro by RNA ligase, suggesting that the RNA is bound by a 5' linkage. The RNA is a long-lived, integral component of p53 rather than a transient reaction intermediate. RNA linkage occurs at an evolutionarily conserved site on p53. We propose that RNA-linked p53 is a major biologically active form of p53 and that its interaction with RNA-linked SV40 T antigen reflects a role in RNA metabolism.
35083311
A DNA binding domain is contained in the C-terminus of wild type p53 protein.
In the present study we evaluated the DNA binding activity of wild type and mutant p53 proteins that were isolated from bacterial expression vectors. A comparison of the binding activities of the various purified p53 proteins, assessed by their ability to bind DNA cellulose columns, indicated that wild type p53 has a higher affinity to DNA than have mutant p53 forms. Furthermore, only wild type p53 was able to bind genomic DNA upon electrophoretic protein blotting. As specific deletion of the C-terminal region of wild type p53 totally abolished binding to genomic DNA, it was concluded that the 47 C-terminal amino acids contain the DNA binding region. The fact that the N-terminus contains a transcription activation region whereas the C-terminus contains a DNA binding domain places p53 in the family of typical transcription factors. Our experiments show that the topographical positioning of these domains plays an important role in the activity of wild type p53.
7819330
p53‐catalyzed annealing of complementary single‐stranded nucleic acids.
p53 has been reported to inhibit the DNA helicase intrinsic to simian virus 40 large tumor antigen (T antigen). We found that inhibition is not restricted to T antigen, but also affects several other DNA and RNA helicases. Complexing of the helicases by the p53 protein as a possible inactivation mechanism could be excluded. Instead, the anti‐helicase activity can be explained by our finding that p53 binds with high affinity to single‐stranded nucleic acids and has a strong DNA.DNA and RNA.RNA annealing activity. We could also show that p53 is able to alter the secondary structure of RNA and/or to influence dynamic RNA‐RNA interactions. These results, and the fact that the affinity of p53 to RNA is about one order of magnitude higher than to single‐stranded DNA, imply an RNA‐specific function of p53 in vivo.
24751716
Distinct modulatory role of RNA in the aggregation of the tumor suppressor protein p53 core domain
Inactivation of the tumor suppressor protein p53 by mutagenesis, chemical modification, protein-protein interaction, or aggregation has been associated with different human cancers. Although DNA is the typical substrate of p53, numerous studies have reported p53 interactions with RNA. Here, we have examined the effects of RNA of varied sequence, length, and origin on the mechanism of aggregation of the core domain of p53 (p53C) using light scattering, intrinsic fluorescence, transmission electron microscopy, thioflavin-T binding, seeding, and immunoblot assays. Our results are the first to demonstrate that RNA can modulate the aggregation of p53C and full-length p53. We found bimodal behavior of RNA in p53C aggregation. A low RNA:protein ratio (∼1:50) facilitates the accumulation of large amorphous aggregates of p53C. By contrast, at a high RNA:protein ratio (≥1:8), the amorphous aggregation of p53C is clearly suppressed. Instead, amyloid p53C oligomers are formed that can act as seeds nucleating de novo aggregation of p53C. We propose that structured RNAs prevent p53C aggregation through surface interaction and play a significant role in the regulation of the tumor suppressor protein.
25693818
Recognition of RNA by the p53 tumor suppressor protein in the yeast three-hybrid system.
The p53 tumor suppressor protein is a homotetrameric transcription factor whose gene is mutated in nearly half of all human cancers. In an unrelated screen of RNA/protein interactions using the yeast three-hybrid system, we inadvertently detected p53 interactions with several different RNAs. A literature review revealed previous reports of both sequence-specific and -non-specific interactions between p53 and RNA. Using yeast three-hybrid selections to identify preferred RNA partners for p53, we failed to identify primary RNA sequences or obvious secondary structures required for p53 binding. The cationic p53 C-terminus was shown to be required for RNA binding in yeast. We show that while p53 strongly discriminates between certain RNAs in the yeast three-hybrid assay, the same RNAs are bound equally by p53 in vitro. We further show that the p53 RNA-binding preferences in yeast are mirrored almost exactly by a recombinant tetrameric form of the HIV-1 nucleocapsid (NC) protein thought to be a sequence-nonspecific RNA-binding protein. However, the possibility of specific RNA binding by p53 could not be ruled out because p53 and HIV-1 NC displayed certain differences in RNA-binding preference. We conclude that (1) p53 binds RNA in vivo, (2) RNA binding by p53 is largely sequence-nonspecific in the yeast nucleus, (3) some structure-specific RNA binding by p53 cannot be ruled out, and (4) caution is required when interpreting results of RNA screens in the yeast three-hybrid system because sequence-dependent differences in RNA folding and display can masquerade as sequence-dependent differences in protein recognition.
21970634
Mechanistic insights into p53-promoted RNA-RNA annealing.
The tumour suppressor protein p53 promotes the annealing of complementary nucleic acids in vitro. We observed an up to 1600-fold increase of RNA-RNA annealing by recombinant p53 protein which was shown to bind to RNA in sequence-independent way. Nuclease mapping experiments suggest that p53 binds to intramolecular duplex portions and only marginally changes the overall secondary structure of RNA at conditions of increased annealing. Thus, the mechanism of p53-promoted RNA-RNA annealing does not seem to be dependent on an activity that melts or changes RNA structure. The activation enthalpy of RNA-RNA annealing is decreased in the presence of p53, i.e. the p53 protein could stabilize the transition state whereas the activation entropy is unfavourable. A comparison with thermodynamic data measured for other facilitators strongly suggests that the mechanism of p53-promoted RNA-RNA annealing is distinct from the mechanism by which other facilitators work. The annealing activity of p53 is almost abolished in the presence of magnesium indicating that it can be sharply regulated in vitro and, in principle, could also be regulated in vivo.
12110609
Alternatively spliced forms in the carboxy-terminal domain of the p53 protein regulate its ability to promote annealing of complementary single strands of nucleic acids
The carboxy-terminal domain of the p53 protein comprising amino acid residues 311 to 393 is able to promote the reassociation of single-stranded RNA or DNA into duplex hybrids. This domain is as efficient as the intact p53 protein in both the rate and the extent of the double-stranded product produced in this reaction. Both wild-type and mutant p53 proteins from cancerous cells carry out this reaction. The monoclonal antibody PAb421, which detects an epitope between residues 370 and 378, blocks the ability of p53 to reassociate single strands of RNA or DNA. Similarly, the alternative splice form of the murine p53 protein, which removes amino acid residues 364 to 390 and replaces them with 17 new amino acids, does not carry out the reassociation reaction with RNA or DNA. This is the first indication of functionally distinct properties of the alternative splice forms of p53. These results suggest that this splice alternative can regulate a p53-mediated reaction that may be related to the functions of this protein.
46293271
Ribonuclease activity of p53 in cytoplasm in response to various stress signals
The tumor suppressor p53 protein is expressed at low levels under normal conditions. The subcellular localization and functional activation of p53 are influenced by diverse stress signals. p53 in cytoplasm exerts intrinsic 3'→5' exonuclease activity with various RNA and DNA substrates. ssRNAs containing an adenosine and uridine-rich (ARE) element are permissive targets for p53-mediated degradation. The analysis of the exonuclease activity in cytoplasm with activated p53 induced by various drug treatments or following γ-irradiation revealed that the expression of p53 exonuclease activity in response to stress signals is heterogeneous. Various genotoxic and non-genotoxic agents upregulate p53 yet have different effects on expression of exonuclease activity with ARE RNA but not with DNA substrate. Ribonuclease activity is enhanced in cytoplasmic extracts of HCT116 (p53+/+) cells exposed to γ-irradiation or treated by the non-genotoxic drug AS101 but decreased following treatment by genotoxic (e.g., doxorubicin) or non-genotoxic (e.g., DFMO) agents, thus indicating that p53 exonuclease activity is dependent on the specific stress and nature of the substrate. Apparently, the disparity in expression of p53 ribonuclease activity after each treatment is attributable to the different post-treatment response and to two posttranscriptional events: the interaction of RNA-binding HuR protein with ARE RNA protects the substrate from degradation by p53 and/or decrease in p53 ARE RNA binding capacity due to phosphorylation at Ser392 leads to reduction in p5 ribonuclease activity. Our results provide new insights into p53 exonuclease function and into the mechanisms behind the regulation ARE-RNA degradation by p53 under different cellular conditions.
208495614
Negative feedback regulation of wild‐type p53 biosynthesis.
When growth‐arrested mouse fibroblasts re‐entered the cell‐cycle, the rise in tumour suppressor p53 mRNA level markedly preceded the rise in expression of the p53 protein. Furthermore, gamma‐irradiation of such cells led to a rapid increase in p53 protein biosynthesis even in the presence of the transcription inhibitor actinomycin D. Both findings strongly suggest that p53 biosynthesis in these cells is regulated at the translational level. We present evidence for an autoregulatory control of p53 expression by a negative feed‐back loop: p53 mRNA has a predicted tendency to form a stable stem‐loop structure that involves the 5′‐untranslated region (5′‐UTR) plus some 280 nucleotides of the coding sequence. p53 binds tightly to the 5′‐UTR region and inhibits the translation of its own mRNA, most likely mediated by the p53‐intrinsic RNA re‐annealing activity. The inhibition of p53 biosynthesis requires wild‐type p53, as it is not observed with MethA mutant p53, p53‐catalysed translational inhibition is selective; it might be restricted to p53 mRNA and a few other mRNAs that are able to form extensive stem‐loop structures. Release from negative feed‐back regulation of p53 biosynthesis, e.g. after damage‐induced nuclear transport of p53, might provide a means for rapidly increasing p53 protein levels when p53 is required to act as a cell‐cycle checkpoint determinant after DNA damage.
24564430
Two internal ribosome entry sites mediate the translation of p53 isoforms
The p53 tumour suppressor protein has a crucial role in cell‐cycle arrest and apoptosis. Previous reports show that the p53 messenger RNA is translated to produce an amino‐terminal‐deleted isoform (ΔN‐p53) from an internal initiation codon, which acts as a dominant‐negative inhibitor of full‐length p53. Here, we show that two internal ribosome entry sites (IRESs) mediate the translation of both full‐length and ΔN‐p53 isoforms. The IRES directing the translation of full‐length p53 is in the 5′‐untranslated region of the mRNA, whereas the IRES mediating the translation of ΔN‐p53 extends into the protein‐coding region. The two IRESs show distinct cell‐cycle phase‐dependent activity, with the IRES for full‐length p53 being active at the G2–M transition and the IRES for ΔN‐p53 showing highest activity at the G1–S transition. These results indicate a novel translational control of p53 gene expression and activity.
23285812
The identification of an internal ribosomal entry site in the 5′-untranslated region of p53 mRNA provides a novel mechanism for the regulation of its translation following DNA damage
The tumor suppressor p53 plays a crucial role in maintaining the genetic integrity of the cell and in suppressing cell transformation. Its cellular levels are usually low and rise substantially in response to DNA damage. Although research on p53 induction following DNA damage has mainly focused on the post-translational modification of p53 by Mdm2, it is known that protein translation also contributes to p53 induction. However, the mechanisms underlying translational regulation of the p53 protein in response to DNA damage are still poorly understood. We show that p53 synthesis increases dramatically in MCF-7 cells treated with etoposide. Interestingly, this increase is accompanied by an increase in the association of the translation initiation factor eIF-4E with its binding protein 4E-BP1, an inhibitor of cap-dependent protein translation. We further identified an internal ribosomal entry site (IRES) located in the 5′-untranslated region of the p53 transcript, that is capable of driving the cap-independent translation of a downstream cistron encoding Firefly luciferase in a dicistronic expression vector. Moreover, we found that the activity of the IRES element increases in response to etoposide-induced DNA damage in MCF-7 cells. These findings provide a novel mechanism for the regulation of p53 translation in response to DNA damage.
20969376
p53 directs conformational change and translation initiation blockade of human fibroblast growth factor 2 mRNA
Tumour suppressor p53 has been shown to inhibit fibroblast growth factor 2 expression post-transcriptionally in cultured cells. Here we have investigated the mechanism responsible for this post-transcriptional blockade. Deletion mutagenesis of the FGF-2 mRNA leader revealed the requirement of at least four RNA cis-acting elements to mediate the inhibitory effect of p53 in SK-Hep-1 transfected cells, suggesting the involvement of RNA secondary or tertiary structures. Recombinant wild-type, but not Ala143 mutant p53, was able to specifically repress FGF-2 mRNA translation in rabbit reticulocyte lysate, in a dose dependent manner. Sucrose gradient experiments showed that p53 blocks translation initiation by preventing 80S ribosome formation on an mRNA bearing the FGF-2 mRNA leader sequence. Interaction of wild-type and mutant p53 with different RNAs showed no significant correlation between p53 RNA binding activity and its translational inhibiting effect. However, by checking the accessibility of the FGF-2 mRNA leader to complementary oligonucleotide probes, we showed that the binding to RNA of wild-type, but not mutant p53, induced RNA conformational changes that might be responsible for the translational blockade. This strongly suggests that p53 represses FGF-2 mRNA translation by a direct mechanism involving its nucleic acid unwinding–annealing activity.
14046610
Tumour suppressor p53 inhibits human fibroblast growth factor 2 expression by a post-transcriptional mechanism
Fibroblast growth factor-2 (FGF-2) is a powerful mitogen and angiogenic factor whose expression is strongly regulated at the translational level. The constitutive upregulation of FGF-2 isoforms in transformed cells prompted us to investigate the post-transcriptional effects of a tumour suppressor, p53, on FGF-2 expression. We show here in human primary skin fibroblasts that the cell density-dependent variation of FGF-2 mRNA translatability was inversely correlated with endogenous p53 expression. Transient cell transfection revealed an inhibitory effect of wild-type p53 on the expression of chimeric FGF–CAT proteins. RNAse mapping experiments ruled out any effect of p53 on FGF–CAT mRNA accumulation, suggesting a translational inhibition. This inhibition was mediated by the FGF-2 mRNA leader, but not by vascular endothelial growth factor or platelet derived growth factor mRNA leaders. Neither p53-like protein p73, nor p21/waf had any inhibitory activity. Furthermore a set of hot spot mutants of p53 bearing mutations in the DNA binding domain had no post-transcriptional inhibitory effect. In contrast a p53 mutant of the transactivating domain was still able to block FGF–CAT expression, indicating that the post-transcriptional activity of p53 described here was independent of the trans-activation of target genes. Such data reveal a novel mechanism by which p53 efficiently blocks the expression of a major proliferating, anti-apoptotic and angiogenic gene.
18387152
Alternative translation of human fibroblast growth factor 2 mRNA occurs by internal entry of ribosomes
Alternative initiations of translation of the human fibroblast growth factor 2 (FGF-2) mRNA, at three CUG start codons and one AUG start codon, result in the synthesis of four isoforms of FGF-2. This process has important consequences on the fate of FGF-2: the CUG-initiated products are nuclear and their constitutive expression is able to induce cell immortalization, whereas the AUG-initiated product, mostly cytoplasmic, can generate cell transformation. Thus, the different isoforms probably have distinct targets in the cell. We show here that translation initiation of the FGF-2 mRNA breaks the rule of the cap-dependent ribosome scanning mechanism. First, translation of the FGF-2 mRNA was shown to be cap independent in vitro. This cap-independent translation required a sequence located between nucleotides (nt) 192 and 256 from the 5' end of the 318-nt-long 5' untranslated region. Second, expression of bicistronic vectors in COS-7 cells indicated that the FGF-2 mRNA is translated through a process of internal ribosome entry mediated by the mRNA leader sequence. By introducing additional AUG codons into the RNA leader sequence, we localized an internal ribosome entry site to between nt 154 and 318 of the 5' untranslated region, just upstream of the first CUG. The presence of an internal ribosome entry site in the FGF-2 mRNA suggests that the process of internal translation initiation, by controlling the expression of a growth factor, could have a crucial role in the control of cell proliferation and differentiation.
40713369
p53 Binds Selectively to the 5′ Untranslated Region ofcdk4, an RNA Element Necessary and Sufficient for Transforming Growth Factor β- and p53-Mediated Translational Inhibition of cdk4
ABSTRACT One consequence of transforming growth factor β (TGF-β) treatment is inhibition of Cdk4 synthesis, and this is dependent on p53. Here, we show that the 5′ untranslated region (UTR) of thecdk4 mRNA is both necessary and sufficient for wild-type p53-dependent TGF-β-regulated translational inhibition of cdk4. Wild-type p53 bound selectively to the 5′ UTR of the cdk4 mRNA and inhibited translation of RNAs that contain this region. RNA binding and translational control are two genetically separable functions of p53, as are specific and nonspecific RNA binding. Moreover, transactivation-defective mutants of p53 retain the ability to regulate cdk4 translation. Our findings suggest that p53 functions as a regulator of translation in response to TGF-β in vivo.
3355357
Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them
RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5′ untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5′ UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms.
23783678
Re‐programming of translation following cell stress allows IRES‐mediated translation to predominate
There is now an overwhelming body of evidence to suggest that internal ribosome entry is required to maintain the expression of specific proteins during patho‐physiological situations when cap‐dependent translation is compromised, for example, following heat shock or during mitosis, hypoxia, differentiation and apoptosis. Translational profiling has been used by several groups to assess the extent to which alternative mechanisms of translation initiation selectively recruit mRNAs to polysomes during cell stress. The data from these studies have shown that under each condition 3–5% of coding mRNAs remain associated with the polysomes. Importantly, the genes identified in each of these studies do not show a significant amount of overlap, suggesting that 10–15% of all mRNAs have the capability for their initiation to occur via alternative mechanism(s).
878318
Transcriptional repression of the eukaryotic initiation factor 4E gene by wild type p53.
The eukaryotic initiation factor 4E (eIF4E) plays important roles in transformation and cancer progression. It is frequently overexpressed in malignant cells, one mechanism of which is through transcriptional activation by c-myc. Here, we report that high level of eIF4E expression and its tumorigenicity could be alternatively associated with defects of p53, since we found that induction of wt-p53 repressed eIF4E expression. Gene transfection of p53 inhibited eIF4E promoter activity, while inactivation of p53 either by mutation or by over-expression of MDM2 resulted in stimulation of eIF4E promoter activity. We demonstrated that p53-repression of eIF4E was regulated by c-myc. The wt-p53 can physically bind to c-myc, which inhibited binding of c-myc to eIF4E promoter and c-myc-stimulated promoter activity. These results suggest that the expression of eIF4E is reciprocally regulated by p53 and c-myc, and loss of p53-mediated control over c-myc-dependent transactivation of eIF4E may represent a novel mechanism for eIF4E-mediated neoplastic transformation and cancer progression.
5474331
The p53 target gene TRIM22 directly or indirectly interacts with the translation initiation factor eIF4E and inhibits the binding of eIF4E to eIF4G
The interferon (IFN)‐inducible protein TRIM22 (Staf50) is a member of the tripartite motif protein family and has been suggested a role in the regulation of viral replication as well as of protein ubiquitylation. In addition, we have previously shown that TRIM22 is a direct target gene for the tumour suppressor p53. Consistently, over‐expression of TRIM22 inhibits the clonogenic growth of monoblastic U937 cells, suggesting anti‐proliferative or cell death‐inducing effects.
28569256
Staf50 is a novel p53 target gene conferring reduced clonogenic growth of leukemic U-937 cells
The tumor suppressor gene p53 is a transcription factor that mediates both cell cycle arrest and apoptosis. Interestingly, p53 also induces differentiation of a number of tissues, including leukemic cells. However, although p53-mediated differentiation of leukemic U-937 cells depends on the transcriptional activity of p53, a p53 target gene mediating differentiation has hitherto not been identified. To screen for novel p53 target genes in leukemic cells, a cDNA microarray analysis was performed with U-937-4/ptsp53 cells, expressing a temperature-sensitive p53 mutant. We report that transcription of the Staf50 (stimulated transacting factor of 50 kDa) gene is upregulated in response to wild-type p53 in U-937-4, K562 and MCF-7 cells. Staf50 was directly activated by p53, as determined by the independence of de novo protein synthesis. Moreover, while the proximal promoter of Staf50 was found not to be p53 responsive, a functional enhancer-like p53-response element in intron 1 of the Staf50 gene was identified that was also transactivated by the p53-family member p73. Direct binding of p53 to the response element was shown by electrophoretic mobility shift analysis. Ectopic expression of Staf50 in U-937 cells resulted in reduced clonogenic growth. Moreover, levels of endogenous Staf50 mRNA correlated to all-trans retinoic acid-induced differentiation of promyelocytic NB-4 and HL60 cells, suggesting that Staf50 could be involved in proliferation and/or differentiation of leukemic cells.
36535169
Repression of RNA Polymerase I Transcription by the Tumor Suppressor p53
ABSTRACT The tumor suppressor protein p53 is frequently inactivated in tumors. It functions as a transcriptional activator as well as a repressor for a number of viral and cellular promoters transcribed by RNA polymerase II (Pol II) and by RNA Pol III. Moreover, it appears that p53 also suppresses RNA Pol I transcription. In this study, we examined the molecular mechanism of Pol I transcriptional inhibition by p53. We show that wild-type, but not mutant, p53 can repress Pol I transcription from a human rRNA gene promoter in cotransfection assays. Furthermore, we show that recombinant p53 inhibits rRNA transcription in a cell-free transcription system. In agreement with these results, p53-null epithelial cells display an increased Pol I transcriptional activity compared to that of epithelial cells that express p53. However, both cell lines display comparable Pol I factor protein levels. Our biochemical analysis shows that p53 prevents the interaction between SL1 and UBF. Protein-protein interaction assays indicate that p53 binds to SL1, and this interaction is mostly mediated by direct contacts with TATA-binding protein and TAFI110. Moreover, template commitment assays show that while the formation of a UBF-SL1 complex can partially relieve the inhibition of transcription, only the assembly of a UBF-SL1-Pol I initiation complex on the rDNA promoter confers substantial protection against p53 inhibition. In summary, our results suggest that p53 represses RNA Pol I transcription by directly interfering with the assembly of a productive transcriptional machinery on the rRNA promoter.
27736966
p53 is a general repressor of RNA polymerase III transcription
p53 is a major tumour suppressor that is inactivated in a large proportion of human cancers. We show that p53 serves as a general repressor of transcription by RNA polymerase (pol) III. It can inhibit the synthesis of a range of essential small cellular RNAs including tRNA, 5S rRNA and U6 snRNA, as well as viral products such as the adenovirus VAI RNA. Fibroblasts derived from p53 knock‐out mice display a substantial increase in pol III transcriptional activity. Endogenous cellular p53 is shown to interact with the TATA‐binding protein (TBP)‐containing general factor TFIIIB, thereby compromising its function severely. However, assembly of TFIIIB into a pre‐initiation complex confers substantial protection against the inhibitory effects of p53. Since TFIIIB is an essential determinant of the biosynthetic capacity of cells, its release from repression by p53 may contribute to a loss of growth control during the development of many tumours.
6012114
p53 inhibits RNA polymerase III-directed transcription in a promoter-dependent manner
Wild-type p53 represses Alu template activity in vitro and in vivo. However, upstream activating sequence elements from both the 7SL RNA gene and an Alu source gene relieve p53-mediated repression. p53 also represses the template activity of the U6 RNA gene both in vitro and in vivo but has no effect on in vitro transcription of genes encoding 5S RNA, 7SL RNA, adenovirus VAI RNA, and tRNA. The N-terminal activation domain of p53, which binds TATA-binding protein (TBP), is sufficient for repressing Alu transcription in vitro, and mutation of positions 22 and 23 in this region impairs p53-mediated repression of an Alu template both in vitro and in vivo. p53's N-terminal domain binds TFIIIB, presumably through its known interaction with TBP, and mutation of positions 22 and 23 interferes with TFIIIB binding. These results extend p53's transcriptional role to RNA polymerase III-directed templates and identify an additional level of Alu transcriptional regulation.
7525825
Several regions of p53 are involved in repression of RNA polymerase III transcription
The tumour suppressor p53 has been shown to regulate RNA polymerase (pol) III transcription both in vitro and in vivo. We have characterized the regions of p53 that contribute to this effect. Repression of pol III transcription in vivo does not require residues 13–19 near the N-terminus of p53 that are highly conserved through evolution. However, amino acids 22 and 23 in the adjacent transactivation domain do contribute to the inhibition of pol III activity. Deletions within the central DNA-binding core domain (residues 102–292) of p53 can entirely abolish the repression function in these assays, despite the fact that pol III templates contain no recognized p53 binding site. Deletion or substitution within the C-terminal domain of p53 can also compromise its ability to repress pol III activity in vitro and in transfected cells. These observations reveal that repression of pol III transcription is a complex function involving multiple regions of p53 extending throughout much of the protein.
44043597
Ect2-Dependent rRNA Synthesis Is Required for KRAS-TRP53-Driven Lung Adenocarcinoma.
The guanine nucleotide exchange factor (GEF) epithelial cell transforming sequence 2 (Ect2) has been implicated in cancer. However, it is not clear how Ect2 causes transformation and whether Ect2 is necessary for tumorigenesis in vivo. Here, we demonstrate that nuclear Ect2 GEF activity is required for Kras-Trp53 lung tumorigenesis in vivo and that Ect2-mediated transformation requires Ect2-dependent rDNA transcription. Ect2 activates rRNA synthesis by binding the nucleolar transcription factor upstream binding factor 1 (UBF1) on rDNA promoters and recruiting Rac1 and its downstream effector nucleophosmin (NPM) to rDNA. Protein kinase Cι (PKCι)-mediated Ect2 phosphorylation stimulates Ect2-dependent rDNA transcription. Thus, Ect2 regulates rRNA synthesis through a PKCι-Ect2-Rac1-NPM signaling axis that is required for lung tumorigenesis.
11161620
MicroRNAs in the p53 network: micromanagement of tumour suppression
In recent years, microRNAs (miRNAs) have been identified as mediators of tumour suppression and stress responses exerted by the p53 tumour suppressor. p53-regulated miRNAs contribute to tumour suppression by controlling the expression of central components of multiple processes, including cell cycle progression, epithelial–mesenchymal transition, stemness, metabolism, cell survival and angiogenesis. The expression and activity of p53 itself is also under the control of miRNAs. Finally, genetic and epigenetic alterations identified in the p53–miRNA network indicate that these pathways are important for the initiation and progression of tumours. In the future, knowledge about the p53–miRNA network may be able to be exploited for diagnostic and therapeutic approaches in cancer prevention and treatment.
4704022
MicroRNAs as Key Effectors in the p53 Network.
The guardian of the genome p53 is embedded in a fine-spun network of MicroRNAs. p53 is able to activate or repress directly the transcription of MicroRNAs that are participating in the tumor-suppressive mission of p53. On the other hand, the expression of p53 is under tight control of MicroRNAs that are either targeting directly p53 or factors that are modifying its protein level or activity. Although the most important function of p53 is suggested to be transcriptional regulation, there are several nontranscriptional functions described. One of those regards the modulation of MicroRNA biogenesis. Wild-type p53 is increasing the maturation of selected MicroRNAs from the primary transcript to the precursor MiRNA by interacting with the Microprocessor complex. Furthermore, p53 is modulating the mRNA accessibility for certain MicroRNAs by association with the RISC complex and transcriptional regulation of RNA-binding proteins. In this way p53 is able to remodel the MiRNA-mRNA interaction network. As wild-type p53 is employing MicroRNAs to suppress cancer development, gain-of-function mutant p53 proteins use MicroRNAs to confer oncogenic properties like chemoresistance and the ability to drive metastasis. Like its wild-type counterpart mutant p53 is able to regulate MicroRNAs transcriptionally and posttranscriptionally. Mutant p53 affects the MiRNA processing at two cleavage steps through interfering with the Microprocessor complex and by downregulating Dicer and KSRP, a modulator of MiRNA biogenesis. Thus, MicroRNAs are essential components in the p53 pathway, contributing substantially to combat or enhance tumor development depending on the wild-type or mutant p53 context.
13310626
p53-Mediated Activation of miRNA34 Candidate Tumor-Suppressor Genes
BACKGROUND
In response to varied cell stress signals, the p53 tumor-suppressor protein activates a multitude of genes encoding proteins with functions in cell-cycle control, DNA repair, senescence, and apoptosis. The role of p53 in transcription of other types of RNAs, such as microRNAs (miRNAs) is essentially unknown.


RESULTS
Using gene-expression analyses, reporter gene assays, and chromatin-immunoprecipitation approaches, we present definitive evidence that the abundance of the three-member miRNA34 family is directly regulated by p53 in cell lines and tissues. Using array-based approaches and algorithm predictions, we define genes likely to be directly regulated by miRNA34, with cell-cycle regulatory genes being the most prominent class. In addition, we provide functional evidence, obtained via antisense oligonucleotide transfection and the use of mouse embryonic stem cells with loss of miRNA34a function, that the BCL2 protein is regulated directly by miRNA34. Finally, we demonstrate that the expression of two miRNA34s is dramatically reduced in 6 of 14 (43%) non-small cell lung cancers (NSCLCs) and that the restoration of miRNA34 expression inhibits growth of NSCLC cells.


CONCLUSIONS
Taken together, the data suggest the miRNA34s might be key effectors of p53 tumor-suppressor function, and their inactivation might contribute to certain cancers.
4338896
A microRNA component of the p53 tumour suppressor network
A global decrease in microRNA (miRNA) levels is often observed in human cancers, indicating that small RNAs may have an intrinsic function in tumour suppression. To identify miRNA components of tumour suppressor pathways, we compared miRNA expression profiles of wild-type and p53-deficient cells. Here we describe a family of miRNAs, miR-34a–c, whose expression reflected p53 status. Genes encoding miRNAs in the miR-34 family are direct transcriptional targets of p53, whose induction by DNA damage and oncogenic stress depends on p53 both in vitro and in vivo. Ectopic expression of miR-34 induces cell cycle arrest in both primary and tumour-derived cell lines, which is consistent with the observed ability of miR-34 to downregulate a programme of genes promoting cell cycle progression. The p53 network suppresses tumour formation through the coordinated activation of multiple transcriptional targets, and miR-34 may act in concert with other effectors to inhibit inappropriate cell proliferation.
24974694
Transcriptional activation of miR-34a contributes to p53-mediated apoptosis.
p53 is a potent tumor suppressor, whose biological effects are largely due to its function as a transcriptional regulator. Here we report that, in addition to regulating the expression of hundreds of protein-coding genes, p53 also modulates the levels of microRNAs (miRNAs). Specifically, p53 can induce expression of microRNA-34a (miR-34a) in cultured cells as well as in irradiated mice, by binding to a perfect p53 binding site located within the gene that gives rise to miR-34a. Processing of the primary transcript into mature miR-34a involves the excision of a 30 kb intron. Notably, inactivation of miR-34a strongly attenuates p53-mediated apoptosis in cells exposed to genotoxic stress, whereas overexpression of miR-34a mildly increases apoptosis. Hence, miR-34a is a direct proapoptotic transcriptional target of p53 that can mediate some of p53's biological effects. Perturbation of miR-34a expression, as occurs in some human cancers, may thus contribute to tumorigenesis by attenuating p53-dependent apoptosis.
13763195
LincRNA-p21 suppresses target mRNA translation.
Mammalian long intergenic noncoding RNAs (lincRNAs) are best known for modulating transcription. Here we report a posttranscriptional function for lincRNA-p21 as a modulator of translation. Association of the RNA-binding protein HuR with lincRNA-p21 favored the recruitment of let-7/Ago2 to lincRNA-p21, leading to lower lincRNA-p21 stability. Under reduced HuR levels, lincRNA-p21 accumulated in human cervical carcinoma HeLa cells, increasing its association with JUNB and CTNNB1 mRNAs and selectively lowering their translation. With elevated HuR, lincRNA-p21 levels declined, which in turn derepressed JunB and β-catenin translation and increased the levels of these proteins. We propose that HuR controls translation of a subset of target mRNAs by influencing lincRNA-p21 levels. Our findings uncover a role for lincRNA as a posttranscriptional inhibitor of translation.
33758683
Long noncoding RNAs in the p53 network
The tumor‐suppressor protein p53 is activated in response to numerous cellular stresses including DNA damage. p53 functions primarily as a sequence‐specific transcription factor that controls the expression of hundreds of protein‐coding genes and noncoding RNAs, including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). While the role of protein‐coding genes and miRNAs in mediating the effects of p53 has been extensively studied, the physiological function and molecular mechanisms by which p53‐regulated lncRNAs act is beginning to be understood. In this review, we discuss recent studies on lncRNAs that are directly or indirectly regulated by p53 and how they contribute to the biological outcomes of p53 activation. WIREs RNA 2017, 8:e1410. doi: 10.1002/wrna.1410
18675430
Insights into p53 transcriptional function via genome-wide chromatin occupancy and gene expression analysis
The tumor-suppressor p53 can induce various biological responses. Yet, it is not clear whether it is p53 in vivo promoter selectivity that triggers different transcription programs leading to different outcomes. Our analysis of genome-wide chromatin occupancy by p53 using chromatin immunoprecipitation (ChIP)-seq revealed ‘p53 default program’, that is, the pattern of major p53-bound sites that is similar upon p53 activation by nutlin3a, reactivation of p53 and induction of tumor cell apoptosis (RITA) or 5-fluorouracil in breast cancer cells, despite different biological outcomes. Parallel analysis of gene expression allowed identification of 280 novel p53 target genes, including p53-repressed AURKA. We identified Sp1 as one of the p53 modulators, which confer specificity to p53-mediated transcriptional response upon RITA. Further, we found that STAT3 antagonizes p53-mediated repression of a subset of genes, including AURKA.
1565266
Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells
The effects of diverse stresses on promoter selectivity and transcription regulation by the tumor suppressor p53 are poorly understood. We have taken a comprehensive approach to characterizing the human p53 network that includes p53 levels, binding, expression and chromatin changes under diverse stresses. Human osteosarcoma U2OS cells treated with anti-cancer drugs Doxorubicin (DXR) or Nutlin-3 (Nutlin) led to strikingly different p53 gene binding patterns based on chromatin immunoprecipitation with high-throughput sequencing experiments. Although two contiguous RRRCWWGYYY decamers is the consensus binding motif, p53 can bind a single decamer and function in vivo. Although the number of sites bound by p53 was six times greater for Nutlin than DXR, expression changes induced by Nutlin were much less dramatic compared with DXR. Unexpectedly, the solvent dimethylsulphoxide (DMSO) alone induced p53 binding to many sites common to DXR; however, this binding had no effect on target gene expression. Together, these data imply a two-stage mechanism for p53 transactivation where p53 binding only constitutes the first stage. Furthermore, both p53 binding and transactivation were associated with increased active histone modification histone H3 lysine 4 trimethylation. We discovered 149 putative new p53 target genes including several that are relevant to tumor suppression, revealing potential new targets for cancer therapy and expanding our understanding of the p53 regulatory network.
4963059
Trans‐acting translational regulatory RNA binding proteins
The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans‐acting regulatory RNA‐binding proteins (RBPs) are necessary to provide mRNA‐specific translation, and these interact with 5′ and 3′ untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans‐acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans‐acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans‐acting ribosomal proteins in directing the translation of specific mRNAs.
8142922
Hypoxia-inducible factor 1 alpha is regulated by RBM38, a RNA-binding protein and a p53 family target, via mRNA translation
Hypoxia-inducible factor 1 (HIF1), a heterodimeric transcription factor, consists of HIF1α and HIF1β and is necessary for cell growth and survival under a hypoxic condition. Thus, the level and activity of HIF1α needs to be tightly controlled. Indeed, HIF1α protein stability is controlled by prolyl hydroxylase and von Hippel-Lindau-mediated proteosomal degradation. However, it remains unclear whether HIF1α expression is controlled by other pathways. Here, we showed that RNA-binding protein RBM38, a target of the p53 family, regulates HIF1α expression via mRNA translation. Specifically, we showed that under a hypoxic condition, ectopic expression of RBM38 decreased, whereas knockdown of RBM38 increased, the level of HIF1α protein. We also showed that the rate of de novo HIF1α protein synthesis was increased by knockdown of RBM38. Additionally, we showed that RBM38 directly bound to HIF1α 5′ and 3′UTRs. Consistently, we showed that the rate of mRNA translation for a heterologous reporter that carries HIF1α 5′and/or 3′UTRs was increased upon knockdown of RBM38. Furthermore, we showed that knockdown of RBM38 increased, whereas ectopic expression of RBM38 decreased, the binding of eIF4E to HIF1α mRNA. Together, our data suggest that RBM38 is a novel translational regulator of HIF1α under a hypoxic condition.
15549438
Ribosome heterogeneity in tumorigenesis: the rRNA point of view
The "specialized ribosome" concept proposes that ribosome variants are produced and differentially regulate translation. Examples supporting this notion demonstrated heterogeneity of ribosomal protein composition. However, ribosome translational activity is carried out by rRNA. We, and others, recently showed that rRNA heterogeneity regulates translation to generate distinct translatomes promoting tumorigenesis.
2012282
p53 Acts as a Safeguard of Translational Control by Regulating Fibrillarin and rRNA Methylation in Cancer

 Summary
 
 Ribosomes are specialized entities that participate in regulation of gene expression through their rRNAs carrying ribozyme activity. Ribosome biogenesis is overactivated in p53-inactivated cancer cells, although involvement of p53 on ribosome quality is unknown. Here, we show that p53 represses expression of the rRNA methyl-transferase fibrillarin (FBL) by binding directly to FBL. High levels of FBL are accompanied by modifications of the rRNA methylation pattern, impairment of translational fidelity, and an increase of internal ribosome entry site (IRES)-dependent translation initiation of key cancer genes. FBL overexpression contributes to tumorigenesis and is associated with poor survival in patients with breast cancer. Thus, p53 acts as a safeguard of protein synthesis by regulating FBL and the subsequent quality and intrinsic activity of ribosomes.
 

7089710
Evidence for rRNA 2′-O-methylation plasticity: Control of intrinsic translational capabilities of human ribosomes
Significance Translational control is a cornerstone of gene-expression regulation in physiological and pathological contexts. The contribution of nonribosomal factors, including messenger RNAs (mRNAs) and mRNA-bound factors, to translational control have been extensively studied. Recently, the hypothesis of a ribosome-mediated regulation emerged, which proposes that cells produce ribosomes of different composition and displaying different translational properties. This work reveals that ribosomal RNA 2′-O-methylation can be modulated in human ribosomes, including at key functional sites for translation, and that changes in the 2′-O-methylation pattern control the intrinsic capabilities of ribosomes to translate mRNAs. This work directly demonstrates the existence of composition-modified ribosomes and their associated change in translational activity as conceptualized by the specialized ribosome concept. Ribosomal RNAs (rRNAs) are main effectors of messenger RNA (mRNA) decoding, peptide-bond formation, and ribosome dynamics during translation. Ribose 2′-O-methylation (2′-O-Me) is the most abundant rRNA chemical modification, and displays a complex pattern in rRNA. 2′-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2′-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2′-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin in human cells. Using RiboMethSeq, a nonbiased quantitative mapping of 2′-O-Me, we identified a repertoire of 2′-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2′-O-Me plasticity. Using the cricket paralysis virus internal ribosome entry site element, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through a 2′-O-Me pattern and not by nonribosomal actors of the translational machinery. Our data establish rRNA 2′-O-Me plasticity as a mechanism providing functional specificity to human ribosomes.
9077957
A systematic RNAi synthetic interaction screen reveals a link between p53 and snoRNP assembly
TP53(tumour protein 53) is one of the most frequently mutated genes in human cancer and its role during cellular transformation has been studied extensively. However, the homeostatic functions of p53 are less well understood. Here, we explore the molecular dependency network of TP53 through an RNAi-mediated synthetic interaction screen employing two HCT116 isogenic cell lines and a genome-scale endoribonuclease-prepared short interfering RNA library. We identify a variety of TP53 synthetic interactions unmasking the complex connections of p53 to cellular physiology and growth control. Molecular dissection of the TP53 synthetic interaction with UNRIP indicates an enhanced dependency of TP53-negative cells on small nucleolar ribonucleoprotein (snoRNP) assembly. This dependency is mediated by the snoRNP chaperone gene NOLC1 (also known as NOPP140), which we identify as a physiological p53 target gene. This unanticipated function of TP53 in snoRNP assembly highlights the potential of RNAi-mediated synthetic interaction screens to dissect molecular pathways of tumour suppressor genes.
3356723
Mutant mice lacking the p53 C-terminal domain model telomere syndromes.
Mutations in p53, although frequent in human cancers, have not been implicated in telomere-related syndromes. Here, we show that homozygous mutant mice expressing p53Δ31, a p53 lacking the C-terminal domain, exhibit increased p53 activity and suffer from aplastic anemia and pulmonary fibrosis, hallmarks of syndromes caused by short telomeres. Indeed, p53Δ31/Δ31 mice had short telomeres and other phenotypic traits associated with the telomere disease dyskeratosis congenita and its severe variant the Hoyeraal-Hreidarsson syndrome. Heterozygous p53+/Δ31 mice were only mildly affected, but decreased levels of Mdm4, a negative regulator of p53, led to a dramatic aggravation of their symptoms. Importantly, several genes involved in telomere metabolism were downregulated in p53Δ31/Δ31 cells, including Dyskerin, Rtel1, and Tinf2, which are mutated in dyskeratosis congenita, and Terf1, which is implicated in aplastic anemia. Together, these data reveal that a truncating mutation can activate p53 and that p53 plays a major role in the regulation of telomere metabolism.
45258360
Profiling of ribose methylations in RNA by high-throughput sequencing.
Ribose methylations are the most abundant chemical modifications of ribosomal RNA and are critical for ribosome assembly and fidelity of translation. Many aspects of ribose methylations have been difficult to study due to lack of efficient mapping methods. Here, we present a sequencing-based method (RiboMeth-seq) and its application to yeast ribosomes, presently the best-studied eukaryotic model system. We demonstrate detection of the known as well as new modifications, reveal partial modifications and unexpected communication between modification events, and determine the order of modification at several sites during ribosome biogenesis. Surprisingly, the method also provides information on a subset of other modifications. Hence, RiboMeth-seq enables a detailed evaluation of the importance of RNA modifications in the cells most sophisticated molecular machine. RiboMeth-seq can be adapted to other RNA classes, for example, mRNA, to reveal new biology involving RNA modifications.
14198894
Profiling of 2′-O-Me in human rRNA reveals a subset of fractionally modified positions and provides evidence for ribosome heterogeneity
Ribose methylation is one of the two most abundant modifications in human ribosomal RNA and is believed to be important for ribosome biogenesis, mRNA selectivity and translational fidelity. We have applied RiboMeth-seq to rRNA from HeLa cells for ribosome-wide, quantitative mapping of 2′-O-Me sites and obtained a comprehensive set of 106 sites, including two novel sites, and with plausible box C/D guide RNAs assigned to all but three sites. We find approximately two-thirds of the sites to be fully methylated and the remainder to be fractionally modified in support of ribosome heterogeneity at the level of RNA modifications. A comparison to HCT116 cells reveals similar 2′-O-Me profiles with distinct differences at several sites. This study constitutes the first comprehensive mapping of 2′-O-Me sites in human rRNA using a high throughput sequencing approach. It establishes the existence of a core of constitutively methylated positions and a subset of variable, potentially regulatory positions, and paves the way for experimental analyses of the role of variations in rRNA methylation under different physiological or pathological settings.
24416588
Impaired Control of IRES-Mediated Translation in X-Linked Dyskeratosis Congenita
The DKC1 gene encodes a pseudouridine synthase that modifies ribosomal RNA (rRNA). DKC1 is mutated in people with X-linked dyskeratosis congenita (X-DC), a disease characterized by bone marrow failure, skin abnormalities, and increased susceptibility to cancer. How alterations in ribosome modification might lead to cancer and other features of the disease remains unknown. Using an unbiased proteomics strategy, we discovered a specific defect in IRES (internal ribosome entry site)–dependent translation in Dkc1m mice and in cells from X-DC patients. This defect results in impaired translation of messenger RNAs containing IRES elements, including those encoding the tumor suppressor p27(Kip1) and the antiapoptotic factors Bcl-xL and XIAP (X-linked Inhibitor of Apoptosis Protein). Moreover, Dkc1m ribosomes were unable to direct translation from IRES elements present in viral messenger RNAs. These findings reveal a potential mechanism by which defective ribosome activity leads to disease and cancer.
3931075
Integrative genome analysis of somatic p53 mutant osteosarcomas identifies Ets2-dependent regulation of small nucleolar RNAs by mutant p53 protein.
TP53 is the most frequently mutated gene in human cancer. Many mutant p53 proteins exert oncogenic gain-of-function (GOF) properties that contribute to metastasis, but the mechanisms mediating these functions remain poorly defined in vivo. To elucidate how mutant p53 GOF drives metastasis, we developed a traceable somatic osteosarcoma mouse model that is initiated with either a single p53 mutation (p53R172H) or p53 loss in osteoblasts. Our study confirmed that p53 mutant mice developed osteosarcomas with increased metastasis as compared with p53-null mice. Comprehensive transcriptome RNA sequencing (RNA-seq) analysis of 16 tumors identified a cluster of small nucleolar RNAs (snoRNAs) that are highly up-regulated in p53 mutant tumors. Regulatory element analysis of these deregulated snoRNA genes identified strong enrichment of a common Ets2 transcription factor-binding site. Homozygous deletion of Ets2 in p53 mutant mice resulted in strong down-regulation of snoRNAs and reversed the prometastatic phenotype of mutant p53 but had no effect on osteosarcoma development, which remained 100% penetrant. In summary, our studies identify Ets2 inhibition as a potential therapeutic vulnerability in p53 mutant osteosarcomas.
9761210
Ribosomal protein S27L is a direct p53 target that regulates apoptosis
Ribosomal proteins were recently shown to regulate p53 activity by abrogating Mdm2-induced p53 degradation (L23, L11, L5) or by enhancing p53 translation (L26). Here, we report that a novel ribosomal protein, RPS27L (S27-like protein), is a direct p53 target. RPS27L, but not its family member RPS27, was identified as a p53 inducible gene in a genome-wide chip-profiling study. Further characterization revealed a p53-dependent induction of RPS27L in multiple cancer cell models. Indeed, a consensus p53-binding site was identified in the first intron of the RPS27L gene and a direct binding of p53 to this site was demonstrated both in vitro and in vivo. Characterization of a luciferase reporter driven by the RPS27L intron fragment revealed a p53-binding site-dependent transaction by wild-type p53, but not by several transactivating-deficient p53 mutants. This transactivation was enhanced by etoposide, a DNA damaging agent that activates p53 and was completely blocked by a dominant-negative p53 mutant. Functionally, overexpression of RPS27L within the physiological inducible levels promoted, whereas siRNA silencing of RPS27L inhibited, apoptosis induced by etoposide. This is the first report, to our knowledge, that p53 directly induces the expression of a ribosomal protein, RPS27L, which in turn promotes apoptosis.
899179
Ribosomal protein S27-like, a p53-inducible modulator of cell fate in response to genotoxic stress.
Activation of the p53 tumor suppressor upon DNA damage elicits either cell cycle arrest or apoptosis, and the precise mechanism governing cell fate after p53 response has not been well defined. Through genomic analysis, we have identified the ribosomal protein S27-like (RPS27L) as a novel p53 transcriptional target gene. Although RPS27L mRNA levels were consistently induced after diverse p53 activating signals, its change in protein level was stimuli-dependent: it was up-regulated when cells were arrested in response to DNA-damaging agents Adriamycin or VP16 but was down-regulated when cells underwent apoptosis in response to antimetabolite agent 5-fluorouracil. RPS27L is a nuclear protein that forms nuclear foci upon DNA damage. Depletion of RPS27L resulted in deficiency in DNA damage checkpoints, leading to conversion of DNA damage-induced p53 response from cell cycle arrest to apoptosis. We further show that RPS27L positively regulates p21 protein expression. Through this mechanism, RPS27L induction by p53 facilitates p21-mediated cell cycle arrest and protects against DNA damage-induced apoptosis. Thus, RPS27L modulates DNA damage response and functions as a part of the control switch to determine cell fate to DNA damage-p53 response.