In this program, we focused on the following projects: (i) RNA oxidation. Previously we have shown that mRNA oxidation leads to a reduction of its translation fidelity. Further study, we and others revealed that RNA oxidation or abasic site formation could have profound effects on gene expression and this is emerging as a common feature in different human pathologies, including cancer. The mammalian AP-endonuclease, APE1, is a ubiquitous multifunctional protein. It plays a central role on DNA base excision repair pathway. In addition, APE1 catalyzes cleavage of oxidized RNA at 8-oxo-guanosine. This enzymatic activity is thought to maintain RNA quality homeostasis by eliminating the damaged RNA. Recent investigation reveal that under oxidative stress conditions, APE1 in the nucleus interacts with the precursor of miRNAs including miR-221/222, which are known to be involved in tumorigenesis. However, the molecular mechanism underlying APE1s role in these processes remains to be elucidated, and it was not known whether APE1 regulates their oxidation levels. In collaboration with Dr. Tells group, we investigated the role of APE1 in oxidizing the precursor of miR-221/222. In this study, APE1 was knockdown by siRNA to reduce more than 80% of APE1 protein in HeLa cells. In addition, the APE1 knockdown cells were transfected with APE1 expression vector to revert the expression in the cells. Total RNA was isolated from these cells, and non-treated cells. After total RNA was derivatized with ARP, the oxidized RNA was pulled down with strep-avidin magnetic beads. Pool of either oxidized RNA or total RNA was examined for the quantity of pri-miR-221 or pri-miR-222 by qRT-PCR using their specific TaqMan probes. Their oxidation levels were determined by the ratio of relative amount in oxidized RNA pool relative to that in total RNA pool. Results showed that oxidation levels of pri-miR-221 was increased in the APE1 knockdown cells, while that in the exogenously expressed APE1 cells was reverted to the same levels as non-treated cells. These results indicated that APE1 plays a crucial role in oxidation of the pri-miR-221/222, suggesting that APE1 induces functional miRNA precursors under oxidative stress by eliminating oxidatively damaged RNA. (ii) Covalent modification of proteins such as methylation, is known to alter their enzymatic activities and/or affinity to interact with other proteins, membranes or nucleic acids. Rickettsiae are obligatory intracellular infectious Gram-negative bacteria that responsible for major rickettsiosis, which include epidemic typhus, spotted fever, and scrub typhus, without the availability of vaccine or early detection method. The outer membrane of Rickettsia is largely composed of an outer membrane protein called OmpB that accounts for up to 15 % of its total cellular proteins. OmpB is known to involve in cell adhesion, attachment, and invasion. We and others showed that methylation of lysyl residues in rickettsial OmpB correlated with bacterial virulence. Methylation profiles analysis using LC-MS/MS methods revealed a high correlation in methylation sites between those detected in native proteins purified from bacteria and those attained from in vitro methylation, except the methylation level was found significantly higher in the native proteins relative to those methylated using purified methtyltransferases with overexpressed OmpB fragments as substrates. In addition, the natively purified OmpB from the avirulent strain Madrid E of Rickettsia prowazekii does not contain any trimethyllysine. An observation consistent with the report that the gene encoding the trimethyltransferase, RP027-028, in Madrid E has been interrupted by a frameshift mutation to generate the inactive trimethyltransferase, the RP027 and RP028 fragments. However, OmpB from the highly virulent Rickettsia prowazekii strains Breinl, and RP22 each contains clusters of trimethyllysines located at a relatively close proximity. Thus, the state and type of OmpB methylation is correlated with rickettsial pathogenicity. To this end, knowledge on the enzyme(s) that catalyzes OmpB methylation and on the nature of the methylated OmpB could provide new insight on OmpB-methylation and its role on virulent effect. Through bioinformatics analysis of genomic DNA sequences of Rickettsia, Dr. Yang's lab revealed five potential sequences of putative protein lysine methyltransferases. Synthesis and expression of these genes, follow with purification and characterization of these genes products revealed then presence of two distinct types of protein lysine methyltransferases. They are the PKMT1 and PKMT2, which catalyzes predominantly monomehtylation and trimethylation, respectively. Among known protein lysine methyltransferases, rickettsial PKMT1 and PKMT2 are unique in that their substrates appear to be limited to OmpB and both methyltransferases are capable of methylating multiple lysyl residues with broad sequence specificity. To better understand the mechanism by which PKMT1 and PKMT2 differentially catalyze OmpB methylation, we carry out crystal structural analysis for PKMT1 from Rickettsia prowazekii, both the apo-form and in complex with its cofactor, S-adenosylmethionine or S-adenosylhomocystine, and for PKMT2 from Rickettsia typhi. The structure of PKMT1 in complex with S-adenosylhomosystine is solved to a resolution of 1.9 . Both enzymes are dimeric, with each monomer containing an S-adenosylmethionine binding domain with a seven-strand Rossmann fold, a dimerization domain, a middle domain, a C-terminal domain, and a centrally located open cavity. Based on the crystal structures, residues involved in catalysis, cofactor binding and substrate interactions were examined using site-directed mutagenesis followed by steady-state kinetic analysis to ascertain their catalytic functions in solution. Together, our data provide new structural and mechanistic insights on how rickettsial methyltransferases catalyze OmpB methylation. has