hnRNPM and ESRP1 show common motif enrichment downstream from discordantly regulated exons and compete for shared binding sites. ( A ) K-mer enrichment analysis showing the top three enriched 6-mers in introns flanking hnRNPM and ESRP1 coregulated cassette exons. Two identical GU-rich motifs (GUGUGU and GUGGUG, black box) were enriched downstream from cassette exons in ESRP1-enhanced and hnRNPM-repressed events. The RBFOX motif (UGCAUG, underlined) was enriched downstream from ESRP1-repressed and hnRNPM-enhanced events. ( B , C ) <t>RNA</t> motif map analysis of GU-rich motifs in introns flanking hnRNPM-ESRP1 coregulated exons with respect to ESRP1 regulation ( B ) and hnRNPM regulation ( C ) reveals enrichment of GU-rich motifs in downstream introns of ESRP1-enhanced and hnRNPM-repressed cassette exon splicing events. Inclusion events (green). Skipping events (red). Control events (black). ( D ) Genome browser plot of RNA sequencing data sets showing hnRNPM knockdown promotes APLP2 exon 7 inclusion and ESRP1 depletion promotes skipping. Black bars indicate constitutive exons. Green bar indicates variable exon 7. Yellow bars indicate location of two clusters of GU-rich motifs within 250 nucleotides (nt) downstream from APLP2 exon 7. ( E ) Immunoblot of HA-tagged ESRP1 overexpression in MDA-MB-231 cells and endogenous hnRNPM. ( F ) ESRP1-HA overexpression in MDA-MB-231 cells results in increased APLP2 exon 7 inclusion. (*) Indicates nonspecific band. ( G ) ( Upper panel) The two GU-rich regions identified within 250 nt downstream from APLP2 exon 7 were used to design RNA probes GU1 and GU2 containing stretches of GU nucleotides underlined and in red with mutant probes GU1-mut and GU2-mut with mutated sequences colored in blue. ( Lower panel) RNA pull-down analysis using RNA probes blotting for endogenous hnRNPM and overexpressed ESRP1-HA in MDA-MB-231 cells shows that hnRNPM and ESRP1 bind common GU-rich sequences. ( H ) RNA pull-down experiments using a static amount of the <t>biotinylated</t> GU2 RNA probe and cell lysate assaying for hnRNPM in the MDA-MB-231 cell line, which does not express ESRP1, and the same line with ESRP1-HA overexpression. A total of 2.5% input was provided as a loading control for both samples. Overexpression of ESRP1 leads to less hnRNPM binding, suggesting that ESRP1 competes for the same GU2 binding site. (Error bars = S.E.M, n = 3, [***] P -value