ROLE OF HNRNP H IN A REVERSIBLE FINE-TUNING MECHANISM OF SPLICING RESPONSE UPON TRANSCRIPTIONAL PAUSING

Alternative splicing is a common way of diversifying the proteome and regulating protein functions without increasing genome size. Chemotherapeutic agents, such as cisplatin, cause changes in the splicing of proliferation-associated exons, which contribute to the overall proliferative phenotype of cancer cells. Transcriptional pausing, which is seen in embryonic stem cells as a timing regulator of gene expression, also impacts splicing. The mechanisms by which transcriptional pausing and pause-release alter the functions of splicing machinery are largely unknown. Thus, I tested the effects of the transcription elongation inhibitor DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole) on the splicing of a set of inducible exons with a common UAGG-GGGG splicing code. These exons were previously identified through a genomewide search for the transcripts sensitive to hnRNP A1 and hnRNP H. I found that the endogenous human HNRNPH1 Exon 4 and its paralog HNRNPH3 Exon 3 had increased skipping upon DRB treatment, as did the pA+ reporter-cloned GRIN1 CI, a brain-region-specific rat exon with this splicing code, previously studied in our lab. I used single and combined mutations to monitor the effect of DRB-induced transcriptional pausing and pause-release on splicing, since the shared motifs include two exonic UAGG motifs and a GGGG close to 5´ splice site. GGGG mutants had increased skipping, compared to wild type, upon DRB treatment. UAGG-GGGG code is necessary for the response, as the triple mutant lost its ability to respond to DRB. These data point to a role of hnRNP H in response to transcription stress