TRANSCRIPTION ELONGATION RATE MODULATES RESPONSE TO DNA DAMAGE OF ALTERNATIVE EXONS WITH SHORT UPSTREAM INTRONS

PREGO A; SCHOR IE; MUÑOZ MJ

Salta

Congreso; Joint LV Annual SAIB Meeting and XIV PABMB Conference; 2019

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular - Panamerican Association for Biochemistry and Molecular Biology

DNA damage affects gene expression patterns by different mechanisms, one of these being the regulation of alternative splicing (AS). In previous reports (Muñoz et al., 2009, 2017) a pathway that modulates the AS patterns in human keratinocytes exposed to UV light was characterized, which includes the generation of pyrimidine dimers, the ATR kinase and the phosphorylation of the C-terminal domain of RNA polymerase II (RNAPII), an important mediator of the response. From an RNA-seq experiment assessing the effect of DNA damage and its repair on global AS regulation, we found that the response of the alternative exons to the damage is correlated with the length of the flanking introns. In particular, shorter upstream introns favor higher inclusion of alternative exons, which can be related to changes on the RNAPII elongation rate. We selected exons with short upstream introns from those that showed regulation by UV light on the RNA-seq and performed a validation of this effect by semi-quantitative RT-PCR, shower higher reproducibility for those cases where the UV-effect was reverted after damage repair. To corroborate whether DNA damage causes changes in the transcriptional kinetics for these genes, the elongation properties of RNAPII were analyzed in one of them using a biochemical approach. Also, the influence of elongation changes on the AS patterns of these endogenous genes, and in the UV-exerted regulation, was tested through the use of drugs that affect RNAPII elongation. This work proposes regulatory mechanisms within the response to genotoxic damage, through combination of genomic analysis with experimental validation.