CONICET | Buscador de Institutos y Recursos Humanos

Alternative splicing (AS) affects the expression of 65% of human genes, generates high protein diversity and is implicated in human disease. The rate of transcription elongation determines the outcome of two competing splicing reactions that occur co-transcriptionally. Rapid transcription favors exon skipping, whereas slower transcriptionfavors exon inclusion. We present here evidence for three different mechanisms that control AS through the kinetic coupling. 1. UV radiation affects AS of many genes, including the upregulation of the pro-apoptotic isoform of Bcl-x, an expected physiological response to DNA damage. The UV effect does not require p53, is not caused by the damage of the DNA template in cis and only affects co-transcriptional splicing. UV light causes hyperphosphorylation of the carboxy terminal domain of RNA polymerase II, which is responsible for the alteration in AS through the inhibition of transcriptional elongation. Pol II mutants engineered to mimic the hyperphosphorylated state duplicate the effects of UV light on AS. 2. The chromatin context affects Pol II elongation rates and in turn, AS. We report here the mechanism by which membrane depolarization in nerve cells affects AS of the NCAM pre-mRNA. Depolarization promotes local heterochromatinization around the alternative exon on the NCAM gene, which creates roadblocks to Pol II elongation. 3. In search for new tools to control AS at the chromatin level, we found that small interfering RNAs (siRNAs) targeted to the intron located downstream of an alternative exon affect AS through a mechanism known as transcriptional gene silencing (TGS). The intronic siRNAs trigger heterochromatinization on DNA target sequences by causing histone H3 Lys9 dimethylation. The effects of intronic siRNAs on AS are not caused by conventional post-transcriptional gene silencing (PTGS), are abolished by inhibitors of histone deacetylation and methylation and depend on the presence of the protein Ago2, known to be necessary for TGS.