CONICET | Buscador de Institutos y Recursos Humanos

Alternative splicing affects the expression of 70% of human genes, generates high protein diversity and is implicated in human disease. The rate of transcription elongation determines the outcome of competing splicing reactions that occur co-transcriptionally. For example, in many cases rapid transcription favors exon skipping, whereas slower transcription favors exon inclusion. The chromatin context affects RNA polymerase II (pol II) elongation rates and in turn, alternative splicing. We report here that membrane depolarization of neuronal cells triggers the skipping of exon 18 from the neural cell adhesion molecule (NCAM) mRNA. We show that this exon responds to RNA pol II elongation, because its inclusion is increased by a slow pol II mutant. Depolarization affects the chromatin template in a specific way, by causing H3K9 hyperacetylation, restricted to an internal region of the NCAM gene surrounding the alternative exon. This intragenic histone hyperacetylation is not paralleled by acetylation at the promoter, is associated with chromatin relaxation and is linked to increases in pol II processivity and H3K36 tri-methylation. On the other hand, when searching for new tools to control alternative splicing at the chromatin level, we found that small interfering RNAs (siRNAs) targeting the intron located downstream of the fibronectin EDI alternative exon affect alternative splicing through a mechanism known as transcriptional gene silencing (TGS). The intronic siRNAs trigger heterochromatinization on DNA target sequences by causing histone H3 Lys9 dimethylation and reduce pol II processivity. This suggests that the effect on alternative splicing is probably due to the creation of roadblocks to pol II elongation. The effects of intronic siRNAs on alternative splicing 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.