CONICET | Buscador de Institutos y Recursos Humanos

Regulation of alternative splicing through its kinetic coupling with transcription can occur via changes in chromatin structure or modulation of RNA polymerase II (pol II) elongation rates. As an example of the first mode we found that siRNAs targeted at intronic sequences located downstream of the fibronectin EDI exon are able to regulate AS through a mechanism known as transcriptional gene silencing (TGS). The effect needs both AGO-1 and AGO-2 and is abolished by factors that favor an open chromatin structure or increase transcriptional elongation. The mechanism involves the formation of facultative heterochromatin epigenetic marks (H3K9me2 and H3K27me3) at the target site that creates a roadblock to pol II elongation. Changes in chromatin structure affecting AS can be triggered by physiological external signals. We found that depolarization of nerve cells causes lower inclusion of NCAM alternatively spliced exon 18. Inhibition of histone deacetylation by trichostatin A causes a similar effect, opposite to the one of inhibition of pol II elongation by flavopiridol. When inducing membrane depolarization, the effects of these drugs are abolished, which suggests that depolarization promotes histone acetylation, facilitating pol II elongation leading to exon 18 exclusion. ChIP analysis confirmed our predictions. The second mode is illustrated by the effects of UV light on AS. The UV effect does not require p53 or damage of the DNA template in cis. It is observed only when splicing is co-transcriptional and is the consequence of inhibition of pol II elongation caused by hyperphosphorylation of the carboxy terminal domain of pol II at serines 2 and 5.