CONICET | Buscador de Institutos y Recursos Humanos

Changes in RNA polymerase II (pol II) elongation rates modulate alternative splicing choices, affecting the timing at which nascent pre-mRNA splice sites and regulatory sequences are presented to the spliceosome (an effect known as kinetic coupling). In search for physiological pathways affecting alternative splicing through its kinetic coupling with transcription, we found that membrane depolarization of neuronal cells triggers the skipping of exon 18 from the neural cell adhesion molecule (NCAM) mRNA, whose inclusion is modulated during differentiation and synaptic plasticity. The effect is seen in both primary cultured neurons and N2a murine neuroblastoma cells, and is independent of the calcium/calmodulin protein kinase (CaMK) pathway. We show that this exon responds to pol II elongation, because its inclusion is increased by a slow pol II mutant. Analysis of changes in histone acetylation across the endogenous NCAM locus, using nucleosomal Chromatin Inmunoprecipitation (nChIP), revealed a local decrease in marks of transcriptional elongation and active transcription, such as H3K36 tri-methylation and H3K9 acetylation, in the neighbourhood of exon 18. Depolarization affects the chromatin template in a specific way, by causing an increase in H3K9 acetylation on 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 H3K36 tri-methylation. The effects on acetylation and splicing are reverted when the depolarizing conditions are withdrawn and can be both duplicated and potentiated by the histone deacetylase inhibitor trichostatin A. Based on these evidences, we postulate that depolarization acts by promoting changes in the intragenic chromatin structure of the NCAM gene, which allows higher pol II elongation rates and the subsequent skipping of exon 18, pointing at a physiological role of intragenic chromatin modulation in mRNA biogenesis.