Search for endogenous small RNAs affecting alternative splicing by TGS

MARIANO ALLÓ1*, PAOLA BERTUCCI1, LUCIANA GOMEZ ACUÑA1, VALERIA BUGGIANO1, ENERITZ AGIRRE2, EDUARDO EYRAS2, JUAN VALCARCEL3 AND ALBERTO R. KORNBLIHTT

Boston, Massachusetts, EEUU

Congreso; 7th Special Interest Group meeting on Alternative Splicing; 2010

Intelligent systems for molecular biology

Small interfering RNAs (siRNAs) are known to mediate post-transcriptional gene silencing (PTGS) by promoting degradation of target mRNAs. When targeted at promoter regions, siRNAs participate in an alternative pathway known as transcriptional gene silencing (TGS) by promoting histone methylation, heterochromatin formation and inhibition of transcription1. We have recently shown that siRNAs targeting sequences located close to an alternative exon are able to regulate its alternative pre-mRNA splicing (AS) in human cells. The effect needs both AGO-1 and AGO-2 (key players of TGS) and is abolished or reduced by factors that favor an open chromatin structure or increase transcriptional elongation. The mechanism involves the presence of facultative heterochromatin epigenetic marks (H3K9me2 and H3K27me3) at the target site and the hetorochromatin associated protein HP1 alpha. The effect is not only triggered by intronic but also by exonic siRNAs2. The use of the cancer-related panel for alternative splicing in cells depleted of AGO1 and Dicer shed light on a possible endogenous pathway of this process that we called TGS-AS. In the search for endogenous examples of TGS-AS we performed ChIP-seq using antibodies against AGO proteins (key players of TGS) and histone H3 covalent modifications (H3K9me2, H3K27me3, H3K36me3 and H3) in MCF7 culture cells. Moreover, we’ve used already published data from Pol II ChIP-seq in MCF7. Using bioinformatic tools we’ve filtered and analyzed all these raw data.