Search for endogenous small RNAs affecting alternative splicing by TGS

MARIANO ALLÓ1,PAOLA BERTUCCI1, LUCIANA GOMEZ ACUÑA1, VALERIA BUGGIANO1,ENERITZ AGIRRE2, EDUARDO EYRAS2, JUAN VALCÁRCEL3 AND ALBERTO R. KORNBLIHTT1

Salve Regina University, Newport, RI, EEUU

Congreso; Gordon Conferences; 2010

Gordon Research Conferences

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 transcription. 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, named TGS-AS, 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 siRNAs. Using ChIP-seq with antobodies to AGO proteins and different histone marks we identified 24,000 target regions for AGO1, 36,615 for AGO2, 23,000 for H3K9me2 and 51,000 for H3K27me3 in the genome of the human mammary cell line MCF7. Approximately 50% of the targets for each mark map within genes. Moreover, we detected a significant enrichment of AGO1 peaks in promoters and 5’ UTRs of highly transcribed genes that it is not associated with histone marks, but shows overlapping with two small RNAs families:  PASRs and tiRNAs. On the contrary, inside of genes AGO1, as well as AGO2 and H3K9me2 are enriched in exons, preferentially in genes with low expression levels. Several histone marks display from 5% to 20% overlapping when assessed at the whole genome level. However the convergence increases to 55% when only AGO1 target sites are considered. Consistently, we have found a set of candidate alternative splicing events (ASE) to be affected by TGS-AS according to AGO1 binding, the presence of facultative heterochromatin histone marks and/or Pol II and antisense transcription. We are also investigating a potential role for miRNAs as mediators of TGS-AS by introducing miRNA target sites in AS reporter minigenes.