RRM
2013 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
Pre-mRNA splicing requires proper splice site selection mediated by many factors including snRNPs and serine-arginine rich (SR) splicing factors. Son is the largest known SR splicing factor, and it has several putative functional domains including an RS domain, a glycine rich patch (G-patch) and double stranded RNA binding domain (DSRBD). One-third of Son’s amino acid sequence consists of novel repetitive sequence motifs of unknown function. Son is essential for organization of pre-mRNA processing factors in nuclear speckles and for cell cycle progression. We previously reported an exon array analysis of Son-depleted HeLa cells that revealed changes in 1061 transcripts showing exon inclusion or exclusion, and a total of 2067 splicing events that are potentially regulated by Son. We validated that Son is required for appropriate splice site choice in transcripts for several chromatin-modifying enzymes, including HDAC6, ADA and SETD8. However, the mechanism by which Son maintains accurate splicing is unknown. We are systematically generating model minigene cassettes for molecular and in situ analysis of Son-dependent splicing regulation. We have constructed a HDAC6 minigene reporter that contains the genomic sequence spanning exons 26 through 29. Following Son depletion in HeLa cells transfected with the HDAC6 minigene reporter construct, we observed skipping of exons 27 and 28 on both the reporter and endogenous HDAC6 transcripts. We hypothesize that the C-terminal RS domain of Son is required to maintain proper splicing of HDAC6 transcripts, and we are evaluating siRNA-refractory Son deletion mutants to determine which domains of Son can rescue splicing of HDAC6 minigene transcripts.
Keywords: Spicing, mimigene reporter, Son
