Abstract
Pre-mRNA splicing requires proper splice site selection mediated by many factors including snRNPs and serine-arginine rich (SR) splicing factors. Our lab previously reported that the SR-like protein SON maintains organization of pre-mRNA splicing factors in nuclear speckles as well as splicing of many human transcripts including mRNAs coding for the chromatin-modifying enzymes HDAC6, ADA and SETD8. However, the mechanism by which SON maintains accurate splicing is unknown. To build tools for understanding SON-dependent pre-mRNA splicing, we constructed a minigene reporter plasmid driving expression of the genomic sequence spanning exons 26 through 29 of HDAC6. Following SON depletion, we observed altered splicing of HDAC6 reporter transcripts that showed exclusion of exons 27 and 28, reflecting the splicing patterns of endogenous HDAC6 mRNA. Importantly, loss of HDAC6 biological function was also observed, as indicated by truncated HDAC6 protein and corresponding absence of aggresome assembly activities of HDAC6 binding-of-ubiquitin zinc finger (BUZ) domain. We therefore propose that SON-mediated splicing regulation of HDAC6 is essential for supporting protein degradation pathways that prevent human disease.
Highlights
Pre-mRNA splicing regulation in eukaryotes is critical for maintaining proper gene expression as well as protein biodiversity
SON Is Required for Retention of Exons 27 and 28 in Endogenous HDAC6 Transcripts and Exogenous HDAC6 Minigene Reporter Transcripts
Misregulated splicing was shown in a subset of human transcripts including skipping of exons 27 and 28 in endogenous HDAC6 transcripts ([9] and Figure 1B)
Summary
Pre-mRNA splicing regulation in eukaryotes is critical for maintaining proper gene expression as well as protein biodiversity. RNA fluorescence in situ hybridization using splice junction probes demonstrated that nascent transcripts from a stably expressed beta-tropomyosin reporter gene were alternatively spliced in SON-depleted cells, consistent with a function for SON in co-transcriptional pre-mRNA processing [9]. Three independent studies later demonstrated that SON maintains accurate splicing of human mRNAs and pointed toward an important role for SON in regulating the expression of genes involved in cell cycle progression and pluripotency [9,10,11]. Microarray analysis from SON-depleted HeLa cells altered the expression of transcripts that fall into pathways and functional categories such as apoptosis, cell cycle, cancer, DNA replication/recombination/repair, amino acid metabolism, integrin mediated cell adhesion, smooth muscle contraction and G protein signaling [9,10]. SON RS domain and G-patch were important for proper splicing of TUBG1 pre-mRNA, and the C-terminal region containing an RS domain, G-patch and DSRM rescued splicing to a significant extent [10]
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