Abstract
Alternative polyadenylation (APA) has been implicated as an important regulator of gene expression. In some cases APA is known to couple with alternative splicing to influence last intron removal, however it is unknown whether APA events influence alternative splicing decisions at upstream exons. A genome‐wide approach was used to examine the correlation between APA and upstream alternative splicing. CstF64 knockdown in HeLa cells coupled with Pas‐Seq was used to trigger and identify APA events. APA genes were then evaluated for changes in alternative splicing using RNA‐seq analyses of the same knockdown samples. Although a significant number of alternative splicing events were identified, no general correlation between APA and upstream alternative splicing events were observed. These results suggest that the coupling and diversification achieved between APA and alternative splicing in general is fixed to defining the last exon. iClip‐Seq experiments identified CstF64 binding to be predominantly in intronic regions. Interestingly, the genome‐wide binding analysis also showed that CstF64 density is elevated upstream of skipped exons indicating a potential role for CstF64 in alternative splicing. We conclude that while the influence of APA on alternative splicing is generally limited to terminal introns, CstF64 binding to nascent pre‐mRNA may contribute to the modulation of splicing patterns.Grant Funding Source: NIH
Highlights
Eukaryotic gene expression and proteomic diversity is dependent on the appropriate removal of introns from pre-mRNAs, a process orchestrated by the spliceosome
To determine if changes in APA correlate with changes in alternative splicing (AS), RT-PCR analysis was performed along the TIMP-2 gene using cDNA from CF1m25 KD and control cells
Proximal poly(A) selection increases terminal splicing efficiency in TIMP-2. These results demonstrate that proximal poly(A) site selection influences the efficiency of terminal intron removal
Summary
Eukaryotic gene expression and proteomic diversity is dependent on the appropriate removal of introns from pre-mRNAs, a process orchestrated by the spliceosome. Multiple mRNA isoforms are generated through alternative splicing (AS), a highly regulated process that has been identified in »95% of human genes.[1,2] AS requires the use of different combinations of splice sites resulting in several classes of splicing patterns, such as: alternative 50 splice site selection (Alt5), alternative 30 splice site selection (Alt3), the skipping of complete exons (SE), or the retention of introns (RI). Many splicing decisions are believed to occur co-transcriptionally,[3,4,5,6,7] both through the use of alternative promoters[8,9,10,11,12] as well as in defining the first[13] and last exons. The U1 snRNP component U1A has been shown to stimulate polyadenylation through interaction with CPSF160,19 U1
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