Abstract
Splicing of the FGFR2 K-SAM exon is repressed by hnRNP A1 bound to the exon and activated by TIA-1 bound to the downstream intron. Both proteins are expressed similarly by cells whether they splice the exon or not, so it is important to know which one is dominant. To answer this question, we used bacteriophage PP7 and bacteriophage MS2 coat fusions to tether hnRNP A1 and TIA-1 to distinct sites on the same pre-mRNA molecule. hnRNP A1 fused to one coat protein was tethered to a K-SAM exon containing the corresponding coat protein's binding site. TIA-1 fused to the other coat protein was tethered to the downstream intron containing that coat protein's binding site. This led to efficient K-SAM exon splicing. Our results show that TIA-1 is dominant for K-SAM exon splicing control and validate the combined use of PP7 and MS2 coat proteins for studying posttranscriptional events.
Highlights
Alternative splicing control is vital for correct gene expression, and it is important to understand how it works
Two further downstream intron splicing enhancers (ISEs) IAS2 and IAS3 are implicated in K-SAM exon splicing activation [4]. hnRNP A1 bound to an exon splicing silencer (ESS) represses K-SAM exon splicing [6]
The vectors contain a polylinker for the insertion of additional coding sequences between the FLAG tag and the nuclear localization signal and were used to make additional vectors coding for hnRNP A1 fusions (A1-PP and A1-MM) and TIA-1 fusions (TIAPP and TIA-MM)
Summary
Alternative splicing control is vital for correct gene expression, and it is important to understand how it works. The KSAM exon is spliced in epithelial cells, while the BEK exon is spliced in mesenchymal cells. Deleting the BEK exon does not lead to efficient K-SAM exon splicing in mesenchymal cells, and deleting the K-SAM exon does not lead to efficient BEK exon splicing in epithelial cells [1]. Both exons are subject to at least partially independent splicing control. We have investigated the independent splicing control of the KSAM exon and identified a major activator (TIA-1) and a major repressor (hnRNP A1) of this exon’s splicing. Two further downstream intron splicing enhancers (ISEs) IAS2 and IAS3 are implicated in K-SAM exon splicing activation [4]. hnRNP A1 bound to an exon splicing silencer (ESS) represses K-SAM exon splicing [6]
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