Regulation of mRNA Abundance by Polypyrimidine Tract-Binding Protein-Controlled Alternate 5′ Splice Site Choice

摘要

Alternative splicing (AS) provides a potent mechanism for increasing protein diversity and modulating gene expression levels. How alternate splice sites are selected by the splicing machinery and how AS is integrated into gene regulation networks remain important questions of eukaryotic biology. Here we report that polypyrimidine tract-binding protein 1 (Ptbp1/PTB/hnRNP-I) controls alternate 5′ and 3′ splice site (5′ss and 3′ss) usage in a large set of mammalian transcripts. A top scoring event identified by our analysis was the choice between competing upstream and downstream 5′ss (u5′ss and d5′ss) in the exon 18 of the Hps1 gene. Hps1 is essential for proper biogenesis of lysosome-related organelles and loss of its function leads to a disease called type 1 Hermansky-Pudlak Syndrome (HPS). We show that Ptbp1 promotes preferential utilization of the u5′ss giving rise to stable mRNAs encoding a full-length Hps1 protein, whereas bias towards d5′ss triggered by Ptbp1 down-regulation generates transcripts susceptible to nonsense-mediated decay (NMD). We further demonstrate that Ptbp1 binds to pyrimidine-rich sequences between the u5′ss and d5′ss and activates the former site rather than repressing the latter. Consistent with this mechanism, u5′ss is intrinsically weaker than d5′ss, with a similar tendency observed for other genes with Ptbp1-induced u5′ss bias. Interestingly, the brain-enriched Ptbp1 paralog Ptbp2/nPTB/brPTB stimulated the u5′ss utilization but with a considerably lower efficiency than Ptbp1. This may account for the tight correlation between Hps1 with Ptbp1 expression levels observed across mammalian tissues. More generally, these data expand our understanding of AS regulation and uncover a post-transcriptional strategy ensuring co-expression of a subordinate gene with its master regulator through an AS-NMD tracking mechanism. Author Summary Mammalian gene expression is extensively controlled at the post-transcriptional level and understanding of the underlying mechanisms can provide important biomedical insights. Here we identified a number of novel alternate splicing (AS) events where the choice between competing splice sites (ss) is regulated by polypyrimidine tract-binding protein 1 (Ptbp1/PTB/hnRNP-I). A top-scoring event was the choice between alternate upstream and downstream 5′ss (u5′ss and d5′ss) in the Hps1 gene mutated in patients with type 1 Hermansky-Pudlak Syndrome (HPS). Preferential utilization of the u5′ss in the presence of Ptbp1 gives rise to stable mRNAs encoding a full-length Hps1 protein, whereas the d5′ss bias triggered by Ptbp1 down-regulation generates RNA species cleared by nonsense-mediated decay (NMD). We show that Ptbp1 functions in this circuitry by activating the intrinsically weaker u5′ss. Brain-enriched Ptbp1 paralog Ptbp2/nPTB/brPTB stimulated the u5′ss utilization but with a considerably lower efficiency than Ptbp1. We propose that this mechanism accounts for a tight correlation between Hps1 with Ptbp1 expression levels observed in mammalian tissues. Overall, these data expand our understanding of AS regulation and uncover an AS-NMD-mediated tracking mechanism ensuring co-expression of master regulator and its subordinate gene.