Regulation and consequences of cryptic splicing

ABSTRACT Our research aims to comprehensively understand the molecular mechanisms and functions of RNA splicing in regulating cellular phenotypes. RNA splicing is a critical process that removes introns from pre-mRNA and is essential to gene regulation and transcriptome diversity. In humans, introns constitute approximately one-fourth of the whole genome, representing a vast expansion compared to lower eukaryotes and other mammals. Long introns contain a high number of pseudo splice sites that closely resemble those of coding exons. Misuse of pseudo splice sites gives rise to cryptic splicing, which leads to the erroneous insertion of intronic elements into mRNA. Consequently, cryptic splicing results in disruption of transcriptome integrity and is associated with numerous pathologies including neuronal degeneration and cancer. Our proposed work aims to build on our recent discoveries in investigating the regulatory mechanisms of cryptic splicing and its consequences. We recently conducted a survey of RNA binding protein (RBP) interactions with introns and identified a group of RBPs, including heterogenous nuclear ribonucleoprotein M (hnRNPM), whose binding is highly enriched in introns. We found that depletion of hnRNPM by shRNA causes over a thousand cryptic splicing events, suggesting that hnRNPM is a crucial repressor of cryptic splicing. Our bioinformatics analysis shows that hnRNPM binding is enriched in regions that contain repetitive sequences. It further predicts that hnRNPM- repressed cryptic splicing products can be targets of nonsense-mediated decay or templates to generate truncated proteins. The cryptic splicing products also have the potential to form double-stranded RNAs (dsRNAs) that trigger an interferon response, leading to significant increases in the expression of interferon-stimulated genes. Building on these findings, we will first comprehensively investigate the underlying mechanisms by which RBPs repress cryptic splicing to maintain transcriptome integrity. We will investigate the role of RBPs that preferentially bind to introns, including hnRNPM interaction partners, in repressing cryptic splicing. We will also delineate the nature of interactions between these RBPs and hnRNPM. Furthermore, we will determine the molecular details of how loss of hnRNPM affects spliceosome factor binding at pseudo splice sites, resulting in cryptic splicing. Second, we will investigate the fate of cryptic splicing transcripts by experimentally characterizing them at both the mRNA and protein levels and evaluating their impact on cellular activities. Given our finding that hnRNPM depletion leads to a dsRNA-dependent upregulation of interferon-stimulated genes, we plan to further investigate the pathways mediating the crosstalk between dsRNA of cryptic transcripts and upregulation of interferon-stimulated genes and their impact on cell fitness. Successful completion of these studies will shed light on the regulatory mechanisms for cryptic splicing suppression and how failure to control cryptic splicing impacts cellular behavior. With the centrality of cryptic splicing to disease states, the fundamental insights learned from this research may pave the way for novel therapeutic interventions.