Deciphering Regulatory Factors and Mechanisms Driving Disease-Associated Alternative Splicing

Division Of Basic Sciences - Nci

Investigators

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Abstract

Alternative splicing is a pervasive mechanism of gene regulation that diversifies the transcriptome by generating multiple mRNA isoforms from a single gene. This process is critical for development, differentiation, and cellular stress responses, and its dysregulation is implicated in a wide range of diseases, including genetic syndromes, neurodegeneration, and cancer. Despite detailed understanding of the spliceosome's core mechanics, the regulatory networks and pathways that govern context-specific alternative splicing decisions remain largely undefined. This gap is especially pronounced for splicing events that are misregulated in disease, where the upstream regulators and mechanisms often remain elusive due to the lack of efficient, high-throughput functional screening methods. To address this challenge, we developed CRASP-Seq (CRISPR-based RNA and Splicing Perturbation sequencing), an innovative screening platform that combines genome-wide CRISPR-mediated genetic perturbations with deep sequencing of splicing reporters. CRASP-Seq captures splice junctions directly, allowing for precise and quantitative assessment of how the knockout of each human gene affects a given splicing event. This approach is highly scalable and cost-effective, requiring only a single RNA extraction from a pooled, transduced cell population-overcoming major limitations of traditional arrayed screening platforms. Using CRASP-Seq, we have generated and optimized a panel of splicing reporters that span a wide range of alternative splicing modes, including cassette exon inclusion/skipping, alternative 5' and 3' splice site selection, and intron retention. Many of these events are directly implicated in disease contexts, including LMNA in Hutchinson-Gilford Progeria Syndrome (HGPS), PKM and FAS in cancer metabolism and apoptosis, EZH2 in epigenetic regulation, and XBP1, a critical effector of the unfolded protein response (UPR). Our application of CRASP-Seq to LMNA aberrant splicing identified ZNF207 as a key positive regulator of progerin production. Depletion of ZNF207 in HGPS-derived fibroblasts restores normal splicing of LMNA and significantly reduces progerin expression. Mechanistically, ZNF207 directly binds pre-mRNA targets and regulates a broad network of alternative splicing events. Through base editor-mediated high-throughput mutagenesis, we identified a single point mutation in ZNF207's second zinc finger domain that disrupts its splicing regulatory activity and impairs its interaction with U1 snRNP components. We have also applied CRASP-Seq to dissect stress-responsive splicing programs, including the IRE1a-XBP1 signaling axis of the UPR. In this context, our screen uncovered RBM39-a coactivator of U2 snRNP assembly-as an essential regulator of XBP1 cytoplasmic splicing. RBM39 loss triggers exon skipping in ERN1 (IRE1a), generating a dominant-negative isoform that impairs downstream XBP1 activation. Notably, genotoxic and thermal stress also induce ERN1 exon-18 skipping, suggesting a conserved stress-responsive mechanism that attenuates UPR signaling via alternative splicing regulation. Overall, this project seeks to systematically map the trans-acting regulators and upstream pathways that control disease-relevant alternative splicing events, using CRASP-Seq as a versatile and quantitative discovery platform. By integrating CRISPR screening, splicing reporter engineering, and targeted mutagenesis, we aim to define new mechanistic principles of splicing regulation, uncover functionally relevant disease drivers, and establish experimental foundations for therapeutic modulation of RNA processing.