Systematic Identification and Functional Characterization of Alternatively Spliced Exons with Phenotypic Relevance

Division Of Basic Sciences - Nci

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Abstract

Our primary objective is to systematically identify and characterize alternative exons with significant phenotypic impact. Using innovative functional genomics platforms-including orthogonal exon deletion and splice site mutagenesis-we are dissecting the functional relevance of alternative exons at genome scale. By integrating massively parallel exon perturbation with cellular fitness assays, we have uncovered a large set of alternative exons that either promote or suppress cell proliferation. Fitness-promoting exons tend to occur in essential, highly expressed genes and frequently overlap protein domains or interaction interfaces. In contrast, fitness-suppressing exons are enriched in non-essential, low-expression genes, often exhibit low inclusion levels, and tend to map to intrinsically disordered regions and sites of clinical mutations. As a case study, we investigated TAF5 alternative exon 8, a fitness-promoting exon whose inclusion regulates assembly of the TFIID transcription initiation complex and downstream gene expression. We further showed that exon 8 inclusion is modulated by the splicing factor SRSF1 and is dynamically regulated in response to genotoxic stress. To expand the resolution and throughput of exon functional studies, we developed scCHyMErA-Seq-a scalable CRISPR-based exon deletion screening platform integrated with 10x Genomics single-cell transcriptomic profiling. This technology enables efficient exon deletion while simultaneously capturing Cas9/Cas12a guide RNAs and polyadenylated transcripts at single-cell resolution. Applying scCHyMErA-Seq to the high-throughput profiling of alternative cassette exons, we identified numerous splicing events with strong regulatory effects on gene expression. For instance, analysis of NRF1 alternative exon 7 revealed that its inclusion modulates NRF1's recruitment to target gene promoters and thereby shapes transcriptional output. Importantly, transcriptional profiles derived from scCHyMErA-Seq closely mirror those from orthogonal, low-throughput approaches-offering both accuracy and scalability. Overall, scCHyMErA-Seq provides a powerful platform to systematically uncover the functional impact of alternative splicing by linking specific exon usage to transcriptional phenotypes. Moving forward, our goals are to: 1. Engineer advanced tools for high-throughput exon perturbation with diverse phenotypic readouts; 2. Identify phenotypically relevant splicing events through screens in clinically relevant models, including patient-derived renal cell carcinoma and bladder cancer systems; 3. Elucidate the functions of key alternative exons in gene expression regulators.