Project description:Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB (HJAY)
Project description:Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB (Exon array)
Project description:Despite advances in splicing mechanics, the precise regulatory factors and pathways governing most alternative splicing events remain elusive. We developed CRASP-Seq, a method combining pooled CRISPR-based genetic perturbations with deep sequencing of splicing reporters, to quantitively assess the impact of every gene on alternative splicing, from a single RNA extraction. Applying CRASP-Seq to disease-related splicing events we identified both known and novel regulators, validated through focused assays. These regulators are enriched for RNA splicing, spliceosome biogenesis, and gene expression terms. Application of CRASP-Seq to LMNA cryptic splicing event that causes progeria uncovered ZNF207, primarily known for mitotic spindle assembly, as a regulator of progerin splicing. Depletion of ZNF207 enhances canonical LMNA splicing and decreases progerin levels in patient-derived cells. Transcriptomic and proteomic analyses reveal that ZNF207 has a broad impact on alternative splicing by binding to RNA targets and interacting with spliceosome complexes, underscoring its direct role in splicing regulation.