Spliceosomal Proofreading Factors Safeguard 3′ Splice Site Fidelity and Prevent Proteotoxicity and Inflammation
Description
Precise intron removal by RNA splicing is essential for faithful gene expression, yet the mechanisms ensuring splicing fidelity in mammals remain unclear. Using a systematic knockdown RNA-seq screen, we uncover widespread splicing errors and identify AQR, SYF1, and SYF3 as cooperative safeguards of 3′ splice site (3′ss) fidelity in human and mouse. These factors act during spliceosome assembly to correct U2AF-mediated mis-recognition of non-canonical 3′ss bearing AG dinucleotides embedded within pyrimidine-rich sequences and lacking canonical branch points (BPs), likely through kinetic proofreading. Their loss triggers pervasive 3′ss mis-splicing, resulting in accumulation of misfolded proteins, proteotoxic stress, unfolded protein response activation, and ultimately cell death and intestinal inflammation. Together, our study reveals a previously unrecognized layer of splicing fidelity control in mammals that links aberrant splice site selection to proteostasis and inflammation.