Abstract

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 sequencing (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 misrecognition 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 the 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.