QKI ensures splicing fidelity during cardiogenesis by engaging the U6 tri-snRNP to activate splicing at weak 5'...

During organogenesis, precise pre-mRNA splicing is essential to assemble tissue architecture. Many developmentally essential exons bear weak 5' splice sites (5'SS) yet are spliced with high precision, implying unknown yet active splicing fidelity mechanisms. By combining transcriptome and alternative splicing profiling with temporal eCLIP mapping of RNA interactions across development, we identify the RNA-binding protein QKI as an essential direct regulator of splicing fidelity in key cardiac transcripts. Although QKI is dispensable for cardiac specification, its loss disrupts sarcomere assembly despite intact expression of sarcomere mRNAs through exon skipping and nuclear retention of mis-spliced RNAs. QKI-dependent exons in essential cardiac genes have weak 5'SS and frequently show poor complementarity with U6 snRNA. We show that QKI directly interacts with U6 snRNA using an overlapping interface to its traditional intronic binding activity, securing U4/U6·U5 tri-snRNP to ensure splicing fidelity. Thus, QKI exemplifies how context-aware RBPs enforce splicing fidelity at structurally vulnerable splice sites during organogenesis.