QKI ensures splicing fidelity during cardiogenesis by engaging the U6 tri-snRNP to activate splicing at weak 5'splice sites [RNA-Seq]

During organogenesis, the establishment of tissue architecture requires precise RNA processing. Many developmentally essential exons bear weak 5' splice sites (5'SS) yet are spliced with high precision, implying unknown yet active splicing fidelity mechanisms. Combining transcriptome and alternative splicing profiling and 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 lineage specification, its loss disrupts sarcomere assembly despite intact expression of sarcomere mRNAs through exon skipping and intron retention in key sarcomeric mRNAs. 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 recruitment to ensure exon inclusion. Thus, QKI exemplifies how context-aware RBPs enforce splicing fidelity at structurally vulnerable splice sites by coupling local intronic cues to spliceosome engagement, thereby securing accurate exon inclusion during organogenesis. Overall design: Human embryonic stem cells (WT and QKI KO) were utilzed to perform RNA-seq at D0, D4, and D8 timepoints in cardiomyocyte differentiation