Journal: bioRxiv
Article Title: QKI ensures splicing fidelity during cardiogenesis by engaging the U6 tri-snRNP to activate splicing at weak 5ʹ splice sites
doi: 10.1101/2025.09.04.674271
Figure Lengend Snippet: a , QKI eCLIP-seq reveals robust and selective binding to U6 snRNA across all stages of human cardiac differentiation (D0, D4, D8). Significance was determined by the Mann-Whitney U test. b , QKI shows negligible enrichment for other spliceosomal snRNAs (U1, U2, U4, U5, U6ATAC, U11, U12, U4atac) compared to U6 snRNA across all stages of human cardiac differentiation. Significance was determined by the Mann-Whitney U test. c, Comparative enrichment analysis of QKI on U6 snRNA (log₂ fold change over size-matched input) relative to 155 ENCODE-profiled RBPs, showing selective U6 engagement (comparable to canonical U6-binding factor SMNDC1) but not to U1 d , QKI shows selective binding to U6 snRNA, but not to the minor spliceosomal U6ATAC snRNA, in contrast to SMNDC1, which binds to both snRNAs. e , QKI binds specifically to the 5ʹ region of U6 snRNA across all stages and replicates, distinct from canonical U6-binding splicing factors (e.g., PRPF8, RBM22), which predominantly bind the 3ʹ region. f , Domain architecture of QKI and schematic of the KH domain double mutant (K120A/R124A) used to assess functional contributions of canonical RNA binding to U6 engagement. g , Normalized read density in eCLIP of transgenic MYC-tagged wild-type and KH domain mutant QKI in HEK293T cells, shown for eCLIP peaks identified in independent experiments with anti-QKI antibody in HEK293T. h , Quantification of ( g ) shows a significant loss of eCLIP signal with the KH domain mutant. i , Enrichment of QKI binding at indicated snRNAs in cells expressing WT or KH mutant QKI indicates that U6 binding is preserved upon mutation of the canonical RNA recognition domain. j , Base-resolution eCLIP mapping of QKI binding to U6 snRNA reveals that the selective enrichment at the 5ʹ stem-loop and adjacent single-stranded region critical for 5ʹSS engagement is preserved in the QKI KH domain mutant. k , RT-PCR using a NIN exon 18 minigene shows that the KH-domain mutant QKI fails to restore splicing despite retaining U6 binding. l, Schematic showing secondary structure of human U6 snRNA with key regions including the 5ʹ stem-loop, 5ʹSS interacting domain, internal stem-loop, and telestem. Colors indicate regions used for NMR analysis. m-o , NMR titration of recombinant QKI KH–QUA2 protein with in vitro transcribed U6 RNA fragments. Residues affected based on chemical shift perturbations overlap with the canonical RNA-binding surfaces. m , The U6 fragment containing both the 13-mer sequence with a partial QRE motif and the 5ʹ stem-loop induces the strongest chemical shift changes, consistent with an extended RNA-binding interface and a higher affinity interaction. n , The 13-mer alone also triggers pronounced shifts, yet to a lesser extent compared to ( m ). o , In contrast, the 5ʹ stem-loop alone causes only minor perturbations, consistent with a weak interaction. Error bars represent ±SEM; p- values are calculated using Student’s t -test; biological replicates n = 3
Article Snippet: During cell lysis incubation, beads were coupled to antibodies as follows; 125 μl per sample of sheep anti-rabbit Dynabeads M-280 (Cat# 11204D, Life Technologies) was washed twice in co-IP lysis buffer and incubated with 10 μg/sample (10 cm plate) or 15 μg/sample (15 cm plate) of either QKI (Cat# A300-183A), RBM42 (Cat# A305-138A, Bethyl Laboratories), SNRNP27 (Cat# HPA034541, Sigma-Aldrich) or TXNL4A (Cat# PA5-120069, Thermo Fischer Scientific) antibodies for 45 minutes at room temperature on a rotator.
Techniques: Binding Assay, MANN-WHITNEY, Mutagenesis, Functional Assay, RNA Binding Assay, Transgenic Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Titration, Recombinant, In Vitro, Sequencing
Bethyl is a verified supplier 
