Haenel, H. | bioRxiv Science

bioRxiv2026

RNA G-quadruplexes mediate cooperativity in HNRNPH binding and splicing regulation

Alternative splicing, regulated by RNA-binding proteins (RBPs), enables the generation of diverse transcript isoforms critical for cellular function. However, how RNA secondary structure impacts RBP binding and function remains poorly understood. Here, we unravel how RNA G-quadruplexes (rG4s) facilitate cooperativity in splicing regulation by the RBP heterogeneous nuclear ribonucleoprotein H (HNRNPH). Through high-throughput in vivo and in vitro studies combined with theoretical modeling, we dissect how rG4s mediate cooperative HNRNPH binding to RNA, ultimately modulating the splicing of hundreds of exons. rG4 unfolding by HNRNPH exposes multiple G-rich binding sites, thereby establishing indirect cooperativity, which is further amplified to achieve switch-like splicing regulation. HNRNPH-mediated regulation is evident in breast cancer patients, with tumors showing rG4-disrupting variants and global HNRNPH alterations, driving distinct splicing patterns that distinguish tumor subtypes. Overall, our findings offer valuable insights into the mechanistic role of RNA secondary structures in cooperative RBP binding and splicing regulation and highlight the clinical relevance of HNRNPH-dependent splicing in cancer. HighlightsO_LIHundreds of cassette exons are cooperatively regulated by HNRNPH. C_LIO_LIUnfolding of RNA G-quadruplexes (rG4s) at HNRNPH binding sites facilitates indirect cooperativity in RNA binding. C_LIO_LIMulti-step splicing amplifies the response into highly switch-like regulation. C_LIO_LIrG4-disrupting variants and changing HNRNPH expression are associated with breast cancer phenotypes. C_LI

molecular biology↗

bioRxiv2024

Long-read transcriptome sequencing of CLL and MDS patients uncovers molecular effects of SF3B1 mutations

BackgroundMutations in splicing factor 3B subunit 1 (SF3B1) frequently occur in patients with chronic lymphocytic leukemia (CLL) and myelodysplastic syndromes (MDS). These mutations have a different effect on the disease prognosis with beneficial effect in MDS and worse prognosis in CLL patients. A full-length transcriptome approach can expand our knowledge on SF3B1 mutation effects on RNA splicing and its contribution to patient survival and treatment options. ResultsWe applied long-read transcriptome sequencing to 44 MDS and CLL patients with and without SF3B1 mutations and found > 60% of novel isoforms. Splicing alterations were largely shared between cancer types and specifically affected the usage of introns and 3 splice sites. Our data highlighted a constrained window at canonical 3 splice sites in which dynamic splice site switches occurred in SF3B1-mutated patients. Using transcriptome-wide RNA binding maps and molecular dynamics simulations, we showed multimodal SF3B1 binding at 3 splice sites and predicted reduced RNA binding at the second binding pocket of SF3B1K700E. ConclusionsOur work presents the hitherto most complete long-read transcriptome sequencing study in CLL and MDS and provides a resource to study aberrant splicing in cancer. Moreover, we showed that different disease prognosis results most likely from the different cell types expanded during cancerogenesis rather than different mechanism of action of the mutated SF3B1. These results have important implications for understanding the role of SF3B1 mutations in hematological malignancies and other related diseases. HighlightsO_LILong-read transcriptome sequencing data enables the identification of > 60% of novel isoforms in the transcriptomes of CLL and MDS patients and isogenic cell lines. C_LIO_LISF3B1 mutations trigger common splicing alterations upon SF3B1 mutations across patient cohorts, most frequently decreased intron retention and increased alternative 3 splice site usage. C_LIO_LIMutation effect depends on alternative 3 splice site and branch point positioning that coincide with bimodal SF3B1 binding at these sites C_LIO_LIMolecular dynamics simulations predict reduced binding of SF3B1K700E to mRNA at the second binding pocket harboring the polypyrimidine tract. C_LI

cancer biology↗