RNA secondary structures modulate hnRNPH1-mediated alternative splicing of cold-shock protein RBM3

Abstract

Alternative splicing is regulated not only by sequence-specific RNA-protein interactions, but also by dynamic RNA secondary structures that remodel under varying cellular conditions. hnRNPH1, a G-rich motif-binding protein, promotes exon skipping across numerous transcripts, yet how RNA structure shapes its regulatory activity remains unclear. Building on our previous work showing that cooler temperature enhances hnRNPH1 binding to RBM3 pre-mRNA and drives poison exon (PE) skipping, we now identify RNA G-quadruplex (rG4) remodelling as the principal cis-acting mechanism underlying this temperature-sensitive regulation. Using small-molecule stabilizers and destabilizers, we show that rG4 folding reduces PE skipping, whereas rG4 destabilization enhances hnRNPH1-mediated PE exclusion. These effects are conserved across neuronal and non-neuronal cells. Integrating proteomics, phosphoproteomics, kinase perturbation assays, and candidates emerging from our prior genome-wide CRISPR screen, we further uncover contributions from temperature-sensitive CDC2-like kinase signalling and the RNA helicase DDX5, a known rG4 resolver and an hnRNPH1/hnRNPF co-factor. Together, our findings establish rG4 remodelling as the dominant driver of hnRNPH1-dependent RBM3 splicing, while implicating helicase activity and kinase signalling as modulatory layers that fine-tune this response. More broadly, this work illustrates how environmentally driven RNA structural transitions interface with enzymatic regulators to achieve temperature-responsive control of alternative splicing.