The human branchpoint-interacting stem-loop sequence and structure regulates U2 snRNA expression, branchpoint...

During pre-mRNA splicing, the branch helix forms when U2 snRNP engages with introns to initiate spliceosome assembly. The branch helix is mutually exclusive with the U2 snRNA branchpoint-interacting stem loop (BSL). In yeast, BSL alteration affects branchpoint recognition, but its role in human cells, where branchpoint usage is more flexible, is unknown. To examine the impact of perturbing BSL base pairing, we used a self-contained orthogonal splicing system that pairs an engineered U2 snRNA and splicing reporter. Our results show that BSL mutations affect both U2 snRNA accumulation and splicing in human cells. We also examined the relationship between BSL stability and U2 snRNA complementarity to branchpoint sequence. The results indicate that pairing between the branchpoint sequence and BSL loop links branchpoint fidelity and intron-mediated unwinding of the BSL stem, which supports and extends a toehold-mediated strand invasion model of branch helix formation advanced by Pena and coworkers from cryo-EM structures. Finally, we investigated transcriptome-wide effects of expressing U2 snRNA with either a cancer-associated BSL mutation or with an altered branchpoint recognition sequence. Similarities in both splicing and gene expression changes between the mutants suggest a shared cellular response mechanism leading to gene upregulation linked to oncogenic pathways.