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

The branch helix is a structure that forms when U2 snRNP engages with introns to initiate spliceosome assembly, and its formation is mutually exclusive with the branchpoint-interacting stem loop (BSL) present in U2 snRNA. While BSL structure impacts splicing with the constrained branchpoint sequence in yeast introns, its influence in the flexible context of human branchpoints is unknown. We employed an orthogonal U2 snRNA and splicing reporter to examine effects of perturbing BSL sequence. Changes to BSL base-pairing differentially affect both orthogonal U2 snRNA expression and reporter splicing. High complementarity between the branchpoint sequence and U2 snRNA increases splicing efficiency with WT and stabilized BSL, but not when BSL base-pairing is reduced. Together our data suggest that BSL structure influences the biogenesis of U2 snRNP and that after base pairs are established with the loop of the BSL, the intron drives unwinding of the BSL stem. Finally, we investigated the transcriptome-wide changes when U2 snRNA with either a cancer-associated BSL mutation or with an altered branchpoint recognition sequence is expressed. In both cases, similar changes in splicing and gene expression suggests that while altered U2 snRNA is tolerated, cells respond to their presence by upregulating genes linked to oncogenic pathways.