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

Abstract

During pre-mRNA splicing, the branch helix forms when U2 snRNP engages with introns to initiate spliceosome assembly. Its formation is mutually exclusive with the branchpoint-interacting stem loop (BSL) present in U2 snRNA. While BSL structure impacts splicing with the strict consensus branchpoint sequence of yeast introns, its influence in the flexible context of human branchpoints is unknown. We employed an orthogonal U2 snRNA and splicing reporter to examine the impact of perturbing BSL base-pairing and found differential effects on both orthogonal U2 snRNA expression and reporter splicing, indicating that BSL structure influences the biogenesis of U2 snRNP and its function in splicing. Furthermore, high complementarity between the branchpoint sequence and U2 snRNA increases splicing efficiency with wildtype and stabilized BSL, but not when BSL base-pairing is reduced. These data are consistent with complementarity between the intron and the loop of the BSL driving intron-mediated unwinding of the BSL stem. Finally, we investigated transcriptome-wide effects of expressing U2 snRNA with either a cancer-associated BSL mutation or with an altered branchpoint recognition sequence. Similar changes in splicing and gene expression suggests that while altered U2 snRNA is tolerated, cells respond by upregulating genes linked to oncogenic pathways. Main ConclusionsO_LIU2 snRNA BSL base-pairing influences branchpoint sequence recognition and splicing of an orthogonal splicing reporter C_LIO_LIPropose a model for branchpoint sequence complementarity inducing BSL unwinding to promote splicing efficiency C_LIO_LIU2 snRNA BSL and BPRS mutations alter cellular gene expression regulation and splicing C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/673180v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@19daa92org.highwire.dtl.DTLVardef@295f40org.highwire.dtl.DTLVardef@2c7f8eorg.highwire.dtl.DTLVardef@16a562f_HPS_FORMAT_FIGEXP M_FIG C_FIG