Divergent RNA structures support accurate splicing of the SF3B1-sensitive MAP3K7 intron

Splicing is governed by interactions between the spliceosome and precursor RNA sequence and structural elements. However, the relative contributions of RNA sequence and structural elements remain unclear. Here, we systematically dissect these determinants using a high-throughput mutagenesis approach with the MAP3K7 intron reporter. The MAP3K7 gene encodes a serine/threonine kinase involved in response to environmental stress. MAP3K7 precursor RNA contains a cryptic 3' splice site that increases in use when the core spliceosomal protein SF3B1 is mutated. SF3B1 mutations are known to promote aberrant splicing and are associated with cancer, particularly the lysine 700 to glutamate mutation (K700E). We designed a pooled library of 249 MAP3K7 mutants targeting branch points, RNA-binding protein motifs, nucleotide composition and predicted structural elements. The impact of these mutants on splicing was measured in the context of normal and SF3B1 K700E expression. RNA structure was assessed in parallel using in vitro high-throughput SHAPE-MAP chemical probing. We found that branchpoint mutations drive the strongest increases in cryptic splice-site use. There is no overall correlation between cryptic splice-site use and structural similarity to the wild-type MAP3K7 RNA. However, mutants within an RNA binding protein hotspot (containing U2AF2, U2AF1, KHSRP and SRSF2 sites) are associated with cryptic splice-site use and structural similarity to wild-type MAP3K7 RNA. These structural changes are associated with increased ensemble diversity. Our results demonstrate that although there are key structured regions within an RNA, there is also extensive variability where divergent RNA structures allow for accurate splicing.