CDK-dependent phosphorylation and disease-associated mutations in distinct regions of splicing factor 3B1 (SF3B1) influence its interaction with chromatin and RNA.

Murthy, Tushar

doi:https://doi.org/10.21985/n2-6rde-d089

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CDK-dependent phosphorylation and disease-associated mutations in distinct regions of splicing factor 3B1 (SF3B1) influence its interaction with chromatin and RNA.

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Splicing factor 3B1 (SF3B1) is a core splicing protein that stabilizes the interaction between the U2 snRNA and the branch point (BP) in the RNA target during splicing. SF3B1 is heavily phosphorylated at its N terminus and a substrate of cyclin-dependent kinases (CDKs). Although SF3B1 phosphorylation coincides with splicing catalysis, the functional significance of SF3B1 phosphorylation is largely undefined. Here, we show that SF3B1 phosphorylation follows a dynamic pattern during cell cycle progression that depends on CDK activity. SF3B1 is known to interact with chromatin, and we found that SF3B1 maximally interacts with nucleosomes during G1/S and that this interaction requires CDK2 activity. In contrast, SF3B1 disassociated from nucleosomes at G2/M, coinciding with a peak in CDK1-mediated SF3B1 phosphorylation. Thus, CDK1 and CDK2 appear to have opposing roles in regulating SF3B1–nucleosome interactions. Importantly, these interactions were modified by the presence and phosphorylation status of linker histone H1, particularly the H1.4 isoform. Performing genome-wide analysis of SF3B1–chromatin binding in synchronized cells, we observed that SF3B1 preferentially bound exons. Differences in SF3B1 chromatin binding to specific sites, however, did not correlate with changes in RNA splicing, suggesting that the SF3B1–nucleosome interaction does not determine cell cycle–dependent changes to mRNA splicing. Our results define a cell cycle stage–specific interaction between SF3B1 and nucleosomes that is mediated by histone H1 and depends on SF3B1 phosphorylation. Importantly, this interaction does not seem to be related to SF3B1’s splicing function and, rather, points toward its potential role as a chromatin modifier. The HEAT domain-containing C terminus of SF3B1 contains frequent mutations that have been discovered in several neoplastic processes including myelodysplastic syndrome (MDS). These mutations are almost always hemizygous and mutually exclusive with other splicing factor mutations, suggesting their role as driver mutations during disease pathogenesis. SF3B1 mutations are associated with the selection of a novel BP and cryptic 3’ splice site (ss) upstream of the canonical sequences. However, the precise molecular mechanism by which mutant SF3B1 expression results in altered BP and 3’ss selection remains unclear. While current hypotheses imply that mutant SF3B1 expression results in mis-splicing due to obligate changes in BP and 3’ss selection, such hypotheses are not supported by transcriptomic analyses of clinical samples, cell lines and isogenic murine models. In order to better define factors that influence BP selection by mutant SF3B1-containing spliceosomes, we generated an isogenic murine embryonic stem cell (mESC) line with the hemizygous Sf3b1 K700E mutation using CRISPR-Cas9-based gene editing. A control homozygous Sf3b1 K700K line was also generated for comparison. To enable a statistically robust and unbiased evaluation of BP choice by wild-type (WT) and mutant SF3B1, mESC lines were combined with a massively parallel reporter assay (MPRA) containing millions of splicing reporter minigenes with degenerate sequences in the 3’ intronic region. Our results demonstrate that while both WT and mutant SF3B1 utilize canonical BP sequences (YTAAY) at similar frequencies, mutant SF3B1 is capable of preferentially utilizing non-canonical BP sequences that vary at -1 position relative to the BP adenosine. Our findings provide crucial insights into the biochemical mechanism by which mutant SF3B1 expression results in altered splicing.

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