Faculty Collaboration Database

Degenerate minigene library analysis enables identification of altered branch point utilization by mutant splicing factor 3B1 (SF3B1). Nucleic Acids Res 2019 Jan 25;47(2):970-980

Date

11/22/2018

Pubmed ID

30462273

Pubmed Central ID

PMC6344872

DOI

10.1093/nar/gky1161

Scopus ID

2-s2.0-85060616422 (requires institutional sign-in at Scopus site)   8 Citations

Abstract

Cancer-associated mutations of the core splicing factor 3 B1 (SF3B1) result in selection of novel 3' splice sites (3'SS), but precise molecular mechanisms of oncogenesis remain unclear. SF3B1 stabilizes the interaction between U2 snRNP and branch point (BP) on the pre-mRNA. It has hence been speculated that a change in BP selection is the basis for novel 3'SS selection. Direct quantitative determination of BP utilization is however technically challenging. To define BP utilization by SF3B1-mutant spliceosomes, we used an overexpression approach in human cells as well as a complementary strategy using isogenic murine embryonic stem cells with monoallelic K700E mutations constructed via CRISPR/Cas9-based genome editing and a dual vector homology-directed repair methodology. A synthetic minigene library with degenerate regions in 3' intronic regions (3.4 million individual minigenes) was used to compare BP usage of SF3B1K700E and SF3B1WT. Using this model, we show that SF3B1K700E spliceosomes utilize non-canonical sequence variants (at position -1 relative to BP adenosine) more frequently than wild-type spliceosomes. These predictions were confirmed using minigene splicing assays. Our results suggest a model of BP utilization by mutant SF3B1 wherein it is able to utilize non-consensus alternative BP sequences by stabilizing weaker U2-BP interactions.

Author List

Gupta AK, Murthy T, Paul KV, Ramirez O, Fisher JB, Rao S, Rosenberg AB, Seelig G, Minella AC, Pillai MM

Author

Sid Rao PhD, MD Associate Professor in the Pediatrics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Base Pairing
Cells, Cultured
Embryonic Stem Cells
Gene Library
HEK293 Cells
Humans
Mice
Mutation
Nucleotide Motifs
Phosphoproteins
RNA Splice Sites
RNA Splicing Factors
RNA, Messenger