Abstract
Splicing factor 3b subunit 1 (SF3B1) is an essential protein in spliceosomes and mutated frequently in many cancers. While roles of SF3B1 in single intron splicing and roles of its cancer-linked mutant in aberrant splicing have been identified to some extent, regulatory functions of wild-type SF3B1 in alternative splicing (AS) are not well-understood yet. Here, we applied RNA sequencing (RNA-seq) to analyze genome-wide AS in SF3B1 knockdown (KD) cells and to identify a large number of skipped exons (SEs), with a considerable number of alternative 5′ splice-site selection, alternative 3′ splice-site selection, mutually exclusive exons (MXE), and retention of introns (RI). Among altered SEs by SF3B1 KD, survival motor neuron 2 (SMN2) pre-mRNA exon 7 splicing was a regulatory target of SF3B1. RT-PCR analysis of SMN exon 7 splicing in SF3B1 KD or overexpressed HCT116, SH-SY5Y, HEK293T, and spinal muscular atrophy (SMA) patient cells validated the results. A deletion mutation demonstrated that the U2 snRNP auxiliary factor 65 kDa (U2AF65) interaction domain of SF3B1 was required for its function in SMN exon 7 splicing. In addition, mutations to lower the score of the polypyrimidine tract (PPT) of exon 7, resulting in lower affinity for U2AF65, were not able to support SF3B1 function, suggesting the importance of U2AF65 in SF3B1 function. Furthermore, the PPT of exon 7 with higher affinity to U2AF65 than exon 8 showed significantly stronger interactions with SF3B1. Collectively, our results revealed SF3B1 function in SMN alternative splicing.
Highlights
Introns are removed from pre-mRNA during splicing by the spliceosome, a protein–RNA complex that catalyzes the excision of introns and the ligation of exons to form mature mRNA [1,2].The spliceosome assembles onto pre-mRNA with five small nuclear ribonucleoproteins(U1, U2, U4, U5, and U6) and non-snRNPs [1]
SF3B1-interacting U2 snRNP auxiliary factor kDa (U2AF65) protein expression level was not affected by SF3B1 KD
SF3B1 overexpression can promote cassette exon inclusion of survival motor neuron 2 (SMN2) pre-mRNA. These results indicated that SF3B1 is a regulatory factor of survival of motor neuron (SMN) pre-mRNA splicing
Summary
Introns are removed from pre-mRNA during splicing by the spliceosome, a protein–RNA complex that catalyzes the excision of introns and the ligation of exons to form mature mRNA [1,2]. The first support is from splicing factor 3b (SF3B), a multiprotein component of the U2 snRNP that can interact with pre-mRNA at or near the BPS to reinforce the base-pairing between U2 snRNP and BPS [6,7]. A duplicate of SMN1 gene called SMN2 is found in all of the SMA patients It mostly produces an mRNA with cassette exon 7 exclusion (~80–90%) that encodes a truncated protein and highly unstable isoform (SMN∆7) that does not support SMN function [39,40]. A deletion mutation demonstrated that the U2AF65 interaction domain of SF3B1 was required for its function in SMN exon 7 splicing. Our results revealed the important function of SF3B1 in SMN alternative splicing
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