Altered RNA export by SF3B1 mutants confers sensitivity to nuclear export inhibition
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ABSTRACT:
PROVIDER: PRJNA1073893 | ENA |
REPOSITORIES: ENA
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Project description:SF3B1 mutations are the most frequent spliceosomal alterations across cancers, yet no successful therapy exists to target this pathway. Previous findings from a phase 2 clinical trial of the XPO1 inhibitor selinexor in patients with high-risk myelodysplastic neoplasms (MDS) relapsed or refractory to hypomethylating agents (HMA) revealed increased activity in patients with SF3B1 mutations. XPO1 (Exportin-1) is responsible for the export of over 200 proteins, but also plays a role in the transport of multiple RNA species, including small nuclear RNAs (snRNAs), ribosomal RNAs (rRNAs), and select messenger RNAs (mRNAs) out of the nucleus. We therefore hypothesized that XPO1 inhibition perturbs RNA export and may preferentially affect SF3B1 mutants via altered splicing, given the role of XPO1 in exporting snRNAs, which form the catalytic portion of the spliceosome. To evaluate the mechanism underlying preferentially sensitivity of SF3B1-mutants to XPO1 inhibition, we performed nuclear and cytoplasmic fraction followed by RNA sequencing before and after XPO1 inhibition in SF3B1 wildtype and SF3B1 K666N cells (subcellular RNA-seq). Whole transcriptomic analysis of subcellular RNA-seq data revealed more nuclear retention of global RNA transcripts after XPO1 inhibition in the SF3B1 mutant cell line. Similarly, we performed subcellular RNA-seq for small RNAs and found snRNAs to be increased in the nucleus after XPO1 inhibition in the SF3B1 mutant cells. We then performed total cellular RNA sequencing to understand the effect of XPO1 inhibition on global RNA expression and RNA splicing. Differential gene expression analysis identified that XPO1 inhibition had the greatest effect on cell cycle in SF3B1 wildtype cells but in SF3B1-mutant cells differentiation pathways were more significantly affected. Alternative splicing analysis showed increased 3’ alternative splicing events in SF3B1 mutant after XPO1 inhibition. These results signify the mechanistic basis for preferential sensitivity of SF3B1 mutant cells to nuclear export inhibition arises through nuclear retention of spliceosomal snRNAs and select mRNAs that result in perturbation of differentiation pathways.
2024-08-05 | GSE255179 | GEO
Project description:Pre-mRNA splicing and nuclear export are coordinated processes that ensure efficient and accurate gene expression. In recent years, disruptions in this process have been found to result in substantial gene expression aberrations, with potential contributions from SF3B1 mutations, whose mechanism remains elusive. The present study reveals that the K700E mutation in SF3B1 attenuates its interaction with THOC5, resulting in reduced mRNA binding by THOC5, and ultimately, the inhibition of mRNA nuclear export. Interestingly, overexpressing THOC5 can restore the attenuated interaction, facilitating mRNA nuclear export. Importantly, other cancer-associated SF3B1 mutations may exert a similar impact. Our research highlights the critical role of the THOC5–SF3B1 interaction in regulating mRNA nuclear export and provides valuable insights into the impact of SF3B1 mutations on this process.
2024-09-12 | GSE268355 | GEO
Project description:Cancer-associated SF3B1 Mutations Inhibit mRNA Nuclear Export by Disrupting SF3B1–THOC5 Interactions
| PRJNA1116549 | ENA
Project description:Knockdown of mutant and/or wild-type SF3B1 in MEL202 cell line by Degron knock-in, followed by RNA-seq, to identify splicing events governed by mutant SF3B1. Control: parental MEL202 cell line. Experiments: mutant-SF3B1 knockdown; wildtype-SF3B1 knockdown; mutant SF3B1 knockout. Treatments: each of these four conditions plus and minus shld.
2015-03-11 | E-GEOD-66719 | biostudies-arrayexpress
Project description:Much remains unknown concerning the mechanism by which the splicing machinery pinpoints short exons within intronic sequences and how splicing factors are directed to their pre-mRNA targets. Part of the explanation probably lies in differences in chromatin organization between exons and introns. Proteomic, co-immunoprecipitation, and sedimentation analyses described here indicated that SF3B1, an essential splicing component of the U2 snRNP complex, is strongly associated with nucleosomes. ChIP-seq and RNA-seq analyses revealed that SF3B1 is specifically bound to nucleosomes located at exonic positions. SF3B1 binding is enriched at nucleosomes positioned over short exons flanked by long introns that are also characterized by differential GC content between exons and introns. Disruption of SF3B1 binding to such nucleosomes affected the splicing of these exons similarly to inhibition of SF3B1 expression. Our findings suggest that the association of SF3B1 with nucleosomes is functionally important for splice site recognition and that SF3B1 conveys splicing-relevant information embedded in chromatin structure. MNase-seq on Input and SF3B1 pull-down, mRNA-seq on control and SF3B1 si-RNA treated cells as well as on TSA (Trichostatin A) treated and untreated cells.
2015-04-22 | E-GEOD-65644 | biostudies-arrayexpress
Project description:Using RNA-Seq, we determined changes to gene expression and splicing on inducible expression of SF3B1-WT and SF3B1-MUT (K700E) in K562 cells. Using RNA-Seq, we determined changes to gene expression and splicing on inducible expression of SF3B1-WT and SF3B1-MUT (K700E) in K562 cells.
2016-07-15 | E-GEOD-70959 | biostudies-arrayexpress
Project description:Liver lineage confers sensitivity of cancer cells to sulfonation-dependent alkylators
| PRJNA714004 | ENA
Project description:Recent studies have shown that multiple components of the mRNA splicing machinery are mutated in myelodysplastic syndrome (MDS) patients. SF3B1 is frequently mutated in refractory anemia with ringed sideroblasts (RARS)-MDS patients, however, the pathophysiological role of SF3B1 mutations has not been elucidated yet. In this study, we examined the function of Sf3b1 in murine hematopoiesis. Since Sf3b1 null homozygotes died during preimplantation development, in this study, we utilized Sf3b1 heterozygous mice showing grossly normal growth. We harvested bone marrow stem/progenitor (LSK) cells from wild type (WT) and Sf3b1+/- mice (n=4) at 20 weeks old. In addition, to exclude the possibility of indirect effect from bone marrow environment, we transplanted total bone marrow cells from WT or Sf3b1+/- (CD45.2+) mice into lethally irradiated CD45.1+ recipient mice, and then harvested (CD45.2+) LSK cells from the recipients (n=5) at 9 months-post transplantation.
2013-09-20 | E-GEOD-51038 | biostudies-arrayexpress
Project description:The production of noncanonical mRNA transcripts is associated with cell transformation. Driven by our previous findings on the sensitivity of T cell acute lymphoblastic leukemia (T-ALL) cells to SF3B1 inhibitors, we identified that SF3B1 inhibition blocks T-ALL growth in vivo with no notable associated toxicity. We also revealed protein stabilization of the U2 complex component SF3B1 via deubiquitination. Our studies showed that SF3B1 inhibition perturbs exon skipping, leading to nonsense-mediated decay and diminished levels of DNA damage response–related transcripts, such as the serine/threonine kinase CHEK2, and impaired DNA damage response. We also identified that SF3B1 inhibition leads to a general decrease in R-loop formation. We further demonstrate that clinically used SF3B1 inhibitors synergize with CHEK2 inhibitors and chemotherapeutic drugs to block leukemia growth. Our study provides the proof of principle for posttranslational regulation of splicing components and associated roles and therapeutic implications for the U2 complex in T cell leukemia.
2022-03-13 | GSE198392 | GEO