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Spliceostatin A targets SF3b and inhibits both splicing and nuclear retention of pre-mRNA

Abstract

The removal of intervening sequences from transcripts is catalyzed by the spliceosome, a multicomponent complex that assembles on the newly synthesized pre-mRNA. Pre-mRNA translation in the cytoplasm leads to the generation of aberrant proteins that are potentially harmful. Therefore, tight control to prevent undesired pre-mRNA export from the nucleus and its subsequent translation is an essential requirement for reliable gene expression. Here, we show that the natural product FR901464 (1) and its methylated derivative, spliceostatin A (2), inhibit in vitro splicing and promote pre-mRNA accumulation by binding to SF3b, a subcomplex of the U2 small nuclear ribonucleoprotein in the spliceosome. Importantly, treatment of cells with these compounds resulted in leakage of pre-mRNA to the cytoplasm, where it was translated. Knockdown of SF3b by small interfering RNA induced phenotypes similar to those seen with spliceostatin A treatment. Thus, the inhibition of pre-mRNA splicing during early steps involving SF3b allows unspliced mRNA leakage and translation.

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Figure 1: FR901464-induced accumulation of p27*.
Figure 2: Identification of the splicing factor SF3b as the target.
Figure 3: SSA-mediated inhibition of in vitro and in vivo splicing.
Figure 4: Subcellular localization of poly(A) RNA and SC35.
Figure 5: Pre-mRNA translation in SSA-treated cells.
Figure 6: Phenotypic changes induced by SF3b knockdown.
Figure 7: p27* as a functional CDK inhibitor.

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Acknowledgements

We thank A. Krainer (Cold Spring Harbor Laboratory) for pSP64-HβΔ6 and pμC3-C4, K. Nagata for kind advice regarding preparation of nuclear extracts, and A. Kulozik (University of Heidelberg) for the NMD detection system. We are grateful to the RIKEN Brain Science Institute's Research Resources Center for DNA sequencing analysis and mass spectrometry. This work was supported in part by the CREST Research Project, the Japan Science and Technology Agency, The Strategic Research Programs for R&D, RIKEN, and a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan. SF3b image in Graphical Abstract from Golas et al. Science 300, 980–984 (2003). Reprinted with permission from AAAS.

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M.Y. is responsible for project planning and experimental design, with support from M.H., T.T. and S.H.; D.K. performed most of the experiments; H.M., K.I., H.W. and T.K. synthesized chemical compounds; E.T. carried out in vitro kinase assays; T.N. performed in vitro splicing assays; T.Y. carried out pilot study; H.N. prepared FR901464.

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Correspondence to Minoru Yoshida.

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Kaida, D., Motoyoshi, H., Tashiro, E. et al. Spliceostatin A targets SF3b and inhibits both splicing and nuclear retention of pre-mRNA. Nat Chem Biol 3, 576–583 (2007). https://doi.org/10.1038/nchembio.2007.18

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