Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA; Integrated Program in Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
Cell Chem Biol. 2019 Mar 21;26(3):443-448.e3. doi: 10.1016/j.chembiol.2018.11.008. Epub 2019 Jan 10.
The spliceosome mediates precursor mRNA splicing in eukaryotes, including the model organism Saccharomyces cerevisiae (yeast). Despite decades of study, no chemical inhibitors of yeast splicing in vivo are available. We have developed a system to efficiently inhibit splicing and block proliferation in living yeast cells using compounds that target the human spliceosome protein SF3B1. Potent inhibition is observed in yeast expressing a chimeric protein containing portions of human SF3B1. However, only a single point mutation in the yeast homolog of SF3B1 is needed for selective inhibition of splicing by pladienolide B, herboxidiene, or meayamycin in liquid culture. Mutations that enable inhibition also improve splicing of branch sites containing mismatches between the intron and small nuclear RNA-suggesting a link between inhibitor sensitivity and usage of weak branch sites in humans. This approach provides powerful new tools for manipulating splicing in live yeast and studies of spliceosome inhibitors.
剪接体在真核生物中介导前体 mRNA 的剪接，包括模式生物酿酒酵母（酵母）。尽管经过几十年的研究，目前还没有可用于体内抑制酵母剪接的化学抑制剂。我们开发了一种使用靶向人类剪接体蛋白 SF3B1 的化合物在活酵母细胞中有效抑制剪接和阻断增殖的系统。在表达含有部分人 SF3B1 的嵌合蛋白的酵母中观察到强烈的抑制作用。然而，在液体培养中，只有 SF3B1 的酵母同源物中的单个点突变就足以选择性地抑制 pladienolide B、herboxidiene 或 meayamycin 的剪接。使抑制作用成为可能的突变还改善了包含内含子和小核 RNA 之间错配的分支位点的剪接，这表明抑制剂敏感性与人类中弱分支位点的使用之间存在联系。这种方法为在活酵母中操纵剪接以及研究剪接体抑制剂提供了强大的新工具。
