Wang Qiang, Zhang Li, Lynn Bert, Rymond Brian C
Department of Biology and Department of Chemistry, University of Kentucky, Lexington, KY 40506-0225, USA.
Nucleic Acids Res. 2008 May;36(8):2787-98. doi: 10.1093/nar/gkn144. Epub 2008 Mar 29.
The 3' end of mammalian introns is marked by the branchpoint binding protein, SF1, and the U2AF65-U2AF35 heterodimer bound at an adjacent sequence. Baker's yeast has equivalent proteins, branchpoint binding protein (BBP) (SF1) and Mud2p (U2AF65), but lacks an obvious U2AF35 homolog, leaving open the question of whether another protein substitutes during spliceosome assembly. Gel filtration, affinity selection and mass spectrometry were used to show that rather than a U2AF65/U2AF35-like heterodimer, Mud2p forms a complex with BBP without a third (U2AF35-like) factor. Using mutants of MUD2 and BBP, we show that the BBP-Mud2p complex bridges partner-specific Prp39p, Mer1p, Clf1p and Smy2p two-hybrid interactions. In addition to inhibiting Mud2p association, the bbpDelta56 mutation impairs splicing, enhances pre-mRNA release from the nucleus, and similar to a mud2::KAN knockout, suppresses a lethal sub2::KAN mutation. Unexpectedly, rather than exacerbating bbpDelta56, the mud2::KAN mutation partially suppresses a pre-mRNA accumulation defect observed with bbpDelta56. We propose that a BBP-Mud2p heterodimer binds as a unit to the branchpoint in vivo and serves as a target for the Sub2p-DExD/H-box ATPase and for other splicing factors during spliceosome assembly. In addition, our results suggest the possibility that the Mud2p may enhance the turnover of pre-mRNA with impaired BBP-branchpoint association.
哺乳动物内含子的3'端由分支点结合蛋白SF1以及结合在相邻序列上的U2AF65 - U2AF35异二聚体标记。酿酒酵母有等效的蛋白质，即分支点结合蛋白（BBP）（SF1）和Mud2p（U2AF65），但缺乏明显的U2AF35同源物，这使得在剪接体组装过程中是否有其他蛋白质替代成为一个悬而未决的问题。凝胶过滤、亲和选择和质谱分析表明，Mud2p不是形成类似U2AF65/U2AF35的异二聚体，而是与BBP形成一个没有第三个（类似U2AF35）因子的复合物。利用MUD2和BBP的突变体，我们表明BBP - Mud2p复合物桥接了伴侣特异性的Prp39p、Mer1p、Clf1p和Smy2p双杂交相互作用。除了抑制Mud2p的结合外，bbpDelta56突变还损害剪接，增强前体mRNA从细胞核的释放，并且与mud2::KAN敲除类似，抑制致死性的sub2::KAN突变。出乎意料的是，mud2::KAN突变不是加剧bbpDelta56，而是部分抑制了bbpDelta56观察到的前体mRNA积累缺陷。我们提出，BBP - Mud2p异二聚体在体内作为一个整体结合到分支点，并在剪接体组装过程中作为Sub2p - DExD/H盒ATP酶和其他剪接因子的靶点。此外，我们的结果表明，Mud2p可能增强与BBP - 分支点结合受损的前体mRNA的周转。
