Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
Nucleic Acids Res. 2012 May;40(10):4539-52. doi: 10.1093/nar/gks049. Epub 2012 Jan 28.
Saccharomyces cerevisiae Msl5 (branchpoint binding protein) orchestrates spliceosome assembly by binding the branchpoint sequence 5'-UACUAAC and establishing cross intron-bridging interactions with other components of the splicing machinery. Reciprocal tandem affinity purifications verify that Msl5 exists in vivo as a heterodimer with Mud2 and that the Msl5-Mud2 complex is associated with the U1 snRNP. By gauging the ability of mutants of Msl5 to complement msl5Δ, we find that the Mud2-binding (amino acids 35-54) and putative Prp40-binding (PPxY(100)) elements of the Msl5 N-terminal domain are inessential, as are the C-terminal proline-rich domain (amino acids 382-476) and two zinc-binding CxxCxxxxHxxxxC motifs (amino acids 273-286 and 299-312). A subset of conserved branchpoint RNA-binding amino acids in the central KH-QUA2 domain (amino acids 146-269) are essential pairwise (Ile198-Arg190; Leu256-Leu259) or in trios (Leu169-Arg172-Leu176), whereas other pairs of RNA-binding residues are dispensable. We used our collection of viable Msl5 mutants to interrogate synthetic genetic interactions, in cis between the inessential structural elements of the Msl5 polypeptide and in trans between Msl5 and yeast splicing factors (Mud2, Nam8 and Tgs1) that are optional for vegetative growth. The results suggest a network of important but functionally buffered protein-protein and protein-RNA interactions between the Mud2-Msl5 complex at the branchpoint and the U1 snRNP at the 5' splice site.
酿酒酵母 Msl5（分支点结合蛋白）通过结合分支点序列 5'-UACUAAC，并与剪接体其他成分建立交叉内含子桥接相互作用，来协调剪接体的组装。相互串联的亲和纯化验证了 Msl5 在体内以 Msl5-Mud2 异二聚体的形式存在，并且 Msl5-Mud2 复合物与 U1 snRNP 相关。通过衡量 Msl5 突变体补充 msl5Δ 的能力，我们发现 Msl5 N 端结构域中的 Mud2 结合（氨基酸 35-54）和假定的 Prp40 结合（PPxY(100))元件、C 端富含脯氨酸的结构域（氨基酸 382-476）以及两个锌结合 CxxCxxxxHxxxxC 基序（氨基酸 273-286 和 299-312）都是非必需的。中央 KH-QUA2 结构域（氨基酸 146-269）中一组保守的分支点 RNA 结合氨基酸对于形成二聚体至关重要（Ile198-Arg190；Leu256-Leu259）或三聚体（Leu169-Arg172-Leu176），而其他 RNA 结合残基则是可有可无的。我们使用了一系列可行的 Msl5 突变体来探究顺式（在 Msl5 多肽的非必需结构元件之间）和反式（在 Msl5 和酵母剪接因子之间（Mud2、Nam8 和 Tgs1），这些因子对于营养生长是可选的）的合成遗传相互作用。结果表明，在分支点处的 Mud2-Msl5 复合物和 5' 剪接位点处的 U1 snRNP 之间存在着一个重要但功能上有缓冲作用的蛋白质-蛋白质和蛋白质-RNA 相互作用网络。
