Microbiology and Immunology Department, Weill Cornell Medical College, New York, NY 10065, USA and Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
Nucleic Acids Res. 2013 Aug;41(15):7485-500. doi: 10.1093/nar/gkt490. Epub 2013 Jun 10.
Yeast pre-mRNA splicing initiates via formation of a complex comprising U1 snRNP bound at the 5' splice site (5'SS) and the Msl5•Mud2 heterodimer engaged at the branchpoint (BP). Here, we present a mutational analysis of the U1 snRNA, which shows that although enlarging the 5' leader between the TMG cap and the (3)ACUUAC(8) motif that anneals to the 5'SS is tolerated, there are tight constraints on the downstream spacer between (3)ACUUAC(8) and helix 1 of the U1 fold. We exploit U1 alleles with 5' extensions, variations in the (3)ACUUAC(8) motif, downstream mutations and a longer helix 1 to discover new intra-snRNP synergies with U1 subunits Nam8 and Mud1 and the trimethylguanosine (TMG) cap. We describe novel mutations in U1 snRNA that bypass the essentiality of the DEAD-box protein Prp28. Structure-guided mutagenesis of Msl5 distinguished four essential amino acids that contact the BP sequence from nine other BP-binding residues that are inessential. We report new synthetic genetic interactions of the U1 snRNP with Msl5 and Mud2 and with the nuclear cap-binding subunit Cbc2. Our results fortify the idea that spliceosome assembly can occur via distinct genetically buffered microscopic pathways involving cross-intron-bridging interactions of the U1 snRNP•5'SS complex with the Mud2•Msl5•BP complex.
酵母前体 mRNA 剪接通过形成一种复合物启动，该复合物包含结合在 5' 剪接位点（5'SS）的 U1 snRNP 和结合在分支点（BP）的 Msl5•Mud2 异二聚体。在这里，我们对 U1 snRNA 进行了突变分析，结果表明，尽管在 TMG 帽和与 5'SS 退火的（3）ACUUAC（8）基序之间的 5' 前导区放大是可以容忍的，但在（3）ACUUAC（8）和 U1 折叠的螺旋 1 之间的下游间隔区存在严格的限制。我们利用具有 5' 延伸、（3）ACUUAC（8）基序变异、下游突变和更长的螺旋 1 的 U1 等位基因，发现与 U1 亚基 Nam8 和 Mud1 以及三甲基鸟苷（TMG）帽的新的内 snRNP 协同作用。我们描述了 U1 snRNA 中的新突变，这些突变绕过了 DEAD 盒蛋白 Prp28 的必需性。对 Msl5 的结构导向诱变区分了与 BP 序列接触的四个必需氨基酸和与 BP 结合无关的九个其他 BP 结合残基。我们报告了 U1 snRNP 与 Msl5 和 Mud2 以及与核帽结合亚基 Cbc2 的新的合成遗传相互作用。我们的结果强化了这样一种观点，即剪接体组装可以通过涉及 U1 snRNP•5'SS 复合物与 Mud2•Msl5•BP 复合物之间的跨内含子桥接相互作用的不同遗传缓冲微观途径发生。
