Noble S M, Guthrie C
Department of Biochemistry and Biophysics, University of California, San Francisco 94143, USA.
EMBO J. 1996 Aug 15;15(16):4368-79.
Vertebrate spliceosomal snRNAs associate with a conserved set of proteins, the Sm proteins, via a conserved RNA sequence, the Sm site. Assembly of this complex is required for the accumulation of stable snRNPs, hypermethylation of the 5' cap structure and nuclear import of the resultant particles. The function of individual core snRNP proteins is poorly understood, in part because of the difficulty of selectively inactivating individual polypeptides in vivo. Using a transcriptional pulse-chase method we have defined for the first time the steps of snRNP biogenesis in Saccharomyces cerevisiae. We describe a novel component of spliceosomal snRNPs, Brr1, which is distinct in sequence from Sm core proteins and yet which shares many of their properties, as well as a genetic interaction with the yeast homolog of Sm D1 core protein. Through a kinetic analysis of snRNP formation in wild-type and brr1 mutant cells we demonstrate specific defects in a subset of steps in the brr1 mutant: newly synthesized snRNAs are destabilized and 3'-end processing is slowed, whereas the cap hypermethylation reaction is unaffected. Notably, the stability of mature particles, as measured by promoter shut-off experiments, is normal in the absence of the Brr1 snRNP protein.
脊椎动物剪接体snRNA通过保守的RNA序列（Sm位点）与一组保守的蛋白质（Sm蛋白）结合。这种复合物的组装对于稳定snRNP的积累、5'帽结构的超甲基化以及所得颗粒的核输入是必需的。单个核心snRNP蛋白的功能了解甚少，部分原因是在体内选择性地使单个多肽失活存在困难。我们使用转录脉冲追踪方法首次确定了酿酒酵母中snRNP生物发生的步骤。我们描述了剪接体snRNP的一个新组分Brr1，其序列与Sm核心蛋白不同，但具有许多共同特性，并且与Sm D1核心蛋白的酵母同源物存在遗传相互作用。通过对野生型和brr1突变体细胞中snRNP形成的动力学分析，我们证明brr1突变体中一部分步骤存在特定缺陷：新合成的snRNA不稳定，3'端加工减慢，而帽超甲基化反应不受影响。值得注意的是，通过启动子关闭实验测量，在没有Brr1 snRNP蛋白的情况下，成熟颗粒的稳定性正常。
