Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6-U5] tri-snRNP
Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6-U5] tri-snRNP
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DOI：
10.1093/emboj/18.16.4535
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发表时间：
1999-08-16
期刊：
影响因子：
11.4
通讯作者：
Fabrizio, P
中科院分区：
文献类型：
--
作者：
Gottschalk, A;Neubauer, G;Fabrizio, P
25S [U4/U6][5] tri-snRNP（小核核糖核蛋白）是核前mrna剪接机制的中心单位。U4、U5和U6 snRNAs在剪接体中经历了多次重排，了解所有三snrnp蛋白对于剪接体周期中RNA动力学的详细研究至关重要。本文用质谱方法对从酿酒酵母中纯化的[U4/U6 U5] tri-snRNP蛋白进行了表征，除了已知的tri-snRNP蛋白（只有一个，Lsm3p，未被检测到），我们还鉴定了8个以前未被表征的蛋白。这些蛋白包括4种sm样蛋白（Lsm2p、Lsm5p、Lsm6p和Lsm7p）和4种特异性蛋白（Snu13p、Dib1p、Snu23p和Snu66p），其中Snu13p包含一个假定的rna结合域。有趣的是，Dib1p的Schizosaccharomyces pombe同源物Dim1p先前被认为在细胞周期进程中起作用。在体外和/或体内研究了Snu23p、Snu66p和Spp381p在mrna前剪接中的作用。最后，我们发现tri-snRNP和U2 snRNP都与通过甘油梯度离心分离的萃取物中蛋白a标记的Snu23p、Snu66p和sp381p共沉淀。这表明这些蛋白质，至少部分地，也存在于[U2(.)U4/U6(.)]中。[5] 4 - snrnp复合物。
The 25S [U4/U6(.)U5] tri-snRNP (small nuclear ribonucleoprotein) is a central unit of the nuclear pre-mRNA splicing machinery. The U4, U5 and U6 snRNAs undergo numerous rearrangements in the spliceosome, and knowledge of all of the tri-snRNP proteins is crucial to the detailed investigation of the RNA dynamics during the spliceosomal cycle. Here we characterize by mass spectrometric methods the proteins of the purified [U4/U6 U5] tri-snRNP from the yeast Saccharomyces cerevisiae, In addition to the known tri-snRNP proteins (only one, Lsm3p, eluded detection), we identified eight previously uncharacterized proteins. These include four Sm-like proteins (Lsm2p, Lsm5p, Lsm6p and Lsm7p) and four specific proteins named Snu13p, Dib1p, Snu23p and Snu66p, Snu13p comprises a putative RNA-binding domain. Interestingly, the Schizosaccharomyces pombe orthologue of Dib1p, Dim1p, was previously assigned a role in cell cycle progression. The role of Snu23p, Snu66p and, additionally, Spp381p in pre-mRNA splicing was investigated in vitro and/or in vivo. Finally, we show that both tri-snRNPs and the U2 snRNP are co-precipitated with protein A-tagged versions of Snu23p, Snu66p and Spp381p from extracts fractionated by glycerol gradient centrifugation. This suggests that these proteins, at least in part, are also present in a [U2(.)U4/U6(.)U5] tetra-snRNP complex.