An in vivo and in vitro structure-function analysis of the Saccharomyces cerevisiae U3A snoRNP: Protein-RNA contacts and base-pair interaction with the pre-ribosomal RNA
An in vivo and in vitro structure-function analysis of the Saccharomyces cerevisiae U3A snoRNP: Protein-RNA contacts and base-pair interaction with the pre-ribosomal RNA
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DOI：
10.1006/jmbi.1997.1320
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发表时间：
1997-10-31
影响因子：
5.6
通讯作者：
Branlant, C
中科院分区：
文献类型：
--
作者：
Mereau, A;Fournier, R;Branlant, C
介绍了S.用化学探针和酶促探针在半纯化的U3 A snoRNP中研究了酿酒酵母U3 A snoRNA，并用DMS作探针在体内研究了U3 A snoRNA。酿酒酵母U3 A snoRNA由短的5'结构域组成，两个茎-环结构，含有遗传学保守的盒A1和A，和一个大的十字形3'结构域，含有盒B、C、C'和D。提供了RNA-蛋白质接触的精确鉴定。保护蛋白的snoRNP和ii？vivo几乎相同，并且仅在3'结构域中发现。存在两个不同的蛋白质锚定位点：（i）盒C'及其周围区域，该位点可能包括盒D，（ii）盒B和C桶以及茎环2和4的碱基。盒C'被蛋白质包裹，RNA-蛋白质相互作用在盒C和D的水平上更松散，并且在snoRNP中保留了盒C和D相互作用。在雅阁的这个位置上有一个蛋白结合位点吗？体内突变分析显示，盒Ct对于U3 A snoRNA积累是重要的，而5 ′结构域中的突变对RNA稳定性几乎没有影响。我们的体内探测实验强烈表明，在指数生长的细胞中，大多数U3 A snoRNA分子参与了与5 '-ETS区域的10-bp相互作用以及最近提出的与18 S rRNA序列的两种相互作用。OW实验研究导致了由J.休斯提出的相互作用模型的一个稍微修改的版本。连接异源螺旋的单链片段在体内对DMS高度敏感，并且通过突变分析测试其功能重要性。(C)出版社：Academic Press Limited。
The structure and accessibility of the S. cerevisiae U3A snoRNA was studied In semi-purified U3A snoRNPs using both chemical and enzymatic probes and in vivo using DMS as the probe, The results obtained show that S. cerevisiae U3A snoRNA is composed of a short 5' domain with. two stem-loop structures containing the phylogenetically conserved boxes Al and A and a large cruciform 3' domain containing boxes B, C, C' and D. A precise identification of RNA-protein contacts is provided. Protection by proteins in the snoRNP and ii? vivo are nearly identical and were exclusively found in the 3' domain. There are two distinct protein anchoring sites: (i), box C' and its surrounding region, this site probably includes box D, (ii) the boxes B and C pail and the bases of stem-loop 2 and 4. Box C' is wrapped by the proteins, RNA-protein interactions are more loose at the level of boxes C and D and a box C and D interaction is preserved in the snoRNP. In accord with this location of the protein binding sites, an it? vivo mutational analysis showed that box Ct is important for U3A snoRNA accumulation, whereas mutations in the 5' domain have little effect on RNA stability. Our in viva probing experiments strongly suggest that, in exponentially growing cells, most of the U3A snoRNA molecules are involved in the 10-bp interaction with the 5'-ETS region and in two of the interactions recently proposed with 18 S rRNA sequences. Ow experimental study leads to a slightly revised version of the model of interaction proposed by J. Hughes. Single-stranded segments linking the heterologous helices are highly sensitive to DMS in vivo and their functional importance was tested by a mutational analysis. (C) 1997 Academic Press Limited.