Genetic evidence for 18S rRNA binding and an Rps19p assembly function of yeast nucleolar protein Nep1p
Genetic evidence for 18S rRNA binding and an Rps19p assembly function of yeast nucleolar protein Nep1p
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DOI：
10.1007/s00438-006-0132-x
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发表时间：
2006-09-01
影响因子：
3.1
通讯作者：
Entian, Karl-Dieter
中科院分区：
文献类型：
--
作者：
Buchhaupt, Markus;Meyer, Britta;Entian, Karl-Dieter
核仁蛋白Nep 1及其人类同源物此前已被证明参与18 S rRNA的成熟，并直接或间接干扰甲基化反应。在这里，我们报告，功能丧失突变三角洲snr 57和核糖体40 S亚基蛋白19（Rps 19 p）的多拷贝表达可以部分抑制酿酒酵母三角洲nep 1生长缺陷。SnR 57介导18 S rRNA中G（1570）的2 '-O-核糖甲基化。通过进行三杂交筛选，我们分离出几个短的RNA序列与Nep 1 p的强结合亲和力。所有分离的RNA共享一个六核苷酸的一致基序C/UUCAAC。此外，其中一个分离的RNA完全对应于18 S rRNA的核苷酸1553-1577，其包括G（1570），snR 57依赖性18 S rRNA甲基化的位点。从蛋白质-蛋白质交联数据和S.酿酒酵母小核糖体亚基，我们建议Rps 19 p是本地化的Nep 1 p 18 S rRNA结合位点附近。我们的研究结果表明，Nep 1 p结合相邻的螺旋47的18 S rRNA，并可能支持协会的Rps 19 p前核糖体颗粒。
The nucleolar protein Nep1 and its human homologue were previously shown to be involved in the maturation of 18S rRNA and to interfere directly or indirectly with a methylation reaction. Here, we report that the loss-of-function mutation Delta snr57 and multicopy expression of the ribosomal 40S subunit protein 19 (Rps19p) can partially suppress the Saccharomyces cerevisiae Delta nep1 growth defect. SnR57 mediates 2'-O-ribose-methylation of G(1570) in the 18S rRNA. By performing a three-hybrid screen, we isolated several short RNA sequences with strong binding affinity to Nep1p. All isolated RNAs shared a six-nucleotide consensus motif C/UUCAAC. Furthermore, one of the isolated RNAs exactly corresponded to nucleotides 1553-1577 of the 18S rRNA, which includes G(1570), the site of snR57-dependent 18S rRNA methylation. From protein-protein crosslink data and the cryo-EM map of the S. cerevisiae small ribosomal subunit, we suggest that Rps19p is localized in close vicinity to the Nep1p 18S rRNA binding site. Our results suggest that Nep1p binds adjacent to helix 47 of the 18S rRNA and possibly supports the association of Rps19p to pre-ribosomal particles.