Pseudouridylation (Ψ) of U2 snRNA in S-cerevisiae is catalyzed by an RNA-independent mechanism
Pseudouridylation (Ψ) of U2 snRNA in S-cerevisiae is catalyzed by an RNA-independent mechanism
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DOI：
10.1093/emboj/cdg191
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发表时间：
2003-04-15
期刊：
影响因子：
11.4
通讯作者：
Yu, YT
中科院分区：
文献类型：
--
作者：
Ma, XJ;Zhao, XL;Yu, YT
脊椎动物中snRNA的假尿苷化由小核仁/卡哈尔体特异性RNA（sno/scaRNA）指导。我们开发了一种体外系统，使用细胞提取物和单位点放射性标记的U2 snRNAs研究酿酒酵母中的假尿苷酸化。微球菌核酸酶处理的细胞提取物完全有能力催化U2假尿苷化，这表明一个RNA独立的过程。通过筛选酿酒酵母GST-ORF蛋白质文库鉴定酵母U2内Psi（35）的假尿苷酶活性。该活性与YOR 243 c ORF相关，该ORF先前未被指定功能。当GST-YOR 243 c蛋白在大肠杆菌中表达时，假尿苷化活性与在酿酒酵母中表达的活性相当，证明该蛋白（命名为Pus 7）单独可以催化U2中的Psi（35）形成。使用野生型和pus 7-Delta菌株的体外和体内分析均表明，Pus 7对于U2中Psi（35）的形成是必不可少的。使用位点特异性放射性标记的U2和U2片段，我们表明Pus 7活性对Psi（35）是特异性的，并且U2茎环II区对于假尿苷化反应是必需的。BLAST搜索揭示了多种生物体中的Pus 7同源物。
Pseudouridylation of snRNAs in vertebrates is guided by small nucleolar/Cajal body-specific RNAs (sno/scaRNAs). We developed an in vitro system using cell extracts and single site-radiolabeled U2 snRNAs to study pseudouridylation in Saccharomyces cerevisiae. Micrococcal nuclease-treated cell extracts are fully competent to catalyze U2 pseudouridylation, suggesting an RNA-independent process. A pseudouridylase activity for Psi(35) within yeast U2 is identified via a screen of an S.cerevisiae GST-ORF protein library. This activity is associated with YOR243c ORF, which has not previously been assigned function. When the GST-YOR243c protein is expressed in Escherichia coli, pseudouridylation activity is comparable to that expressed in S.cerevisiae, demonstrating that this protein (designated Pus7) alone can catalyze Psi(35) formation in U2. Both in vitro and in vivo analyses using wild-type and pus7-Delta strains show that Pus7 is indispensable for Psi(35) formation in U2. Using site-specific radiolabeled U2 and U2 fragments, we show that Pus7 activity is specific for Psi(35) and that the U2 stem- loop II region is essential for the pseudouridylation reaction. A BLAST search revealed Pus7 homologs in various organisms.