Youssef Osama A, Terns Rebecca M, Terns Michael P
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
Nucleic Acids Res. 2007;35(18):6196-206. doi: 10.1093/nar/gkm673. Epub 2007 Sep 12.
H/ACA RNP complexes change uridines to pseudouridines in target non-coding RNAs in eukaryotes and archaea. H/ACA RNPs are comprised of a guide RNA and four essential proteins: Cbf5 (pseudouridine synthase), L7Ae, Gar1 and Nop10 in archaea. The guide RNA captures the target RNA via two antisense elements brought together to form a contiguous binding site within the pseudouridylation pocket (internal loop) of the guide RNA. Cbf5 and L7Ae interact independently with the guide RNA, and here we have examined the impacts of these proteins on the RNA in nucleotide protection assays. The results indicate that the interactions observed in a fully assembled H/ACA RNP are established in the sub-complexes, but also reveal a unique Cbf5-guide RNA interaction that is displaced by L7Ae. In addition, the results indicate that L7Ae binding at the kink (k)-turn of the guide RNA induces the formation of the upper stem, and thus also the pseudouridylation pocket. Our findings indicate that L7Ae is essential for formation of the substrate RNA binding site in the archaeal H/ACA RNP, and suggest that k-turn-binding proteins may remodel partner RNAs with important effects distant from the protein-binding site.
在真核生物和古细菌中，H/ACA核糖核蛋白复合物可将靶标非编码RNA中的尿苷转变为假尿苷。古细菌中的H/ACA核糖核蛋白由一条引导RNA和四种必需蛋白组成：Cbf5（假尿苷合酶）、L7Ae、Gar1和Nop10。引导RNA通过两个反义元件捕获靶标RNA，这两个反义元件汇聚在一起，在引导RNA的假尿苷化口袋（内环）内形成一个连续的结合位点。Cbf5和L7Ae分别与引导RNA相互作用，我们在此通过核苷酸保护试验研究了这些蛋白质对RNA的影响。结果表明，在完全组装的H/ACA核糖核蛋白中观察到的相互作用在亚复合物中就已建立，但也揭示了一种独特的Cbf5-引导RNA相互作用，该相互作用会被L7Ae取代。此外，结果表明L7Ae结合在引导RNA的扭结（k）转角处会诱导上部茎环的形成，进而也诱导假尿苷化口袋的形成。我们的研究结果表明，L7Ae对于古细菌H/ACA核糖核蛋白中底物RNA结合位点的形成至关重要，并表明k转角结合蛋白可能会重塑伴侣RNA，其重要影响远及蛋白结合位点之外。
