SHQ1 is required prior to NAF1 for assembly of H/ACA small nucleolar and telomerase RNPs
SHQ1 is required prior to NAF1 for assembly of H/ACA small nucleolar and telomerase RNPs
复制标题
DOI：
10.1261/rna.1532109
复制
发表时间：
2009-06-01
期刊：
影响因子：
4.5
通讯作者：
Meier, U. Thomas
中科院分区：
文献类型：
--
作者：
Grozdanov, Petar N.;Roy, Sujayita;Meier, U. Thomas
酵母中H/ACA RNPs的组装至少需要两个辅助因子，Naf1p和Shq1p。虽然Naf1p及其人类同源基因NAF1的功能已被详细描述，但Shq1p及其推定的人类同源基因SHQ1的功能仍不清楚。我们证明SHQ1确实在人类H/ACA RNPs的生物发生中起作用，并剖析了其作用机制。像NAF1一样，SHQ1结合主要的H/ACA核心蛋白和伪尿嘧啶合成酶NAP57（又名dyskerin），但在新生H/ACA RNA的组装作用之前，因为SHQ1与NAP57在体内和体外的相互作用排除了NAF1和其他H/ACA核心蛋白在H/ACA RNA转录位点的相互作用。SHQ1的n端热休克蛋白20样CS结构域对于NAP57的结合是不可缺少的。与其作为组装因子的作用一致，SHQ1定位于核质，而不存在于核核和Cajal小体（成熟的H/ACA RNPs的位置）中。在依赖于NAF1的功能性H/ACA RNPs的体外组装系统中，过量的重组SHQ1会干扰组装。重要的是，敲低细胞SHQ1可阻止新合成的H/ACA报告RNA的积累，并通常降低内源性H/ACA RNA（包括端粒酶RNA）的水平。综上所述，体内和体外H/ACA RNPs的功能组装需要SHQ1和NAF1的顺序作用。该分步过程可作为H/ACA RNP组装过程中有效的质量控制手段。
Assembly of H/ACA RNPs in yeast is aided by at least two accessory factors, Naf1p and Shq1p. Although the function of Naf1p and its human ortholog NAF1 has been delineated in detail, that of Shq1p and its putative human ortholog SHQ1 remains obscure. We demonstrate that SHQ1 indeed functions in the biogenesis of human H/ACA RNPs and we dissect its mechanism of action. Like NAF1, SHQ1 binds the major H/ACA core protein and pseudouridine synthase NAP57 ( aka dyskerin) but precedes the assembly role of NAF1 at nascent H/ACA RNAs because the interaction of SHQ1 with NAP57 in vivo and in vitro precludes that of NAF1 and of the other H/ACA core proteins that are present at the sites of H/ACA RNA transcription. The N-terminal heat shock protein 20-like CS domain of SHQ1 is dispensable for NAP57 binding. Consistent with its role as an assembly factor, SHQ1 localizes to the nucleoplasm and is excluded from nucleoli and Cajal bodies, the sites of mature H/ACA RNPs. In an in vitro assembly system of functional H/ACA RNPs that is dependent on NAF1, excess recombinant SHQ1 interferes with assembly. Importantly, knockdown of cellular SHQ1 prevents accumulation of a newly synthesized H/ACA reporter RNA and generally reduces the levels of endogenous H/ACA RNAs including telomerase RNA. In summary, the sequential action of SHQ1 and NAF1 is required for functional assembly of H/ACA RNPs in vivo and in vitro. This step-wise process could serve as an efficient means of quality control during H/ACA RNP assembly.