H/ACA ribonucleoproteins (H/ACA RNPs) are responsible for introducing many pseudouridines into RNAs, but are also involved in other cellular functions. Utilizing a purified and reconstituted yeast H/ACA RNP system that is active in pseudouridine formation under physiological conditions, we describe here the quantitative characterization of H/ACA RNP formation and function. This analysis reveals a surprisingly tight interaction of H/ACA guide RNA with the Cbf5p–Nop10p–Gar1p trimeric protein complex whereas Nhp2p binds comparably weakly to H/ACA guide RNA. Substrate RNA is bound to H/ACA RNPs with nanomolar affinity which correlates with the GC content in the guide-substrate RNA base pairing. Both Nhp2p and the conserved Box ACA element in guide RNA are required for efficient pseudouridine formation, but not for guide RNA or substrate RNA binding. These results suggest that Nhp2p and the Box ACA motif indirectly facilitate loading of the substrate RNA in the catalytic site of Cbf5p by correctly positioning the upper and lower parts of the H/ACA guide RNA on the H/ACA proteins. In summary, this study provides detailed insight into the molecular mechanism of H/ACA RNPs.

中文翻译：

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真核H / ACA核糖核蛋白的有效RNA伪尿苷化需要高亲和力结合和指导RNA的正确定位

H / ACA核糖核蛋白（H / ACA RNP）负责将许多假尿苷导入RNA，但也参与其他细胞功能。利用在生理条件下在假尿苷形成中具有活性的纯化和重构的酵母H / ACA RNP系统，我们在此描述H / ACA RNP形成和功能的定量表征。该分析揭示了H / ACA指导RNA与Cbf5p–Nop10p–Gar1p三聚体蛋白复合物之间的紧密紧密相互作用，而Nhp2p与H / ACA指导RNA的结合较弱。底物RNA以纳摩尔亲和力与H / ACA RNP结合，这与引导底物RNA碱基配对中的GC含量相关。指导RNA中的Nhp2p和保守的Box ACA元件都是有效的假尿苷形成所必需的，但指导RNA或底物RNA的结合则不需要。这些结果表明，Nhp2p和Box ACA基序可通过将H / ACA指导RNA的上部和下部正确定位在H / ACA蛋白上，间接促进底物RNA在Cbf5p催化位点的装载。总而言之，这项研究为H / ACA RNP的分子机制提供了详细的见识。