Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada.
Alberta RNA Research and Training Institute (ARRTI), Department of Chemistry & Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada.
RNA Biol. 2022 Jan;19(1):764-773. doi: 10.1080/15476286.2022.2078092.
snR30/U17 is a highly conserved H/ACA RNA that is required for maturation of the small ribosomal subunit in eukaryotes. By base-pairing to the expansion segment 6 (ES6) of 18S ribosomal RNA (rRNA), the snR30 H/ACA Ribonucleoprotein (RNP) indirectly facilitates processing of the precursor rRNA (pre-rRNA) together with other proteins such as Utp23 and other RNAs acting as ribosome assembly factors. However, the details of the molecular interaction network of snR30 and its binding partners and how these interactions contribute to pre-rRNA processing remains unknown. Here, we report the reconstitution of a snR30 RNP and quantitative characterization of the interactions of snR30, H/ACA proteins, the Utp23 protein and ES6 of the 18S rRNA. The snR30 RNA is bound tightly by both H/ACA proteins and Utp23. We dissected the importance of different 18S rRNA regions for snR30 RNP binding and demonstrated that the snR30 complex is tightly anchored on the pre-rRNA through base-pairing to ES6 whereas other reported rRNA binding sites do not contribute to the affinity of the snR30 RNP. On its own, the ribosome assembly factor Utp23 binds in a tight, but unspecific manner to RNA. However, in complex with the snR30 RNP, Utp23 increases the affinity of the RNP for rRNA revealing synergies between snR30 RNP and Utp23 which are enhancing specificity and affinity for rRNA, respectively. Together, these findings provide mechanistic insights how the snR30 RNP and Utp23 cooperate to interact tightly and specifically with rRNA during the early stages of ribosome biogenesis.
snR30/U17 是一种高度保守的 H/ACA RNA，对于真核生物小核糖体亚基的成熟是必需的。通过与 18S 核糖体 RNA（rRNA）的扩展段 6（ES6）碱基配对，snR30 H/ACA 核糖核蛋白（RNP）与其他蛋白质（如 Utp23）一起间接促进前体 rRNA（pre-rRNA）的加工，其他 RNA 作为核糖体组装因子。然而，snR30 及其结合伙伴的分子相互作用网络的细节以及这些相互作用如何促进 pre-rRNA 加工仍然未知。在这里，我们报告了 snR30 RNP 的重建以及 snR30、H/ACA 蛋白、Utp23 蛋白和 18S rRNA 的 ES6 之间相互作用的定量特征。snR30 RNA 被 H/ACA 蛋白和 Utp23 紧密结合。我们剖析了不同 18S rRNA 区域对 snR30 RNP 结合的重要性，并证明 snR30 复合物通过与 ES6 碱基配对紧密锚定在 pre-rRNA 上，而其他报道的 rRNA 结合位点对 snR30 RNP 的亲和力没有贡献。单独的核糖体组装因子 Utp23 以紧密但非特异性的方式结合 RNA。然而，在与 snR30 RNP 复合物中，Utp23 增加了 RNP 对 rRNA 的亲和力，揭示了 snR30 RNP 和 Utp23 之间的协同作用，分别增强了 rRNA 的特异性和亲和力。总之，这些发现提供了机制见解，说明 snR30 RNP 和 Utp23 如何在核糖体生物发生的早期阶段紧密且特异性地相互作用与 rRNA 结合。
