Maturation of the 90S pre-ribosome requires Mrd1 dependent U3 snoRNA and 35S pre-rRNA structural rearrangements

In eukaryotes, biogenesis of ribosomes requires folding and assembly of the precursor rRNA (pre-rRNA) with a large number of proteins and snoRNPs into huge RNA-protein complexes. In spite of intense genetic, biochemical and high resolution cryo-EM studies in Saccharomyces cerevisiae, information about the conformation of the earliest 35S pre-rRNA is limited. To overcome this, we performed high-throughput SHAPE chemical probing on the 35S pre-rRNA associated with 90S pre-ribosomes. We focused our analyses on external (5´ETS) and internal (ITS1) transcribed spacers as well as the 18S region. We show that in the 35S pre-rRNA, the central region of the 18S is in a more open configuration compared to 20S pre-rRNA and that the central pseudoknot is not formed. The essential ribosome biogenesis protein Mrd1 influences the structure of the 18S part locally and is involved in organizing the central pseudoknot and surrounding structures. Our results demonstrate that the U3 snoRNA dynamically interacts with the 35S pre-rRNA and that Mrd1 is required for disrupting U3 snoRNA base-pairing interactions in the 5'ETS. We propose that the dynamic U3 snoRNA interactions and Mrd1 are essential for establishing the structure of the central region of 18S that is required for processing and 40S subunit function.在真核生物中，核糖体生物发生（ribosome biogenesis）需要将前体核糖体RNA（precursor rRNA, pre-rRNA）与大量蛋白质及小核仁核糖核蛋白颗粒（snoRNPs）进行折叠与组装，形成庞大的核糖核酸蛋白复合物。尽管针对酿酒酵母（Saccharomyces cerevisiae）已开展了大量深入的遗传学、生物化学及高分辨率冷冻电镜（cryo-EM）研究，但有关最早出现的35S前体核糖体RNA（35S pre-rRNA）构象的相关信息仍较为有限。为解决这一研究局限，我们对与90S前核糖体（90S pre-ribosomes）结合的35S pre-rRNA开展了高通量SHAPE化学探测实验。本研究的分析重点聚焦于外部转录间隔区（5´ETS）、内部转录间隔区（ITS1）以及18S区域。研究结果显示，在35S pre-rRNA中，18S的中心区域相较于20S前体核糖体RNA（20S pre-rRNA）处于更为开放的构型，且中心假结（central pseudoknot）尚未形成。必需核糖体生物发生蛋白Mrd1可局部调控18S区域的结构，并参与组织中心假结及其周边结构。我们的研究证实，U3小核仁RNA（U3 snoRNA）可与35S pre-rRNA发生动态相互作用，且Mrd1是破坏5'ETS中U3 snoRNA碱基配对相互作用的必需因子。我们提出，动态的U3 snoRNA相互作用与Mrd1对于建立18S中心区域的结构至关重要，而该结构是核糖体加工过程及40S亚基功能发挥所必需的。