RNA helicase-mediated regulation of snoRNP dynamics on pre-ribosomes and rRNA 2’-O-methylation

RNA helicases play important roles in diverse aspects of RNA metabolism through their functions in remodelling ribonucleoprotein complexes (RNPs), such as pre-ribosomes. Here, we show that the DEAD box helicase Dbp3 is required for efficient processing of the U18 and U24 intron-encoded snoRNAs and 2'-O-methylation of various sites within the 25S ribosomal RNA (rRNA) sequence. Furthermore, numerous box C/D snoRNPs accumulate on pre-ribosomes in the absence of Dbp3. Many snoRNAs guiding Dbp3-dependent rRNA modifications have overlapping pre-rRNA basepairing sites and therefore form mutually exclusive interactions with pre-ribosomes. Analysis of the distribution of these snoRNAs between pre-ribosome-associated and 'free' pools demonstrated that many are almost exclusively associated with pre-ribosomal complexes. Our data suggest that retention of such snoRNPs on pre-ribosomes when Dbp3 is lacking may impede rRNA 2'-O-methylation by reducing the recycling efficiency of snoRNPs and by inhibiting snoRNP access to proximal target sites. The observation of substoichiometric rRNA modification at adjacent sites suggests that the snoRNPs guiding such modifications likely interact stochastically rather than hierarchically with their pre-rRNA target sites. Together, our data provide new insights into the dynamics of snoRNPs on pre-ribosomal complexes and the remodelling events occurring during the early stages of ribosome assembly.RNA解旋酶（RNA helicase）通过重塑核糖核蛋白复合物（ribonucleoprotein complexes, RNPs，如前核糖体）的功能，在RNA代谢的诸多方面发挥关键作用。本研究证实，DEAD盒解旋酶（DEAD box helicase）Dbp3对于U18和U24内含子编码的核仁小RNA（small nucleolar RNAs, snoRNAs）的高效加工，以及25S核糖体RNA（25S ribosomal RNA, rRNA）序列内多位点的2'-O-甲基化修饰不可或缺。进一步研究发现，在缺失Dbp3的情况下，大量C/D盒核仁小核糖核蛋白复合物（C/D box snoRNPs）会在预核糖体上积累。诸多介导Dbp3依赖性rRNA修饰的snoRNAs，其前核糖体RNA（pre-rRNA）碱基配对位点存在重叠，因此它们与预核糖体的结合存在互斥性。对这些snoRNAs在预核糖体结合池与“游离”池之间的分布分析显示，其中多数几乎仅与预核糖体复合物结合。我们的数据表明，缺失Dbp3时此类snoRNPs在预核糖体上的滞留，会通过降低snoRNPs的循环效率以及阻碍snoRNPs接近邻近靶位点，进而抑制rRNA的2'-O-甲基化修饰。相邻位点的rRNA修饰呈现亚化学计量特征，这提示介导此类修饰的snoRNPs，大概率以随机而非层级的方式与其pre-rRNA靶位点结合。综上，本研究为预核糖体复合物上snoRNPs的动态变化过程，以及核糖体组装早期阶段发生的重塑事件提供了全新的见解。