Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, United States of America.
PLoS Genet. 2020 Dec 11;16(12):e1009215. doi: 10.1371/journal.pgen.1009215. eCollection 2020 Dec.
The first metastable assembly intermediate of the eukaryotic ribosomal small subunit (SSU) is the SSU Processome, a large complex of RNA and protein factors that is thought to represent an early checkpoint in the assembly pathway. Transition of the SSU Processome towards continued maturation requires the removal of the U3 snoRNA and biogenesis factors as well as ribosomal RNA processing. While the factors that drive these events are largely known, how they do so is not. The methyltransferase Bud23 has a role during this transition, but its function, beyond the nonessential methylation of ribosomal RNA, is not characterized. Here, we have carried out a comprehensive genetic screen to understand Bud23 function. We identified 67 unique extragenic bud23Δ-suppressing mutations that mapped to genes encoding the SSU Processome factors DHR1, IMP4, UTP2 (NOP14), BMS1 and the SSU protein RPS28A. These factors form a physical interaction network that links the binding site of Bud23 to the U3 snoRNA and many of the amino acid substitutions weaken protein-protein and protein-RNA interactions. Importantly, this network links Bud23 to the essential GTPase Bms1, which acts late in the disassembly pathway, and the RNA helicase Dhr1, which catalyzes U3 snoRNA removal. Moreover, particles isolated from cells lacking Bud23 accumulated late SSU Processome factors and ribosomal RNA processing defects. We propose a model in which Bud23 dissociates factors surrounding its binding site to promote SSU Processome progression.
真核核糖体小亚基 (SSU) 的第一个亚稳组装中间体是 SSU 加工体，这是一个由 RNA 和蛋白质因子组成的大型复合物，被认为代表了组装途径中的早期检查点。SSU 加工体向持续成熟的转变需要去除 U3 snoRNA 和生物发生因子以及核糖体 RNA 加工。虽然驱动这些事件的因素在很大程度上是已知的，但它们是如何做到的尚不清楚。甲基转移酶 Bud23 在这个转变过程中起作用，但除了非必需的核糖体 RNA 甲基化之外，其功能尚未确定。在这里，我们进行了全面的遗传筛选以了解 Bud23 的功能。我们确定了 67 个独特的外显子 bud23Δ 抑制突变，这些突变映射到编码 SSU 加工体因子 DHR1、IMP4、UTP2（NOP14）、BMS1 和 SSU 蛋白 RPS28A 的基因上。这些因子形成了一个物理相互作用网络，将 Bud23 的结合位点与 U3 snoRNA 连接起来，并且许多氨基酸取代削弱了蛋白-蛋白和蛋白-RNA 相互作用。重要的是，该网络将 Bud23 与必需的 GTPase Bms1 联系起来，Bms1 在解体途径的后期起作用，而 RNA 解旋酶 Dhr1 则催化 U3 snoRNA 的去除。此外，从缺乏 Bud23 的细胞中分离出的颗粒积累了晚期 SSU 加工体因子和核糖体 RNA 加工缺陷。我们提出了一个模型，其中 Bud23 解离其结合位点周围的因子以促进 SSU 加工体的进展。
