Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712, USA.
RNA. 2022 Mar;28(3):371-389. doi: 10.1261/rna.079025.121. Epub 2021 Dec 21.
The two subunits of the eukaryotic ribosome are produced through quasi-independent pathways involving the hierarchical actions of numerous -acting biogenesis factors and the incorporation of ribosomal proteins. The factors work together to shape the nascent subunits through a series of intermediate states into their functional architectures. One of the earliest intermediates of the small subunit (SSU or 40S) is the SSU processome which is subsequently transformed into the pre-40S intermediate. This transformation is, in part, facilitated by the binding of the methyltransferase Bud23. How Bud23 is released from the resultant pre-40S is not known. The ribosomal proteins Rps0, Rps2, and Rps21, termed the Rps0-cluster proteins, and several biogenesis factors bind the pre-40S around the time that Bud23 is released, suggesting that one or more of these factors could induce Bud23 release. Here, we systematically examined the requirement of these factors for the release of Bud23 from pre-40S particles. We found that the Rps0-cluster proteins are needed but not sufficient for Bud23 release. The atypical kinase/ATPase Rio2 shares a binding site with Bud23 and is thought to be recruited to pre-40S after the Rps0-cluster proteins. Depletion of Rio2 prevented the release of Bud23 from the pre-40S. More importantly, the addition of recombinant Rio2 to pre-40S particles affinity-purified from Rio2-depleted cells was sufficient for Bud23 release in vitro. The ability of Rio2 to displace Bud23 was independent of nucleotide hydrolysis. We propose a novel role for Rio2 in which its binding to the pre-40S actively displaces Bud23 from the pre-40S.
真核核糖体的两个亚基是通过涉及众多因子的准独立途径产生的，这些因子的作用是通过一系列中间状态将新生亚基塑造成其功能结构。小亚基（SSU 或 40S）的最早中间产物之一是 SSU 加工体，随后转化为前 40S 中间产物。这种转化部分是由甲基转移酶 Bud23 的结合所促进的。Bud23 如何从产生的前 40S 中释放出来尚不清楚。核糖体蛋白 Rps0、Rps2 和 Rps21 被称为 Rps0 簇蛋白，以及几个生物发生因子在 Bud23 释放的同时结合前 40S，这表明这些因子中的一个或多个可能诱导 Bud23 释放。在这里，我们系统地检查了这些因子对于 Bud23 从前 40S 颗粒释放的要求。我们发现，Rps0 簇蛋白是必需的，但不足以释放 Bud23。非典型激酶/ATPase Rio2 与 Bud23 具有结合位点，并且被认为是在 Rps0 簇蛋白之后被招募到前 40S 的。Rio2 的耗竭阻止了 Bud23 从前 40S 中的释放。更重要的是，将重组 Rio2 添加到 Rio2 耗尽的细胞中亲和纯化的前 40S 颗粒足以在体外释放 Bud23。Rio2 置换 Bud23 的能力不依赖于核苷酸水解。我们提出了 Rio2 的一个新作用，即它与前 40S 的结合主动将 Bud23 从前 40S 中置换出来。
