18SrRNA序列是高度保守的,特别是在它的3'端,其由核酸内切酶Nob1形成。Nob1如何识别其目标序列尚不清楚,体外实验表明Nob1容易出错。此外,3'端周围的序列是退化的,附近有类似的地点。这里,我们用酵母遗传学,生物化学,和下一代测序,以研究ATPaseRio1在监测18SrRNA3'端的准确性中的作用。我们证明Nob1可以错切其rRNA底物,并且错切的rRNA在绕过Rio1介导的质量控制(QC)步骤时积累,但不在具有完整QC机制的健康细胞中。机械上,我们显示Rio1与错切的rRNA的结合弱于其与精确处理的18SrRNA的结合。因此,过量的Rio1导致错切的rRNA的积累。含有错切rRNA的核糖体可以翻译,尽管更慢,从而邀请与尾随核糖体的碰撞。这些碰撞利用mRNAQC的部分机制导致有缺陷的核糖体的降解。总之,数据支持一个模型,其中Rio1检查新生18SrRNA的3'-末端,以防止含有18SrRNA的核糖体错误地参与翻译,它们诱导核糖体碰撞。数据还表明核糖体碰撞如何纯化具有不同功能的改变的核糖体的细胞,对核糖体异质性的概念具有重要意义。
The 18S rRNA sequence is highly conserved, particularly at its 3\'-end, which is formed by the endonuclease Nob1. How Nob1 identifies its target sequence is not known, and in vitro experiments have shown Nob1 to be error-prone. Moreover, the sequence around the 3\'-end is degenerate with similar sites nearby. Here, we used yeast genetics, biochemistry, and next-generation sequencing to investigate a role for the ATPase Rio1 in monitoring the accuracy of the 18S rRNA 3\'-end. We demonstrate that Nob1 can miscleave its rRNA substrate and that miscleaved rRNA accumulates upon bypassing the Rio1-mediated quality control (QC) step, but not in healthy cells with intact QC mechanisms. Mechanistically, we show that Rio1 binding to miscleaved rRNA is weaker than its binding to accurately processed 18S rRNA. Accordingly, excess Rio1 results in accumulation of miscleaved rRNA. Ribosomes containing miscleaved rRNA can translate, albeit more slowly, thereby inviting collisions with trailing ribosomes. These collisions result in degradation of the defective ribosomes utilizing parts of the machinery for mRNA QC. Altogether, the data support a model in which Rio1 inspects the 3\'-end of the nascent 18S rRNA to prevent miscleaved 18S rRNA-containing ribosomes from erroneously engaging in translation, where they induce ribosome collisions. The data also demonstrate how ribosome collisions purify cells of altered ribosomes with different functionalities, with important implications for the concept of ribosome heterogeneity.