The kinase Rio1 and a ribosome collision-dependent decay pathway survey the integrity of 18S rRNA cleavage
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 step, but not in healthy cells with intact quality control 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 quality control. 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. Overall design: To examine the accuracy of 18S rRNA 3'-end formation in vivo, we performed 3'-RACE (rapid amplification of cDNA ends) sequencing on 18S rRNA and 25S rRNA to survey the 3'-ends of 18S rRNA and 25S rRNA. Saccharomyces cerevisaea strains were engineered and grown in conditions to deplete endogenous Pno1 and Dim1 or Rps14 and Dim1 protein expression, while expressing plasmid-derived Pno1 or Pno1-KKKF mutant proteins and Dim1 or Dim1-E85A mutant proteins, or plasmid-derived Rps14 and Dim1 proteins. 18S rRNA 3'-end formation was investigated using 8 biological replicates of yeast cells expressing plasmid-derived Pno1 and Dim1-E85A (wild type control), 8 biological replicates of yeast cells expressing plasmid-derived Pno1-KKKF and Dim1-E85A (ribosome assembly quality control bypass condition), and 2 biological replicates of yeast cells expressing plasmid-derived Rps14 and Dim1 (control for the effects of the Dim1-E85A mutant on 18S rRNA 3'-end miscleavage frequency). 25S rRNA 3'-end formation was investigated using 2 biological replicates of yeast cells expressing plasmid-derived Pno1 and Dim1-E85A and 2 biological replicates of yeast cells expressing plasmid-derived Pno1-KKKF and Dim1-E85A as controls to test whether bypass conditions specifically altered 18S rRNA miscleavage frequency. Yeast strains were grown to early stationary phase (between OD600 1.2-1.8) at 25°C or 30°C, 40S and 60S ribosomal subunits were purified, and 18S and 25S rRNAs were extracted for cDNA library preparation and 3'-RACE sequencing.