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. In contrast, the sequence around the 3-end is degenerate with similar sites nearby. How RNA is correctly processed by the endonuclease Nob1 is not known, especially because in vitro experiments have shown it to be error-prone. Here we used yeast genetics, biochemistry, and next generation sequencing to investigate a role for Rio1 in monitoring the 3-end of 18S rRNA. 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 weakened. Accordingly, excess Pno1 results in accumulation of miscleaved rRNA. Ribosomes containing miscleaved rRNA enter the polysomes and produce dominant negative growth defects, suggesting that they cause defects during translation. Our data strongly suggest that ribosome collisions identify these miscleaved 18S rRNA-containing ribosomes as partially functional and target them for degradation. Altogether, the data support a model in which Rio1 inspects the 3-end of the nascent 18S rRNA, only removing Nob1 and Pno1 from the ribosomes with precisely cleaved 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" the cells of altered ribosomes with different functionalities, with important implications for the concept of ribosome heterogeneity.