Erroneous ribosomal RNAs promote the generation of antisense ribosomal siRNA

Ribosome biogenesis is a multistep process, during which mistakes can occur at any step of pre-rRNA processing, modification, and ribosome assembly. Misprocessed rRNAs are usually detected and degraded by surveillance machineries. Recently, we identified a class of antisense ribosomal siRNAs (risiRNAs) that down-regulate pre-rRNAs through the nuclear RNAi pathway. To further understand the biological roles of risiRNAs, we conducted both forward and reverse genetic screens to search for more suppressor of siRNA (susi) mutants. We isolated a number of genes that are broadly conserved from yeast to humans and are involved in pre-rRNA modification and processing. Among them, SUSI-2(ceRRP8) is homologous to human RRP8 and engages in m1A methylation of the 26S rRNA. C27F2.4(ceBUD23) is an m7G-methyltransferase of the 18S rRNA. E02H1.1(ceDIMT1L) is a predicted m6(2)Am6(2)A-methyltransferase of the 18S rRNA. Mutation of these genes led to a deficiency in modification of rRNAs and elicited accumulation of risiRNAs, which further triggered the cytoplasmic-to-nuclear and cytoplasmic-to-nucleolar translocations of the Argonaute protein NRDE-3. The rRNA processing deficiency also resulted in accumulation of risiRNAs. We also isolated SUSI-3(RIOK-1), which is similar to human RIOK1, that cleaves the 20S rRNA to 18S. We further utilized RNAi and CRISPR-Cas9 technologies to perform candidate-based reverse genetic screens and identified additional pre-rRNA processing factors that suppressed risiRNA production. Therefore, we concluded that erroneous rRNAs can trigger risiRNA generation and subsequently, turn on the nuclear RNAi-mediated gene silencing pathway to inhibit pre-rRNA expression, which may provide a quality control mechanism to maintain homeostasis of rRNAs.