SummaryThe structural and functional repertoire of small non-protein-coding RNAs (ncRNAs) is central for establishing gene regulation networks in cells and organisms. Here, we show that an mRNA-derived 18-nucleotide-long ncRNA is capable of downregulating translation in Saccharomyces cerevisiae by targeting the ribosome. This 18-mer ncRNA binds to polysomes upon salt stress and is crucial for efficient growth under hyperosmotic conditions. Although the 18-mer RNA originates from the TRM10 locus, which encodes a tRNA methyltransferase, genetic analyses revealed the 18-mer RNA nucleotide sequence, rather than the mRNA-encoded enzyme, as the translation regulator. Our data reveal the ribosome as a target for a small regulatory ncRNA and demonstrate the existence of a yet unkown mechanism of translation regulation. Ribosome-targeted small ncRNAs are found in all domains of life and represent a prevalent but so far largely unexplored class of regulatory molecules.

Ribosome-associated non-coding RNAs (rancRNAs) have been recognized as an emerging class of regulatory molecules capable of fine-tuning translation in all domains of life. RancRNAs are ideally suited for allowing a swift response to changing environments and are therefore considered pivotal during the first wave of stress adaptation. Previously, we identified an mRNA-derived 18 nucleotides long rancRNA (rancRNA_18) in Saccharomyces cerevisiae that rapidly downregulates protein synthesis during hyperosmotic stress. However, the molecular mechanism of action remained enigmatic. Here, we combine biochemical, genetic, transcriptome-wide and structural evidence, thus revealing rancRNA_18 as global translation inhibitor by targeting the E-site region of the large ribosomal subunit. Ribosomes carrying rancRNA_18 possess decreased affinity for A-site tRNA and impaired structural dynamics. Cumulatively, these discoveries reveal the mode of action of a rancRNA involved in modulating protein biosynthesis at a thus far unequalled precision.