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Summary: N6-methyladenosine (m6A) is a conserved ribonucleoside modification that regulates many facets of RNA metabolism. Using quantitative mass spectrometry, we find that the universally conserved tandem adenosines at the 3′ end of 18S rRNA, thought to be constitutively di-methylated (m62A), are also mono-methylated (m6A). Although present at substoichiometric amounts, m6A at these positions increases significantly in response to sulfur starvation in yeast cells and mammalian cell lines. Combining yeast genetics and ribosome profiling, we provide evidence to suggest that m6A-bearing ribosomes carry out translation distinctly from m62A-bearing ribosomes, featuring a striking specificity for sulfur metabolism genes. Our work thus reveals methylation multiplicity as a mechanism to regulate translation.

Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be noncoding, including 5′ UTRs and long noncoding RNAs (lncRNAs). Apparent ribosome footprints outside of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs). Here, we show hallmarks of translation in these footprints: copurification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including the induction of immune responses following human cytomegalovirus (HCMV) infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts at understanding how cells manage and exploit its consequences.