Ribose methylation is one of the two most abundant modifications in human ribosomal RNA and is believed to be important for ribosome biogenesis, mRNA selectivity and translational fidelity. We have applied RiboMeth-seq to rRNA from HeLa cells for ribosome-wide, quantitative mapping of 2′-O-Me sites and obtained a comprehensive set of 106 sites, including two novel sites, and with plausible box C/D guide RNAs assigned to all but three sites. We find approximately two-thirds of the sites to be fully methylated and the remainder to be fractionally modified in support of ribosome heterogeneity at the level of RNA modifications. A comparison to HCT116 cells reveals similar 2′-O-Me profiles with distinct differences at several sites. This study constitutes the first comprehensive mapping of 2′-O-Me sites in human rRNA using a high throughput sequencing approach. It establishes the existence of a core of constitutively methylated positions and a subset of variable, potentially regulatory positions, and paves the way for experimental analyses of the role of variations in rRNA methylation under different physiological or pathological settings.

Prokaryotic toxin – antitoxin (TA) loci encode mRNA interferases that inhibit translation, either by cleaving mRNA codons at the ribosomal A site or by cleaving any RNA site-specifically. So far, seven mRNA interferases of Escherichia coli have been identified, four of which cleave mRNA by a translation-dependent mechanism. Here, we experimentally confirmed the presence of three novel TA loci in E. coli. We found that the yafNO, higBA (ygjNM) and ygiUT loci encode mRNA interferases related to RelE. YafO and HigB cleaved translated mRNA only, while YgiU cleaved RNA site-specifically at GC[A/U], independently of translation. Thus, YgiU is the first RelE-related mRNA interferase that cleaves mRNA independently of translation, in vivo. All three loci were induced by amino acid starvation, and inhibition of translation although to different degrees. Carbon starvation induced only two of the loci. The yafNO locus was induced by DNA damage, but the transcription originated from the dinB promoter. Thus, our results showed that the different TA loci responded differentially to environmental stresses. Induction of the three loci depended on Lon protease that may sense the environmental stresses and activate TA loci by cleavage of the antitoxins. Transcription of the three TA operons was autoregulated by the antitoxins.

Ribose methylations are the most abundant chemical modifications of ribosomal RNA and are critical for ribosome assembly and fidelity of translation. Many aspects of ribose methylations have been difficult to study due to lack of efficient mapping methods. Here, we present a sequencing-based method (RiboMeth-seq) and its application to yeast ribosomes, presently the best-studied eukaryotic model system. We demonstrate detection of the known as well as new modifications, reveal partial modifications and unexpected communication between modification events, and determine the order of modification at several sites during ribosome biogenesis. Surprisingly, the method also provides information on a subset of other modifications. Hence, RiboMeth-seq enables a detailed evaluation of the importance of RNA modifications in the cells most sophisticated molecular machine. RiboMeth-seq can be adapted to other RNA classes, for example, mRNA, to reveal new biology involving RNA modifications.

SummaryVibrio cholerae codes for 13 toxin-antitoxin (TA) loci all located within the superintegron on chromosome II. We show here that the two higBA TA loci of V. cholerae encode functional toxins, HigB-1 and HigB-2, whose ectopic expression inhibits cell growth of Escherichia coli, and functional antitoxins, HigA-1 and HigA-2, which counteract the toxicity of the cognate toxins. Three hours of ectopic expression of the HigB toxins resulted in bacteriostasis without any detectable loss of cell viability. The HigB toxins inhibited translation by cleavage of mRNA. Efficient mRNA cleavage occurred preferentially within the translated part of a model mRNA and only when the mRNA was translatable. Promoter analysis in V. cholerae and E. coli showed that the two higBA loci are both transcribed into bi-cistronic mRNAs and that the higBA-2 mRNA is leaderless. Transcription of the two higBA loci was strongly induced by amino acid (aa) starvation in V. cholerae and E. coli, indicating that the regulatory mechanisms of transcriptional induction are conserved across the two species. Both higBA loci stabilized a test-plasmid very efficiently in E. coli, raising the possibility that the loci contribute to maintain genetic stability of the V. cholerae superintegron. Based on these results we discuss the possible biological functions of the TA loci of V. cholerae.

Ribose methylations are the most abundant chemical modifications of ribosomal RNA and are critical for ribosome assembly and fidelity of translation. Many aspects of ribose methylations have been difficult to study due to lack of efficient mapping methods. Here, we present a sequencing‐based method (RiboMeth‐seq) and its application to yeast ribosomes, presently the best‐studied eukaryotic model system. We demonstrate detection of the known as well as new modifications, reveal partial modifications and unexpected communication between modification events, and determine the order of modification at several sites during ribosome biogenesis. Surprisingly, the method also provides information on a subset of other modifications. Hence, RiboMeth‐seq enables a detailed evaluation of the importance of RNA modifications in the cells most sophisticated molecular machine. RiboMeth‐seq can be adapted to other RNA classes, for example, mRNA, to reveal new biology involving RNA modifications.