Satoshi Kimura

Satoshi Kimura works in the field of RNA modification. In this mSphere of Influence article, he reflects on how the paper “Efficient and quantitative high-throughput tRNA sequencing” by G. Zheng, Y. Qin, W. C. Clark, Q. Dai, et al. (Nat. Methods 12:835–837, 2015, https://doi.org/10.1038/nmeth.3478 ) has impacted him by demonstrating that deep sequencing technology is harnessed to profile tRNA modifications.

Trypanosoma cruzi, the etiological agent of Chagas disease, infects millions of people in the Americas. This parasite undergoes drastic changes in its morphology and metabolism between infective and noninfective forms through global remodeling of its proteome. Chemical modification of tRNA (tRNA modification) contributes to the control of protein expression by modulating the codon decoding process. However, knowledge of tRNA modification profiles, the enzymes that create modifications and their regulation in different cellular conditions is largely restricted to relatively few model organisms. Here, we profile tRNA modifications in both infective and noninfective forms of T. cruzi to probe their dynamic changes. Genome mining of tRNA modifying enzymes identified 65 putative tRNA-modifying enzymes in T. cruzi for 27 species of tRNA modifications, most of which were detected in T. cruzi tRNA by liquid chromatography mass spectrometry analyses. tRNA sequencing detected reverse transcription-derived signatures at 170 sites in T. cruzi tRNAs that are likely derived from 19 tRNA modifications. tRNA modifications and tRNA modification enzymes are differentially modulated across the life stages of T. cruzi. We found that hydroxywybutosine (OHyW) at position 37 on tRNA ^Phe(GAA) had a reduced level in the infective form (metacyclic trypomastigote) and the associated modification enzyme Tyw1a exhibited reduced expression in this stage. Knockout of Tyw1a increased the differentiation from epimastigote (noninfective form) to metacyclic trypomastigote, suggesting that changes in OHyW37 modification levels alter the rate of metacyclogenesis. Overall, our findings suggest that tRNA modification changes during the life stages of T. cruzi contribute to the differentiation of this parasite.

Modified tRNA anticodons are critical for proper mRNA translation during protein synthesis. It is generally thought that almost all bacterial tRNAs^Ile use a modified cytidine—lysidine (L)—at the first position (34) of the anticodon to decipher the AUA codon as isoleucine (Ile). Here we report that tRNAs^Ile from plant organelles and a subset of bacteria contain a new cytidine derivative, designated 2-aminovaleramididine (ava²C). Like L34, ava²C34 governs both Ile-charging ability and AUA decoding. Cryo-electron microscopy structural analyses revealed molecular details of codon recognition by ava²C34 with a specific interaction between its terminal amide group and an mRNA residue 3′-adjacent to the AUA codon. These findings reveal the evolutionary variation of an essential tRNA modification and demonstrate the molecular basis of AUA decoding mediated by a unique tRNA modification.

Diverse chemical modifications fine-tune the function and metabolism of tRNA. Although tRNA modification is universal in all kingdoms of life, profiles of modifications, their functions, and physiological roles have not been elucidated in most organisms including the human pathogen, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To identify physiologically important modifications, we surveyed the tRNA of Mtb, using tRNA sequencing (tRNA-seq) and genome-mining. Homology searches identified 23 candidate tRNA modifying enzymes that are predicted to create 16 tRNA modifications across all tRNA species. Reverse transcription-derived error signatures in tRNA-seq predicted the sites and presence of nine modifications. Several chemical treatments prior to tRNA-seq expanded the number of predictable modifications. Deletion of Mtb genes encoding two modifying enzymes, TruB and MnmA, eliminated their respective tRNA modifications, validating the presence of modified sites in tRNA species. Furthermore, the absence of mnmA attenuated Mtb growth in macrophages, suggesting that MnmA-dependent tRNA uridine sulfation contributes to Mtb intracellular growth. Our results lay the foundation for unveiling the roles of tRNA modifications in Mtb pathogenesis and developing new therapeutics against tuberculosis.

Diverse chemical modifications fine-tune the function and metabolism of tRNA. Although tRNA modification is universal in all kingdoms of life, profiles of modifications, their functions, and physiological roles have not been elucidated in most organisms including the human pathogen, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To identify physiologically important modifications, we surveyed the tRNA of Mtb, using tRNA sequencing (tRNA-seq) and genome-mining. Homology searches identified 23 candidate tRNA modifying enzymes that are predicted to create 16 tRNA modifications across all tRNA species. Reverse transcription-derived error signatures in tRNA-seq predicted the sites and presence of 9 modifications. Several chemical treatments prior to tRNA-seq expanded the number of predictable modifications. Deletion of Mtb genes encoding two modifying enzymes, TruB and MnmA, eliminated their respective tRNA modifications, validating the presence of modified sites in tRNA species. Furthermore, the absence of mnmA attenuated Mtb growth in macrophages, suggesting that MnmA-dependent tRNA uridine sulfation contributes to Mtb intracellular growth. Our results lay the foundation for unveiling the roles of tRNA modifications in Mtb pathogenesis and developing new therapeutics against tuberculosis.

This study probed in vitro the mechanisms of competition/coexistence between Streptococcus sanguinis (known for being correlated with health in the oral cavity) and Streptococcus mutans (responsible for aciduric oral environment and formation of caries) by means of quantitative Raman spectroscopy and imaging. In situ Raman assessments of live bacterial culture/coculture focusing on biofilm exopolysaccharides supported the hypothesis that both species engaged in antagonistic interactions. Experiments of simultaneous colonization always resulted in coexistence, but they also revealed fundamental alterations of the biofilm with respect to their water-insoluble glucan structure. Raman spectra (collected at fixed time but different bacterial ratios) showed clear changes in chemical bonds in glucans, which pointed to an action by Streptococcus sanguinis to discontinue the impermeability of the biofilm constructed by Streptococcus mutans. The concurrent effects of glycosidic bond cleavage in water-insoluble α − 1,3–glucan and oxidation at various sites in glucans’ molecular chains supported the hypothesis that secretion of oxygen radicals was the main “chemical weapon” used by Streptococcus sanguinis in coculture.

Diverse chemical modifications fine-tune the function and metabolism of tRNA. Although tRNA modification is universal in all kingdoms of life, profiles of modifications, their functions, and physiological roles have not been elucidated in most organisms including the human pathogen, Mycobacterium tuberculosis ( Mtb ), the causative agent of tuberculosis. To identify physiologically important modifications, we surveyed the tRNA of Mtb , using tRNA sequencing (tRNA-seq) and genome-mining. Homology searches identified 23 candidate tRNA modifying enzymes that are predicted to create 16 tRNA modifications across all tRNA species. Reverse transcription-derived error signatures in tRNA-seq predicted the sites and presence of 9 modifications. Several chemical treatments prior to tRNA-seq expanded the number of predictable modifications. Deletion of Mtb genes encoding two modifying enzymes, TruB and MnmA, eliminated their respective tRNA modifications, validating the presence of modified sites in tRNA species. Furthermore, the absence of mnmA attenuated Mtb growth in macrophages, suggesting that MnmA-dependent tRNA uridine sulfation contributes to Mtb intracellular growth. Our results lay the foundation for unveiling the roles of tRNA modifications in Mtb pathogenesis and developing new therapeutics against tuberculosis.