Pseudouridine at position 55 (Ψ55) in eubacterial tRNA is produced by TruB. To clarify the role of the Ψ55 modification, we constructed a truB gene disruptant (ΔtruB) strain of Thermus thermophilus which is an extreme-thermophilic eubacterium. Unexpectedly, the ΔtruB strain exhibited severe growth retardation at 50°C. We assumed that these phenomena might be caused by lack of RNA chaperone activity of TruB, which was previously hypothetically proposed by others. To confirm this idea, we replaced the truB gene in the genome with mutant genes, which express TruB proteins with very weak or no enzymatic activity. However the growth retardation at 50°C was not rescued by these mutant proteins. Nucleoside analysis revealed that Gm18, m5s2U54 and m1A58 in tRNA from the ΔtruB strain were abnormally increased. An in vitro assay using purified tRNA modification enzymes demonstrated that the Ψ55 modification has a negative effect on Gm18 formation by TrmH. These experimental results show that the Ψ55 modification is required for low-temperature adaptation to control other modified. 35S-Met incorporation analysis showed that the protein synthesis activity of the ΔtruB strain was inferior to that of the wild-type strain and that the cold-shock proteins were absence in the ΔtruB cells at 50°C.

In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNA Cys has an m 2 2 G26m 2 G27 or m 2 2 G26m 2 2 G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m 2 G26 formation is faster than the m 2 G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme.

Background: Topologically knotted tRNA methyltransferases specifically recognize substrate tRNA. Results: Site-directed mutagenesis studies, chimeric protein analysis, and pre-steady state kinetics clarify the tRNA recognition sites of TrmH. Conclusion:The N-and C-terminal regions function in the initial binding process, and substrate tRNA is discriminated by the catalytic domain in an induced-fit process. Significance: Study of how proteins recognize RNA is crucial for understanding RNA maturation processes.

Transfer RNA (Gm18) methyltransferase (TrmH) catalyzes methyl transfer from S-adenosyl-L-methionine to a conserved G18 in tRNA. We investigated the recognition mechanism of Thermus thermophilus TrmH for its guanosine target. Thirteen yeast tRNA Phe mutant transcripts were prepared in which the modification site and/or other nucleotides in the D-loop were substituted by dG, inosine, or other nucleotides. We then conducted methyl transfer kinetic studies, gel shift assays, and inhibition experiments using these tRNA variants. Sites of methylation were confirmed with RNA sequencing or primer extension. Although the G18G19 sequence is not essential for methylation by TrmH, disruption of G18G19 severely reduces the efficiency of methyl transfer. There is strict recognition of guanosine by TrmH, in that methylation occurs at the adjacent G19 when the G18 is replaced by dG or adenosine. The fact that TrmH methylates guanosine in D-loops from 4 to 12 nucleotides in length suggests that selection of the position of guanosine within the D-loop is relatively flexible. Our studies also demonstrate that the oxygen 6 atom of the guanine base is a positive determinant for TrmH recognition. The recognition process of TrmH for substrate is inducible and product-inhibited, in that tRNAs containing Gm18 are excluded by TrmH. In contrast, substitution of G18 with dG18 results in the formation of a more stable TrmH-tRNA complex. To address the mechanism, we performed the stopped-flow presteady state kinetic analysis. The result clearly showed that the binding of TrmH to tRNA is composed of at least three steps, the first bi-molecular binding and the subsequent two uni-molecular induced-fit processes.

Objectives The Coronavirus disease (COVID-19) pandemic has imposed restrictions on people’s social behavior. However, there is limited evidence regarding the relationship between changes in social participation and depressive symptom onset among older adults during the pandemic. We examined the association between changes in social participation and the onset of depressive symptoms among community-dwelling older adults during the COVID-19 pandemic. Design This was a longitudinal study. Setting Communities in Minokamo City, a semi-urban area in Japan. Participants We recruited community-dwelling older adults aged ≥ 65 years using random sampling. Participants completed a questionnaire survey at baseline (March 2020) and follow-up (October 2020). Measurements Depressive symptoms were assessed using the Two-Question Screen. Based on their social participation status in March and October 2020, participants were classified into four groups: “continued participation,” “decreased participation,” “increased participation,” and “consistent non-participation.” Results A total of 597 older adults without depressive symptoms at baseline were analyzed (mean age = 79.8 years; 50.4% females). Depressive symptoms occurred in 20.1% of the participants during the observation period. Multivariable Poisson regression analysis showed that decreased social participation was significantly associated with the onset of the depressive symptoms, compared to continued participation, after adjusting for all covariates (incidence rate ratio = 1.59, 95% confidence interval = 1.01–2.50, p = 0.045). Conclusion Older adults with decreased social participation during the COVID-19 pandemic demonstrated a high risk of developing depressive symptoms. We recommend that resuming community activities and promoting the participation of older adults, with sufficient consideration for infection prevention, are needed to maintain mental health among older adults. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s12603-021-1674-7 and is accessible for authorized users.