Loss of N1-methylation of G37 in tRNA induces ribosome stalling and reprograms gene expression

  1. Isao Masuda
  2. Jae-Yeon Hwang
  3. Thomas Christian
  4. Sunita Maharjan
  5. Fuad Mohammad
  6. Howard Gamper
  7. Allen R Buskirk
  8. Ya-Ming Hou

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    Evaluation Summary:

    This manuscript provides evidence that loss of N1-methylation of G37 of tRNAs in bacteria on depletion of TrmD results in defects in aminoacylation and peptidyl-transfer, leading to ribosome stalling and phenotypes indicating activation of the stringent response.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

N 1 -methylation of G37 is required for a subset of tRNAs to maintain the translational reading-frame. While loss of m 1 G37 increases ribosomal +1 frameshifting, whether it incurs additional translational defects is unknown. Here, we address this question by applying ribosome profiling to gain a genome-wide view of the effects of m 1 G37 deficiency on protein synthesis. Using E coli as a model, we show that m 1 G37 deficiency induces ribosome stalling at codons that are normally translated by m 1 G37-containing tRNAs. Stalling occurs during decoding of affected codons at the ribosomal A site, indicating a distinct mechanism than that of +1 frameshifting, which occurs after the affected codons leave the A site. Enzyme- and cell-based assays show that m 1 G37 deficiency reduces tRNA aminoacylation and in some cases peptide-bond formation. We observe changes of gene expression in m 1 G37 deficiency similar to those in the stringent response that is typically induced by deficiency of amino acids. This work demonstrates a previously unrecognized function of m 1 G37 that emphasizes its role throughout the entire elongation cycle of protein synthesis, providing new insight into its essentiality for bacterial growth and survival.

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  1. Version published to 10.7554/elife.70619 on eLife
  2. eLife

    Author Response:

    Reviewer #2:

    This manuscript explored the effect N1-methylation of G37 of tRNAs in bacteria. The authors found that loss of methylation, through the depletion of trmD, results in defects in aminoacylation and peptidyl-transfer, leading to ribosome stalling and activation of the stringent response (as mediated by accumulation of deacylated tRNAs). Briefly, the authors conducted ribosome profiling on trmD conditional-knockout E coli cells and compared it to "wild-type" cells, and documented increased ribosome stalling on codons decoded by tRNAs modified by trmD. Stalling occurs when the ribosome is decoding these codons, i.e. when they occupy the A site. Further biochemical characterization showed that stalling is likely to occur due to defects in aminoacylation and peptide-bond formation for the trmD-substrate tRNAs, primarily for tRNAPro. Finally, analysis of gene expression shows that loss of trmD results in the activation of the stringent response as well as rewiring of central-carbon metabolism.

    Overall, this is a comprehensive study of an essential and universally conserved tRNA methylation. The manuscript expands on the role of m1G37 in translation, beyond its established role in reading-frame maintenance. However, the novelty of the findings was not immediately clear to me, and in particular whether they significantly advance our understanding of tRNA modification. For instance, it is known that defects in tRNA methylation (albeit different than N1-methylation of G37, discussed here) activates Gcn2 in yeast, which arguably is equivalent to the stringent response in bacteria.

    We thank this reviewer for the overall positive comments of our work. To address the concern about novelty, we have revised the fourth paragraph in Discussion (pp24-25) to emphasize the novelty of our finding.

    Specifically, most of the published genome-wide studies of tRNA modifications, leading to a stress response, are performed in eukaryotes (e.g., Saccharomyces, Neurospora, and Drosophila) (cited in the revised manuscript). Although we have shown that loss of the s2 group from the cmnm5s2U34-state in E. coli tRNAGln led to reduced aminoacylation and reduced tRNA binding and accommodation to the ribosome A site, we did not investigate whether it induces a stress response in E. coli (Rodriguez-Hernandez et al, 2013). While there are studies in bacteria that demonstrate changes of tRNA modifications in response to stress, these are not in the same theme as the focus of this work, which is to determine how changes of tRNA modifications induce a stress response. Thus, our work here provides an important example showing that m1G37 deficiency leads to the stringent response in E. coli, which is in parallel with the results of studies in eukaryotes showing that loss of tRNA modifications turns on the GCN4 response in yeast and the mTOR-like response in Drosophila. This parallel provides a framework for understanding the evolution of a common cellular priority that activates amino acid biosynthesis in response to deficiency of amino acids or to deficiency of tRNA modification, both of which would prevent active protein synthesis and compromise cell viability.

    Furthermore, the authors made the claim "In contrast, while m1 G37 deficiency reduces peptide bond formation for some tRNAs at the A site, it consistently reduces the rate of aminoacylation for all tRNAs examined, which has not been shown for other metabolically deficient tRNAs." in the discussion section, which is inaccurate. Previous data, some from the same group, has shown that thiolation of the wobble base in tRNAGln is important for aminoacylation, tRNA selection by the ribosome and reading-frame maintenance. The argument that m1G37's pleiotropic effect on translation is unique is not convincing.

    Yes, we agree with the reviewer and have removed the claim from Discussion. We apologize for our over-statement in the previous submission. We have also cited our own work on E. coli tRNAGln (Rodriguez-Hernandez et al, 2013), and explained that we did not investigate the possibility of a stress response in that work (pp24-25). These considerations demonstrate the novelty of this present work on m1G37 deficiency in E. coli, providing an example of a stress response in bacteria that is in parallel with the stress response in eukaryotes that is activated by changes of the post-transcriptional modification state of tRNA.

    Reviewer #3:

    The study expands upon the previous findings of the Hou lab that the lack of TrmD-catalyzed modification in the anticodons of several bacterial tRNAs leads to +1 frameshifting when the undermodified tRNA is positioned in the ribosomal P site. In the current study, the authors show that a number of other aspects of translation are affected when the m1G modification in the tRNA anticodon is lacking.

    Specifically, the study shows that undermodified tRNAs are less efficiently aminoacylated by the corresponding aminoacyl-tRNA synthetases leading to excessive presence of deacylated tRNAs. One of the consequences is ribosome pausing when the respective codons need to be decoded. The shift in the balance of aminoacyl-tRNA relative to deacyl-tRNA resembles the one caused by amino acid starvation. Indeed, the authors show that changes in the transcriptome triggered by reduced tRNA modification resemble those observed at stringent response.

    We thank this reviewer for the positive comments on our manuscript.

    While the paper is generally good and interesting in its current version it is not perfectly focused: discussion of the metabolic changes resulting from transcriptome remodeling are relatively fuzzy and do not contribute much to the main story.

    We agree with the reviewer that the discussion of metabolic changes resulting from transcriptome remodeling is preliminary. We have substantially shortened the Results section “Metabolic changes” (pp 21-22) and have removed a previous figure that illustrated metabolic changes.

    Another problem is that some of the claims (e.g. that the lack of anticodon modification affects peptide bond formation) are not properly termed and thus, misleading. In fact, the lack of tRNA modification affects dipeptide formation (possibly by interfering with decoding or tRNA accommodation) rather than influencing the rate of peptidyl transfer per se.

    We agree with the reviewer that our measurement of peptide-bond formation encompasses all of the reaction steps up to and including peptide-bond formation in the A site. The kobs of each of our measurements is a composite kinetic term that reports on the overall rate of peptide-bond formation. We have carefully revised the text to reflect this point in Results “Reduced aminoacylation and A-site peptide-bond formation of m1G37-deficient tRNAs” (pp13-14).

  3. eLife

    Evaluation Summary:

    This manuscript provides evidence that loss of N1-methylation of G37 of tRNAs in bacteria on depletion of TrmD results in defects in aminoacylation and peptidyl-transfer, leading to ribosome stalling and phenotypes indicating activation of the stringent response.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    This study shows using ribosome profiling that m1G37 deficiency in E. coli achieved by depletion of TrmD leads to ribosome stalling at codons specific for tRNAs that are methylated by TrmD, including strong pauses at all Pro CCN codons and at the Arg CGG codon, and weak pauses at the Leu CUA codon (in one of two mutants studied). The stalling occurs primarily when the affected codons are in the ribosomal A site, indicating defects in decoding. Biochemical experiments show that m1G37 deficiency reduces rates of tRNA aminoacylation in all isoacceptors examined, and rates of peptide-bond formation in some of the isoacceptors, suggesting that the reduced rates of one or both of these reactions lead to ribosome stalling at the A site. RNA-seq in the mutants shows changes in mRNA expression, some of which can be rationalized by translational pausing in leader peptides of the Leu and Ilv operons at the TrmD-dependent Leu or Pro codons, respectively, leading to loss of attenuation and derepressed transcription of the operons. Many of the other transcriptional changes exhibit the signature of the stringent response, known to be activated by binding of deacylated tRNA to the A site of translating ribosomes, which would be consistent with reduced aminoacylation of the TrmD-dependent tRNAs in vivo. Some of the mRNA changes shared by the TrmD mutant and the stringent response are expected to up-regulate enzymes of the glyoxylate cycle, which could help cells deal with amino acid deficiency-the biological goal of the stringent response. Other changes involving down-regulation of mRNAs encoding enzymes of glycolysis and up-regulation of mRNAs for enzymes of fatty acid metabolism, are predicted to shift metabolism from catabolism of glucose to fatty acids, which would support the glyoxylate cycle. These latter changes in mRNA levels are not shared by the stringent response, however, and the mechanism for the role of TrmD and m1G37 modification in tRNA is unknown.

    The evidence is strong from the ribosome profiling data that loss of m1G37 in tRNAs leads to slow decoding of the triplets in the ribosome A site that are decoded by the known TrmD-modified Pro, Arg and Leu tRNA isoacceptors. Similar results have been published previously for certain mutants of tRNA modifying enzymes in yeast. It is particularly interesting here because most previous work apparently implicated m1G37 in suppressing +1 frameshifting by influencing codon-anticodon interactions in the P site, whereas here they found no evidence for increased +1 frameshifting in their profiling data on the trmD mutants. The biochemical data indicating reduced rates of aminoacylation and peptide bond formation by the unmodified tRNAs appear to be sound, and they have demonstrated reduced aminoacylation of tRNAPro(UGG), which could be mitigated by overexpressing the Pro tRNA synthetase, providing strong evidence that reduced charging of this tRNA is a major determinant of the changes of gene expression on depletion of TrmD. Overexpressing the Pro and Arg synthetases also improved cell viability, particularly for ProRS. They also performed genetic experiments to show that the derepression of the Leu and Ilv operons results from stalling at the relevant codons in the leader peptides, involving replacing the CUA Leu codons with other Leu codons in the case of the Leu operon, and replacing the Pro codons with Ala codons in the leader peptide sequence of the Ilv operon, in operon reporter constructs. Again, overexpressing the ProRS dampened derepression of the Ilv operon reporter, strongly supporting their model. Thus, the authors have provided strong evidence to support all of their major claims.

  5. eLife

    Reviewer #2 (Public Review):

    This manuscript explored the effect N1-methylation of G37 of tRNAs in bacteria. The authors found that loss of methylation, through the depletion of trmD, results in defects in aminoacylation and peptidyl-transfer, leading to ribosome stalling and activation of the stringent response (as mediated by accumulation of deacylated tRNAs). Briefly, the authors conducted ribosome profiling on trmD conditional-knockout E coli cells and compared it to "wild-type" cells, and documented increased ribosome stalling on codons decoded by tRNAs modified by trmD. Stalling occurs when the ribosome is decoding these codons, i.e. when they occupy the A site. Further biochemical characterization showed that stalling is likely to occur due to defects in aminoacylation and peptide-bond formation for the trmD-substrate tRNAs, primarily for tRNAPro. Finally, analysis of gene expression shows that loss of trmD results in the activation of the stringent response as well as rewiring of central-carbon metabolism.

    Overall, this is a comprehensive study of an essential and universally conserved tRNA methylation. The manuscript expands on the role of m1G37 in translation, beyond its established role in reading-frame maintenance. However, the novelty of the findings was not immediately clear to me, and in particular whether they significantly advance our understanding of tRNA modification. For instance, it is known that defects in tRNA methylation (albeit different than N1-methylation of G37, discussed here) activates Gcn2 in yeast, which arguably is equivalent to the stringent response in bacteria.

    Furthermore, the authors made the claim "In contrast, while m1 G37 deficiency reduces peptide bond formation for some tRNAs at the A site, it consistently reduces the rate of aminoacylation for all tRNAs examined, which has not been shown for other metabolically deficient tRNAs." in the discussion section, which is inaccurate. Previous data, some from the same group, has shown that thiolation of the wobble base in tRNAGln is important for aminoacylation, tRNA selection by the ribosome and reading-frame maintenance. The argument that m1G37's pleiotropic effect on translation is unique is not convincing.

  6. eLife

    Reviewer #3 (Public Review):

    The study expands upon the previous findings of the Hou lab that the lack of TrmD-catalyzed modification in the anticodons of several bacterial tRNAs leads to +1 frameshifting when the undermodified tRNA is positioned in the ribosomal P site. In the current study, the authors show that a number of other aspects of translation are affected when the m1G modification in the tRNA anticodon is lacking.

    Specifically, the study shows that undermodified tRNAs are less efficiently aminoacylated by the corresponding aminoacyl-tRNA synthetases leading to excessive presence of deacylated tRNAs. One of the consequences is ribosome pausing when the respective codons need to be decoded. The shift in the balance of aminoacyl-tRNA relative to deacyl-tRNA resembles the one caused by amino acid starvation. Indeed, the authors show that changes in the transcriptome triggered by reduced tRNA modification resemble those observed at stringent response.

    While the paper is generally good and interesting in its current version it is not perfectly focused: discussion of the metabolic changes resulting from transcriptome remodeling are relatively fuzzy and do not contribute much to the main story. Another problem is that some of the claims (e.g. that the lack of anticodon modification affects peptide bond formation) are not properly termed and thus, misleading. In fact, the lack of tRNA modification affects dipeptide formation (possibly by interfering with decoding or tRNA accommodation) rather than influencing the rate of peptidyl transfer per se.

  7. Version published to 10.1101/2021.05.27.446073v1 on bioRxiv