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Loss of N1-Methylation of G37 in tRNA Induces Ribosome Stalling and Reprograms Gene Expression

ABSTRACT: N1-methylation of G37 is required for a subset of tRNAs to maintain the translational reading-frame. While loss of m1G37 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 m1G37 deficiency on protein synthesis. Using E. coli as a model, we show that m1G37 deficiency induces ribosome stalling at codons that are normally translated by m1G37-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 m1G37 deficiency reduces tRNA aminoacylation and in some cases peptide-bond formation. We observe changes of gene expression in m1G37 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 m1G37 that emphasizes its role throughout the entire elongation cycle of protein synthesis, providing new insight into its essentiality for bacterial growth and survival.

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

PROVIDER: GSE165592 | GEO | 2021/08/19

REPOSITORIES: GEO

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Project description:Many tumors escape by activating multiple cellular pathways that induce immunosuppression. One pivotal immune-suppressive mechanism is the production of tryptophan metabolites along the kynurenine pathway by IFNγ-induced IDO1 enzyme production 4-8. Phase III clinical trials using chemical inhibition of IDO1 in combination with PD1 pathway blockade, however, failed to improve melanoma treatment 9-12. This points at an incomplete understanding of the role of IDO1 and the consequent tryptophan degradation on mRNA translation and cancer progression. Here, we investigated the effects of prolonged IFNγ treatment on mRNA translation in melanoma cells by ribosome profiling. Surprisingly, we observed a massive accumulation of ribosomes ~20 amino acids downstream of tryptophan codons (termed here as W-Bumps) along with their expected stalling at the tryptophan codon itself. This indicated ribosomal bypass of the tryptophan codons in the absence of tryptophan. Detailed examination of W-Bumps position and its corresponding peptide sequences pinpointed towards ribosomal frameshifting events and their effects in the ribosome exit tunnel. In particular, W-Bumps strength was associated with the disorderedness level of potential out-of-frame peptides predicted downstream of tryptophan codons. Indeed, reporter assays demonstrated the induction of ribosomal frameshifting, and the generation of trans-frame proteins and their presentation at the cell surface after IFNγ treatment. Proteomics and immunopeptidomics analyses verified the production of IFNγ-induced trans-frame and out-of-frame aberrant peptides and their presentation on HLA class I molecules. Priming of naïve T cells from healthy donors with aberrant peptides resulted in identification of reactive, peptide-specific T cells. Altogether, our results suggest that IFNγ-induced IDO1-mediated tryptophan depletion plays a role in the immune recognition of melanoma cells by contributing to the diversity of the peptidome landscape, and by inducing the presentation of aberrant peptides.

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Loss of N1-Methylation of G37 in tRNA Induces Ribosome Stalling and Reprograms Gene Expression

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