METTL18-mediated histidine methylation of RPL3 modulates translation elongation for proteostasis maintenance

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    Evaluation Summary:

    This work examines how METTLL18-mediated RPL3 histidine methylation on 245 position regulates translation elongation and protects cells from cellular aggregation of Tyr-rich proteins. The study hints at the existence of a "ribosome code" and how posttranslational modification of ribosomal proteins could affect translation.

    (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. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

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Abstract

Protein methylation occurs predominantly on lysine and arginine residues, but histidine also serves as a methylation substrate. However, a limited number of enzymes responsible for this modification have been reported. Moreover, the biological role of histidine methylation has remained poorly understood to date. Here, we report that human METTL18 is a histidine methyltransferase for the ribosomal protein RPL3 and that the modification specifically slows ribosome traversal on Tyr codons, allowing the proper folding of synthesized proteins. By performing an in vitro methylation assay with a methyl donor analog and quantitative mass spectrometry, we found that His245 of RPL3 is methylated at the τ- N position by METTL18. Structural comparison of the modified and unmodified ribosomes showed stoichiometric modification and suggested a role in translation reactions. Indeed, genome-wide ribosome profiling and an in vitro translation assay revealed that translation elongation at Tyr codons was suppressed by RPL3 methylation. Because the slower elongation provides enough time for nascent protein folding, RPL3 methylation protects cells from the cellular aggregation of Tyr-rich proteins. Our results reveal histidine methylation as an example of a ribosome modification that ensures proteome integrity in cells.

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  1. Author Response

    Reviewer #1 (Public Review):

    Overall, this study is well designed with convincing experimental data. The following critiques should be considered:

    1. It is important to examine whether the phenotype of METTL18 KO is mediated through change with RPL3 methylation. The functional link between METTL18 and RPL3 methylation on regulating translation elongation need to be examined in details.

    We truly thank the reviewer for the suggestion. Accordingly, we set up experiments combined with hybrid in vitro translation (Panthu et al. Biochem J 2015 and Erales et al. PNAS 2017) and the Renilla–firefly luciferase fusion reporter system (Kisly et al. NAR 2021) (see Figure 5A).

    To test the impact of RPL3 methylation on translation directly, we purified ribosomes from METTL18 KO cells or naïve HEK293T cells supplemented with …

  2. Evaluation Summary:

    This work examines how METTLL18-mediated RPL3 histidine methylation on 245 position regulates translation elongation and protects cells from cellular aggregation of Tyr-rich proteins. The study hints at the existence of a "ribosome code" and how posttranslational modification of ribosomal proteins could affect translation.

    (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. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

  3. Reviewer #1 (Public Review):

    Overall, this study is well designed with convincing experimental data. The following critiques should be considered:

    1. It is important to examine whether the phenotype of METTL18 KO is mediated through change with RPL3 methylation. The functional link between METTL18 and RPL3 methylation on regulating translation elongation need to be examined in details.

    2. The obvious discrepancy between the recent NAR an this study lies in the ribosomal profiling results (such as Fig.S5). The cell line specific regulation between HAP1 (previously used in NAR) vs 293T cell used here ( in this study) needs to be explored. For example, would METLL18 KO in HAP1 cells cause polysome profiling difference in this study? Some of negative findings in this study (such as Fig.S3B, Fig.S5A) would need some kind of positive control …

  4. Reviewer #2 (Public Review):

    The manuscript by Matsura-Suzuki et al. characterizes the role of METTL18 histidine methyltransferase in protein synthesis. The authors used genetical manipulation, affinity purification and mass spectrometry to indicate METTL18 protein as methyltransferase that specifically modifies ribosomal protein RPL3 during ribosome maturation. Using METTL18 based in vitro methylation system they annotate His245 residue in RPL3 protein as a methylhistidine and confirmed their results using a mass spectrometry on immunopurified RPL3 protein from HEK293 cells. The authors use ribosome profiling techniques, luciferase aggregation assays and mass spectrometry analyses of cellular aggregates to argue for Tyrosine specific effects during protein synthesis that influence protein folding.
    While METTL18 was recently …

  5. Reviewer #3 (Public Review):

    In this article, Matsuura-Suzuki et al provided strong evidence that the mammalian protein METTL18 methylates a histidine residue in the ribosomal protein RPL3 using a combination of Click chemistry, quantitative mass spectrometry, and in vitro methylation assays. They showed that METTL18 was associated with early sucrose gradient fractions prior to the 40S peak on a polysome profile and interpreted that as evidence that RPL3 is modified early in the 60S subunit biogenesis pathway. They performed cryo-EM of ribosomes from a METTL18-knockout strain, and show that the methyl group on the histidine present in published cryo-EM data was missing in their new cryo-EM structure. The missing methyl group gave minor changes in the residue conformation, in keeping with the minor effects observed on translation. They …