# Cracking the case of m7G modification in human tRNAs

Pierre Barraud, Carine Tisné

► To cite this version:

Pierre Barraud, Carine Tisné. Cracking the case of m7G modification in human tRNAs. Nature Structural and Molecular Biology, 2023, 30 (3), pp.242-243. 10.1038/s41594-023-00937-5. hal-04200247

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# Cracking the Case of m<sup>7</sup>G Modification in Human tRNAs

Pierre Barraud<sup>a</sup>, Carine Tisné<sup>a,*</sup>

<sup>a</sup>Expression génétique microbienne, Université Paris Cité, CNRS, Institut de biologie physico-chimique, IBPC, Paris, France

To cite this article: Barraud, P., Tisné, C. Nat Struct Mol Biol 30, 242-243 (2023).

This is a postprint of an article published in Nature Structural and Molecular Biology. The final authenticated version is available online at: https://doi.org/10.1038/s41594-023-00937-5

The RNA methyltransferase METTL1 catalyzes N7-methylguanosine (m<sup>7</sup>G) modification at position 46 in human transfer RNAs (tRNAs). Its dysregulation drives tumorigenesis in many cancer types. Two papers now reveal the structural basis of this tRNA maturation event.

Transfer RNA (tRNA) is an adapter molecule that links a specific codon in messenger RNA (mRNA) with its corresponding amino acid during protein synthesis. tRNAs are thus essential components of the cellular protein synthesis machineries, and are also implicated in many roles outside translation. The biogenesis of tRNAs typically includes the maturation of their extremities, the removal of intronic sequences if present, the addition of the 3'-CCA amino-acid accepting sequence, the aminoacylation, and all along this process, the enzymatic incorporation of a large number of post-transcriptional chemical modifications, including multiple methylations catalyzed by methyltransferases (MTases). These modifications occur at specific sites in a tightly controlled manner, which ensures that the tRNA biogenesis effectively leads to the formation of functional tRNAs [1]. To date, about 150 types of RNA modifications have been identified in all domains of life [2,3], and about 80% of them were identified in tRNAs, which are thus the RNA family bearing the largest number and diversity of RNA modifications. Among these, the N7-methylguanosine at position 46 in the variable loop (m<sup>7</sup>G46, Fig.1 a,b) is a prevalent modification in a large subset of tRNAs from bacteria to humans. m<sup>7</sup>G46 interacts with the base pair 13-22 in the D-arm to stabilize tRNA tertiary structure. In humans, m<sup>7</sup>G46 is catalysed by METTL1 and the essential cofactor WDR4 that is required for METLL1 MTase activity [4]. METTL1, which shares the Rossmann-like fold of class I MTases, uses the S-adenosyl-L-methionine (SAM) as a cofactor methyl donor and the S-adenosyl-L-homocysteine (SAH) is the cofactor product of the methylation reaction.

<footnote>*Corresponding author
Email address: carine.tisne@cnrs.fr (Carine Tisné)</footnote>
In recent years, deficiencies and dysregulation of tRNA modifications have been linked to many human diseases, including mitochondrial diseases, neurological disorders and cancer [5,6], highlighting the importance of understanding the mechanisms underlying modification deposition. In two recent studies published in Nature, Li et al. [7] and Ruiz-Arroyo et al. [8] have made significant progress in this direction by providing structural and biochemical insights into the mechanisms of tRNA methylation by the human m<sup>7</sup>G46 MTase complex METTL1-WDR4. In these studies, cryo-electron microscopy (cryo-EM) structures of complexes between the human METTL1-WDR4 complex and different tRNAs (tRNA<sup>Phe</sup>, tRNA<sup>Val</sup> and tRNA<sub>3</sub><sup>Lys</sup>) in different states of SAM/SAH presence in the active site of METTL1 are solved: in presence of SAH for the structure with tRNA<sup>Phe</sup> at 3.3 Å resolution; with no ligand for the structure with tRNA<sup>Val</sup> at 3.6 Å resolution; and with no cofactor (3.7 Å resolution), with SAM (4.0 Å resolution) or with SAH (3.5 Å resolution) for the three structures solved with tRNA<sub>3</sub><sup>Lys</sup>. METTL1 and WDR4 form a stable complex with extensive intermolecular interactions crucial for optimal MTase activity.

The authors of the two studies observed similar binding of tRNAs by the METTL1-WDR4 complex in all the different structural states they obtained, which overall enabled visualization of the structural rearrangements of both the MTase complex and the tRNA during the catalytic progression. METTL1 binds the variable loop and interacts with the tRNA elbow (formed by the intricate assembly of the T- and D- loops), whereas WDR4 interacts with METTL1 and directly participates in the interaction with tRNAs at the level of the T-loop. The presence of SAM or SAH renders the N-terminal tail of METTL1 observable and helps to better define the catalytic loop of METTL1, which extends towards the tRNA (Fig. 1c). Indeed, METTL1 N-terminus, unstructured in absence of either SAM or SAH and not visible in X-ray structures, makes important specific contacts with the tRNA, the catalytic loop of METTL1 and the C-terminal helix of WDR4 when SAM or SAH are bound in the active site. This network of interactions is mediated by METTL1 N-terminus to stabilize the assembly near the catalytic pocket of METTL1. The deletion of the first 19 amino acids of METTL1 or mutation of Lys18 significantly alters the MTase activity of METTL1 in vitro [7,8] and in vivo [7]. NMR spectroscopy was further used to confirm that the N-terminus segment (1-45) is largely disordered in solution and interacts with the METTL1 core [7]. Among all these cryo-EM structures, the most extensive RNA-protein interaction and protein rearrangements are observed in the state captured in the structure of the SAH-bound METTL1-WDR4 complex with tRNA<sub>3</sub><sup>Lys</sup> [8]. In this structure, the G46 base is flipped out in the catalytic pocket of METTL1, the tRNA variable loop structure is modified and stabilized by contacts with residues of METTL1. METTL1 is poised for methylation at the N7 position, thus providing a view of the pre-catalytic state (Fig. 1c). Li et al. [7] did not observe the G46 flipping, probably because in their structures, they used either a fully modified tRNA<sup>Phe</sup> from yeast (already bearing the m<sup>7</sup>G46 modification) or a synthetic tRNA<sup>Val</sup> without modification, but in this latter case, neither the SAH nor the SAM is present in the active site. These structures provide structural information on the initial tRNA binding and recognition prior to G46 flipping and accommodation for methyl transfer, and are therefore essential for the understanding of the molecular rearrangements
at work in this system (Fig. 1c). Overall, these studies provide a comprehensive mechanis-
tic model for how METTL1 and WDR4 cooperate to recognize tRNAs and catalyze m<sup>7</sup>G46
modification (Fig. 1c). One key finding from both studies is the importance of flexible and
disordered regions of RNA modification enzymes for the recognition of RNA and catalysis of
RNA modifications. These findings highlight the versatility of disordered regions in enzymes
and their crucial role in substrate recognition and catalysis.

Phosphorylation of Ser27 has been shown to inhibit the ability of METTL1 to modify
tRNA [9]. Ser27 is located within the N-terminus of METTL1, for which no structural
information was previously available. In the pre-catalytic complex, Ser27 lies between the
cofactor SAM/SAH and the tRNA backbone, and its phosphorylation would likely perturb
the interaction through steric clash and charge repulsion, which was further validated by
mutagenesis and MTase activity assays. These data show that this phosphorylation event
in the N-terminus of METTL1 disrupts the catalytic center of the enzyme, leading to its in-
activation. In both papers, Ser27 phosphorylation is thus proposed to play a regulatory role
in the tRNA modification process. This regulatory mechanism is thought to be important
in controlling tRNA modification levels in the cell.

Recent studies highlight that the METTL1-WDR4 complex promotes cancer in multiple contexts [10–13]. The structural data described in these two papers help to understand why some mutations in the METTL1-WDR4 complex associated with human diseases cause defect in tRNA m<sup>7</sup>G46 deposition. The development of RNA methyltransferase inhibitors as a potential avenue for discovering new drugs is an exciting prospect. Inhibiting the activity of these enzymes could potentially disrupt the development and progression of cancer cells. However, it is important to note that tRNA methylation plays important roles in normal cellular processes, and disrupting these could potentially have unintended consequences. Therefore, the development of RNA methyltransferase inhibitors as potential cancer treatments will likely require careful consideration and further research to fully understand their potential risks and benefits.

In conclusion, the recent structural studies on RNA modification enzymes by Li et al. [7] and Ruiz-Arroyo et al. [8] provide important insights into the molecular mechanisms by which these enzymes recognize and modify their tRNA substrates. Both studies highlight the important role played by flexible and disordered regions in these processes (Fig. 1c), and demonstrate the power of combining cryo-EM with the use of AlphaFold [14,15] to provide high-resolution structures of RNA-protein complexes.

Until now, most RNA-modifying enzymes have been crystallized alone or with their co-
factor SAM, but researchers have been unable to elucidate how they recognize and modify
their RNA substrate. This field is now entering exciting times, and we anticipate structural
biologists being able to catch these enzymes in action, revealing their secrets, in the years
to come.
## References

1. Barraud, P. & Tisné, C. To be or not to be modified: Miscellaneous aspects influencing nucleotide modifications in tRNAs. IUBMB Life 71, 1126-1140 (2019).

2. Boccaletto, P. et al. MODOMICS: a database of RNA modification pathways. 2021 update. Nucleic Acids Res 50, D231-D235 (2022).

3. Oerum, S., Meynier, V., Catala, M. & Tisné, C. A comprehensive review of m6A/m6Am RNA methyltransferase structures. Nucleic Acids Res 49, 7239-7255 (2021).

4. Alexandrov, A., Martzen, M. R. & Phizicky, E. M. Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA. RNA 8, 1253-1266 (2002).

5. Suzuki, T. The expanding world of tRNA modifications and their disease relevance. Nat Rev Mol Cell Biol 22, 375-392 (2021).

6. Orellana, E. A., Siegal, E. & Gregory, R. I. tRNA dysregulation and disease. Nat Rev Genet 23, 651-664 (2022).

7. Li, J. et al. Structural basis of regulated m7G tRNA modification by METTL1-WDR4. Nature (2023) doi:10.1038/s41586-022-05566-4.

8. Ruiz-Arroyo, V. M. et al. Structures and mechanisms of tRNA methylation by METTL1-WDR4. Nature (2023) doi:10.1038/s41586-022-05565-5.

9. Cartlidge, R. A. et al. The tRNA methylase METTL1 is phosphorylated and inactivated by PKB and RSK in vitro and in cells. EMBO J 24, 1696-1705 (2005).

10. Chen, J. et al. Aberrant translation regulated by METTL1/WDR4-mediated tRNA N7-methylguanosine modification drives head and neck squamous cell carcinoma progression. Cancer Commun (Lond) 42, 223-244 (2022).

11. Cheng, W., Gao, A., Lin, H. & Zhang, W. Novel roles of METTL1/WDR4 in tumor via m7G methylation. Mol Ther Oncolytics 26, 27-34 (2022).

12. Lin, S. et al. Mettl1/Wdr4-Mediated m7G tRNA Methylome Is Required for Normal mRNA Translation and Embryonic Stem Cell Self-Renewal and Differentiation. Mol Cell 71, 244-255.e5 (2018).

13. Ma, J. et al. METTL1/WDR4-mediated m7G tRNA modifications and m7G codon usage promote mRNA translation and lung cancer progression. Mol Ther 29, 3422-3435 (2021).

14. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583-589 (2021).

15. Varadi, M. et al. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res 50, D439-D444 (2022).

## Acknowledgments

The authors acknowledge support from the CNRS, the ANR (ANR-19-CE07-0028 and ANR-19-CE44-0013) and the LABEX DYNAMO (ANR-11-LABX-0011).

## Competing interests

The authors declare no competing interests.
<figure><img src="image_2.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_4.png" /></figure>

**Figure 1.** (a) Chemical structure of 7-methylguanosine (m<sup>7</sup>G). (b) Cloverleaf representation of tRNA secondary structure. The five main regions of tRNAs, namely the acceptor stem, the D-arm, the anticodon-arm, the variable loop, and the T-arm are labelled. The position of the m<sup>7</sup>G46 modification in the variable loop is represented with a red circle. (c) Mechanistic model of METTL1-WDR4 catalytic cycle for m<sup>7</sup>G46 methylation in tRNA. METTL1, WDR4 and tRNA are schematically represented as surfaces in yellow, green, and blue, respectively. The main mobile elements important for catalysis and that experience structural rearrangements in the different catalytic steps are represented as sketched ribbon elements. In the free METTL1-WDR4, the METTL1 N-terminal and catalytic loop are flexible, as well as the WDR4 C-terminal helix. Upon binding of tRNA and SAM, the METTL1 N-terminal and catalytic loop change conformation and become ordered to accommodate both the tRNA and the cofactor. Then, structural rearrangements occur, which lead to the G46 base flipping and structuration of the WDR4 C-terminal helix interacting across the METTL1 catalytic center with the tRNA D- and anticodon-arms. After methyl transfer, the m<sup>7</sup>G46-tRNA and the SAH cofactor are released.