Trm112, a unique methyltransferase at the interface between ribosome synthesis and function – TrMTases

This project uses innovative methodological approaches to address these aims. One of the greatest challenges of this project is the production and purification of transient enzyme-substrate complexes (protein-protein but also protein-RNA complexes) in quantity suitable for structural studies and/or biochemical studies. The structural study of protein-RNA complexes is still a highly challenging task and several techniques will be combined to optimize our chances of success and trap these transient complexes. Appropriate methods will be selected according to the characteristics of the target proteins, taking into account our results obtained concomitantly.
To understand the function of Mtq2 and Bud23 in ribosome biogenesis, we will use Northern blotting and polysome profile analysis. We will also use affinity purification protocols optimized to detect transient interactions followed by mass spectrometry. We will also use microscopic analyses and flow cytometry to decipher the role of these MTases in cell cycle progression.
Regarding regulation of Trm112-MTases interaction network, we will quantify production and stability of the mRNA/proteins. We will also investigate for post-translation modifications which could affect the affinity of any of the four MTase for Trm112.

Crystal structure of Trm9-Trm112 complex, involved in mcm5U modification in tRNA, was determined. The mechanism of action of this protein has been deciphered in details through in vitro enzymatic assays and in vivo studies of several mutants (Letoquart et al, Nucleic Acids Res 2015).
As major conclusions, we showed that all Trm112-MTase complexes rely on the same molecular bases and explained why all Trm112 dependent MTases compete to interact with this partner. Trm112 shows high structural plasticity, allowing interaction with various MTase partners sharing less than 20% sequence identity. We also have investigated the mechanisms used by Trm12 to activate the tRNA MTase Trm11 (manuscript under revision in Nucleic Acids Res).
We showed in collaboration with Denis Lafontaine (ULB, Charleroi) that human WBSCR22-TRMT112 are the functional homologues of yeast Bud23-Trm112. This complex is required for rRNA processing reactions leading to the synthesis of 18S rRNA. In both organisms ribosome biogenesis requires only the presence of the modification enzyme rather than its RNA modifying catalytic activity (Zorbas et al, Mol Biol Cell, 2015).

Many proteins involved in translation do not function alone but belong to dynamic multiprotein assemblies. Despite significant achievements such as the crystal structure of yeast ribosomes, only few information is available about how these complexes form and function: our work will contribute to this research field.
Investigation of the co-activator Trm112 and its associated methyltransférases (MTases), which are conserved from yeast to man will not only be essential for the understanding of MTases function, but will also provide novel insights into the mechanisms of translation, ribosome synthesis, and their regulation. The role of post-transcriptional/post-translational modifications on the translation apparatus are very much in their infancy, and their impacts are yet to be determined. There are also increasing evidences that in eukaryotes and particularly in metazoans, proteins are not expressed in all cell tissues, and enzymes are not constitutively active during cell cycle. Hence, this study may document the mechanisms used by cells to regulate protein interaction networks.
In human, 1% of the proteome is constituted by S-adenosyl-L-methionine-dependent MTases, 30 % being linked to disease state. Human Mtq2 has been suggested to be linked to Down syndrome. It is also well known that defects in ribosome biogenesis have been linked to human diseases (ribosomopathies) and neurodegenerative disorders. WBSCR22, the human Bud23 ortholog was recently identified as a novel metastasis promoter. Finally, ABH8, the human Trm9 ortholog is involved in the cellular response to genotoxic stresses, is highly expressed in a variety of human cancer cells while its down-regulation results in suppression of tumour growth, angiogenesis and metastasis. Hence, the results obtained could also be used in future studies on the role of these complexes in cell biology and tumorigenesis and/or on drug design approaches.

Conferences
1. Létoquart, J., Huvelle, H., Wacheul, L., Bourgeois, G., Zorbas, G., Graille, M., Lafontaine, DL & Heurgué-Hamard, V. 10th EMBO conference on ribosome synthesis, Brussels, Belgium, 19-23 August 2015 (poster)
2. Létoquart, J., Huvelle, H., Wacheul, L., Bourgeois, G., Zorbas, C., Nicolas, E., Graille, M., Lafontaine, DL & Heurgué-Hamard, V. How the evolutionarily conserved Bud23-Trm112 methylation complex acts during ribosome biogenesis in budding yeast and human cells ?. 10th SifrARN meeting, Toulouse, March 8-10th 2016 (poster)
3. Létoquart, J., Bourgeois, G., van tran, N., Marcoux, J., Saliou, JM., Huvelle, E., Wacheul, L., Zorbas, C., van Tilbeurgh, H., Liger, D., Cianferani, S., Lafontaine, DL., Heurgué-Hamard, V. & Graille, M Structural and functional studies of four Trm112-Methyltransferase holoenzymes modifying RNA and proteins involved in translation. 10th SifrARN meeting, Toulouse, March 8-10th 2016 (poster)
4. Létoquart, J., Bourgeois, G., van tran, N., Marcoux, J., Saliou, JM., Huvelle, E., Wacheul, L., Zorbas, C., van Tilbeurgh, H., Liger, D., Cianferani, S., Lafontaine, DL., Heurgué-Hamard, V. & Graille, M. EMBO conference “Ribosome structure and function”, Strasbourg, July 6-10th 2016 (poster)

Publications
1. Zorbas, C., Nicolas, E., Wacheul, L., Huvelle, E., Heurgué-Hamard, V & Lafontaine D.L.J. The human 18S rRNA base methyltransferases (2015) DIMT1L and WBSCR22-TRMT112 but not rRNA modification are required for ribosome biogenesis. Mol. Biol. Cell, 26, 2080-2095.
2. Létoquart, J., van Tran, N., Caroline, V., Aleksandrov, A., Lazar, N., van Tilbeurgh, H., Liger D & Graille M. (2015)Insights into molecular plasticity in protein complexes from Trm9-Trm112 tRNA modifying enzyme crystal structure. Nucleic Acids Res, 43, 10989-1002
3. Bourgeois, G.; Marcoux, J.; Saliou, JM.; Cianferani, S.; Graille, M. Activation mode of the eukaryotic m2G10 tRNA methyltransferase Trm11 by its partner protein Trm112. Nucleic Acids Res; Under revision.

Translation is a multi-step process leading to protein synthesis from messenger RNA (mRNA) templates. It is carried out by the ribosome, a macromolecular complex made of proteins and ribosomal RNAs (rRNA), together with the help of transfer RNAs (tRNA) and translation factors. For correct biosynthesis and function, these translation components are subjected to post-transcriptional/ and post-translational modifications (PTM), which influence the efficiency and accuracy of ribosome biogenesis and/or translation with established consequences on cell cycle progression.
Methylation is one of the most common PTM found on RNAs and proteins. The effects of methylation on translation are perfectly illustrated by the Trm112 protein, which is unique acting as an activating platform of four methyltransferases (MTases) modifying rRNA (Bud23), tRNA (Trm9 and Trm11) or translation termination factor (Mtq2). Trm112 is thus ideally located at the interface between ribosome biosynthesis and function.

In this project, we propose to understand the central role of these Trm112-MTases in fine-tuning translation as well as in cell biology. We have planned a highly synergistic workflow, based on our complementary experimental expertises (yeast genetics, biochemistry, biophysics, structural biology) to address the following specific goals:
i) Determination the 3-D structures of these four Trm112-MTases holoenzymes in complex with their substrate (protein or RNA fragments) to understand their catalytic mechanism as well as the molecular basis for recognition.
ii) Deciphering the role of the Bud23-Trm112 and Mtq2-Trm112 complexes in ribosome biosynthesis and in cell cycle.
iii) Study of the regulation and dynamics of the Trm112-MTases interaction network during cell cycle and in response to various stimuli.

The feasibility of all these goals is supported by preliminary results that we have recently gathered and that are presented in details in the application.

This project will yield new insight into the physiological function of these Trm112-MTase complexes, which are conserved in higher eukaryotes and involved in human diseases.