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Mammalian methionine adenosyltransferase 2A promotes rRNA processing and methylation of translation factors to induce translation in an mTORC1-independent manner

MAHABUB ALAM 東北大学

2022.03.25

概要

Background: Translation, one of the essential steps of gene expression, consumes a huge amount of energy of the cells. Therefore, translation is a tightly controlled process to maintain cell energy homeostasis. Due to the high demand for growth, protein synthesis is induced in rapidly proliferative cells such as in cancers. In cancer, the activity of mammalian mechanistic target of rapamycin complex (mTOR) particularly mTORC1 induces translation. Therefore, mTORC1 inhibitor have been used as anti-cancer therapy. One of the major problems of mTORC1 inhibitor treatment is that many cancers develop resistant to such inhibition. Therefore, it is essential to discover mTORC1 independent translational regulation to invent new drug targets. Interestingly, cancer cells are highly dependent on methionine and other methionine cycle metabolites for proliferation which links the methionine metabolic cycle with cancer. The enzyme Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethonine (SAM) in the first reaction of the methionine cycle. SAM is the essential methyl-donor required for the methylation of various biomolecules including DNA, RNA, histone and non-histone proteins. The level of intracellular SAM affects gene expression by changing methylation of these molecules. Gene expression control by histone proteins or mRNA methylation have been extensively studied. However, little is known about influence of gene expression by the methylation of structural and functional components of translation machinery. Various studies identified multiple methylation sites on rRNA, ribosomal proteins and translation factors. Although the biological significance of ribosomal protein methylation is still unknown, some specific methylation of rRNA and translation factors appears important for translation. In mammals, three MATs isozymes are liver specific MAT1 and 3, and the ubiquitous MAT2. Expression of MAT2A, the catalytic subunit of the MAT2 isozyme, is positively correlated with proliferation of various cancer cells. However, the specific functions of MAT2A in cancer cells is unclear. Considering this upregulation in cancer and methionine dependency of cancer cells, it is assumable that MAT2A might be required for methylation of translation machinery by the production of SAM to facilitate translation in proliferating cancer cells.

Hypothesis: Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we hypothesized that MAT2 and its catalytic product SAM are necessary for protein synthesis.

Methods: In various experiments, MAT2A was depleted by RNAi or was chemically inhibited by cycloleucine (cLEU). The gene expression was quantified either by RT-qPCR or high throughput sequencing. The polysome analysis was performed by sucrose gradient ultra-centrifugation. We used quantitative mass spectrometry for proteomic studies and methylated peptides quantification.

Results: In this study we discovered that MAT2A and SAM stimulate protein synthesis by facilitating the biogenesis of 18S rRNA and by dynamic methylation of translation factors in an mTORC1-independent manner. We revealed that both depletion and inhibition of MAT2A reduced protein synthesis in mammalian cells. Overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under the normal culture conditions. These effects of MAT2 manipulations did not accompany a reduction in the mTORC1 activity. Moreover, MAT2A inhibition reduced polysome formation. Sequencing of the polysome- bound RNA revealed that MAT2 inhibition decreased the translation efficiency (TE) of a fraction of mRNAs. We found that MAT2A interacts with proteins involved in rRNA processing and ribosome biogenesis. Depletion and inhibition of MAT2 reduced the 18S rRNA abundance. In addition, we observed that some translation factors were dynamically methylated in response to the activity of MAT2A or the availability of SAM.

Conclusion: These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the amount of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced whereas it may support proliferation when SAM is sufficient. Therefore, MAT2A could be a targetable molecule for cancer therapy to suppress translation in cancer cells in an mTORC1-independent manner.

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