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Cracking the epitranscriptome

Periodic Reporting for period 3 - CrackEpitranscriptom (Cracking the epitranscriptome)

Reporting period: 2019-11-01 to 2021-04-30

The goal of our project is to decipher the function of a chemical modification imposed upon the basic building blocks of our genetic code - the mRNA molecules - following their formation. For years it was thought that the sequence of the mRNA - that encodes the productions of proteins - essentially mirrors the DNA sequence (with some exceptions). In recent years it has turned out, however, that following the production of the mRNA it can acquire chemical modifications, that can then alter its stability, localization and ability to encode proteins. These modifications have meanwhile been implicated in a large number of both physiological and pathological conditions, and hence understanding where they are, what their roles are, and how they bring these about are critical questions that will impact our understanding of health and disease.

In our proposal, we set to explore the roles of a particularly abundant modification called m6A, involving the methylation of an adenosine. We are studying this modification in the context of a specific environmental response in yeast called meiosis, during which we have previously found that m6A is induced across >1000 mRNA molecules. We have three key goals: First, to identify unknown components that are involved in mRNA methylation, and to identify specific m6A sites that are important for meiosis. Second, to understand the function of mRNA methylation, by looking at the molecular consequences to having versus not having methylation. Third, to understand how such functional roles are mediated.
We have made substantial progress on all three aims. First, we have been able to succesfully perform a genetic screen, to identify a novel factor involved in the methylation pathway in yeast. We are currently extensively following up on this candidate, and working on pinpointing its role in the context of meiosis. In parallel, we have been working hard to understand the m6A code, that is why certain sites undergo methylation and others do not. Deciphering such codes has critical bearing on our ability to assess the conservation of m6A across evolution, which in turn can shed critical insight on which m6A sites are important for meiosis, versus ones that are not. By deciphering this code, we could also generate hundreds of distinct yeast strains, in which this code is disrupted in various manners, which has allowed us to address the role played by m6A in yeast and to uncover that it is involved in mRNA destabilization. Both these findings have been accepted for publication in Cell. Finally, using the tools developed in the above two aims, we are currently working on dissect the mechanisms of action underlying the role played by m6A primarily in the context of mRNA degradation.
A major leap forward beyond the state of the art, already made by our study, is the ability to precisely quantify m6A levels at a systematic scale. So far it has only been possible to get a qualitative sense of m6A, which has severely limited our ability to study this modification, and to measure changes that it can undergo over time, in cellular responses, and across disease states. It was also difficult to connect m6A with direct, functional consequences, given that it was not possible to obtain dose-response relationships, given the inability to quantify it. With our method we can now address these questions.

A second leap forward is our ability to generate large pools of designed mutants varying in their methylation levels in a programmable way. This allows us to to obtain much more direct relationships between the methylation and distinct, functional readouts that we can obtain.

These two leaps in our technological toolkit will allow us to now address the functions and mechanisms of action of m6A at unprecedented resolution and by the end of this project we hope to be able to make major breakthroughs into these questions.