Periodic Reporting for period 4 - CrackEpitranscriptom (Cracking the epitranscriptome)
Período documentado: 2021-05-01 hasta 2022-04-30
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 one particularly abundant modification called m6A, involving the methylation of an adenosine, with the goals of identifying the pathways involved in its installation and recognition, and dissecting the function and mechanisms of action of this modification. This modification is deeply connected to the fate of an mRNA, and understanding this modification, the machinery depositing it and the underlying mechanisms therefore has the potential to unlock questions concerning the forces dictating the lifespan and regulation to which mRNAs are subjected in health and in disease. More generally, we sought to address similar questions also in the context of additional post-transcriptional modifications on mRNA. Over 170 such modifications exist, many of which implicated in diverse human diseases, and therefore understanding their distribution and function is of utmost importance, as well as value to society.
We have made substantial progress on all three aims. Our work has allowed to dissect the code governing the formation of m6A, to unravel novel components of the RNA methylation machinery, to explore the functional ramifications of m6A in both yeast and mammalian contexts and to unravel a strong-quantitative link with mRNA stability, and to work on defining the forces underlying the evolution of this modifications.
In parallel, on the basis of the methods-development aspect of this proposal, we were also able to establish approaches allowing transcriptome-wide mapping of additional modifications. Our work was disseminated in the form of multiple publications (as listed in the publication section).
In parallel, on the basis of the methods-development aspect of this proposal, we were also able to establish approaches allowing transcriptome-wide mapping of additional modifications. Our work was disseminated in the form of multiple publications (as listed in the publication section).
A major advance made possible by this funding source is enhanced ability to monitor m6A at a transcriptome-wide scale. Methodologies that had existed were limited in their quantitative abilities, in their resolution, and in the scale at which they could be applied. These limited the ability to connect m6A with direct, functional consequences, and to explore the extent to which this modification was dynamic. This project has given rise to two methods (m6A-seq2 and MAZTER-seq, Garcia-Campos et al, Cell, 2019 and Dierks et al, Nature Methods, 2021) that mark major steps forward in our ability to quantitatively map this modification at substantially broader scales and resolutions than had been possible.
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, as well as to systematically explore the determinants giving rise to their formation.
These two leaps were of huge relevance both for the mechanistic dissection of roles played by m6A and of additional modifications.
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, as well as to systematically explore the determinants giving rise to their formation.
These two leaps were of huge relevance both for the mechanistic dissection of roles played by m6A and of additional modifications.