Decapping

The flow of genetic information in all cells progresses from DNA to messenger RNA (mRNA) to protein. Many events must occur precisely to generate the protein product accurately and efficiently. As a consequence, eukaryotic cells have evolved a finely tuned “gene-expression factory” that encompasses the routing of a nascent transcript through multimeric mRNA–protein complexes that mediate its splicing, polyadenylation, nuclear export, translation and ultimate degradation. Traditionally, mRNA decay was considered a simple passive destruction step of mRNA but this view has been challenged in the recent years. Eukaryotic mRNA decay now appears as a highly regulated process that allows cells to rapidly modulate protein production in response to environmental factors. Regulation of mRNA decay rates is an important control point in determining the abundance of cellular transcripts. Decay rates of individual mRNAs differ extensively and the half-lives of certain mRNAs are known to change markedly throughout the cell cycle or in response to environmental cues. These differences in mRNA decay rates have notable effects on the expression of specific genes, and provide the cell with flexibility in effecting rapid change in transcript abundance as an essential step in the regulation of gene expression. To understand the control of gene expression, it is thus necessary to understand the regulation and mechanisms of mRNA degradation. Over the past years, most of the enzymes involved in mRNA decay have been identified, yielding to a well documented view of the general cytoplasmic mRNA decay. Although the proteins involved in mRNA decay are known, they are all part of dynamic and multifunctional protein assemblies. The importance of these protein-protein interactions is becoming increasingly evident. It is therefore essential to understand in molecular details the dynamics of these complexes, and the interactions involved, in order to unravel the mechanisms of mRNA decay and the logic of its regulation. Mature translatable eukaryotic mRNAs are protected from fast and uncontrolled degradation in the cytoplasm by two cis-acting stability determinants: a methylguanosine cap and a poly(A) tail at the 5’ and 3’ extremities, respectively. As a consequence, mRNA degradation necessitates a subsequent remodelling of the mRNP structure that is triggered by specific signals and leads to the recruitment of activators of decay and enzymes. Eukaryotic mRNA degradation typically initiates with deadenylation followed by the removal of the cap structure protecting the 5’ end of mRNAs. In yeast, this key step is accomplished by the recruitment of a decapping complex composed of the Dcp2 catalytic subunit and its activator Dcp1 that eliminate the cap structure protecting the 5’ end of mRNAs. The Dcp1-Dcp2 complex has a low intrinsic decapping activity. Several accessory factors activate this decapping enzyme by a mechanism that remains largely obscure. Once mRNAs are uncapped at their 5’ end, they are rapidly degraded in the 5’?3’ direction by the cytoplasmic exonuclease Xrn1 that interacts with the decapping machinery. Our goal is to unravel the mechanisms underlying the activation of the Dcp1-Dcp2 decapping enzyme by structural and functional approaches and using yeast S. cerevisiae as a model organism. We propose to focus on the various proteins known to influence decapping so as to determine their precise role on (1) Dcp1-Dcp2 activation; (2) mRNP remodelling and (3) relation between inhibition of translation initiation and mRNA degradation. To reach this goal, we have organized a highly complementary consortium with expertise in structural biology, biophysics, biochemistry, and molecular genetics.