Vicente Pelechano Garcia

Genomes encode instructions for cells to regulate gene activity in response to their environment. However, and despite its importance for biology, medicine and biotechnology, the underpinning regulatory code remains undeciphered. Gene regulation consists of two major steps. First, genes are transcribed into mRNA. Second, post-transcriptional mechanisms regulate mRNA stability and the rate at which it is translated into proteins. This second step of gene regulation is still poorly understood because relevant parameters such as mRNA half-life, mRNA protein binding and subcellular localization are difficult to assay. The lack of understanding of post-transcriptional regulation implies that we still do not have a complete picture of the regulatory code. In EPIC, we exploit the advantages of the model eukaryote Saccharomyces cerevisiae and other species covering a broad evolutionary range to derive the first comprehensive sequence-based model of eukaryotic gene regulation. EPIC integrates the complementary expertise of 3 teams. It combines innovative high-throughput technologies (Pelechano) to probe post-transcriptional regulation at an unprecedented scale across a broad range of species and conditions with synthetic biology to massively test regulatory sequences (Verstrepen). Deep learning on these data allows us to build predictive models and unravel complex regulatory instructions (Gagneur). Ultimately, EPIC will enable us to decipher the actual language of gene regulation and facilitate (re)writing genomes. EPIC will enable understanding and predicting regulation, and ultimately phenotype, from DNA, closing a major gap in basic biology, while also opening exciting avenues for applications in biotechnology and medicine, from pinpointing disease-causing mutations to rational design of genes, RNAs and cells.

Appropriate regulation of gene expression, namely the production of functional proteins, is essential to the appropriate and timely development of all organisms. In eukaryotic cells, genes are transcribed in the nucleus, and the mRNAs are translated in the cytoplasm to produce proteins. Despite the separation of these different steps of gene expression in two different cellular compartments, evidence from the last decades has accumulated to indicate that the nuclear and cytoplasmic phases of gene expression are physically connected to ensure appropriate coordination and regulation. This project deals with the study of one complex, the Ccr4-Not complex, conserved across the eukaryotic kingdom, and a master regulator connecting the nuclear and cytoplasmic phases of gene expression. In the nucleus the Ccr4-Not complex promotes transcription elongation, impacts transcription-coupled repair mechanisms, regulates transcription factors and chromatin modifying components, regulates silencing and binds mRNAs during transcription to define their subsequent translation and turnover in the cytoplasm. In the cytoplasm it is important during translation for the production of soluble proteins, for co-translational processes, for deadenylation and for deadenylation-independent repression of translation initiation. The interconnections between all of these functions are still far from understood. This proposal has three defined aims centered towards understanding our recent finding that Not proteins form condensates and regulate translation elongation dynamics for production of soluble, functional and well assembled proteins. The first aim is to understand how the Not proteins regulate solubility of mRNAs, focusing on two mRNAs whose solubility is oppositely regulated by Not1 and Not4, then extending findings to global regulation of mRNA solubility by Not1 and Not4. The second aim is to define if Not proteins impact translation elongation dynamics directly, which functional domains of the Not proteins are relevant, if ribosome-associated targets of the Not4 E3 ligase contribute, the role of Not proteins in the nucleus, and how this correlates with changes in mRNA solubility. It will also explore the role of the RNA binding protein Puf3 for regulation of solubility and translation elongation dynamics of its targets by the Not proteins. Finally, the third aim is to characterize further Not protein condensates, their regulation, their dynamism and how they relate to translation elongation dynamics and mRNA solubility.These studies will be done in the model organism, the yeast S.cerevisiae, because of the simple and powerful genetic tools that we can combine with global approaches, basic molecular biology and biochemistry to confirm global findings. Moreover, over the years we have accumulated a large number of relevant strains and plasmids for this project, as well as expertise with yeast genetics. Our work should improve our understanding of how the Not subunits of the Ccr4-Not complex contribute to regulation of translational elongation, and gain new understanding on how this stage of gene expression is regulated in eukaryotic cells.