Vicente Pelechano Garcia

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.