Blanc SVSE 2 - Sciences de la vie, de la santé et des écosystèmes : Biologie cellulaire, développement

Ubiquitylation and spatiotemporal control of gene expression – UBIGENEX

This project is realized in yeast, a simple organism in which these basic functions are preserved and easily and safely manipulated genetically, We use experimental approaches combining genetics, biochemistry (or chemistry of life), molecular biology and microscopy approaches that allow us to track molecules such as RNAs in cells.

Recent studies, some performed in our laboratory showed that ubiquitylation plays an important role in this coupling. Ubiquitin is a small molecule that, by binding specifically to target proteins, dictates the functions of these proteins. We have shown that the protein responsible for the export of mRNA in the cytoplasm, specifically interacted with ubiquitylated proteins involved in certain stages of the synthesis of mRNA. Through this project, we have now shown that the ubiquitylation of proteins intimately associated with the genes may dictate the fate of an mRNA. Furthermore, we demonstrated that this small molecule is also capable of modulating the function of the nuclear pore, a multimolecular structure that strictly control the exchange of molecules between the nucleus and the rest of the cell.

The knowledge acquiredin the yeast model can now be used to understand the mechanisms of dysfunction of mRNA synthesis observed in certain pathologies, such as cancer.

This project is only halfway but has already led to four publications and a fifth well under way in international journals, peer-reviewed, and highly respected in the field.

Submission summary

Transcripts generated by RNA polymerase II undergo a carefully orchestrated series of processing steps which involve the machineries responsible for chromatin modifications, transcription initiation, elongation and 3’ end maturation before being exported to the cytoplasm. However the precise mechanisms responsible for this intimate coordinatioon remains to be elucidated. Ubiquitin conjugation, capable of controlling both protein degradation but also protein/protein interactions, appears as a good candidate to favor dynamic molecular scaffolds responsible for functional coupling events.The major goal of this proposal is thus to decipher the role of modifications by ubiquitin on the spatiotemporal control of gene expression and in particular on the coordination between different steps of mRNA biogenesis, DNA damage and the structural and transport functions of the nuclear pore complex.
We propose to concentrate our efforts on four major objectives. The first one concerns the roles of ubiquitylation in the coupling between 3’end processing and nuclear export of mRNP, an objective based on previous and original results obtained during our last ANR financed project. We identified Mex67, the mRNA export receptor, and some of its adaptors as ubiquitylated proteins or Ubiquitin-Associated domain (UBA)-containing proteins and proposed that UBA/ubiquitin interactions could precisely control essential remodelling steps of the release, quality control and export of mature mRNPs. More precisely, we propose to identify ubiquitylated factors and effector proteins that could transduce the ubiquitin signal in the coupling between 3’end processing and nuclear export of mRNP.
Our second objective is to determine the precise roles of ubiquitylation in the coupling between 3’ end and upstream transcriptional processes. We’ll focus our attention on two partners of Mex67-UBA, Swd2, a component of both the H3K4 methylation complex COMPASS and the 3’end processing holoCPF complex, and Ioc2, a component of an Isw1 containing chromatin remodelling complex that acts in coordination with COMPASS. We propose to determine how their ubiquitylation control their coordinated function on gene expression through transcription initiation and termination of both sense and antisense transcripts.
The aim of the third topics is to extend the role of ubiquitylation to the coordination between mRNP synthesis (transcription and posttranscriptional events) and transcription-dependent DNA damage and repair. In particular, our preliminary results indicate that double-strand breaks are induced by mutating ubiquitylation sites of Swd2 but independently of their effects on COMPASS function. We therefore propose to further characterize this DNA damage and to test whether it is correlated to a transcription-dependent replication defect due to a co- or postrancriptional event (mRNA processing or export).
Finally, we recently analyzed the ubiquitylation pattern of the Nuclear Pore Complex (NPC), the first systematic analysis of the post-translational modifications of the NPC. To our surprise, we found that half of the nucleoporins are modified, mainly by monoubiquitylation. We thus propose to determine how ubiquitylation of the NPC controls its diverses function, and in particular in nucleocytoplasmic transport but also in the gene gating and eroded telomere relocalization.
This project will be performed by the collaboration between C. Dargemont’s and V. Géli’s team, whose expertise in transcription related processes, ubiquitylation mechanisms and mRNA export are fully complementary. Both teams already developed technological approaches and biological tools and obtained preliminary data that will guarantee the success of this proposal. A combination of multiscale approaches, from single gene and molecular structures to genome-wide profiling, biochemistry and genetic technologies should led to a better understanding of such coupling and synchronization mechanisms.

Project coordination

CATHERINE DARGEMONT (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B) – catherine.dargemont@inserm.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CNRS CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B
CNRS DR12-IGC CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE PROVENCE

Help of the ANR 500,000 euros
Beginning and duration of the scientific project: - 36 Months

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