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     Macromolecular Interaction Genetics


  Director : Jacquier Alain (jacquier@pasteur.fr)


  abstract

 

We study various aspects of RNA metabolism in the yeast Saccharomyces cerevisiae that we use as an eukaryotic model. During year 2003, we particularly focused on: 1) the study of the role of the Mlp proteins in the nuclear retention of unspliced pre-mRNAs, 2) the study of a new mechanism of mRNA decay regulation that acts at the level of decapping, 3) the study of the maturation and export of ribosomal particles and, in particular, the assembly of the pre-60S complexes.



  report

cale

The main domain of interest of the laboratory is the study of RNA metabolism (maturation, transport, degradation) using the yeast Saccharomyces cerevisiae as a model organism. These metabolic pathways involve numerous steps from transcription within the nucleus to degradation in the cytoplasm and include RNA maturation and transport. We use different generic approaches such as two-hybrid (RNA three-hybrid) screens, biochemical affinity purifications (TAP) and genetic screens (co-lethality screens for example) in order to identify the functions of new factors involved in these pathways. The combination of the three approaches lead to hypotheses on the pathways in which these factors might be involved. Additional functional assays, more specific of given pathways, can then be applied in order to characterise in more details the role of these proteins.

Nucleoplasmic architecture and retention of non-mature mRNAs

The Mlp1 and Mlp2 proteins form intranuclear filaments localized at the nuclear periphery. Our two-hybrid data had led us to identify the Nup60 nucleoporine as a main nucleopore anchor point for the Mlp proteins. In collaboration with the group of Ulf Nehrbass at the Institut Pasteur, we have now shown that Mlp1 is involved in the retention of unspliced pre-mRNAs that reached the nuclear periphery. This mechanism involves the recognition of the 5' splice site consensus sequence. In absence of Nup60, which results in the delocalization of the Mlp proteins, the nuclear retention of pre-mRNAs is strongly affected. These observations define the Mlp proteins as factors involved in the quality control of mRNAs before nuclear export ()

Regulated mRNA decay

Modulating the rate of mRNA degradation is a fast and efficient way to control gene expression. In eukaryotes, mRNA decay involves the degradation of the poly(A) tail (deadenylation) followed, in the major pathway, of the decapping and 5' to 3' exonucleolitic degradation. In the vast majority of the studied examples, modulation of this process occurs at the level of deadenylation. We have now shown that the ribosomal protein Rps28b autoregulates the degradation of its own transcript by a new mechanism that activates mRNA decapping, bypassing deadenylation. This mechanism requires the presence of the decapping enhancer factor Edc3, which is associated with the decapping complex. Rps28b specifically activates decapping of its own mRNA by binding both to Edc3 and a conserved cis-regulatory element present in the 3'UTR of its transcript. These results show that specific modulation of decapping efficiency on natural transcripts can control mRNA turnover (Badis et al., submitted).

Assembly of preribosomal complexes

Ribosome biogenesis is a highly coordinated process, involving many preribosomal complexes. We and others have recently determined the global composition of preribosomal complexes in Saccharomyces cerevisiae (Saveanu et al., 2001; ). From all the available data, we generated a list of the preribosomal factors involved in this pathway (). To get insight into the poorly understood mechanisms involved in the coordinated assembly of the ribosomal proteins, we combine several approaches . 1- Proteomic approaches: we determined the composition of preribosomal complexes which are blocked at specific steps. 2- Genetic approaches: from the proteomic results, we select candidates for a functional analysis. We generate mutants that we use for genetic screens (synthetic lethals, high-copy suppressors, two-hybrid) in order to find genetic and physical links between preribosomal factors. We have recently shown that genetic links can be correlated with physical links. For example, genetic suppression of a Rlp24 mutant by the overexpression of NOG1 may be explained by a direct physical interaction. These results allowed us to propose a model of sequential protein association.

Keywords: ARN, Saccharomyces cerevisiae, nucleus, nucleolus, ribosome



  web site

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  publications

puce Publications 2003 of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Labouise, Odile, (labouise@pasteur.fr) Jacquier, Alain, CNRS, DR2, (jacquier@pasteur.fr)

Fromont-Racine, Micheline, CNRS, DR2, (mfromont@pasteur.fr)

Saveanu Cosmin , Institut Pasteur, Chargé de recherche, 2ème échelon, (csaveanu@pasteur.fr)
Badis-Breard, Gwenaël, Post-doc

Lebreton, Alice, étudiante en thèse

Teixeira, Teresa, Post-doc

Torchet, Claire, Post-doc
Hantraye, Florence, Ingénieur position 1, (fricard@pasteur.fr)

Decourty, Laurence, Technicienne supérieure de laboratoire, (decourty@pasteur.fr)

Activity Reports 2003 - Institut Pasteur
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