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  Director : Jacquier Alain (jacquier@pasteur.fr)


  abstract

 

We study various aspects of RNA metabolism in the yeast S. cerevisiae (maturation, transport, and degradation). During year 2002, we particularly focused on ribosome biogenesis by studying: 1) a new small nucleolar RNA responsible for the formation of two universally conserved pseudouridines within rRNA and the role of these two pseudouridines; 2) a novel mechanism of auto-regulation of expression of a ribosomal protein that involves the degradation of its own mRNA; 3) the characterization and the assembly of pre-60S particles.



  report

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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.

In 2002, we more specifically studied several aspects related to ribosomal particle biogenesis. We can recognize three different topics connected to this theme: 1) the study of a new mechanism of post-transcriptional auto-regulation of the expression of a ribosomal protein; 2) identification of a new small nucleolar RNA (snoRNA) of the H/ACA type and targeting the modification of the only two universally conserved pseudouridines within the rRNA; 3) the characterization and study of the assembly of pre-60S particles, precursors of the large ribosomal subunit.

Identification in yeast of the small nucleolar RNA guiding de modification of the two universally conserved pseudouridines within the RNA of the large ribosomal subunit.

Ribosomal RNAs, especially eukaryotic rRNAs, contain a number of modified nucleotides. The most abundant nucleotide modifications found within rRNAs fall into two classes: 2'-O-ribose methylations and pseudouridylations. In eukaryotes, small nucleolar guide RNAs - the snoRNAs - specify the position of these modifications. The 2'-O-ribose methylations and pseudouridylations are guided by the box C/D and box H/ACA snoRNAs respectively. The role of these modifications remains poorly understood as no clear phenotype has yet been assigned to the absence of specific 2'-O-ribose methylations or pseudouridylations. We have now identified and characterised in yeast a novel intronic H/ACA snoRNA, snR191, which guides pseudouridylation at positions 2258 and 2260 in the 25S large ribosomal subunit rRNA. Most interestingly, these two modified bases are the only pseudouridines universally conserved from bacteria to human in rRNA. The corresponding human snoRNA is hU19. We have now found that, in yeast, the presence of this snoRNA is not essential for viability but provides a growth advantage to the cell. Additional experiments will be required to identify the biochemical role played by these modifications within the ribosome.

A novel mechanism of post-transcriptional auto-regulation of the expression of a ribosomal protein.

In growing yeasts, ribosome synthesis consumes an enormous amount of the resources of the cells. Ribosomal protein expression must thus be tightly regulated. In yeast, and in contrast to prokaryotes, the major part of this regulation is done at the level of transcription. Yet, several mechanisms of fine-tuning post-transcriptional regulation have been observed for few ribosomal proteins in yeast. The mechanisms employed operate at diverse steps such as splicing or translation.

Analysis of a two-hybrid network elaborated in our laboratory reveals that a protein of yet unknown function, Yel015p, is linked specifically and multiple times with cytoplasmic proteins involved in decapping and mRNA degradation (Fromont-Racine et al. (2000) Yeast, 17, 95-110). Yet, the absence of this protein does not affect the global stability of mRNAs in the cell. We thus made the hypothesis that it is involved in the degradation of specific messenger RNAs. Analysis of the transcriptome of cells lacking Yel015p with DNA micro-arrays allowed us to identify a target for this protein. It consists of the mRNA coding for a ribosomal protein. The level of this mRNA increases when Yel015p is absent and decreases when Yel015p is over-expressed. In addition, northern blot analyses show that the ribosomal protein is able to auto-regulate the level of its own mRNA, but only when Yel015p is present. The mRNA of this ribosomal protein possess a long 3'-UTR carrying a stem-loop structure conserved between several yeast species. We could show that this stem-loop is the target of the machinery involved in the auto-regulation mechanism. In addition, a two-hybrid link exists between this ribosomal protein and Yel015p. Our working hypothesis is thus that this ribosomal protein regulates the level of its own mRNA by binding to the stem-loop within its 3'UTR. The interaction of this bound protein with Yel015p would allow the recruitment of the decapping-mRNA degradation complex. In this new mechanism of auto-regulation, the polyA degradation step would be bypassed, a prediction that we have verified experimentally.

Dynamic of assembly of pre-ribosomal complexes and characterization of associated factors.

In eukaryotes, ribosome biogenesis takes place not only in the nucleolus, but also in the nucleoplasm and the cytoplasm. During maturation, pre-rRNAs are modified and cleaved into large ribonucleoparticles.

Very recently, by affinity purification and identification by mass spectrometry, we have characterized several new intermediates of ribosome maturation in yeast. We have identified about 50 new pre-ribosomal factors physically associated to these pre-60S complexes. Using conditional mutants, we purified complexes blocked at specific steps to determine the order of assembly and dissociation of the pre-ribosomal factors during pre-60S formation. In addition, by classical analyses, we study the role of some newly identified factors in the ribosome biogenesis.

Our data allowed us to establish a model in which the association of some pre-ribosomal factors to pre-60S particles is dependent upon the presence of other pre-ribosomal factors into earlier complexes.

Keywords: ribosome biogenesis, snoRNA, mRNA degradation, TAP-purification, rRNA maturation



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  LABOUISE Odile (labouise@pasteur.fr) FROMONT-RACINE Micheline, CNRS, CR2, (mfromont@pasteur.fr)

SAVEANU Cosmin , Institut Pasteur, Chargé de recherche, 2ème échelon, (csaveanu@pasteur.fr)

BADIS-BREARD Gwenaël, graduate student

LEBRETON Alice, graduate student





HANTRAYE Florence, Ingénieur position 1, (fricard@pasteur.fr)

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

LABOUISE Odile, Secrétaire, (labouise@pasteur.fr)

Activity Reports 2002 - Institut Pasteur
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