Unit: Macromolecular Interaction Genetics

Director: Jacquier Alain

We study various aspects of RNA metabolism in the yeast Saccharomyces cerevisiae that we use as an eukaryotic model. During year 2004, we particularly focused on: 1) the complete set of H/ACA snoRNAs that guide rRNA pseudouridylations in Saccharomyces cerevisiae, 2) the study of the maturation and export of ribosomal particles and, in particular, the assembly of the pre-60S complexes.

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 characterize in more details the role of these proteins.

Exhaustive search of the H/ACA snoRNAs that guide rRNA pseudouridylations in Saccharomyces cerevisiae.

Conversion of uridines into pseudouridines (Ψs) is the most frequent base modification in ribosomal RNAs (rRNAs). In eukaryotes, the pseudouridylation sites are specified by base pairing with specific target sequences within H/ACA small nucleolar RNAs (snoRNAs). The yeast rRNAs harbour 44 Ψs, but, when this work begun, 15 Ψs had completely unknown guide snoRNAs. This suggested that many snoRNAs remained to be discovered. To address this problem and further complete the snoRNA assignment to Ψ sites, we identified the complete set of RNAs associated with the H/ACA snoRNP specific proteins Gar1p and Nhp2p by coupling TAP-tag purifications with genomic DNA microarrays experiments. Surprisingly, while we identified all the previously known H/ACA snoRNAs, we selected only three new snoRNAs. This suggested that most of the missing Ψ guides were present in previously known snoRNAs but had been overlooked. We confirmed this hypothesis by systematically investigating the role of previously known, as well as of the newly identified snoRNAs, in specifying rRNA Ψ sites and found all but one missing guide RNAs. During the completion of this work, another study, based on bioinformatic predictions, also reported the identification of most missing guide RNAs. Altogether, all Ψ guides are now identified and we can tell that, in budding yeast, the 44 Ψs are guided by 28 snoRNAs. Finally, aside from snR30, an atypical small RNA of heterogeneous length and three ORF encoded RNAs, all Gar1p and Nhp2p associated RNAs characterized by our work turned out to be snoRNAs involved in rRNA Ψ specification.

Assembly of preribosomal complexes

Ribosome biogenesis is a highly complex and co-ordinated process that occurs not only in the nucleolus but also in the nucleoplasm and the cytoplasm of eukaryotic cells. We have recently characterised the protein composition of several ribosomal subunit precursors involved in the 60S biogenesis, in yeast . An almost complete set of stably associated pre-ribosomal factors is now available (see for review Fromont-Racine et al., 2003).

To gain insights in the mechanisms involved in the dynamic of ribosome assembly, (i) we characterised the successive complexes (proteomic approach) and (ii) we determined the role of some key factors (functional analyses).

(i) We purified preribosomal complexes by two successive affinity purifications (TAP), using preribosomal factors as baits. However, taking account that a given factor is involved in several preribosomal complexes, we used mutant strains that accumulate stalled complexes to purify these as homogenously as possible. To compare the composition of these various complexes, we use SILAC, a quantitative approach based on mass spectrometry. In a first study, we identified early and late groups of preribosomal factors relative to the stage at which the Nog1 protein functions. We currently refine this study using various mutant strains involved in different steps all along of the pre-60S maturation pathway.

(ii) We characterize in more details the role of several proteins such as: 1) Mak11, an early factor transiently associated to the pre-60S and present in a small complex; 2) Nsa2, involved in the central steps of the pre-60S biogenesis and the cellular amount of which seems highly regulated; 3) Ybr267w (Rei1), which is committed to late cytoplasmic steps and seems involved in recycling of other factors via specific karyopherins.

Jacquier Alain

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

Activity Reports 2004 - Institut Pasteur

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