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 2005, we particularly focused on: 1) the exhaustive identification of H/ACA snoRNAs that guide each rRNA pseudouridylations in Saccharomyces cerevisiae, 2) the study of late maturation and export events during 60S ribosomal particle maturation, 3) the description of new cryptic intergenic transcripts rapidly degraded by a nuclear machinery that involves a new poly(A)-polymerase.
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 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 identification of the H/ACA snoRNAs that guide rRNA pseudouridylations in Saccharomyces cerevisiae.
Conversion of uridines into pseudouridines (Ys) 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 Y s, but, when this work begun, 15 Ys 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 Y 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 Y 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 Y 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 Y guides are now identified and we can tell that, in budding yeast, the 44 Y s 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 Y specification (Torchet et al., 2005). This work was done in collaboration with Frédérique Devaux at ENS Paris.
Nuclear RNA degradation and control of genetic expression
The fraction of the genome that is expressed is generally considered to parallel the complexity of the transcriptome. In this report we show that several supposedly silent intergenic regions in the genome of S. cerevisiae are actually transcribed by RNA polymerase II, suggesting that the expressed fraction of the genome is higher than anticipated. Surprisingly, however, RNAs originating from these regions are rapidly degraded by the combined action of the exosome and (reminiscent of what is observed in bacteria) a new poly(A) polymerase activity that is defined by the Trf4 protein and one of two RNA-binding proteins, Air1p and Air2p. We show that such polyadenylation-assisted degradation mechanism is also responsible for the degradation of several Pol I and Pol III transcripts. Our data strongly support the existence of a post-transcriptional quality control mechanism limiting inappropriate expression of genetic information (Wyers et al., 2005; La Cava et al., 2005). This work was performed in collaboration with the groups of Domenico Libri and Bertrand Séraphin at the CNRS, Gif-sur-Yvette and the group of David Tollervey at the University of Edinburgh.
Assembly of preribosomal complexes
In 2005, we pursued the characterisation of the protein composition of several pre-60S complexes in yeast. We used quantitative approaches, based on mass spectrometry, such as SILAC and iTRAQ, in order to determine the dynamics of ribosome assembly.
In 2005, we particularly focused these analyses on late pre-60S maturation events. We have characterized a new pre-60S cytoplasmic factor, Ybr267w/Rei1, involved in these late maturation events. The absence of this factor affects the reimport in the nucleus of at least three shuttling factors: Arx1, Tif6 and a new factor, Yjl122w/Alb1. In the absence of Rei1, the cytoplasmic accumulation of a small complex containing Arx1 and Alb1 inhibits the dissociation of Tif6 from the preribosomal particles and its recycling to the nucleus. Tif6 has previously been described as a factor inhibiting the association between the small and large ribosomal subunits. We thus propose a model in which Rei1 coordinates the dissociation and recycling of the last pre-60S factors, allowing the entry of the newly synthesized mature large subunits in translation (Lebreton et al., 2006).
Keywords: ARN, Saccharomyces cerevisiae, RNA degradation, non-coding RNAs, ribosome maturation