Dynamic nuclear organization

We have projects for undergraduates, masters students, PhD students and post doctoral fellows in the following areas :

Study of intra-nuclear degradation of pre-rRNA (C. Dez) :

The production of ribosomes, responsible for protein synthesis, is clearly of paramount importance to any cell. Ribosome biogenesis consumes a vast amount of the resources in rapidly growing cells. It requires up to 80% of the total energy and represents about 95% of transcriptional activity (Moss and Stefanovsky, 2002). A surveillance pathway that eliminates defective pre-ribosomal particles was recently identified (Dez et al., 2006). This process involves the addition of poly(A) tails to the targeted rRNAs moieties of the aberrant pre-ribosomes by the TRAMP complex (Dez et al., 2006; Kadaba et al., 2004; Lacava et al., 2005; Vanacova et al., 2005; Wyers et al., 2005) prior to exosome-dependent degradation. A specific nucleolar substructure was unraveled, in which surveillance is likely to take place, and designated this region the “No-Body” (Dez et al., 2006; Houseley and Tollervey, 2006). Using quantitative mass spectrometry (SILAC and iTRAQ) and synthetic lethality screens, we will focus on large-scale identification of putative “sensor factors”, responsible for rapid detection and targeting of aberrant (and potentially deleterious) pre-ribosomes to the degradation machinery. The second step will be to characterize the function of each of these candidates in the surveillance process. We will precisely define if these factors are involved in the detection of aberrant pre-ribosomal particles, their targeting for degradation and/or their addressing to the No-Body where degradation is supposed to take place.

Dynamic nuclear organization: the functional relationship between transcription of genes encoding ribosomal components and chromatin dynamics (O. Gadal; C; Normand)

Epigenetic control of gene expression, understood as heritable change in transcription occurring without changes in nucleotide sequence, is the subject of intense research. One level of this epigenetic regulation involves the information encoded by the three-dimensional distributions of genes within a given nuclear volume (Gilbert et al., 2004). Spatial positioning of genes is correlated with regulation of transcription (Cremer and Cremer, 2001). In addition, recent research has shown that active genes dynamically colocalize to shared transcription sites (Osborne et al., 2004). However, how specific gene positioning is achieved and what nuclear components affect the transcriptional state are still open questions (Misteli, 2004).

In this project, we will investigate how the Pol I machinery can orchestrate the transcription of genes encoding ribosomal components and translational machineries, and how this regulation is linked to spatial positioning. Starting from genetic screens, we will identify components of the Pol I machinery functionally connected with the expression of other ribosomal components. Using quantitative analysis of genes position labeled with fluorescent tags, we will characterize in vivo position and dynamics of genes involved in ribosome biogenesis and of the translational machineries. We will study chromatin dynamics in mutant backgrounds that affect the co-regulation of ribosomal components. Finally, we will characterize the function of the factors affecting chromatin dynamics using biochemical tools available in S.cerevisiae.

Study of rRNA synthesis by the Miller spreading method (I. Leger-Sylvestre) :

Electron microscopy visualization of active rRNA genes by the Miller spreading method typically shows genes heavily packed with polymerases from which lateral fibres (nascent ribonucleoproteins RNP) extend. The fibres gradually increase in length within each of the transcript unit leading to the well-known "Christmas tree" configuration [Figure 2]. The gradient of fibres length represents a "time series" in the maturation of the nascent transcripts. I. Léger-Silvestre succeeded to establish the visualisation of individual spread yeast rRNA genes by electron microscopy and is establishing immunolocalisation of tagged factors in these spread genes.

Figure 2 : The yeast rRNA gene repeat unit (adapted from French S et al., MCB, 2003).
A. Schematic of four tandem rRNA gene repeats
B. Schematic of one gene spacer unit, including the Pol I-transcribed 35S rRNA gene (long thick line), the Pol III-transcribed 5S rRNA gene (short thick line), and nontranscribed spacers (thin lines).
C. One transcription unit observed in electron microscopy (unpublished data, Toulouse).

References :

Cremer, T., and Cremer, C. (2001). Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nat Rev Genet 2, 292-301.
Dez, C., Houseley, J., and Tollervey, D. (2006). Surveillance of nuclear-restricted pre-ribosomes within a distinct sub-nucleolar region of S.cerevisiae. Embo J 25, 1534-1546.
French, S. L., Osheim, Y. N., Cioci, F., Nomura, M., and Beyer, A. L. (2003). In exponentially growing saccharomyces cerevisiae cells, rRNA synthesis Is determined by the summed RNA polymerase I loading rate rather than by the number of active genes. Mol Cell Biol 23, 1558-1568.
Gilbert, N., Boyle, S., Fiegler, H., Woodfine, K., Carter, N. P., and Bickmore, W. A. (2004). Chromatin architecture of the human genome: gene-rich domains are enriched in open chromatin fibers. Cell 118, 555-566.
Houseley, J., and Tollervey, D. (2006). Yeast Trf5p is a nuclear poly(A) polymerase. EMBO Rep 7, 205-211.
Kadaba, S., Krueger, A., Trice, T., Krecic, A. M., Hinnebusch, A. G., and Anderson, J. (2004). Nuclear surveillance and degradation of hypomodified initiator tRNAMet in S. cerevisiae. Genes Dev.
Lacava, J., Houseley, J., Saveanu, C., Petfalski, E., Thompson, E., Jacquier, A., and Tollervey, D. (2005). RNA degradation by the exosome is promoted by a nuclear polyadenylation complex. Cell 121, 713-724.
Misteli, T. (2004). Spatial positioning; a new dimension in genome function. Cell 119, 153-156.
Moss, T., and Stefanovsky, V. Y. (2002). At the center of eukaryotic life. Cell 109, 545-548.
Osborne, C. S., Chakalova, L., Brown, K. E., Carter, D., Horton, A., Debrand, E., Goyenechea, B., Mitchell, J. A., Lopes, S., Reik, W., and Fraser, P. (2004). Active genes dynamically colocalize to shared sites of ongoing transcription. Nat Genet 36, 1065-1071.
Vanacova, S., Wolf, J., Martin, G., Blank, D., Dettwiler, S., Friedlein, A., Langen, H., Keith, G., and Keller, W. (2005). A new yeast poly(A) polymerase complex involved in RNA quality control. PLoS Biol 3, e189.
Wyers, F., Rougemaille, M., Badis, G., Rousselle, J. C., Dufour, M. E., Boulay, J., Regnault, B., Devaux, F., Namane, A., Seraphin, B., et al. (2005). Cryptic pol II transcripts are degraded by a nuclear quality control pathway involving a new poly(A) polymerase. Cell 121, 725-737.