Susan J Baserga | Maria Carmo-Fonseca | Ingrid Grummt | Danièle Hernandez-Verdun |
Julian A Hiscox | Denis L J Lafontaine | Angus I Lamond | David A Matthews | Jo Milner |
Mark O J Olson| Craig S Pikaard | John J Rossi | Carlos Rubbi | Peter Shaw |
Michael Taliansky | Robert Y Tsai | Elisa Varela | Robert J White | Adrian Whitehouse |
Mark O J Olson
The Role of Phosphorylation in the Dynamics and Location of Nucleolar Proteins during the Cell Cycle
Department of Biochemistry
The University of Mississippi Medical Center
Jackson MS 39216
USA
Email: molson@biochem.umsmed.edu
Biography
Mark Olson received a B.A. in Chemistry from St. Olaf College in Minnesota and a Ph.D. in Biochemistry from the University of Minnesota. He was a post-doctoral fellow in Biochemistry at the University of Alberta in Canada. Before moving to the University of Mississippi Medical Center, where he is now Professor and Chair of the Biochemistry Department, he was a faculty member in the Pharmacology Department at Baylor College of Medicine in Houston, Texas. Mark’s current research is focused on the role of nonribosomal proteins in ribosome biogenesis and on the regulation of nucleolar structure during the cell cycle. His research has been funded by the National Institutes of Health, the National Science Foundation and various private agencies.
Abstract
The nucleolus is an exceedingly complex subnuclear body that is composed of several hundred different macromolecules. Nucleolar structure, which is governed by the cell cycle and by the synthetic requirements of the cell, is highly variable and dynamic. Ultimately, the nucleolar structure is dependent on the affinities of the nucleolar components for each other. We have studied the regulation of these affinities by examining the dynamics of nucleolar protein B23/NPM. This is an abundant multifunctional nucleolar phosphoprotein that has nucleic acid binding, ribonuclease and molecular chaperone activities. It is phosphorylated by casein kinase 2 (CK2) during interphase and by a cyclin dependant kinase 1 (cdk1) during mitosis. Previous studies suggest that CK2 phosphorylation modulates interactions with other proteins and that cdk1 phosphorylation regulates interactions with RNA. We have utilized mutations in these sites to study the effects of phosphorylation on the dynamics of B23 in the nucleolus using fluorescence recovery after photobleaching (FRAP) and also examined the locations of the protein and its mutants during the cell cycle. Our results support the idea that phosphorylation at the CK2- and the cdk1 sites reduce the affinity of protein B23 for other proteins and for RNA, respectively.
Key primary references
Huang, N., Negi, S., Szebeni, A. & Olson, M.O. (2005) Protein NPM3 interacts with the multifunctional nucleolar protein B23/nucleophosmin and inhibits ribosome biogenesis. J. Biol. Chem. 280; 5496-5502.
Szebeni, A., Hingorani, K., Negi, S. & Olson, M.O. (2003) Role of protein kinase CK2 phosphorylation in the molecular chaperone activity of nucleolar protein B23. J. Biol. Chem. 278; 9107-9115.
Dundr, M., Misteli,T. & Olson, M.O. (2000) The dynamics of postmitotic reassembly of the nucleolus. J. Cell Biol. 150; 433-446.
Dundr, M. & Olson, M.O. (1998) Partially processed pre-rRNA is preserved in association with processing components in nucleolus-derived foci during mitosis. Mol. Biol. Cell 9; 2407-2422.
Key reviews
Olson, M.O. & Dundr, M. (2005) The moving parts of the nucleolus. Histochem. Cell Biol. 123; 203-216.
Olson, M.O.J. (2004) The Nucleolus. M.O.J. Olson, Editor, Landes Bioscience, Georgetown, TX, USA.
Epigenetic mechanisms of rRNA gene silencing in nucleolar dominance.
Biology Department
Washington University
1 Brookings Drive, Saint Louis, MO 63130
USA
Email: pikaard@biology2.wustl.edu
Biography
Craig S. Pikaard earned a B.S. degree in Horticulture from the Pennsylvania State University in 1980 and a PhD in Plant Physiology from Purdue University in 1985. He then conducted postdoctoral research with Ronald Reeder at the Fred Hutchinson Cancer Research Center as an NIH fellow. Craig joined the faculty of Washington University in 1990 where he now holds the rank of professor in the Biology Department. Craig’s lab is currently interested in chromatin-mediated gene silencing. One sub-group within the lab is focused on understanding the genetic and epigenetic mechanisms responsible for nucleolar dominance. A second sub-group is focused on understanding the role of the newly discovered nuclear RNA polymerase IV in siRNA-mediated DNA methylation and heterochromatin dynamics in plants.
Abstract
In most offspring there are genes that are expressed from the chromosomes inherited from only one parent. Often a maternal or paternal imprint dictates which allele will be active. However, this is not the case for the uniparental expression of ribosomal RNA (rRNA) genes in genetic hybrids. This epigenetic phenomenon, known as nucleolar dominance, occurs both in plants and animals but is best studied in plants because non-sterile hybrids can be generated. Previous work from the lab has shown that rRNA gene silencing involves concerted changes in both DNA methylation and histone modification and supports a model whereby DNA and histone modifications are each upstream of one another in a self-reinforcing, circular pathway. The chromatin modifying activities involved in this repression cycle are being identified using transgene-induced RNA interference (RNAi) to knock down the expression of targeted genes. Two histone deacetylases, one DNA methyltransferase and several methylcytosine binding proteins have been identified in the screens thus far. A variety of genetic, cytogenetic and biochemical approaches are being employed to understand the mechanism(s) of action of these chromatin modifying activities and to understand how their actions are intertwined to comprise an epigenetic on-off switch.
Key Primary references
Lawrence, R.J., Earley, K., Pontes, O., Silva, M., Chen, Z.J., Neves, N., Viegas. W, & Pikaard C.S. (2004) A concerted DNA methylation/histone methylation switch regulates rRNA gene dosage control and nucleolar dominance. Mol. Cell 13; 599-609.
Lewis, M.S., Cheverud, J.M. & Pikaard, C.S. (2004) Evidence for NORs as the units of regulation in nucleolar dominance in Arabidopsis thaliana inter-ecotype hybrids. Genetics 167; 931-939.
Lewis, M.S. & Pikaard, C.S. (2001) Restricted chromosomal silencing in nucleolar dominance. Proc. Natl. Acad. Sci. U.S.A. 98;14536-14540.
Frieman, M., Chen, Z.J., Saez-Vasquez, J., Shen, L.A. & Pikaard C.S. (1999) RNA polymerase I transcription in a Brassica interspecific hybrid and its progenitors: tests of transcription factor involvement in nucleolar dominance. Genetics 152; 451-460.
Key Reviews
Grummt, I. & Pikaard, C.S. (2003) Epigenetic silencing of RNA polymerase I transcription. Nature Reviews Mol. Cell. Biol. 4; 641-649.
Pikaard, Craig S. (2000) The epigenetics of nucleolar dominance.Trends in Genetics 16; 495-500.
HIV and the Nucleolus
Division of Molecular Biology
Graduate School of Biological Sciences
Beckman Research Institute of the City of Hope
Duarte, CA 91010
USA
Email: jrossi@coh.org
Biography
Dr. Rossi received his doctoral training under Dr. Claire Berg in microbial genetics from the University of Connecticut at Storrs. He carried out his postdoctoral training at Brown University in the laboratory of Dr. Arthur Landy studying the gene structure and processing pathways of the E. coli tyrosyl tRNA gene clusters. From there he moved to a position in the Beckman Research Institute of the City of Hope in Durate CA, and began a research program using synthetic DNAs in the studies of RNA splicing. As an outgrowth of a long-term interest in RNA processing, his laboratory began to develop and test the idea of utilizing catalytic RNAs or ribozymes for inhibition of HIV infection. This research program has led to two clinical trials in which ribozyme genes have been transduced into hematopoietic stem cells for autologous transplant in HIV infected individuals. Work in his laboratory continues to focus upon enhancing the intracellular efficacy of ribozymes and RNA decoys via RNA trafficking and target co-localization approaches, including nucleolar localization. Work with a nucleolar localized anti-HIV ribozyme provided strong evidence for nucleolar trafficking of singly spliced and unspliced HIV RNAs. Recently, the lab has begun to explore the exciting new molecular pathway for targeted gene inhibition using RNA interference as a potential therapeutic approach for targeting HIV. Combinations of a nucleolar trafficking HIV TAR decoy with an antiviral shRNA have been developed for use in a human clinical trial of genetically modified hematopoietic stem cells in HIV infected patients. In addition to studies on the potential applications of various RNAs for disease therapy, his laboratory also studies the biochemistry and molecular mechanisms of these RNA based therapeutic agents.
Abstract
Ectopically expressed HIV-1 proteins Tat and Rev accumulate in the nucleolus of cells in which the virus can replicate. This observation prompted us to explore the possibility that HIV RNA itself can traffic through the nucleolus via its association with Tat and Rev. To investigate this possibility we constructed a hybrid U16 small nucleolar RNA/anti-HIV-1 hammerhead ribozyme under the transcriptional control of the human U6 promoter. The ribozyme target is a sequence in the 5’ UTR of HIV-1 within a highly conserved sequence. Cells expressing the chimeric U16Rz were tested for localization of the ribozyme via in situ hybridization. The ribozyme was shown to accumulate within the nucleoli of HEK 293 cells and partially co-localize with the U3 snoRNA. To further explore the possibility that HIV-1 traffic’s through the nucleolus, the chimeric U16/Rz was inserted in a retroviral vector, which was used to transduce CEM T-lymphocytes. As a control, a mutation in the catalytic core of the ribozyme was made that abrogates ribozyme function and this was also inserted into the retroviral vector and transduced into CEM cells. When cells expressing either the wild type or mutant ribozymes were challenged with HIV-1, potent inhibition of HIV antigen production was observed only by the wild type ribozyme. RNA analyses of the restricted HIV transcripts suggest that only the fully unspliced and singly spliced transcripts were downregulated. These results suggest that Rev-Crm1 interactions direct the RRE containing transcripts through the nucleolus. To further exploit these observations, we have inserted the HIV TAR binding region or the Rev Binding element into the U16 apical loop, and used these as antiviral decoys. Both strategies have proven to be successful, and one of these is currently be used for a human gene therapy trial in combination with a small hairpin RNA and ribozyme in HIV infected individuals. Further work in this area has employed the use of a nucleolar localizing ribozyme library to find both cellular and viral targets for inhibition of HIV-1. These results will be discussed as well.
Key Primary References
Michienzi, A., Cagnon, L., Bahner, I. & Rossi, J.J. (2000) Ribozyme-mediated inhibition of HIV 1 suggests nucleolar trafficking of HIV-1 RNA. Proc. Natl. Acad. Sci. U.S.A. 97; 8955-60.
Michienzi, A., Castanotto, D., Lee, N., Li, S., Zaia, J.A. and Rossi, J.J. (2003) RNA-mediated inhibition of HIV in a gene therapy setting. Ann. N. Y. Acad. Sci. 100; 63-71.
Michienzi, A., Li, S., Zaia, J.A. & Rossi, J.J. (2002) A nucleolar TAR decoy inhibitor of HIV-1 replication. Proc. Natl. Acad. Sci. U.S.A. 99; 14047-52.
Key Reviews
Michienzi, A., Castanotto, D., Lee, N., Li, S., Zaia, J.A. and Rossi J.J. (2003) RNA-mediated inhibition of HIV in a gene therapy setting. Ann. N. Y. Acad. Sci. 1002; 63-71.
|
The nucleolus as a controller YCR p53 Research Group, |
|
Biography
Carlos obtained his degree in Biochemistry at the University of La Plata, Argentina, where he also got a PhD degree in Immunology of bacterial infections. Shortly afterwards, he moved to the University of Essex, UK, to work on cell separation, acquiring experience in digital and confocal microscopy. In 1996 Carlos moved to the University of York, to work in Prof. Milner's YCR p53 Research Group. Here, he focused on applying digital microscopy to nuclear processes that involved the p53 tumour suppressor protein. Within this line of research he became interested in p53 stabilisation as well as its role in genomic instability and developed two models that comprise current research interests: the role of the nucleolus in p53 stabilisation and modulation of DNA repair processes through chromatin control of p53.
Abstract
The levels of the p53 tumour suppressor protein are controlled by ubiquitination and proteasomal degradation of the protein and determine its capacity to transactivate target genes involved in cell cycle arrest and apoptosis. We have recently demonstrated that the nucleolus participates in the regulation of the levels of. In this model the nucleolus constantly promotes degradation of p53 such that any stress that impairs nucleolar function will cause stabilisation of p53. In the present work I evaluate the relative importance of the nucleolus model for p53 stabilisation in comparison with models based on stabilisation cofactors. Heterokaryon experiments demonstrate that the stability/degradation of p53 can only be controlled by a long-lived, nucleus-associated component, which cannot translocate to the cytoplasm. Experiments with micronucleated cells unambiguously confirm that this component is the nucleolus. FRET experiments using His-tagged ubiquitin and EGFP-tagged p53 demonstrate that ubiquitinated p53 is associated with the nucleolus. However, the combination with micronucleation experiments and bition of proteasomal degradation indicates that rather than being involved in the ubiquitination step of p53, the nucleolus is more likely to participate in the export of ubiquitin-tagged p53. This export also appears to be linked to cytoplasmic degradation of p53. These results firmly establish the nucleolus as a necessary route for degradation ubiquitinated p53.
Key Primary References
Rubbi, C.P. & Milner, J (2003) Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses. EMBO J. 22; 6068-6077.
Lohrum, M.A., Ludwig, R.L., Kubbutat, M.H., Hanlon, M. & Vousden, K.H (2003) Regulation of HDM2 activity by the ribosomal protein L11. Cancer Cell 3; 577-587.
Dai, M.S., Zeng, S.X., Jin, Y., Sun, X.X., David, L. & Lu, H (2004) Ribosomal protein L23 activates p53 by inhibiting MDM2 function in response to ribosomal perturbation but not to translation inhibition. Mol. Cell. Biol. 24; 7654-7668.
Bhat, K.P., Itahana, K., Jin, A., and Zhang, Y (2004) Essential role of ribosomal protein L11 in mediating growth inhibition-induced p53 activation. EMBO J. 23; 2402-2412.
Jin, A., Itahana, K., O'Keefe, K. & Zhang, Y (2004) Inhibition of HDM2 and activation of p53 by ribosomal protein L23. Mol. Cell. Biol. 24; 7669-7680.
Zhang, Y., Wolf, G. W., Bhat, K., Jin, A., Allio, T., Burkhart, W.A., and Xiong, Y (2003) Ribosomal protein L11 negatively regulates oncoprotein MDM2 and mediates a p53-dependent ribosomal-stress checkpoint pathway. Mol. Cell. Biol. 23; 8902-8912.
|
Novel functions for the nucleolus from proteomic and RNomic Analysis of Arabidopsis nucleoli John Innes Centre |
|
Biography
Peter Shaw received a BA in Natural Sciences from Cambridge University (1972) and a PhD in Protein X-ray Crystallography from Bristol University (1975). After post-doctoral research in protein crystallography at Birmingham and Bristol Universities, he joined the scientific faculty at the John Innes Centre in 1979, initially working on high-resolution biological electron microscopy and the analysis of 2 dimensional crystalline arrays. For the past 20 years Peter’s research has centred on the functional organization of the nucleus and nucleolus and on chromosome architecture. He is currently Associate Head of the Department of Cell and Developmental Biology at the John Innes Centre, and is an honorary professor at the University of East Anglia.
Abstract
The nucleolus is the site of rDNA transcription, rRNA processing and ribosomal subunit assembly, but is also involved in a range of other RNA/RNP functions, cell cycle control and stress responses. To investigate the wider functions of the nucleolus in plants, we carried out a partial proteomic analysis of Arabidopsis nucleoli, in collaboration with John Brown’s group at SCRI, Dundee, in which we identified 217 proteins of which ca. 7% were also present in the human nucleolus. The proteins include many expected nucleolar proteins involved in ribosome biogenesis, ribosomal proteins, snoRNP core proteins, DEAD box helicases, RNA/DNA/chromatin interacting proteins, splicing and translation factors and RNA transport factors. In addition, proteins with unknown function were identified, some of which were plant specific proteins or proteins found in both the Arabidopsis and human nucleolar proteomes.
Full length cDNAs for 76 of the identified proteins were expressed as GFP-protein fusions in Arabidopsis cells. Nearly 90% of the proteins are clearly nucleolar or associated with the nucleolus. Many show labelling of other nuclear bodies. Surprisingly, the Arabidopsis nucleolar proteome contained six components of the exon junction complex (EJC): Aly/Ref, UAP56, RNPS1, Y14, Magoh and eIF4A-III. EJC Proteins link transcription and splicing with mRNA export, surveillance and decay. GFP-fusions for these and a number of other EJC proteins confirmed an association with the nucleolus. This is in contast to animal EJC proteins, which are usually excluded from the nucleolus. The nucleolar association of plant EJC proteins may reflect accumulation/ storage/ assembly of EJC components, or that spliced or aberrant mRNAs move through the nucleolus, perhaps as part of the mRNA export process or in nuclear/nucleolar surveillance and nonsense-mediated decay (NMD). Evidence for a role for the plant nucleolus in NMD has been obtained from an examination of nucleolar mRNAs.
Key Primary References
Pendle, A.F., Clark, G.P., Boon, R., Lewandowska, D., Lam, Y.W., Andersen, J., Mann, M., Lamond, A.I., Brown, J.W. & Shaw P.J. (2005) Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions. Mol. Biol. Cell. 16; 260-269.
Brown, J.W., Shaw, P.J., Shaw, P. & Marshall, D.F. (2005) Arabidopsis nucleolar protein database. (AtNoPDB). Nucleic Acids Res. 33; Database Issue, D633-636.
Koroleva, O.A., Tomlinson, M.L., Leader, D., Shaw, P. & Doonan, J.H. (2005) High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions. Plant J. 41;162-174.
Gonzalez-Melendi, P., Wells, B., Beven, A.F. & Shaw, P.J. (2001) Single ribosomal transcription units are linear, compacted Christmas trees in plant nucleoli. Plant J. 27; 223-233.
Key Reviews
Shaw, P. & Doonan, J. (2005) The Nucleolus. Playing by Different Rules?
Cell Cycle 4; 102-105.
Shaw, P.J. (2005) The Nucleolus. In: Encyclopedia of Life Sciences, Vol 2005, Chichester: John Wiley & Sons, Ltd: Chichester. http://www.els.net/ [doi:10.1038/npg.els.0003958]
Shaw, P.J. & Brown, J.W. (2004) Plant Nuclear Bodies. Curr. Opin. Plant Biol. 7; 614-620.
Involvement of the nucleolus in plant virus systemic infection
Scottish Crop Research Institute
Invergowrie
Dundee DD2 5DA
UK
Email: Michael.Taliansky@scri.ac.uk
Biography
Michael Taliansky received a Biology Diploma (equiv. of MSc.)in Biochemistry (1971) and a PhD in Virology (1976) from Moscow State University (MSU, Russia). Since then he worked at the Department of Virology of MSU as a research scientist (1975-1987) and head of the Laboratory of Molecular Virology (1984-1994). In 1994 Michael moved to the Scottish Crop Research Institute (SCRI, Dundee) where he works as a Principal Scientist (Band IMP3). The focus of his research is on structure and functions of plant viruses (virus assembly, transport, interaction with a plant host cell). Recently Michael has made significant advances in a new area of plant molecular virology: nucleolar targeting as a novel function of plant viruses.
Abstract
The umbravirus ORF3 protein is involved in long-distance movement of viral RNA via the phloem in the form of RNP particles. In addition to the cytoplasm, where the ORF3 protein of umbravirus, Groundnut rosette virus (GRV), forms RNP particles, this protein also accumulates in the nucleus preferentially targeting the nucleolus. The aim of this work is to investigate links between nucleolar functions and long-distance virus movement. At present there is virtually no information on this aspect of virology.
The umbraviral ORF3 protein contains two conserved domains one of which includes an R-rich sequence and another, which contains invariant L residues. Both these domains were involved in the localization of the ORF3 protein to the nucleolus. The L-rich domain also functioned as a nuclear export signal, suggesting that the ORF3 protein shuttles between the nucleus/nucleolus and cytoplasm. Functional analysis of ORF3 protein mutants revealed a correlation between the ORF3 protein nucleolar localization and its ability to form the RNP particles and transport viral RNA long distances. It was also shown that the ORF3 protein interacts with a nucleolar protein, fibrillarin, re-distributing it from the nucleolus to cytoplasm. Data on a role of these interactions in the assembly of movement–competent umbravirus RNP particles will be presented. Functional implications of the nucleolar involvement in umbravirus biology that may also apply to other viruses will be discussed.
Key primary references
Ryabov, E.V., Robinson, D.J. & Taliansky, M.E. (1999) A plant virus-encoded protein facilitates long-distance movement of heterologous viral RNA.Proc. Natl. Acad. Sci. U.S.A.96; 1212-1217.
Taliansky, M., Roberts, I.M., Kalinina, N., Ryabov, E.V., Raj, S.K., Robinson, D.J. & Oparka, K.J. (2003) An umbraviral protein, involved in long-distance RNA movement, binds viral RNA and forms unique, protective ribonucleoprotein complexes. J. Virol. 77; 3031-3040.
Kim, S.H., Ryabov, E.V., Brown, J.W.S. & Taliansky, M. (2004) Involvement of the nucleolus in plant virus systemic infection. Biochemical Society Transactions,32; 557-560.
Ryabov, E.V., Kim, S.H. & Taliansky M. (2004) Identification of nuclear localisation signal and nuclear export signal of the umbraviral long-distance RNA movement protein. J. Gen. Virol. 85; 1329-1333.
Haupt, S., Stroganova, T., Ryabov, E.V., Kim, S.H., Fraser, G., Duncan, G., Mayo, M., Barker, H. & Taliansky, M. (2005) Nucleolar localisation of Potato leafroll virus capsid proteins.J. Gen. Virol. 86; 2891-2896.
Key review
Taliansky, M.E. & Robinson, D.J. (2003) Molecular biology of umbraviruses: phantom warriors. J. Gen. Virol. 84; 1951-1960.
|
Nucleostemin: a nucleolar mechanism controlling stem cell proliferation and protecting against ageing Texas A&M Health Science Centre, IBT |
|
Biography
Robert Tsai received an M.D. from the National Taiwan University in 1988 and a Ph.D. (Neuroscience) from the Johns Hopkins University School of Medicine in 1996. He finished his Neurology residency training at The National Taiwan University Hospital and a post-doctoral training at National Institutes of Health with Ronald McKay. Robert is currently an Assistant Professor at the Texas A and M University Health Science Center, Institute of Biosciences and Technology. The focus of Robert’s research is on the mechanism of stem cell self-renewal, mediated by nucleostemin, and its roles in tumorigenesis and ageing. His research is principally funded by the National Institutes of Health and TIRR/Mission Connect.
Abstract
Nucleostemin (NS) encodes a nucleolar GTP-binding protein highly enriched in the cancer and stem cells [1, 2]. To determine its biological activity in vivo, we generated an NS loss-of-function mouse model using the gene targeting approach. We showed that the embryogenesis of homozygous NS-null (NS-/-) mice was aborted before the blastula stage, consistent with its high expression in embryonic stem cells. Although the growth and fertility of heterozygous NS+/- mice appeared normal in the early generations, mouse embryonic fibroblast (MEF) cells derived from these animals had less NS protein, slower population growth rates and more senescent cells than their wild-type litter mates. Conversely, transgenic overexpression of NS could prolong the mitotic window of MEF cells, reduce the percentage of senescent cells, and rescue the NS-/- mice. After four generations of heterozygous intercrosses, both the NS+/- and wild-type offspring began to develop an early hair-loss phenotype. This work demonstrates the roles of NS in establishing early embryogenesis, and preventing premature ageing in MEF cells and mice.
Key Primary References
Tsai, R.Y. & McKay, R.D. (2002) A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells. Genes Dev. 16; 2991-3003.
Tsai, R.Y. & McKay. R.D. (2005) A multistep, GTP-driven mechanism controlling the dynamic cycling of nucleostemin. J.Cell Biol. 168; 179-184
Condensin and nucleolar dynamics through mitosis
Friedrich Miescher Institute for Biomedical Research
Novartis Research Foundation
Gasser Group
Maulbeerstrasse 66
4058 Basel
Switzerland
Email: elisa.varela@fmi.ch
Biography
Elisa Varela obtained her Ph D. in microbiology on Dec. 1988, at the “Universidad Autonoma” University of Madrid, Spain. She had postdoctoral training in the Department of Biochemistry and Molecular Biology, in Pennsylvania State University, (Pennsylvania, USA). In 2004 she joined the group led by Dr. S. M. Gasser, as a postdoctoral fellow funded by the Spanish Government and the Swiss Cancer League, at the Friedrich Miescher Institute for Biomedical Research (Basel, Switzerland).
Abstract
In S. cerevisiae the nucleolus is a single nuclear compartment, found adjacent to the nuclear envelope and opposite to the spindle pole body, during interphase. It contains the tandemly repeated ribosomal DNA array located in the right arm of chromosome XII. The interphase nuclear architecture is modified during mitosis and has to be reestablished after telophase, when chromosomes have segregated to the opposite poles of daughter cells. The rDNA array, is the last piece of the genome to segregate and Condensin Complex is required for it, as well as for the compaction, resolution of tangles and separation of the array. The end of mitosis is orchestrated by the mitotic exit network, a kinase protein cascade that causes release of Cdc14 phosphatase from the nucleolus, promoting entry into G1. At this stage, release of Condensin from the rDNA array allows decondensation to occur. We are interested in the mechanism that controls the release of Condensin from the rDNA array at the end of mitosis and how these changes in chromatin compaction influence the re-establishment of proper G1 nuclear architecture.
Key References
Bystricky, K., Laroche T., Van Houwe, G., Blaszczyk M. & Gasser, S.M. (2005) Chromosome looping in yeast: telomere pairing and coordinated movement reflect anchoring efficiency and territorial organization. J. Cell Biol. 168; 375-387.
Bystricky, K., Heun, P., Gehlen, L., Langowski, J. & Gasser, S.M. (2004) Long-range compaction and flexibility of interphase chromatin in budding yeast analyzed by high-resolution imaging techniques. Proc. Natl. Acad. Sci. U.S.A., 101; 16495-16500.
Gotta, M., Strahl-Bolsinger, S., Renauld, H., Laroche, T., Kennedy, B.K., Grunstein M. & Gasser, S.M. (1997) Localization of Sir2p: the nucleolus as a compartment for silent information regulators. EMBO J. 16; 3243-3255.
Laroche, T., Martin, S.G., Tsai-Pflugfelder, M. and Gasser, S.M. (2000)The dynamics of yeast telomeres and silencing proteins through the cell cycle. J. Struct. Biol. 129; 159-174.
Key Reviews
Gasser, S.M., Hediger, F., Taddei, A., Neumann, F.R. & Gartenberg, M.R. (2004) The function of telomere clustering in yeast: the circe effect. Cold Spring Harb Symp. Quant Biol. 69; 327-37.
Taddei, A., Hediger, F., Neumann, F.R. & Gasser, S.M. (2004) The function of nuclear architecture: a genetic approach. Anu. Rev. Genet. 38; 305-345.
RNA polymerase III transcription, cell growth and cancer
Institute of Biomedical and Life Sciences
University of Glasgow, Glasgow, G12 8QQ.
& Beatson Institute for Cancer Research,
Bearsden, Glasgow, G61 1BD.
UK
Email: rwhite@udcf.gla.ac.uk
Biography
Having graduated with first class honours from Oxford University, Bob White worked with Peter Rigby at the National Institute for Medical Research, London, investigating transcription by RNA polymerase (pol) III. He received his PhD in 1990 and continued to study pol III transcription at the Wellcome/CRC Institute, Cambridge, with Stephen Jackson. In 1995, Bob set up his own lab at Glasgow University’s Institute of Biomedical and Life Sciences. He received the Jenner Research Fellowship in 1996 from the Lister Institute of Preventive Medicine and the Young Scientist Award in 1999 from the British Association for Cancer Research. At the age of 35, he became Professor of Gene Transcription at Glasgow University. In 2003 he received the Young Cancer Researcher Award from the European Association for Cancer Research and in 2004 he was awarded the Tenovus Medal and elected a Fellow of the Royal Society of Edinburgh. He became a Fellow of the Academy of Medical Sciences in 2005. His lab has demonstrated many unexpected links between pol III and key growth-controllers that can explain the hyperactivity of pol III transcription in cancer cells. In 2006/7, Bob’s lab will move to the Beatson Institute for Cancer Research.
Abstract
Transcription by pol III is abnormally active in transformed cells. Our work aims to characterize the mechanisms responsible for this deregulation. The tumour suppressor RB inhibits pol III transcription by binding and inactivating TFIIIB, a factor that recruits pol III onto promoters. RB function is compromised in cancers through mutation of the Rb gene, hyperphosphorylation by cyclin-dependent kinases or binding of oncoproteins; each of these mechanisms can derepress TFIIIB and increase pol III activity. TFIIIB is also bound and repressed by p53 in healthy cells. Mutations in p53 contribute to the deregulation of pol III transcription in some cancers and in Li-Fraumeni syndrome. As well as being regulated by these tumour suppressors, TFIIIB is also bound and activated by several oncogenic proteins, including c-Myc, CK2 and the Erk MAP kinases. TFIIIB therefore lies at the centre of a complex regulatory network of conflicting influences. The fact that it is targeted directly by oncoproteins and tumour suppressors provides a clear indication of the importance of controlling pol III output. In support of this, pol III-specific transcription factors are frequently overexpressed in human tumours. For example, the DNA-binding factor TFIIIC, which recruits TFIIIB to promoters, is produced at high levels in ovarian carcinomas. HPV16-infected cervical carcinomas overexpress Brf1, a specific subunit of TFIIIB. Remarkably, Brf1 overexpression can be sufficient to accelerate cell growth and proliferation. Clearly, there is strong selective pressure to raise pol III output during tumour development; this can be achieved through a range of molecular mechanisms and may have dramatic effects on proliferative capacity.
Key primary references
White, R.J., Trouche, D., Martin, K., Jackson, S.P. & Kouzarides, T. (1996) Repression of RNA polymerase III transcription by the retinoblastoma protein. Nature 382; 88-90.
Cairns, C.A. & White, R.J. (1998) p53 is a general repressor of RNA polymerase III transcription. EMBO J. 17; 3112-3123.
Winter, A.G., Sourvinos, G., Allison, S.J., Tosh, K., Scott, P.H., Spandidos, D.A. & White, R.J. (2000) RNA polymerase III transcription factor TFIIIC2 is overexpressed in ovarian tumours. Proc. Natl. Acad. Sci. U.S.A. 97; 12619-12624.
Gomez-Roman, N., Grandori, C., Eisenman, R.N. & White, R.J. (2003) Direct activation of RNA polymerase III transcription by c-Myc. Nature 421; 290-294.
Grandori, C., Gomez-Roman, N., Felton-Edkins, Z.A., Ngouenet, C., Galloway, D.A., Eisenman, R.N. & White, R.J. (2005) c-Myc binds to human ribosomal DNA and stimulates transcription of rRNA genes by RNA polymerase I. Nature Cell Biol. 7; 311-318.
Key review
White, R.J. (2005) RNA polymerases I and III, growth control and cancer. Nature Rev. Mol. Cell. Biol. 6; 69-78.
Role of the nucleolus in herpesvirus mRNA export
Institute of Molecular and Cellular Biology
University of Leeds
Leeds
LS2 9JT
UK
Email: a.whitehouse@leeds.ac.uk
Biography
Adrian Whitehouse obtained his BSc from the University of Sheffield in 1991 and his D.Phil in Molecular Virology from the University of Oxford in 1994. Following postdoctoral work at the Molecular Medicine Unit, St James’s Hospital in Leeds he was awarded a Medical Research Council Non-clinical Fellowship in 1998, and joined the former School of Biochemistry & Molecular Biology, University of Leeds, as a Lecturer in 2002. He was appointed to Reader in Molecular Virology in 2005. The focus of Adrian’s research is investigating the virus-host cell interactions, which regulate gamma-2 herpesvirus replication.
Abstract
ORF 57 is a RNA-binding, nucleocytoplasmic shutting protein that functions to export intronless viral mRNAs during herpesvirus lytic-replication. ORF 57 localises to spliceosomes and the nucleolus. In order to determine the functional significance of ORF 57’s nucleolar targeting a series of ORF 57 deletion mutants were constructed. Our data demonstrate that ORF 57 contains two distinct nuclear localisation signals (NLS) and that either of these NLS was sufficient for nuclear localisation of ORF 57. However, loss of either NLS was enough to prevent localisation of ORF 57 to the nucleolus. In order to determine if a loss of nucleolar localisation affected ORF 57-mediated mRNA export, a viral mRNA export assay was performed. Results demonstrated that ORF 57 mutants, which localised to the nucleus but not the nucleolus were unable export viral mRNA. This suggests a possible role for the nucleolus in viral mRNA export.
References
Williams, B., Boyne, J.R., Goodwin, D.J., Roaden, L.R., Wilson, S.A. & Whitehouse, A. (2005) The prototype gamma-2 herpesvirus nucleocytoplasmic shuttle protein, ORF 57, transports viral RNA via the cellular mRNA export pathway. Biochemical J. 87; 295-308.
Goodwin, D.J. & Whitehouse, A. (2001) A g-2 herpesvirus nucleocytoplasmic shuttle protein interacts with importin a-1 and importin a-5. J. Biol. Chem. 276; 19905-19912.
Goodwin, D.J., Hall, K.T., Stevenson, A.J., Markham, A.F. & Whitehouse, A. (1999); The ORF 57 gene product of Herpes virus saimiri shuttles between the nucleus and cytoplasm and is involved in viral RNA nuclear export. J. Virol. 73; 10519-10524.