Maria Carmo-Fonseca | Ingrid Grummt | Julian Hiscox | David A. Matthews |Mark O. J . Olson
Craig S. Pikaard | John J. Rossi | Michael Taliansky |Elisa Varela | Robert J.White | Adrian Whitehouse
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 | Michael Taliansky | Elisa Varela |
Carlos Rubbi | Peter Shaw | Robert Tsai | Robert J White | Adrian Whitehouse |
The nucleolus and RNA
editing
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de
Lisboa
Av. Prof. Egas Moniz 1649-028 Lisboa, Portugal
E-mail: carmo.fonseca@fm.ul.pt
Biography
Maria Carmo-Fonseca obtained her MD
in 1983 and the PhD in Cell Biology in 1988. Since 1998, she is Professor at
the Faculty of Medicine, University of Lisbon. Carmo-Fonseca did a post-doc at
EMBL (Heidelberg, Germany) from 1989 to 1992. Since 2002 she serves as
Executive Director of the Institute of Molecular Medicine in Lisbon, a national
center of excellence for biomedical research. Carmo-Fonseca is also head of the
Cell Biology Department at this Institute. The focus of Carmo’s research is the regulation of gene expression
within the sophisticated environment that is the nucleus of a living cell. The
group is particularly interested in understanding how mRNA biogenesis is
regulated and how errors in this process lead to human disease.
Abstract
A defining feature of eukaryotic cells
is the generation of protein diversity either post-transcriptionally by
alternative splicing and RNA editing or post-translationally by modification of
amino acids in proteins. One of the most recently discovered post-translational
modification mechanism in eukaryotes involves the covalent attachment of the
small ubiquitin-like modifier, SUMO, to target proteins. We have recently
described that proteins modified by SUMO-1 are present in the nucleolus, that
SUMO-1 in the nucleolus co-localizes with the RNA editing enzyme ADAR1 and that
this enzyme represents a novel substrate for sumoylation. ADAR1 (adenosine
deaminase that acts on RNA) is a member of the family of enzymes that catalyse
the conversion of adenosine to inosine in double-stranded RNA. In mammals there
are three ADAR enzymes, termed ADAR1, ADAR2, and ADAR3. Inactivation
of editing enzymes in mice and in the fruit fly has resulted in profound
neurological phenotypes. Several lines of evidence suggest that ADAR activity is
tightly controlled in the cell. In our work we demonstrate that ADAR1 is
modified by SUMO at lysine residue 418. Substitution of this amino acid residue
by arginine, which cannot be modified by SUMO, affects the editing activity of
the enzyme. Our finding that ADAR1 co-localizes with SUMO-1 in the nucleolus
and that sumoylation of ADAR1 reduces editing activity suggests that the
nucleolus represents a “sink” for inactive ADAR1 in the cell.
Key primary references
Desterro, J., Keegan, L., Lafarga,
M., Berciano, M.T., O’Connell, M., and Carmo-Fonseca, M. (2003) Dynamic
association of RNA editing enzymes with the nucleolus. J. Cell Sci. 116:
1805-1818.
Desterro JM, Keegan LP, Jaffray E,
Hay RT, O’Connell MA, Carmo-Fonseca M (2005) SUMO-1 Modification Alters ADAR1
Editing Activity. Mol. Biol. Cell 16: 5115-5126.
Key review
Carmo-Fonseca, M., Mendes-Soares,
L., and Campos, I. (2000) To be or not to be in the nucleolus. Nature Cell
Biology 2, E107-E112.
Regulation of ribosomal RNA gene
transcription
Molecular Biology
of the Cell II,
German Cancer
Research Center,
Im Neuenheimer
Feld 280
D-69120
Heidelberg, Germany
Email: I.Grummt@DKFZ-Heidelberg.de
Biography.
Ingrid Grummt received her PhD in 1970 at the Humboldt-University in Berlin, worked as a Postdoc at the German Academy of Sciences in
Berlin-Buch and the Max-Planck-Institute of
Biochemistry in Munich, led a research group since 1977, and is Head of a
Research Division at
the German Cancer Research Center in Heidelberg. Her
research focuses on the molecular mechanisms that regulate RNA polymerase I
transcription.
Abstract
Regulation of rRNA synthesis is a key factor that controls cell growth
and balances the effects of growth factors to prevent uncontrolled cell
proliferation. The key factor that directs growth-dependent regulation of rDNA
transcription is TIF-IA, a transcription factor whose activity fluctuates in
response to cell growth and serves a key function in adapting cellular
biosynthetic activities to cell growth. We have inactivated the murine TIF-IA gene by homologous
recombination in mice and embryonic fibroblasts (MEFs). TIF-IA-/-
embryos die before/at embryonic day 9.5, displaying retardation of growth and
development. In MEFs, Cre-mediated depletion of TIF-IA leads to disruption of nucleoli, cell cycle arrest, upregulation of
p53, and induction of apoptosis. The striking correlation between perturbation
of nucleolar function, elevated levels of p53 and induction of cell suicide
supports the view that the nucleolus is a stress sensor that regulates p53
activity.
In addition, we study rDNA regulation at the epigenetic level. Ribosomal
genes exist in two distinct types of chromatin, an “open” one that is
permissive to transcription and a “closed” one that is transcriptionally
refractive. In exploring the mechanisms that establish and propagate the active
and silent state of rRNA genes, we have identified and functionally
characterized a chromatin remodeling complex, termed NoRC, that mediates rRNA
gene silencing. NoRC recruits DNA methyltransferase and histone deacetylase to
the rDNA promoter, thereby triggering heterochromatin formation and silencing
of rDNA transcription. Heterochromatin formation appears to be mediated through
interaction of TIP5, the large subunit of NoRC, with 100-250 nucleotide RNAs
that match the rDNA promoter sequence. Mutations that abrogate RNA binding of
TIP5 impair the association of NoRC with rDNA and fail to induce
heterochromatin formation. Knockdown of IGS transcripts abolishes the nucleolar
localization of NoRC, decreases DNA methylation and enhances rDNA
transcription. The results reveal an important contribution of small IGS
transcripts in alterations of chromatin structure and epigenetic control of the
rDNA locus.
Key primary references
Li,
Y., Santoro, R., Grummt, I.: The chromatin remodeling complex NoRC controls
replication timing of ribosomal RNA genes. EMBO J. (2005) 24, 120-127.
Mayer,
C., Bierhoff, H., Grummt, I.: The nucleolus as a stress sensor: JNK inactivates
the transcription factor TIF-IA and down-regulates rRNA synthesis.
Genes&Dev. (2005) 19, 933-941.
Yuan,
X., Zhou, Y., Casanova, E., Chai, M., Kiss, E., Gröne, H.-J., Schütz, G.,
Grummt, I.: Genetic inactivation of the transcription factor TIF-IA leads to
nucleolar disruption, cell cycle arrest and p53-mediated apoptosis. Mol. Cell.
(2005) 19, 77-89.
Zhou,
Y., Grummt, I.: The PHD finger/bromodomain of NoRC
interacts with acetylated histone H4K16 and is sufficient for rDNA silencing.
Current Biol. (2005) 15, 1434-1438.
Key reviews
Grummt,
I. Pikaard, C.S.: Epigenetic control of ribosomal RNA gene transcription. Nature
Review Mol. Cell. Biol. (2003) 4, 641-649.
Grummt,
I.: Life on a planet of its own: regulation of RNA polymerase I transcription
in the nucleolus. Genes&Dev. (2003) 17, 1691-1702.
Mayer,
C, Grummt, I.: Cellular stress and nucleolar function. Cell Cycle (2005) 4,
1036-1038.
Nucleolar assembly and the cell
cycle.
Nuclei and
cell cycle, Institut Jacques Monod,
CNRS,
Université Paris VI and Paris VII,
2 place
Jussieu, 75251 Paris Cedex 05, France.
E-mail :
dhernand@ccr.jussieu.fr
Biography.
Danièle Hernandez-Verdun received a
BSc in Biology and a PhD in Development and Reproduction from Faculty of
Science, Paris (1974). She spent a
post-doctoral training period in the Karolinska Institutet Stockholm, Sweden.
She obtained a permanent research position in the CNRS on a mouse developmental
project in Paris, and then moved to a new group and has been interested in
nuclear, mostly nucleolar organization. Joining the Cell Biology Department of
the IJM in 1988, she developed a research team focussed on the nucleolus during
the cell cycle. She used successively many cell biology approaches with
presently a particular interest for living cell investigations. She was
president of the French Society of Electron Microscopy. The research funding is
mainly from the CNRS and from a Cancer Research grant (ARC).
Abstract.
One of the fundamental features of
nuclear organization is that many components of the RNA synthesis and
processing machineries are organized in compartments. This implies that the
recruitment of dedicated machineries and formation of discrete nuclear domains
are crucial events at the beginning of interphase. The nucleolus is the first
active domain to be assembled after mitosis. Its functions depend on
recruitment of the nucleolar machineries involved in processing of ribosomal
RNAs (rRNA) as well as rDNA transcription activation. During mitosis, the RNA polymerase
I transcription machinery is assembled on ribosomal genes and repressed by the
CDK1-cyclin B pathway. The proteins involved in rRNA processing are distributed
around the chromosomes. New results concerning the translocation of processing
machinery on transcription sites will be presented. We examined the pathway of
the rRNA processing machinery in living cells, and the role of the prenucleolar
bodies (PNBs), on the migration of the machinery from the chromosome periphery
to sites of rDNA transcription. Protein
interactions along the recruitment pathway were investigated by time-lapse
analysis of fluorescence resonance energy transfer (FRET). Interestingly
interactions between processing proteins occurred first in PNBs before being
recruited in nucleoli. The dynamics of these interactions suggest that PNBs are
pre-assembly platforms for rRNA processing complexes.
Key primary references.
Angelier
N., Tramier M., Louvet E., Coppey-Moisan M., Savino T. M., De Mey J. R. and
Hernandez-Verdun D (2005). Tracking the interactions of rRNA processing
proteins during nucleolar assembly in living cells. Mol. Biol. Cell, 16, 2862-71.
Sirri V., Hernandez-Verdun D. and Roussel P. (2002). Cyclin-dependent kinases govern formation and
maintenance of the nucleolus. J. Cell Biol. 156, 969-981.
Savino T. M., Gébrane-Younès
J., De Mey J., Sibarita, J-B
and Hernandez-Verdun D. (2001). Nucleolar
assembly of the rRNA processing machinery in living cells. J. Cell Biol 153, 1097-1110.
Verheggen C., Almouzni G. and Hernandez-Verdun D. (2000).
The ribosomal RNA processing machinery is recruited to the nucleolar domain
before RNA polymerase I during Xenopus laevis. development. J. Cell Biol. 149, 293-305.
Key reviews.
Hernandez-Verdun
D., Roussel P., Gébranne-Younès J. (2002). Emerging concepts of nucleolar
assembly. J. Cell Sci. 115, 2265-2270.
Hernandez-Verdun D. and
Roussel P. (2003) Regulators of nucleolar functions. Progress in Cell Cycle
Research, Vol 5, 301-308.
Eds : Meijer L., Jézéquel A. and Roberge M.
Trafficking of viral proteins to
the nucleolus.
Institute of
Cellular and Molecular Biology,
Astbury Centre
for Structural Molecular Biology,
University of
Leeds, Leeds, LS2 9JT, UK.
E-mail:
j.a.hiscox@leeds.ac.uk
Biography.
Julian A. Hiscox received a BSc in
Genetics from University College London (1991) and a PhD in Microbiology from
the University of Reading (1994).
He conducted post-doctoral training in the Department of Microbiology
University of Alabama at Birmingham (USA) and the Division of Molecular Biology
at the Institute for Animal Health (Compton, UK). Julian is a Senior Lecturer in Virology at the University of
Leeds; prior to joining the faculty at Leeds in 2003 he was a Lecturer in Virology
at the University of Reading. The
focus of Julian’s research is on the coronavirus nucleocapsid protein, both in
terms of trafficking to the nucleolus, affect on the host cell cycle and
biophysical characterisation.
His research is principally funded by the Biotechnology and Biological
Sciences Research Council (BBSRC).
Abstract.
The nucleolus is a dynamic
sub-nuclear structure involved in ribosome biogenesis, RNA processing, control
of cell growth and mediating responses to cell stress and is therefore crucial
to the normal operation of the eukaryotic cell. In order to traffic to the nucleolus proteins must usually
contain a targeting motif.
However, unlike cellular nuclear localisation signals there is no
obvious consensus sequence for the nucleolar targeting/retention of proteins to
the nucleolus. Studying these
signals can be problematic as many nucleolar retention motifs are part of
nuclear localisation signals and in some cases nuclear export signals. Utilising the coronavirus nucleocapsid
(N) protein we have been investigating nucleolar localisation with the avian
and severe acute respiratory (SARS) coronavirus N proteins and the subsequent
consequences for the host cell function and virus replication. We have delineated nucleolar and nuclear
targeting and also nuclear export signals in the N protein and have mapped an
eight amino acid arginine rich motif which can direct an exogenous protein to
the nucleolus. Molecular modelling
of the N protein nucleolar retention signal revealed that this motif is
available for interaction with cellular factors which may mediate nucleolar
localisation. Our data suggests
that not only is the primary sequence of a nucleolar localisation signal
important for its function but also the context with regard to the three
dimensional structure of the protein.
Key primary references.
Chen, H., Gill, A., Dove, B. K., Emmett, S. R.,
Kemp, F. C., Ritchie, M. A., Dee, M. & Hiscox, J. A. (2005). Mass
spectroscopic characterisation of the coronavirus infectious bronchitis virus
nucleoprotein and elucidation of the role of phosphorylation in RNA binding
using surface plasmon resonance. Journal of Virology 79, 1164-1179.
Chen, H.,
Wurm, T., Britton, P., Brooks, G. & Hiscox, J. A. (2002). Interaction of
the coronavirus nucleoprotein with nucleolar antigens and the host cell. Journal
of Virology 76, 5233-5250.
Wurm, T.,
Chen, H., Britton, P., Brooks, G. & Hiscox, J. A. (2001). Localisation to
the nucleolus is a common feature of coronavirus nucleoproteins and the protein
may disrupt host cell division. Journal of Virology 75, 9345-9356.
You,
J.-H., Dove, B. K., Enjuanes, L., DeDiego, M. L., Alvarez, E., Howell, G.,
Heinen, P., Zambon, M. & Hiscox, J. A. (2005). Sub-cellular localisation of
the severe acute respiratory syndrome coronavirus nucleocapsid protein. Journal
of General Virology 86, 3303-3310.
Key reviews.
Hiscox,
J. A. (2002). Brief review: The nucleolus - a gateway to viral infection? Archives
of Virology 147, 1077-1089.
Hiscox,
J. A. (2003). The interaction of animal cytoplasmic RNA viruses with the
nucleus to facilitate replication. Virus Research 95, 13-22.
The role of the nucleolus in
viral infection
Department of
Cellular and Molecular Medicine
University of
Bristol
Bristol
BS8 1TD
UK
d.a.matthews@bristol.ac.uk
Biography.
Dr
Matthews received a B.Sc. in Microbiology and Virology from the University of
Warwick, a M.Sc. in Biological Sciences by Research from the University of
Warwick and a PhD in Biochemistry from the University of St Andrews. He
initially worked on the molecular epidemiology of respiratory syncytial virus
at Warwick University before moving on to adenovirus research at St Andrews for
his PhD. His post-doctoral training was originally on adenovirus gene therapy
vector development at McMaster University (Canada) and later on adenovirus-host
cell interactions at the University of Leeds where he secured a personal
fellowship from the Medical Research Council. Dr Matthews is currently a
Lecturer in Virology at the University of Bristol where the focus of his
research is on adenovirus interactions with the host cell. In particular the
work focuses on interactions with components of the nucleolus and with the
nuclear import machinery. His research has been primarily funded by the Medical
Research Council and the Wellcome Trust.
Abstract.
During
infection with adenovirus, rRNA processing is disrupted and eventually rRNA
synthesis is down regulated. In addition, major nucleolar antigens are
redistributed within the infected nucleus. We have been using this viral system
to ask where the nucleolar antigens are localised relative to the viral
replication proteins and what role they might play during a viral infection. In
addition we are interested in which viral proteins are targeted to the
nucleolus and for what purpose. So far we have identified three viral proteins
that are targeted to the nucleolus and established the molecular basis of this
phenomenon. We have also shown that at least one is capable of disrupting the
nucleolus. In addition we have been examining the potential for nucleolar
antigens to play a role in the replication of viral DNA.
References
Lee,
T.W.R, Lawrence, F.J., Dauksaite, V. Blair, G.E., Akusjärvi, G. & Matthews,
D.A. (2004).
Precursor
of human adenovirus core polypeptide Mu targets the nucleolus and modulates the
expression of E2 proteins. J Gen Virol. 85, 185-196.
Lee T.W.R., Blair, G.E. & Matthews, D.A.
(2003) Adenovirus core protein VII contains distinct sequences that mediate
targeting to the nucleus, nucleolus, and colocalisation with human chromosomes.
J Gen Virol. 84, 3423-3428.
Matthews,
D. A.
(2001). Adenovirus protein V induces redistribution of nucleolin and B23 from
nucleolus to cytoplasm. J Virol 75, 1031-8.
Matthews,
D. A.
& Russell, W. C. (1998). Adenovirus core protein V is delivered by the
invading virus to the nucleus of the infected cell and later in infection is
associated with nucleoli. J Gen Virol 79, 1671-5.
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
E-mail:
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. Journal of
Biological Chemistry 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. Journal of Biological Chemistry 278, 9107-9115.
Dundr,M., Misteli,T., Olson,M.O.J.
(2000). The dynamics of postmitotic reassembly of the nucleolus. Journal of
Cell Biology 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.
The Nucleolus, M.O.J. Olson, Editor, Landes
Bioscience, Georgetown, TX, 2004.
Epigenetic mechanisms of rRNA
gene silencing in nucleolar dominance.
Biology Department,
Washington University
1 Brookings
Drive, Saint Louis, MO 63130
USA, e-mail:
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 RJ, Earley K, Pontes O,
Silva M, Chen ZJ, Neves N, Viegas W, Pikaard CS (2004). A concerted DNA
methylation/histone methylation switch regulates rRNA gene dosage control and
nucleolar dominance. Molecular Cell 13:599-609.
Lewis MS, Cheverud JM, Pikaard CS
(2004). Evidence for NORs as the units of regulation in nucleolar dominance in Arabidopsis
thaliana
inter-ecotype hybrids. Genetics 167:931-939.
Lewis, Michelle S. and Craig S.
Pikaard. Restricted chromosomal silencing in nucleolar dominance (2001). Proc.
Natl. Acad. Sci. USA 98:14536-14540
Frieman, Matthew, Z. Jeffrey Chen,
Julio Saez-Vasquez, L. Annie Shen, and Craig S. Pikaard (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 CS (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
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
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., L. Cagnon, I. Bahner, and J. J.
Rossi.
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., D. Castanotto, N. Lee, S. Li, J. A. Zaia, and J. J.
Rossi. 2003. RNA-mediated inhibition of HIV in a gene therapy
setting. Ann N Y Acad Sci 1002:63-71.
Michienzi, A., S. Li, J. A. Zaia, and J. J. Rossi. 2002. A nucleolar TAR
decoy inhibitor of HIV-1 replication. Proc Natl Acad Sci USA 99:14047-52.
Key
Reviews:
Michienzi, A., D.
Castanotto, N. Lee, S. Li, J. A. Zaia, and J. J. Rossi. 2003. RNA-mediated
inhibition of HIV in a gene therapy setting. Ann N Y Acad Sci 1002:63-71.
Involvement of the nucleolus in
plant virus systemic infection.
Scottish Crop
Research Institute, Invergowrie,
Dundee, DD2
5DA, UK
E-mail: 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., and Taliansky M.E. (1999). A plant virus-encoded protein
facilitates long-distance movement of heterologous viral RNA. Proceedings of
the National Academy of Sciences of USA
96, 1212-1217.
Taliansky,
M, Roberts, I.M., Kalinina, N., Ryabov, E.V., Raj, S.K., Robinson, D.J., and
Oparka, K.J. (2003). An umbraviral protein, involved in long-distance RNA
movement, binds viral RNA and forms unique, protective ribonucleoprotein
complexes. Journal of Virology 77, 3031-3040.
Kim,
S.H., Ryabov, E.V., Brown, J.W.S. and Taliansky, M. (2004). Involvement of the nucleolus in plant virus systemic
infection. Biochemical Society
Transactions, 32, 557-560.
Ryabov, E.V., Kim,
S.H. and Taliansky M. (2004). Identification of nuclear localisation signal and
nuclear export signal of the umbraviral long-distance RNA movement protein. Journal
of General Virology, 85, 1329-1333.
Haupt,S.,
Stroganova, T., Ryabov, E.V., Kim, S.H., Fraser, G., Duncan, G., Mayo, M., Barker, H. and Taliansky, M. (2005). Nucleolar
localisation of Potato leafroll virus capsid proteins. Journal of General Virology, 86, 2891-2896.
Key review.
Taliansky,
M. E. & Robinson, D. J. (2003). Molecular biology of umbraviruses: phantom
warriors. Journal of General Virology, 84, 1951-1960.
Condensin
and nucleolar dynamics through mitosis
Friedrich
Miescher Institute for Biomedical Research
Novartis
Research Foundation
Gasser Group
Maulbeerstrasse
66
4058 Basel,
Switzerland
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 reestablishment of proper G1 nuclear
architecture.
Key
References
Bystricky K., Laroche T., Van Houwe
G., Blaszczyk M. and Gasser S. M. (2005).Chromosome looping in yeast: telomere
pairing and coordinated movement reflect anchoring efficiency and territorial
organization. Journal of Cell Biology 168, 375-387.
Bystricky K., Heun P. Gehlen L.
Langowski J. and 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. USA, 101: 16495-16500.
Gotta M., Strahl-Bolsinger S.,
Renauld H., Laroche T., Kennedy BK., Grunstein M. and 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.
Angela Taddei, Florance Hediger,
Frank R. Neumann, and Susan M. Gasser (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
and
Beatson Institute for Cancer Research
Bearsden, Glasgow, G61 1BD, U.K.
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. and Kouzarides, T. (1996).
Repression of RNA polymerase III transcription by the retinoblastoma protein. Nature 382, 88-90.
Cairns,
C. A. and 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. and
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. and 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. and 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 Journal, 387,
295-308.
Goodwin, D.J. & Whitehouse, A. (2001). A g