Table Of ContentProgress in Molecular and Subcellular Biology
35
Series Editors:W.E.G.Müller (Managing Editor),Ph.Jeanteur,
I.Kostovic,Y.Kuchino,A.Macieira-Coelho,R.E.Rhoads
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Protein Degradation in Health Sponges (Porifera)
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V. Matranga (Ed.)
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Regulation of Alternative Splicing Antifouling Compounds
Ph. Jeanteur (Ed.) N. Fusetani and A. Clare (Eds.)
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W.E.G. Müller (Ed.)
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RNA Trafficking and Nuclear Structure
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Ph. Jeanteur (Ed.)
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Ph. Jeanteur (Ed.)
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Alternative Splicing and Disease
Ph. Jeanteur (Ed.)
Philippe Jeanteur (Ed.)
RNA Trafficking
and Nuclear Structure
Dynamics
With 28 Figures,8 in Color
123
Professor Dr.PHILIPPEJEANTEUR
Institute of Molecular Genetics
of Montpellier
BP 5051
1919 Route de Mende
34293 Montpellier Codex 05
France
ISSN 0079-6484
ISBN 978-3-540-74265-4 e-ISBN 978-3-540-74266-1
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Preface
Recent years have witnessed an extraordinary explosion in our knowledge of
the dynamics ofnuclear structure.The constantly growing interest in the mol-
ecular aspects of RNA trafficking has received a dramatic stimulus from the
synergistic boost oftwo rapidly developing technologies over recent years.On
the one hand,the use of nontoxic fluorescent proteins (GFP,green fluorescent
protein and its derivatives) has allowed the visualization ofmoving molecules
in living cells due to computerized 3-D cell imaging techniques.These,in com-
bination with FRAP (fluorescence recovery after photobleaching) methods,
make it possible to follow the dynamic interactions between individual mole-
cules.On the other hand,the development of proteomics has led to the mole-
cular identification of proteins in motion within the nucleus.
In the face ofthe wealth ofnew observations (and reviews thereof) dealing
with nuclear structure,it appeared timely to narrow the scope ofthis book and
to restrict it to dynamic aspects,as they involve or are of relevance to RNA.It
came with the realization that nuclear structure is much more dynamic than
previously anticipated; this is probably the major general message of this
volume.
The first three chapters deal with the structural organization of different
subnuclear compartments. Nuclear compartments, unlike those in the cyto-
plasm,are not delimited by surrounding membranes.The chapter by Platani
and Lamond surveys the chromatin compartment, the nucleolus and
perinucleolar compartment, Cajal bodies and gems, the speckles containing
splicing factors, as well as the PML bodies characteristic of promyelocytic
leukemia.
The second chapter by Raˇska concentrates on the structure – function
relationship of the nucleolus, in a search for active ribosomal genes. He
first reviews the results obtained by a combination of electron microscopy,
cytochemistry and immunocytochemistry approaches to refine nucleolar
structure. He then goes on with the mapping of its DNA and RNA moie-
ties by in situ hybridization together with its protein components by
immunocytochemistry.
The next chapter by Nykamp and Swanson touches on RNA-mediated
pathogenesis by presenting an original view ofdiseases involving trinucleotide
expansion.They argue that,in addition to the protein being altered by the pres-
ence of unusual glutamic acid repeats,RNA can have an intrinsic toxic effect
due to the (CUG)n expansions themselves.
VI Preface
The next four chapters impinge on more dynamic aspects of RNA traffick-
ing.Bertrand and Bordonné concentrate on small nuclear and nucleolar RNPs
(snRNPs and snoRNPs) with special emphasis on the latter.They draw atten-
tion to similarities in their biogenesis and argue for the possibility of a
common origin between snoRNAs and snRNAs.
The other chapters focus on mRNA trafficking toward the nuclear pore.
Kiesler and Visa further the monumental work of Daneholt’s lab,using as a
model system the Balbiani rings of Chironomus tentans.They track BR RNP
particles from the gene to the nuclear pore,arguing for a free diffusion process
with transient interactions with nonchromatin nucleoplasmic structures.They
also discuss the functional significance of nuclear retention of mRNA. The
paper by Braga,Rino and Carmo-Fonseca reaches the same conclusion about
a passive diffusion mechanism for nuclear mRNPs,but also suggests the pos-
sibility of additional energy-dependent reactions.
Finally,Fusco,Bertrand and Singer review the latest technologies for live cell
imaging of mRNA which were pioneered in Singer’s lab and have already
proven so useful.
Obviously,this blooming field is still in its infancy and has yet to yield its
full harvest of new mechanisms and concepts.
PHILIPPEJEANTEUR
Contents
Nuclear Organisation and Subnuclear Bodies
Melpomeni Platani and Angus I.Lamond
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2 Chromatin and Nuclear Compartments . . . . . . . . . . . . . . . . . . . 3
3 The Nucleolus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
4 Perinucleolar Compartment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5 Cajal Bodies,gems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
6 Speckles – Nuclear Bodies Enriched in Splicing Factors . . . . . . 9
7 Promyelocytic Leukemia Bodies . . . . . . . . . . . . . . . . . . . . . . . . . 12
8 Other Forms of Nuclear Bodies . . . . . . . . . . . . . . . . . . . . . . . . . . 13
9 Conclusion,Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Searching for Active Ribosomal Genes
Ivan Raˇska
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2 Historical Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3 The Ups and Downs ofAffinity Cytochemistry:
the Case of the Nucleolus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4 Mapping ofActive Ribosomal Genes . . . . . . . . . . . . . . . . . . . . . 33
4.1 Electron Microscopy and Cytochemistry Approaches . . . . . . . . 35
4.2 Immunocytochemical Mapping of Nucleolar
Chromatin Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4.3 In Situ Hybridization Mapping of rDNA . . . . . . . . . . . . . . . . . . 37
4.4 In Situ Hybridization Mapping of rRNA . . . . . . . . . . . . . . . . . . . 38
4.5 Immunocytochemical and In Situ Hybridization Mapping
of Nucleolar Proteins and Ribonucleoproteins . . . . . . . . . . . . . . 40
4.6 Mapping of the Nucleolar Transcription Signal . . . . . . . . . . . . . 41
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Toxic RNA in the Nucleus:Unstable Microsatellite
Expression in Neuromuscular Disease
Keith R.Nykamp and Maurice S.Swanson
1 Overvi.e .w . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
VIII Contents
2 Unstable Microsatellites in Neurological
and Neuromuscular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2.1 Microsatellites:Definition and Potential Functions . . . . . . . . . . 58
2.2 Coding Region Expansions and Potential Effects
on Transcriptional Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2.3 Myotonic Dystrophy and RNA-Mediated Pathogenesis . . . . . . . 61
3 RNA-Mediated Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.1 Nuclear RNA Foci in Myotonic Dystrophy . . . . . . . . . . . . . . . . . 63
3.2 (CUG) RNA Structures and Gain-of-Function Models . . . . . . . 65
n
3.3 Guilt by Association:Proteins that Bind CUG
and CCUG Repeat Expansions . . . . . . . . . . . . . . . . . . . . . . . . . . 66
3.4 Cellular Functions Affected by (CUG) Expansion . . . . . . . . . . . 69
n
3.5 All Together Now:Loss of Myogenic Differentiation
in Congenital DM1 Requires (CUG) Expansion
n
and the DMPK3¢-UTR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
4 Unstable Microsatellites,Toxic RNA and Implications
for Nuclear Structure and Function . . . . . . . . . . . . . . . . . . . . . . 72
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Assembly and Traffic ofSmall Nuclear RNPs
Edouard Bertrand and Rémy Bordonné
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
2 Biogenesis of Small Nuclear RNPs . . . . . . . . . . . . . . . . . . . . . . . 79
2.1 U1,U2,U4 and U5 snRNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
2.2 U6 snRNP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
2.3 U7 snRNP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
3 Biogenesis of Small Nucleolar RNPs (snoRNPs) . . . . . . . . . . . . . 85
3.1 Principal Features and Functions of snoRNA Families . . . . . . . 85
3.2 snoRNA Synthesis and Genomic Organization . . . . . . . . . . . . . . 87
3.3 Trafficking of snoRNA and snoRNP Proteins . . . . . . . . . . . . . . . 88
4 Noncanonical Types of Box C/D and Box H/ACA RNAs . . . . . . 89
5 Similarities Between snRNA and snoRNA Biogenesis . . . . . . . . 90
6 snRNAs and snoRNAs:a Common Origin? . . . . . . . . . . . . . . . . 91
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Intranuclear Pre-mRNA Trafficking in an Insect Model System
Eva Kiesler and Neus Visa
1 The Balbiani Ring Genes of Chironomus tentans:
a Model System for Studying Gene Expression in Situ . . . . . . . . 99
1.1 The Active Balbiani Ring Genes . . . . . . . . . . . . . . . . . . . . . . . . . 100
1.1.1 Processing of BR pre-mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
1.2 Proteins Associated with the BR (pre-)mRNA . . . . . . . . . . . . . . 102
1.2.1 DExD/H Box Proteins in the BR RNP Particle . . . . . . . . . . . . . . 104
2 The Movement of the (pre)mRNP Particles
from the Gene to the NPC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Contents IX
2.1 Three-Dimensional-Analysis of Nucleoplasmic BR RNP
Particles by Electron Tomography . . . . . . . . . . . . . . . . . . . . . . . 106
2.2 Protein Components of CFs and FGCs . . . . . . . . . . . . . . . . . . . . 107
2.2.1 Hrp65 and Other DBHS Proteins . . . . . . . . . . . . . . . . . . . . . . . . 108
2.2.2 The hrp65 Isoforms:Specialized Roles in Gene Expression? . . . 109
2.3 Intranuclear Movement of (pre-)mRNPs . . . . . . . . . . . . . . . . . . 109
2.4 Nuclear Retention of mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
2.5 CFs and FGCs in Mammalian Cells? . . . . . . . . . . . . . . . . . . . . . . 112
3 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Photobleaching Microscopy Reveals the Dynamics
ofmRNA-Binding Proteins Inside Live Cell Nuclei
José Braga,José Rino and Maria Carmo-Fonseca
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
2 General Concepts of Quantitative FRAP . . . . . . . . . . . . . . . . . . . 120
3 FRAP Reveals the Dynamics of GFP-PABP2 and GFP-TAP
in the Living Cell Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
4 Estimating the Fraction of GFP Fusion Proteins Bound
to mRNP Complexes by FLIP . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
5 Simulation of mRNP Mobility Within the Nucleus Using
a Random Walk Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
6 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Imaging ofSingle mRNAs in the Cytoplasm ofLiving Cells
Dahlene Fusco,Edouard Bertrand and Robert H.Singer
1 Rationale for Live Cell Imaging of mRNA . . . . . . . . . . . . . . . . . 135
2 Existing Technologies for the Visualization of RNA
in Living Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
2.1 Microinjection of Fluorescent RNA . . . . . . . . . . . . . . . . . . . . . . . 137
2.2 Fluorescent Oligonucleotide Probes (FIVH-Fluorescent
In Vivo Hybridization) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
2.3 Cell Permeant Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
2.4 RNA Tagged with Sequence-Specific GFP Fusion Proteins . . . . . 139
3 Analysis of Single RNA Dynamics in Living Cells . . . . . . . . . . . 140
4 Time-Lapse Microscopy/Particle Tracking . . . . . . . . . . . . . . . . . 141
5 In Vivo mRNA Analyses in the Cytoplasm . . . . . . . . . . . . . . . . . 142
6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Nuclear Organisation and Subnuclear Bodies
Melpomeni Platani1 and Angus I. Lamond1
1
Introduction
The cell nucleus, described by Franz Bauer in 1802, was one of the first intra-
cellular structures to be identified by early microscopists. The nucleus is sur-
rounded by a double membrane, of which the outer membrane is continuous
with the endoplasmic reticulum, and this serves to separate the sites of gene
transcription from the sites of mRNA translation in the cytoplasm. Transport
of molecules between the nuclear and cytoplasmic compartments of the cell
occurs via specific protein complexes located in the nuclear envelope, termed
nuclear pore complexes (NPCs, see Fig. 1).
The nucleus contains the genomic information of the cell and is also the site
of major metabolic activities, including DNA synthesis, transcription, pre-
mRNA splicing and ribosome subunit synthesis and assembly. The genome is
organised in chromosomes that are packaged into large-scale domains, termed
“chromosome territories”, which occupy distinct regions of the nucleus (Fig. 1;
Cremer et al. 1982; Cremer and Cremer 1988, 2001). It is now recognised that
much of the internal structure of the nucleus, like the cytoplasm, is highly
compartmentalised. As discussed below, multiple classes of subnuclear
organelles, or nuclear bodies (NBs), are formed that act as compartments for
specific groups of nuclear factors. A major difference between nuclear and
cytoplasmic organelles, however, is that compartments in the nucleus are
formed without surrounding membranes. The localisation of nuclear factors
in NBs can serve several functions, including enhancing the efficiency of reac-
tions by creating a high local concentration of requisite factors involved in a
common process, particularly when multiple activities must act upon a single
substrate, as is the case during ribosome subunit synthesis. NBs can also
partition specific factors, control access of enzymes and receptors to their
substrates and may play a role in intranuclear transport and in the control of
gene expression.
In this review we will discuss the organisation of the cell nucleus and, in
particular, consider the structure, composition and properties of some of the
1Wellcome Trust Biocentre, MSI/WTB Complex, DD1 5EH, Dundee, Scotland, United Kingdom.
Tel. +44-1382-345473; Fax. +44-1382-345695, e-mail: [email protected]
Progress in Molecular and Subcellular Biology
P. Jeanteur (Ed.): RNA Trafficking and Nuclear Structure Dynamics
© Springer-Verlag Berlin Heidelberg 2003
