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Experimental Cell Research 292 (2004) 312–321

Ultrastructural characterisation of a nuclear domain highly enriched in

survival of motor neuron (SMN) protein

Manuela Malatesta,a,b Catia Scassellati,a Gunter Meister,c Oliver Plottner,c Dirk Buhler,c

Gabriele Sowa,c Terence E. Martin,d Eva Keidel,c Utz Fischer,c and Stanislav Fakana,*

aCentre of Electron Microscopy, University of Lausanne, 1005 Lausanne, Switzerlandb1nstitute of Histology and Laboratory Analyses, University of Urbino, 61029 Urbino, Italy

cMax-Planck-Institute for Biochemistry, 82152 Martinsried, GermanydDepartment of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA

Received 5 February 2003, revised version received 20 August 2003

Abstract

Mutations in the survival of motor neuron (SMN) gene are the major cause of spinal muscular atrophy (SMA). The SMN gene encodes a

38-kDa protein that localises in the cytoplasm and in nuclear bodies termed Gemini of coiled bodies (gems). When visualised by

immunofluorescence microscopy, gems often appeared either in close proximity to, or entirely overlapping with coiled (Cajal) bodies (CBs)

implying a possible functional relationship between these nuclear domains. With the aim of identifying subnuclear compartments

corresponding to gems, we have investigated the intranuclear localisation of SMN and of its interacting protein Gemin2 by immunoelectron

microscopy in cultured cells and in liver cells of hibernating dormouse. These antigens are highly enriched in round-shaped electron-dense

fibro-granular clusters (EFGCs), which also display a biochemical composition similar to gems visualised by immunofluorescence

microscopy. Our data reveal a novel SMN/Gemin2 containing nuclear domain and support the idea that it represents the structural counterpart

of gems seen in the light microscope.

D 2003 Elsevier Inc. All rights reserved.

Keywords: SMN protein; Gems; Subnuclear domains; Immunoelectron microscopy

Introduction

Spinal muscular atrophy (SMA) is a common autosomal

recessive disease characterised by degeneration of motor

neurons in the spinal cord (Ref. [1] and references therein).

SMA is caused by mutations in the survival of motor neuron

(SMN) gene that is duplicated in a 500-kb inverted repeat on

chromosome 5p13 [2]. The primary transcript of the telo-

meric copy encodes full-length SMN. Reduced levels of the

full-length SMN protein causes apoptotic cell death of

motor neurons in the spinal cord that consequently leads

to SMA.

The SMN gene encodes a 294-amino-acid protein that

forms high molecular weight complexes (SMN-complexes)

in the nucleus and the cytoplasm [3–5]. Functional studies

0014-4827/$ - see front matter D 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.yexcr.2003.08.022

* Corresponding author. Centre of Electron Microscopy, University of

Lausanne, 27 Bugnon, CH-1005 Lausanne, Switzerland. Fax: +41-21-692-

5055.

E-mail address: sfakan@cme.unil.ch (S. Fakan).

revealed a critical role for SMN (as part of a macromolec-

ular complex) during the assembly of spliceosomal snRNPs

U1, U2, U4 and U5 [5–9]. During this process, the nuclear-

encoded m7G-capped U snRNA is transiently transferred to

the cytoplasm, where the common Sm proteins B, BV, D1,D2, D3, E, F and G are stored. Hereafter, the Sm proteins

assemble onto a U snRNA and form the Sm core domain.

This domain is common to spliceosomal U snRNPs, and

may serve as a structural framework for the fully assembled

particles. Subsequent to Sm core assembly, the cap structure

of U snRNAs is hypermethylated to the m3G-cap. The

assembled and modified U snRNP particle is then trans-

ported to the nucleus, where it functions in pre-mRNA

splicing (Refs. [10,11] and references therein). Apart from

its function in the cytosol, SMN may also be involved in

specific nuclear activities. Thus, biochemical and genetic

studies indicated that SMN might act as a pre-mRNA

splicing factor [4,7,12]. SMN may also function in tran-

scription as it interacts with the transcription factors and can

stimulate transcription in vivo [13,14].

M. Malatesta et al. / Experimental Cell Research 292 (2004) 312–321 313

SMN and other components of SMN-complexes, such as

Gemin2, dpl03/Gemin3 and Gemin4/GIPl exhibit a charac-

teristic intracellular localisation as determined by immuno-

fluorescence microscopy. These proteins show a general

cytoplasmic distribution but are also highly concentrated in

a nuclear structure termed gems (gemini of coiled (Cajal)

bodies (CBs)) [15–17] since, in HeLa cells, gems are often

found in association with CBs, although they do not contain

p80-coilin [15]. Moreover, gems and CBs respond in a very

similar manner to metabolic changes and during the cell

cycle [3]. This indicated that both types of nuclear bodies

may be related in structure and/or function. The initial

studies clearly indicated that gems represent a novel type

of nuclear domain [15], although this domain has not been

well characterised up to this time. SMN protein was later

also localised in CBs [18–20]; however, the functional

relationships of gems and CBs with regard to intranuclear

pathways of SMN remain unclear.

In this study we analysed the subnuclear distribution of

SMN by immunoelectron microscopy, identifying a novel

type of nuclear domain highly enriched in this protein,

termed electron-dense fibro-granular clusters (EFGCs).

Moreover, to better characterise such structural constituent,

we analysed its content of several nuclear factors involved

in pre-mRNA processing. Our data strongly support that this

nuclear domain is equivalent to gems visualised by immu-

nofluorescence microscopy.

Fig. 1. Western blot analysis of HeLa whole cell extract with anti-SMN

monoclonal antibody 7B10 (lane 1) and affinity-purified anti-Gemin2

antiserum (lane 2).

Materials and methods

Cells and tissues

HeLa and 3T3 cells were grown as monolayers in

DMEM supplemented with 2 mM L-glutamine, 100 IU/ml

penicillin, 100 mg/ml streptomycin and 5% or 10% heat-

inactivated foetal calf serum (Life Technologies), respec-

tively. Cultures took place in an incubator at 37jC under a

5% CO2, atmosphere. Samples of liver were obtained from

euthermic and hibernating hazel dormice (Muscardinus

avellanarius). This experimental model was chosen since

previous studies showed that hepatocyte nuclei contain a

large number of CBs and other unusual structural constitu-

ents during hibernation [21]. Euthermic dormice were

anaesthetised with ether and sacrificed by decapitation,

while dormant animals were taken from the cage and

immediately sacrificed. This dormouse is protected by law

and only few individuals could be employed upon permis-

sion from local authorities.

Western blotting

HeLa whole cell extracts were separated on a 12% SDS–

polyacrylamide gel and transferred to Hybond C-Nitrocel-

lulose (Amersham-Pharmacia, Germany) using a standard

blot apparatus. Blots were incubated in blocking solution

(Tris-buffered saline (TBS), 5% non-fat milk) for 1 h at

room temperature and then incubated with the anti-SMN

monoclonal antibody 7B10 directed against the N terminus

of the SMN protein [4] or with the affinity-purified antise-

rum against recombinant human Gemin2 protein, at a

1:1000 dilution, for 1 h, at room temperature. After exten-

sive washing with TBS containing 0.1% Tween 20, blots

were incubated with horseradish peroxidase-coupled anti-

rabbit IgG or anti-mouse IgG (Sigma-Aldrich, USA) for 1

h at room temperature. Blots were subsequently washed

three times in TBS containing 0.1% Tween 20. Protein

bands were visualised by enhanced chemoluminescence

ECL (Amersham-Pharmacia, Germany).

Immunofluorescence microscopy

Cells were fixed either in 3.7% paraformaldehyde in

PBS, 5 min, permeabilized with Triton X-100 (0.2%, 10

min) and washed three times in PBS, or were fixed and

permeabilized in 100% methanol (� 20jC) for 2 min,

followed by three washing steps with phosphate-buffered

saline (PBS, pH 7.4). Primary and secondary antibody

incubations were performed for 60 min at room temperature

in blocking buffer containing PBS with 3% bovine serum

albumin (Boehringer Mannheim). The primary antibodies

were a mouse monoclonal anti-SMN antibody (7B10, used

at a 1:2000 dilution), a mouse monoclonal anti-SMN anti-

body (2B1, BD Transduction Laboratories) and a mouse

monoclonal anti-Coilin antibody (56, BD Transduction

Fig. 2. Localisation of SMN by immunofluorescence. HeLa cells were stained by anti-SMN antibody 2B1 (B), anti-SMN 7B10 (A, C and G) and anti-coilin (D

and H). Double labelling experiments are shown in E and I (7B10 and anti-coilin). Arrows indicate CBs not labelled with 7B10 in image D, and coilin-negative

structures enriched in SMN in image G. Phase-contrast images of the analysed cells are shown in F and K. Scale bar, 5 Am.

M. Malatesta et al. / Experimental Cell Research 292 (2004) 312–321314

M. Malatesta et al. / Experimental Cell Research 292 (2004) 312–321 315

Laboratories, both used at a 1:l000 dilution). Before immu-

nofluorescence, the anti-SMN 7B10 was covalently coupled

to fluorescein-5-isothiocyanate (FITC, Molecular Probes).

The rhodamine-conjugated anti-mouse secondary antibody

was used at 1:1000 dilutions (Alexa Fluor 546 goat anti-

mouse IgG conjugate, Molecular Probes). Images were

captured using a Microview camera (Princeton Instruments)

on a Zeiss Axioskop 2.

Immunoelectron microscopy

Cell monolayers were fixed in situ with 4% paraformal-

dehyde in 0.1 M Sorensen phosphate buffer (pH 7.4) for 1

h at 4jC. The cells were then scraped off the flask bottom,

centrifuged to form a pellet (2000 rpm for 5 min) and

embedded in 2% low-viscosity agarose. Free aldehydes

were blocked by 0.5 M NH4Cl in PBS for 10 min at 4jC.The specimens were then dehydrated in ethanol, embedded

in LR White resin and polymerised for 24 h at 60jC. Small

fragments of liver were fixed with 4% paraformaldehyde in

0.1 M Sorensen phosphate buffer (pH 7.4) for 2 h at 4jC.After washing in Sorensen buffer and in 0.1 M PBS, free

aldehydes were blocked with 0.5 M NH4Cl in PBS for 45

min at 4jC. Following washing in PBS, the samples were

dehydrated in ethanol at progressively lower temperature,

embedded in Lowicryl K4M and UV-polymerised [22].

Immunocytochemical analyses were carried out using the

following primary antibodies: mouse monoclonal anti-SMN

(7B10, Ref. [4]) diluted 1:20, rabbit polyclonal anti-Gemin2

1:20 [5]; mouse monoclonal anti-Sm snRNP (Y12) 1:50

[23]; rabbit polyclonal anti-fibrillarin 1:10 [24]; rabbit

polyclonal anti-coilin 1:50 [25]; and mouse monoclonal

anti-SC-35 1:200 [26].

Ultrathin sections on formvar-carbon-coated nickel grids

were incubated on a drop of 1% normal goat serum (NGS,

Nordic Immunology Laboratories, Tilburg, The Nether-

lands) in PBS for 3 min and immunoreacted for 17 h at

4jC with primary antibodies diluted in PBS containing

0.5% bovine serum albumin (BSA, Fluka, Buchs, Switzer-

land) and 0.05% Tween 20 (Sigma, St. Louis, MO, USA).

After rinsing with PBS/Tween and incubating with PBS for

15 min, grids were treated for 3 min with 1% NGS in PBS

and incubated for 30 min at room temperature with colloidal

gold particle-conjugated secondary antibodies diluted in

PBS: goat anti-mouse 12nm 1:10 (GAM 12, Jackson

Immunoresearch Laboratories, West Grove, PA); goat anti-

Table 1

Anti-SMN labelling densities (mean values F SE) over different cellular compar

EFGCs

(gold grains/Am2)

CBs

(gold grains/Am2)

Nucleoplasm

(gold grains/Am2)

HeLa 396.96 F 19.60 34.62 F 14.49* 27.13 F 2.31*

3T3 186.90 F 18.21 – 20.42 F 1.82

Liver 169.95 F 11.33 36.02 F 13.39* 33.60 F 1.41*

In each row, values identified by common symbols are not significantly different f

EFGCs, while the high standard error values of CBs reflect a large variability in

rabbit 12nm 1:10 (GAR 12, Jackson Immunoresearch Lab-

oratories); goat anti-mouse 6nm (GAM IgG 6, Aurion,

Wageningen, The Netherlands). The grids were finally

rinsed with PBS and distilled water and then air-dried.

The preparations embedded in LR White were stained by

the regressive technique [27], which is preferential for

nuclear ribonucleoproteins. The sections of tissues embed-

ded in Lowicryl K4M were conventionally stained using

uranyl acetate and lead citrate because the chromatin in

Lowicryl K4M resin tends to exhibit a bleached aspect

without EDTA treatment. Controls consisted in omitting

the primary antibodies. Observations were carried out with

Philips CM 10 or CM 12 electron microscopes at 80 kV,

using a 30- to 40-Am objective aperture.

To assess the presence of the SMN protein quantitatively,

the labelling density over some cellular compartments was

evaluated. The surface area of each compartment consid-

ered—EFGCs, CBs, nucleoplasm devoid of EFGCs and

CBs, nucleoli, cytoplasm—was measured on 25 randomly

selected electron micrographs (�35000) from each experi-

mental group by using a computerised image analysis

system (Image Pro-Plus for Windows 95). For background

evaluation, resin outside the tissue or cells was considered.

The gold grains present over the investigated compartments

were counted and the labelling density was expressed as the

number of gold grains per square micrometer.

Statistical comparisons between different cellular com-

partments (within the same experimental group) were per-

formed by the Kruskal–Wallis one-way ANOVA test and

relative procedures of multiple comparison. Statistical sig-

nificance was set at P V 0.05.

In addition, the labelling densities of anti-SC-35 and anti-

Sm snRNP probes were evaluated on 30 EFGCs (�35000)

and compared with the background level as described above.

Results

We have produced two antibodies, the monoclonal anti-

SMN antibody 7B10 [4,5] and an antiserum against Gemin2

to investigate the intranuclear localisation of these antigens.

They both specifically recognised their respective antigen in

HeLa whole cell extracts, as determined by Western blotting

(Fig. 1).

The anti-SMN antibody 7B10 was next used to detect

SMN in HeLa cells by immunofluorescence (Fig. 2).

tments in the three experimental models

Nucleolus

(gold grains/Am2)

Cytoplasm

(gold grains/Am2)

Resin

(gold grains/Am2)

6.76 F 2.03# 8.30 F 0.51# 1.44 F 0.69

4.85 F 0.94# 7.32 F 1.15# 1.10 F 0.76

9.42 F 1.41 12.51 F 1.12 0.95 F 0.58

rom each other. Note that the highest labelling densities always occur in the

labelling densities.

Fig. 3. Distribution of SMN and Gemin2 proteins in cell nuclei. (A) HeLa cell; an EFGC (thin arrow) and perichromatin fibrils (PFs, arrowheads) are strongly

labelled with the anti-SMN antibody, while a cluster of interchromatin granules (large arrows) shows SMN localisation rather on its periphery (12 nm particles).

(B) HeLa cell; two EFGCs (arrows) are double labelled with anti-SMN (6 nm particles) and anti-Gemin2 (12 nm particles) antibodies. (C) Hibernating

dormouse hepatocyte; an EFGC (arrow) and perichromatin fibrils (arrowheads) are labelled with anti-SMN antibodies (12 nm particles). Scale bar, 0.5 Am.

M. Malatesta et al. / Experimental Cell Research 292 (2004) 312–321316

M. Malatesta et al. / Experimental Cell Research 292 (2004) 312–321 317

This antibody stained the cytoplasm and also specific dots in

the nucleus (A), consistent with earlier observations. Impor-

tantly, a commercially available monoclonal antibody,

termed 2B1, exhibited exactly the same nuclear staining

pattern (B). To determine whether the nuclear structures

detected by 7B10 correspond to Gems and/or CBs, we

performed double labelling experiments with an antiserum

that specifically detects coilin, a marker protein of CBs, and

7B10. As shown in Fig. 2, and consistent with earlier

findings [18,20], the majority of coilin positive nuclear

structures completely overlapped with the 7B10 signal.

However, we repeatedly found CBs devoid of SMN-label

and SMN-positive dots without detectable amounts of coilin

(arrows in images D and G). The two domains are not

necessarily close to each other and can be distant. Thus,

although the majority of SMN appears to reside in CBs,

Fig. 4. Relative proximity of an EFGC and a CB in HeLa cell nuclei. An EFGC

particles). Scale bar, 0.5 Am.

there is clearly a separate pool of this antigen in (a) discrete

structure(s). Identical labeling results were obtained after the

two different fixation procedures.

Next, we wished to determine the localisation of SMN

and Gemin2 at the ultrastructural level. The immunoelectron

microscopic analysis carried out using the above specific

antibodies allowed us to demonstrate that SMN protein is

highly enriched in nuclear domains appearing as EFGCs

with thin fibrils spreading out at their periphery (Figs. 3–5).

EFGCs, of which we find up to six per nuclear section, have

an average size of 0.1–0.3 Am and were observed in HeLa

and 3T3 cells as well as in hepatocytes of hibernating

dormice, where they have previously been described as

‘‘dense granular bodies’’ [28]. Virtually every EFGC

detected showed a high concentration of SMN, that quan-

titative labelling evaluation demonstrated to be the strongest

(thin arrow) and a CB (open arrow) labelled by anti-SMN probe (12 nm

Fig. 5. An EFGC (thin arrow) contains low amount of SC-35 protein (A) and of snRNPs (B), whereas IGs are significantly labelled (large arrows). Insert in B

shows significant labeling for snRNPs in a coiled (Cajal) body. (C) The SMN protein localises mostly on the dense fibrillar component of the HeLa cell

nucleolus (N) in addition to an EFGC (large arrow) (12 nm particles). Small arrows indicate all labeled areas in the nucleolus. Scale bar, 0.5 Am.

M. Malatesta et al. / Experimental Cell Research 292 (2004) 312–321318

M. Malatesta et al. / Experimental Cell Research 292 (2004) 312–321 319

one among the different cellular compartments investigated

(Table 1). EFGCs also contained the SMN-interacting

Gemin2 protein (Fig. 3B), the non-snRNP splicing factor

SC-35 (Fig. 5A) and spliceosomal U snRNPs (Fig. 5B), but

are virtually devoid of coilin and fibrillarin (not shown). The

quantification of labelling demonstrated the significant

presence of SC-35 factor (62.00 F 4.05 grains/Am2 in

EFGCs vs. 0.23 F 0.12 grains/Am2 over free resin) and U

snRNPs (64.14 F 15.17 grains/Am2 in EFGCs vs. 0.22 F0.06 grains/Am2 over free resin) in these novel nuclear

domains.

CBs also contained SMN protein (Fig. 4); however, CBs

were characterised by a rather variable content of this

protein, some of them being heavily labelled, others weakly

labelled and others even devoid of labelling with anti-SMN

antibody. EFGCs and CBs were sometimes detected in close

proximity to each other in cultured cells as well as in

hibernating dormouse hepatocytes. SMN also showed a

general localisation in PFs previously characterized as in

situ forms of pre-mRNA transcripts [29,30], whereas clus-

ters of interchromatin granules were labelled only on the

periphery (Fig. 3). Some interchromatin granule associated

zones (IGAZ) were also found to contain SMN (not shown).

Finally, some SMN was also observed in the nucleolus,

especially in the dense fibrillar component (Fig. 5C).

Discussion

Previous light microscopic analyses have localised the

SMN protein in discrete nuclear domains termed Gemini of

coiled bodies (gems) because of their close proximity to

CBs [15]. Gems were considered as nuclear domains

distinct from CBs, although CBs are so far the only

structurally defined nuclear constituents known to accumu-

late SMN and its interacting protein Gemin2 [19]. With the

aim of identifying other subnuclear compartments enriched

in SMN and Gemin2, we have analysed, in the present

work, the localisation of these proteins at the immunoelec-

tron microscopic level. Our observations define a new

domain within the nucleus in which SMN was always found

to be highly concentrated and associated to Gemin2. This

domain, which we termed the electron-dense fibro-granular

cluster (EFGC), appears as an electron-dense granular, oval-

or round-shaped nuclear compartment with thin fibrils

spreading out at its periphery. Virtually every EFGC that

we have detected contained both SMN and Gemin2. How-

ever, EFGCs are clearly different from CBs as their mor-

phological appearance differs considerably and also their

biochemical composition is partially different. In fact,

EFGCs do not contain p-80 coilin, the marker protein of

CBs, or fibrillarin [31]. Moreover, EFGCs contain only

small amounts of spliceosomal U snRNPs, whereas CBs

are generally highly enriched in these splicing factors

[21,31–35]. On the other hand, EFGCs contain some SC-

35, a non-snRNP splicing factor [26] absent from CBs

[31,33]. Finally, while EFGCs are always characterised by

high density of anti-SMN labelling, only some CBs are

heavily labelled with this probe, while others remain weakly

labelled or unlabeled. We therefore conclude that EFGCs

are novel SMN-rich nuclear domains that are distinct from

CBs. It should be noted that we observed a similar subnu-

clear localisation of SMN in three different cell types (i.e.

HeLa, 3T3 and hepatocytes) from three different species

(human, mouse and dormouse). Hence, these observations

rule out the possibility that the localisation of SMN in

EFGCs is due to cell-type and/or species specific differences

in the nuclear architecture. We note that the composition of

EFGCs is very similar to what has been reported previously

for Gems. However, EFGCs contain small amounts of

snRNPs while previous immunofluorescence data suggested

that snRNPs may be absent from gems [15]. Such a

discrepancy may be explained by the higher resolution of

immunoelectron microscopy, which allows to identify small

amounts of molecules undetectable by immunofluorescence

microscopy. Furthermore, a possible inaccessibility of anti-

gens within dense domains such as EFGCs to antibodies

when used in immunofluorescence microscopic protocols

could further account for the different results obtained by

the two immunolabelling techniques. The high resolution of

transmission electron microscope could also explain the

higher number of EFGCs/gems observed in our study in

comparison to immunofluorescence observations. In addi-

tion, EFGCs appear more frequent than the coilin negative

nuclear structures observed by immunofluorescence. The

discrepancy in the number of EFGCs visualized by EM and

of gems seen by fluorescence microscopy can probably be

explained by a local concentration of SMN protein. Indeed,

while electron microscopy easily reveals a structural domain

exhibiting even low immunocytochemical labeling signal, a

weak signal would not be visualized above the background

level by fluorescence microscopy.

Other nuclear structural constituents, such as CBs and

IGAZs [36], contain some SMN protein; however, this

occurrence is quite variable, the protein sometimes being

absent from these domains. Moreover, both CBs [25] and

IGAZs [37] were reported to contain p80-coilin, whereas

gems are devoid of this protein [15]. We therefore conclude

that CBs and IGAZs are nuclear domains immunocyto-

chemically distinct from gems, although functional relation-

ships between them cannot be excluded. In fact, gems have

been sometimes found in association with either interchro-

matin granules or CBs. In particular, EFGCs were some-

times observed in intimate contact with CBs in HeLa cells

(present findings) as well as in hibernating dormouse

hepatocytes [28]. Accordingly, previous light microscopic

studies report that gems and CBs generally occur in close

proximity to each other, frequently even overlapping

[15,19,20].

Recent observations on knockout mice suggest that the

presence of full-length coilin molecules in CBs is necessary

for recruiting Sm snRNPs and SMN to CBs [38]. However,

M. Malatesta et al. / Experimental Cell Research 292 (2004) 312–321320

since some CBs do not contain SMN (present study) or

UlsnRNP [39], coilin does seem to be necessary but may not

be sufficient for the recruitment of such factors to this

nuclear domain. Moreover, in nuclei from tissues of animals

arousing from hibernation, CBs are rapidly disintegrating

while still containing significant amounts of coilin within the

residual reticulated structures [21,40]. This suggests that the

presence of coilin, at least under such physiological con-

ditions, is not in itself sufficient for the maintenance of CBs.

Our findings, showing occurrence of SMN in PFs,

strongly suggest that these fibrils, previously demonstrated

as in situ forms of pre-mRNA transcripts and probably also

of pre-mRNA splicing [30], are the nuclear domain where

SMN fulfils its function in transcription and/or splicing,

Given the compact appearance of EFGCs and their high

content in SMN protein, we favour the idea that these

nuclear domains represent storage pools for SMN and its

associated proteins. The presence of some snRNPs could

also suggest for EFGCs a more complex role than a mere

SMN storage site, maybe as nuclear domains where some

late step in splicing factor assembly occurs. On the other

hand, the variable content of SMN protein in CBs suggests a

transient accumulation in these nuclear structural constitu-

ents, possibly related to the cell cycle and/or metabolic rate.

In any case, the close association of gems and CBs,

although occasionally observed, indicates possible direct

functional relationships. Further experiments will be re-

quired to understand the role of these different subnuclear

domains in SMN pathways.

Some SMN protein has also been found in the nucleoli of

all cell types investigated, in particular in the dense fibrillar

component. The dense fibrillar component is considered to

be the site of ribosomal RNA transcription and of at least

first steps of pre-rRNA processing [41]. Biochemically, the

dense fibrillar component is characterised by fibrillarin, a

U3 snoRNP-associated protein involved in several steps of

ribosomal RNA processing [42]. Interestingly, SMN inter-

acts tightly with fibrillarin and another recently identified

SMN-interactor, termed Gemin4/GIP1, can also be detected

in the nucleolus [17,43,44]. Previous immunofluorescence

observations showed frequent distribution of SMN in nu-

cleoli [45]. Thus, together these data suggest that SMN and

other SMN-associated proteins fulfil a role associated with

the nucleolus, such as maturation of rRNA and/or biogen-

esis of ribosomes.

Acknowledgments

We express our gratitude for kindly providing us with

their antibodies to Dr. E.K.L. Chan (anti-coilin), Dr. X.-D. Fu

(anti-SC-35) and Dr. F. Amalric (anti-fibrillarin). We thank

Mrs. J. Fakan, V. Mamin and F. Voinesco for excellent

technical assistance and Mr. W. Blanchard for photographic

work. M.M. was the recipient of a fellowship from the

University of Urbino in the frame of the ‘‘Progetto Giovani

Ricercatori’’. C.S. benefited from a fellowship of the

Fondation du 450e Anniversaire de 1’Universite de Lau-

sanne. S.F. received support from the Swiss National Science

Foundation (grant 3 l-53944.98 and 3 l-64977.01). U.F. was

supported by the Max-Planck Society and by a grant from the

Deutsche Forschungsgemeinschaft (DFG Fi 573/2-2).

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