We have studied in HeLa cells at the electron microscope level the response to adenovirus infection of the RNA processing machinery. Components of the spliceosomes were localized by in situ hybridization with biotinylated U1 and U2 DNA probes and by immunolabeling with Y12 anti-Sm monoclonal antibody, whereas poly(A)+ RNAs were localized by specific binding of biotinylated poly(dT) probe. At early stages of nuclear transformation, the distribution of small nuclear RNPs was similar to that previously described in non-infected nuclei (Visa, N., Puvion-Dutilleul, F., Bachellerie, J.P. and Puvion, E., Eur. J. Cell Biol. 60, 308–321, 1993; Visa, N., Puvion-Dutilleul, F., Harper, F., Bachellerie, J. P. and Puvion, E., Exp. Cell Res. 208, 19–34, 1993). As the infection progresses, the large virus-induced inclusion body consists of a central storage site of functionally inactive viral genomes surrounded by a peripheral shell formed by clusters of interchromatin granules, compact rings and a fibrillogranular network in which are embedded the viral single-stranded DNA accumulation sites. Spliceosome components and poly(A)+ RNAs were then exclusively detected over the clusters of interchromatin granules and the fibrillogranular network whereas the viral single-stranded DNA accumulation sites and compact rings remained unlabeled, thus appearing to not be directly involved in splicing. Our data, therefore, suggest that the fibrillogranular network, in addition to being the site of viral transcription, is also a major site of viral RNA splicing. Like the clusters of interchromatin granules, which had been already involved in spliceosome assembly, they could also have a role in the sorting of viral spliced polyadenylated mRNAs before export to the cytoplasm. The compact rings, which contain non-polyadenylated viral RNA, might accumulate the non-used portions of the viral transcripts resulting from differential poly(A)+ site selection.

REFERENCES

Adami
G.
,
Nevins
J. R.
(
1988
).
Splicing site selection dominates over poly(A) site choice in RNA production from complex adenovirus transcription units.
EMBO J
7
,
2107
2116
Bachellerie
J. P.
,
Puvion
E.
,
Zalta
J. P.
(
1975
).
Ultrastructural organization and biochemical characterization of chromatin-RNA-protein complexes isolated from mammalian cell nuclei.
Eur. J. Biochem
58
,
327
337
Besse
S.
,
Puvion-Dutilleul
F.
(
1994
).
Compartmentalization of cellular and viral DNAs in adenovirus type 5 infection as revealed by ultrastructural in situ hybridization.
Chromosome Res
2
,
123
135
Beyer
A. L.
,
Bouton
A. H.
,
Miller
O. L.
Jr.
(
1981
).
Correlation of hnRNP structure and nascent transcript cleavage.
Cell
26
,
155
165
Beyer
A. L.
(
1983
).
Ultrastructural analysis of the ribonucleoprotein structure of nascent hnRNA.
Mol. Biol. Rep
9
,
49
58
Beyer
A. L.
,
Osheim
Y. N.
(
1988
).
Splice site selection, rate of splicing, and alternative splicing on nascent transcripts.
Genes Dev
2
,
754
765
Bridge
E.
,
Carmo-Fonseca
M.
,
Lamond
A.
,
Pettersson
U.
(
1993
).
Nuclear organization of splicing small nuclear ribonucleoproteins in adenovirus-infected cells.
J. Virol
67
,
5792
5802
Carmo-Fonseca
M.
,
Pepperkok
R.
,
Sproat
B.
,
Ansorge
W.
,
Swanson
M.
,
Lamond
A. I.
(
1991
).
In vivo detection of snRNP-rich organelles in the nuclei of mammalian cells.
EMBO J
10
,
1863
1873
Carmo-Fonseca
M.
,
Pepperkok
R.
,
Carvalho
M. T.
,
Lamond
A. I.
(
1992
).
Transcription-dependent colocalization of the U1, U2, U4/U6 and U5 snRNPs in coiled bodies.
J. Cell Biol
117
,
1
14
Fakan
S.
,
Puvion
E.
,
Spohr
G.
(
1976
).
Localization and characterization of newly synthesized nuclear RNA in isolated rat hepatocytes.
Exp. Cell Res
99
,
155
164
Fakan
S.
,
Leser
G.
,
Martin
T. E.
(
1984
).
Ultrastructural distribution of nuclear ribonucleoproteins as visualized by immunocytochemistry on thin sections.
J. Cell Biol
98
,
358
363
Fakan
S.
,
Leser
G.
,
Martin
T. E.
(
1986
).
Immunoelectron microscope visualization of nuclear ribonucleoprotein antigens within spread transcription complexes.
J. Cell Biol
103
,
1153
1157
Gallinaro
H.
,
Puvion
E.
,
Kister
L.
,
Jacob
M.
(
1983
).
Nuclear matrix and hnRNP share a common structural constituent associated with premessenger RNA.
EMBO J
2
,
953
960
Greenberg
J. R.
(
1972
).
High stability of mRNA in growing cultured cells.
Nature
240
,
102
104
Jimenez-Garcia
L. F.
,
Spector
D. L.
(
1993
).
In vivo evidence that transcription and splicing are coordinated by a recruiting mechanism.
Cell
73
,
47
59
Lamond
A. I.
,
Carmo-Fonseca
M.
(
1993
).
Localization of splicing mRNPs in mammalian cells.
Mol. Biol. Rep
18
,
127
133
Larsson
S.
,
Svensson
C.
,
Akusjäevi
G.
(
1992
).
Control of adenovirus major late gene expression at multiple levels.
J. Mol. Biol
225
,
287
298
Lerner
E. A.
,
Lerner
M. R.
,
Janeway
C. A.
,
Steitz
J. A.
(
1981
).
Monoclonal antibodies to nucleic acid-containing cellular constituents. Probes for molecular biology and autoimmune disease.
Proc. Nat. Acad. Sci. USA
78
,
2737
2741
Nash
R. E.
,
Puvion
E.
,
Bernhard
W.
(
1975
).
Perichromatin fibrils as components of rapidly labeled extranucleolar RNA.
J. Ultrastruct. Res
53
,
395
405
Nevins
J. R.
,
Darnell
J. E.
(
1978
).
Groups of adenovirus type 2 mRNA's derived from a large primary transcript: probable nuclear origin and possible common 3ends.
J. Virol
25
,
811
823
Nevins
J. R.
(
1982
).
Adenovirus gene expression: control at multiple steps of mRNA biogenesis.
Cell
28
,
1
2
Osheim
Y. N.
,
Miller
O. L.
,
Beyer
A. L.
(
1985
).
RNP particles at splice junction sequences on Drosophila chorion transcripts.
Cell
43
,
143
151
Prescott
J. C.
,
Falck-Pedersen
E. F.
(
1992
).
Varied poly(A) site efficiency in the adenovirus major late transcription unit.
J. Biol. Chem
267
,
8175
8181
Puvion
E.
,
Moyne
G.
(
1978
).
Intranuclear migration of newly synthesized extranuleolar ribonucleoproteins. A high resolution quantitative autoradiographical and cytochemical study.
Exp. Cell Res
115
,
79
88
Puvion
E.
,
Viron
A.
(
1981
).
In situ structural and functional relationship between chromatin pattern and RNP structures involved in non-nucleolar chromatin transcription.
J. Ultrastruct. Res
74
,
351
360
Puvion
E.
,
Viron
A.
,
Assens
C.
,
Leduc
E. H.
,
Jeanteur
Ph
(
1984
).
Immunocytochemical identification of nuclear structures containing snRNPs in isolated rat liver cells.
J. Ultrastruct. Res
87
,
180
189
Puvion-Dutilleul
F.
,
Puvion
E.
(
1990
).
Replicating single-stranded adenovirus type 5 DNA molecules accumulate within well-delimited intranuclear areas of lytically infected HeLa cells.
Eur. J. Cell Biol
52
,
379
388
Puvion-Dutilleul
F.
,
Puvion
E.
(
1990
).
Analysis by in situ hybridization and autoradiography of sites of replication and storage of single-and double-stranded adenovirus type 5 DNA in lytically infected HeLa cells.
J. Struct. Biol
103
,
280
289
Puvion-Dutilleul
F.
(
1991
).
Simultaneous detection of highly phosphorylated proteins and major DNA-binding protein distribution in the nucleus of adenovirus type 5 infected HeLa cells.
J. Histochem. Cytochem
39
,
669
680
Puvion-Dutilleul
F.
,
Puvion
E.
(
1991
).
Sites of transcription of adenovirus type 5 genomes in relation to early viral DNA replication in infected HeLa cells. A high resolution in situ hybridization and autoradiographical study.
Biol. Cell
71
,
135
147
Puvion-Dutilleul
F.
,
Pichard
E.
(
1992
).
Segregation of viral double-stranded and single-stranded DNA molecules in nuclei of adenovirus infected cells as revealed by electron microscope in situ hybridization. Biol.
Cell
76
,
139
150
Puvion-Dutilleul
F.
,
Roussev
R.
,
Puvion
E.
(
1992
).
Distribution of viral RNA molecules during the adenovirus type 5 infectious cycle in HeLa cells.
J. Struct. Biol
108
,
354
364
Puvion-Dutilleul
F.
(
1993
).
Protocol of electron microscope in situ nucleic acid hybridization for the exclusive detection of double-stranded DNA sequences in cells containing large amounts of homologous single-stranded DNA and RNA sequences. Application to adenovirus type 5 infected HeLa cells. Microsc.
Res. Tech
25
,
2
11
Singer
R.
,
Penman
S.
(
1972
).
Messenger RNA in HeLa cells: kinetics of formation and decay.
J. Mol. Biol
78
,
321
328
Visa
N.
,
Puvion-Dutilleul
F.
,
Bachellerie
J. P.
,
Puvion
E.
(
1993
).
Intranuclear distribution of U1 and U2 snRNAs visualized by high resolution in situ hybridization: revelation of a novel compartment containing U1 but not U2 snRNA in HeLa cells.
Eur. J. Cell Biol
60
,
308
321
Visa
N.
,
Puvion-Dutilleul
F.
,
Harper
F.
,
Bachellerie
J. P.
,
Puvion
E.
(
1993
).
Intranuclear distribution of poly(A) RNA determined by electron microscope in situ hybridization.
Exp. Cell Res
208
,
19
34
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