The route taken by transcripts from synthetic sites in the nucleus to the cytoplasm has been under scrutiny for years, but details of the pathway remain obscure. A new high-resolution method for mapping the pathway is described; HeLa cells are grown in Br-U so that the analogue is incorporated into RNA and exported to the cytoplasm, before Br-RNA is localized by immuno-electron microscopy. After exposure to low concentrations of Br-U for short periods, cells grow normally. Br-RNA is first found in several thousand extra-nucleolar transcription sites or factories (diameter 50–80 nm), before appearing in several hundred new downstream sites (diameter 50–80 nm) each minute; subsequently, progressively more downstream sites become labelled. These sites can be isolated on sucrose gradients as large nuclear ribonucleoprotein particles of approximately 200 S. Later, Br-RNA is seen docked approximately 200 nm away from approximately 20% nuclear pores, before exiting to the cytoplasm. Individual downstream sites are unlikely to contain individual transcripts; rather, results are consistent with groups of transcripts being shipped together from synthetic sites to pores. A subset of SR proteins are excellent markers of this pathway; this subset is concentrated in tens of thousands of sites, which include transcription, downstream and docking sites. Growth in high concentrations of Br-U for long periods is toxic, and Br-RNA accumulates just inside nuclear pores.

REFERENCES

Ainger
K.
,
Avossa
D.
,
Morgan
F.
,
Hill
S. J.
,
Barry
C.
,
Barbarese
E.
,
Carson
J. H.
(
1993
).
Transport and localization of exogenous MBP mRNA microinjected into oligodendrocytes.
J. Cell Biol
123
,
431
441
Alzhanova-Ericsson
A. T.
,
Sun
X.
,
Visa
N.
,
Kiseleva
E.
,
Wurtz
T.
,
Daneholt
B.
(
1996
).
A protein of the SR family of splicing factors binds extensively to exonic Balbiani ring pre-mRNA and accompanies the RNA from the gene to the nuclear pore.
Genes Dev
10
,
2881
2893
Amberg
D. C.
,
Goldstein
A. L.
,
Cole
C. N.
(
1992
).
Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA.
Genes Dev
6
,
1173
1189
Barbarese
E.
,
Koppel
D. E.
,
Deutscher
M. P.
,
Smith
C. L.
,
Ainger
K.
,
Morgan
F.
,
Carson
J. H.
(
1995
).
Protein translocation components are colocalized in granules of oligodendrocytes.
J. Cell Sci
108
,
2781
2790
Bastos
R.
,
Pante
N.
,
Burke
B.
(
1995
).
Nuclear pore complex proteins.
Int. Rev. Cytol
162
,
257
302
Buss
F.
,
Stewart
M.
(
1995
).
Macromolecular interactions in the nucleoporin p62 complex of rat nuclear pores: binding of nucleoporin p54 to the rod domain of p62.
J. Cell Biol
128
,
251
261
Cáceres
J. F.
,
Misteli
T.
,
Screaton
G. R.
,
Spector
D. L.
,
Krainer
A. R.
(
1997
).
Role of the modular domains of SR proteins in subnuclear localization and alternative splicing specificity.
J. Cell Biol
138
,
225
238
Colwill
K.
,
Pawson
T.
,
Andrews
B.
,
Prasad
J.
,
Manley
J. L.
,
Bell
J. C.
,
Duncan
P. I.
(
1996
).
The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution.
EMBO J
15
,
265
275
Cox
R. F.
(
1976
).
Quantitation of elongating form A and B RNA polymerases in chick oviduct nuclei and effects of estradiol.
Cell
7
,
455
465
Daneholt
B.
(
1997
).
A look at messenger RNP moving through the nuclear pore.
Cell
88
,
585
588
Davis
L. I.
(
1995
).
The nuclear pore complex.
Annu. Rev. Biochem
64
,
865
896
Dreyfuss
G.
,
Matunis
M. J.
,
Pinol-Roma
S.
,
Burd
C. G.
(
1993
).
hnRNP proteins and the biogenesis of mRNA.
Annu. Rev. Biochem
62
,
289
321
Dworetzky
S. I.
,
Feldherr
C. M.
(
1988
).
Translocation of RNA-coated gold particles through the nuclear pores of oocytes.
J. Cell Biol
106
,
575
584
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
Fay
F. S.
,
Taneja
K. L.
,
Shenoy
S.
,
Lifshitz
L.
,
Singer
R. H.
(
1997
).
Quantitative digital analysis of diffuse and concentrated nuclear distributions of nascent transcripts, SC35 and poly(A).
Exp. Cell Res
231
,
27
37
Harris
H.
,
Watts
J. W.
(
1962
).
The relationship between nuclear and cytoplasmic ribonucleic acid.
Proc. Roy. Soc. Ser. B
156
,
109
121
Hesketh
J. E.
,
Pryme
I. F.
(
1991
).
Interaction between mRNA, ribosomes and the cytoskeleton.
Biochem. J
277
,
1
10
Hozák
P.
,
Hassan
A. B.
,
Jackson
D. A.
,
Cook
P. R.
(
1993
).
Visualization of replication factories attached to a nucleoskeleton.
Cell
73
,
361
373
Hozák
P.
,
Cook
P. R.
,
Schöfer
C.
,
Mosgöller
W.
,
Wachtler
F.
(
1994
).
Site of transcription of ribosomal RNA and intra-nucleolar structure in HeLa cells.
J. Cell Sci
107
,
639
648
Huang
S.
,
Spector
D. L.
(
1991
).
Nascent pre-mRNA transcripts are associated with nuclear regions enriched in splicing factors.
Genes Dev
5
,
2288
2302
Huang
S.
,
Deerinck
T. J.
,
Ellisman
M. H.
,
Spector
D. L.
(
1994
).
In vivo analysis of the stability and transport of nuclear poly(A)+RNA.
J. Cell Biol
126
,
877
899
Iborra
F. J.
,
Pombo
A.
,
Jackson
D. A.
,
Cook
P. R.
(
1996
).
Active RNA polymerases are localized within discrete transcription ‘factories’ in human nuclei.
J. Cell Sci
109
,
1427
1436
Izaurralde
E.
,
Mattaj
I. W.
(
1995
).
RNA export.
Cell
81
,
153
159
Jackson
D. A.
,
Cook
P. R.
(
1985
).
Transcription occurs at a nucleoskeleton.
EMBO J
4
,
919
925
Jackson
D. A.
,
Hassan
A. B.
,
Errington
R. J.
,
Cook
P. R.
(
1993
).
Visualization of focal sites of transcription within human nuclei.
EMBO J
12
,
1059
1065
Jackson
D. A.
,
Iborra
F. J.
,
Manders
E. M. M.
,
Cook
P. R.
(
1998
).
Numbers and organization of RNA polymerases, nascent transcripts and transcription units in HeLa nuclei.
Mol. Biol. Cell
9
,
1523
1536
Jacobson
A.
,
Peltz
S. W.
(
1996
).
Interrelationships of the pathways of mRNA decay and translation in eukaryotic cells.
Annu. Rev. Biochem
65
,
693
739
Knowles
R. B.
,
Sabry
J. H.
,
Martone
M. A.
,
Ellisman
M.
,
Bassell
G. J.
,
Kosik
K. S.
(
1996
).
Translocation of RNA granules in living neurons.
J. Neurosci
16
,
7812
7820
Kramer
A.
(
1996
).
The structure and function of proteins involved in mammalian pre-mRNA splicing.
Annu. Rev. Biochem
65
,
367
409
Lawrence
J. B.
,
Singer
R. H.
,
Marselle
L. M.
(
1989
).
Highly localised tracks of specific transcripts within interphase nuclei visualised by in situ hybridisation.
Cell
57
,
493
502
Miriami
E.
,
Sperling
J.
,
Sperling
R.
(
1994
).
Heat shock affects 5′ splice selection, cleavage and ligation of CAD pre-mRNA in hamster cells, but not its packaging in lnRNP particles.
Nucl. Acids Res
22
,
3084
3091
Neugebauer
K. M.
,
Roth
M. B.
(
1997
).
Distribution of pre-mRNA splicing factors at sites of RNA polymerase II transcription.
Genes Dev
11
,
1148
1159
Nigg
E. A.
(
1997
).
Nucleocytoplasmic transport: signals, mechanisms and regulation.
Nature
386
,
779
787
Pante
N.
,
Aebi
U.
(
1996
).
Molecular dissection of the nuclear pore complex.
Crit. Rev. Biochem. Mol. Biol
31
,
153
199
Pante
N.
,
Jarmolowski
J.
,
Izaurralde
E.
,
Sauder
U.
,
Baschong
W.
,
Mattaj
I. W.
(
1997
).
Visualizing nuclear export of different classes of RNA by electron microscopy.
RNA
1
,
498
513
Penman
S.
(
1995
).
Rethinking cell structure.
Proc. Nat. Acad. Sci. USA
92
,
5251
5257
Puvion
E.
,
Puvion-Dutilleul
F.
(
1996
).
Ultrastructure of the nucleus in relation to transcription and splicing: roles of perichromatin fibrils and interchromatin granules.
Exp. Cell Res
229
,
217
225
Romac
J. M.
,
Keene
J. D.
(
1995
).
Overexpression of the arginine-rich carboxy-terminal region of U1 snRNP 70K inhibits both splicing and nucleocytoplasmic transport of mRNA.
Genes Dev
9
,
1400
1410
Rosbash
M.
,
Singer
R. H.
(
1993
).
RNA travel: tracks from DNA to cytoplasm.
Cell
75
,
399
401
Ross
J.
(
1995
).
mRNA stability in mammalian cells.
Microbiol. Rev.
59
,
423
450
Roth
M. B.
,
Zahler
A. M.
,
Gall
J. G.
(
1990
).
A monoclonal antibody that recognizes a phosphorylated epitope stains lampbrush chromosomes and small granules in the amphibian germinal vesicle.
J. Cell Biol
111
,
2217
2223
Schmittgen
T. D.
,
Danenberg
K. D.
,
Horikoshi
T.
,
Lenz
H.-J.
,
Dannenberg
P. V.
(
1994
).
Effects of 5-fluoro-and 5-bromouracil substitution on the translation of human thymidylate synthase mRNA.
J. Biol. Chem
269
,
16269
16275
Sharp
P. A.
(
1994
).
Split genes and RNA splicing.
Cell
77
,
805
815
Sierakowska
H.
,
Shuklas
R. R.
,
Dominski
Z.
,
Kole
R.
(
1989
).
Inhibition of pre-mRNA splicing by 5-fluoro-, 5-chloro-, and 5-bromouridine.
J. Biol. Chem
264
,
19185
19191
Spector
D. L.
(
1993
).
Macromolecular domains within the cell nucleus.
Annu. Rev. Cell. Biol
9
,
265
315
Sperling
R.
,
Sperling
J.
,
Levine
A. D.
,
Spann
P.
,
Stark
G. R.
,
Kornberg
R. D.
(
1985
).
Abundant nuclear ribonucleoprotein form of CAD RNA.
Mol. Cell Biol
5
,
569
575
Sperling
R.
,
Koster
A. J.
,
Melamed-Bussudo
C.
,
Rubinstein
A.
,
Angenitzki
M.
,
Berkovitch-Yellin
Z.
,
Sperling
J.
(
1997
).
Three-dimensional image reconstruction of large nuclear RNP (lnRNP) particles by automated electron tomography.
J. Mol. Biol
267
,
570
583
Steinmetz
E. J.
(
1997
).
Pre-mRNA processing and the CTD of RNA polymerase II: the tail that wags the dog?.
Cell
89
,
491
494
Verheijen
R.
,
van Venrooij
W.
,
Ramaekers
F.
(
1988
).
The nuclear matrix: structure and composition.
J. Cell Sci
90
,
11
36
Wansink
D. G.
,
Schul
W.
,
van der Kraan
I.
,
van Steensel
B.
,
van Driel
R.
,
de Jong
L.
(
1993
).
Fluorescent labelling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus.
J. Cell Biol
122
,
283
293
Wansink
D. G.
,
Nelissen
R. L.
,
de Jong
L.
(
1994
).
In vitro splicing of pre-mRNA containing bromouridine.
Mol. Biol. Rep
19
,
109
113
Yitzhaki
S.
,
Miriami
E.
,
Sperling
R.
,
Sperling
J.
(
1996
).
Phosphorylated ser/arg-rich proteins: limiting factors in the assembly of 200S large nuclear ribonucleoprotein particles.
Proc. Nat. Acad. Sci. USA
93
,
8830
8835
Zachar
Z.
,
Kramer
J.
,
Mims
I. K.
,
Bingham
P. M.
(
1993
).
Evidence for channeled diffusion of pre-mRNAs during nuclear RNA transport in metazoans.
J. Cell Biol
121
,
729
742
Zahler
A. M.
,
Lane
W. S.
,
Stolk
J. A.
,
Roth
M. B.
(
1992
).
SR proteins: a conserved family of pre-mRNA splicing factors.
Genes Dev
6
,
837
847
This content is only available via PDF.