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Vol. 10, Issue 1, 211-223, January 1999
and
*Centre of Electron Microscopy, University of Lausanne, 1005 Lausanne, Switzerland;
In this study we demonstrate, at an ultrastructural level, the in
situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of
living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover,
immunocytochemical localization of several pre-mRNA transcription and
processing factors has been carried out in the same cells. This
high-resolution approach allowed us to reveal perichromatin regions as
the most important sites of nucleoplasmic RNA transcription and the
perichromatin fibrils (PFs) as in situ forms of nascent transcripts.
Furthermore, we show that transcription takes place in a rather diffuse
pattern, without notable local accumulation of transcription sites. RNA polymerase II, heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins, general transcription factor TFIIH, poly(A) polymerase, splicing factor SC-35, and Sm complex of small nuclear
ribonucleoproteins (snRNPs) are associated with PFs. This
strongly supports the idea that PFs are also sites of major pre-mRNA
processing events. The absence of nascent transcripts, RNA polymerase
II, poly(A) polymerase, and hnRNPs within the clusters of
interchromatin granules rules out the possibility that this domain
plays a role in pre-mRNA transcription and polyadenylation; however,
interchromatin granule-associated zones contain RNA polymerase II,
TFIIH, and Sm complex of snRNPs and, after longer periods of BrUTP
incubation, also Br-labeled RNA. Their role in nuclear functions still
remains enigmatic. In the nucleolus, transcription sites occur in the
dense fibrillar component. Our fine structural results show that PFs
represent the major nucleoplasmic structural domain involved in active
pre-mRNA transcriptional and processing events.
E.C. Slater Instituut, University
of Amsterdam, 1018 TV Amsterdam, The Netherlands;
Department of Molecular Genetics and Cell Biology,
University of Chicago, Chicago, Illinois 60637;
§Division
of Biological Sciences, University of California, Davis, California
95616;
Department of Cell Biology, University of Basel,
4056 Basel, Switzerland; and
¶Division of Cellular and
Molecular Medicine, University of California, La Jolla, California
92093
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