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Three-Dimensional Visualization of Transcription Sites and Their Association with Splicing Factor–Rich Nuclear Speckles

Three-Dimensional Visualization of Transcription Sites and Their Association with Splicing... Transcription sites are detected by labeling nascent transcripts with BrUTP in permeabilized 3T3 mouse fibroblasts followed by laser scanning confocal microscopy. Inhibition and enzyme digestion studies confirm that the labeled sites are from RNA transcripts and that RNA polymerase I (RP I) and II (RP II) are responsible for nucleolar and extranucleolar transcription, respectively. An average of 2,000 sites are detected per nucleus with over 90% in the extranucleolar compartment where they are arranged in clusters and three-dimensional networklike arrays. The number of transcription sites, their three-dimensional organization and arrangement into functional zones ( Wei et al. 1998 ) is strikingly maintained after extraction for nuclear matrix. Significant levels of total RP II mediated transcription sites (45%) were associated with splicing factor–rich nuclear speckles even though the speckles occupied <10% of the total extranucleolar space. Moreover, the vast majority of nuclear speckles (>90%) had moderate to high levels of associated transcription activity. Transcription sites were found along the periphery as well as inside the speckles themselves. These spatial relations were confirmed in optical sections through individual speckles and after in vivo labeling of nascent transcripts. Our results demonstrate that nuclear speckles and their surrounding regions are major sites of RP II-mediated transcription in the cell nucleus, and support the view that both speckle- and nonspeckle-associated regions of the nucleus contain sites for the coordination of transcription and splicing processes. transcription sites nuclear matrix nuclear speckles splicing factors laser scanning confocal microscopy Footnotes 1.used in this paper: AS, ammonium sulfate; CTD, carboxy-terminal domain of RNA polymerase II large subunit; RP I and II, RNA polymerase I and II; RP IIo, hyperphosphorylated RNA polymerase II; snRNP, small nuclear ribonucleoprotein; SR, serine-arginine Dr. Samarabandu's present address is Life Imaging Systems Inc., 195 Dufferin Avenue, Suite 300, London, ON N6A 1K7, Canada. Dr. Wei's current address is Department of Ophthalmology, Harvard Medical School, 243 Charles Street, Boston, MA 02114. Submitted: 23 September 1998 Revision requested 2 June 1999 Accepted: 25 June 1999 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Cell Biology Rockefeller University Press

Three-Dimensional Visualization of Transcription Sites and Their Association with Splicing Factor–Rich Nuclear Speckles

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References (102)

Publisher
Rockefeller University Press
Copyright
© 1999 The Rockefeller University Press
ISSN
0021-9525
eISSN
1540-8140
DOI
10.1083/jcb.146.3.543
Publisher site
See Article on Publisher Site

Abstract

Transcription sites are detected by labeling nascent transcripts with BrUTP in permeabilized 3T3 mouse fibroblasts followed by laser scanning confocal microscopy. Inhibition and enzyme digestion studies confirm that the labeled sites are from RNA transcripts and that RNA polymerase I (RP I) and II (RP II) are responsible for nucleolar and extranucleolar transcription, respectively. An average of 2,000 sites are detected per nucleus with over 90% in the extranucleolar compartment where they are arranged in clusters and three-dimensional networklike arrays. The number of transcription sites, their three-dimensional organization and arrangement into functional zones ( Wei et al. 1998 ) is strikingly maintained after extraction for nuclear matrix. Significant levels of total RP II mediated transcription sites (45%) were associated with splicing factor–rich nuclear speckles even though the speckles occupied <10% of the total extranucleolar space. Moreover, the vast majority of nuclear speckles (>90%) had moderate to high levels of associated transcription activity. Transcription sites were found along the periphery as well as inside the speckles themselves. These spatial relations were confirmed in optical sections through individual speckles and after in vivo labeling of nascent transcripts. Our results demonstrate that nuclear speckles and their surrounding regions are major sites of RP II-mediated transcription in the cell nucleus, and support the view that both speckle- and nonspeckle-associated regions of the nucleus contain sites for the coordination of transcription and splicing processes. transcription sites nuclear matrix nuclear speckles splicing factors laser scanning confocal microscopy Footnotes 1.used in this paper: AS, ammonium sulfate; CTD, carboxy-terminal domain of RNA polymerase II large subunit; RP I and II, RNA polymerase I and II; RP IIo, hyperphosphorylated RNA polymerase II; snRNP, small nuclear ribonucleoprotein; SR, serine-arginine Dr. Samarabandu's present address is Life Imaging Systems Inc., 195 Dufferin Avenue, Suite 300, London, ON N6A 1K7, Canada. Dr. Wei's current address is Department of Ophthalmology, Harvard Medical School, 243 Charles Street, Boston, MA 02114. Submitted: 23 September 1998 Revision requested 2 June 1999 Accepted: 25 June 1999

Journal

The Journal of Cell Biology – Rockefeller University Press

Published: Aug 9, 1999

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