Three-dimensional visualization of transcription sites and their association with splicing factor-rich nuclear speckles

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 label ed sites are from RNA transcripts and that RNA polymerase I (RP I) and II ( RP II) are responsible for nucleolar and extranucleolar transcription, resp ectively. An average of 2,000 sites are detected per nucleus with over 90% in the extranucleolar compartment where they are arranged in clusters and t hree-dimensional networklike arrays. The number of transcription sites, the ir three-dimensional organization and arrangement into functional zones (We i et al,, 1998) is strikingly maintained after extraction for nuclear matri x. Significant levels of total RP II mediated transcription sites (45%) wer e associated with splicing factor-rich nuclear speckles even though the spe ckles occupied <10% of the total extranucleolar space. Moreover, the vast m ajority of nuclear speckles (>90%) had moderate to high levels of associate d transcription activity. Transcription sites were found along the peripher y as well as inside the speckles themselves. These spatial relations were c onfirmed in optical sections through individual speckles and after in vivo labeling of nascent transcripts. Our results demonstrate that nuclear speck les and their surrounding regions are major sites of RP II-mediated transcr iption in the cell nucleus, and support the view that both speckle- and non speckle-associated regions of the nucleus contain sites for the coordinatio n of transcription and splicing processes.