Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection

Abstract

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.

Figure 1

Confocal optical section of a microinjected T24 cell showing immunocytochemical signal for Br-RNA, after 10 min incubation, and PANA marker of clusters of IGs. Large unlabeled areas correspond to nucleoli. (A) The typical diffuse distribution of sites of nascent RNA is demonstrated. (B) Several anti-PANA–labeled speckled regions correspond to clusters of IGs. (C) Merged colocalization image shows very little overlapping between anti-PANA–labeled regions (green) and Br-RNA (red). Bar, 2.0 μm.

Figure 4

A high-power micrograph shows, after pulse of 7 min 40 sec, anti-BrU label (mouse anti-BrdU antibody) on PFs arranged in an array resembling a transcription complex. Bar, 0.1 μm.

Figure 6

Double-labeling with mouse anti-BrdU antibody (small grains, 6 nm) and chicken anti-RNA polymerase II antibody (large grains, 15 nm) after incubation of 7 min 20 sec. Colocalization is visible on PFs (arrows). Bar, 0.2 μm.

Figure 9

Colocalization of brominated RNA (mouse anti-BrdU antibody; 6-nm grains) and RNA polymerase II (chicken anti-pol II antibody; 15-nm grains) in the IG-associated zone (asterisk) after 30-min incubation. Bar, 0.2 μm.

Figure 11

BrUTP (mouse anti-BrdU antibody; 6-nm grains) and RNA polymerase II (rabbit anti-pol II antibody; 15-nm grains) double-labeling after 90-min incubation. Even after a prolonged incubation time, the center of the IG cluster remains devoid of label. Some BrU signal can be observed on the periphery of the cluster, whereas no RNA polymerase II is associated with the IG cluster. Bar, 0.2 μm.