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 premRNA transcriptional and processing events.

Subnuclear Localization and Dynamics of the Pre-mRNA 3′ End Processing Factor Mammalian Cleavage Factor I 68-kDa Subunit

“…Together, these results show that both the diffuse nucleoplasmic staining and the overlap of CF I m staining with the periphery of nuclear speckles reflect CF I m association with sites of ongoing transcription and with pre-mRNA molecules. These results confirm and extend previous ultrastructural observations and support the idea that not only splicing (Cmarko et al, 1999;Lamond and Spector, 2003) but also 3Ј end processing of pre-mRNAs may occur on PFs.…”

“…Together, these results show that both the diffuse nucleoplasmic staining and the overlap of CF I m staining with the periphery of nuclear speckles reflect CF I m association with sites of ongoing transcription and with pre-mRNA molecules. These results confirm and extend previous ultrastructural observations and support the idea that not only splicing (Cmarko et al, 1999;Lamond and Spector, 2003) but also 3Ј end processing of pre-mRNAs may occur on PFs.In addition, our data suggest that paraspeckles observed by immunofluorescence may correspond to IGAZs. To clar- ify this point, we examined by immunoelectron microscopy the localization pattern of PSF, a protein that was recently localized to paraspeckles (Fox et al, 2005).…”

“…Previous studies have shown that PFs are the in situ form of nascent transcripts and have suggested that they also represent sites of pre-mRNAs processing (Cmarko et al, 1999). The association of CF I m with PFs was tested by colocalization with an anti-RNA polymerase II antibody, which recognizes the active form of the enzyme ( Figure 6A).…”

“…This is further confirmed by double labeling with anti-SC35. As shown in Figure 5C, SC35 localized within IGCs as well as on the PFs present at the periphery of the granules and in the nucleoplasm, whereas CF I m could only be found on PFs.Previous studies have shown that PFs are the in situ form of nascent transcripts and have suggested that they also represent sites of pre-mRNAs processing (Cmarko et al, 1999). The association of CF I m with PFs was tested by colocalization with an anti-RNA polymerase II antibody, which recognizes the active form of the enzyme ( Figure 6A).…”

Dynamics and three-dimensional localization of ribosomal RNA within the nucleolus

“…This work describes a nonisotopic method for investigating the kinetics of RNAs in cells at light and electron microscopic levels+ Surprisingly, previous studies suggest that BrUTP is poorly incorporated into RNA and that the presence of bromine inhibits its transport and/or processing+ For instance, microinjection of high concentrations of BrUTP into living cells has been proved to be toxic and prevents transport to the cytoplasm (Fay et al+, 1997), and complete substitution of uridine inhibited splicing and translation in vitro (Sierakowska et al+, 1989;Schmittgen et al+, 1994;Wansink et al+, 1994)+ However, in these previous studies, the introduction of nucleotide analogs was generally achieved by cell membrane permeabilization using Triton X-100 (Dundr & Raska, 1993;Jackson et al+, 1993;Wansink et al+, 1993;Hozak et al+, 1994;Masson et al+, 1996;Melcak et al+, 1996) or by microinjection (Wansink et al+, 1993;Masson et al+, 1996;Cmarko et al+, 1999)+ Consequently, these procedures could greatly affect the cell integrity+ Moreover, the permeabilization treatment may cause drastic alteration and partial degradation of subcellular structures+ In contrast, it has been shown recently that when cells are exposed for short periods of time to low concentrations of the nucleoside analog BrU, instead of the nucleotide analog BrUTP, they grow normally and the resulting BrRNAs are transported to the cytoplasm (Iborra et al+, 1998)+ However, this labeling only revealed nonnucleolar RNAs+…”