Discrete nuclear domains of poly(A) RNA and their relationship to the functional organization of the nucleus.

Abstract

Abstract. The functional organization of the nucleus was studied using a fluorescence microscopy approach which allowed integration of positional information for RNA, DNA, and proteins. In cells from sea urchin to human, nuclear poly(A) RNA was found concentrated primarily within several discrete "transcript domains" which often surrounded nucleoli . Concentrations of poly(A) RNA were coincident with snRNP antigen clusters, providing evidence for the localization of premRNA splicing at these sites . The spatial relationship of transcript domains with respect to various classes of DNA was established, in that the poly(A) RNA-rich regions coincided with discrete regions of low DNA density and were non-randomly distributed with respect T HE cell nucleus performs numerous complex tasks which include packaging the enormous length of DNA, high fidelity replication ofthis DNA, transcription, processing, and transport of different classes of RNA, protein import and export, and precise redistribution of nuclear components during mitosis . Additionally, many of these are done in a cell-type specific manner. It has often been proposed that the extremely dense structure of the nucleus may be spatially compartmentalized as a means to efficiently carry out its multiple functions (see Comings, 1968Comings, , 1980 . However, evidence for such physical partitioning has been modest . For example, while great advances have been made in understanding the biochemical steps involved in pre-mRNA transcription and processing, the subnuclear location ofthese events and the extent to which they are compartmentalized is unknown . In this paper we investigate the potential compartmentalization of these and other nuclear events by analyzing the distribution of poly(A) RNA in relation to several nuclear constituents of known functional significance .Unlike the cytoplasm, there are no lipid membranes in the nucleus, but the confinement to the nucleolus ofrRNA genes originating on several different chromosomes shows that elegant spatial and functional regionalization is possible. Strong evidence supporting the early suggestions that interphase centromeres and telomeres are specifically positioned (Rabl, 1885) and individual chromosomes occupy distinct territories (Boveri, 1909) has been presented recently by several

Deciphering the cellular pathway for transport of poly(A)-binding protein II

“…Immunofluorescence microscopy using affinity-purified antibodies reveals PABP2 exclusively localized in the nucleus (Fig+ 1A), as previously reported by Krause et al+ (1994)+ Within the nucleus, PABP2 appears widespread throughout the nucleoplasm with higher concentration in "speckles" (Fig+ 1A, arrows), which represent domains enriched in poly(A) RNA (Fig+ 1B, arrows; see also Carter et al+, 1991)+ To investigate whether PABP2 is permanently retained in the nucleus or transiently shuttles to the cytoplasm, we have made use of an interspecies heterokaryon assay (Borer et al+, 1989;Piñol-Roma & Dreyfuss, 1992)+ The migration of human PABP2 was monitored in human-Drosophila heterokaryons produced by polyethylene glycol-induced fusion of HeLa and SL2 cells (Fig+ 1C-E)+ Heterokaryons were kept in culture for up to 6 h in the presence or absence of the protein synthesis inhibitor emetine+ Specific identification of human PABP2 was possible because the affinity-purified antibodies do not cross-react with Drosophila proteins (Fig+ 1F,G and data not shown)+ As a control, heterokaryons were double-labeled with a monoclonal antibody specific for human hnRNP C, a protein that is always restricted to the nucleus (Piñol-Roma & Dreyfuss, 1992)+ In the absence of emetine, both human PABP2 and hnRNP C proteins synthesized in the cytoplasm of heterokaryons progressively accumulated in the Drosophila nuclei (data not shown)+ In contrast, when protein synthesis was inhibited, no human hnRNP C protein could be detected in Drosophila nuclei, whereas human PABP2 was readily visible (Fig+ 1C-E)+ Human PABP2 was first detected in Drosophila nuclei at 3 h after fusion and progressively accumulated thereafter (data not shown)+ From this we conclude that during the course of the experiment human PABP2 molecules migrated from the HeLa nucleus to the cytoplasm of the heterokaryon and from there they were rapidly imported into the Drosophila nucleus+ Thus, PABP2 shuttles between nucleus and cytoplasm, as recently reported by others (Chen et al+, 1999)+ FIGURE 1. PABP2 shuttles between nucleus and cytoplasm+ HeLa cells were immunostained with affinity-purified anti-PABP2 antibodies (A) and hybridized with a poly(U) riboprobe (B)+ PABP2 is detected exclusively in the nucleoplasm whereas poly(A) RNA is detected in both cytoplasm and nucleoplasm+ Within the nucleoplasm, PABP2 and poly(A) RNA colocalize in speckles (arrows)+ C-E: HeLa cells were fused with Drosophila SL2 cells to form heterokaryons+ HeLa cells were treated with emetine for 3 h before fusion+ After fusion the cells were kept in culture for 3 h in the presence of emetine+ Heterokaryons were fixed and double-labeled with affinity-purified anti-PABP2 antibodies (C) and monoclonal 4F4 directed against hnRNP C (D)+ E shows the corresponding phase-contrast image+ F: The affinity-purified anti-PABP2 antibodies do not react with SL2 nuclei+ G shows the corresponding phase-contrast image+ Bar ϭ 10 mm+ Nuclear import of PABP2 is carrier mediated PABP2 is a small protein (;33 kDa) that may cross the diffusion channel of nuclear pore complexes+ A possible explanation for the observed nucleocytoplasmic shuttling could therefore be that PABP2 diffuses bidirectionally through the pores but is predominantly retained in the nucleus as it binds to the growing poly(A) tails+ To address this question, we analyzed the nucleocytoplasmic distribution of PABP2 fused to nonnuclear proteins+ As reporter proteins we used GFP (;27 kDa) and a form of firefly luciferase (;60 kDa) cont...…”

Localization of hepatitis delta virus RNA in the nucleus of human cells

“…Based on the data obtained by transient expression of each form of the delta antigen in the absence of viral genome replication, we conclude that dAg-S on its own has the ability to assemble into focal aggregates+ Due to its tight interaction with HDV RNA and dAg-L, dAg-S could therefore be responsible for the appearance of the delta foci+ Because HDV depends on HBV to supply the envelope proteins required for completion of packaging, secretion, and infection (see Monjardino & Lai, 1993), mammalian cell systems such as Huh7-D12, which are solely transfected with HDV cDNA, fail to export viral particles to the cytoplasm+ Thus, the foci may represent depository sites for delta RNA and antigens trapped within the nucleus+ In this regard, we are currently analyzing the distribution of HDV components in cells that express HBV envelope proteins+ At present, it is well known that the nucleus contains discrete subdomains that appear as foci or speckles when viewed with the fluorescence microscope+ These include different types of nuclear bodies (for review, see Spector, 1993;Bohmann et al+, 1995), and we show here that the delta foci are occasionally seen in their close vicinity+ However, more than 50% of delta foci are independent from either coiled bodies or PML bodies+ In contrast, the delta foci are consistently adjacent to so-called nuclear speckles or clusters of interchromatin granules, which are highly enriched in components of the splicing machinery (Spector, 1993)+ Although it was initially proposed that nuclear speckles represent preferential splicing sites in the nucleus, subsequent experiments questioned this view+ In particular, there are now several lines of evidence indicating that splicing occurs co-transcriptionally and that nascent RNA transcripts are localized at many sites in the nucleus other than clusters of interchromatin granules (reviewed in FIGURE 8. Intranuclear distribution of ADAR1 in Huh7 and Huh7-D12 cells+ A: Huh7 cells labeled with affinity-purified anti-ADAR1 antibodies show a diffuse staining of the nucleoplasm+ B,C: Huh7-D12 cells were double-labeled with anti-ADAR1 affinity-purified rabbit antibodies (B) and a monoclonal antibody anti-p24 (C)+ Bar, 10 mm+ Fakan, 1994;Spector, 1996;Singer & Green, 1997)+ In addition to splicing factors, RNA polymerase II is also detected in clusters of interchromatin granules (see Zeng et al+, 1997 and references therein), and it is now believed that these molecules shuttle between the clusters of interchromatin granules and the sites of active RNA synthesis depending upon transcriptional activity (Misteli et al+, 1997)+ This suggests that the speckles may represent a storage compartment for inactive transcription and splicing factors (Spector, 1993(Spector, , 1996+ Alternatively, it has been proposed that transcription and splicing factors localize to clusters of interchromatin granules for some form of recycling or reactivation between rounds of pre-mRNA synthesis (Bohmann et al+, 1995;Zeng et al+, 1997)+ Experiments using oligo(dT) probes revealed that clusters of interchromatin granules contain polyadenylated RNA (Cart...…”

Evidence for a nuclear compartment of transcription and splicing located at chromosome domain boundaries

“…Therefore, they were considered so-called 'transcript domains'. These regions co-localize with splicing snRNPs (Carter et al 1991), and more recently it was shown that one RNA transcript species is preferentially associated with the poly(dA) regions (Xing et al 1993).…”

“…Furthermore, the nuclear RNA species could be delineated in preparations of the nuclear matrix (Xing & Lawrence 1991), a chromatin-depleted nucleoskeleton prepared by certain extraction procedures (for review see Berezney 1991). In situ hybridization of poly(dT) oligonucleotides delineates patch-like areas in cell nudei which are assumed to be concentrations of the nuclear fraction of poly-A + RNA (Carter et al 1991). Therefore, they were considered so-called 'transcript domains'.…”

“…By analogy to the fact that the r-DNA is localized and transcribed in the nucleolus, where the formation of the ribosome subunits takes place, it has been postulated that other functional compartments exist in cell nuclei (Blobel 1985, Nyman et al 1986, Hochstrasser & Sedat 1987, Spector 1990, Manuelidis 1990, Carter et al 1991. Hutchison and Weintraub (1985) found DNAse hypersensitive sites in the nuclear periphery, and cbncluded that active RNApolymerase 11 genes are preferentially localized in these regions.…”