An ATP-dependent step is required for the translocation of microinjected precursor mRNA into nuclear speckles.

Nuclear speckles (speckles) represent a distinct nuclear compartment within the interchromatin space and are enriched in splicing factors. In a previous study (Melcák et al., 2001), it has been shown that the pre-spliceosomal assembly on microinjected splicing-competent precursor mRNA takes place in the speckles, and it has been suggested that the targeting of RNA into speckes consists of two interdependent steps, namely the diffusion process, followed by the energy-dependent translocation of RNA into the speckles. In the present study, we confirm the existence of these two steps and show that this latter translocation is ATP dependent.

Prespliceosomal assembly on microinjected precursor mRNA takes place in nuclear speckles.

Nuclear speckles (speckles) represent a distinct nuclear compartment within the interchromatin space and are enriched in splicing factors. They have been shown to serve neighboring active genes as a reservoir of these factors. In this study, we show that, in HeLa cells, the (pre)spliceosomal assembly on precursor mRNA (pre-mRNA) is associated with the speckles. For this purpose, we used microinjection of splicing competent and mutant adenovirus pre-mRNAs with differential splicing factor binding, which form different (pre)spliceosomal complexes and followed their sites of accumulation. Splicing competent pre-mRNAs are rapidly targeted into the speckles, but the targeting is temperature-dependent. The polypyrimidine tract sequence is required for targeting, but, in itself, is not sufficient. The downstream flanking sequences are particularly important for the targeting of the mutant pre-mRNAs into the speckles. In supportive experiments, the behavior of the speckles was followed after the microinjection of antisense deoxyoligoribonucleotides complementary to the specific domains of snRNAs. Under these latter conditions prespliceosomal complexes are formed on endogenous pre-mRNAs. We conclude that the (pre)spliceosomal complexes on microinjected pre-mRNA are formed inside the speckles. Their targeting into and accumulation in the speckles is a result of the cumulative loading of splicing factors to the pre-mRNA and the complexes formed give rise to the speckled pattern observed.

Nuclear pre-mRNA compartmentalization: trafficking of released transcripts to splicing factor reservoirs.

In the present study, the spatial organization of intron-containing pre-mRNAs of Epstein-Barr virus (EBV) genes relative to location of splicing factors is investigated. The intranuclear position of transcriptionally active EBV genes, as well as of nascent transcripts, is found to be random with respect to the speckled accumulations of splicing factors (SC35 domains) in Namalwa cells, arguing against the concept of the locus-specific organization of mRNA genes with respect to the speckles. Microclusters of splicing factors are, however, frequently superimposed on nascent transcript sites. The transcript environment is a dynamic structure consisting of both nascent and released transcripts, i.e., the track-like transcript environment. Both EBV sequences of the chromosome 1 homologue are usually associated with the track, are transcriptionally active, and exhibit in most cases a polar orientation. In contrast to nascent transcripts (in the form of spots), the association of a post-transcriptional pool of viral pre-mRNA (in the form of tracks) with speckles is not random and is further enhanced in transcriptionally silent cells when splicing factors are sequestered in enlarged accumulations. The transcript environment reflects the intranuclear transport of RNA from the sites of transcription to SC35 domains, as shown by concomitant mapping of DNA, RNA, and splicing factors. No clear vectorial intranuclear trafficking of transcripts from the site of synthesis toward the nuclear envelope for export into the cytoplasm is observed. Using Namalwa and Raji cell lines, a correlation between the level of viral gene transcription and splicing factor accumulation within the viral transcript environment has been observed. This supports a concept that the level of transcription can alter the spatial relationship among intron-containing genes, their transcripts, and speckles attributable to various levels of splicing factors recruited from splicing factor reservoirs. Electron microscopic in situ hybridization studies reveal that the released transcripts are directed toward reservoirs of splicing factors organized in clusters of interchromatin granules. Our results point to the bidirectional intranuclear movement of macromolecular complexes between intron-containing genes and splicing factor reservoirs: the recruitment of splicing factors to transcription sites and movement of released transcripts from DNA loci to reservoirs of splicing factors.

Structural organization of the pre-mRNA splicing commitment: a hypothesis.

Several models have been presented in the past to explain localized distributions of nuclear RNAs from individual genes that range from small foci to more elongated "track-like" structures. We present here a hypothesis which explains that, in the case of regulated splicing, there is in diploid cells a spatial separation of transcription sites from the execution of regulated splicing which we situate to domains of SR protein accumulation. In addition, it explains the presence of poly(A) sequences, and the lack of the autoradiographic label due to short pulses of [3H]uridine, in these domains.

Ultrastructural nonisotopic mapping of nucleolar transcription sites in onion protoplasts.

The post- and preembedding ultrastructural localization of transcribing rRNA genes has been carried out in nucleoli of permeabilized onion growing root tip protoplasts by means of the nonisotopic bromouridine method. By means of both post- and preembedding approaches, major synthetic sites were identified with morphologically distinct subdomains of dense fibrillar components, with some signal also being associated with nucleolar fibrillar centers and vacuoles. Moreover, labeled medusoid fibrils within distinct domains seen in Lowicryl thin sections likely represent the morphological correlate of transcribing nucleolar genes.

Does the synthesis of ribosomal RNA take place within nucleolar fibrillar centers or dense fibrillar components? A critical appraisal.

The localization of transcribing rRNA genes within nucleoli of mammalian cells, although intensively studied, has not been established. Most published papers on this topic situate transcribing ribosomal genes either to nucleolar fibrillar centers or to nucleolar dense fibrillar components. To clarify this point, we have generated the electron microscopic affinity cytochemistry picture of the nucleolus of cultured mammalian cells. Three kinds of affinity probes have been used: (1) probes to nucleolar chromatin, including rDNA sequences; (2) probes to a number of macromolecules (such as RNA polymerase I) which are directly, or indirectly, involved in the synthesis and processing of rRNA and formation of preribosomes; (3) antibodies to bromouridine for a recently standardized nonisotopical method depicting incorporated bromouridine within RNA. The results suggest the localization of transcription sites not only to dense fibrillar components but also to the border region between these components and fibrillar centers. Our data support a hypothesis that in metabolically active mammalian nucleoli, fibrillar centers and dense fibrillar components form a single functional domain for the transcription of rRNA genes, with nascent transcripts generating "automatically" dense fibrillar components. Through the active process of transcription, individual rRNA genes thus become engulfed within dense fibrillar components.

Immunoelectron microscopic localization of small nuclear ribonucleoproteins and interchromatin granules in the 2-cell mouse embryo.

The distribution of U1, U2, U4, U5 and U6 small nuclear ribonucleoproteins (snRNPs) and interchromatin granules (IGs) was studied by electron-microscopic immunocytochemistry (EMI) in early 2-cell mouse embryos at the onset of embryonal transcription. The localization of these antigen structures was evaluated with respect to nucleoplasmic ribonucleoprotein (RPN) regions consisting of interchromatin and perichromatin areas. SnRNP structures of maternal origin (labelled with anti-Sm antibody) were widely distributed throughout the nucleoplasm. Specifically labelled IGs were detected by gold particle clusters distributed in the interchromatin regions of the nucleoplasm. Both immunodetections were negative in nucleolar precursor bodies (NPBs). In addition, the labelling of condensed chromatin blocks with anti-DNA antibody showed heterochromatin topology at this developmental stage. Small condensed chromatin blocks were distributed throughout the nucleus and also appeared in association with the NPB rim. The observed status quo represents a transient state of nuclear structure rearrangement.