Biomedical subjects

M Carmo-Fonseca

Publications and source records attributed to M Carmo-Fonseca.

At least 37 records · Page 2Linked to original sources

PubMed1999

Inefficient processing impairs release of RNA from the site of transcription.

We describe here for the first time the site of retention within the nucleus of pre-mRNA processing mutants unable to be exported to the cytoplasm. Fluorescence in situ hybridization was used to detect transcripts from human beta-globin genes that are either normal or defective in splicing or 3' end formation. Nuclear transcripts of both wild-type and mutant RNAs are detected only as intranuclear foci that colocalize with the template gene locus. The kinetics of transcript release from the site of transcription was assessed by treatment of cells with the transcriptional inhibitors actinomycin D, alpha-amanitin and DRB. These drugs induce the rapid disappearance of nuclear foci corresponding to wild-type human beta-globin RNA. In contrast, pre-mRNA mutants defective in either splicing or 3' end formation and which fail to be transported to the cytoplasm, are retained at the site of transcription. Therefore, 3' end processing and splicing appear to be rate limiting for release of mRNA from the site of transcription.

Amanitins↗

PubMed1999

Intranuclear trafficking of messenger RNA.

Within the nucleus, protein-encoding genes are transcribed into messenger RNA by RNA polymerase II. Messenger RNAs migrate to the cytoplasm, but before reaching their final destination the primary transcripts must undergo a series of modifications that include 5'-capping, splicing, and 3'-cleavage/polyadenylation. Errors in these processing events can originate aberrant products that, if translated, would produce abnormal proteins. Therefore, it is not surprising that eukaryotes have evolved a surveillance mechanism that recognizes and rapidly degrades aberrant mRNAs. Recent experiments provide exciting insights into how proper mRNAs are distinguished and selected for export. Transcription by RNA polymerase II is directly coupled to pre-mRNA processing, and the mechanism that targets the processing machinery to the polymerase complex suggests a model for co-transcriptional proofreading. Furthermore, there is evidence that at least some mRNAs move randomly throughout the nucleus, presumably by free diffusion. In this light, retention of aberrant mRNAs by the transcription/processing machinery is crucial to prevent their diffusion to the nuclear pores and eventual translocation to the cytoplasm.

Animals↗

PubMed1998

Microinjection of anti-coilin antibodies affects the structure of coiled bodies.

The coiled body is a distinct subnuclear domain enriched in small nuclear ribonucleoprotein particles (snRNPs) involved in processing of pre-mRNA. Although the function of the coiled body is still unknown, current models propose that it may have a role in snRNP biogenesis, transport, or recycling. Here we describe that anti-coilin antibodies promote a specific disappearance of the coiled body in living human cells, thus providing a novel tool for the functional analysis of this structure. Monoclonal antibodies (mAbs) were raised against recombinant human coilin, the major structural protein of the coiled body. Four mAbs are shown to induce a progressive disappearance of coiled bodies within approximately 6 h after microinjection into the nucleus of HeLa cells. After their disappearance, coiled bodies are not seen to re-form, although injected cells remain viable for at least 3 d. Epitope mapping reveals that the mAbs recognize distinct amino acid motifs scattered along the complete coilin sequence. By 24 and 48 h after injection of antibodies that promote coiled body disappearance, splicing snRNPs are normally distributed in the nucleoplasm, the nucleolus remains unaffected, and the cell cycle progresses normally. Furthermore, cells devoid of coiled bodies for approximately 24 h maintain the ability to splice both adenoviral pre-mRNAs and transiently overexpressed human beta-globin transcripts. In conclusion, within the time range of this study, no major nuclear abnormalities are detected after coiled body disappearance.

Antibodies, Monoclonal↗

PubMed1998

Cisplatin inhibits synthesis of ribosomal RNA in vivo.

Cis -diammininedichloroplatinum(II) (cisplatin or cis -DDP) is a DNA-damaging agent that is widely used in cancer chemotherapy. Cisplatin crosslinks DNA and the resulting adducts interact with proteins that contain high-mobility-group (HMG) domains, such as UBF(upstream binding factor). UBF is a transcription factor that binds to the promoter of ribosomal RNA (rRNA) genes thereby supporting initiation of transcription by RNA polymerase I. Here we report that cisplatin causes a redistribution of UBF in the nucleolus of human cells, similar to that observed after inhibition of rRNA synthesis. A similar redistribution was observed for the major components of the rRNA transcription machinery, namely TBP, TAFIs and RNA polymerase I. Furthermore, we provide for the first time direct in vivo evidence that cisplatin blocks synthesis of rRNA, while activity of RNA polymerase II continues to be detected throughout the nucleus. The clinically ineffective trans isomer (trans -DDP) does not alter the localization of either UBF or other components of the RNA polymerase I transcription machinery. These results suggest that disruption of rRNA synthesis, which is stimulated in proliferating cells, plays an important role in the clinical success of cisplatin.

Antineoplastic Agents↗

PubMed1998

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

Hepatitis delta virus (HDV) is a human pathogen that can greatly increase the severity of liver damage caused by an hepatitis B infection. HDV contains a circular, single-stranded RNA genome that encodes a unique protein, the delta antigen. Two forms of the delta antigen, deltaAg-S and deltaAg-L, are derived from a single open reading frame by RNA editing. Here we analyze the subcellular distribution of HDV RNA and its spatial relationship to known intranuclear structures. The human hepatoma cell line Huh7 was stably transfected with wild-type HDV cDNA and the viral RNAs were localized by in situ hybridization and fluorescence confocal microscopy. HDV RNA is detected throughout the nucleoplasm, with additional concentration in focal structures closely associated with nuclear speckles or clusters of interchromatin granules. Both the smaller form of the delta antigen (deltaAg-S), which is required for HDV genomic replication, and the larger form of the delta antigen (deltaAg-L), which represses replication, co-localize with delta RNA throughout the nucleoplasm and in the foci. However, the foci do not incorporate bromo-UTP and do not concentrate either RNA polymerase II or cleavage and polyadenylation factors required for viral RNA synthesis and 3' end processing, respectively. Thus, it is unlikely that the delta foci represent major sites of viral transcription or replication. In conclusion, the data show that viral RNA-protein complexes accumulate in structures closely associated with interchromatin granules, a subnuclear domain highly enriched in small nuclear ribonucleoproteins, poly(A+) RNA, and RNA splicing protein factors. This implies a specific compartmentalization of ribonucleoproteins in the nucleus.

Carcinoma, Hepatocellular↗

PubMed1998

Acute osmotic/stress stimuli induce a transient decrease of transcriptional activity in the neurosecretory neurons of supraoptic nuclei.

Administration of hypertonic NaCl solutions by intraperitoneal injection evokes a transient expression of immediate-early genes in the hypothalamic magnocellular neurons of supraoptic nuclei (SON), which is followed by an upregulation of arginine vasopressin synthesis and a general increase in cellular metabolic activity. Here we have analysed the changes that occur in the nucleus of SON neurons during the period of transient Fos expression after injection of hypertonic saline. Within the first 30 minutes after injection, the nuclei become significantly smaller, contain more condensed chromatin and incorporate less 3H-uridine than the controls. By 12 hours these effects are reverting and at 24 hours the nuclei are already more active than the controls. Additionally, we observe an initial decrease in the number of coiled bodies per nucleus within the first 2 hours, followed by a 3-fold increase at 24 hours after injection. As coiled bodies are transcription-dependent subnuclear 'organelles', these results further support the view that injection of hypertonic saline causes a transient inhibition of nuclear activity. Our data show that SON neurons respond to acute osmotic/stress stimuli first with inhibition and then with activation of gene expression. Importantly, inhibition of transcriptional activity occurs simultaneously with maximal accumulation of Fos protein in the nucleus, raising the possibility that activation of c-fos expression may cause repression of target genes.

Animals↗

PubMed1997

Semithin cryosections as a tool to perform high resolution immunofluorescence and in situ hybridization analysis of the nervous tissue: a study in the supraoptic nucleus.

Immunofluorescence and fluorescence in in situ hybridization represent powerful approaches to correlate biochemical and molecular data with the structural organization of cells and tissues. However, the analysis of tissues by fluorescence microscopy is limited by the fact that most methods currently used to preserve the morphological integrity of sectioned samples at high resolution do not allow access of the labeled probes to the target molecules. Here we have made use of semithin cryosections obtained from rat supraoptic nucleus to perform immunofluorescence with antibodies directed against cytoplasmic and nuclear antigens, as well as fluorescence in situ hybridization with antisense oligonucleotide probes complementary to the poly(A) tail of mRNA and to specific mRNAs. In addition, DNA was visualized by incubation of sections with digoxigenin-labeled nucleotides in the presence of Escherichia coli DNA polymerase I. The high resolution of this DNA staining in combination with immunolabeling for nuclear antigens provides a powerful tool to analyze the structural and functional compartmentalization of neuronal cell nuclei. The major conclusion from this study is that performing fluorescence microscopy on 1 micron-thick cryosections provides an important tool to accurately localize proteins, DNA and RNA within nervous tissue in general and particularly in the model of supraoptic nucleus. Moreover, the cryosectioning technique appears particularly suited to the study of the localization of specific mRNA species in the neuronal cytoplasm and represents a useful approach to addressing the functional significance of mRNA localization in protein targeting.

Animals↗

PubMed1997

Targeting of U2AF65 to sites of active splicing in the nucleus.

U2AF65 is an essential splicing factor that promotes binding of U2 small nuclear (sn)RNP at the pre-mRNA branchpoint. Here we describe a novel monoclonal antibody that reacts specifically with U2AF65. Using this antibody, we show that U2AF65 is diffusely distributed in the nucleoplasm with additional concentration in nuclear speckles, which represent subnuclear compartments enriched in splicing snRNPs and other splicing factors. Furthermore, transient expression assays using epitope-tagged deletion mutants of U2AF65 indicate that targeting of the protein to nuclear speckles is not affected by removing either the RNA binding domain, the RS domain, or the region required for interaction with U2AF35. The association of U2AF65 with speckles persists during mitosis, when transcription and splicing are downregulated. Moreover, U2AF65 is localized to nuclear speckles in early G1 cells that were treated with transcription inhibitors during mitosis, suggesting that the localization of U2AF65 in speckles is independent of the presence of pre-mRNA in the nucleus, which is consistent with the idea that speckles represent storage sites for inactive splicing factors. After adenovirus infection, U2AF65 redistributes from the speckles and is prefferentially detected at sites of viral transcription. By combining adenoviral infection with transient expression of deletion mutants, we show a specific requirement of the RS domain for recruitment of U2AF65 to sites of active splicing in the nucleus. This suggests that interactions involving the RS region of U2AF65 may play an important role in targeting this protein to spliceosomes in vivo.

Amino Acid Sequence↗

PubMed1997

The spatial distribution of human immunoglobulin genes within the nucleus: evidence for gene topography independent of cell type and transcriptional activity.

The three-dimensional positioning of immunoglobulin (Ig) genes within the nucleus of human cells was investigated using in situ hybridization and confocal microscopy. The visualization of heavy and light chain genes in B-lymphoid cells showed that the three Ig genes are differentially and nonrandomly distributed in different nuclear subvolumes: the kappa genes were found to be preferentially confined to an outer nuclear volume, whereas the gamma and lambda genes consistently occupied more central positions within the nucleus, the lambda genes being more interior when compared with the gamma genes. The data further show that these overall topographical distributions are independent of gene transcriptional activity and are conserved in different cell types. Although subtle gene movements within those defined topographical regions cannot be excluded by this study, the results indicate that tissue specificity of gene expression is not accompanied by drastic changes in gene nuclear topography, rather suggesting that gene organization within the nucleus may be primarily dependent on structural constraints imposed on the respective chromosomes.

B-Lymphocytes↗

PubMed1997

The cdk7-cyclin H-MAT1 complex associated with TFIIH is localized in coiled bodies.

TFIIH is a general transcription factor for RNA polymerase II that in addition is involved in DNA excision repair. TFIIH is composed of eight or nine subunits and we show that at least four of them, namely cdk7, cyclin H, MAT1, and p62 are localized in the coiled body, a distinct subnuclear structure that is transcription dependent and highly enriched in small nuclear ribonucleoproteins. Although coiled bodies do not correspond to sites of transcription, in vivo incorporation of bromo-UTP shows that they are surrounded by transcription foci. Immunofluorescence analysis using antibodies directed against the essential repair factors proliferating cell nuclear antigen and XPG did not reveal labeling of the coiled body in either untreated cells or cells irradiated with UV light, arguing that coiled bodies are probably not involved in DNA repair mechanisms. The localization of cyclin H in the coiled body was predominantly detected during the G1 and S-phases of the cell cycle, whereas in G2 coiled bodies were very small or not detected. Finally, both cyclin H and cdk7 did not colocalize with P80 coilin after disruption of the coiled body, indicating that these proteins are specifically targeted to the small nuclear ribonucleoprotein-containing domain.

Carrier Proteins↗

PubMed1997

Genome replication in early mouse embryos follows a defined temporal and spatial order.

The spatial and temporal organisation of replication sites during early mouse embryogenesis was analysed using high resolution confocal and video fluorescence microscopy. The results show that distinct replication patterns occur in the transcriptionally inactive pronuclei of 1-cell embryos as well as in the transcriptionally active nuclei from 2- and 16/32-cell embryos. This indicates that specific chromatin regions are replicated at different times during S-phase and provides the first evidence that mechanisms controlling the temporal and spatial replication of DNA are already present in the haploid pronuclei of the mammalian zygote. Furthermore the data demonstrate that the male and female pronuclei in one-cell embryos replicate their genomes asynchronously. Finally, we observe changes in the dynamics of embryonic genome replication during early development which correlate with gross chromatin structure transitions detected at the electron microscope level. Taken together these results indicate that DNA synthesis in the mouse zygote follows a defined four-dimensional order which may evolve during development and differentiation.

Animals↗

PubMed1996

The topography of chromosomes and genes in the nucleus.

At present several lines of evidence indicate that the nucleus is functionally compartmentalized into discrete structures with defined properties. For instance, it is well established that the molecular machines involved in replication, transcription, and RNA processing assemble into morphological entities but it remains unclear whether these correspond to autonomous "organelles" or rather represent temporary accumulations of either active factors recruited onto DNA templates or inactive factors stored in the vicinity of genes. According to the latter view, the spatial distribution of genes during interphase should play a major role in the subnuclear organization of nonchromatin components such as replication, transcription, and processing factors, and in this article we discuss current ideas on the functional compartmentalization of chromosomes and genes within the nucleus.

Binding Sites↗

PubMed1996

GATA transcription factors associate with a novel class of nuclear bodies in erythroblasts and megakaryocytes.

The nuclear distribution of GATA transcription factors in murine haemopoietic cells was examined by indirect immunofluorescence. Specific bright foci of GATA-1 fluorescence were observed in erythroleukaemia cells and primary murine erythroblasts and megakaryocytes, in addition to diffuse nucleoplasmic localization. These foci, which were preferentially found adjacent to nucleoli or at the nuclear periphery, did not represent sites of active transcription or binding of GATA-1 to consensus sites in the beta-globin loci. Immunoelectron microscopy demonstrated the presence of intensely labelled structures likely to represent the GATA-1 foci seen by immunofluorescence. The GATA-1 nuclear bodies differed from previously described nuclear structures and there was no co-localization with nuclear antigens involved in RNA processing or other ubiquitous (Spl, c-Jun and TBP) or haemopoietic (NF-E2) transcription factors. Interestingly, GATA-2 and GATA-3 proteins also localized to the same nuclear bodies in cell lines co-expressing GATA-1 and -2 or GATA-1 and -3 gene products. This pattern of distribution is, thus far, unique to the GATA transcription factors and suggests a protein-protein interaction with other components of the nuclear bodies via the GATA zinc finger domain.

Animals↗

PubMed1996

Nuclear morphogenesis and the onset of transcriptional activity in early hamster embryos.

Coiled bodies and interchromatin granules are distinct subnuclear domains that contain splicing small nuclear ribonucleoproteins (snRNPs) and protein-splicing factors. Here we have studied the morphogenesis of coiled bodies and clusters of interchromatin granules in relation to the onset of transcriptional activity in early hamster embryos. The results indicate that major embryonic transcription by RNA polymerase II is first detected during the early two-cell stage (15-20 h post-fertilization), whereas RNA polymerase I activity and nucleologenesis are only observed in late two-cell embryos (30-40 h postfertilization). Splicing snRNPs and heterogeneous nuclear RNP (hnRNP) proteins are shown to be imported into the pronuclei following fertilization, and prominent clusters of interchromatin granules containing the splicing factor SC-35 are already observed in both maternal and paternal pronuclei of one-cell embryos. Interestingly, these large clusters of interchromatin granules do not appear to concentrate splicing snRNPs. In contrast, coiled bodies are first detected during the two-cell stage after the onset of transcription, and they are clearly enriched in snRNPs. Taken together with results previously obtained in mouse embryos, these data suggest that the assembly of coiled bodies and clusters of interchromatin granules is independent from the onset of embryonic transcriptional activity, and that coiled bodies represent the major snRNP-enriched subnuclear domain in the early mammalian embryo.

Animals↗

PubMed1996

In vivo evidence that TATA-binding protein/SL1 colocalizes with UBF and RNA polymerase I when rRNA synthesis is either active or inactive.

Here we show that the TATA-binding protein (TBP) is localized in the nucleoplasm and in the nucleolus of mammalian cells, consistent with its known involvement in transcription by RNA polymerase I, II, and III. In the nucleolus of actively growing cells, TBP colocalizes with upstream binding factor (UBF) and RNA polymerase I at the sites of rRNA transcription. During mitosis, when rRNA synthesis is down-regulated, TBP colocalizes with TBP-associated factors for RNA polymerase I (TAF(I)s), UBF, and RNA polymerase I on the chromosomal regions containing the rRNA genes. Treatment of cells with a low concentration of actinomycin D inhibits rRNA synthesis and causes a redistribution of the rRNA genes that become concentrated in clusters at the periphery of the nucleolus. A similar redistribution was observed for the major components of the rRNA transcription machinery (i.e., TBP, TAF(I)s, UBF, and RNA polymerase I), which still colocalized with each other. Furthermore, anti-TBP antibodies are shown to coimmunoprecipitate TBP and TAF(I)63 in extracts prepared from untreated and actinomycin D-treated cells. Collectively, the data indicate that in vivo TBP/promoter selectivity factor, UBF, and RNA polymerase I remain associated with both active and inactive rRNA genes.

3T3 Cells↗

PubMed1996

Characterization of a helicase-like transcription factor involved in the expression of the human plasminogen activator inhibitor-1 gene.

A 5.4-kb cDNA encoding the protein that binds to the B Box of the plasminogen activator inhibitor-1 (PAI-1) gene was isolated and sequenced. The protein, named helicase-like transcription factor (HLTF), contains a DNA-binding domain, a RING finger domain, and seven helicase domains and is homologous to SWI/SNF proteins. Two HLTF mRNAs of 5.5 and 4.5 kb were detected in most human tissues, a single gene was located on chromosome 3q24-25, and the protein was located in the nucleoplasm. Two HLTF proteins differing in translation start site (Met-1 or Met-123) were obtained by in vitro translation in reticulocyte lysate or by immunoprecipitation from HeLa cell nuclear extracts. In vitro transcription from the PAI-1 promoter in HeLa cell extracts was inhibited by HLTF antibodies and by the HLTF DNA binding domain. Over-expression of HLTF or HLTFMet123 produced a three-fold induction of PAI-1-LUC transient expression in HeLa cells. Mutation of the PAI-1 B Box led to an eight-fold reduction of basal PAI-1-LUC expression in these cell lines, but did not affect the four- to six-fold induction by phorbol esters.

Amino Acid Sequence↗

PubMed1996

The dynamics of coiled bodies in the nucleus of adenovirus-infected cells.

The coiled body is a specific intranuclear structure of unknown function that is enriched in splicing small nuclear ribonucleoproteins (snRNPs). Because adenoviruses make use of the host cell-splicing machinery and subvert the normal subnuclear organization, we initially decided to investigate the effect of adenovirus infection on the coiled body. The results indicate that adenovirus infection induces the disassembly of coiled bodies and that this effect is probably secondary to the block of host protein synthesis induced by the virus. Furthermore, coiled bodies are shown to be very labile structures, with a half-life of approximately 2 h after treatment of HeLa cells with protein synthesis inhibitors. After blocking of protein synthesis, p80 coilin was detected in numerous microfoci that do not concentrate snRNP. These structures may represent precursor forms of the coiled body, which goes through a rapid cycle of assembly/disassembly in the nucleus and requires ongoing protein synthesis to reassemble.

Adenovirus Infections, Human↗

PubMed1995

Influence of transcription and replication on the in situ resolution of immunoglobulin heavy-chain constant region genes: an interphase cytogenetics analysis.

An interphase cytogenetics analysis was performed to investigate whether replication and transcription could influence in situ resolution of immunoglobulin (Ig) heavy chain constant region genes. A plasmid probe recognizing five C gamma segments separated by known linear DNA distances was hybridized in situ and visualized by digital fluorescence microscopy. In interphase nuclei from phytohemagglutinin (PHA)-stimulated lymphocytes, the gamma genes were resolved as one to three signals per allele in the majority of nuclei, whereas in a minority, complex patterns of several signals per allele could be observed. The latter were restricted to nuclei in an early stage of the S phase, as assessed by hybridization experiments performed in cells grown in the presence of bromodeoxyuridine. To investigate whether the in situ resolution of the C gamma segments could vary as a function of the transcription activity of the locus, the C gamma probe was subsequently hybridized to nuclei from a mature B cell line (JVM-2), which produces gamma transcripts as shown by in situ RNA hybridization experiments. Primary human fibroblasts were further used as representative of a non-lymphoid cell type with transcriptionally inactive Ig genes. When Gl nuclei from the three cell types were compared in terms of the in situ resolution of the C gamma locus, JVM-2 cells were found to include the highest percentage of higher resolution patterns (three to five signals per allele in 28% of nuclei), fibroblasts the lowest (three signals per allele, 2%), while PHA-stimulated lymphocytes occupied an intermediate position between the other two cell types (three or four signals per allele, 15%). The data show that the in situ resolution of Ig C gamma genes varies throughout the cell cycle and is influenced by the transcriptional activity of the locus. The variability of the resolution patterns observed appears to reflect different levels of chromatin packaging, which in turn are likely to influence the probe accessibility to its target. These observations are relevant for the interpretation of data from interphase cytogenetics analysis of independent, but closely spaced, DNA segments.

Cells, Cultured↗