The Experts below are selected from a list of 159 Experts worldwide ranked by ideXlab platform
Luis Eduardo Coelho Andrade - One of the best experts on this subject based on the ideXlab platform.
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the behavior of the Coiled Body in cells infected with adenovirus in vitro
Molecular Biology Reports, 1996Co-Authors: Silvia Helena Rodrigues, Neusa Pereira Da Silva, Luis R Delicio, Celso Francisco Hernandes Granato, Luis Eduardo Coelho AndradeAbstract:The Coiled Body is a phylogenetically conserved nuclear organelle whose function is not known. Probes for detection of p80-coilin, an 80 kDa protein enriched in the Coiled Body, have made possible studies determining the behavior of the Coiled Body during the cell cycle, in proliferating cells, as well as reports suggesting some relationship of the Coiled Body to mRNA splicing and to the nucleolus. The objective of this study is to examine the distribution of p80-coilin and nucleolar proteins in cells infected with adenovirus in vitro. HeLa cells grown as monolayers were infected with successive dilutions of type 5 human adenovirus culture and fixed in methanol/acetone at different time points. Single and double indirect immunofluorescence was performed with human autoantibodies to p80-coilin, fibrillarin, NOR-90/hUBF, RNA polymerase I, PM-Scl, and To, as well as rabbit polyclonal serum to p80-coilin (R288) and mouse monoclonal antiBody to adenovirus 72-kDa DNA-binding protein. Indirect immunofluorescence (IIF) with anti-p80-coilin antibodies showed that the usual bright dot-like Coiled Body staining pattern was replaced in infected cells by 1-5 clusters of tiny dots at the periphery of the nucleus. This phenomenon was first detected within 12 h of infection and affected more severely cells with increased length and load of infection. Cells subjected to heat shock presented no such alteration. Double IIF showed cells with abnormal Coiled Body appearance expressed the viral 72-kDa DNA-binding protein. Nucleolar proteins RNA polymerase I and NOR-90/hUBF became associated with the p80-coilin-enriched clusters and were no longer detected in the nucleolus. Other nucleolar proteins, like PM-Scl and To, remained associated to the nucleolus and were not detected in the newly formed clusters. Fibrillarin had a heterogeneous behavior, being restricted to the nucleolus in some infected cells while in some others it was associated with the p80-coilin-enriched clusters. Thus our results showed that in vitro adenovirus infection induced radical redistribution of nucleolar and Coiled Body constituents into newly formed structures characterized by clusters of tiny dots in the periphery of the nucleus. The fact that three major proteins involved in rRNA synthesis and processing colocalized with p80-coilin in these clusters may bring additional support to the idea that the Coiled Body and p80-coilin may be implicated in functions related to the nucleolus.
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formation of nuclear bodies in hepatocytes of estrogen treated roosters
Molecular Biology of the Cell, 1995Co-Authors: Robert L Ochs, Luis Eduardo Coelho Andrade, Edward K L Chan, Eng M Tan, T W Stein, D Gallo, K BraschAbstract:Abstract As a model for cellular growth and stimulation without accompanying proliferation, we have examined the induction and formation of nuclear bodies (NBs) in hepatocytes of estrogen-treated roosters. Four-week-old roosters were injected with a single intramuscular dose of estradiol and then killed at time points of 8 h, 48 h, and 4 wk post-injection. For immunofluorescence analyses, livers were excised and isolated hepatocyte nuclei were fixed and then labeled with antiBody to the Coiled Body-specific protein, p80-coilin. In control animals (no estradiol) or in animals 8 h post-injection, each hepatocyte nucleus contained an average of 1.0 Coiled Body (CB), which appeared randomly distributed in the nucleoplasm. At 48 h post-injection, there were an average of 2.7 CBs/nucleus and many of these appeared to be in contact with the nucleolus. Pairs of CBs were also observed. By 4 wk post-injection an average of 1.5 CBs/nucleus were detected, with no apparent relationship to the nucleolus observed. By serial-section electron microscopy of intact livers, two different types of round NBs were observed, sometimes in close proximity to each other and to the expanded interchromatin granule region in maximally stimulated cells. One type of NB was a classical CB that averaged 0.35 microns in diameter and the other NB type was ring shaped, averaged 0.25 microns in diameter, was composed of a fibrous shell surrounding a hollow interior, and appeared as a simple NB when sectioned tangentially through its outer shell. Immunoelectron microscopy revealed that CBs were the only class of NBs that contained p80-coilin. From these data, we conclude that CBs proliferate in response to estrogen stimulation, possibly arising from the nucleolar surface and then increasing in number by replicative division.
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immunocytochemical analysis of the Coiled Body in the cell cycle and during cell proliferation
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Luis Eduardo Coelho Andrade, Eng M Tan, Edward K L ChanAbstract:Abstract Coiled bodies (CBs) are small, round structures found in the nucleoplasm of most eukaryotic cells. Human autoantibodies to a 80-kDa protein, p80-coilin, are immunohistologic markers for CBs. A polyclonal rabbit antiserum (R288) raised against recombinant p80-coilin was shown to have similar immunochemical properties as human autoantibodies and was used to analyze the expression of p80-coilin-associated CBs in cell cultures synchronized by double thymidine block, nocodazole arrest, serum starvation, or hormonal deprivation. By employing thymidine block and nocodazole arrest of HeLa cells, CBs were observed in immunofluorescent studies to be largest in size in the S and G2 phases of the cell cycle. These large CBs might have coalesced into one or two such structures per cell from smaller and more numerous CBs of three to eight per cell during the mid G1 phase of the cell cycle. No CB-like structures were observed in mitosis and early G1. However, immunoblotting analyses showed that the total amount of p80-coilin remained approximately the same throughout the cell cycle. When HeLa cells were separated into soluble and particulate fractions, p80-coilin was detected predominantly in the soluble fraction in mitosis and early G1, whereas it was present predominantly in the particulate fraction in late G1, S, and G2 when structurally distinct CBs were observed. In the analysis of CBs in two experimental models of cell proliferation (reversal of 3T3 serum starvation and FRTL-5 thyrotropin deprivation), proliferating cells contained larger, brighter, and more numerous CBs as well as a > 2-fold increase in the total amount of p80-coilin compared to that in quiescent cells. The expression of p80-coilin in quiescent cells induced to proliferate and the cyclic formation and breakdown of CBs might be consistent with the notion that CBs may be specialized centers related to the maturation of mRNA, but this evidence is indirect and needs further definitive study.
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immunological and ultrastructural studies of the nuclear Coiled Body with autoimmune antibodies
Experimental Cell Research, 1991Co-Authors: Ivan Raska, Luis Eduardo Coelho Andrade, Robert L Ochs, Edward K L Chan, Chengming Chang, Goran Roos, Eng M TanAbstract:Abstract Studies with human autoimmune sera identified autoantibodies reacting with a novel antigen of 80 kDa. In interphase mammalian cells, the 80-kDa antigen was enriched in nuclear Coiled bodies and was used as a marker for this nuclear structure. This antigen was subsequently named p80-coilin. By light and electron microscopic immunocytochemistry, a number of other antigens were also localized to the Coiled Body, including components of small nuclear ribonucleoproteins which are involved in the processing of nucleolar and extranucleolar RNA. Although the function of the Coiled Body is unknown, the presence of these subcellular particles might indicate an involvement in RNA metabolism. The identification of a protein highly enriched in this structure and the availability of specific antibodies might help in its isolation and the study of its function.
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human autoantiBody to a novel protein of the nuclear Coiled Body immunological characterization and cdna cloning of p80 coilin
Journal of Experimental Medicine, 1991Co-Authors: Luis Eduardo Coelho Andrade, Ivan Raska, Edward K L Chan, Goran Roos, Carol L Peebles, Eng M TanAbstract:Antibodies producing an unusual immunofluorescent pattern were identified in the sera of patients with diverse autoimmune features. This pattern was characterized by the presence of up to six round discrete nuclear bodies in interphase cell nuclei. Immunoblotting analysis showed that these sera recognized an 80-kD nuclear protein, and affinity-purified anti-p80 antiBody from the protein band reproduced the fluorescent staining of nuclear bodies. Colloidal gold immunoelectron microscopy showed that the affinity-purified anti-p80 antiBody recognized the Coiled Body, an ultramicroscopic nuclear structure probably first described by the Spanish cytologist Ramon y Cajal. Five cDNA clones were isolated from a MOLT-4 cell lambda gt-11 expression library using human antiBody and oligonucleotide probes. The longest cDNA insert was 2.1 kb and had an open reading frame of 405 amino acids. A clone encoding a 14-kD COOH-terminal region of the protein was used for expression of a beta-galactosidase fusion protein. An epitope was present in this COOH-terminal 14-kD region, which was recognized by 18 of 20 sera with anti-p80 reactivity, and affinity-purified antiBody from the recombinant protein also reacted in immunofluorescence to show specific staining of the Coiled Body. This is the first demonstration and molecular cloning of a protein that appears to have particular identification with the Coiled Body, and it was designated p80-coilin. AutoantiBody to p80-coilin may be useful for the elucidation of the structure and function of the Coiled Body, and the availability of a cDNA sequence could be helpful in further studies to clarify the clinical significance of this autoantiBody response.
Edward K L Chan - One of the best experts on this subject based on the ideXlab platform.
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formation of nuclear bodies in hepatocytes of estrogen treated roosters
Molecular Biology of the Cell, 1995Co-Authors: Robert L Ochs, Luis Eduardo Coelho Andrade, Edward K L Chan, Eng M Tan, T W Stein, D Gallo, K BraschAbstract:Abstract As a model for cellular growth and stimulation without accompanying proliferation, we have examined the induction and formation of nuclear bodies (NBs) in hepatocytes of estrogen-treated roosters. Four-week-old roosters were injected with a single intramuscular dose of estradiol and then killed at time points of 8 h, 48 h, and 4 wk post-injection. For immunofluorescence analyses, livers were excised and isolated hepatocyte nuclei were fixed and then labeled with antiBody to the Coiled Body-specific protein, p80-coilin. In control animals (no estradiol) or in animals 8 h post-injection, each hepatocyte nucleus contained an average of 1.0 Coiled Body (CB), which appeared randomly distributed in the nucleoplasm. At 48 h post-injection, there were an average of 2.7 CBs/nucleus and many of these appeared to be in contact with the nucleolus. Pairs of CBs were also observed. By 4 wk post-injection an average of 1.5 CBs/nucleus were detected, with no apparent relationship to the nucleolus observed. By serial-section electron microscopy of intact livers, two different types of round NBs were observed, sometimes in close proximity to each other and to the expanded interchromatin granule region in maximally stimulated cells. One type of NB was a classical CB that averaged 0.35 microns in diameter and the other NB type was ring shaped, averaged 0.25 microns in diameter, was composed of a fibrous shell surrounding a hollow interior, and appeared as a simple NB when sectioned tangentially through its outer shell. Immunoelectron microscopy revealed that CBs were the only class of NBs that contained p80-coilin. From these data, we conclude that CBs proliferate in response to estrogen stimulation, possibly arising from the nucleolar surface and then increasing in number by replicative division.
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is the Coiled Body involved in nucleolar functions
Experimental Cell Research, 1994Co-Authors: Manuela Malatesta, Edward K L Chan, Carlo Zancanaro, Terence E Martin, Francois Amalric, R Luhrmann, Peter Vogel, S FakanAbstract:Coiled bodies (CBs) are structural constituents observed in nuclei of most eukaryotic cells. They usually occur in the nucleoplasm as well as in contact with the nucleolar surface. In this work we studied the hepatocyte nuclei of hibernating dormice in order to investigate possible modifications of CBs along the seasonal cycle. CBs were abundant during hibernation and rapidly disappeared upon arousal from hibernation. Moreover, CBs were frequently found to be integrated into the nucleolar Body. Immunocytochemical analyses showed that CBs contain nucleoplasmic as well as nucleolar RNA-processing factors, suggesting an "ambiguous" role for this organelle in the nuclear functions.
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immunocytochemical analysis of the Coiled Body in the cell cycle and during cell proliferation
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Luis Eduardo Coelho Andrade, Eng M Tan, Edward K L ChanAbstract:Abstract Coiled bodies (CBs) are small, round structures found in the nucleoplasm of most eukaryotic cells. Human autoantibodies to a 80-kDa protein, p80-coilin, are immunohistologic markers for CBs. A polyclonal rabbit antiserum (R288) raised against recombinant p80-coilin was shown to have similar immunochemical properties as human autoantibodies and was used to analyze the expression of p80-coilin-associated CBs in cell cultures synchronized by double thymidine block, nocodazole arrest, serum starvation, or hormonal deprivation. By employing thymidine block and nocodazole arrest of HeLa cells, CBs were observed in immunofluorescent studies to be largest in size in the S and G2 phases of the cell cycle. These large CBs might have coalesced into one or two such structures per cell from smaller and more numerous CBs of three to eight per cell during the mid G1 phase of the cell cycle. No CB-like structures were observed in mitosis and early G1. However, immunoblotting analyses showed that the total amount of p80-coilin remained approximately the same throughout the cell cycle. When HeLa cells were separated into soluble and particulate fractions, p80-coilin was detected predominantly in the soluble fraction in mitosis and early G1, whereas it was present predominantly in the particulate fraction in late G1, S, and G2 when structurally distinct CBs were observed. In the analysis of CBs in two experimental models of cell proliferation (reversal of 3T3 serum starvation and FRTL-5 thyrotropin deprivation), proliferating cells contained larger, brighter, and more numerous CBs as well as a > 2-fold increase in the total amount of p80-coilin compared to that in quiescent cells. The expression of p80-coilin in quiescent cells induced to proliferate and the cyclic formation and breakdown of CBs might be consistent with the notion that CBs may be specialized centers related to the maturation of mRNA, but this evidence is indirect and needs further definitive study.
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immunological and ultrastructural studies of the nuclear Coiled Body with autoimmune antibodies
Experimental Cell Research, 1991Co-Authors: Ivan Raska, Luis Eduardo Coelho Andrade, Robert L Ochs, Edward K L Chan, Chengming Chang, Goran Roos, Eng M TanAbstract:Abstract Studies with human autoimmune sera identified autoantibodies reacting with a novel antigen of 80 kDa. In interphase mammalian cells, the 80-kDa antigen was enriched in nuclear Coiled bodies and was used as a marker for this nuclear structure. This antigen was subsequently named p80-coilin. By light and electron microscopic immunocytochemistry, a number of other antigens were also localized to the Coiled Body, including components of small nuclear ribonucleoproteins which are involved in the processing of nucleolar and extranucleolar RNA. Although the function of the Coiled Body is unknown, the presence of these subcellular particles might indicate an involvement in RNA metabolism. The identification of a protein highly enriched in this structure and the availability of specific antibodies might help in its isolation and the study of its function.
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human autoantiBody to a novel protein of the nuclear Coiled Body immunological characterization and cdna cloning of p80 coilin
Journal of Experimental Medicine, 1991Co-Authors: Luis Eduardo Coelho Andrade, Ivan Raska, Edward K L Chan, Goran Roos, Carol L Peebles, Eng M TanAbstract:Antibodies producing an unusual immunofluorescent pattern were identified in the sera of patients with diverse autoimmune features. This pattern was characterized by the presence of up to six round discrete nuclear bodies in interphase cell nuclei. Immunoblotting analysis showed that these sera recognized an 80-kD nuclear protein, and affinity-purified anti-p80 antiBody from the protein band reproduced the fluorescent staining of nuclear bodies. Colloidal gold immunoelectron microscopy showed that the affinity-purified anti-p80 antiBody recognized the Coiled Body, an ultramicroscopic nuclear structure probably first described by the Spanish cytologist Ramon y Cajal. Five cDNA clones were isolated from a MOLT-4 cell lambda gt-11 expression library using human antiBody and oligonucleotide probes. The longest cDNA insert was 2.1 kb and had an open reading frame of 405 amino acids. A clone encoding a 14-kD COOH-terminal region of the protein was used for expression of a beta-galactosidase fusion protein. An epitope was present in this COOH-terminal 14-kD region, which was recognized by 18 of 20 sera with anti-p80 reactivity, and affinity-purified antiBody from the recombinant protein also reacted in immunofluorescence to show specific staining of the Coiled Body. This is the first demonstration and molecular cloning of a protein that appears to have particular identification with the Coiled Body, and it was designated p80-coilin. AutoantiBody to p80-coilin may be useful for the elucidation of the structure and function of the Coiled Body, and the availability of a cDNA sequence could be helpful in further studies to clarify the clinical significance of this autoantiBody response.
Eng M Tan - One of the best experts on this subject based on the ideXlab platform.
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formation of nuclear bodies in hepatocytes of estrogen treated roosters
Molecular Biology of the Cell, 1995Co-Authors: Robert L Ochs, Luis Eduardo Coelho Andrade, Edward K L Chan, Eng M Tan, T W Stein, D Gallo, K BraschAbstract:Abstract As a model for cellular growth and stimulation without accompanying proliferation, we have examined the induction and formation of nuclear bodies (NBs) in hepatocytes of estrogen-treated roosters. Four-week-old roosters were injected with a single intramuscular dose of estradiol and then killed at time points of 8 h, 48 h, and 4 wk post-injection. For immunofluorescence analyses, livers were excised and isolated hepatocyte nuclei were fixed and then labeled with antiBody to the Coiled Body-specific protein, p80-coilin. In control animals (no estradiol) or in animals 8 h post-injection, each hepatocyte nucleus contained an average of 1.0 Coiled Body (CB), which appeared randomly distributed in the nucleoplasm. At 48 h post-injection, there were an average of 2.7 CBs/nucleus and many of these appeared to be in contact with the nucleolus. Pairs of CBs were also observed. By 4 wk post-injection an average of 1.5 CBs/nucleus were detected, with no apparent relationship to the nucleolus observed. By serial-section electron microscopy of intact livers, two different types of round NBs were observed, sometimes in close proximity to each other and to the expanded interchromatin granule region in maximally stimulated cells. One type of NB was a classical CB that averaged 0.35 microns in diameter and the other NB type was ring shaped, averaged 0.25 microns in diameter, was composed of a fibrous shell surrounding a hollow interior, and appeared as a simple NB when sectioned tangentially through its outer shell. Immunoelectron microscopy revealed that CBs were the only class of NBs that contained p80-coilin. From these data, we conclude that CBs proliferate in response to estrogen stimulation, possibly arising from the nucleolar surface and then increasing in number by replicative division.
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immunocytochemical analysis of the Coiled Body in the cell cycle and during cell proliferation
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Luis Eduardo Coelho Andrade, Eng M Tan, Edward K L ChanAbstract:Abstract Coiled bodies (CBs) are small, round structures found in the nucleoplasm of most eukaryotic cells. Human autoantibodies to a 80-kDa protein, p80-coilin, are immunohistologic markers for CBs. A polyclonal rabbit antiserum (R288) raised against recombinant p80-coilin was shown to have similar immunochemical properties as human autoantibodies and was used to analyze the expression of p80-coilin-associated CBs in cell cultures synchronized by double thymidine block, nocodazole arrest, serum starvation, or hormonal deprivation. By employing thymidine block and nocodazole arrest of HeLa cells, CBs were observed in immunofluorescent studies to be largest in size in the S and G2 phases of the cell cycle. These large CBs might have coalesced into one or two such structures per cell from smaller and more numerous CBs of three to eight per cell during the mid G1 phase of the cell cycle. No CB-like structures were observed in mitosis and early G1. However, immunoblotting analyses showed that the total amount of p80-coilin remained approximately the same throughout the cell cycle. When HeLa cells were separated into soluble and particulate fractions, p80-coilin was detected predominantly in the soluble fraction in mitosis and early G1, whereas it was present predominantly in the particulate fraction in late G1, S, and G2 when structurally distinct CBs were observed. In the analysis of CBs in two experimental models of cell proliferation (reversal of 3T3 serum starvation and FRTL-5 thyrotropin deprivation), proliferating cells contained larger, brighter, and more numerous CBs as well as a > 2-fold increase in the total amount of p80-coilin compared to that in quiescent cells. The expression of p80-coilin in quiescent cells induced to proliferate and the cyclic formation and breakdown of CBs might be consistent with the notion that CBs may be specialized centers related to the maturation of mRNA, but this evidence is indirect and needs further definitive study.
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immunological and ultrastructural studies of the nuclear Coiled Body with autoimmune antibodies
Experimental Cell Research, 1991Co-Authors: Ivan Raska, Luis Eduardo Coelho Andrade, Robert L Ochs, Edward K L Chan, Chengming Chang, Goran Roos, Eng M TanAbstract:Abstract Studies with human autoimmune sera identified autoantibodies reacting with a novel antigen of 80 kDa. In interphase mammalian cells, the 80-kDa antigen was enriched in nuclear Coiled bodies and was used as a marker for this nuclear structure. This antigen was subsequently named p80-coilin. By light and electron microscopic immunocytochemistry, a number of other antigens were also localized to the Coiled Body, including components of small nuclear ribonucleoproteins which are involved in the processing of nucleolar and extranucleolar RNA. Although the function of the Coiled Body is unknown, the presence of these subcellular particles might indicate an involvement in RNA metabolism. The identification of a protein highly enriched in this structure and the availability of specific antibodies might help in its isolation and the study of its function.
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human autoantiBody to a novel protein of the nuclear Coiled Body immunological characterization and cdna cloning of p80 coilin
Journal of Experimental Medicine, 1991Co-Authors: Luis Eduardo Coelho Andrade, Ivan Raska, Edward K L Chan, Goran Roos, Carol L Peebles, Eng M TanAbstract:Antibodies producing an unusual immunofluorescent pattern were identified in the sera of patients with diverse autoimmune features. This pattern was characterized by the presence of up to six round discrete nuclear bodies in interphase cell nuclei. Immunoblotting analysis showed that these sera recognized an 80-kD nuclear protein, and affinity-purified anti-p80 antiBody from the protein band reproduced the fluorescent staining of nuclear bodies. Colloidal gold immunoelectron microscopy showed that the affinity-purified anti-p80 antiBody recognized the Coiled Body, an ultramicroscopic nuclear structure probably first described by the Spanish cytologist Ramon y Cajal. Five cDNA clones were isolated from a MOLT-4 cell lambda gt-11 expression library using human antiBody and oligonucleotide probes. The longest cDNA insert was 2.1 kb and had an open reading frame of 405 amino acids. A clone encoding a 14-kD COOH-terminal region of the protein was used for expression of a beta-galactosidase fusion protein. An epitope was present in this COOH-terminal 14-kD region, which was recognized by 18 of 20 sera with anti-p80 reactivity, and affinity-purified antiBody from the recombinant protein also reacted in immunofluorescence to show specific staining of the Coiled Body. This is the first demonstration and molecular cloning of a protein that appears to have particular identification with the Coiled Body, and it was designated p80-coilin. AutoantiBody to p80-coilin may be useful for the elucidation of the structure and function of the Coiled Body, and the availability of a cDNA sequence could be helpful in further studies to clarify the clinical significance of this autoantiBody response.
Maria Carmofonseca - One of the best experts on this subject based on the ideXlab platform.
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the spinal muscular atrophy disease gene product smn a link between snrnp biogenesis and the cajal Coiled Body
Journal of Cell Biology, 1999Co-Authors: Teresa Carvalho, Fatima Almeida, Alexandre Calapez, Miguel Lafarga, Maria T Berciano, Maria CarmofonsecaAbstract:The spliceosomal snRNAs U1, U2, U4, and U5 are synthesized in the nucleus, exported to the cytoplasm to assemble with Sm proteins, and reimported to the nucleus as ribonucleoprotein particles. Recently, two novel proteins involved in biogenesis of small nuclear ribonucleoproteins (snRNPs) were identified, the Spinal muscular atrophy disease gene product (SMN) and its associated protein SIP1. It was previously reported that in HeLa cells, SMN and SIP1 form discrete foci located next to Cajal (Coiled) bodies, the so-called “gemini of Coiled bodies” or “gems.” An intriguing feature of gems is that they do not appear to contain snRNPs. Here we show that gems are present in a variable but small proportion of rapidly proliferating cells in culture. In the vast majority of cultured cells and in all primary neurons analyzed, SMN and SIP1 colocalize precisely with snRNPs in the Cajal Body. The presence of SMN and SIP1 in Cajal bodies is confirmed by immunoelectron microscopy and by microinjection of antibodies that interfere with the integrity of the structure. The association of SMN with snRNPs and coilin persists during cell division, but at the end of mitosis there is a lag period between assembly of new Cajal bodies in the nucleus and detection of SMN in these structures, suggesting that SMN is targeted to preformed Cajal bodies. Finally, treatment of cells with leptomycin B (a drug that blocks export of U snRNAs to the cytoplasm and consequently import of new snRNPs into the nucleus) is shown to deplete snRNPs (but not SMN or SIP1) from the Cajal Body. This suggests that snRNPs flow through the Cajal Body during their biogenesis pathway.
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the cdk7 cyclin h mat1 complex associated with tfiih is localized in Coiled bodies
Molecular Biology of the Cell, 1997Co-Authors: Peter Jordan, Celso Cunha, Maria CarmofonsecaAbstract:Abstract 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.
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the biogenesis of the Coiled Body during early mouse development
Development, 1995Co-Authors: Joao A Ferreira, Maria CarmofonsecaAbstract:The Coiled Body is an ubiquitous nuclear organelle that contains essential components of the pre-mRNA splicing machinery as well as the nucleolar protein fibrillarin. Here we have studied the biogenesis of the Coiled Body in early mouse embryos. The results show that Coiled bodies form and concentrate splicing snRNPs as early as in the maternal and paternal pronuclei of 1-cell embryos. This argues that the Coiled Body is likely to play a basic role in the nucleus of mammalian cells. In order to correlate the appearance of Coiled bodies with the onset of transcriptional activity, embryos were incubated with brominated UTP and the incorporated nucleotide was visualized by fluorescence microscopy. In agreement with previous studies, transcriptional activity was first observed during the 2-cell stage. Thus, Coiled bodies form before activation of embryonic gene expression. The appearance of Coiled bodies in 1-cell embryos was preceded by the formation of morphologically distinct structures that also contain coilin and which we therefore refer to as pre-Coiled bodies. At the electron microscopic level pre-Coiled bodies have a compact fibrillar structure, whereas Coiled bodies resemble a tangle of Coiled threads. Although both pre-Coiled bodies and Coiled bodies contain the nucleolar protein fibrillarin, the assembly of Coiled bodies is separated both in time and in space from ribosome synthesis. Our results suggest that the embryonic ‘nucleolus-like Body’ is a structural scaffold that nucleates independently the formation of the Coiled Body and the assembly of the machinery responsible for ribosome biosynthesis.
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differential interaction of splicing snrnps with Coiled bodies and interchromatin granules during mitosis and assembly of daughter cell nuclei
Journal of Cell Biology, 1994Co-Authors: Joao A Ferreira, Maria Carmofonseca, Angus I. LamondAbstract:In the interphase nucleus of mammalian cells the U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs), which are subunits of spliceosomes, associate with specific subnuclear domains including interchromatin granules and Coiled bodies. Here, we analyze the association of splicing snRNPs with these structures during mitosis and reassembly of daughter nuclei. At the onset of mitosis snRNPs are predominantly diffuse in the cytoplasm, although a subset remain associated with remnants of Coiled bodies and clusters of mitotic interchromatin granules, respectively. The number and size of mitotic Coiled bodies remain approximately unchanged from metaphase to early telophase while snRNP-containing clusters of mitotic interchromatin granules increase in size and number as cells progress from anaphase to telophase. During telophase snRNPs are transported into daughter nuclei while the clusters of mitotic interchromatin granules remain in the cytoplasm. The timing of nuclear import of splicing snRNPs closely correlates with the onset of transcriptional activity in daughter nuclei. When transcription restarts in telophase cells snRNPs have a diffuse nucleoplasmic distribution. As cells progress to G1 snRNP-containing clusters of interchromatin granules reappear in the nucleus. Coiled bodies appear later in G1, although the Coiled Body antigen, p80 coilin, enters early into telophase nuclei. After inhibition of transcription we still observe nuclear import of snRNPs and the subsequent appearance of snRNP-containing clusters of interchromatin granules, but not Coiled Body formation. These data demonstrate that snRNP associations with Coiled bodies and interchromatin granules are differentially regulated during the cell division cycle and suggest that these structures play distinct roles connected with snRNP structure, transport, and/or function.
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assembly of snrnp containing Coiled bodies is regulated in interphase and mitosis evidence that the Coiled Body is a kinetic nuclear structure
Journal of Cell Biology, 1993Co-Authors: Maria Carmofonseca, Joao A Ferreira, Angus I. LamondAbstract:Coiled bodies (CBs) are nuclear organelles in which splicing snRNPs concentrate. While CBs are sometimes observed in association with the nucleolar periphery, they are shown not to contain 5S or 28S rRNA or the U3 snoRNA. This argues against CBs playing a role in rRNA maturation or transport as previously suggested. We present evidence here that CBs are kinetic structures and demonstrate that the formation of snRNP-containing CBs is regulated in interphase and mitosis. The Coiled Body antigen, p80 coilin, was present in all cell types studied, even when CBs were not prominent. Striking changes in the formation of CBs could be induced by changes in cellular growth temperature without a concomitant change in the intracellular p80 coilin level. During mitosis, CBs disassemble, coinciding with a mitotic-specific phosphorylation of p80 coilin. Coilin is shown to be a phosphoprotein that is phosphorylated on at least two additional sites during mitosis. CBs reform in daughter nuclei after a lag period during which they are not detected. CBs are thus, dynamic nuclear organelles and we propose that cycling interactions of splicing snRNPs with CBs may be important for their participation in the processing or transport of pre-mRNA in mammalian cells.
Jun Cheng - One of the best experts on this subject based on the ideXlab platform.
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methylation of nucleolar and Coiled Body phosphoprotein 1 is associated with the mechanism of tumorigenesis in hepatocellular carcinoma
Oncology Reports, 2013Co-Authors: Xuefei Duan, Qi Wang, Shu Nai Liu, Jinqian Zhang, Mengran Zhang, Jun ChengAbstract:Nucleolar and Coiled-Body phosphoprotein 1 (NOLC1) plays an essential role in the synthesis of rRNA and the biosynthesis of ribosomes. Previous studies suggest that NOLC1 is crucial for normal cell growth, and plays a role in the regulation of tumorigenesis of nasopharyngeal carcinoma (NPC) and demonstrate that both NOLC1 and tumor protein 53 work synergistically to activate the MDM2 promoter in NPC cells. Yet, the functioning of NOLC1 in liver cancer remains unknown. The aim of the present study was to understand how the NOLC1 gene is regulated in liver carcinogenesis. In this study, we showed that NOLC1 was silenced or downregulated in liver tumor tissues when compared with that in the matched non-cancer tissues. In addition, human hepatoma cells weakly expressed NOLC1, whereas cultured human normal liver cell lines expressed abundant levels. The hypermethylation status in the promoter CpG1 start region appeared to be correlated with the NOLC1 expression levels in liver cell lines or liver normal and tissue specimens. We found that four CpG dinucleotides were located at the CpG1 start region. Further molecular analysis of mutagenesis indicated that the four CpG dinucleotides play a role in the promoter activity of the NOLC1 gene. The expression of NOLC1 and DNA methylation of its promoter affected cell proliferation and apoptosis. The expression of NOLC1 in hepatoma cell lines was restored following exposure to the demethylation agent, 5-azacytidine. Low expression of NOLC1 in hepatoma cell lines and liver cancer tissues was associated with cyclin D3. In conclusion, our study demonstrated that DNA methylation is a key mechanism of silenced NOLC1 expression in human hepatocellular carcinoma cells, and NOLC1 gene hypermethylation of the four CpG dinucleotides is a potential biomarker for hepatocellular carcinoma.
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Identification of nucleolar and Coiled-Body phosphoprotein 1 (NOLC1) minimal promoter regulated by NF-κB and CREB.
BMB reports, 2011Co-Authors: Xue Song Gao, Qi Wang, Biao Yang, Hao Song, Shu Nai Liu, Jun ChengAbstract:Nucleolar and Coiled-Body phosphoprotein 1 (NOLC1) is a phosphoprotein that transiently associates with the mature nucleolar H/ACA and C/D box small nucleolar ribonucleoproteins (snoRNPs). Several lines of evidence indicate that NOLC1 plays an important role in the synthesis of rRNA and the biosynthesis of ribosomes. In the present study, we examined the transcriptional regulation mechanisms that govern the expression of NOLC1. We first performed functional dissection of the NOLC1 promoter. We demonstrated that transcription factors NF-κB and CREB could bind to the minimal NOLC1 promoter. This was demonstrated by electrophoretic mobility shift assays and chromatin immunoprecipitation. Mutagenesis and overexpression assays revealed that NF-κB and CREB positively regulated the NOLC1 promoter. These findings may provide new insight into the mechanisms that regulate NOLC1 expression. [BMB reports 2011; 44(1): 70-75]