scholarly journals Disassembly of interchromatin granule clusters alters the coordination of transcription and pre-mRNA splicing

2002 ◽  
Vol 156 (3) ◽  
pp. 425-436 ◽  
Author(s):  
Paula Sacco-Bubulya ◽  
David L. Spector
Keyword(s):  
Mammalian Cell ◽  
Protein Interactions ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Cell Nuclei ◽  
Nuclear Speckles ◽  
Sr Protein ◽  
Interchromatin Granule Clusters ◽  
Interchromatin Granule

To examine the involvement of interchromatin granule clusters (IGCs) in transcription and pre-mRNA splicing in mammalian cell nuclei, the serine-arginine (SR) protein kinase cdc2-like kinase (Clk)/STY was used as a tool to manipulate IGC integrity in vivo. Both immunofluorescence and transmission electron microscopy analyses of cells overexpressing Clk/STY indicate that IGC components are completely redistributed to a diffuse nuclear localization, leaving no residual structure. Conversely, overexpression of a catalytically inactive mutant, Clk/STY(K190R), causes retention of hypophosphorylated SR proteins in nuclear speckles. Our data suggest that the protein–protein interactions responsible for the clustering of interchromatin granules are disrupted when SR proteins are hyperphosphorylated and stabilized when SR proteins are hypophosphorylated. Interestingly, cells without intact IGCs continue to synthesize nascent transcripts. However, both the accumulation of splicing factors at sites of pre-mRNA synthesis as well as pre-mRNA splicing are dramatically reduced, demonstrating that IGC disassembly perturbs coordination between transcription and pre-mRNA splicing in mammalian cell nuclei.

scholarly journals Serine/threonine phosphatase 1 modulates the subnuclear distribution of pre-mRNA splicing factors.

1996 ◽  
Vol 7 (10) ◽  
pp. 1559-1572 ◽  
Author(s):  
T Misteli ◽  
D L Spector
Keyword(s):  
Rna Polymerase Ii ◽  
Mammalian Cell ◽  
Cell Nucleus ◽  
Nuclear Extract ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Cell Nuclei ◽  
Nuclear Speckles ◽  
Permeabilized Cells

HeLa cell nuclei were permeabilized and reconstituted with nuclear extract to identify soluble nuclear factors which play a role in the organization of pre-mRNA splicing factors in the mammalian cell nucleus. Permeabilized nuclei reconstituted with nuclear extract were active in transcription and DNA replication and nuclear speckles containing pre-mRNA splicing factors were maintained over several hours independent of soluble nuclear components. The characteristic rounding up of nuclear speckles in response to inhibition of RNA polymerase II seen in vivo was reproduced in permeabilized cells and was strictly dependent on a catalytic activity present in the nuclear extract. By inhibitor titration experiments and sensitivity to inhibitor 2, this activity was identified as a member of the serine/threonine protein phosphatase 1 family (PP1). Interference with PP1 activity affected the distribution of pre-mRNA splicing factors in transcriptionally active, permeabilized cells, and excess PP1 activity caused increased dephosphorylation of SR proteins in nuclear speckles. These data show that the dynamic reorganization of the mammalian cell nucleus can be studied in permeabilized cells and that PP1 is involved in the rounding up of speckles as well as the overall organization of pre-mRNA splicing factors in the mammalian cell nucleus.

scholarly journals Serine Phosphorylation of SR Proteins Is Required for Their Recruitment to Sites of Transcription In Vivo

1998 ◽  
Vol 143 (2) ◽  
pp. 297-307 ◽  
Author(s):  
Tom Misteli ◽  
Javier F. Cáceres ◽  
Jade Q. Clement ◽  
Adrian R. Krainer ◽  
Miles F. Wilkinson ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Active Sites ◽  
Rna Binding ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Serine Phosphorylation ◽  
Splicing Factors ◽  
Nuclear Speckles

Expression of most RNA polymerase II transcripts requires the coordinated execution of transcription, splicing, and 3′ processing. We have previously shown that upon transcriptional activation of a gene in vivo, pre-mRNA splicing factors are recruited from nuclear speckles, in which they are concentrated, to sites of transcription (Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. Nature. 387:523–527). This recruitment process appears to spatially coordinate transcription and pre-mRNA splicing within the cell nucleus. Here we have investigated the molecular basis for recruitment by analyzing the recruitment properties of mutant splicing factors. We show that multiple protein domains are required for efficient recruitment of SR proteins from nuclear speckles to nascent RNA. The two types of modular domains found in the splicing factor SF2/ ASF exert distinct functions in this process. In living cells, the RS domain functions in the dissociation of the protein from speckles, and phosphorylation of serine residues in the RS domain is a prerequisite for this event. The RNA binding domains play a role in the association of splicing factors with the target RNA. These observations identify a novel in vivo role for the RS domain of SR proteins and suggest a model in which protein phosphorylation is instrumental for the recruitment of these proteins to active sites of transcription in vivo.

scholarly journals The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2

2012 ◽  
Vol 23 (6) ◽  
pp. 1115-1128 ◽  
Author(s):  
Noriko Saitoh ◽  
Chiyomi Sakamoto ◽  
Masatoshi Hagiwara ◽  
Lourdes T. Agredano-Moreno ◽  
Luis Felipe Jiménez-García ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Mammalian Cells ◽  
Nuclear Architecture ◽  
Sr Proteins ◽  
Nucleocytoplasmic Transport ◽  
Dependent Manner ◽  
Small Interfering Rnas ◽  
Nuclear Speckles ◽  
Sr Protein ◽  
Interchromatin Granule

The mammalian cell nucleus is functionally compartmentalized into various substructures. Nuclear speckles, also known as interchromatin granule clusters, are enriched with SR splicing factors and are implicated in gene expression. Here we report that nuclear speckle formation is developmentally regulated; in certain cases phosphorylated SR proteins are absent from the nucleus and are instead localized at granular structures in the cytoplasm. To investigate how the nuclear architecture is formed, we performed a phenotypic screen of HeLa cells treated with a series of small interfering RNAs. Depletion of Ran-binding protein 2 induced cytoplasmic intermediates of nuclear speckles in G1 phase. Detailed analyses of these structures suggested that a late step in the sequential nuclear entry of mitotic interchromatin granule components was disrupted and that phosphorylated SR proteins were sequestered in an SR protein kinase–dependent manner. As a result, the cells had an imbalanced subcellular distribution of phosphorylated and hypophosphorylated SR proteins, which affected alternative splicing patterns. This study demonstrates that the speckled distribution of phosphorylated pre-mRNA processing factors is regulated by the nucleocytoplasmic transport system in mammalian cells and that it is important for alternative splicing.

scholarly journals Human RNPS1 and Its Associated Factors: a Versatile Alternative Pre-mRNA Splicing Regulator In Vivo

2004 ◽  
Vol 24 (3) ◽  
pp. 1174-1187 ◽  
Author(s):  
Eiji Sakashita ◽  
Sawako Tatsumi ◽  
Dieter Werner ◽  
Hitoshi Endo ◽  
Akila Mayeda
Keyword(s):  
Exon Skipping ◽  
Mrna Splicing ◽  
Rna Recognition Motif ◽  
Nuclear Speckles ◽  
Sr Protein ◽  
Potential Component ◽  
Splicing Regulator ◽  
P Domain

ABSTRACT Human RNPS1 was originally purified and characterized as a pre-mRNA splicing activator, and its role in the postsplicing process has also been proposed recently. To search for factors that functionally interact with RNPS1, we performed a yeast two-hybrid screen with a human cDNA library. Four factors were identified: p54 (also called SRp54; a member of the SR protein family), human transformer 2β (hTra2β; an exonic splicing enhancer-binding protein), hLucA (a potential component of U1 snRNP), and pinin (also called DRS and MemA; a protein localized in nuclear speckles). The N-terminal region containing the serine-rich (S) domain, the central RNA recognition motif (RRM), and the C-terminal arginine/serine/proline-rich (RS/P) domain of RNPS1 interact with p54, pinin, and hTra2β, respectively. Protein-protein binding between RNPS1 and these factors was verified in vitro and in vivo. Overexpression of RNPS1 in HeLa cells induced exon skipping in a model β-globin pre-mRNA and a human tra-2β pre-mRNA. Coexpression of RNPS1 with p54 cooperatively stimulated exon inclusion in an ATP synthase γ-subunit pre-mRNA. The RS/P domain and RRM are necessary for the exon-skipping activity, whereas the S domain is important for the cooperative effect with p54. RNPS1 appears to be a versatile factor that regulates alternative splicing of a variety of pre-mRNAs.

scholarly journals Disruption of pre-mRNA splicing in vivo results in reorganization of splicing factors

1994 ◽  
Vol 124 (3) ◽  
pp. 249-260 ◽  
Author(s):  
RT O'Keefe ◽  
A Mayeda ◽  
CL Sadowski ◽  
AR Krainer ◽  
DL Spector
Keyword(s):  
Cell Nucleus ◽  
Functional Significance ◽  
Living Cells ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Interchromatin Granule Clusters ◽  
Interchromatin Granule ◽  
Transcription In Vitro

We have examined the functional significance of the organization of pre-mRNA splicing factors in a speckled distribution in the mammalian cell nucleus. Upon microinjection into living cells of oligonucleotides or antibodies that inhibit pre-mRNA splicing in vitro, we observed major changes in the organization of splicing factors in vivo. Interchromatin granule clusters became uniform in shape, decreased in number, and increased in both size and content of splicing factors, as measured by immunofluorescence. These changes were transient and the organization of splicing factors returned to their normal distribution by 24 h following microinjection. Microinjection of these oligonucleotides or antibodies also resulted in a reduction of transcription in vivo, but the oligonucleotides did not inhibit transcription in vitro. Control oligonucleotides did not disrupt splicing or transcription in vivo. We propose that the reorganization of splicing factors we observed is the result of the inhibition of splicing in vivo.

Organization of transcription and pre-mRNA splicing within the mammalian cell nucleus

1995 ◽  
Vol 53 ◽  
pp. 768-769
Author(s):  
D.L. Spector ◽  
S. Huang ◽  
S. Kaurin
Keyword(s):  
Rna Polymerase Ii ◽  
Cell Nucleus ◽  
Functional Organization ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Interchromatin Granule Clusters ◽  
Interchromatin Granule ◽  
Nuclear Regions ◽  
Relationship Of ◽  
Perichromatin Fibrils

We have been interested in the organization of RNA polymerase II transcription and pre-mRNA splicing within the cell nucleus. Several models have been proposed for the functional organization of RNA within the eukaryotic nucleus and for the relationship of this organization to the distribution of pre-mRNA splicing factors. One model suggests that RNAs which must be spliced are capable of recruiting splicing factors to the sites of transcription from storage and/or reassembly sites. When one examines the organization of splicing factors in the nucleus in comparison to the sites of chromatin it is clear that splicing factors are not localized in coincidence with heterochromatin (Fig. 1). Instead, they are distributed in a speckled pattern which is composed of both perichromatin fibrils and interchromatin granule clusters. The perichromatin fibrils are distributed on the periphery of heterochromatin and on the periphery of interchromatin granule clusters as well as being diffusely distributed throughout the nucleoplasm. These nuclear regions have been previously shown to represent initial sites of incorporation of 3H-uridine.

Monochloramine induces reorganization of nuclear speckles and phosphorylation of SRp30 in human colonic epithelial cells: role of protein kinase C

2003 ◽  
Vol 285 (5) ◽  
pp. C1294-C1303 ◽  
Author(s):  
Ya-Qin Zhu ◽  
Yu Lu ◽  
Xiao-Di Tan
Keyword(s):  
Gastrointestinal Tract ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Morphological Changes ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Intestinal Epithelial ◽  
Nuclear Speckles ◽  
Colonic Epithelial Cells ◽  
Stimulation Of

Intestinal epithelial cells are constantly stimulated by reactive oxidant metabolites (ROMs) in inflamed mucosa. Monochloramine (NH2Cl), a cell-permeant ROM, is particularly relevant to the pathogenesis of inflammation in the gastrointestinal tract. Nuclear speckles, a unique nuclear subcompartment, accumulate a family of proteins, namely, serine- and arginine-rich (SR) proteins. They play important roles in regulation of pre-mRNA splicing. Currently, little is known about the link between inflammatory stimulation and the pre-mRNA splicing process, although gene expression is changed in inflamed tissues. The present study was designed to investigate whether stimulation of human colonic epithelial cells (HT-29 and Caco-2 cell lines) with NH2Cl affects nuclear speckles and their components. By indirect immunofluorescence, nuclear speckles have been shown to undergo rapid aggregation after NH2Cl stimulation. By utilizing Western blotting, SRp30 (a subset of SR proteins) in intestinal epithelial cells was found to be phosphorylated after NH2Cl treatment, whereas other SR proteins were not responsive to NH2Cl stimulation. The cytotoxic effect of NH2Cl was excluded by both negative lactate dehydrogenase assay and propidium iodide staining. Therefore, NH2Cl-induced morphological changes on nuclear speckles and phosphorylated SRp30 do not result from intestinal epithelial injury. Furthermore, the effect of NH2Cl on nuclear speckles and SRp30 was blocked by bisindolylmaleimide I, a selective PKC inhibitor. Together, the available data suggest that stimulation of intestinal epithelial cells with NH2Cl results in a consequent change on pre-mRNA splicing machinery via a distinctive signal pathway involving activation of PKC. This effect may contribute to oxidant-induced pathophysiological changes in the gastrointestinal tract.

scholarly journals Roles for SR Proteins and hnRNP A1 in the Regulation of c-src Exon N1

2003 ◽  
Vol 23 (6) ◽  
pp. 1874-1884 ◽  
Author(s):  
Nanette Rooke ◽  
Vadim Markovtsov ◽  
Esra Cagavi ◽  
Douglas L. Black
Keyword(s):  
Regulatory Element ◽  
Sr Proteins ◽  
Hnrnp A1 ◽  
Sr Protein ◽  
Enhancer Element ◽  
Splicing Regulators ◽  
In Vitro Splicing ◽  
Hnrnp F

ABSTRACT The splicing of the c-src exon N1 is controlled by an intricate combination of positive and negative RNA elements. Most previous work on these sequences focused on intronic elements found upstream and downstream of exon N1. However, it was demonstrated that the 5′ half of the N1 exon itself acts as a splicing enhancer in vivo. Here we examine the function of this regulatory element in vitro. We show that a mutation in this sequence decreases splicing of the N1 exon in vitro. Proteins binding to this element were identified as hnRNP A1, hnRNP H, hnRNP F, and SF2/ASF by site-specific cross-linking and immunoprecipitation. The binding of these proteins to the RNA was eliminated by a mutation in the exonic element. The activities of hnRNP A1 and SF2/ASF on N1 splicing were examined by adding purified protein to in vitro splicing reactions. SF2/ASF and another SR protein, SC35, are both able to stimulate splicing of c-src pre-mRNA. However, splicing activation by SF2/ASF is dependent on the N1 exon enhancer element whereas activation by SC35 is not. In contrast to SF2/ASF and in agreement with other systems, hnRNP A1 repressed c-src splicing in vitro. The negative activity of hnRNP A1 on splicing was compared with that of PTB, a protein previously demonstrated to repress splicing in this system. Both proteins repress exon N1 splicing, and both counteract the enhancing activity of the SR proteins. Removal of the PTB binding sites upstream of N1 prevents PTB-mediated repression but does not affect A1-mediated repression. Thus, hnRNP A1 and PTB use different mechanisms to repress c-src splicing. Our results link the activity of these well-known exonic splicing regulators, SF2/ASF and hnRNP A1, to the splicing of an exon primarily controlled by intronic factors.

scholarly journals Cętki jądrowe jako modelowe ciałka jądrowe. Cętki jądrowe w układzie nerwowym

Kosmos ◽  
2020 ◽  
Vol 69 (1) ◽  
pp. 17-35
Author(s):  
Andrzej Szczepankiewicz
Keyword(s):  
Nuclear Bodies ◽  
Nuclear Speckles ◽  
Interchromatin Granule Clusters ◽  
Interchromatin Granule

Jądro komórkowe oprócz heterochromatyny czyli kompleksu DNA i białek histonowych, zawiera struktury zwane ciałkami jądrowymi (ang. nuclear bodies). Są to niewielkie, zazwyczaj koliste twory ?zawieszone? w nukleoplazmie, składające się z białek lub białek i niekodującego RNA. Znanych jest kilkanaście rodzajów takich struktur. Artykuł przedstawia obecny stan wiedzy na temat ciałek nazywanych w angielskiej literaturze nuclear speckles, czyli cętki jądrowe lub interchromatin granule clusters czyli skupiska ziaren interchromatynowych. Zaobserwowane po raz pierwszy przez Romana y Cajala w neuronach, obecne jednak we wszystkich typach komórek, struktury te uważane są za magazyny i miejsce modyfikacji czynników składania (splicingu) mRNA. Najnowsze badania, prezentowane w artykule pozwalają sądzić, że ich udział w metabolizmie RNA jest bardziej złożony. Świadczą o tym również doniesienia o roli cętek.

scholarly journals HRS/SRp40-mediated inclusion of the fibronectin EIIIB exon, a possible cause of increased EIIIB expression in proliferating liver.

1997 ◽  
Vol 17 (7) ◽  
pp. 4096-4104 ◽  
Author(s):  
K Du ◽  
Y Peng ◽  
L E Greenbaum ◽  
B A Haber ◽  
R Taub
Keyword(s):  
Time Course ◽  
Growth Control ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Cellular Growth ◽  
Liver Growth ◽  
Alternatively Spliced ◽  
Growth Changes ◽  
Possible Cause

Serine-arginine (SR)-rich proteins are believed to be important in mediating alternative pre-mRNA splicing. HRS/SRp40 expression is elevated in liver cell proliferation during development, regeneration, and oncogenesis. We tested whether HRS expression correlates with the appearance of alternatively spliced fibronectin transcripts during liver growth. HRS was highly expressed during the proliferative phase of liver development, correlating with expression of the fibronectin EIIIB alternative exon. In regenerating liver, HRS protein was induced in a time course consistent with the observed increase in fibronectin transcripts containing the EIIIB exon, particularly in nonparenchymal liver cells. Furthermore, in an in vivo assay, HRS, and not other SR proteins, directly mediated EIIIB exon inclusion in the fibronectin transcript. This alternative splicing was dependent on a purine-rich region within the EIIIB exon to which HRS specifically bound. We have established that HRS has the potential to contribute to the regulation of fibronectin pre-mRNA splicing during liver growth. Changes in fibronectin forms may be important in modifying liver architecture during the proliferative response, thus providing a potential mechanism by which SR proteins may participate in cellular growth control.

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