Cytostellin distributes to nuclear regions enriched with splicing factors

1994 ◽  
Vol 107 (3) ◽  
pp. 387-396 ◽  
Author(s):  
D.B. Bregman ◽  
L. Du ◽  
Y. Li ◽  
S. Ribisi ◽  
S.L. Warren
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Splicing Factors ◽  
Macromolecular Complex ◽  
Nuclear Speckles ◽  
Serum Stimulation ◽  
Cell Structures ◽  
Dividing Cells ◽  
Nuclear Regions

Cytostellin, a approximately 240 kDa phosphoprotein found in all cells examined from human to yeast, is predominantly intranuclear in interphase mammalian cells and undergoes continuous redistribution during the cell cycle. Here, mammalian cytostellin is shown to localize to intranuclear regions enriched with multiple splicing proteins, including spliceosome assembly factor, SC-35. Cytostellin and the splicing proteins also co-localize to discrete foci (called ‘dots’), which are distributed throughout the cell during mitosis and part of G1. The cytostellin that is localized to these dots resists extraction by Triton X-100, indicating that it is tightly associated with insoluble cell structures. All immunostainable cytostellin reappears in the nucleus before S-phase. Although cytostellin and the splicing proteins co-localize in interphase and dividing cells, cytostellin is not detected in purified spliceosomes, and it associates with six unidentified proteins, forming a macromolecular complex that is biochemically distinct from the proteins that comprise spliceosomes. This macromolecular complex is detected at constant levels throughout the cell cycle, and the level of cytostellin protein remains constant during the cell cycle. Nevertheless, intranuclear cytostellin immunostaining fluctuates markedly during the cell cycle. The monoclonal antibody (mAb) H5 epitope of cytostellin is ‘masked’ in serum-starved cells, but 60 minutes after serum stimulation intense cytostellin immunoreactivity appears in the nuclear speckles. This rapid induction of cytostellin immunoreactivity in subnuclear regions enriched with many splicing factors, as well as accumulations of RNA polymerase II (Pol II) transcripts, suggests that cytostellin may have a function related to mRNA biogenesis.

scholarly journals Hypophosphorylated SR splicing factors transiently localize around active nucleolar organizing regions in telophase daughter nuclei

2004 ◽  
Vol 167 (1) ◽  
pp. 51-63 ◽  
Author(s):  
Paula A. Bubulya ◽  
Kannanganattu V. Prasanth ◽  
Thomas J. Deerinck ◽  
Daniel Gerlich ◽  
Joel Beaudouin ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Sr Proteins ◽  
Splicing Factors ◽  
Nuclear Speckles ◽  
Sr Protein ◽  
Nucleolar Organizing Regions ◽  
Transcription Sites ◽  
Nuclear Regions ◽  
Rna Polymerase Ii Transcription

Upon completion of mitosis, daughter nuclei assemble all of the organelles necessary for the implementation of nuclear functions. We found that upon entry into daughter nuclei, snRNPs and SR proteins do not immediately colocalize in nuclear speckles. SR proteins accumulated in patches around active nucleolar organizing regions (NORs) that we refer to as NOR-associated patches (NAPs), whereas snRNPs were enriched at other nuclear regions. NAPs formed transiently, persisting for 15–20 min before dissipating as nuclear speckles began to form in G1. In the absence of RNA polymerase II transcription, NAPs increased in size and persisted for at least 2 h, with delayed localization of SR proteins to nuclear speckles. In addition, SR proteins in NAPs are hypophosphorylated, and the SR protein kinase Clk/STY colocalizes with SR proteins in NAPs, suggesting that phosphorylation releases SR proteins from NAPs and their initial target is transcription sites. This work demonstrates a previously unrecognized role of NAPs in splicing factor trafficking and nuclear speckle biogenesis.

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.

scholarly journals A Functional Interaction between the Carboxy-Terminal Domain of RNA Polymerase II and Pre-mRNA Splicing

1997 ◽  
Vol 136 (1) ◽  
pp. 5-18 ◽  
Author(s):  
Lei Du ◽  
Stephen L. Warren
Keyword(s):  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Functional Interaction ◽  
Carboxy Terminal Domain ◽  
Localization Signal ◽  
Nuclear Domains ◽  
Carboxy Terminal

In the preceding study we found that Sm snRNPs and SerArg (SR) family proteins co-immunoprecipitate with Pol II molecules containing a hyperphosphorylated CTD (Kim et al., 1997). The association between Pol IIo and splicing factors is maintained in the absence of pre-mRNA, and the polymerase need not be transcriptionally engaged (Kim et al., 1997). The latter findings led us to hypothesize that a phosphorylated form of the CTD interacts with pre-mRNA splicing components in vivo. To test this idea, a nested set of CTD-derived proteins was assayed for the ability to alter the nuclear distribution of splicing factors, and to interfere with splicing in vivo. Proteins containing heptapeptides 1-52 (CTD52), 1-32 (CTD32), 1-26 (CTD26), 1-13 (CTD13), 1-6 (CTD6), 1-3 (CTD3), or 1 (CTD1) were expressed in mammalian cells. The CTD-derived proteins become phosphorylated in vivo, and accumulate in the nucleus even though they lack a conventional nuclear localization signal. CTD52 induces a selective reorganization of splicing factors from discrete nuclear domains to the diffuse nucleoplasm, and significantly, it blocks the accumulation of spliced, but not unspliced, human β-globin transcripts. The extent of splicing factor disruption, and the degree of inhibition of splicing, are proportional to the number of heptapeptides added to the protein. The above results indicate a functional interaction between Pol II's CTD and pre-mRNA splicing.

scholarly journals Primase p49 mRNA expression is serum stimulated but does not vary with the cell cycle.

1989 ◽  
Vol 9 (5) ◽  
pp. 1940-1945 ◽  
Author(s):  
B Y Tseng ◽  
C E Prussak ◽  
M T Almazan
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Small Subunit ◽  
Mrna Levels ◽  
Proliferating Cells ◽  
Restriction Point ◽  
Serum Stimulation ◽  
G1 Cells

Expression of the small-subunit p49 mRNA of primase, the enzyme that synthesizes oligoribonucleotides for initiation of DNA replication, was examined in mouse cells stimulated to proliferate by serum and in growing cells. The level of p49 mRNA increased approximately 10-fold after serum stimulation and preceded synthesis of DNA and histone H3 mRNA by several hours. Expression of p49 mRNA was not sensitive to inhibition by low concentrations of cycloheximide, which suggested that the increase in mRNA occurred before the restriction point control for cell cycle progression described for mammalian cells and was not under its control. p49 mRNA levels were not coupled to DNA synthesis, as observed for the replication-dependent histone genes, since hydroxyurea or aphidicolin had no effect on p49 mRNA levels when added before or during S phase. These inhibitors did have an effect, however, on the stability of p49 mRNA and increased the half-life from 3.5 h to about 20 h, which suggested an interdependence of p49 mRNA degradation and DNA synthesis. When growing cells were examined after separation by centrifugal elutriation, little difference was detected for p49 mRNA levels in different phases of the cell cycle. This was also observed when elutriated G1 cells were allowed to continue growth and then were blocked in M phase with colcemid. Only a small decrease in p49 mRNA occurred, whereas H3 mRNA rapidly decreased, when cells entered G2/M. These results indicate that the level of primase p49 mRNA is not cell cycle regulated but is present constitutively in proliferating cells.

Pre-mRNA splicing and nuclear organization

1992 ◽  
Vol 50 (1) ◽  
pp. 502-503
Author(s):  
David L. Spector ◽  
Gayle Lark ◽  
Sui Huang
Keyword(s):  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Transformed Cells ◽  
Splicing Factors ◽  
Cell Nuclei ◽  
Small Nuclear Ribonucleoprotein ◽  
Coiled Bodies ◽  
Passage Number ◽  
Immortal Cells ◽  
Labeling Pattern

Several major classes of small nuclear ribonucleoprotein particles (snRNPs) (Ul, U2, U4/U6, U5) as well as other splicing factors have been shown to be involved in the processing of pre-mRNA molecules. For most RNA polymerase II transcripts, such processing includes the addition of a 7-methylguanosine cap structure at the 5’ end of the nascent RNA transcripts, hnRNP assembly, splicing, polyadenylation, and the exchange of hnRNP proteins for mRNP proteins. Splicing of nuclear pre-mRNA molecules occurs in spliceosomes, macromolecular complexes composed of a pre-mRNA, snRNPs and other splicing factors. Since RNA processing is essential to cellular function, we and others have been interested in identifying the organization of splicing factors in cell nuclei. We have examined the localization of snRNPs in a variety of mammalian cells and have observed differences in the organization of these factors in transformed cells, immortal cells, and cells of defined passage number. Cells of defined passage number exhibit a speckled staining pattern after immunolabeling with anti-Sm, anti-B’ or anti-m3G antibodies. Furthermore, 2 to 3% of the cells, in a given population, exhibit 1 to 2 round “foci” in addition to the speckled labeling pattern. However, transformed cells exhibited 1 to 4 intensely stained round foci, in 81 to 99% of the cells, in addition to the speckled labeling pattern. Immortal cells exhibited 1 to 4 intensely stained smaller foci in 4 to 40% of the cells, in addition to the speckled labeling pattern. When immortal cells (REF-52) which had been transformed by adenovirus (REF-52 Ad5.4) were examined, these cells exhibited an increase in the percentage of cells containing 1 to 2 intensely stained foci, in addition to the speckled labeling, from 24% to 99%. We have identified these intensely stained foci as coiled bodies which can be visualized in the nucleoplasm of cells with or without antibody labeling. This study is the first to directly correlate an increase in the number of cells containing coiled bodies in a given cell population with the transformed phenotype. Based on this study, we conclude that the organization of snRNPs within the mammalian cell nucleus is a reflection of the physiology of the cell which may change upon transformation or immortalization.

Localization of poly(A)-binding protein 2 (PABP2) in nuclear speckles is independent of import into the nucleus and requires binding to poly(A) RNA

2000 ◽  
Vol 113 (12) ◽  
pp. 2309-2318 ◽  
Author(s):  
A. Calado ◽  
M. Carmo-Fonseca
Keyword(s):  
Nuclear Import ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Splicing Factors ◽  
Additional Component ◽  
Nuclear Speckles ◽  
High Affinity

The nuclei of mammalian cells contain domains, termed nuclear speckles, which are enriched in splicing factors and poly(A) RNA. Although nuclear speckles are thought to represent reservoirs from which splicing factors are recruited to sites of transcription and splicing, the presence of poly(A) RNA in these structures remains enigmatic. An additional component of the speckles is poly(A) binding protein 2 (PABP2), a protein that binds with high affinity to nascent poly(A) tails, stimulating their extension and controlling their length. In this work we investigated whether PABP2 contributes to the targeting of poly(A) RNA to the speckles. The results show that localization of PABP2 in speckles is independent of import of the protein into the nucleus. Inhibition of transcription or poly(A) synthesis at the end of mitosis does not affect nuclear import of PABP2 but prevents its localization to speckles. Furthermore, PABP2 mutants with decreased ability to bind to poly(A) fail to localize to speckles. Taken together the results show that PABP2 localizes to the nuclear speckles as a consequence of its binding to poly(A) RNA, contrasting to splicing factors which assemble into speckles in the absence of newly synthesized transcripts.

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 Intracellular Dynamics of the Ubiquitin-Proteasome-System

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 367 ◽  
Author(s):  
Maisha Chowdhury ◽  
Cordula Enenkel
Keyword(s):  
Cell Cycle ◽  
Protein Degradation ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Current Knowledge ◽  
Ubiquitin Proteasome System ◽  
Yeast Cells ◽  
Ubiquitin Chain ◽  
Dividing Cells ◽  
Ubiquitin Proteasome

The ubiquitin-proteasome system is the major degradation pathway for short-lived proteins in eukaryotic cells. Targets of the ubiquitin-proteasome-system are proteins regulating a broad range of cellular processes including cell cycle progression, gene expression, the quality control of proteostasis and the response to geno- and proteotoxic stress. Prior to degradation, the proteasomal substrate is marked with a poly-ubiquitin chain. The key protease of the ubiquitin system is the proteasome. In dividing cells, proteasomes exist as holo-enzymes composed of regulatory and core particles. The regulatory complex confers ubiquitin-recognition and ATP dependence on proteasomal protein degradation. The catalytic sites are located in the proteasome core particle. Proteasome holo-enzymes are predominantly nuclear suggesting a major requirement for proteasomal proteolysis in the nucleus. In cell cycle arrested mammalian or quiescent yeast cells, proteasomes deplete from the nucleus and accumulate in granules at the nuclear envelope (NE) / endoplasmic reticulum ( ER) membranes. In prolonged quiescence, proteasome granules drop off the nuclear envelopeNE / ER membranes and migrate as droplet-like entitiesstable organelles  throughout the cytoplasm, as thoroughly investigated in yeast. When quiescence yeast cells are allowed to resume growth, proteasome granules clear and proteasomes are rapidly imported into the nucleus.Here, we summarize our knowledge about the enigmatic structure of proteasome storage granules and the trafficking of proteasomes and their substrates between the cyto- and nucleoplasm.Most of our current knowledge is based on studies in yeast. Their translation to mammalian cells promises to provide keen insight into protein degradation in non-dividing cells, which comprise the majority of our body’s cells.

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 A novel mammalian protein, p55CDC, present in dividing cells is associated with protein kinase activity and has homology to the Saccharomyces cerevisiae cell division cycle proteins Cdc20 and Cdc4.

1994 ◽  
Vol 14 (5) ◽  
pp. 3350-3363 ◽  
Author(s):  
J Weinstein ◽  
F W Jacobsen ◽  
J Hsu-Chen ◽  
T Wu ◽  
L G Baum
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Division ◽  
Protein Kinase ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Yeast Cells ◽  
Protein Kinase Activity ◽  
Growing Cell ◽  
Dividing Cells

A novel protein, p55CDC, has been identified in cycling mammalian cells. This transcript is readily detectable in all exponentially growing cell lines but disappears when cells are chemically induced to fall out of the cell cycle and differentiate. The p55CDC protein appears to be essential for cell division, since transfection of antisense p55CDC cDNA into CHO cells resulted in isolation of only those cells which exhibited a compensatory increase in p55CDC transcripts in the sense orientation. Immunoprecipitation of p55CDC yielded protein complexes with kinase activity which fluctuated during the cell cycle. Since p55CDC does not have the conserved protein kinase domains, this activity must be due to one or more of the associated proteins in the immune complex. The highest levels of protein kinase activity were seen with alpha-casein and myelin basic protein as substrates and demonstrated a pattern of activity distinct from that described for the known cyclin-dependent cell division kinases. The p55CDC protein was also phosphorylated in dividing cells. The amino acid sequence of p55CDC contains seven repeats homologous to the beta subunit of G proteins, and the highest degree of homology in these repeats was found with the Saccharomyces cerevisiae Cdc20 and Cdc4 proteins, which have been proposed to be involved in the formation of a functional bipolar mitotic spindle in yeast cells. The G beta repeat has been postulated to mediate protein-protein interactions and, in p55CDC, may modulate its association with a unique cell cycle protein kinase. These findings suggest that p55CDC is a component of the mammalian cell cycle mechanism.

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