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.

scholarly journals Differences in snRNP localization between transformed and nontransformed cells.

1992 ◽  
Vol 3 (5) ◽  
pp. 555-569 ◽  
Author(s):  
D L Spector ◽  
G Lark ◽  
S Huang
Keyword(s):  
Mammalian Cell ◽  
Cell Nucleus ◽  
Mammalian Cells ◽  
Staining Pattern ◽  
Transformed Cells ◽  
Coiled Bodies ◽  
Passage Number ◽  
Immortal Cells ◽  
Labeling Pattern

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, 2-3% of the cells, in a given population, exhibit labeling of 1 or 2 round coiled bodies in addition to the speckled-labeling pattern. However, transformed cells exhibited 1-4 intensely stained coiled bodies, in 81-99% of the cells, in addition to the speckled-labeling pattern. Immortal cells exhibited 1-4 intensely stained smaller coiled bodies in 4-40% of the cells, in addition to the speckled-labeling pattern. When immortal cells (REF-52) that had been transformed by adenovirus (REF-52Ad5.4) were examined, these cells exhibited an increase in the percentage of cells containing 1 or 2 intensely stained coiled bodies, in addition to the speckled labeling, from 24 to 99%. On the basis of this study, we conclude that the organization of snRNPs within the mammalian cell nucleus is a reflection of the physiology of the cell that may change upon transformation or immortalization.

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 Spatial mapping of splicing factor complexes involved in exon and intron definition

2008 ◽  
Vol 181 (6) ◽  
pp. 921-934 ◽  
Author(s):  
Jonathan D. Ellis ◽  
David Llères ◽  
Marco Denegri ◽  
Angus I. Lamond ◽  
Javier F. Cáceres
Keyword(s):  
Rna Polymerase Ii ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Sr Proteins ◽  
Small Subunit ◽  
Spatial Mapping ◽  
Fluorescence Lifetime Imaging ◽  
Cell Nuclei ◽  
Exon Definition ◽  
Small Nuclear Ribonucleoprotein

We have analyzed the interaction between serine/arginine-rich (SR) proteins and splicing components that recognize either the 5′ or 3′ splice site. Previously, these interactions have been extensively characterized biochemically and are critical for both intron and exon definition. We use fluorescence resonance energy transfer (FRET) microscopy to identify interactions of individual SR proteins with the U1 small nuclear ribonucleoprotein (snRNP)–associated 70-kD protein (U1 70K) and with the small subunit of the U2 snRNP auxiliary factor (U2AF35) in live-cell nuclei. We find that these interactions occur in the presence of RNA polymerase II inhibitors, demonstrating that they are not exclusively cotranscriptional. Using FRET imaging by means of fluorescence lifetime imaging microscopy (FLIM), we map these interactions to specific sites in the nucleus. The FLIM data also reveal a previously unknown interaction between HCC1, a factor related to U2AF65, with both subunits of U2AF. Spatial mapping using FLIM-FRET reveals differences in splicing factors interactions within complexes located in separate subnuclear domains.

scholarly journals A conserved epitope on a subset of SR proteins defines a larger family of Pre-mRNA splicing factors.

1995 ◽  
Vol 129 (4) ◽  
pp. 899-908 ◽  
Author(s):  
K M Neugebauer ◽  
J A Stolk ◽  
M B Roth
Keyword(s):  
Rna Polymerase Ii ◽  
Active Sites ◽  
Nuclear Extract ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Structural Element ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

The removal of introns from eukaryotic pre-mRNA occurs in a large ribonucleoprotein complex called the spliceosome. We have generated a monoclonal antibody (mAb 16H3) against four of the family of six SR proteins, known regulators of splice site selection and spliceosome assembly. In addition to the reactive SR proteins, SRp20, SRp40, SRp55, and SRp75, mAb 16H3 also binds approximately 20 distinct nuclear proteins in human, frog, and Drosophila extracts, whereas yeast do not detectably express the epitope. The antigens are shown to be nuclear, nonnucleolar, and concentrated at active sites of RNA polymerase II transcription which suggests their involvement in pre-mRNA processing. Indeed, most of the reactive proteins observed in nuclear extract are detected in spliceosomes (E and/or B complex) assembled in vitro, including the U1 70K component of the U1 small nuclear ribonucleoprotein particle and both subunits of U2AF. Interestingly, the 16H3 epitope was mapped to a 40-amino acid polypeptide composed almost exclusively of arginine alternating with glutamate and aspartate. All of the identified antigens, including the human homolog of yeast Prp22 (HRH1), contain a similar structural element characterized by arginine alternating with serine, glutamate, and/or aspartate. These results indicate that many more spliceosomal components contain such arginine-rich domains. Because it is conserved among metazoans, we propose that the "alternating arginine" domain recognized by mAb 16H3 may represent a common functional element of pre-mRNA splicing factors.

scholarly journals The WW Domain-Containing Proteins Interact with the Early Spliceosome and Participate in Pre-mRNA Splicing In Vivo

2004 ◽  
Vol 24 (20) ◽  
pp. 9176-9185 ◽  
Author(s):  
Kai-Ti Lin ◽  
Ruei-Min Lu ◽  
Woan-Yuh Tarn
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Extract ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Ww Domain ◽  
Ww Domains ◽  
Small Nuclear Ribonucleoprotein

ABSTRACT A growing body of evidence supports the coordination of mRNA synthesis and its subsequent processing events. Nuclear proteins harboring both WW and FF protein interaction modules bind to splicing factors as well as RNA polymerase II and may serve to link transcription with splicing. To understand how WW domains coordinate the assembly of splicing complexes, we used glutathione S-transferase fusions containing WW domains from CA150 or FBP11 in pull-down experiments with HeLa cell nuclear extract. The WW domains associate preferentially with the U2 small nuclear ribonucleoprotein and with splicing factors SF1, U2AF, and components of the SF3 complex. Accordingly, WW domain-associating factors bind to the 3′ part of a pre-mRNA to form a pre-spliceosome-like complex. We performed both in vitro and in vivo splicing assays to explore the role of WW/FF domain-containing proteins in this process. However, although CA150 is associated with the spliceosome, it appears to be dispensable for splicing in vitro. Nevertheless, in vivo depletion of CA150 substantially reduced splicing efficiency of a reporter pre-mRNA. Moreover, overexpression of CA150 fragments containing both WW and FF domains activated splicing and modulated alternative exon selection, probably by facilitating 3′ splice site recognition. Our results suggest an essential role of WW/FF domain-containing factors in pre-mRNA splicing that likely occurs in concert with transcription in vivo.

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

1997 ◽  
Vol 8 (7) ◽  
pp. 1207-1217 ◽  
Author(s):  
P Jordan ◽  
C Cunha ◽  
M Carmo-Fonseca
Keyword(s):  
Rna Polymerase Ii ◽  
Excision Repair ◽  
Uv Light ◽  
Nuclear Antigen ◽  
Coiled Body ◽  
Small Nuclear Ribonucleoprotein ◽  
Coiled Bodies ◽  
Repair Mechanisms ◽  
Nuclear Ribonucleoprotein

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.

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 Differential interaction of splicing snRNPs with coiled bodies and interchromatin granules during mitosis and assembly of daughter cell nuclei.

1994 ◽  
Vol 126 (1) ◽  
pp. 11-23 ◽  
Author(s):  
J A Ferreira ◽  
M Carmo-Fonseca ◽  
A I Lamond
Keyword(s):  
Nuclear Import ◽  
Mammalian Cells ◽  
Interphase Nucleus ◽  
Transcriptional Activity ◽  
Coiled Body ◽  
Cell Nuclei ◽  
Differential Interaction ◽  
Coiled Bodies ◽  
Interchromatin Granules ◽  
Small Nuclear Ribonucleoproteins

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.

Specific small nuclear RNAs from SV40-transformed cells stimulate transcription initiation in nontransformed isolated nuclei

1982 ◽  
Vol 60 (3) ◽  
pp. 252-262 ◽  
Author(s):  
Maurice J. Ringuette ◽  
Karen Gordon ◽  
Jolanta Szyszko ◽  
Margarida O. Krause
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Transformed Cells ◽  
Regulation Of Transcription ◽  
Cell Nuclei ◽  
Small Nuclear Rnas ◽  
Mouse Cells ◽  
3T3 Cell Lines

Previous studies in our laboratory have implicated small nuclear RNAs (SnRNA) in the regulation of transcription in isolated mammalian cell nuclei. The present investigation was designed to develop a transcription assay system using isolated intact nuclei with optimized RNA polymerase II activity which would be capable of reinitiation in vitro to study the mode of action of the "active" RNA.We used nuclei isolated from either human WI38 or Balb 3T3 mouse cells to test the activity of SnRNA purified from SV40-transformed WI38 or 3T3 cell lines. These systems were found to support transcription up to 60 min, 40–60% of which was polymerase II dependent. In vitro initiations were detected by [γ-32P]ATP incorporation as well as by Hg-Sepharose chromatography using (γ-S)ATP as substrate. Results supported the following conclusions: (a) SnRNA from transformed cells stimulates the transcriptional activity of nontransformed nuclei while homologous SnRNA has little or no activity; (b) the stimulation is NaOH-sensitive and is dependent on RNA polymerase II since it is eliminated by 1 μg/mL α-amanitin; (c) the active subfraction of SnRNA from mouse cells was found to be of identical size (320–350 nucleotides) to that previously identified in human and monkey cells; and (d) analysis of the transcripts obtained from control and stimulated cell nuclei revealed that SnRNA activity is due primarily to an increase in the number of initiated chains.

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