Nuclear organization of pre-mRNA splicing factors and their substrates

1994 ◽  
Vol 52 ◽  
pp. 18-19
Author(s):  
S. Huang ◽  
T. J. Deerinck ◽  
M. H. Ellisman ◽  
D. L. Spector
Keyword(s):  
Mammalian Cells ◽  
Large Body ◽  
Mrna Splicing ◽  
Electron Microscopic Level ◽  
Splicing Factors ◽  
Electron Microscopic ◽  
Speckled Pattern ◽  
Interchromatin Granule Clusters ◽  
Interchromatin Granule ◽  
Perichromatin Fibrils

Previous studies from our laboratory as well as other laboratories have shown that a variety of pre-mRNA splicing factors are localized to a subnuclear speckled domain when mammalian cells are immunolabeled with antibodies against these pre-mRNA splicing factors. At the electron microscopic level the speckled pattern is composed of both interchromatin granule clusters and perichromatin fibrils. A large body of evidence has accumulated from both our laboratory and other laboratories which has suggested that the perichromatin fibrils represent nascent transcripts and the interchromatin granule clusters represent storage and/or assembly sites for pre-mRNA splicing factors. The majority of substrates for these splicing factors are pre-mRNAs which contain a poly(A) tail of approximately 200-300 nucleotides. During the past year we have studied the distribution of poly(A)+ RNA in the mammalian cell nucleus and its transport through nuclear pores by fluorescence and electron microscopic in situ hybridization. Poly(A)+ RNA was detected in the nucleus as a speckled pattern which we have found to totally colocalize with pre-mRNA splicing factors at interchromatin granule clusters and perichromatin fibrils.

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 In vivo analysis of the stability and transport of nuclear poly(A)+ RNA.

1994 ◽  
Vol 126 (4) ◽  
pp. 877-899 ◽  
Author(s):  
S Huang ◽  
T J Deerinck ◽  
M H Ellisman ◽  
D L Spector
Keyword(s):  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Stable Population ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Pores ◽  
Electron Microscopic ◽  
Interchromatin Granule Clusters ◽  
Interchromatin Granule ◽  
Pore Complexes

We have studied the distribution of poly(A)+ RNA in the mammalian cell nucleus and its transport through nuclear pores by fluorescence and electron microscopic in situ hybridization. Poly(A)+ RNA was detected in the nucleus as a speckled pattern which includes interchromatin granule clusters and perichromatin fibrils. When cells are fractionated by detergent and salt extraction as well as DNase I digestion, the majority of the nuclear poly(A)+ RNA was found to remain associated with the nonchromatin RNP-enriched fraction of the nucleus. After inhibition of RNA polymerase II transcription for 5-10 h, a stable population of poly(A)+ RNA remained in the nucleus and was reorganized into fewer and larger interchromatin granule clusters along with pre-mRNA splicing factors. This stable population of nuclear RNA may play an important role in nuclear function. Furthermore, we have observed that, in actively transcribing cells, the regions of poly(A)+ RNA which reached the nuclear pore complexes appeared as narrow concentrations of RNA suggesting a limited or directed pathway of movement. All of the observed nuclear pores contained poly(A)+ RNA staining suggesting that they are all capable of exporting RNA. In addition, we have directly visualized, for the first time in mammalian cells, the transport of poly(A)+ RNA through the nuclear pore complexes.

scholarly journals RCC1 and nuclear organization.

1997 ◽  
Vol 8 (6) ◽  
pp. 1143-1157 ◽  
Author(s):  
S Huang ◽  
A Mayeda ◽  
A R Krainer ◽  
D L Spector
Keyword(s):  
Microscopic Examination ◽  
Nuclear Organization ◽  
Electron Microscopic Examination ◽  
Splicing Factors ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Interchromatin Granule Clusters ◽  
Interchromatin Granule

We have examined the effect of RCC1 function on the nuclear organization of pre-mRNA splicing factors and poly(A)+ RNA in the tsBN2 cells, a RCC1 temperature-sensitive mutant cell line. We have found that at 4-6 h after shifting cells from the permissive temperature (32.5 degrees C) to the restrictive temperature (39.5 degrees C), both small nuclear ribonucleoprotein particles and a general splicing factor SC35 reorganized into 4-10 large round clusters in the nucleus, as compared with the typical speckled distribution seen in cells at the permissive temperature. In situ hybridization to poly(A)+ RNA resulted in a similar pattern. Examination by double labeling demonstrated that the redistribution of splicing factors coincides with that of poly(A)+ RNA. Such changes in the nuclear organization of splicing factors and poly(A)+ RNA were not the result of the temperature shift or of chromatin condensation. Cellular transcription was not significantly altered in these cells and extracts made from both the permissive and restrictive temperature were splicing competent. Electron microscopic examination demonstrated that the large clusters containing both splicing factors and poly(A)+ RNA were fused interchromatin granule clusters. In addition, small electron-dense dot-like structures measuring approximately 80 nm in diameter were also observed, most of which are accumulated in enlarged interchromatin granule clusters in the nucleoplasm of RCC1- cells. In spite of the significant changes observed in the nucleoplasm, relatively little alteration was observed in nucleolar structure by both light and electron microscopic examination. The above observations suggest that the RCC1 protein directly or indirectly regulates the organization of splicing components and poly(A)+ RNA in the cell nucleus and that RCC1 may play a role in nuclear organization.

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.

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 Intron-dependent recruitment of pre-mRNA splicing factors to sites of transcription.

1996 ◽  
Vol 133 (4) ◽  
pp. 719-732 ◽  
Author(s):  
S Huang ◽  
D L Spector
Keyword(s):  
Spatial Association ◽  
Electron Microscopic Examination ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Electron Microscopic ◽  
Rna Transcripts ◽  
Highly Active ◽  
Transcription Sites ◽  
Active Transcription ◽  
Nascent Rna

We have examined the nuclear localization of transiently and stably expressed nascent RNA transcripts containing or lacking introns in order to determine if the spatial association of RNA transcripts and pre-mRNA splicing factors in nuclei is random or functionally significant. Our findings show that the association between nascent RNA and splicing factors in the nucleus is intron-dependent when the RNAs are either transiently or stably expressed. Furthermore, our data indicate that splicing factors are recruited to the transcription sites. The presence of both pre-and mRNA at these locations suggest that pre-mRNA splicing occurs at these sites of transcription. In addition, electron microscopic examination of the highly active transcription sites has revealed a granular appearance which closely resembles, but is functionally different from, interchromatin granule clusters. Our findings demonstrate that the nucleus is highly organized and dynamic with regard to the functions of the transcription and pre-mRNA splicing.

scholarly journals SRPK2: A Differentially Expressed SR Protein-specific Kinase Involved in Mediating the Interaction and Localization of Pre-mRNA Splicing Factors in Mammalian Cells

1998 ◽  
Vol 140 (4) ◽  
pp. 737-750 ◽  
Author(s):  
Huan-You Wang ◽  
Wen Lin ◽  
Jacqueline A. Dyck ◽  
Joanne M. Yeakley ◽  
Zhou Songyang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Splicing Regulation ◽  
Phosphorylation Sites ◽  
Sr Protein ◽  
Random Peptide ◽  
Selection For

Abstract. Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells. Two kinases, SR protein-specific kinase (SRPK1) and Clk/Sty, have been shown to phosphorylate the SR family of splicing factors. We report here the cloning and characterization of SRPK2, which is highly related to SRPK1 in sequence, kinase activity, and substrate specificity. Random peptide selection for preferred phosphorylation sites revealed a stringent preference of SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites in candidate arginine and serine-rich (RS) domain–containing proteins. Phosphorylation of an SR protein (ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain–containing protein (U1 70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus. These observations likely reflect the function of the SRPK family of kinases in spliceosome assembly and in mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities between SRPK1 and 2, however, are in contrast to their differences in expression. SRPK1 is highly expressed in pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues and in many experimental cell lines. Interestingly, SRPK2 also contains a proline-rich sequence at its NH2 terminus, and a recent study showed that this NH2-terminal sequence has the capacity to interact with a WW domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely regulated and targeted, thereby contributing to splicing regulation in different tissues, during development, or in response to signaling.

scholarly journals Nuclear PtdIns(4,5)P2 assembles in a mitotically regulated particle involved in pre-mRNA splicing

2001 ◽  
Vol 114 (13) ◽  
pp. 2501-2511 ◽  
Author(s):  
Shona L. Osborne ◽  
Claire L. Thomas ◽  
Steve Gschmeissner ◽  
Giampietro Schiavo
Keyword(s):  
Rna Polymerase Ii ◽  
Associated Factors ◽  
Mrna Processing ◽  
Mrna Splicing ◽  
Limited Information ◽  
Interchromatin Granule Clusters ◽  
Cellular Processes ◽  
Interchromatin Granule ◽  
Cycle Dependent

Phosphoinositide turnover regulates multiple cellular processes. Compared with their well-known cytosolic roles, limited information is available on the functions of nuclear phosphoinositides. Here, we show that phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P2) stably associates with electron-dense particles within the nucleus that resemble interchromatin granule clusters. These PtdIns(4,5)P2-containing structures have a distribution which is cell-cycle dependent and contain components of both the transcriptional and pre-mRNA processing machinery, including RNA polymerase II and the splicing factor SC-35. Immunodepletion and add-back experiments demonstrate that PtdIns(4,5)P2 and associated factors are necessary but not sufficient for pre-mRNA splicing in vitro, indicating a crucial role for PtdIns(4,5)P2-containing complexes in nuclear pre-mRNA processing.

scholarly journals Kinetochore structure, duplication, and distribution in mammalian cells: analysis by human autoantibodies from scleroderma patients.

1981 ◽  
Vol 91 (1) ◽  
pp. 95-102 ◽  
Author(s):  
S Brenner ◽  
D Pepper ◽  
M W Berns ◽  
E Tan ◽  
B R Brinkley
Keyword(s):  
Immunoelectron Microscopy ◽  
Mammalian Cells ◽  
Specific Antigen ◽  
Cell Types ◽  
Electron Microscopic Level ◽  
Immunoperoxidase Technique ◽  
Mitotic Chromosomes ◽  
Electron Microscopic ◽  
Interphase Nuclei ◽  
Kinetochore Region

The specificity of the staining of CREST scleroderma patient serum was investigated by immunofluorescence and immunoelectron microscopy. The serum was found to stain the centromere region of mitotic chromosomes in many mammalian cell types by immunofluorescence. It also localized discrete spots in interphase nuclei which we have termed "presumptive kinetochores." The number of presumptive kinetochores per cell corresponds to the chromosome number in the cell lines observed. Use of the immunoperoxidase technique to localize the antisera on PtK2 cells at the electron microscopic level revealed the specificity of the sera for the trilaminar kinetochore disks on metaphase and anaphase chromosomes. Presumptive kinetochores in the interphase nuclei were also visible in the electron microscope as randomly arranged, darkly stained spheres averaging 0.22 micrometers in diameter. Preabsorption of the antisera was attended using microtubule protein, purified tubulin, actin, and microtubule-associated proteins. None of these proteins diminished the immunofluorescence staining of the sera, indicating that the antibody-specific antigen(s) is a previously unrecognized component of the kinetochore region. In some interphase cells observed by both immunofluorescence and immunoelectron microscopy, the presumptive kinetochores appeared as double rather than single spots. Analysis of results obtained using a microspectrophotometer to quantify DNA in individual cells double stained with scleroderma serum and the DNA fluorescent dye, propidium iodide, led to the conclusion that the presumptive kinetochores duplicate in G2 of the cell cycle.

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