ARTICLE |
Correspondence to: Roeland W. Dirks, Dept. of Molecular Cell Biology, Lab. for Cytochemistry and Cytometry, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Pre-mRNA synthesis in eukaryotic cells is preceded by the formation of a transcription initiation complex and binding of unphosphorylated RNA polymerase II (Pol II) at the promoter region of a gene. Transcription initiation and elongation are accompanied by the hyperphosphorylation of the carboxy-terminal domain (CTD) of Pol II large subunit. Recent biochemical studies provided evidence that RNA processing factors, including those required for splicing, associate with hyperphosphorylated CTDs forming "transcription factories." To directly visualize the existence of such factories, we simultaneously detected human cytomegalovirus immediate-early (IE) DNA and RNA with splicing factors and Pol II in rat 9G cells inducible for IE gene expression. Combined in situ hybridization and immunocytochemistry revealed that, after induction, both splicing factors and Pol II are present at the sites of IE mRNA synthesis and of IE mRNA processing that extend from the transcribing gene. Noninduced cells revealed no such associations. When IE mRNA-synthesizing cells were treated with a transcription inhibitor, these associations disappeared within 30 min. Our results show that the association of Pol II and splicing factors with IE DNA is dependent on its transcriptional activity and furthermore suggest that splicing factors are still associated with Pol II during active splicing. (J Histochem Cytochem 47:245254, 1999)
Key Words: in situ hybridization, immunocytochemistry, transcription, splicing, monoclonal antibody CC-3
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Synthesis of mRNAs is initiated by binding of a hypophosphorylated form of RNA polymerase II (Pol IIA) to the promoter region. This interaction is mediated by several transcription factors that assemble on the promoter and on Pol II, including the carboxyterminal domain (CTD) of the largest subunit. Concomitant with transcription initiation, Pol II is hyperphosphorylated at the CTD, is released from the promoter protein complex, and forms an elongation complex (
To understand how the different components required for gene transcription and RNA processing are integrated in the cell nucleus, immunofluorescence microscopic studies have been performed using specific antibodies raised against splicing factors and Pol II. These studies revealed that splicing factors are located in 2050 speckles and in many small spots throughout the nucleoplasm, excluding nucleoli (for review see
Interestingly, Pol IIO is also found in speckles and in small foci dispersed throughout the nucleoplasm excluding nucleoli (
The high degree of overlap between sites of newly synthesized RNA and those of splicing factor and Pol IIO localization suggests further that transcription and RNA processing are spatially linked in vivo (see also
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture
Rat 9G cells harboring a tandem repeat of the HCMV-IE gene in one of the rat chromosomes were a generous gift of Rene Boom (
For some experiments, cells were also incubated for 5 min to 3 hr with 25 µg/ml 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB; Sigma) or with 0.5 µg/ml actinomycin D (Sigma) to inhibit RNA Pol II transcription.
In Situ Hybridization
Slides with rat 9G cells were fixed, pretreated, and hybridized with a probe as described previously (
Plasmid pSS containing the 5.0-KB SphI-SalI genomic fragment of the immediate-early region of HCMV (
Ten µl of probe was applied to a slide and covered with an 18 x 18-mm coverslip. Probe and target sequences were denatured simultaneously by placing the slides on an 80C metal plate for 3 min. Hybridizations were done at 37C overnight in a moist chamber.
Posthybridization Washes and Immunocytochemical Detection
After hybridization, cells were washed three times in 50% formamide, 2 x SSC for 10 min each at 37C, and 5 min in Tris-buffered saline (TBS: 150 mM NaCl, 100 mM Tris-HCl, pH 7.4) at RT. Cells hybridized with the oligo (dT) 50 probe were washed three times in 2 x SSC for 3 min each at RT.
For the detection of digoxigenin-labeled probes, cells were incubated with monoclonal antibody (MAb) anti-digoxin (Sigma; dilution1:1000) followed by an incubation with a secondary anti-mouse antibody conjugated with FITC (Sigma), Cy3 (Jackson Immunoresearch; West Grove, PA), or Texas Red (Vector Labs; Burlingame, CA) or with a sheep anti-digoxigenin antibody conjugated with FITC (Boehringer Mannheim). Biotin-labeled probes were detected with streptavidin Cy3(Jackson Immunoresearch), streptavidinTexas Red (Vector), or avidinCy5 (Jackson Immunoresearch).
For the detection of splicing factors, MAb anti-m3G (Oncogene Science; Cambridge, MA), which reacts with the 2,2,7-trimethyl guanosine cap of snRNAs (
Finally, cells were mounted in Vectashield (Vector) containing 4',6'-diamidino-2-phenyl indole (DAPI) as a DNA counterstain.
Microscopy
Images were collected with an epifluorescence microscope (DM; Leica, Oberkochen, Germany) equipped with a 100 W mercury arc lamp, a triple excitation filter for red, green and blue excitation (Omega; Brattleboro, VT), a filter for Cy5 excitation, and PI Fluotar x100, NA 1.30-0.60, PI APO x63, NA 1.40 objectives. Different fluorochromes could be selectively excited and recorded without image shifts by inserting appropriate filters in the excitation way. Digital images were captured with a cooled CCD camera (Photometrics; Tucson, AZ) and processed on a Macintosh computer using SCIL-image (Multihouse; Amsterdam, The Netherlands).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hypo- and Hyperphosphorylated Pol II Have Different Localization Patterns in Rat 9G Cells
Initially, MAb CC-3 was shown to react with a phosphodependent epitope on a 255-kD nuclear matrix protein and possibly with an unidentified 180-kD protein. The 255-kD protein proved to be identical to the phosphorylated form of the largest RNA Pol II subunit (
|
Because it has been suggested that staining of speckles for hyperphosphorylated Pol II is correlated with low transcriptional activity (
Next, rat 9G cells were stained with MAb 8WG16 (
Hyperphosphorylated Pol II Associates with IE Gene Clusters after Induction
Double labeling experiments in which Pol IIO staining patterns were compared with those of Br-UTP incorporation revealed a high degree of overlap between the two (
Next, cells were first induced for IE gene expression, then treated with the transcription inhibitor DRB and finally heat-denatured before hybridization. As illustrated in Figure 2GI, a co-localization of Pol IIO with the IE gene was no longer observed. Analysis of 50 induced cells that were incubated with DRB for 30 min revealed that in more than 90% of the induced cells there was no association between Pol IIO and IE DNA. Cells induced for IE gene expression could still be recognized by the presence of IE mRNA in the cytoplasm after DRB treatment. When the heat-denaturation step of DRB-treated cells before hybridization was omitted, no nuclear signals were observed, whereas cytoplasmic RNA signals were still present. This confirms that the nuclear signals observed after DRB treatment and denaturation of the cells are indeed showing hybridization to IE DNA.
Because DRB inhibits phosphorylation of Pol IIO by inhibiting a CTD kinase (
Recently, we have shown that the association of splicing factors with the IE gene cluster is dependent on its transcriptional activity (
Next, we performed double labeling experiments combining the detection of IE mRNA with that of Pol IIA. The results showed that Pol IIA was present in small foci throughout the nucleoplasm but did not co-localize with IE mRNA (not shown). Often, a small Pol IIA-positive spot was found associated with an IE transcription domain. However, we cannot conclude whether this represents a functional interaction because of the abundance and widespread distribution of Pol IIA in the cell nucleus. In addition, it cannot be excluded that the number of Pol IIA molecules associated with IE mRNA is below the detection threshold of the technique used because we were unable to detect convincingly the association of Pol IIA with the 50 copies of the IE gene. Nevertheless, our results, which suggest that Pol IIA is not associated with accumulated RNA near the IE gene cluster, are in agreement with biochemical studies showing that Pol IIA is present in transcription initiation complexes only and is not involved in transcription elongation.
Hyperphosphorylated Pol II Co-localizes with Splicing Factors and IE Pre-mRNA Beyond the Dimensions of the Gene Cluster
The association of Pol IIO with sites of IE gene expression was analyzed in more detail using probes that are specific for IE mRNA, DNA, and intron sequences. Double labeling of induced cells with a RNA probe specific for IE DNA and MAb CC-3 revealed that Pol IIO not only co-localizes with the IE gene cluster but also accumulates near this cluster (Figure 3AC). This accumulation domain often had the shape of an irregular dot and occasionally of an elongated track. To determine whether Pol IIO co-localizes with IE mRNA in these domains, we performed triple labeling experiments in which IE DNA, IE mRNA, and Pol IIO were visualized simultaneously. Figure 3DI show that Pol IIO co-localizes completely with IE mRNA that accumulated in dot- (Figure 3DF) or track- (Figure 3G-I) like domains near the transcribing gene cluster. This complete co-localization was observed in all cells analyzed showing IE gene expression. The gene cluster was always observed as a small spot at one site or in the middle of a dot of IE RNA or at one site of an elongated track. To prove that splicing factors are also present in these domains, cells were first hybridized with the pSS probe and then incubated with MAb anti-m3G. In accordance with our previous observations (
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous immunocytochemical studies using MAb CC-3 demonstrated that Pol IIO is localized in speckle domains and in addition is more diffuse throughout the nucleoplasm, except for nucleoli, in a variety of cell types (
In agreement with previous data (
Our results taken together with biochemical data support a model in which splicing factors are recruited by the hyperphosphorylated CTD of elongating Pol IIO, after which they associate with this domain and participate in co-transcriptional splicing. When the transcription rate is high and partially or nonspliced transcripts accumulate near the transcribing gene, Pol IIO will dissociate from the gene after transcription but will remain associated with splicing factors while the splicing reaction is being completed. Finally, Pol IIO and splicing factors may return to speckles as a complex where they are recycled and stored.
We can only speculate about the mechanism by which Pol II and splicing factors are recruited from speckles to sites of active gene expression. It is possible that they are recruited as a complex, in which case Pol IIO must be dephosphorylated before association with a promoter region and splicing factors must be released. Alternatively, Pol IIO is converted to Pol IIA just before or after being released from the speckle, in which case Pol II and splicing factors are recruited separately. In this respect, it should be noted that associations of splicing factors with the hypophosphorylated form of RNA Pol II have not been observed (
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baurén G, Jiang W-Q, Bernholm K, Gu F, Wieslander L (1996) Demonstration of a dynamic, transcription-dependent organization of pre-mRNA splicing factors in polytene nuclei. J Cell Biol 133:929-941[Abstract]
Baurén G, Wieslander L (1994) Splicing of Balbiani ring 1 gene pre-mRNA occurs simultaneously with transcription. Cell 76:183-192[Medline]
Beyer AL, Osheim YN (1988) Splice site selection, rate of splicing, and alternative splicing on nascent transcripts. Genes Dev 2:754-765[Abstract]
Bisotto S, Lauriault P, Duval M, Vincent M (1995) Co-localization of a high molecular mass phosphoprotein of the nuclear matrix (p255) with spliceosomes. J Cell Sci 108:1873-1882
Boom R, Geelen JL, Sol CJ, Raap AK, Minnaar RP, Klaver BP, van der Noordana J (1986) Establishment of a rat cell line inducible for the expression of human cytomegalovirus immediate early gene products by protein synthesis inhibitors. J Virol 58:851-859[Medline]
Bregman DB, Du L, van der Zee S, Warren SL (1995) Transcription-dependent redistribution of the large subunit of RNA polymerase II to discrete nuclear domains. J Cell Biol 129:287-298[Abstract]
Carter KC, Bowman D, Carrington W, Fogarty K, McNeil JA, Fay FS, Lawrence JB (1993) A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus. Science 259:1330-1335[Medline]
Carter KC, Taneja KL, Lawrence JB (1991) Discrete nuclear domains of poly(A) RNA and their relationship to the functional organization of the nucleus. J Cell Biol 115:1191-1202[Abstract]
Chabot B, Bisotto S, Vincent M (1995) The nuclear matrix phosphoprotein p255 associates with splicing complexes as part of the [U4/U6.U5] tri-snRNP particle. Nucleic Acids Res 23:3206-3213[Abstract]
Cho E-J, Takagi CR, Moore CR, Buratowski S (1997) mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxyterminal domain. Genes Dev 11:3319-3326
Dahmus ME (1996) Reversible phosphorylation of the C-terminal domain of RNA polymerase II. J Biol Chem 271:19009-19012
Dirks RW (1996) RNA molecules lighting up under the microscope. Histochem Cell Biol 106:151-166[Medline]
Dirks RW, Daniël KC, Raap AK (1995) RNAs radiate from gene to cytoplasm as revealed by fluorescence in situ hybridization. J Cell Sci 108:2565-2572
Dirks RW, de Pauw ESD, Raap AK (1997) Splicing factors associate with nuclear HCMV-IE transcripts after transcriptional activation of the gene, but dissociate upon transcription inhibition: evidence for a dynamic organization of splicing factors. J Cell Sci 110:515-522
Dirks RW, Raap AK (1995) Cell-cycle-dependent gene expression studied by two-colour fluorescent detection of a mRNA and histone mRNA. Histochem Cell Biol 104:391-395[Medline]
Dirks RW, van de Rijke FM, Fujishita S, van der Ploeg M, Raap AK (1993) Methodologies for specific intron and exon localization in cultured cells by haptenized and fluorochromized probes. J Cell Sci 104:1187-1197
Du L, Warren SL (1997) A functional interaction between the carboxy-terminal domain of RNA polymerase II and pre-mRNA splicing. J Cell Biol 136:5-18
Dubois M-F, Bellier S, Seo S-J, Bensaude O (1994a) Phosphorylation of the RNA polymerase II largest subunit during heat shock and inhibition of transcription in HeLa cells. J Cell Physiol 158:417-426[Medline]
Dubois M-F, Nguyen VT, Bellier S, Bensaude O (1994b) Inhibitors of transcription such as 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole and isoquinoline sulfonamide derivatives (H-8 and H-7) promote dephosphorylation of the carboxyl-terminal domain of RNA polymerase II largest subunit. J Biol Chem 269:13331-13336
Fakan S (1994) Perichromatin fibrils are in situ forms of nascent transcripts. Trends Cell Biol 4:86-90
Grande MA, van der Kraan I, de Jong L, van Driel R (1997) Nuclear distribution of transcription factors in relation to sites of transcription and RNA polymerase II. J Cell Sci 110:1781-1791
Greenleaf AL (1993) Positive patches and negative noodles: linking RNA processing to transcription? Trends Biochem Sci 18:117-119[Medline]
Ishov AM, Stenberg RM, Maul GG (1997) Human cytomegalovirus immediate early interaction with host nuclear structures: definition of an immediate transcript environment. J Cell Biol 138:5-16
Jackson DA, Hassan AB, Errington RJ, Cook PR (1993) Visualization of focal sites of transcription within human nuclei. EMBO J 12:1059-1065[Abstract]
JiménezGarcía LF, Spector DL (1993) In vivo evidence that transcription and splicing are coordinated by a recruiting mechanism. Cell 73:47-59[Medline]
Kim E, Du L, Bregman DB, Warren SL (1997) Splicing factors associate with hyperphosphorylated RNA polymerase II in the absence of pre-mRNA. J Cell Biol 136:19-28
Laybourn PJ, Dahmus ME (1990) Phosphorylation of RNA polymerase IIA occurs subsequent to interaction with the promoter and before the initiation of transcription. J Biol Chem 265:13165-13173
Marshall NF, Peng J, Xie Z, Price DH (1996) Control of RNA polymerase II elongation potential by a novel carboxyl-terminal domain kinase. J Biol Chem 271:27176-27183
McCracken S, Fong N, Rosonina E, Yankulov K, Brothers G, Siderovski D, Hessel A, Foster S, Amgen EST Program, Shuman S, Bentley DL (1997a) 5'-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II. Genes Dev 11:33063318
McCracken S, Fong N, Yankulov K, Ballantyne S, Pan G, Greenblatt J, Patterson SD, Wickens M, Bentley DL (1997b) The C-terminal domain of RNA polymerase II couples mRNA processing to transcription. Nature 385:357-361[Medline]
Misteli T, Cáceres JF, Spector DL (1997) The dynamics of a pre-mRNA splicing factor in living cells. Nature 387:523-527[Medline]
Misteli T, Spector DL (1996) Serine/threonine phosphatase 1 modulates the subnuclear distribution of pre-mRNA splicing factors. Mol Biol Cell 7:1559-1572[Abstract]
Moen PT, Smith KP, Lawrence JB (1995) Compartmentalization of specific pre-mRNA metabolism: an emerging view. Hum Mol Genet 4:1779-1789[Abstract]
Mortillaro MJ, Blencowe BJ, Wei X, Nakayasu H, Du L, Warren SL, Sharp PA, Berezney R (1996) A hyperphosphorylated form of the large subunit of RNA polymerase II is associated with splicing complexes and the nuclear matrix. Proc Natl Acad Sci USA 93:8253-8257
Neugebauer KM, Roth MB (1997) Distribution of pre-mRNA splicing factors at sites of RNA polymerase II transcription. Genes Dev 11:1148-1159[Abstract]
O'Keefe RT, Mayeda A, Sadowski CL, Krainer AR, Spector DL (1994) Disruption of pre-mRNA splicing in vivo results in reorganization of splicing factors. J Cell Biol 124:249-260[Abstract]
Payne JM, Dahmus ME (1993) Partial purification and characterization of two distinct protein kinases that differentially phosphorylate the carboxy-terminal domain of RNA polymerase subunit IIa. J Biol Chem 268:80-87
Perry RP, Kelly DE (1970) Inhibition of RNA synthesis by actinomycin D: characteristic dose-response of different RNA species. J Cell Physiol 76:127-139[Medline]
Reuter R, Appel B, Bringmann P, Rinke J, Lührmann R (1984) 5'-Terminal caps of snRNAs are reactive with antibodies specific for 2,2,7-trimethylguanosine in whole cells and nuclear matrices. Exp Cell Res 154:548-560[Medline]
Sisodia SS, SollnerWebb B, Cleveland DW (1987) Specificity of RNA maturation pathways: RNAs transcribed by RNA polymerase III are not substrates for splicing or polyadenylation. Mol Cell Biol 7:3602-3612[Medline]
Spector DL (1993) Macromolecular domains within the cell nucleus. Annu Rev Cell Biol 9:265-315
Spector DL, O'Keefe RT, JiménezGarcía LF (1993) Dynamics of transcription and pre-mRNA splicing within the mammalian cell nucleus. Cold Spring Harbor Symp Quant Biol 58:799-805[Medline]
Steinmetz EJ (1997) Pre-mRNA processing and the CTD of RNA polymerase II: the tail that wags the dog? Cell 89:491-494[Medline]
Thompson NE, Steinberg TH, Aronson DB, Burgess RR (1989) Inhibition of in vivo and in vitro transcription by monoclonal antibodies prepared against wheat germ RNA polymerase II that react with the heptapeptide repeat of eukaryotic RNA polymerase II. J Biol Chem 264:11511-11520
van de Corput MPC, Dirks RW, van Gijlswijk RPM, van de Rijke FM, Raap AK (1998) Fluorescence in situ hybridization using horseradish peroxidase labeled oligonucleotides and tyramide signal amplification for sensitive DNA and mRNA detection. Histochem Cell Biol 110:431-437[Medline]
Vincent M, Lauriault P, Dubois M-F, Lavoie S, Bensaude O, Chabot B (1996) The nuclear matrix protein p255 is a highly phosphorylated form of RNA polymerase II largest subunit which associates with spliceosomes. Nucleic Acids Res 24:4649-4652
Wansink DG, Schul W, van der Kraan I, van Steensel B, van Driel R, de Jong L (1993) Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus. J Cell Biol 122:283-293[Abstract]
Weeks JR, Hardin SE, Shen J, Lee JM, Greenleaf AL (1993) Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: correlations with gene activity and transcript processing. Genes Dev 7:2329-2344[Abstract]
Wuarin Y, Schibler U (1994) Physical isolation of nascent RNA chains transcribed by RNA polymerase II: evidence for cotranscriptional splicing. Mol Cell Biol 14:7219-7225[Abstract]
Xing Y, Johnson CV, Dobner PR, Lawrence JB (1993) Higher level organization of individual gene transcription and RNA splicing. Science 259:1326-1330[Medline]
Xing Y, Johnson CV, Moen PT, McNeil JA, Lawrence JB (1995) Nonrandom gene organization: structural arrangements of specific pre-mRNA transcription and splicing with SC-35 domains. J Cell Biol 131:1635-1647[Abstract]
Yankulov K, Yamashita K, Roy R, Egly J-M, Bentley DL (1995) The transcriptional elongation inhibitor 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole inhibits transcription factor IIH-associated protein kinase. J Biol Chem 270:23922-23925
Yuryev A, Patturajan M, Litingtung Y, Joshi RV, Gentile C, Gebara M, Corden JL (1996) The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins. Proc Natl Acad Sci USA 93:6975-6980
Zeng C, Kim E, Warren SL, Berget SM (1997) Dynamic relocation of transcription and splicing factors dependent upon transcriptional activity. EMBO J 16:1401-1412