E1BF is an essential RNA polymerase I transcription factor with an intrinsic protein kinase activity that can modulate rRNA gene transcription.

Protein kinase(s) and protein phosphatase(s) present in a Xenopus S-100 transcription extract strongly influence promoter-dependent transcription by RNA polymerase I. The protein kinase inhibitor 6-dimethyl-aminopurine causes transcription to increase, while the protein phosphatase inhibitor okadaic acid causes transcription to decrease. Repression is also observed with inhibitor 2, and the addition of extra protein phosphatase 1 stimulates transcription, indicating that the endogenous phosphatase is a type 1 enzyme. Partial fractionation of the system, single-round transcription reactions, and kinetic experiments show that two different steps during ribosomal gene transcription are sensitive to protein phosphorylation: okadaic acid affects a step before or during transcription initiation, while 6-dimethylaminopurine stimulates a process "late" in the reaction, possibly reinitiation. The present results are a clear demonstration that transcription by RNA polymerase I can be regulated by protein phosphorylation.

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Human Nopp140, Which Interacts with RNA Polymerase I: Implications for rRNA Gene Transcription and Nucleolar Structural Organization

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.8536 ◽

1999 ◽

Vol 19 (12) ◽

pp. 8536-8546 ◽

Cited By ~ 63

Author(s):

Hung-Kai Chen ◽

Chi-Yun Pai ◽

Jing-Yi Huang ◽

Ning-Hsing Yeh

Keyword(s):

Amino Acids ◽

Rna Polymerase ◽

Gene Transcription ◽

Ectopic Expression ◽

Rna Polymerase I ◽

Rrna Genes ◽

Rrna Synthesis ◽

Rrna Gene ◽

Amino Terminal ◽

Polymerase I

ABSTRACT Nopp140 is thought to shuttle between nucleolus and cytoplasm. However, the predominant nucleolar localization of Nopp140 homologues from different species suggests that Nopp140 is also involved in events occurring within the nucleolus. In this study, we demonstrated that the largest subunit of RNA polymerase I, RPA194, was coimmunoprecipitated with the human Nopp140 (hNopp140). Such an interaction is mediated through amino acids 204 to 382 of hNopp140. By double immunofluorescence, hNopp140 was colocalized with RNA polymerase I at the rDNA (rRNA genes) transcription active foci in the nucleolus. These results suggest that Nopp140 can interact with RNA polymerase I in vivo. Transfected cells expressing the amino-terminal half of hNopp140, hNopp140N382 (amino acids 1 to 382), displayed altered nucleoli with crescent-shaped structures. This phenotype is reminiscent of the segregated nucleoli induced by actinomycin D treatment, which is known to inhibit rRNA synthesis. Consistently, the hNopp140N382 protein mislocalized the endogenous RNA polymerase I and shut off cellular rRNA gene transcription as revealed by an in situ run-on assay. These dominant negative effects of the mutant hNopp140N382 suggest that Nopp140 plays an essential role in rDNA transcription. Interestingly, ectopic expression of hNopp140 to a very high level caused the formation of a transcriptionally inactive spherical structure occupying the entire nucleolar area which trapped the RNA polymerase I, fibrillarin, and hNopp140 but excluded the nucleolin. The mislocalizations of these nucleolar proteins after hNopp140 overexpression imply that Nopp140 may also play roles in maintenance of nucleolar integrity.

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The yeast nucleolar protein Cbf5p is involved in rRNA biosynthesis and interacts genetically with the RNA polymerase I transcription factor RRN3.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.6175 ◽

1997 ◽

Vol 17 (10) ◽

pp. 6175-6183 ◽

Cited By ~ 65

Author(s):

C Cadwell ◽

H J Yoon ◽

Y Zebarjadian ◽

J Carbon

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Genomic Library ◽

Rna Polymerase I ◽

Nucleolar Protein ◽

Rrna Gene ◽

Temperature Sensitive ◽

Cell Biol ◽

Polymerase I

Yeast Cbf5p was originally isolated as a low-affinity centromeric DNA binding protein (W. Jiang, K. Middleton, H.-J. Yoon, C. Fouquet, and J. Carbon, Mol. Cell. Biol. 13:4884-4893, 1993). Cbf5p also binds microtubules in vitro and interacts genetically with two known centromere-related protein genes (NDC10/CBF2 and MCK1). However, Cbf5p was found to be nucleolar and is highly homologous to the rat nucleolar protein NAP57, which coimmunoprecipitates with Nopp140 and which is postulated to be involved in nucleolar-cytoplasmic shuttling (U. T. Meier, and G. Blobel, J. Cell Biol. 127:1505-1514, 1994). The temperature-sensitive cbf5-1 mutant demonstrates a pronounced defect in rRNA biosynthesis at restrictive temperatures, while tRNA transcription and pre-rRNA and pre-tRNA cleavage processing appear normal. The cbf5-1 mutant cells are deficient in cytoplasmic ribosomal subunits at both permissive and restrictive temperatures. A high-copy-number yeast genomic library was screened for genes that suppress the cbf5-1 temperature-sensitive growth phenotype. SYC1 (suppressor of yeast cbf5-1) was identified as a multicopy suppressor of cbf5-1 and subsequently was found to be identical to RRN3, an RNA polymerase I transcription factor. A cbf5delta null mutant is not rescued by plasmid pNOY103 containing a yeast 35S rRNA gene under the control of a Pol II promoter, indicating that Cbf5p has one or more essential functions in addition to its role in rRNA transcription.

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Extensive purification of a putative RNA polymerase I holoenzyme from plants that accurately initiates rRNA gene transcription in vitro

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.94.22.11869 ◽

1997 ◽

Vol 94 (22) ◽

pp. 11869-11874 ◽

Cited By ~ 41

Author(s):

J. Saez-Vasquez ◽

C. S. Pikaard

Keyword(s):

Rna Polymerase ◽

Gene Transcription ◽

Rna Polymerase I ◽

Rrna Gene ◽

Transcription In Vitro ◽

Polymerase I

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Identification of two steps during Xenopus ribosomal gene transcription that are sensitive to protein phosphorylation.

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.2011 ◽

1994 ◽

Vol 14 (3) ◽

pp. 2011-2020 ◽

Cited By ~ 4

Author(s):

P Labhart

Keyword(s):

Protein Kinase ◽

Rna Polymerase ◽

Protein Phosphorylation ◽

Gene Transcription ◽

Okadaic Acid ◽

Protein Phosphatase ◽

Transcription Initiation ◽

Ribosomal Gene ◽

Rna Polymerase I ◽

Polymerase I

Protein kinase(s) and protein phosphatase(s) present in a Xenopus S-100 transcription extract strongly influence promoter-dependent transcription by RNA polymerase I. The protein kinase inhibitor 6-dimethyl-aminopurine causes transcription to increase, while the protein phosphatase inhibitor okadaic acid causes transcription to decrease. Repression is also observed with inhibitor 2, and the addition of extra protein phosphatase 1 stimulates transcription, indicating that the endogenous phosphatase is a type 1 enzyme. Partial fractionation of the system, single-round transcription reactions, and kinetic experiments show that two different steps during ribosomal gene transcription are sensitive to protein phosphorylation: okadaic acid affects a step before or during transcription initiation, while 6-dimethylaminopurine stimulates a process "late" in the reaction, possibly reinitiation. The present results are a clear demonstration that transcription by RNA polymerase I can be regulated by protein phosphorylation.

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Immunocytochemical localisation of the nucleolar protein fibrillarin and RNA polymerase I during mouse early embryogenesis

Zygote ◽

10.1017/s0967199400002884 ◽

1996 ◽

Vol 4 (1) ◽

pp. 49-58 ◽

Cited By ~ 17

Author(s):

Jorge M. Cuadros-Fernández ◽

Pedro Esponda

Keyword(s):

Rna Polymerase ◽

Early Development ◽

Gene Transcription ◽

Early Embryogenesis ◽

Mouse Embryos ◽

Rna Polymerase I ◽

Nucleolar Protein ◽

Rrna Gene ◽

Polymerase I ◽

Nucleolar Precursor Bodies

SummaryWe have employed immunocytochemical procedures to localise the nucleolar protein fibrillarin and the enzyme RNA polymerase I in the numerous dense fibrillar bodies (nucleolar precursor bodies) which appear in the nuclei of mammalian early embryos. The aim of this study was to search for relationships between the localisation of these proteins, the changes in the structure of the nucleolar precursor bodies and the resumption of rRNA gene transcription during mouse early embryogenesis. Three human autoimmune sera which recognised fibrillarin and a rabbit antiserum created against RNA Polymerase. I were employed for fluorescence and electron microscopic immunocytochemical assays. A statistical analysis was also applied. Immunocytochemistry revealed that fibrillarin and RNA polymerase I showed the same localisation in the nucleolar precursor bodies. These proteins were immunolocalised only from the late 2-cell stage onward. Fibrillarin was initially detected at the periphery of the nucleolar pricursor bodies and the labelling gradually increased until the morula and blastocyst stages, where normally active nucleoli are found. The pattern of increase of fibrillarin during early embryogenesis shows a parallelism with the rise in rRNA gene transcription occurring during these embryonic stages, and a possible correlation between these two phenomena is suggested. Results demonstrated that nucleolar precursor bodies differ in their biochemical composition from the nucleolus and also from the prenucleolar bodies which appear during mitosis. When anti-fibrillarin antibodies were microinjected into the male pronucleus of mouse embryos to analyse the functions of fibrillarin during early development, they partially blocked the early development of mouse embryos and only 23.8% of injected embryos reach the blastocyst stage.

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Replication of an rRNA gene origin plasmid in the Tetrahymena thermophila macronucleus is prevented by transcription through the origin from an RNA polymerase I promoter.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3372 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3372-3381 ◽

Cited By ~ 14

Author(s):

W J Pan ◽

R C Gallagher ◽

E H Blackburn

Keyword(s):

Rna Polymerase ◽

Tetrahymena Thermophila ◽

Rna Polymerase I ◽

Rrna Gene ◽

Wild Type ◽

Independent Evidence ◽

Origin Region ◽

Origin Function ◽

Polymerase I ◽

Wild Type Promoter

In the somatic macronucleus of the ciliate Tetrahymena thermophila, the palindromic rRNA gene (rDNA) minichromosome is replicated from an origin near the center of the molecule in the 5' nontranscribed spacer. The replication of this rDNA minichromosome is under both cell cycle and copy number control. We addressed the effect on origin function of transcription through this origin region. A construct containing a pair of 1.9-kb tandem direct repeats of the rDNA origin region, containing the origin plus a mutated (+G), but not a wild type, rRNA promoter, is initially maintained in macronuclei as an episome. Late, linear and circular replicons with long arrays of tandem repeats accumulate (W.-J. Pan and E. H. Blackburn, Nucleic Acids Res, in press). We present direct evidence that the +G mutation inactivates this rRNA promoter. It lacks the footprint seen on the wild-type promoter and produces no detectable in vivo transcript. Independent evidence that the failure to maintain wild-type 1.9-kb repeats was caused by transcription through the origin came from placing a short DNA segment containing the rRNA gene transcriptional termination region immediately downstream of the wild-type rRNA promoter. Insertion of this terminator sequence in the correct, but not the inverted, orientation restored plasmid maintenance. Hence, origin function was restored by inactivating the rRNA promoter through the +G mutation or causing termination before transcripts from a wild-type promoter reached the origin region. We propose that transcription by RNA polymerase I through the rDNA origin inhibits replication by preventing replication factors from assembling at the origin.

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Stimulation of the mouse rRNA gene promoter by a distal spacer promoter.

Molecular and Cellular Biology ◽

10.1128/mcb.15.8.4648 ◽

1995 ◽

Vol 15 (8) ◽

pp. 4648-4656 ◽

Cited By ~ 14

Author(s):

M H Paalman ◽

S L Henderson ◽

B Sollner-Webb

Keyword(s):

Rna Polymerase ◽

Gene Promoter ◽

Rna Polymerase I ◽

Rrna Gene ◽

Transcriptional Terminator ◽

Wide Range ◽

Polymerase I ◽

Enhancer Sequences ◽

Stimulation Of

We show that the mouse ribosomal DNA (rDNA) spacer promoter acts in vivo to stimulate transcription from a downstream rRNA gene promoter. This augmentation of mammalian RNA polymerase I transcription is observed in transient-transfection experiments with three different rodent cell lines, under noncompetitive as well as competitive transcription conditions, over a wide range of template concentrations, whether or not the enhancer repeats alone stimulate or repress expression from the downstream gene promoter. Stimulation of gene promoter transcription by the spacer promoter requires the rDNA enhancer sequences to be present between the spacer promoter and gene promoter and to be oriented as in native rDNA. Stimulation also requires that the spacer promoter be oriented toward the enhancer and gene promoter. However, stimulation does not correlate with transcription from the spacer promoter because the level of stimulation is not altered by either insertion of a functional mouse RNA polymerase I transcriptional terminator between the spacer promoter and enhancer or replacement with a much more active heterologous polymerase I promoter. Further analysis with a series of mutated spacer promoters indicates that the stimulatory activity does not reside in the major promoter domains but requires the central region of the promoter that has been correlated with enhancer responsiveness in vivo.

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