Different populations of RNA polymerase II in living mammalian cells

Transcriptional cyclin-dependent kinases (CDKs) regulate RNA polymerase II initiation and elongation as well as cotranscriptional mRNA processing. In this report, we describe an important role for CDK12 in the epidermal growth factor (EGF)-induced c-FOS proto-oncogene expression in mammalian cells. This kinase was found in the exon junction complexes (EJC) together with SR proteins and was thus recruited to RNA polymerase II. In cells depleted of CDK12 or eukaryotic translation initiation factor 4A3 (eIF4A3) from the EJC, EGF induced fewer c-FOS transcripts. In these cells, phosphorylation of serines at position 2 in the C-terminal domain (CTD) of RNA polymerase II, as well as levels of cleavage-stimulating factor 64 (Cstf64) and 73-kDa subunit of cleavage and polyadenylation specificity factor (CPSF73), was reduced at the c-FOS gene. These effects impaired 3′ end processing of c-FOS transcripts. Mutant CDK12 proteins lacking their Arg-Ser-rich (RS) domain or just the RS domain alone acted as dominant negative proteins. Thus, CDK12 plays an important role in cotranscriptional processing of c-FOS transcripts.

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1Chapter 6 MicroRNA-Based RNA Polymerase II Expression Vectors for RNA Interference in Mammalian Cells

Regulation of Gene Expression by Small RNAs ◽

10.1201/9781420008708-21 ◽

2009 ◽

pp. 319-334

Keyword(s):

Rna Interference ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Mammalian Cells ◽

Expression Vectors

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DNA methylation directs microRNA biogenesis in mammalian cells

Nature Communications ◽

10.1038/s41467-019-13527-1 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 13

Author(s):

Ohad Glaich ◽

Shivang Parikh ◽

Rachel E. Bell ◽

Keren Mekahel ◽

Maya Donyo ◽

...

Keyword(s):

Dna Methylation ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Mammalian Cells ◽

Sequence Conservation ◽

Mirna Biogenesis ◽

Coding Sequence ◽

Microrna Biogenesis ◽

Flanking Regions

AbstractMicroRNA (miRNA) biogenesis initiates co-transcriptionally, but how the Microprocessor machinery pinpoints the locations of short precursor miRNA sequences within long flanking regions of the transcript is not known. Here we show that miRNA biogenesis depends on DNA methylation. When the regions flanking the miRNA coding sequence are highly methylated, the miRNAs are more highly expressed, have greater sequence conservation, and are more likely to drive cancer-related phenotypes than miRNAs encoded by unmethylated loci. We show that the removal of DNA methylation from miRNA loci leads to their downregulation. Further, we found that MeCP2 binding to methylated miRNA loci halts RNA polymerase II elongation, leading to enhanced processing of the primary miRNA by Drosha. Taken together, our data reveal that DNA methylation directly affects miRNA biogenesis.

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Conditional Expression of RNA Polymerase II in Mammalian Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m001883200 ◽

2000 ◽

Vol 275 (32) ◽

pp. 24375-24382 ◽

Cited By ~ 57

Author(s):

Mark Meininghaus ◽

Rob D. Chapman ◽

Manuela Horndasch ◽

Dirk Eick

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Mammalian Cells ◽

Conditional Expression

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Alteration of nuclear lamin organization inhibits RNA polymerase II–dependent transcription

The Journal of Cell Biology ◽

10.1083/jcb.200112047 ◽

2002 ◽

Vol 156 (4) ◽

pp. 603-608 ◽

Cited By ~ 170

Author(s):

Timothy P. Spann ◽

Anne E. Goldman ◽

Chen Wang ◽

Sui Huang ◽

Robert D. Goldman

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Mammalian Cells ◽

Protein A ◽

Selective Inhibition ◽

Chromatin Organization ◽

Dominant Negative ◽

Gene Activity ◽

Structural Components ◽

Nuclear Lamins

RTegulation of gene activity is mediated by alterations in chromatin organization. In addition, chromatin organization may be governed in part by interactions with structural components of the nucleus. The nuclear lamins comprise the lamina and a variety of nucleoplasmic assemblies that together are major structural components of the nucleus. Furthermore, lamins and lamin-associated proteins have been reported to bind chromatin. These observations suggest that the nuclear lamins may be involved in the regulation of gene activity. In this report, we test this possibility by disrupting the normal organization of nuclear lamins with a dominant negative lamin mutant lacking the NH2-terminal domain. We find that this disruption inhibits RNA polymerase II activity in both mammalian cells and transcriptionally active embryonic nuclei from Xenopus laevis. The inhibition appears to be specific for polymerase II as disruption of lamin organization does not detectably inhibit RNA polymerases I and III. Furthermore, immunofluorescence observations indicate that this selective inhibition of polymerase II–dependent transcription involves the TATA binding protein, a component of the basal transcription factor TFIID.

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RNA Polymerase II Accumulation in the Promoter-Proximal Region of the Dihydrofolate Reductase and γ-Actin Genes

Molecular and Cellular Biology ◽

10.1128/mcb.23.6.1961-1967.2003 ◽

2003 ◽

Vol 23 (6) ◽

pp. 1961-1967 ◽

Cited By ~ 61

Author(s):

Chonghui Cheng ◽

Phillip A. Sharp

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Dihydrofolate Reductase ◽

Mammalian Cells ◽

Proximal Region ◽

Heptad Repeat ◽

Pol Ii ◽

Actin Genes ◽

Carboxyl Terminal ◽

Promoter Proximal Region

ABSTRACT The carboxyl-terminal domain (CTD) of RNA polymerase II (Pol II) can be phosphorylated at serine 2 (Ser-2) and serine 5 (Ser-5) of the CTD heptad repeat YSPTSPS, and this phosphorylation is important in coupling transcription to RNA processing, including 5′ capping, splicing, and polyadenylation. The mammalian endogenous dihydrofolate reductase and γ-actin genes have been used to study the association of Pol II with different regions of transcribed genes (promoter-proximal compared to distal regions) and the phosphorylation status of its CTD. For both genes, Pol II is more concentrated in the promoter-proximal regions than in the interior regions. Moreover, different phosphorylation forms of Pol II are associated with distinct regions. Ser-5 phosphorylation of Pol II is concentrated near the promoter, while Ser-2 phosphorylation is observed throughout the gene. These results suggest that the accumulation of paused Pol II in promoter-proximal regions may be a common feature of gene regulation in mammalian cells.

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Transcription of brain chromatin by ribonucleic acid polymerases from brain nucleic and from Escherichia coli

Biochemical Journal ◽

10.1042/bj1301095 ◽

1972 ◽

Vol 130 (4) ◽

pp. 1095-1099 ◽

Cited By ~ 6

Author(s):

Vijendra K. Singh ◽

S. C. Sung

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Mammalian Cells ◽

Transcriptional Control ◽

Polymerase Activity ◽

Control Mechanisms ◽

E Coli ◽

Bacterial Enzyme ◽

Rna Polymerase Activity

1. Transcription of ox brain chromatin by brain nuclear RNA polymerase II and Escherichia coli RNA polymerase was studied. 2. The soluble chromatin prepared from brain nuclei contained DNA, RNA, histone and non-histone proteins. Such chromatin preparations did not display any endogenous RNA polymerase activity, when assayed in the presence of concentrations of KCl as high as 0.4m. 3. The chromatin-templated activity of brain nuclear polymerase II was stimulated by KCl, with an optimum around 0.25m. 4. The template activity of brain chromatin for brain nuclear polymerase II and E. coli enzyme was about 20–25% of that of pure DNA. This greatly repressed templatecapacity of chromatin was probably due to the acid-soluble chromosomal proteins. 5. Brain nuclear polymerase II was 3–4 times more active with dehistonized chromatin than with pure DNA as template, whereas bacterial enzyme was almost equally active with either of these two templates, reflecting the specificity of the transcriptional control mechanisms in mammalian cells.

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Apoptosis and Autophagy Induction in Mammalian Cells by Small Interfering RNA Knockdown of mRNA Capping Enzymes

Molecular and Cellular Biology ◽

10.1128/mcb.00021-08 ◽

2008 ◽

Vol 28 (19) ◽

pp. 5829-5836 ◽

Cited By ~ 22

Author(s):

Chun Chu ◽

Aaron J. Shatkin

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Mammalian Cells ◽

Small Interfering Rna ◽

Fluorescent Protein ◽

Mrna Translation ◽

Proteolytic Processing ◽

Tunel Staining ◽

Interfering Rna ◽

Capping Enzymes

ABSTRACT Addition of a 5′ cap to RNA polymerase II transcripts, the first step of pre-mRNA processing in eukaryotes from yeasts to mammals, is catalyzed by the sequential action of RNA triphosphatase, guanylyltransferase, and (guanine-N-7)methyltransferase. The effects of knockdown of these capping enzymes in mammalian cells were investigated using T7 RNA polymerase-synthesized small interfering RNA and also a lentivirus-based inducible, short hairpin RNA system. Decreasing either guanylyltransferase or methyltransferase resulted in caspase-3 activation and elevated terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining characteristic of apoptosis. Induction of apoptosis was independent of p53 tumor suppressor but dependent on BAK or BAX. In addition, levels of the BH3 family member Bim increased, while Mcl-1 and Bik levels remained unchanged during apoptosis. In contrast to capping enzyme knockdown, apoptosis induced by cycloheximide inhibition of protein synthesis required BAK but not BAX. Both Bim and Mcl-1 levels decreased in cycloheximide-induced apoptosis while Bik levels were unchanged, suggesting that apoptosis in siRNA-treated cells is not a direct consequence of loss of mRNA translation. siRNA-treated BAK−/− BAX−/− double-knockout mouse embryonic fibroblasts failed to activate capase-3 or increase TUNEL staining but instead exhibited autophagy, as demonstrated by proteolytic processing of microtubule-associated protein 1 light chain 3 (LC3) and translocation of transfected green fluorescent protein-LC3 from the nucleus to punctate cytoplasmic structures.

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