1Chapter 6 MicroRNA-Based RNA Polymerase II Expression Vectors for RNA Interference in 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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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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RNA-interference-directed chromatin modification coupled to RNA polymerase II transcription

Nature ◽

10.1038/nature03652 ◽

2005 ◽

Vol 435 (7046) ◽

pp. 1275-1279 ◽

Cited By ~ 52

Author(s):

Vera Schramke ◽

Daniel M. Sheedy ◽

Ahmet M. Denli ◽

Carolina Bonila ◽

Karl Ekwall ◽

...

Keyword(s):

Rna Interference ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Chromatin Modification ◽

Rna Polymerase Ii Transcription

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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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