Optimized reaction conditions and specific inhibitors for initiation of transcription by RNA polymerase II in nuclei from cultured 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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Complete, 12-subunit RNA polymerase II at 4.1-A resolution: Implications for the initiation of transcription

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1130601100 ◽

2003 ◽

Vol 100 (12) ◽

pp. 6969-6973 ◽

Cited By ~ 180

Author(s):

D. A. Bushnell ◽

R. D. Kornberg

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Initiation Of Transcription

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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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