title: In vivo processing assay based on a dual-luciferase reporter system to evaluate DROSHA enzymatic activity.
abstract: Luciferase reporter assays are widely used to study promoter activity, transcription factors, intracellular signaling, protein interactions (Jia et al., PloS One 6:e26414), miRNA processing (Allegra and Mertens, Biochem Biophys Res Commun 406:501-505), and target recognition (Jin et al., Methods Mol Biol 936:117-127). Here we describe the use of a dual-luciferase reporter system to evaluate the enzymatic activity of a key enzyme involved in RNA maturation-DROSHA. This dual system is a simple and fast method for the quantification of the DROSHA processing activity in live cells.
authors.affiliation: Internal Medicine III, University of Ulm, Ulm, Germany.
authors.forename: Vera
authors.initials: V
authors.lastname: Bilan
authors.forename: Danilo
authors.initials: D
authors.lastname: Allegra
authors.forename: Florian
authors.initials: F
authors.lastname: Kuchenbauer
authors.forename: Daniel
authors.initials: D
authors.lastname: Mertens
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D002448:Cell Adhesion; D057075:Enzyme Assays / Q000379:methods*; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D055029:Inverted Repeat Sequences; D008156:Luciferases / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D059367:RNA Cleavage; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D043244:Ribonuclease III / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D020413:3' Untranslated Regions; C505362:MIRN16 microRNA, human; D035683:MicroRNAs; D012322:RNA Precursors; D034741:RNA, Small Interfering; D008156:Luciferases; C517918:DROSHA protein, human; D043244:Ribonuclease III