Journal: Genes & Development
Article Title: DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs
doi: 10.1101/gad.282616.116
Figure Lengend Snippet: DUSP11 directly dephosphorylates the BLV pre-miRNAs and 5p miRNAs. ( A ) Immunoblot analysis to confirm expression of DUSP11 and DUSP11 catalytic mutant proteins generated using in vitro transcription/translation. The membrane was probed using anti-DUSP11 and anti-tubulin antibodies. ( B ) In vitro phosphatase reactions on the [γ-32P]-BLV-pre-miR-B5 mimic and [γ-32P]-BLV-miR-B5-5p miRNA mimic using CIP (positive control) or the in vitro translated DUSP11, DUSP11 catalytic mutant, or luciferase (negative control) from A . Reactions were fractionated on 15% PAGE/8 M urea, and RNAs were stained with EtBr. RNAs were then transferred to a membrane, exposed to a storage phosphor screen, and imaged on a Typhoon bimolecular imager. ( C ) Northern blot analysis from wild-type and DUSP11 knockout HEK293T cells transfected with a 5′ triphosphorylated BLV-B5 pre-miRNA mimic pretreated with (+) or without (−) RNA 5′ polyphosphatase. The blot was first probed for the 5p miRNA arm (green), stripped, and reprobed for the 3p arm (orange). Note that a lighter exposure for the input RNA is shown as compared with the RNA recovered from cells.
Article Snippet: Ten picomoles of each mimic was incubated in a 20-µL reaction (50 mM Tris, 10 mM KCl, 5 mM DTT, protease inhibitor [Roche], 1 U of SUPERase In RNase inhibitor [ThermoFisher]) that contained 3.3 µL of in vitro translated products (DUSP11-3xFlag, DUSP11-CM-3x-Flag, or the negative control luciferase) or 1 µg of CIP (New England Biolabs) for 1 h at 37°C.
Techniques: Expressing, Mutagenesis, Generated, In Vitro, Positive Control, Luciferase, Negative Control, Polyacrylamide Gel Electrophoresis, Staining, Northern Blot, Knock-Out, Transfection