Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse.
“PMEFs were grown to 50–60% confluency, starved in medium without FCS for 16 h and then treated with 50 ng/ml FGF2 (Peprotech) for 30 min. .. PMEFs were irradiated for 10 min with a total dose of 20 Gy using a RS 2000 biological irradiator(Rad Source Technologies) and then incubated at 37°C for another 30 min. .. Cells were seeded onto glass coverslips, fixed in 4% formaldehyde for 10 min and permeabilized in 0.5% NP-40 for 10 min at room temperature.”