1. Nuclear re-localization of Dicer in primary mouse embryonic fibroblast nuclei following DNA damage
- Author
-
Burger, Kaspar and Gullerova, Monika
- Subjects
Ribonuclease III ,0301 basic medicine ,Cytoplasm ,Cancer Research ,genetic processes ,Biochemistry ,DEAD-box RNA Helicases ,Gene Knockout Techniques ,Mice ,RNA interference ,Post-Translational Modification ,Phosphorylation ,Cells, Cultured ,Genetics (clinical) ,Staining ,Cell Staining ,food and beverages ,Precipitation Techniques ,Cell biology ,Chromatin ,Nucleic acids ,Protein Transport ,medicine.anatomical_structure ,Female ,Cellular Structures and Organelles ,Research Article ,lcsh:QH426-470 ,DNA damage ,Immunoblotting ,Active Transport, Cell Nucleus ,Molecular Probe Techniques ,Biology ,Research and Analysis Methods ,03 medical and health sciences ,Genetics ,medicine ,Immunoprecipitation ,Animals ,Humans ,Molecular Biology Techniques ,Cytoplasmic Staining ,Molecular Biology ,Ecology, Evolution, Behavior and Systematics ,Cell Nucleus ,HEK 293 cells ,fungi ,Biology and Life Sciences ,Proteins ,RNA ,DNA ,Cell Biology ,Fibroblasts ,Embryo, Mammalian ,Nuclear Staining ,lcsh:Genetics ,Cell nucleus ,enzymes and coenzymes (carbohydrates) ,HEK293 Cells ,030104 developmental biology ,Specimen Preparation and Treatment ,biology.protein ,Dicer - Abstract
Dicer is a key component of RNA interference (RNAi) and well-known for its role in biogenesis of micro (mi)RNA in the cytoplasm. Increasing evidence suggests that mammalian Dicer is also present and active in the nucleus. We have previously shown that phosphorylated human Dicer associates with chromatin in response to DNA damage and processes double-stranded (ds)RNA in the nucleus. However, a recent study by Much et al. investigated endogenously tagged HA-Dicer both in primary mouse embryonic fibroblasts (PMEFs) as well as adult homozygous viable and fertile HA-Dicer mice under physiological conditions and concluded that murine Dicer is exclusively cytoplasmic. The authors challenged several findings, reporting functions of Dicer in mammalian nuclei. We have re-investigated this issue by applying subcellular fractionation, super-resolution microscopy followed by 3D reconstitution, and phospho-Dicer-specific antibodies using the same HA-Dicer PMEF cell line. Our data show that a small fraction of the murine HA-Dicer pool, approximately 5%, localises in the nucleus and is phosphorylated upon DNA damage. We propose that Dicer localisation is dynamic and not exclusively cytoplasmic, particularly in cells exposed to DNA damage., Author summary Cytoplasmic Dicer is a key component of the canonical micro (mi)RNA biogenesis pathway. However, a growing body of evidence points toward localisation and activity of mammalian Dicer in the nucleus. A recent study by Much et al., employed an endogenously HA-tagged Dicer knock-in mouse cell line to show that Dicer is exclusively cytoplasmic. This paper challenges several studies reporting various RNA metabolic functions of Dicer in human nuclei. Given the controversy about Dicer’s subcellular localisation, it is essential to address this issue. Employing the same cells as used by Much and colleagues, we combined super-resolution microscopy followed by 3D reconstitution and biochemical assays to show that endogenously tagged HA-Dicer prominently localises in the nucleus under physiological conditions. We demonstrate that DNA damage triggers accumulation of phosphorylated HA-Dicer in the nucleus, confirming previous observations in human cells. Our data indicate evolutionary conservation of nuclear Dicer localisation and function in mammals in response to DNA damage.
- Published
- 2018