Dicer as a Repressor of Antiviral Responses in Mouse Embryonic Stem Cells

Abstract

Recent studies have demonstrated that pluripotent stem cells (PSCs), including human and mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have underdeveloped antiviral innate immunity. Specifically, they fail to express IFN in response to viral stimuli. This finding has led to the hypothesis that the lack of IFN response is an intrinsic property of PSCs. It has also inspired the investigation of the underlying molecular mechanisms. In an effort to conduct a holistic analysis of antiviral responses in ESCs, we have developed an assay that can simultaneously measure RNA‐mediated gene expression and RNA‐induced antiviral responses. In this system, GFP is expressed from functionalized synthetic GFP‐mRNA and is used to measure the cellular translation activity, whereas dsRNA and 5’‐triphosphate‐ssRNA act as viral RNA analogs. Transfection of ESCs with these synthetic RNAs can potently activate all common antiviral signaling pathways, including RNA interference (RNAi), dsRNA activated protein kinase (PKR), OAS‐RNase L, and IFN responses. Our data revealed that RNAi activity was detected in ESCs, but not in Dicer knockout ESCs (D‐/‐ESCs) as expected since Dicer is a key enzyme in miRNA and siRNA processing. The antiviral responses mediated by PKR, OAS‐RNase L, and IFN were either not detected or detected at low levels in ESCs, but they increased dramatically in D‐/‐ESCs, as indicated by increased activity of PKR (measured by low level GFP expression and PKR phosphorylation) and RNase L (determined by RNA degradation). Furthermore, the components of the IFN signaling pathway are expressed at higher basal levels in D‐/‐ESCs than in normal ESCs and are further up‐regulated by RNA transfection. The most notable effect of RNA transfection into D‐/‐ESCs is the induction of cell death and decreased cell proliferation, which is at least partly attributed to PKR activation and increased expression of the cell cycle inhibitors p21 and p19. Our findings suggest that ESCs are exquisitely sensitive to cellular damages caused by antiviral response and that Dicer acts as a repressor of antiviral responses in ESCs under normal conditions.Support or Funding InformationThis work was in part supported by the National Institute of General Medical Sciences (R15GM128196‐01). We thank Mississippi‐IDeA Network of Biomedical Research Excellence for the use of the imaging facility (funded by the National Institute of General Medical Sciences P20 GM103476‐11).