Date of Award

Fall 12-9-2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

School

Biological, Environmental, and Earth Sciences

Committee Chair

Dr. Yan-Lin Guo

Committee Chair School

Biological, Environmental, and Earth Sciences

Committee Member 2

Dr. Faqing Huang

Committee Member 2 School

Mathematics and Natural Sciences

Committee Member 3

Dr. Fengwei Bai

Committee Member 3 School

Biological, Environmental, and Earth Sciences

Committee Member 4

Dr. Alex Flynt

Committee Member 4 School

Biological, Environmental, and Earth Sciences

Committee Member 5

Dr. Shahid Karim

Committee Member 5 School

Biological, Environmental, and Earth Sciences

Abstract

Recent studies have demonstrated that embryonic stem cells (ESCs) are deficient in expressing type I interferons (IFN), the cytokines that play key roles in antiviral responses. However, the underlying molecular mechanisms and biological implications of this finding are poorly understood. In this study, I used a synthetic RNA-based assay that can simultaneously assess multiple forms of antiviral responses in ESCs. Dicer is an enzyme essential for RNA interference (RNAi), which is used as a major antiviral mechanism in invertebrates but not clear in vertebrates. RNAi activity is detected in wild-type ESCs but is abolished in Dicer knockout ESCs (D−/−ESCs) as expected. D-/-ESCs have two unique phenotypes that distinguish it from wild-type ESCs: slow proliferation rate and the inability to exit the pluripotent state. Surprisingly, D−/−ESCs have gained the ability to express IFN, which is otherwise deficient in wild-type ESCs. Furthermore, D−/−ESCs have constitutively active double-stranded RNA (dsRNA)-activated protein kinase (PKR), an enzyme that is also involved in antiviral response. D−/−ESCs show increased sensitivity to the cytotoxicity resulting from synthetic RNA transfection that mimics virus infection. The effects of synthetic dsRNA can be partly replicated with a synthetic B2RNA corresponding to the retrotransposon B2 short interspersed nuclear element. B2RNA has secondary structure features of dsRNA and accumulates in D−/−ESCs, suggesting that B2RNA could be a cellular RNA that activates PKR and contributes to the decreased cell proliferation and viability of D−/−ESCs. Treatment of D−/−ESCs with a PKR inhibitor and IFNβ-neutralizing antibodies increased cell proliferation rate and cell viability. Based on these findings, we propose that, in ESCs, Dicer acts as a repressor of antiviral responses and plays a key role in the maintenance of proliferation, viability, and pluripotency of ESCs.

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