Expression and Function of Downstream-of-Gene RNAs in Innate Immunity
Date of Award
Spring 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Immunobiology
First Advisor
Steitz, Joan
Abstract
Over the past decade, there has been an explosion in our understanding of the many different types of non-coding RNAs (ncRNAs), their mechanisms of actions, and their remarkable variety of functions in gene expression and genome maintenance . However, the functional assignments of ncRNAs have lagged behind the detection of thousands of transcribed units. Downstream-of-Gene containing RNAs (DoG RNAs), produced by transcriptional readthrough of a subset of expressed genes in stressed cells, are one such class of ncRNAs whose function has remained enigmatic. In this thesis, I investigate the expression and function of DoG RNAs in the innate immune response and in the human brain, with the overarching goal of understanding their physiological significance to human health and disease. First, I examined the role of innate immune sensing in the production of DoG RNAs. Activating the cytosolic dsRNA sensor MDA5 with poly(I:C) was sufficient to induce DoG RNAs during the interferon (IFN) antiviral response. Many IFN-stimulated genes (ISGs), including the gene encoding IFN-β (Ifnb1), produced high levels of DoG RNAs in response to poly(I:C). Blocking the synthesis of Ifnb1-DoG RNA or depleting it with CRISPR revealed its dual function in restricting IFN-β activation at baseline while enabling its efficient induction upon innate viral recognition. These findings demonstrate that the host immune response contributes to the DoG RNA landscape in viral infections and that DoG RNAs are potential gene regulators that modulate the antiviral response. Next, I comprehensively characterized the expression of DoG RNAs across 47 human tissues to understand their contributions to the tissue-specific transcriptome and their physiological relevance. I discovered that a significantly greater number of genes express DoG RNAs in the cerebellum compared to any other tissue in the basal state. The high density of neurons drives heightened DoG RNA expression in the cerebellum, which is further induced upon in vivo hyperosmotic stress. This work re-defines DoG RNAs as a class of ncRNAs with distinct and versatile expression profile across different tissues, cell types, and physiological conditions. Finally, I sought to understand why basal DoG RNA expression is elevated in neurons. Analyses of genomic sequences transcribed upon readthrough revealed an abundance of transcriptionally active transposable elements in DoG regions. Retrotransposon transcripts from DoG regions, which account for a large fraction of all intergenic retroelement transcripts, were retained in the nucleus. I show that DoG RNAs and their anti-sense retroelement transcripts form nuclear dsRNAs and that expressing DoG RNA anti-sense to retroelements can block the retroelement-induced IFN response. The inverse correlation between DoG RNA and ISG expression in cerebella of healthy and ataxic patients further supports the model that DoG RNAs restrict retrotransposon transcripts escape from the nucleus and their triggering of the IFN response. High DoG RNA production in neurons could act to dampen the IFN-dependent neuroinflammation in this critical non-replicating cell type. In conclusion, these studies highlight the complexities of DoG RNA expression and function. DoG RNAs in viral infections are induced by both viral and host factors, likely contributing to pro-viral and anti-viral gene expression pathways, respectively. DoG RNAs have cell type- and context-dependent expression profiles, and constitutive and inducible DoG expression in immune cells and neurons likely support different functions. Collectively, these findings reveal new functions for a large class of ncRNAs, implicating DoG RNAs as both potential gene and transposon regulators that contribute to innate immunity against viruses and retrotransposons.
Recommended Citation
Park, Annsea, "Expression and Function of Downstream-of-Gene RNAs in Innate Immunity" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1044.
https://elischolar.library.yale.edu/gsas_dissertations/1044
