Studies on cellular and viral double-stranded RNAs: from mechanism to application

Abstract

Beyond the canonical role of RNA in conveying sequence-embeded genetic information, recent studies have shown that RNA also transmit a biological signal through its structure. Double-stranded RNA (dsRNA) is an important pathogen-associated molecule pattern (PAMP) as viruses produce long dsRNA molecules as a byproduct during viral replication. In vertebrates, the innate immune system recognizes the dsRNA structure rather than a specific nucleotide sequence and induces an antiviral state to defend against viral infection. This structure-mediated recognition allows innate immune proteins to bind endogenous dsRNAs as well and induce subsequent signaling transductions. In this thesis, I will discuss the capturing method of dsRNA using their structural characteristic as well as its application to the detection of viral dsRNA and studying the biological role of endogenous dsRNA structure. In Chapter 1, reactive poly(pentafluorophenyl acrylate) (PPFPA)-grafted surfaces were used to immobilize dsRNA recognizing J2 antibody to construct a universal virus detection platform with enhanced sensitivity. The PPFPA-grafted silicon substrate was optimized for biomolecule immobilization by modulating surface hydrophilicity. To increase the long dsRNA detection sensitivity, a two-step method is devised where the captured dsRNAs are visualized with multiple fluorophore-tagged J2 antibodies. This universal virus detection platform successfully detected the dsRNAs from the lysate of hepatitis A or C virus-infected cells. Chapter 2 will discuss the biological role of inverted repeats of Alu (IRAlus) elements, which form dsRNA structure that can lead to nuclear sequestration and subsequent translational suppression of the host mRNA. Genes that contain functional IRAlus elements were initially identified, and by utilizing J2 formaldehyde-mediated cross-linking immunoprecipitation and sequencing (fCLIP-seq), I greatly expanded the number of genes that contain IRAlus elements that can induce gene silencing effects. With this method, I found a new mechanism for the crosstalk between alternative polyadenylation and endogenous dsRNAs in the post-transcriptional regulation of numerous biological processes including MDM2-p53 associated tumorigenesis and neurodegenerative diseases. Collectively, this thesis will extend our knowledge of the structural characteristics and biological role of the viral dsRNA as well as the endogenous dsRNAs.