Wednesday, May 15, 2024
5/15/2024
Project Summary/Abstract To prevent or limit infection, upon sensing foreign nucleic acids (e.g., viral RNA) interferon (IFN) is produced and elicits a potent innate immune response orchestrated by hundreds of IFN-stimulated genes. However, cells face several challenges when attempting to differentiate between foreign and self nucleic acids. For instance, cells often produce double-stranded RNAs (dsRNA) that can be mistakenly recognized as foreign and lead to autoimmunity. This is exemplified by the fact that naturally occurring mutations in an RNA-editing enzyme called ADAR1 (adenosine deaminase acting on RNA 1) cause an autoinflammatory disorder named Aicardi-Goutières syndrome characterized by an aberrant IFN response in the absence of infection. In addition to this link to autoimmunity, recent studies have demonstrated that depleting ADAR1 sensitizes tumor cells to innate or therapy-induced immune responses and ADAR1 may therefore be a promising target for anti-cancer therapies. Unfortunately, there is a gap in our basic understanding of how ADAR1 enables cells to differentiate self from non-self RNAs. With this grant, we aim to address this knowledge gap. ADAR1 belongs to a family of proteins that modifies adenosines (A) to inosines (I) on dsRNA. These A- to-I modifications, through an unknown mechanism, prevent the sensing of dsRNAs by receptor proteins including PKR (protein kinase R), which normally triggers translational inhibition and cell death. It has been speculated that these modifications alter the secondary structures of dsRNAs and prevent them from activating PKR. However, ADAR1 frequently targets unpaired adenosine bases, and therefore A-to-I conversion is unlikely to cause major disruption to dsRNA structures. Here, we propose a novel hypothesis that one or more unknown RNA-binding proteins (RBPs) are involved in differentiating ADAR1-edited vs. unedited dsRNAs. This RBP may preferentially bind to inosine-containing RNAs and prevent them from activating PKR. We will use a combination of genome-wide and high-coverage RBP-targeted CRISPR knockout screens to identify protein factors, which together with ADAR1, prevent self RNA from triggering immune responses and cell death. We will evaluate potential hits using a semi-arrayed siRNA library and include additional readouts such as PKR activation and IFN production to prioritize candidates for further investigation. Lastly, we will initiate preliminary mechanistic studies to determine their RNA interactions and potential protein interactions with ADAR1 and PKR. This work will lay the foundation for future detailed mechanistic studies. In conclusion, our proposal has the potential to open new avenues of research to understand the biological functions and importance of inosines within RNA. Our study may reveal novel and paradigm-shifting mechanistic insights into how cells differentiate self vs. non-self RNA. It will also provide critical information for developing ADAR1-based cancer immunotherapies.
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