1 Project Summary 2 RNA editing of cellular RNAs helps the cell distinguish between self and non-self RNAs. This 3 editing of adenosines into inosines is generally catalyzed by the ‘adenosine deaminase acting on 4 RNA-1’ protein (ADAR1). A>I editing is augmented in tumors and upon infection, primarily 5 through the interferon-induced longer isoform of ADAR1 that comprises a Z-DNA/Z-RNA binding 6 domain named ‘Zα’ at its N-terminus. Misediting is implicated in neurological diseases such as 7 Aicardi-Goutières syndrome. Z-RNA in the form of repeats of cytosine and guanosine (CpG) in a 8 left-handed double-helical conformation has been proposed in cells, but the prevalence of such 9 structures and their exact role are unknown. In addition, many —if not most— regions proposed 10 to adopt a Z conformation do not resemble regular (CpG)n. How these local Z-RNA conformations 11 are generated within A-form helices, stabilized and regulated by Zα of ADAR1, as well as their 12 exact role in the function of these RNAs, remain unknown. Our hypothesis is that the binding 13 of Zα to Z-RNA plays an essential role during the editing process. Here, we propose to 14 answer the following questions: What is the mechanism for Z-RNA formation at CpG but also 15 non-CpG sequences? How widespread are transitions to Z-RNA across transcriptomes? Is Z- 16 RNA sampled in the free form or only adopted upon binding by Zα? What are the structural 17 features of Z-RNA recognition by Zα at non CpG sequences? We will first dynamically 18 characterize the propensity of various sequence contexts to adopt Z-RNA conformations. 19 This aim will use advanced NMR methods to characterize the sequence of events that lead an 20 RNA region from A-form to Z-form. Second, we will determine the unbiased 3D structure of 21 RNA fragments bound to Zα in solution. Finally, we will identify and localize Zα binding 22 sites and Zα-dependent A>I editing events. This aim will take advantage of the robust 23 expertise and support for next-generation sequencing on our campus and at a contracted 24 company. Overall, our joint work as co-PIs will provide a structural rationale for the formation of 25 Z-RNA and the resulting formation of A-Z junctions across a variety of RNAs. We ultimately aim 26 to explain how the Z-RNA binding domain of ADAR1 increases the specificity and activity of 27 ADAR1. Our findings will help beyond this application with proposing a comprehensive 28 mechanism for ADAR1 editing and its RNA-mediated transcriptome-wide regulation, and 29 contribute to understanding disease such as cancer or auto-immune deficiencies.