UC Davis

The innate immune system relies on molecular sensors to detect specific patterns, like viral double-stranded RNA (dsRNA), triggering responses such as apoptosis and immune infiltration. Adenosine Deaminases Acting on RNA (ADARs) play a critical role by catalyzing site-selective hydrolytic deamination of adenosine (A) to inosine (I). This mechanism helps distinguish self from non-self RNA, preventing erroneous immune activation. However, ADAR1 loss-of-function mutations lead to Aicardi Goutières Syndrome (AGS), a severe autoimmune disorder in children. While most AGS-associated mutations in ADAR1 are within its catalytic domain, their precise effects on adenosine deamination remain unclear. This dissertation investigates four AGS-causing mutations (G1007R, R892H, K999N, and Y1112F) in ADAR1 p110 and truncated variants. Measurement of adenosine deamination rates using RNA substrates derived from human transcripts edited by ADAR1 (hGli1, 5-HT2cR) reveals that mutations affecting amino acids stabilizing the base-flipped conformation of the ADAR-RNA complex (G1007R and R892H) have the most significant impact on catalysis. The K999N mutation, near the RNA binding interface, contextually alters catalytic activity, while the Y1112F mutation exerts minimal effects. The characterization of ADAR1 disease-associated mutations necessitated several optimizations in purification conditions, construct design, and protein-RNA binding assay. These findings shed light on the context-dependent effects of AGS-causing mutations on adenosine deamination, advancing our structural understanding of ADAR1-mediated RNA editing and how mutations in ADAR1 catalytic domain may lead to disease phenotype.From another perspective, ADAR-mediated RNA editing can rectify disease-causing mutations using complementary guide RNAs. However, ADARs exhibit a preference against RNA sites containing 5’G or 5’C adjacent to the edited adenosine (5’GA and 5’CA sites). Several structural studies in our lab have suggested a steric clash between the exocyclic amine of guanosine and a critical glycine residue (G489) in ADAR’s flipping loop. Recent work from our lab showcases the restoration of ADAR activity using guanosine or adenosine nucleosides that pair with 5’G in a syn conformation. This is supported by two high-resolution crystal structures demonstrating how a Gsyn-Ganti and Gsyn-AH+anti pairs alleviate clashes. Following on this work this thesis outlines efforts to discover guide RNAs enabling editing at 5’CA sites by evading G489 in a similar manner. We identified guanosine and adenosine analogs capable of robustly and selectively editing these sites through non-Watson-Crick pairing. In addition, a high-throughput method was implemented to identify unique sequence motifs that enable more precise editing at 5’GA sites. This dissertation also highlights the efforts to structurally characterize these motifs through X-ray crystallography. These combined efforts inform guide RNA design for disease targets featuring unfavorable 5’ sequence contexts.