Characterizing the RNA Editing Specificityof ADAR Isoforms and Deaminase Domains In Vitro

Author

Mariel Therese Bartley

Date of Award

2020

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Rice Laboratory

Abstract

Adenosine deaminases acting on RNA (ADARs) convert adenosine-to-inosine in double stranded RNA. Selectivity of editing sites depends on the sequence of the RNA as well as the secondary structure. Identification of sites of ADAR editing by editome analysis is skewed due to the different abundance of each adenosine-containing triplet, as well as the presence of complex RNA structures. To determine the editing specificity of each ADAR protein, a high throughput sequencing based assay was developed to measure editing in a synthetic dsRNA substrate with one of each of the 16 different adenosine-containing triplets, so that each possible editing site was equally represented. The ADAR1- and ADAR2-deaminases, as well as the full-length ADAR2 and isoforms ADAR1- p150 and ADAR1-p110, were purified and activity was measured for each, so that the inherent activity of the deaminase domains could be characterized and then compared to the editing patterns seen for the longer proteins containing dsRNA binding domains and Z-RNA binding domains. The ADAR1 deaminase was found to skew slightly to favoring 5’A editing sites, while ADAR2- deaminase favored 5’U. From homology modelling of ADAR1 onto the ADAR2 crystal structure, this difference in editing specificity could be due to the ADAR1 protein having a weaker interaction with the orphan base of the RNA substrate. Characterization of full-length ADAR1-p110 and ADAR2 found that each full-length protein had less editing of the UAG triplet than the respective deaminases, while ADAR1-p110 increased editing of the AAG triplet and ADAR2 increased editing of the CAG triplet. Comparing ADAR1 isoforms p110 and p150 found no significant differences in editing specificity. The in vitro assay was also used to confirm the inactivity of the ADAR3 deaminase, and probe and characterize the editing specificity of the ADAR3 deaminase mutant A389V, which rescued editing activity. The pattern of editing was similar to that of the ADAR1-deaminase, despite ADAR3 sharing sequence similarity with ADAR2. Insights into the different patterns of substrate selectivity by ADAR deaminases and the likely causes of these differences, can provide insight for future development of ADAR deaminase constructs for site-directed RNA editing. A pilot experiment for characterizing in vitro editing in complex RNA was also performed, using HEK 293T total RNA and reovirus T1L RNA as substrates for the purified ADAR constructs. An adapted sequencing library preparation was successfully used to identify and count individual editing events in reovirus T1L RNA, and further improvements are required to generate enough editing sites to compare editing frequencies in both substrates to the 50bp in vitro substrate. The goal of characterizing editing in these complex substrates will be to identify RNA secondary structures which are differentially edited by the full-length ADAR1-p110 and ADAR2, when compared to the dsRBD-lacking deaminase constructs.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Recommended Citation

Bartley, Mariel Therese, "Characterizing the RNA Editing Specificityof ADAR Isoforms and Deaminase Domains In Vitro" (2020). Student Theses and Dissertations. 712.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/712