RNA binding candidates for human ADAR3 from substrates of a gain of function mutant expressed in neuronal cells.

Human ADAR3 is a catalytically inactive member of the Adenosine Deaminase Acting on RNA (ADAR) protein family, whose active members catalyze A-to-I RNA editing in metazoans. Until now, the reasons for the catalytic incapability of ADAR3 has not been defined and its biological function rarely explored. Yet, its exclusive expression in the brain and involvement in learning and memory suggest a central role in the nervous system. Here we describe the engineering of a catalytically active ADAR3 enzyme using a combination of computational design and functional screening. Five mutations (A389V, V485I, E527Q, Q549R and Q733D) engender RNA deaminase in human ADAR3. By way of its catalytic activity, the ADAR3 pentamutant was used to identify potential binding targets for wild type ADAR3 in a human glioblastoma cell line. Novel ADAR3 binding sites discovered in this manner include the 3'-UTRs of the mRNAs encoding early growth response 1 (EGR1) and dual specificity phosphatase 1 (DUSP1); both known to be activity-dependent immediate early genes that respond to stimuli in the brain. Further studies reveal that the wild type ADAR3 protein can regulate transcript levels for DUSP1 and EGR1, suggesting a novel role ADAR3 may play in brain function.

Chemical Profiling of A-to-I RNA Editing Using a Click-Compatible Phenylacrylamide.

Straightforward methods for detecting adenosine-to-inosine (A-to-I) RNA editing are key to a better understanding of its regulation, function, and connection with disease. We address this need by developing a novel reagent, N-(4-ethynylphenyl)acrylamide (EPhAA), and illustrating its ability to selectively label inosine in RNA. EPhAA is synthesized in a single step, reacts rapidly with inosine, and is "click"-compatible, enabling flexible attachment of fluorescent probes at editing sites. We first validate EPhAA reactivity and selectivity for inosine in both ribonucleosides and RNA substrates, and then apply our approach to directly monitor in vitro A-to-I RNA editing activity using recombinant ADAR enzymes. This method improves upon existing inosine chemical-labeling techniques and provides a cost-effective, rapid, and non-radioactive approach for detecting inosine formation in RNA. We envision this method will improve the study of A-to-I editing and enable better characterization of RNA modification patterns in different settings.

Synthesis of native-like crosslinked duplex RNA and study of its properties.

A variety of enzymes have been found to interact with double-stranded RNA (dsRNA) in order to carry out its functions. We have endeavored to prepare the covalently crosslinked native-like duplex RNA, which could be useful for biochemical studies and RNA nanotechnology. In this study, the interstrand covalently linked duplex RNA was formed by a crosslinking reaction between vinylpurine (VP) and the target cytosine or uracil in RNA. We measured melting temperatures and CD spectra to identify the properties of the VP crosslinked duplex RNA. The crosslinking formation increased the thermodynamic stability without disturbing the natural conformation of dsRNA. In addition, a competitive binding experiment with the duplex RNA binding enzyme, ADAR2, showed the crosslinked dsRNA bound the protein with nearly the same binding affinity as the natural dsRNA, confirming that it has finely preserved the natural traits of duplex RNA.

A Bump-Hole Approach for Directed RNA Editing.

Molecules capable of directing changes to nucleic acid sequences are powerful tools for molecular biology and promising candidates for the therapeutic correction of disease-causing mutations. However, unwanted reactions at off-target sites complicate their use. Here we report selective combinations of mutant editing enzyme and directing oligonucleotide. Mutations in human ADAR2 (adenosine deaminase acting on RNA 2) that introduce aromatic amino acids at position 488 reduce background RNA editing. This residue is juxtaposed to the nucleobase that pairs with the editing site adenine, suggesting a steric clash for the bulky mutants. Replacing this nucleobase with a hydrogen atom removes the clash and restores editing activity. A crystal structure of the E488Y mutant bound to abasic site-containing RNA shows the accommodation of the tyrosine side chain. Finally, we demonstrate directed RNA editing in vitro and in human cells using mutant ADAR2 proteins and modified guide RNAs with reduced off-target activity.