RNA editing, the changing of a nucleotide sequence from that encoded by the gene, can both diversify and re-code the genome. Adenosine deaminases that act on RNA (ADARs) are one class of enzymes that edit RNA by causing adenosine to inosine (functionally similar to guanine) mutations in RNA that is largely double stranded. These mutations are essential to neural receptor activity, and have been linked to clinical depression, schizophrenia, epileptic seizures, and amyotrophic lateral sclerosis. The ADAR enzyme consists of a catalytic domain, whose structure has been previously determined, as well as one or more double stranded RNA binding motifs (dsRBMs) depending on the organism and type of ADAR. Given the high specificity of RNA editing by ADARs, dsRBM structure plays a key role in substrate recognition. By understanding the mechanism of RNA editing, specifically why ADARs recognize only certain double stranded RNA substrates, this project will shed light on the functional implications of RNA editing and could possibly lead to further understanding of multiple clinical diseases. The goal of this study is to determine the structure of dsRBMs from various species in both the absence and presence of RNA. The RBMs have been truncated from the rest of the protein due to the highly dynamic nature of the ADAR enzyme. A unique construct utilizing a cysteine protease domain has been employed in the cloning process to improve the solubility of the RBM as well as provide a means to better track the small RBM (7-8 kDA) during purification.The structure will be determined by X-ray diffraction of crystals of the dsRBM constructs. This determined structure will be critical in understanding why RNA editing is highly specific.

Double-stranded RNA binding motifs and recognition of RNA-editing substrates

ACS Spring 2022

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