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Download Improving site-directed RNA editing by screening RNA editing
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Improving site-directed RNA editing by screening RNA editing enzymes from different organisms Edgar O. Correa-Colón, Joshua J. C. Rosenthal, Ph.D. Department of Biological Sciences, University of Puerto Rico, Rio Piedras Campus, PR Institute of Neurobiology, University of Puerto Rico, Medical Sciences Campus, PR Recoding genetic information through RNA editing is a process catalyzed by adenosine deaminases that act on RNA (ADAR). ADARs are an evolutionarily conserved family of enzymes that convert adenosines to inosines within mRNA transcripts. Because inosine is read as guanosine during translation, RNA editing may lead to recoding during translation, resulting in proteins with altered functions. Our group has also demonstrated that RNA editing can be exploited to correct genetic mutations. Because ADAR enzymes are present in all metazoans, we hypothesize that ADARs from different organisms possess distinctive functional characteristics, leading to variable editing efficiencies on different targets. To test this idea, we investigated the differences between the sequence of human ADAR's deaminase domain and that of different organisms’, which may lead to distinctive activities. Experimentally, we engineered a recombinant editase that consists of a λ-phage RNA-binding protein fused to ADARs’ deaminase domain from different organisms. This λ-phage RNA-binding protein naturally recognizes with high affinity a BoxB RNA hairpin in bacteriophages. In order to direct this recombinant editase to a target adenosine, we fused the BoxB RNA hairpin to an antisense guide RNA oligo that is complementary to the target. This complex will allow site-specific mRNA editing. Therefore, using a human embryonic cell line (HEK293T cells), we transfect the antisense guide RNA, our engineered editase from different species, and the target eGFP reporter sequence with a premature termination codon (W58X). This study has the potential to provide a better understanding of sitedirected RNA editing through targeted nucleotide deamination by ADAR enzymes and could enable possible intervention of genetic diseases and associated disorders of mRNA mutations.