Download Improving site-directed RNA editing by screening RNA editing

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.