Gabra-3 Gene A To I Of Rna Editing Sites In The Identification Of Its Molecular Mechanism

In eukaryotes, A-to-I RNA editing catalyzed by ADARs is an increasingly concerned mechanism in academic world as an important post-transcriptional modificative processing. ADARs could recognize a site-selective adenosine (A) in double strand RNA of pre-mRNA using its double strand binding domain and modify it to inosine (I). Through this processing, the genetic information is changed in mRNA level. For a protein-coding gene, the changed amino acid dramatically alters the functional properties of the encoded protein product and increases the diversity of protein and the complexity of expression regulation in organism. Many ion channels in mammalian neuronal undergo A-to-I RNA editing; GABAA receptorα3 subunit is one of them. ADAR1 and ADAR2 have the potential to efficiently recognize the RNA stem-loop structure which is formed by its exon9. A-to-I RNA editing results in a conversion of genomically encoded isoleucine (I) to methionine (M) codon, it is important for the function of GABAA receptorα3 subunits.Previous studies of GABAA receptorα3 subunit have showed that mammalian animals human(Homo sapiens), mouse(Mus musculus) and aves animal chicken(Gallus gallus) all undergo editing, while frog(Xenopus tropicalis) and pufferfish(Tetraodon nigroviridis) have a genomically encoded methionine at the equivalent position. So in order to further determine the exact arisen location of A-to-I RNA editing during the evolution, we cloned the GABAA receptorα3 subunit from brains of three reptiles animals:chinese three-keeled pond turtle (Chinemys reevesii), siamese crocodile(Crocodylus siamensis), little striped whiptail (Aspidoscelis inornata) and two amphibian animals:newt (Cynops orientalis), axolotl (Ambystoma mexicanum) to examine the editing efficiency of I/M site. Our results showing I/M site is edited in brain of three reptilian animals, but no editing was observed in newt and axolotl. Based on the data, we summarized the evolutionary mechanism of Gabra-3 A-to-I RNA editing:During the evolution, G which represents the ancestral state, was converted into A when the separation of caudata and raniformes, but A-to-I RNA editing occurred immediately after the G-to-A conversion event when reptilian present, maintaining similarity at the protein level to reduce negative selective pressure. We also detected I/M site editing level of different species of mammalia, aves and reptile. We found that the editing efficiency of this site is decreased from mammalian to amphibian span 450 million years.The small exonic RNA duplex structure which is formed by its exon9 encoding 16 amino acids, is the smallest naturally stem-loop structure suffering A-to-I RNA editing till now. So in this study, we were mimicking a lot of duplex structures like Gabra-3 RNA duplex by mutation, construction of minigene and in vitro transcription by T7 RNA polymerase. Using Xenopus laevis oocytes expression system, detected the editing situation of mutant mini-gene I/M site in vivo. Our data showing the sequence near editing site affect editing efficiency and mutation for the Gabra-3 stem-loop structure influence the occurrence of RNA editing. Using chemical mapping, we analyzed the secondary structure of human wildtype Gabra-3 and GC/UC,GC/CU,GC/CC. The data showing the real secondary structures of these four RNA molecules supported the energetically most favorable folding predictions of Mfold. These experiments ensured our conclusion which is proposed base on the reliability of secondary structure. But we couldn't summarize a rule from the differences between edited structures and no-edited structures to infer the editing occurrence or not at I/M site. Therefore, we speculated that sequence and secondary structure of RNA molecular influence the ADARs recognition in a certain extent, but may be the conformation of RNA tertiary structures is the key to decide whether interact with ADARs.In addition, we also designed a temperature control experiment to regulate the RNA editing of I/M site through changing RNA structure which is caused by different RNA folding temperature. We found that when the temperature rises to 37℃, no-edited mutant minigenes can recognized by ADARs. And the results show that the structures of GC/UC, GC/CC folded at 37℃are different with 25℃.To investigate functional consequences of Gabra-3 mRNA editing, we used whole-cell voltage-clamp recording and a rapid drug delivery system to apply GABA to lifted HEK293T cells that had been transiently cotransfected with plasmids containing mousep,γand either nonedited a3(I) or edited a3 (M) cDNAs, Experimental data showing the peak current amplitudes were significantly smaller from the edited receptors and edited a3 (M)βγcurrents deactivated much more faster after GABA releasing. We speculated RNA editing alter the amino acids on TM3 of Gabra-3 and may influence conformational stability, its location is therefore consistent with a role in channel gating. In addition, RNA editing may regulate the type of neurotransmitter in the nervous system during development.