A computational screen for site selective A-to-I editing detects novel sites in neuron specific Hu proteins

Background  

Several bioinformatic approaches have previously been used to find novel sites of ADAR mediated A-to-I RNA editing in human. These studies have discovered thousands of genes that are hyper-edited in their non-coding intronic regions, especially in alu retrotransposable elements, but very few substrates that are site-selectively edited in coding regions. Known RNA edited substrates suggest, however, that site selective A-to-I editing is particularly important for normal brain development in mammals.     

A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing

Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo.     

Faithful editing of a tomato-specific mRNA editing site in transgenic tobacco chloroplasts

RNA editing sites and their site-specific trans-acting recognition factors are thought to have coevolved. Hence, evolutionary loss of an editing site by a genomic mutation is normally followed by the loss of the specific recognition factor for this site, due to the absence of selective pressure for its maintenance. Here, we have tested this scenario for the only tomato-specific plastid RNA editing site. A single C-to-U editing site in the tomato rps12 gene is absent from the tobacco and nightshade plastid genomes, where the presence of a genomic T nucleotide obviates the need for editing of the rps12 mRNA. We have introduced the tomato editing site into the tobacco rps12 gene by plastid transformation and find that, surprisingly, this heterologous site is efficiently edited in the transplastomic plants. This suggests that the trans-acting recognition factor for the rps12 editing site has been maintained, presumably because it serves another function in tobacco plastids. Bioinformatics analyses identified an editing site in the rpoB gene of tobacco and tomato whose sequence context exhibits striking similarity to that of the tomato rps12 editing site. This may suggest that requirement for rpoB editing resulted in maintenance of the rps12 editing activity or, alternatively, the pre-existing rpoB editing activity facilitated the evolution of a novel editing site in rps12.     

Identification of the chloroplast adenosine-to-inosine tRNA editing enzyme

Plastids (chloroplasts) of higher plants exhibit two types of conversional RNA editing: cytidine-to-uridine editing in mRNAs and adenosine-to-inosine editing in at least one plastid genome-encoded tRNA, the tRNA-Arg(ACG). The enzymes catalyzing RNA editing reactions in plastids are unknown. Here we report the identification of the A-to-I tRNA editing enzyme from chloroplasts of the model plant Arabidopsis thaliana. The protein (AtTadA) has an unusual structure in that it harbors a large N-terminal domain of >1000 amino acids, which is not required for catalytic activity. The C-terminal region of the protein displays sequence similarity to tadA, the tRNA adenosine deaminase from Escherichia coli. We show that AtTadA is imported into chloroplasts in vivo and demonstrate that the in vitro translated protein triggers A-to-I editing in the anticodon of the plastid tRNA-Arg(ACG). Suppression of AtTadA gene expression in transgenic Arabidopsis plants by RNAi results in reduced A-to-I editing in the chloroplast tRNA-Arg(ACG). The RNAi lines display a mild growth phenotype, presumably due to reduced chloroplast translational efficiency upon limited availability of edited tRNA-Arg(ACG).     

ADAR-mediated RNA editing in non-coding RNA sequences

Adenosine to inosine (A-to-I) RNA editing is the most abundant editing event in animals. It converts adenosine to inosine in double-stranded RNA regions through the action of the adenosine deaminase acting on RNA (ADAR) proteins. Editing of pre-mRNA coding regions can alter the protein codon and increase functional diversity. However, most of the A-to-I editing sites occur in the non-coding regions of pre-mRNA or mRNA and non-coding RNAs. Untranslated regions (UTRs) and introns are located in pre-mRNA non-coding regions, thus A-to-I editing can influence gene expression by nuclear retention, degradation, alternative splicing, and translation regulation. Non-coding RNAs such as microRNA (miRNA), small interfering RNA (siRNA) and long non-coding RNA (lncRNA) are related to pre-mRNA splicing, translation, and gene regulation. A-to-I editing could therefore affect the stability, biogenesis, and target recognition of non-coding RNAs. Finally, it may influence the function of non-coding RNAs, resulting in regulation of gene expression. This review focuses on the function of ADAR-mediated RNA editing on mRNA non-coding regions (UTRs and introns) and non-coding RNAs (miRNA, siRNA, and lncRNA).     

Computational analysis of RNA editing sites in plant mitochondrial genomes reveals similar information content and a sporadic distribution of editing sites

A computational analysis of RNA editing sites was performedon protein-coding sequences of plant mitochondrial genomes fromArabidopsis thaliana, Beta vulgaris, Brassica napus, and Oryzasativa. The distribution of nucleotides around edited and uneditedcytidines was compared in 41 nucleotide segments and included1481 edited cytidines and 21,390 unedited cytidines in the 4genomes. The distribution of nucleotides was examined in 1,2, and 3 nucleotide windows by comparison of nucleotide frequencyratios and relative entropy. The relative entropy analyses indicatethat information is encoded in the nucleotide sequences in the5 prime flank (–18 to –14, –13 to –10,–6 to –4, –2/–1) and the immediate 3prime flanking nucleotide (+1), and these regions may be importantin editing site recognition. The relative entropy was largewhen 2 or 3 nucleotide windows were analyzed, suggesting thatseveral contiguous nucleotides may be involved in editing siterecognition. RNA editing sites were frequently preceded by 2pyrimidines or AU and followed by a guanidine (HYCG) in themonocot and dicot mitochondrial genomes, and rarely precededby 2 purines. Analysis of chloroplast editing sites from a dicot,Nicotiana tabacum, and a monocot, Zea mays, revealed a similardistribution of nucleotides around editing sites (HYCA). Thesimilarity of this motif around editing sites in monocots anddicots in both mitochondria and chloroplasts suggests that amechanistic basis for this motif exists that is common in thesedifferent organelle and phylogenetic systems. The preferredsequence distribution around RNA editing sites may have an importantimpact on the acquisition of editing sites in evolution becausethe immediate sequence context of a cytidine residue may rendera cytidine editable or uneditable, and consequently determinewhether a T to C mutation at a specific position may be correctedby RNA editing. The distribution of editing sites in many protein-codingsequences is shown to be non-random with editing sites clusteredin groups separated by regions with no editing sites. The sporadicdistribution of editing sites could result from a mechanismof editing site loss by gene conversion utilizing edited sequenceinformation, possibly through an edited cDNA intermediate.     

Regulation of ion channel/neurotransmitter receptor function by RNA editing

RNA editing by select adenosine deamination (A-to-I editing) alters functional determinants in certain ion channels and neurotransmitter receptors in vertebrates and invertebrates. In most cases, edited and unedited versions of a given receptor/channel co-exist to expand the functional space of the receptor population. Recent studies have characterized K(+) channels in squid that are edited at multiple positions, revealed a role for Q/R site editing in AMPA receptor assembly, and demonstrated a link between serotonin levels and the extent of editing of a mammalian serotonin receptor.