Editing Frequency Research Articles

RNA Editing Sites Exist in Protein-coding Genes in the Chloroplast Genome of Cycas taitungensis

RNA editing is a post-transcriptional process that results in modifications of ribonucleotides at specific locations. In land plants editing can occur in both mitochondria and chloroplasts and most commonly involves C-to-U changes, especially in seed plants. Using prediction and experimental determination, we investigated RNA editing in 40 protein-coding genes from the chloroplast genome of Cycas taitungensis. A total of 85 editing sites were identified in 25 transcripts. Comparison analysis of the published editotypes of these 25 transcripts in eight species showed that RNA editing events gradually disappear during plant evolution. The editing in the first and third codon position disappeared quicker than that in the second codon position. ndh genes have the highest editing frequency while serine and proline codons were more frequently edited than the codons of other amino acids. These results imply that retained RNA editing sites have imbalanced distribution in genes and most of them may function by changing protein structure or interaction. Mitochondrion protein-coding genes have three times the editing sites compared with chloroplast genes of Cycas, most likely due to slower evolution speed.

Deep sequencing of small RNAs from human skin reveals major alterations in the psoriasis miRNAome

Psoriasis is a chronic and complex inflammatory skin disease with lesions displaying dramatically altered mRNA expression profiles. However, much less is known about the expression of small RNAs. Here, we describe a comprehensive analysis of the normal and psoriatic skin miRNAome with next-generation sequencing in a large patient cohort. We generated 6.7 × 10(8) small RNA reads representing 717 known and 284 putative novel microRNAs (miRNAs). We also observed widespread expression of isomiRs and miRNA*s derived from known and novel miRNA loci, and a low frequency of miRNA editing in normal and psoriatic skin. The expression and processing of selected novel miRNAs were confirmed with qRT-PCR in skin and other human tissues or cell lines. Eighty known and 18 novel miRNAs were 2-42-fold differentially expressed in psoriatic skin. Of particular significance was the 2.7-fold upregulation of a validated novel miRNA derived from the antisense strand of the miR-203 locus, which plays a role in epithelial differentiation. Other differentially expressed miRNAs included hematopoietic-specific miRNAs such as miR-142-3p and miR-223/223*, and angiogenic miRNAs such as miR-21, miR-378, miR-100 and miR-31, which was the most highly upregulated miRNA in psoriatic skin. The functions of these miRNAs are consistent with the inflammatory and hyperproliferative phenotype of psoriatic lesions. In situ hybridization of differentially expressed miRNAs revealed stratified epidermal expression of an uncharacterized keratinocyte-derived miRNA, miR-135b, as well as the epidermal infiltration of the hematopoietic-specific miRNA, miR-142-3p, in psoriatic lesions. This study lays a critical framework for functional characterization of miRNAs in healthy and diseased skin.

Complete chloroplast genome sequence of Magnolia kwangsiensis (Magnoliaceae): implication for DNA barcoding and population genetics

Here, we report a completely sequenced plastome using Illumina/Solexa sequencing-by-synthesis (SBS) technology. The plastome of Magnolia kwangsiensis Figlar & Noot. is 159 667 bp in length with a typical quadripartite structure: 88 030 bp large single-copy (LSC) and 18 669 bp small single-copy (SSC) regions, separated by two 26484 bp inverted repeat (IR) regions. The overall predicted gene number is 129, among which 17 genes are duplicated in IR regions. The plastome of M. kwangsiensis is identical in its gene order to previously published plastomes of magnoliids. Furthermore, the C-to-U type RNA editing frequency of 114 seed plants is positively correlated with plastome GC content and plastome length, whereas plastome length is not correlated with GC content. A total of 16 potential putative barcoding or low taxonomic level phylogenetic study markers in Magnoliaceae were detected by comparing the coding and noncoding regions of the plastome of M. kwangsiensis with that of Liriodendron tulipifera L. At least eight markers might be applied not only to Magnoliaceae but also to other taxa. The 86 mononucleotide cpSSRs that distributed in single-copy noncoding regions are highly valuable to study population genetics and conservation genetics of this endangered rare species.

Gene editing activity on extrachromosomal arrays in C. elegans transgenics

Gene editing by modified single-stranded oligonucleotides is a strategy aimed at inducing single base changes into the genome, generating a permanent genetic change. The work presented here explores gene editing capabilities in the model organism Caenorhabditis elegans. Current approaches to gene mutagenesis in C. elegans have been plagued by non-specificity and thus the ability to induce precise, directed alterations within the genome of C. elegans would offer a platform upon which structure/function analyses can be carried out. As such, several in vivo assay systems were developed to evaluate gene editing capabilities in C. elegans. Fluorescence was chosen as the selectable endpoint as fluorescence can be easily detected through the transparent worm body even from minimal expression. Two tissue specific fluorescent expression vectors containing either a GFP or mCherry transgene were mutagenized to create a single nonsense mutation within the open reading frame of each respective fluorescent gene. These served as the target site to evaluate the frequency of gene editing on extrachromosomal array transgenic lines. Extrachromosomal arrays can carry hundreds of copies of the transgene, therefore low frequency events (like those in the gene editing reaction) may be detected. Delivery of the oligonucleotide was accomplished by microinjection into the gonads of young adult worms in an effort to induce repair of the mutated fluorescent gene in the F1 progeny. Despite many microinjections on the transgenic strains with varying concentrations of ODNs, no gene editing events were detected. This result is consistent with the previous research, demonstrating the difficulties encountered in targeting embryonic stem cells and the pronuclei of single-celled embryos.

Are substitution rates and RNA editing correlated?

BackgroundRNA editing is a post-transcriptional process that, in seed plants, involves a cytosine to uracil change in messenger RNA, causing the translated protein to differ from that predicted by the DNA sequence. RNA editing occurs extensively in plant mitochondria, but large differences in editing frequencies are found in some groups. The underlying processes responsible for the distribution of edited sites are largely unknown, but gene function, substitution rate, and gene conversion have been proposed to influence editing frequencies.ResultsWe studied five mitochondrial genes in the monocot order Alismatales, all showing marked differences in editing frequencies among taxa. A general tendency to lose edited sites was observed in all taxa, but this tendency was particularly strong in two clades, with most of the edited sites lost in parallel in two different areas of the phylogeny. This pattern is observed in at least four of the five genes analyzed. Except in the groups that show an unusually low editing frequency, the rate of C-to-T changes in edited sites was not significantly higher that in non-edited 3rd codon positions. This may indicate that selection is not actively removing edited sites in nine of the 12 families of the core Alismatales. In all genes but ccmB, a significant correlation was found between frequency of change in edited sites and synonymous substitution rate. In general, taxa with higher substitution rates tend to have fewer edited sites, as indicated by the phylogenetically independent correlation analyses. The elimination of edited sites in groups that lack or have reduced levels of editing could be a result of gene conversion involving a cDNA copy (retroprocessing). If so, this phenomenon could be relatively common in the Alismatales, and may have affected some groups recurrently. Indirect evidence of retroprocessing without a necessary correlation with substitution rate was found mostly in families Alismataceae and Hydrocharitaceae (e.g., groups that suffered a rapid elimination of all their edited sites, without a change in substitution rate).ConclusionsThe effects of substitution rate, selection, and/or gene conversion on the dynamics of edited sites in plant mitochondria remain poorly understood. Although we found an inverse correlation between substitution rate and editing frequency, this correlation is partially obscured by gene retroprocessing in lineages that have lost most of their edited sites. The presence of processed paralogs in plant mitochondria deserves further study, since most evidence of their occurrence is circumstantial.

Increased accumulation of intron-containing transcripts in rice mitochondria caused by low temperature: is cold-sensitive RNA editing implicated?

An increase in the unspliced cox2 transcript and accompanying decrease in the frequency of RNA editing near the exon/intron junction (intron binding site 1, IBS1) have been reported in cold-treated wheat. Here, an attempt was made to clarify whether a similar phenomenon occurs in rice. Levels of unspliced cox2 transcript increased and its editing at the IBS was abolished after cold treatment. The accumulation of COXII protein remained unaffected. The accumulation of intron-containing transcripts of another eight mitochondrial genes, 23 introns in total, was analyzed by Northern blotting and semi-quantitative RT-PCR. An increase in 14 of the 23 intron-adjoining cDNA after cold treatment was observed. Six RNA editing sites in the IBS of four genes were tested as to their status by sequencing cDNA. One of these sites in the nad7 transcript showed a close association with splicing, with editing and splicing occurring simultaneously, irrespective of cold treatment. Two other sites in the intron-containing cox2 and rps3 transcripts were sensitive to cold, where editing frequency began to decrease 1 day after cold treatment, and finally exhibited a tight association with splicing 14 days later. The other sites were efficiently edited. The intron-spliced transcripts were fully edited at all six sites.

Increased A-to-I RNA editing of the transcript for GABAA receptor subunit α3 during chick retinal development

Adenosine-to-inosine (A-to-I) RNA editing is a cotranscriptional or posttranscriptional gene regulatory mechanism that increases the diversity of the proteome in the nervous system. Recently, the transcript for GABA type A receptor subunit α3 was found to be subjected to RNA editing. The aim of this study was to determine if editing of the chicken α3 subunit transcript occurs in the retina and if the editing is temporally regulated during development. We also raised the question if editing of the α3 transcript was temporally associated with the suggested developmental shift from excitation to inhibition in the GABA system. The editing frequency was studied by using Sanger and Pyrosequencing, and to monitor the temporal aspects, we studied the messenger RNA expression of the GABAA receptor subunits and chloride pumps, known to be involved in the switch. The results showed that the chick α3 subunit was subjected to RNA editing, and its expression was restricted to cells in the inner nuclear and ganglion cell layer in the retina. The extent of editing increased during development (after embryonic days 8-9) concomitantly with an increase of expression of the chloride pump KCC2. Expression of several GABAA receptor subunits known to mediate synaptic GABA actions was upregulated at this time. We conclude that editing of the chick GABAA subunit α3 transcript in chick retina gives rise to an amino acid change that may be of importance in the switch from excitatory to inhibitory receptors.

Large 3′ UTR of sugar beet rps3 is truncated in cytoplasmic male-sterile mitochondria

Genomic alteration near or within mitochondrial gene is often associated with cytoplasmic male sterility (CMS). Its influence on the expression of the mitochondrial gene was proposed as one of the possible causes of CMS. In sugar beet mitochondrial rps3, whose downstream 1,056-bp region contains Norf246, an apparently non-functional open reading frame (ORF), was deleted in CMS mitochondria. In our previous study, normal rps3 (3.8 kb), CMS rps3 (2.7 kb), and Norf246 (3.8 and 0.9 kb) were shown to be transcribed. The present study was conducted to determine whether the deletion affected gene expression. Reverse transcription (RT)-PCR analysis revealed the co-transcription of rps3 and Norf246. By circularized RNA (CR) RT-PCR analysis, the 5' and 3' termini of the 3.8- and the 0.9-kb transcripts were determined. The results suggested that the 3.8-kb transcripts were the rps3 mRNA bearing ~464-base 5' untranslated region (UTR) and ~1,508-base 3' UTR, whereas no functional ORF was observed in the 0.9-kb transcripts. CR-RT-PCR revealed that the 3' UTR of the 2.7-kb transcripts was reduced to ~460 bases. However, no difference in the accumulation of RPS3 polypeptide and RNA editing was detected by protein gel blot analysis and cDNA sequencing. Although the deleted region encoded the truncated-atp9 that was edited, no influence on the pattern and frequency of RNA editing of genuine atp9 was evident. The results eliminated rps3 as a candidate for the CMS gene, making preSatp6, a unique ORF fused with CMS atp6, the sole CMS-associated region in sugar beet.

Extensive Loss of RNA Editing Sites in Rapidly Evolving Silene Mitochondrial Genomes: Selectionvs. Retroprocessing as the Driving Force

Theoretical arguments suggest that mutation rates influence the proliferation and maintenance of RNA editing. We identified RNA editing sites in five species within the angiosperm genus Silene that exhibit highly divergent mitochondrial mutation rates. We found that mutational acceleration has been associated with rapid loss of mitochondrial editing sites. In contrast, we did not find a significant difference in the frequency of editing in chloroplast genes, which lack the mutation rate variation observed in the mitochondrial genome. As found in other angiosperms, the rate of substitution at RNA editing sites in Silene greatly exceeds the rate at synonymous sites, a pattern that has previously been interpreted as evidence for selection against RNA editing. Alternatively, we suggest that editing sites may experience higher rates of C-to-T mutation than other portions of the genome. Such a pattern could be caused by gene conversion with reverse-transcribed mRNA (i.e., retroprocessing). If so, the genomic distribution of RNA editing site losses in Silene suggests that such conversions must be occurring at a local scale such that only one or two editing sites are affected at a time. Because preferential substitution at editing sites appears to occur in angiosperms regardless of the mutation rate, we conclude that mitochondrial rate accelerations within Silene have "fast-forwarded" a preexisting pattern but have not fundamentally changed the evolutionary forces acting on RNA editing sites.

Informed Investors and the Internet

Abstract: During the last decade the Internet has become an increasingly important source for gathering company related information. We employ Wikipedia editing frequency as an instrument that captures the degree in which the population is engaged with the processing of company‐related information. We find that firms whose information is processed by the population more frequently are associated with lower analysts' forecast errors, smaller analysts' forecast dispersions, and significant changes in bid‐ask spreads on analysts' recommendation days. These results indicate that information processing over the Internet is related to the degree to which investors and analysts are informed about companies.

612 Metastasis – gene expression differences associated with site and treatment sensitivity

Although an increasing number of RNA adenosine-to-inosine (A-to-I) editing sites are being discovered, how the editing frequencies of these sites are modulated to fine-tune protein function in adaptive responses is not well understood. A previous study screening for heat tolerance in Drosophila mutants discovered a hypnos-2 mutant strain that was later found to be defective in dADAR, the Drosophila gene encoding the A-to-I editing enzyme. This supports the hypothesis that cells and organisms respond to stressful environments by ADAR (adenosine deaminase acting on RNA)-mediated RNA editing. Here, we investigated changes in the RNA A-to-I editing frequencies of 30 Drosophila nervous system targets in response to heat shock, a stress acclimatization that requires the dADAR function. To our surprise, most of these nervous system editing targets showed reduced editing. Our results suggest that a change in RNA editing pattern is a mechanism by which organisms acclimate to drastic environmental change. However, how RNA editing confers heat resistance is more complicated and requires further investigation.

Massive APOBEC3 Editing of Hepatitis B Viral DNA in Cirrhosis

DNA viruses, retroviruses and hepadnaviruses, such as hepatitis B virus (HBV), are vulnerable to genetic editing of single stranded DNA by host cell APOBEC3 (A3) cytidine deaminases. At least three A3 genes are up regulated by interferon-α in human hepatocytes while ectopic expression of activation induced deaminase (AICDA), an A3 paralog, has been noted in a variety of chronic inflammatory syndromes including hepatitis C virus infection. Yet virtually all studies of HBV editing have confined themselves to analyses of virions from culture supernatants or serum where the frequency of edited genomes is generally low (≤10−2). We decided to look at the nature and frequency of HBV editing in cirrhotic samples taken during removal of a primary hepatocellular carcinoma. Forty-one cirrhotic tissue samples (10 alcoholic, 10 HBV+, 11 HBV+HCV+ and 10 HCV+) as well as 4 normal livers were studied. Compared to normal liver, 5/7 APOBEC3 genes were significantly up regulated in the order: HCV±HBV>HBV>alcoholic cirrhosis. A3C and A3D were up regulated for all groups while the interferon inducible A3G was over expressed in virus associated cirrhosis, as was AICDA in ∼50% of these HBV/HCV samples. While AICDA can indeed edit HBV DNA ex vivo, A3G is the dominant deaminase in vivo with up to 35% of HBV genomes being edited. Despite these highly deleterious mutant spectra, a small fraction of genomes survive and contribute to loss of HBeAg antigenemia and possibly HBsAg immune escape. In conclusion, the cytokine storm associated with chronic inflammatory responses to HBV and HCV clearly up regulates a number of A3 genes with A3G clearly being a major restriction factor for HBV. Although the mutant spectrum resulting from A3 editing is highly deleterious, a very small part, notably the lightly edited genomes, might help the virus evolve and even escape immune responses.

Assessing serotonin receptor mRNA editing frequency by a novel ultra high-throughput sequencing method

RNA editing is a post-transcriptional modification of pre-mRNA that results in increased diversity in transcriptomes and proteomes. It occurs in a wide variety of eukaryotic organisms and in some viruses. One of the most common forms of pre-mRNA editing is A-to-I editing, in which adenosine is deaminated to inosine, which is read as guanosine during translation. This phenomenon has been observed in numerous transcripts, including the mammalian 5-HT2C receptor, which can be edited at five distinct sites. Methods used to date to quantify 5-HT2C receptor editing are labor-intensive, expensive and provide limited information regarding the relative abundance of 5-HT2C receptor editing variants. Here, we present a novel, ultra high-throughput method to quantify 5-HT2C receptor editing, compare it to a more conventional method, and use it to assess the effect of a range of genetic and pharmacologic manipulations on 5-HT2C editing. We conclude that this new method is powerful and economical, and we provide evidence that alterations in 5-HT2C editing appear to be a result of regional changes in brain activity, rather than a mechanism to normalize 5-HT2C signaling.

Insights into the Evolution of Mitochondrial Genome Size from Complete Sequences of Citrullus lanatus and Cucurbita pepo (Cucurbitaceae)

The mitochondrial genomes of seed plants are unusually large and vary in size by at least an order of magnitude. Much of this variation occurs within a single family, the Cucurbitaceae, whose genomes range from an estimated 390 to 2,900 kb in size. We sequenced the mitochondrial genomes of Citrullus lanatus (watermelon: 379,236 nt) and Cucurbita pepo (zucchini: 982,833 nt)--the two smallest characterized cucurbit mitochondrial genomes--and determined their RNA editing content. The relatively compact Citrullus mitochondrial genome actually contains more and longer genes and introns, longer segmental duplications, and more discernibly nuclear-derived DNA. The large size of the Cucurbita mitochondrial genome reflects the accumulation of unprecedented amounts of both chloroplast sequences (>113 kb) and short repeated sequences (>370 kb). A low mutation rate has been hypothesized to underlie increases in both genome size and RNA editing frequency in plant mitochondria. However, despite its much larger genome, Cucurbita has a significantly higher synonymous substitution rate (and presumably mutation rate) than Citrullus but comparable levels of RNA editing. The evolution of mutation rate, genome size, and RNA editing are apparently decoupled in Cucurbitaceae, reflecting either simple stochastic variation or governance by different factors.

Large-scale analysis of structural, sequence and thermodynamic characteristics of A-to-I RNA editing sites in human Alu repeats

BackgroundAlu repeats in the human transcriptome undergo massive adenosine to inosine RNA editing. This process is selective, as editing efficiency varies greatly among different adenosines. Several studies have identified weak sequence motifs characterizing the editing sites, but these alone do not account for the large diversity observed.ResultsHere we build a dataset of 29,971 editing sites and use it to characterize editing preferences. We focus on structural aspects, studying the double-stranded RNA structure of the Alu repeats, and show the editing frequency of a given site to depend strongly on the micro-structure it resides in. Surprisingly, we find that interior loops, and especially the nucleotides at their edges, are more likely to be edited than helices. In addition, the sequence motifs characterizing editing sites vary with the micro-structure. Finally, we show that thermodynamic stability of the site is important for its editing.ConclusionsAnalysis of a large dataset of editing events reveals more information on sequence and structural motifs characterizing the A-to-I editing process

Assessing serotonin receptor mRNA editing frequency by a novel ultra high-throughput sequencing method

Assessing serotonin receptor mRNA editing frequency by a novel ultra high-throughput sequencing method

Large-scale mRNA sequencing determines global regulation of RNA editing during brain development

RNA editing by adenosine deamination has been shown to generate multiple isoforms of several neural receptors, often with profound effects on receptor function. However, little is known about the regulation of editing activity during development. We have developed a large-scale RNA sequencing protocol to determine adenosine-to-inosine (A-to-I) editing frequencies in the coding region of genes in the mammalian brain. Using the 454 Life Sciences (Roche) Amplicon Sequencing technology, we were able to determine even low levels of editing with high accuracy. The efficiency of editing for 28 different sites was analyzed during the development of the mouse brain from embryogenesis to adulthood. We show that, with few exceptions, the editing efficiency is low during embryogenesis, increasing gradually at different rates up to the adult mouse. The variation in editing gave receptors like HTR2C and GABA(A) (gamma-aminobutyric acid type A) a different set of protein isoforms during development from those in the adult animal. Furthermore, we show that this regulation of editing activity cannot be explained by an altered expression of the ADAR proteins but, rather, by the presence of a regulatory network that controls the editing activity during development.

TaqMan-based, real-time quantitative polymerase chain reaction method for RNA editing analysis

Abnormal adenosine to inosine (A-to-I) messenger RNA (mRNA) editing has been linked to several disease states afflicting the central nervous system. Here we report an assay to determine RNA editing frequencies at specific sites that is based on quantitative polymerase chain reaction (qPCR) with TaqMan probes. The assay was tested by measuring the frequency of the A-to-I mRNA editing at the Q/R site of the human kainate receptor subunit GluR5 and was compared with two established methods of assessing RNA editing: sequencing of individual clones and restriction analysis. The qPCR assay displayed high sensitivity and reproducibility, demonstrated exceptional discrimination between edited and unedited transcript variants, and proved to have several advantages over the other editing methods. Due to the fact that TaqMan-based qPCR technology can be easily adapted to different editing targets, the increased capabilities afforded by this new technique should facilitate various RNA editing studies that aim to elucidate the role of this process in normal physiology and in disease.

Nipah Virus Edits Its P Gene at High Frequency To Express the V and W Proteins

Nipah virus (NiV) is predicted to encode four proteins from its P gene (P, V, W, and C) via mRNA editing and an alternate open reading frame. By use of specific antibodies, the expression of the V, W, and C proteins in NiV-infected cells has now been confirmed. Analysis of the P-gene transcripts shows a ratio of P:V:W mRNA of 1:1:1, but this differs over time, with greater proportions of V and W transcripts observed as the infection progresses. Eighty-two percent of transcripts are edited, with up to 11 G insertions observed. This exceptionally high editing frequency ensures expression of the V and W proteins.

Determination of the henipavirus phosphoprotein gene mRNA editing frequencies and detection of the C, V and W proteins of Nipah virus in virus-infected cells

The henipaviruses, Nipah virus (NiV) and Hendra virus (HeV), are highly pathogenic zoonotic paramyxoviruses. Like many other paramyxoviruses, henipaviruses employ a process of co-transcriptional mRNA editing during transcription of the phosphoprotein (P) gene to generate additional mRNAs encoding the V and W proteins. The C protein is translated from the P mRNA, but in an alternate reading frame. Sequence analysis of multiple, cloned mRNAs showed that the mRNA editing frequencies of the P genes of the henipaviruses are higher than those reported for other paramyxoviruses. Antisera to synthetic peptides from the P, V, W and C proteins of NiV were generated to study their expression in infected cells. All proteins were detected in both infected cells and purified virions. In infected cells, the W protein was detected in the nucleus while P, V and C were found in the cytoplasm.