Absence Of Editing Research Articles

Attenuation of Dopaminergic Neurodegeneration in a C. elegans Parkinson's Model through Regulation of Xanthine Dehydrogenase (XDH-1) Expression by the RNA Editase, ADR-2.

Differential RNA editing by adenosine deaminases that act on RNA (ADARs) has been implicated in several neurological disorders, including Parkinson's disease (PD). Here, we report results of a RNAi screen of genes differentially regulated in adr-2 mutants, normally encoding the only catalytically active ADAR in Caenorhabditis elegans, ADR-2. Subsequent analysis of candidate genes that alter the misfolding of human α-synuclein (α-syn) and dopaminergic neurodegeneration, two PD pathologies, reveal that reduced expression of xdh-1, the ortholog of human xanthine dehydrogenase (XDH), is protective against α-synuclein-induced dopaminergic neurodegeneration. Further, RNAi experiments show that WHT-2, the worm ortholog of the human ABCG2 transporter and a predicted interactor of XDH-1, is the rate-limiting factor in the ADR-2, XDH-1, WHT-2 system for dopaminergic neuroprotection. In silico structural modeling of WHT-2 indicates that the editing of one nucleotide in the wht-2 mRNA leads to the substitution of threonine with alanine at residue 124 in the WHT-2 protein, changing hydrogen bonds in this region. Thus, we propose a model where wht-2 is edited by ADR-2, which promotes optimal export of uric acid, a known substrate of WHT-2 and a product of XDH-1 activity. In the absence of editing, uric acid export is limited, provoking a reduction in xdh-1 transcription to limit uric acid production and maintain cellular homeostasis. As a result, elevation of uric acid is protective against dopaminergic neuronal cell death. In turn, increased levels of uric acid are associated with a decrease in ROS production. Further, downregulation of xdh-1 is protective against PD pathologies because decreased levels of XDH-1 correlate to a concomitant reduction in xanthine oxidase (XO), the form of the protein whose by-product is superoxide anion. These data indicate that modifying specific targets of RNA editing may represent a promising therapeutic strategy for PD.

DEK48 Is Required for RNA Editing at Multiple Mitochondrial Sites and Seed Development in Maize.

In flowering plants, C-to-U RNA editing can be critical to normal functions of mitochondrion-encoded proteins. Mitochondrial C-to-U RNA editing is facilitated by many factors from diverse protein families, of which the pentatricopeptide repeat (PPR) proteins play an important role. Owing to their large number and frequent embryo lethality in mutants, functions of many PPRs remain unknown. In this study, we characterized a mitochondrion-localized DYW-type PPR protein, DEK48, functioning in the C-to-U RNA editing at multiple mitochondrial transcripts in maize. Null mutation of Dek48 severely arrests embryo and endosperm development, causing a defective kernel (dek) phenotype, named dek48. DEK48 loss of function abolishes the C-to-U editing at nad3-185, -215, and nad4-376, -977 sites and decreases the editing at 11 other sites, resulting in the alteration of the corresponding amino acids. Consequently, the absence of editing caused reduced assembly and activity of complex I in dek48. Interestingly, we identified a point mutation in dek48-3 causing a deletion of the Tryptophan (W) residue in the DYW motif that abolishes the editing function. In sum, this study reveals the function of DEK48 in the C-to-U editing in mitochondrial transcripts and seed development in maize, and it demonstrates a critical role of the W residue in the DYW triplet motif of DEK48 for the C-to-U editing function in vivo.

Separable Digitally Signed Video

A very simple scheme of a video stream electronic signature is proposed allowing us to isolate any continuous fragment from the video stream in respect of which the property of an electronic signature is preserved not only with respect to establishing authorship and the absence of modification of each individual frame but also the absence of editing i.e. changing the sequence of frames.

PPR-DYW Protein EMP17 Is Required for Mitochondrial RNA Editing, Complex III Biogenesis, and Seed Development in Maize

The conversion of cytidines to uridines (C-to-U) at specific sites in mitochondrial and plastid transcripts is a post-transcriptional processing event that is important to the expression of organellar genes. Pentatricopeptide repeat (PPR) proteins are involved in this process. In this study, we report the function of a previously uncharacterized PPR-DYW protein, Empty pericarp17 (EMP17), in the C-to-U editing and kernel development in maize. EMP17 is targeted to mitochondria. The loss-function of EMP17 arrests maize kernel development, abolishes the editing at ccmFC-799 and nad2-677 sites, and reduces the editing at ccmFC-906 and -966 sites. The absence of editing causes amino acid residue changes in CcmFC-267 (Ser to Pro) and Nad2-226 (Phe to Ser), respectively. As CcmFC functions in cytochrome c (Cytc) maturation, the amount of Cytc and Cytc1 protein is drastically reduced in emp17, suggesting that the CcmFC-267 (Ser to Pro) change impairs the CcmFC function. As a result, the assembly of complex III is strikingly decreased in emp17. In contrast, the assembly of complex I appears less affected, suggesting that the Nad2-226 (Phe to Ser) change may have less impact on Nad2 function. Together, these results indicate that EMP17 is required for the C-to-U editing at several sites in mitochondrial transcripts, complex III biogenesis, and seed development in maize.

The Pentatricopeptide Repeat Protein MEF100 Is Required for the Editing of Four Mitochondrial Editing Sites in Arabidopsis.

In Arabidopsis thaliana there are more than 600 C-to-U RNA editing events in the mitochondria and at least 44 in the chloroplasts. Pentatricopeptide repeat (PPR) proteins provide the specificity for these reactions. They recognize RNA sequences in a partially predictable fashion via key amino acids at the fifth and last position in each PPR motif that bind to individual ribonucleotides. A combined approach of RNA-Seq, mutant complementation, electrophoresis of mitochondrial protein complexes and Western blotting allowed us to show that MEF100, a PPR protein identified in a genetic screen for mutants resistant to an inhibitor of γ -glutamylcysteine synthetase, is required for the editing of nad1-493, nad4-403, nad7-698 and ccmFN2-356 sites in Arabidopsis mitochondria. The absence of editing in mef100 leads to a decrease in mitochondrial Complex I activity, which probably explains the physiological phenotype. Some plants have lost the requirement for MEF100 at one or more of these sites through mutations in the mitochondrial genome. We show that loss of the requirement for MEF100 editing leads to divergence in the MEF100 binding site.

What do editors do? Understanding the physiological functions of A-to-I RNA editing by adenosine deaminase acting on RNAs.

Adenosine-to-inosine (A-to-I) editing is a post-transcriptional modification of RNA which changes its sequence, coding potential and secondary structure. Catalysed by the adenosine deaminase acting on RNA (ADAR) proteins, ADAR1 and ADAR2, A-to-I editing occurs at approximately 50 000–150 000 sites in mice and into the millions of sites in humans. The vast majority of A-to-I editing occurs in repetitive elements, accounting for the discrepancy in total numbers of sites between species. The species-conserved primary role of editing by ADAR1 in mammals is to suppress innate immune activation by unedited cell-derived endogenous RNA. In the absence of editing, inverted paired sequences, such as Alu elements, are thought to form stable double-stranded RNA (dsRNA) structures which trigger activation of dsRNA sensors, such as MDA5. A small subset of editing sites are within coding sequences and are evolutionarily conserved across metazoans. Editing by ADAR2 has been demonstrated to be physiologically important for recoding of neurotransmitter receptors in the brain. Furthermore, changes in RNA editing are associated with various pathological states, from the severe autoimmune disease Aicardi-Goutières syndrome, to various neurodevelopmental and psychiatric conditions and cancer. However, does detection of an editing site imply functional importance? Genetic studies in humans and genetically modified mouse models together with evolutionary genomics have begun to clarify the roles of A-to-I editing in vivo. Furthermore, recent developments suggest there may be the potential for distinct functions of editing during pathological conditions such as cancer.

Efficient Targeted Mutagenesis in Apple and First Time Edition of Pear Using the CRISPR-Cas9 System.

Targeted genome engineering has emerged as an alternative to classical plant breeding and transgenic methods to improve crop plants. Among other methods (zinc finger nucleases or TAL effector nucleases) the CRISPR-Cas system proved to be the most effective, convenient and least expensive method. In this study, we optimized the conditions of application of this system on apple and explored its feasibility on pear. As a proof of concept, we chose to knock-out the Phytoene Desaturase (PDS) and Terminal Flower 1 (TFL1) genes. To improve the edition efficiency, two different single guide RNAs (gRNAs) were associated to the Cas9 nuclease for each target gene. These gRNAs were placed under the control of the U3 and U6 apple promoters. Characteristic albino phenotype was obtained for 85% of the apple transgenic lines targeted in MdPDS gene. Early flowering was observed in 93% of the apple transgenic lines targeted in MdTFL1.1 gene and 9% of the pear transgenic lines targeted in PcTFL1.1. Sequencing of the target zones in apple and pear CRISPR-PDS and CRISPR-TFL1.1 transgenic lines showed that the two gRNAs induced mutations but at variable frequencies. In most cases, Cas9 nuclease cut the DNA in the twenty targeted base pairs near the protospacer adjacent motif and insertions were more frequent than deletions or substitutions. The most frequent edition profile of PDS as well as TFL1.1 genes was chimeric biallelic. Analysis of a sample of potential off-target sequences of the CRISPR-TFL1.1 construct indicated the absence of edition in cases of three mismatches. In addition, transient transformation with the CRISPR-PDS construct produced two T-DNA free edited apple lines. Our overall results indicate that, despite the frequent occurrence of chimerism, the CRISPR-Cas 9 system is a powerful and precise method to induce targeted mutagenesis in the first generation of apple and pear transgenic lines.

RNA editing status of nad7 intron domains in wheat mitochondria.

The most highly conserved structures of group II introns are the helical domains V and VI near the 3'splice site. Within this region of each of the four introns in the wheat mitochondrial nad7 gene encoding NADH dehydrogenase subunit 7, there are A-C mispairs. To determine whether C-to-U type RNA editing restores conventional A-U pairing, we sequenced RT-PCR products from partially-spliced nad7 template RNA and gel-fractionated, excised intron RNA. We examined transcripts from germinating wheat embryos and seedlings because these two stages of development show pronounced differences in steady state levels of nad7 intronic RNAs. We observed editing at only two of the six predicted sites, and they were located at homologous positions within domain V of the third and fourth introns. A third site was found to be edited within the unmodelled domain VI loop of the fourth intron. Similar patterns of RNA editing were seen in wheat embryos and seedlings. These observations, and the presence of other non-conventional base pairs particularly within domain V of plant mitochondrial introns, indicate weaker helical core structure than in ribozymic group II introns. Moreover, the incompleteness or absence of editing in wheat nad7 excised intron RNA suggests that, although editing may contribute to splicing efficiency, it is not essential for splicing.

Complete Phenotypic Characterization of apobec-1 Knockout Mice with a Wild-type Genetic Background and a Human Apolipoprotein B Transgenic Background, and Restoration of Apolipoprotein B mRNA Editing by Somatic Gene Transfer of Apobec-1

We have produced gene knockout mice by targeted disruption of the apobec-1 gene. As recently reported by Hirano et al. (Hirano, K.-I., Young, S. G., Farese, R. V., Jr., Ng, J., Sande, E., Warburton, C., Powell-Braxton, L. M., and Davidson, N. O. (1996) J. Biol. Chem. 271, 9887-9890), these animals do not edit apolipoprotein (apo) B mRNA or produce apoB-48. In this study we have performed a detailed analysis of the lipoprotein phenotypic effects of apobec-1 gene disruption that were not examined in the previous study. We first analyzed the plasma lipoproteins in knockout animals with a wild-type genetic background. Although there was no difference in plasma cholesterol between apobec-1(-/-), +/-, or +/+ mice, there was a marked (176%) increase in plasma apoB-100, from 1.8 +/- 1.2 mg/dl in apobec-1(+/+) mice to 2.7 +/- 0.6 mg/dl in apobec-1(+/-) and 5.0 +/- 1.4 mg/dl in apobec-1(-/-) mice. Plasma apoE was similar in these animals. By fast protein liquid chromatography (FPLC) analysis, there was a significant decrease in plasma high density lipoprotein (HDL) cholesterol in apobec-1(-/-) mice. We further fractionated the plasma lipoproteins into d < 1.006, 1.006-1.02, 1.02-1.05, 1.05-1.08, 1.08-1.10, and 1.10-1.21 g/ml classes, and found a marked (30-40%) reduction in the cholesterol and protein content in the (d 1.08-1.10 and 1.10-1.21) HDL fractions, corroborating the FPLC data. SDS-gel analysis revealed an absence of apoB-48, an increase in apoB-100 in the very low density lipoprotein (VLDL) and low density lipoprotein (LDL) fractions, and a small decrease in apoA-I in the HDL fractions in the apobec-1(-/-) samples. We next raised the basal plasma apoB levels in the apobec-1(-/-) animals by cross-breeding them with human apoB transgenic (TgB) mice. The plasma apoB-100 was 3-fold higher in apobec-1(-/-)/TgB+/- mice (26.6 +/- 18.3 mg/dl) than in apobec-1(+/+)/TgB+/- mice (9.8 +/- 3.9 mg/dl, p < 0.05). The apobec-1(-/-)/TgB+/- mice had a plasma cholesterol levels of 170 +/- 28 mg/dl and triglyceride levels of 106 +/- 31 mg/dl, which are 80% and 58% higher, respectively, than the corresponding values of 94 +/- 21 mg/dl and 67 +/- 11 mg/dl in apobec+/+/TgB+/- mice. By FPLC, the apobec-1(-/-)/TgB+/- animals developed markedly elevated plasma LDL cholesterol (518.5 +/- 329.5 microg/ml) that is 373% that of apobec1(+/+)/TgB+/- mice (139.0 +/- 87.0 microg/ml) (p < 0.05). The elevated plasma triglyceride was accounted for mainly by a 97% increase in VLDL triglyceride in the apobec1(-/-)/TgB+/- mice. We conclude that apobec-1(-/-) animals have a distinctive lipoprotein phenotype characterized by significant hyperapoB-100 and HDL deficiency in mice with a wild-type genetic background. Furthermore, the abolition of apoB mRNA editing elevates plasma total cholesterol and LDL cholesterol in apobec-1(-/-) animals with a TgB background. Finally, to exclude the possibility that absence of apoB mRNA editing was a secondary effect of chronic Apobec-1 deficiency, we treated apobec-1(-/-) mice with a replication-defective mouse Apobec-1 adenoviral vector and found that we could acutely restore apoB mRNA editing in the liver. These experiments indicate that Apobec-1 is an essential component of the apoB mRNA editing machinery and absence of editing in the knockout animals is a direct consequence of the absence of functional Apobec-1.

Maxicircle DNA and edited mRNA sequences of closely related trypanosome species: implications of kRNA editing for evolution of maxicircle genomes.

kRNA editing produces functional mRNAs by uridine insertion and deletion. We analyzed portions of the apocytochrome b and NADH dehydrogenase subunits 7 and 8 (ND7 and 8) genes and their edited mRNAs in Trypanosoma congolense and compared these to the corresponding sequences in T.brucei. We find that these genes are highly diverged between the two species, especially in the positions of thymidines and in nucleotide transitions. Editing eliminates differences in encoded uridines producing edited mRNAs that are identical except for the nucleotide substitutions. The resulting predicted proteins are identical since all nucleotide substitutions are silent. A T.congolense minicircle-encoded gRNA which can specify editing of ND8 mRNA was identified. This gRNA can basepair with both T.congolense and T.brucei ND8 mRNA despite nucleotide transitions due to the flexibility of G:U base-pairing. These results illustrate how editing affects the characteristics of maxicircle sequence divergence and allows protein sequence conservation despite a level of DNA sequence divergence which would be predicted to be intolerable in the absence of editing.

Enzymic editing mechanisms and the origin of biological information transfer

Current knowledge of enzymic editing mechanisms in DNA replication, transcription and translation can be used to predict error rates in the absence of editing. Primitive enzymes which possessed synthetic activity but not yet editing mechanisms would have had extremely high error rates resulting in heterogeneous proteins. Based on present knowledge of molecular biology and biochemistry, it is concluded that the evolution of contemporary information transfer systems from primitive systems lacking such editing mechanisms remains an unsolved problem in theoretical biology.