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PubMed2001

A recombinant newcastle disease virus with low-level V protein expression is immunogenic and lacks pathogenicity for chicken embryos.

Newcastle disease virus (NDV) edits its P-gene mRNA by inserting a nontemplated G residue(s) at a conserved editing site (3'-UUUUUCCC-template strand). In the wild-type virus, three amino-coterminal P-gene-derived proteins, P, V, and W, are produced at frequencies of approximately 68, 29, and 2%, respectively. By applying the reverse genetics technique, editing-defective mutants were generated in cell culture. Compared to the wild-type virus, mutants lacking either six nucleotides of the conserved editing site or the unique C-terminal part of the V protein produced as much as 5, 000-fold fewer infectious progeny in vitro or 200,000-fold fewer in 6-day-old embryonated chicken eggs. In addition, both mutants were unable to propagate in 9- to 11-day-old embryonated specific-pathogen-free (SPF) chicken eggs. In contrast, a mutant (NDV-P1) with one nucleotide substitution (UUCUUCCC) grew in eggs, albeit with a 100-fold-lower infectious titer than the parent virus. The modification in the first two mutants described above led to complete abolition of V expression, whereas in NDV-P1 the editing frequency was reduced to less than 2%, and as a result, V was expressed at a 20-fold-lower level. NDV-P1 showed markedly attenuated pathogenicity for SPF chicken embryos, unlike currently available ND vaccine strains. These findings indicate that the V protein of NDV has a dual function, playing a direct role in virus replication as well as serving as a virulence factor. Administration of NDV-P1 to 18-day-old embryonated chicken eggs hardly affected hatchability. Hatched chickens developed high levels of NDV-specific antibodies and were fully protected against lethal challenge, demonstrating the potential use of editing-defective recombinant NDV as a safe embryo vaccine.

Animals↗
PubMed2025

An advanced cytosine base editor enabled the generation of cattle with a stop codon in the β-lactoglobulin gene.

β-Lactoglobulin (BLG) is an allergen present in milk that can induce an acute immune response in certain individuals. The successful use of cytosine base editors (CBEs) can introduce stop codons into premature mRNA, thereby generating animals with disrupted genes that negatively regulate target traits. In this study, we employed a CBE system to target the major milk allergen BLG in bovine embryos, mammary epithelial cells, and live cattle. First, the precise single-base editing of the BLG gene in bovine embryos was achieved by designing an effective sgRNA to induce a c.61C > T substitution in the coding region, converting codon 21Gln (p.21Gln) to a premature stop codon. Sanger sequencing revealed an editing efficiency of 83.3% (20 out of 24 embryos), including two homozygous edits. Second, a bovine mammary epithelial cell line harboring BLG edits was constructed using the same CBE system. Sequencing showed that the designed sgRNA1 enabled the simultaneous conversion of three consecutive cytosines (c.59-61CCC > TTT) to thymines. At position c.61, single-cell clones exhibited monoallelic or biallelic editing (BLGc.61C > T), with monoallelic edits at positions c.59 and c.60 (CC > TT). Gene expression analysis confirmed that the BLGc.61C > T mutation effectively suppressed BLG expression at both the mRNA and protein levels, even in monoallelically edited cells. Finally, we successfully generated a heterozygous BLGc.61C > T single-base-edited dairy cow that despite its heterozygosity, showed significantly reduced BLG expression in the mammary epithelial cells and milk. Collectively, this study demonstrates the feasibility of using CBEs to disrupt BLG expression in dairy cows and provides a foundation for application in generating hypoallergenic dairy products.

Animals↗
PubMed2026

Diversity at the HYP1 locus in potato cyst nematodes does not result from developmentally-programmed somatic mutations.

Most genetic diversity stems from spontaneous mutations, that is, errors in DNA repair or replication. But for dozens of organisms across the tree of life, mutations at specific loci are not spontaneous but developmentally programmed: effectively, some organisms edit their own DNA sequences. This is perhaps most common among pathogens and parasites, many of which use editing to diversify genes that produce important antigens. Plant-parasitic potato cyst nematodes are damaging agricultural pests that establish a lifelong feeding site inside the root of their host plant. We previously observed extensive diversity of rare alleles at HYP1, the most highly expressed gene that encodes a protein secreted by potato cyst nematodes during parasitism. Importantly, HYP1 alleles differ from each other by complex, in-frame rearrangements of short repeated sequence motifs within a single exon. Combining several lines of evidence, we previously hypothesized that potato cyst nematodes use developmentally-programmed mutations, or editing, to diversify HYP1 alleles in the soma. In the current work, we now test this hypothesis. We employ highly accurate long-read DNA sequencing of a simplified genetic system to identify potential rare edited alleles, we use a transgenic yeast system to describe large de novo mutations at HYP1, and we interpret our findings in light of key population genetic parameters as well as the genetic diversity surrounding HYP1 and across the genome.

Animals↗
PubMed1990

Ribosomal protein S14 transcripts are edited in Oenothera mitochondria.

The gene encoding ribosomal protein S14 (rps14) in Oenothera mitochondria is located upstream of the cytochrome b gene (cob). Sequence analysis of independently derived cDNA clones covering the entire rps14 coding region shows two nucleotides edited from the genomic DNA to the mRNA derived sequences by C to U modifications. A third editing event occurs four nucleotides upstream of the AUG initiation codon and improves a potential ribosome binding site. A CGG codon specifying arginine in a position conserved in evolution between chloroplasts and E. coli as a UGG tryptophan codon is not edited in any of the cDNAs analysed. An inverted repeat 3' of an unidentified open reading frame is located upstream of the rps14 gene. The inverted repeat sequence is highly conserved at analogous regions in other Oenothera mitochondrial loci.

Amino Acid Sequence↗
PubMed2026

The TANG cluster comprising ten nitrate transporter genes controls fruit sweetness and size in tomato.

Sucrose is a major transport form of photoassimilated carbon in tomato, Arabidopsis, and many other plant species, and plays a critical regulatory role in plant growth, development, and fruit quality. Plant vacuoles function as storage organelles, accumulating substantial quantities of metabolically inactive nitrates as a nitrogen reserve and soluble sugars as a carbon reserve. Consequently, the balance between nitrate and sucrose accumulation determines plant growth dynamics and fruit taste. In this study, we identified a gene cluster designated TANG (Total soluble solidsAccumulation viaNitrate transporterGene cluster), comprising ten nitrate transporter genes that are significantly associated with sucrose accumulation in tomato. This gene cluster mediates the transport of nitrate between the cytoplasm and vacuole, thereby influencing its storage. Functional disruption of TANG8, a member of the gene cluster, results in either enhanced sugar accumulation or increased fruit size. Selective disruption of multiple TANG cluster members yields fruits with elevated sweetness and increased fruit size in S. pimpinellifolium. The interaction between the TANG members and a tonoplast localized Sucrose Transporter 4 provides insight into the competitive accumulation of nitrate and sugar. The multiplex editing of a gene cluster provides a successful example of engineering crops with high quality and yield.

Gene cluster↗
PubMed1994

Adenovirus-mediated gene transfer of rat apolipoprotein B mRNA-editing protein in mice virtually eliminates apolipoprotein B-100 and normal low density lipoprotein production.

Apolipoprotein (apo) B-100 is the major protein component in low density lipoprotein (LDL); it contains the binding domain for the LDL receptor and the attachment site for apolipoprotein(a) in lipoprotein(a). ApoB-48 is colinear with the amino-terminal half of apoB-100 and misses the part of the molecule required for LDL receptor interaction and lipoprotein(a) formation. ApoB-48 mRNA is produced by the editing of apoB-100 mRNA, a process by which the codon CAA for Gln-2153 is changed to UAA, an in-frame stop codon. We used the cloned catalytic component of the rat apoB mRNA-editing enzyme (REPR) to construct a replication-defective recombinant adenoviral vector containing REPR cDNA (AvREPR) and a control vector (Av1LacZ4) containing a beta-galactosidase cDNA to investigate the effect of REPR gene delivery in C57BL/6 mice. Intravenous injection of AvREPR in mice resulted in efficient transduction of liver cells, where REPR mRNA and protein were overexpressed, reaching a peak at 7 and 12 days, returning toward control levels at 39 days after AvREPR administration. ApoB mRNA editing activity in liver extracts showed changes parallel to those of REPR mRNA expression; the proportion of edited apoB mRNA in the total hepatic apoB mRNA increased from approximately 60% to more than 90% at the peak of REPR expression. The proportion of plasma apoB-100 in AvREPR-transduced animals decreased from approximately 50% to < 10% of total plasma apoB concentration. Plasma very low density lipoproteins were polydisperse in control animals with an average diameter of 54.9 +/- 20.6 nm (uninjected control) and 54.7 +/- 16.8 nm (Av1LacZ4-treated), respectively. They became much smaller (average diameter 39.3 +/- 12.7 nm) and more uniform in size at day 12 following AvREPR administration. On the same day, the normal plasma LDL (26.2-25.5 nm) was almost completely eliminated in treated animals. Adenovirus-mediated transfer of the REPR cDNA is an efficient method to reduce plasma apoB-100 and normal LDL production.

APOBEC-1 Deaminase↗
PubMed2025

Design of optimized epigenetic regulators for durable gene silencing with application to PCSK9 in nonhuman primates.

Epigenetic editing is a promising strategy for modifying gene expression while avoiding the permanent alterations and potential genotoxicity of genome-editing technologies. Here we designed optimized epigenetic regulators (EpiRegs) by testing combinations of transcription activator-like effector (TALE)-based and catalytically deactivated Cas9 (dCas9)-based epigenetic modification effectors and fusion protein structures. TALE-based EpiReg (EpiReg-T) achieved a final efficiency of 98% in mice, surpassing the initial dCas9-based efficiency of 64%. We demonstrated the approach in macaques by introducing DNA methylation and histone modifications to inhibit proprotein convertase subtilisin/kexin type 9 (PCSK9) expression, thereby lowering low-density lipoprotein cholesterol levels. A single dose of EpiReg-T delivered with lipid nanoparticles achieved efficient (>90%) and long-lasting (343&#x2009;days) silencing of PCSK9 in the liver. Integrative multiomic analyses revealed minimal off-target effects in EpiReg-T-treated monkeys, mice and human-derived cells. EpiReg can be redirected to other genes by reengineering the DNA-binding domain. Our findings represent a step toward the clinical application of epigenetic editing for the treatment of human diseases.

Animals↗
PubMed2005

Nuclear localization of the Nipah virus W protein allows for inhibition of both virus- and toll-like receptor 3-triggered signaling pathways.

The Nipah virus V and W proteins, which are encoded by the P gene via RNA editing, have a common N-terminal domain but unique C-terminal domains. They localize to the cytoplasm and nucleus, respectively, and have both been shown to function as inhibitors of JAK/STAT signaling. Here we report that V and W proteins also block virus activation of the beta interferon (IFN-beta) promoter and the IFN regulatory factor 3 (IRF3)-responsive IFN-stimulated gene 54 promoter. Surprisingly, only W protein shows strong inhibition of promoter activation in response to stimulation of Toll-like receptor 3 (TLR3) by extracellular double-stranded RNA. This activity is dependent on the nuclear localization of W protein. Within the unique C-terminal domain of W protein, we have identified a nuclear localization signal (NLS) that requires basic residues at positions 439, 440, and 442. This NLS is responsible for mediating the preferential interaction of W protein with karyopherin-alpha 3 and karyopherin-alpha 4. Nuclear localization of W protein therefore enables it to target both virus and TLR3 pathways, whereas the cytoplasmic V protein is restricted to inhibiting the virus pathway. We propose that this discrepancy is in part due to the V protein being less able to block signaling in response to the kinase, TBK-1, whereas both V and W can prevent promoter activation in response to IKKepsilon. We demonstrate that, when the TLR3 pathway is stimulated, the levels of phosphorylated IRF3 are reduced in the presence of W protein but not V protein, confirming the differential effects of these proteins and illustrating that W protein-mediated inhibition is due to a loss of active IRF3.

Amino Acid Sequence↗
PubMed1996

Developmental- and tissue-specificity of RNA editing in mitochondria of suspension-cultured maize cells and seedlings.

C to U editing of apt9, nad3, and cox2 mRNAs was investigated in maize seedlings at various developmental stages as well as in suspension-cultured cells. Heterogeneity of mRNAs that result from incomplete editing was analyzed for each gene and from five tissues or developmental conditions. The editing status of approximately 30 cDNA clones was determined by digestion with a restriction enzyme that discriminates between unedited and edited DNA sequences. The atp9 and spliced cox2 cDNAs were essentially completely edited in all samples examined. Analysis of three editing sites of nad3 cDNAs indicated that incompletely edited cDNAs were detected in all tissues and treatments with a temporal increase in the overall editing status, from 50% at 3 days to about 75% at 7 days. These results indicate that incompletely edited mRNAs are prevalent for some plant mitochondrial genes, and can change with developmental or growth conditions.

Amino Acid Sequence↗
PubMed2002

Chemical modification of nucleotide bases and mRNA editing depend on hexamer or nucleoprotein phase in Sendai virus nucleocapsids.

The minus-strand genome of Sendai virus is an assembly of the nucleocapsid protein (N) and RNA, in which each N subunit is associated with precisely 6 nt. Only genomes that are a multiple of 6 nt long replicate efficiently or are found naturally, and their replication promoters contain sequence elements with hexamer repeats. Paramyxoviruses that are governed by this hexamer rule also edit their P gene mRNA during its synthesis, by G insertions, via a controlled form of viral RNA polymerase "stuttering" (pseudo-templated transcription). This stuttering is directed by a cis-acting sequence (3' UNN UUUUUU CCC), whose hexamer phase is conserved within each virus group. To determine whether the hexamer phase of a given nucleotide sequence within nucleocapsids affected its sensitivity to chemical modification, and whether hexamer phase of the mRNA editing site was important for the editing process, we prepared a matched set of viruses in which a model editing site was displaced 1 nt at a time relative to the genome ends. The relative abilities of these Sendai viruses to edit their mRNAs in cell culture infections were examined, and the ability of DMS to chemically modify the nucleotides of this cis-acting signal within resting viral nucleocapsids was also studied. Cytidines at hexamer phases 1 and 6 were the most accessible to chemical modification, whereas mRNA editing was most extensive when the stutter-site C was in positions 2 to 5. Apparently, the N subunit imprints the nucleotide sequence it is associated with, and affects both the initiation of viral RNA synthesis and mRNA editing. The N-subunit assembly thus appears to superimpose another code upon the genetic code.

Base Sequence↗
PubMed1993

Ribosomal protein gene rpl5 is cotranscribed with the nad3 gene in Oenothera mitochondria.

The rpl5 ribosomal protein gene was identified in the mitochondrial genome of the higher plant Oenothera berteriana. The gene is present in a unique genomic location upstream of the gene encoding subunit 3 of the NADH dehydrogenase (nad3). Both genes are cotranscribed, and the mRNA is modified at several cytidine residues by RNA editing. Analysis of the editing profiles of both genes by direct cDNA analysis and polymerase chain reaction (PCR) revealed that not all transcripts are fully edited at all sites. Eight of the nine C to U conversions in the rpl5 reading frame are non-silent and change the deduced amino acid sequence. The genes of the prokaryotic-like cistron that includes the rpsl9, rps3, rpl16, rpl5, and rpsl4 genes, which is at least partially conserved in the mitochondrial genomes of other higher and lower plants, are dispersed in the Oenothera mitochondrial genome.

Amino Acid Sequence↗
PubMed2004

Plastid transformation in higher plants.

Plastids of higher plants are semi-autonomous organelles with a small, highly polyploid genome and their own transcription-translation machinery. This review provides an overview of the technology for the genetic modification of the plastid genome including: vectors, marker genes and gene design, the use of gene knockouts and over-expression to probe plastid function and the application of site-specific recombinases for excision of target DNA. Examples for applications in basic science include the study of plastid gene transcription, mRNA editing, photosynthesis and evolution. Examples for biotechnological applications are incorporation of transgenes in the plastid genome for containment and high-level expression of recombinant proteins for pharmaceutical and industrial applications. Plastid transformation is routine only in tobacco. Progress in implementing the technology in other crops is discussed.

DNA, Plant↗
PubMed1992

The P gene of the porcine paramyxovirus LPMV encodes three possible polypeptides P, V and C: the P protein mRNA is edited.

The nucleotide sequence of the P gene of the porcine paramyxovirus La-Piedad-Michoacan-Mexico virus (LPMV) was analysed. Three long open reading frames (ORFs) were found in the mRNA sense. Insertion of two G residues is necessary to obtain an ORF encoding the P protein, which gives a P protein of 404 amino acids with a calculated Mr of 42475. This form of editing was demonstrated, two non-templated G residues being added in a portion of the mRNA transcripts. The LPMV V protein, which has a conserved cysteine-rich C-terminal region, is encoded by an exact copy of the P gene. The third ORF has the capacity to encode a protein of 126 amino acids, which may resemble the C proteins found in some paramyxoviruses. The ORF starts from an AUG codon down-stream of the first AUG codon of the P/V ORF.

Amino Acid Sequence↗
PubMed2004

Non-functional immunoglobulin G transcripts in a case of hyper-immunoglobulin M syndrome similar to type 4.

Summary 86% of immunoglobulin G (IgG) heavy-chain gene transcripts were found to be non-functional in the peripheral blood B cells of a patient initially diagnosed with common variable immunodeficiency, who later developed raised IgM, whereas no non-functionally rearranged transcripts were found in the cells of seven healthy control subjects. All the patient's IgM heavy-chain and kappa light-chain transcripts were functional, suggesting that either non-functional rearrangements were being selectively class-switched to IgG, or that receptor editing was rendering genes non-functional after class-switching. The functional gamma-chain sequences showed a normal rate of somatic hypermutation while non-functional sequences contained few somatic mutations, suggesting that most came from cells that had no functional gene and therefore were not receiving signals for hypermutation. However, apoptosis of peripheral blood lymphocytes was not impaired. No defects have been found in any of the genes currently known to be responsible for hyper-IgM syndrome but the phenotype fits best to type 4.

Adult↗
PubMed1996

Paramyxovirus RNA editing and the requirement for hexamer genome length.

Paramyxoviruses cotranscriptionally edit their P gene mRNA by the programmed insertion of G residues into a short G run contained within a larger purine run, via pseudo-templated transcription. The templates for paramyxovirus transcription are genome nucleocapsids in which each nucleoprotein subunit is associated with 6 nt, and only genomes whose lengths are multiples of 6 are found naturally or are replicated efficiently in transfected cell systems. We have examined the effect of varying total genome length on the frequency and number of insertions into the mRNA editing site in a transfected cell system, using constructs that generate mini-genome analogues. We found that, as long as the purine run sequence and the region immediately upstream were unaltered, editing occurred during mRNA synthesis independent of the precise length of the minigenome. However, when mini-genome constructs whose lengths were not multiples of 6 were used, insertions (or deletions) occurred during antigenome synthesis within the purine run, which strikingly restored the hexamer length. Genome length correction due to changes in the antigenome purine run length occurred only when the mini-genome was not a multiple of 6, and these changes were only poorly affected by mutations in the mRNA editing site and the region immediately upstream. Our results suggest that the mRNA editing site is a natural hotspot for viral polymerase slippage during genome replication, and that this site serves the dual and complementary function of maintaining hexamer genome length. The unusual requirement of paramyxoviruses for genomes of precise hexamer length may have evolved to maintain genome stability against insertions in the mRNA editing site during replication.

Base Composition↗
PubMed2006

Preferential interaction of a 25kDa protein with an A6 pre-mRNA substrate for RNA editing in Trypanosoma brucei.

Mitochondrial gene expression in kinetoplastids is controlled after transcription, potentially at the levels of RNA maturation, stability and translation. Among these processes, RNA editing by U-insertion/deletion catalysed by multi-subunit editing complexes is best characterised at the molecular level. Nevertheless, mitochondrial RNA metabolism overall remains poorly understood, including the potential regulatory factors that may interact with the relevant catalytic molecular machines and/or RNA substrates. Here we report on a approximately 25kDa polypeptide in mitochondrial extracts that exhibits a preferential "zero-distance" photo-crosslinking interaction with an A6 pre-mRNA model substrate for RNA editing containing a single [(32)P] at the first editing site. The approximately 25kDa polypeptide purified away from editosomes upon ion-exchange chromatography and glycerol gradient sedimentation. Competition assays with homologous and heterologous transcripts suggest that the preferential recognition of the A6 substrate is based on relatively low-specificity RNA-protein contacts. Our mapping and substrate truncation analyses suggest that the crosslinking activity primarily targeted a predicted stem-loop region containing the first editing sites. Consistent with the notion that pre-mRNA folding may be required, pre-annealing with guide RNA abolished crosslinking. Interestingly, this preferential protein interaction with the A6 substrate seemed to require adenosine 5'-triphosphate but not hydrolysis. As in other biological systems, fine regulation in vivo may be brought about by transient networks of relatively low-specificity interactions in which multiple auxiliary factors bind to mRNAs and/or editing complexes in unique higher-order assemblies.

Adenosine Triphosphate↗
PubMed2006

RNA editing level in the mouse is determined by the genomic repeat repertoire.

A-to-I RNA editing is the conversion of adenosine to inosine in double-stranded cellular and viral RNAs. Recently, abundant editing of human transcripts affecting thousands of genes has been reported. Most editing sites are confined to the primate-specific Alu repeats. Notably, the editing level in mouse was shown to be much lower. In order to find the reason for this dramatic difference, here we identify editing sites within mouse repeats and analyze the sequence properties required for RNA editing. Our results show that the overall rate of RNA editing is determined by specific properties of different repeat families such as abundance, length, and divergence. We show that the striking difference in editing levels between human and mouse is mostly due to the higher divergence of the different mouse repeats.

Animals↗
PubMed2004

Computational prediction of RNA editing sites.

MOTIVATION: Some organisms edit their messenger RNA resulting in differences between the genomic sequence for a gene and the corresponding messenger RNA sequence. This difference complicates experimental and computational attempts to find and study genes in organisms with RNA editing even if the full genomic sequence is known. Nevertheless, knowledge of these editing sites is crucial for understanding the editing machinery of these organisms. RESULTS: We present a computational technique that predicts the position of editing sites in the genomic sequence. It uses a statistical approach drawing on the protein sequences of related genes and general features of editing sites of the organism. We apply the method to the mitochondrion of the slime mold Physarum polycephalum. It correctly predicts over 90% of the amino acids and over 70% of the editing sites.

Algorithms↗