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PubMed2005

Editing of the grapevine mitochondrial cytochrome b mRNA and molecular modeling of the protein.

Cytochrome b (COB), the central catalytic subunit of ubiquinol cytochrome c reductase, is a component of the transmembrane electron transfer chain that generates proton motive force. Some plant COB mRNAs are processed by RNA editing, which changes the gene coding sequence. This report presents the sequences of the grapevine (Vitis vinifera L.) mitochondrial gene for apocytochrome b (cob), the edited mRNA and the deduced protein. Grapevine COB is 393 amino acids long and is 98% identical to homologs in rapeseed, Arabidopsis thaliana and Oenothera sp. Twenty-one C-U editing sites were identified in the grapevine cob mRNA, resulting in 20 amino acid changes. These changes increase the overall hydrophobicity of the protein and result in a more conserved protein. Molecular modeling of grapevine COB shows that residues changed by RNA editing fit the secondary structure characteristic of an integral membrane protein. This is the first complete mitochondrial gene reported for grapevine. Novel RNA editing sites were identified in grapevine cob, which have not been previously reported for other plants.

Amino Acid Sequence↗
PubMed2002

Phylogenetic comparison of the pre-mRNA adenosine deaminase ADAR2 genes and transcripts: conservation and diversity in editing site sequence and alternative splicing patterns.

Adenosine deaminase that acts on RNA -2 (ADAR2) is a member of a family of vertebrate genes that encode adenosine (A)-to-inosine (I) RNA deaminases, enzymes that deaminate specific A residues in specific pre-mRNAs to produce I. Known substrates of ADAR2 include sites within the coding regions of pre-mRNAs of the ionotropic glutamate receptors, GluR2-6, and the serotonin receptor, 5HT2C. Mammalian ADAR2 expression is itself regulated by A-to-I editing and by several alternative splicing events. Because the biological consequences of ADAR2 function are significant, we have undertaken a phylogenetic comparison of these features. Here we report a comparison of cDNA sequences, genomic organization, editing site sequences and patterns of alternative splicing of ADAR2 genes from human, mouse, chicken, pufferfish and zebrafish. Coding sequences and intron/exon organization are highly conserved. All ADAR2 genes show evidence of transcript editing with required sequences and predicted secondary structures very highly conserved. Patterns and levels of editing and alternative splicing vary among organisms, and include novel N-terminal exons and splicing events.

Adenosine Deaminase↗
PubMedDate not supplied

[Reduction of the edited domain of the mitochondrial A6 gene for ATPase subunit 6 in Trypanosomatidae].

The sequence of mitochondrial A6 (MURF4) was compared for several trypanosomatids in order to assess the reduction of the edited domain (ED). The association between the ED reduction and the phylogenetic position of a species proved to be less tight than believed earlier. Compared with digenetic species, monogenetic ones more often displayed ED reduction and had smaller ED.

Adenosine Triphosphatases↗
PubMed2006

Extensive RNA editing in transcripts from the PsbB operon and RpoA gene of plastids from the enigmatic moss Takakia lepidozioides.

RNA editing is a post-transcriptional process that changes individual nucleotides in transcripts, and usually occurs in the plastids of land plants. The number of RNA editing sites in a plastid is significantly divergent in bryophytes, ranging from zero in liverworts to almost 1,000 sites in hornworts. In this study, we identified 132 RNA editing sites in the transcripts of six genes from the psbB operon and the rpoA of the moss Takakia lepidozioides. This is the highest number of RNA editing sites known in this region among land plant species. All were cytidine-to-uridine conversions. More than 91% of RNA editing occurred at the first or second codon positions, and it altered amino acid identity. Six editing sites created new translation initiation codons or stop codons. Thirty-two sites were commonly observed in the hornwort Anthoceros angustus. This finding suggests that the enigmatic bryophyte Takakia is closely related to hornworts with respect to RNA editing events.

Base Sequence↗
PubMed2002

Transcript abundance supercedes editing efficiency as a factor in developmental variation of chloroplast gene expression.

In maize plastids, transcripts are known to be modified at 27 C-to-U RNA editing sites, affecting the expression-of 15 different genes. The relative contribution of editing efficiency versus transcript abundance in regulation of chloroplast gene expression has previously been analyzed for only a few genes. We undertook a comprehensive analysis of the editing efficiency of each of the 27 maize editing sites in 10 different maize tissues, which contain a range of plastid types including chloroplasts, etioplasts, and amyloplasts. Using a reproducible poisoned primer extension assay, we detected variation between RNA editing extent of different sites in the same transcript in the same tissue, and between the same site in different tissues. The most striking editing deficiency is in an editing site in ndhB that is edited at only 8% and 1% in roots and callus plastids respectively, whereas green leaf chloroplasts edit this site at 100%. Editing efficiencies of some sites are not affected by the developmental stages we examined and are always edited close to 80-100%. The relative amounts of transcripts of each of the 10 genes that exhibited variable editing extents were determined by real-time PCR. Seven genes exhibited over 100 times lower transcript abundance in either roots or tissue-cultured cells relative to green leaf tissue. The quantitative analysis indicates that a particular editing site can be efficiently edited over a large range of transcript abundance, resulting in no general correlation of transcript abundance and editing extent. The independent variation of editing efficiency of different sites within the same transcript fits with a model that postulates individual trans-acting factors specific to each editing site. Because tissues where editing efficiency at certain sites is low invariably also exhibited greatly decreased abundance of the transcripts carrying those sites, decrease in the amounts of particular RNAs rather than a lack of editing is predicted to have the most significant impact on gene expression under steady-state conditions. Our data is consistent with the hypothesis that the role of editing in angiosperm plastids is to correct otherwise detrimental mutations rather than to generate significant protein diversity.

Amino Acid Sequence↗
PubMed1994

RNA editing in the Wilms' tumor susceptibility gene, WT1.

Rat kidney WT1 cDNAs contain either a thymidine or a cytosine residue at position 839. Genomic WT1 DNA contains only T839. To explain these results, we propose the WT1 transcript undergoes RNA editing in which U839 is converted to C, resulting in the replacement of leucine 280 in WT1 by proline. RNA editing at the same nucleotide was observed in WT1 cDNAs from human testis. In functional assays, the WT1-leucine280 polypeptide repressed the EGR-1 promoter in in vitro assays approximately 30% more efficiently than WT1-proline. Edited WT1-C839 mRNA was barely detectable in neonatal kidney, whereas adult rat kidneys contained both U839 and C839-WT1 mRNA, suggesting a role for the two protein isoforms in growth and differentiation.

Animals↗
PubMed1992

The polarity of editing within a multiple gRNA-mediated domain is due to formation of anchors for upstream gRNAs by downstream editing.

Seventeen kinetoplast minicircle-encoded and nine maxicircle-encoded gRNA genes have been identified. Six overlapping minicircle-encoded gRNAs mediate editing for the 5'-pan-edited MURF4 gene and two for the 5'-edited COIII gene. The pan-edited RPS12 mRNA is edited by seven minicircle-encoded gRNAs and one maxicircle-encoded gRNA. The 3'-most gRNA in each domain forms an anchor with unedited mRNA, whereas upstream gRNAs form anchors only with edited mRNA, thereby explaining the observed 3' to 5' polarity of editing within an editing domain. We suggest that a role of G-U base pairs is to allow breathing of the edited mRNA-gRNA hybrid and formation of the upstream anchor hybrid.

Animals↗
PubMed2002

Regulation of the K channels by cytoplasmic domains.

Regulation of intracellular potassium levels is one of the basic functions of all cells, controlling cellular osmolarity and transmitting information. In higher organisms, elaborate control of transmembrane potassium flux has evolved to endow nervous systems with the remarkable ability to transmit electrical signals between cells. Multiple genes, gene splicing, mRNA editing, and selective tetrameric assembly of K channel genes provide the basis for creating distinct electrophysiological properties at varying developmental and cellular stages. This assembly mechanism, primarily governed by the T1 domain, is under the control of intracellular signals. Atomic structures of the isolated T1 domains of Shaker and Shaw subfamilies provided us with valuable structural insights into understanding both channel assembly and functional regulation of the entire channel molecule through conformational changes.

Binding Sites↗
PubMed2025

A CRISPR/Cas9 mutant resource for OsSm RNA-binding genes in rice.

Pre-mRNA, produced by eukaryotic DNA transcription, undergoes splicing by the spliceosome, which removes introns and joins exons to form mRNA. The spliceosome is a large and highly dynamic molecular machine. Its core components include five small nuclear ribonucleoproteins (snRNPs) and the various spliceosome-related proteins. The conserved Smith (Sm) complex and the Sm-like proteins (LSm) serve as primary components of the snRNPs. Sm proteins are involved in processes such as pre-mRNA splicing and mRNA degradation, which can regulate gene expression, thereby influencing plant growth, development, and stress responses. While 25 Sm proteins have been identified in rice, their specific roles in regulating rice growth and development remain unclear. In this study, we employed the CRISPR/Cas9 system to edit 15 OsSm genes, and 13 mutants were obtained, with mutation rates ranging from 20.83 to 83.87%. In comparison to the wild type (WT), the mutants exhibited dwarfism, reduced tiller numbers, lower seed-setting rates or sterility, and increased susceptibility to diseases. One Sm mutant, ossmf-2, exhibited dwarfism, delayed flowering, and small grains. Through transcriptome analysis, three target genes, OsMRG702, OsRGG2, and OsLA1, were identified. Mutations of the OsSmF protein may lead to the abnormal splicing of these genes and finally lead to the inhibition of growth and development. Our study first edited the OsSm genes and generated a mutant library in rice. Most of the mutants exhibited abnormal growth and development, underscoring the essential roles of OsSm proteins in rice physiology. Furthermore, this work addresses a critical gap in the functional characterization of Sm proteins in rice. The resulting mutant collection offers valuable germplasm resources and lays a theoretical foundation for elucidating the molecular regulatory networks involving spliceosomal components and their target genes in the control of crop growth, development, and reproduction.

Oryza↗
PubMed2026

CRISPR/Cas9-Mediated Editing of Bsr-d1 and Pi21 Enhances Blast Resistance in a High-Quality Rice Maintainer Line.

Rice (Oryza sativa L.) is a staple food crop worldwide, and improving disease resistance is a core target in rice breeding. In this study, we employed CRISPR/Cas9 genome editing to modify the coding sequence (CDS) of two susceptibility genes, Bsr-d1 and Pi21, in the elite maintainer line Gengxiang B to enhance its blast resistance. We generated Bsr-d1/Pi21 double homozygous mutants via Agrobacterium-mediated genetic transformation. Quantitative RT-PCR revealed significantly suppressed transcript accumulation of both target genes in the edited lines compared with the wild type Gengxiang B. Upon inoculation with Magnaporthe oryzae, multiple defense-related marker genes were markedly upregulated in the double mutants. Phenotypic assays demonstrated significantly reduced disease severity for both leaf and panicle blast in the edited lines compared with the wild type. Importantly, no statistically detectable differences were found between the double mutants and wild-type plants for key agronomic or grain quality traits. Collectively, these results demonstrate that CRISPR/Cas9-mediated editing of susceptibility loci generates genetically stable blast-resistant rice germplasm without compromising agronomic traits or grain quality, providing valuable genetic resources for future rice varietal improvement.

Bsr-d1↗
PubMed2003

Serotonin 2C receptors: suicide, serotonin, and runaway RNA editing.

Transcripts of the gene encoding the serotonin 2C receptor are modified by RNA editing, a posttranscriptional process that converts adenosines to inosines. This editing changes up to three genomically encoded amino acids located in the second intracellular loop of the G-protein-coupled receptor. Compared with nonedited receptors, extensively edited receptor isoforms activate G protein less efficiently. Studies on mice revealed that 5-HT2C pre-mRNA editing is regulated in a serotonin-dependent manner, and postmortem studies on brain tissues of patients with schizophrenia and major depression found distinct site-specific alterations of this editing in the prefrontal cortex, a brain region expressing a large number of differently edited 5-HT2C mRNA isoforms. At present, the most complex alterations in 5-HT2C pre-mRNA editing were found in brains of depressed suicide victims. In these brains, 5-HT2C receptor isoforms with reduced function are expressed at significantly increased levels, suggesting that the regulation of editing by synaptic serotonin is defective.

Animals↗
PubMed1996

Preferential RNA editing at specific sites within transcripts of two plant mitochondrial genes does not depend on transcriptional context or nuclear genotype.

Transcripts of most plant mitochondrial protein-coding genes exhibit C-to-U RNA editing events. In Petunia, two co-transcribed genes, nad3 and rps12, exhibit transcripts which are not fully edited at all potential editing sites. We investigated the nad3/rps12 transcript population in four different genotypes. In one pair of genotypes, the nuclear genome is identical but the nad3/rps12 genes are in different transcriptional contexts. Both the nad3/ rps12 genes and the plant mitochondrial genomes are identical in a second pair of genotypes, but the nuclear background is derived from two different Petunia species. We found that the overall extent of editing varied greatly between genotypes and is affected by nuclear genotype but not by the global transcriptional context. Local sequence context around a particular site does affect editing frequency. In all genotypes, certain sites exhibit high editing frequency, but these sites do not share obvious primary sequence characteristics. In all genotypes examined, editing sites which do not affect the encoded amino acid are less frequently edited than sites which alter codons to non-synonymous forms. All these data indicate that an unidentified property of the sequences immediately surrounding a cytosine affect its selection as a target in the editing process.

Amino Acid Sequence↗
PubMed2025

Engraftment and persistence of HBB base-edited hematopoietic stem cells in nonhuman primates.

Sickle cell disease (SCD) is caused by a single nucleotide change in the β-globin gene that adenine base editors can convert to the nonpathogenic Makassar β-globin variant. Here, we evaluated the long-term efficiency and off-target editing potential of autologous Makassar base editing in three rhesus macaques as a step toward human translation. Base editing of CD34+CD90+ hematopoietic stem cells (HSCs) at the Makassar locus reached greater than 60% efficiency using a bystander nucleotide as a proxy for the sickle cell target in cells from healthy macaques. No impact on myeloid and erythroid colony formation was seen, and clonal analysis revealed that >90% of HSCs were edited, >20% with biallelic editing. After transplantation of autologous gene-edited HSCs, all three macaques rapidly recovered neutrophils, red blood cells, and platelets with stable editing of 25.6%, on average, observed across nucleated blood cells. Similarly, the bone marrow stem cell compartment maintained over 20% of cells harboring mono- or biallelic edits. Off-target editing was assessed at over 900 candidate sites, with editing observed at eight sites, but no selection for or impact of these edits was observed throughout engraftment. These data support further translation of base editing of autologous HSCs for the treatment of patients with SCD.

Animals↗
PubMed1994

Ancient origin of RNA editing in kinetoplastid protozoa.

A rooted phylogenetic tree of the kinetoplastid protozoa has been constructed that, together with a comparative analysis of editing of several genes, leads to the surprising conclusion that extensive or pan-editing with multiple overlapping guide RNAs is more ancient than 5'-editing. The mechanism of editing is still uncertain, but multiple ribonucleoprotein complexes have been identified that contain components of the enzymatic machinery.

Animals↗
PubMed2001

A single alteration 20 nt 5' to an editing target inhibits chloroplast RNA editing in vivo.

Transcripts of typical dicot plant plastid genes undergo C-->U RNA editing at approximately 30 locations, but there is no consensus sequence surrounding the C targets of editing. The cis-acting elements required for editing of the C located at tobacco rpoB editing site II were investigated by introducing translatable chimeric minigenes containing sequence -20 to +6 surrounding the C target of editing. When the -20 to +6 sequence specified by the homologous region present in the black pine chloroplast genome was incorporated, virtually no editing of the transcripts occurred in transgenic tobacco plastids. Nucleotides that differ between the black pine and tobacco sequence were tested for their role in C-->U editing by designing chimeric genes containing one or more of these divergent nucleotides. Surprisingly, the divergent nucleotide that had the strongest negative effect on editing of the minigene transcript was located -20 nt 5' to the C target of editing. Expression of transgene transcripts carrying the 27 nt sequence did not affect the editing extent of the endogenous rpoB transcripts, even though the chimeric transcripts were much more abundant than those of the endogenous gene. In plants carrying a 93 nt rpoB editing site sequence, transgene transcripts accumulated to a level three times greater than transgene transcripts in the plants carrying the 27 nt rpoB editing sites and resulted in editing of the endogenous transcripts from 100 to 50%. Both a lower affinity of the 27 nt site for a trans-acting factor and lower abundance of the transcript could explain why expression of minigene transcripts containing the 27 nt sequence did not affect endogenous editing.

Base Sequence↗
PubMed2002

Striking differences in RNA editing requirements to express the rps4 gene in magnolia and sunflower mitochondria.

The ribosomal protein S4 gene (rps4) has been identified as a single copy sequence in the mitochondrial genomes of two distant higher plants, Magnolia and Helianthus. Sequence analysis revealed that the rps4 genes present in the magnolia and sunflower mitochondrial genomes encode S4 polypeptides of 352 and 331 amino acids, respectively, longer than their counterparts in liverwort and bacteria. Expression of the rps4 genes in the investigated higher plant mitochondria was confirmed by Western blot analysis. In Helianthus, one of two short nucleotide insertions at the 3'-end introduces in the coding region a premature termination codon. Northern hybridizations and reverse transcription-polymerase chain reaction analysis demonstrated that the monocistronic RNA transcripts generated from the rps4 locus in Magnolia and Helianthus mitochondria are modified by RNA editing at 28 and 13 positions, respectively. Although evolutionarily conserved, RNA editing requirements of the rps4 appear more extensive in Magnolia than in Helianthus and in the other higher plants so far investigated. Furthermore, our analysis also suggests that selection of editing sites is RNA sequence-specific in a duplicated sequence context.

Amino Acid Sequence↗
PubMed2025

CRISPR/Cas9-Mediated Gene-Knockout and In Situ Complementation System for Phytophthora sojae.

The establishment of reliable and efficient systems for genome editing in Phytophthora is very important for studying gene functions. Here, step-by-step methods for CRISPR/Cas9-based gene knockout and in situ complementation for Phytophthora sojae are presented. These steps include the sgRNA design, Cas9-sgRNA plasmid construction, homologous replacement, complementation vector construction, P. sojae transformation, and detection of mutations for both gene knockout and in situ complementation. These methods may also potentially be adapted for other Phytophthora species.

Phytophthora↗
PubMedDate not supplied

Comparative analysis of RNA editing sites in higher plant chloroplasts.

Transcripts of land plant chloroplast genomes undergo C-to-U RNA editing. Systematic search disclosed 31 editing sites in tobacco, 27 in maize, and 21 in rice. Based on these identified sites, potential editing sites have been predicted in the transcripts from four angiosperm chloroplast genomes which have been completely sequenced. Most RNA editing events occur in internal codons, which result in amino-acid substitutions. The initiation codon AUG was found to be created from ACG by RNA editing in the transcripts from rpl2, psbL, and ndhD genes. Comparison of editing patterns raises a possibility that many editing sites were acquired in the evolution of angiosperms.

Binding Sites↗