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PubMed2000

Identification and characterization of the trnS/pseudo-tRNA/nad3/rps12 gene cluster from Coix lacryma-jobi L: organization, transcription and RNA editing.

During a study of mitochondrial sequence conservation between the liverwort Marchantia polymorpha and several Angiosperm species, as revealed by heterologous hybridization experiments, the trnS/pseudo-tRNA/nad3/rps12 gene cluster in Coix lacryma-jobi L., an Asian grass species from the Andropogoneae, was identified using the mitochondrial probe orf167 from M. polymorpha. The Coix gene cluster was cloned and sequenced, and its expression analyzed. The gene sequence and gene locus organization were found to be similar to the corresponding cluster in wheat and maize. Northern hybridization and reverse transcription-polymerase chain reaction analyses indicated that nad3 and rps12 genes were co-transcribed as a 1.25 kb RNA molecule. The transcript displayed 20 and six RNA edition sites, in the nad3 and rps12 genes, respectively, that changed the codon identities to amino acids, which are better conserved in different organisms. Twenty-three cDNA clones were analysed for the edition process and revealed different partial editing patterns without apparent sequential processing.

Journal Article↗
PubMed2026

Emerging Nucleic Acid-Based Therapies for Hypercholesterolemia with Focus on a New Modality, Liver-Directed miR-30c Analog C2.

Despite major advances in lipid-lowering therapies, a significant unmet need remains, particularly for patients with homozygous familial hypercholesterolemia (HoFH), severe heterozygous familial hypercholesterolemia (HeFH), and those who fail to achieve guideline-recommended LDL-C targets. Nucleic acid-based therapeutics have emerged as a transformative approach for treating hypercholesterolemia. Antisense oligonucleotides and small interfering RNAs (siRNAs) have demonstrated durable hepatic gene silencing and have led to approved therapies, while gene replacement and in vivo genome-editing strategies offer the potential for long-lasting, and possibly one-time, interventions. In parallel, microRNAs (miRNAs) have attracted increasing interest because of their ability to coordinately regulate multiple genes involved in lipoprotein metabolism, cholesterol transport, and lipid homeostasis. Human genetic studies further support the importance of miRNA-mediated regulation, exemplified by a rare ~2.5 kb deletion in the distal LDLR 3'UTR ("del2.5") that disrupts miRNA-binding sites and is associated with lifelong low LDL-C levels. This review summarizes recent advances, mechanisms of action, clinical progress, and remaining challenges across antisense oligonucleotides, siRNAs, gene therapy, genome editing, and emerging miRNA-based therapeutics for hypercholesterolemia. As an example of the latter approach, the liver-directed miR-30c analog C2 has demonstrated preclinical activity by coordinately reducing hepatic lipoprotein secretion and lipogenesis while enhancing cholesterol elimination, resulting in reduced LDL-C and atherosclerosis. However, it must be noted that these findings remain preclinical, and further optimization of delivery, pharmacokinetics, safety, and long-term efficacy will be required before clinical evaluation. Continued advances in RNA chemistry, targeted delivery, and genome engineering are expected to further expand the therapeutic landscape for dyslipidemia and cardiovascular disease.

Humans↗
PubMed2005

Edit, cut and paste in the nicotinic acetylcholine receptor gene family of Drosophila melanogaster.

Nicotinic acetylcholine receptors (nAChRs) are important for fast synaptic cholinergic transmission. They are targets of drugs/chemicals for human and animal health as well as for pest control. With the advent of genome sequencing, entire nAChR gene families have now been described for vertebrates and invertebrates. Mostly, these are extensive with a large number of distinct subunits, making possible many nAChR subtypes differing in transmitter affinity, channel conductance, ion selectivity, desensitization, modulation and pharmacology. The smallest nAChR gene family to date is that of the fruit fly, Drosophila melanogaster, with only 10 members. This apparently compact family belies its true diversity as 4 of the 10 subunits show alternative splicing. Also, using Drosophila, A-to-I pre-mRNA editing has been demonstrated for the first time in nAChRs. Such is the extent of this variation, that one subunit alone (Dalpha6) can potentially generate far more isoforms than seen in entire gene families from other species. We present here three-dimensional models constructed for insect nAChRs, which show that many variations introduced by alternative splicing and RNA editing may influence receptor function.

Alternative Splicing↗
PubMed2026

From the Microscope to the Genome: A New Era in the Molecular Genetics of Epidermolysis Bullosa.

Epidermolysis bullosa (EB) is a heterogeneous group of inherited disorders characterised by skin fragility, caused by pathogenic variants in genes encoding structural components of the dermo-epidermal junction. With the advent of next-generation sequencing (NGS), the diagnostic paradigm has shifted from a morphological to a genotype-oriented approach. This review summarises the genetic architecture of EB, the types of mutations and genotype-phenotype relationships, the challenges in interpreting variants of unknown significance (VUS), and therapeutic strategies targeting specific mutational mechanisms, including read-through approaches, exon skipping and genome editing. The role of modifier genes and epigenetic factors in clinical variability is also discussed. The focus is on the translational potential of genomics for personalized therapy in EB. Overall, this review synthesizes the molecular basis of all four major EB types across 16+ classical genes, highlights the paradigm shift where NGS achieves a diagnostic yield exceeding 90%, and critically assesses recent therapeutic milestones-ranging from the first FDA-approved topical gene therapy to precision RNA and genome-editing modalities.

Humans↗
PubMed2006

The evolution of chloroplast RNA editing.

RNA editing alters the nucleotide sequence of an RNA molecule so that it deviates from the sequence of its DNA template. Different RNA-editing systems are found in the major eukaryotic lineages, and these systems are thought to have evolved independently. In this study, we provide a detailed analysis of data on C-to-U editing sites in land plant chloroplasts and propose a model for the evolution of RNA editing in land plants. First, our data suggest that the limited RNA-editing system of seed plants and the much more extensive systems found in hornworts and ferns are of monophyletic origin. Further, although some eukaryotic editing systems appear to have evolved to regulate gene expression, or at least are now involved in gene regulation, there is no evidence that RNA editing plays a role in gene regulation in land plant chloroplasts. Instead, our results suggest that land plant chloroplast C-to-U RNA editing originated as a mechanism to generate variation at the RNA level, which could complement variation at the DNA level. Under this model, many of the original sites, particularly in seed plants, have been subsequently lost due to mutation at the DNA level, and the function of extant sites is merely to conserve certain codons. This is the first comprehensive model for the evolution of the chloroplast RNA-editing system of land plants and may also be applicable to the evolution of RNA editing in plant mitochondria.

Adiantum↗
PubMed2006

Rap1 signal controls B cell receptor repertoire and generation of self-reactive B1a cells.

We previously reported that the mice deficient for SPA-1, a Rap1 GTPase-activating protein, developed hematopoietic stem cell disorders. Here, we demonstrate that SPA-1(-/-) mice show an age-dependent increase in B220(high) B1a cells producing anti-dsDNA antibody and lupus-like nephritis. SPA-1(-/-) peritoneal B1 cells revealed the altered Vkappa gene repertoire, including skewed Vkappa4 usage and the significant Igkappa/Iglambda isotype inclusion indicative of extensive receptor editing. Rap1GTP induced OcaB gene activation via p38MAPK-dependent Creb phosphorylation, and consistently, SPA-1(-/-) immature BM B cells showing high Rap1GTP exhibited the augmented expression of OcaB and Vkappa4 genes. SPA-1(-/-) BM cells could transfer the autoimmunity in association with the generation of peritoneal B220(high) B1a cells in Rag-2(-/-) recipients. Finally, a portion of SPA-1(-/-) mice developed B1 cell leukemia with hemolytic autoantibody. Present results suggest that the regulated Rap1 signal in the immature B cells plays a role in modifying the B cell receptor repertoire and in maintaining the self-tolerance.

Animals↗
PubMed2002

Rewriting the information in DNA: RNA editing in kinetoplastids and myxomycetes.

RNA editing has a major impact on the genes and genomes that it modifies. Editing by insertion, deletion and base conversion exists in nuclear, mitochondrial and viral genomes throughout the eukaryotic lineage. Editing was first discovered in kinetoplastids, and recent work has resulted in the characterization of some components of the editing machinery. Two proteins with ligase activity have been identified in Trypanosoma brucei, and other proteins in the editosome complex are yielding to the probe of research. A second group of protists, myxomycetes, are unique in their use of four different types of editing within a single transcript. Phylogenetic analysis of editing in representative myxomycetes revealed a different history of the four types of editing in this lineage. Development of a soluble in vitro editing system has provided further support for the co-transcriptional nature of editing in Physarum polycephalum, and will certainly provide future opportunities for understanding this mysterious process.

Cytidine↗
PubMed2004

Tissue-specific modification of gld-2 mRNA in C. elegans: likely C-to-U editing.

Seventeen years after the discovery of tissue-specific apoB mRNA editing, only three nucleus-encoded mRNAs have been shown to undergo C-to-U editing. All three mRNAs occur in mammals. apoB mRNA editing is tissue-specific and occurs normally, whereas NF1 and NAT1 mRNA editing is found largely in tumors. Here we report the first example of C-to-U RNA editing in Caenorhabditis elegans. The gld-2 gene encodes an atypical poly(A) polymerase that governs the mitosis/meiosis decision in the germ line as well as progression through meiosis and early embryogenesis. At least two of its alternatively spliced transcripts are germline-specific. We find that most and perhaps all germline-specific transcripts generated by the gld-2 gene undergo C-to-U editing, but that somatic transcripts show no detectable editing. The gld-2 C-to-U editing event changes the codon from CCG to CUG, which is predicted to cause a proline to leucine substitution in the protein sequence. Our findings suggest the presence of a sequence- and tissue-specific cytidine deaminase acting on RNA, or CDAR. This CDAR modifies a specific base in gld-2 mRNA, and acts only in the germline.

Animals↗
PubMed1989

Internal frameshifts within the mitochondrial genes for cytochrome oxidase subunit II and maxicircle unidentified reading frame 3 of Leishmania tarentolae are corrected by RNA editing: evidence for translation of the edited cytochrome oxidase subunit II mRNA.

The Leishmania tarentolae cytochrome oxidase (EC 1.9.3.1) subunit II (COII) and maxicircle unidentified reading frame 3 (MURF3) mRNAs are edited internally by the addition of four and five uridine residues, respectively, which eliminate -1 and +1 reading frameshifts in the gene sequences. The editing events in COII are conserved in three kinetoplastid species, and those in MURF3 are conserved in two species. A primer extension assay showed that the ratio of edited to unedited RNA differed for each gene: 89% of the COII and 36% of the MURF3 transcripts are edited. Preliminary evidence was obtained for translation of the edited COII transcript into protein: antibodies generated against a synthetic peptide with the predicted carboxyl-terminal amino acid sequence reacted with a polypeptide of the correct molecular weight in immunoblots of a mitochondrial lysate.

Amino Acid Sequence↗
PubMed1993

RNA editing as a source of genetic variation.

Kinetoplastid RNA editing alters mitochondrial RNA transcripts by addition and deletion of uridine residues, producing open reading frames that may be twice as long as the original RNA. Although the COIII gene encoding cytochrome c oxidase subunit III in Trypanosoma brucei is edited along its entire length, the presumably homologous genes in two related trypanosomes, Leishmania tarentolae and Crithidia fasciculata, are only modestly edited at their 5' ends. We used a comparative approach to investigate the evolution of an edited gene and to determine how well editing creates conserved protein sequences. As RNA editing probably involves the pairing of several guide RNA molecules with the messenger RNA, we expected the edited proteins to be resistant to evolutionary change. Here we report that RNA editing is extensive in the mitochondria of four species of the insect parasite Herpetomonas, which is possibly an evolutionary precursor of T. brucei and L. tarentolae, and the discovery that RNA editing is a novel source of frameshift mutations over evolutionary time. The edited proteins accumulate mutations nearly twice as rapidly as the unedited versions.

Animals↗
PubMed2024

A PLA2 deletion mutant using CRISPR/Cas9 coupled to RNASeq reveals insect immune genes associated with eicosanoid signaling.

Eicosanoids mediate insect immune responses and synthesized by the catalytic activity of phospholipase A2 (PLA2). A uniquely encoded secretory PLA2 (sPLA2) is associated with immune responses of a lepidopteran insect, Spodoptera exigua. Its deletion mutant was generated using a CRISPR/Cas9 genome editing technology. Both wild and mutant lines were then immune-challenged, and the resulting transcripts were compared with their naïve transcripts by RNASeq using the Illumina-HiSeq platform. In total, 12,878 unigenes were further analyzed by differentially expressed gene tools. Over 69% of the expressed genes in S. exigua larvae are modulated in their expression levels by eicosanoids, recorded from CRISPR/Cas9 mutagenesis against an eicosanoid-synthetic gene, Se-sPLA2. Further, about 36% of the immune-associated genes are controlled by the eicosanoids in S. exigua. Indeed, the deletion mutant suffered significant immunosuppression in both cellular and humoral responses in response to bacterial challenge as well as severely reduced developmental and reproductive potentials.

Animals↗
PubMed2025

Programmable promoter editing for precise control of transgene expression.

Subtle changes in gene expression direct cells to distinct cellular states. Identifying and controlling dose-dependent transgenes require tools for precisely titrating expression. Here, we develop a highly modular, extensible framework called DIAL for building editable promoters that allow for fine-scale, heritable changes in transgene expression. Using DIAL, we increase expression by recombinase-mediated excision of spacers between the binding sites of a synthetic zinc finger transcription factor and the core promoter. By nesting varying numbers and lengths of spacers, DIAL generates a tunable range of unimodal setpoints from a single promoter. Through small-molecule control of transcription factors and recombinases, DIAL supports temporally defined, user-guided control of transgene expression that is extensible to additional transcription factors. Lentiviral delivery of DIAL generates multiple setpoints in primary cells and induced pluripotent stem cells. As promoter editing generates stable states, DIAL setpoints are heritable, facilitating mapping of transgene levels to phenotype and fate in direct conversion to induced motor neurons. The DIAL framework opens opportunities for tailoring transgene expression and improving the predictability and performance of gene circuits across diverse applications.

Promoter Regions, Genetic↗
PubMed2006

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Relaxation of functional constraint on light-independent protochlorophyllide oxidoreductase in Thuja.

The light-independent protochlorophyllide oxidoreductase (DPOR) plays a key role in the ability of nonflowering plants and algae to synthesize chlorophyll in darkness. This enzyme consists of three subunits encoded by the chlB, chlL, and chlN genes in the plastid genome. Previously, we found a high nonsynonymous substitution rate (dN) of the chlL gene in the lineage of Thuja standishii, a conifer belonging to the Cupressaceae. Here we revealed that the acceleration of dN in the chlL occurred as well in other species of Thuja, Thuja occidentalis and Thuja plicata. In addition, dark-grown seedlings of T. occidentalis were found to exhibit a pale yellowish color, and their chlorophyll concentration was much lower than that of other species of Cupressaceae. The results suggested that the species of Thuja have lost the ability to synthesize chlorophyll in darkness, and the functional constraint on the DPOR would thus be expected to be relaxed in this genus. Therefore, we expected to find that the evolutionary rates of all subunits of DPOR would in this case be accelerated. Sequence analyses of the chlN and chlB (encoding the other subunits of DPOR) in 18 species of Cupressaceae revealed that the dN of the chlN gene was accelerated in Thuja as was the dN of the chlL gene, but the dN of the chlB gene did not appear to differ significantly among the species of Cupressaceae. Sequencing of reverse transcription-polymerase chain reaction (RT-PCR) products of these genes showed that RNA editing was rare and unlikely to have contributed to the acceleration. Moreover, the RT-PCR analysis indicated that all chl genes were still transcriptionally active in T. occidentalis. Based on these results, it appears that species of Thuja still bear the DPOR protein, although the enzyme has lost its activity because of nonsynonymous mutations of some of the chl genes. The lack of acceleration of the dN of the chlB gene might be accounted for by various unknown functions of its gene product.

Evolution, Molecular↗
PubMed1992

Transcript-specific developmental regulation of polyadenylation in Trypanosoma brucei mitochondria.

Transcripts from many mitochondrial genes in kinetoplastids are heterogeneous in size, often occurring as 2 distinct size classes, but this cannot be accounted for by RNA editing alone. Analyses of transcripts from 6 mitochondrial genes of Trypanosoma brucei indicates that the size variation is due to poly(A) tail length. A larger fraction of CYb, COI and COII transcripts have longer poly(A) tails in procyclic than in bloodstream forms. These transcripts are also more abundant in the procyclic forms. In contrast, a more substantial fraction of CR1 transcripts have longer poly(A) tails in bloodstream than in procyclic forms and these transcripts tend to be more abundant in bloodstream forms. Both ND4 and MURF1 transcripts show a similar size distribution of poly(A) tail lengths in these life cycle states although both transcripts are more abundant in bloodstream forms. Furthermore, genes with edited transcripts tend to have longer poly(A) tails than unedited transcripts. Transcript abundance is not strictly correlated with longer poly(A) tails. Thus, poly(A) length variation appears to be developmentally regulated in a transcript-specific fashion in T. brucei. This regulation of polyadenylation may influence mitochondrial gene expression as polyadenylation can regulate cytoplasmic gene expression in eukaryotes.

Animals↗
PubMed2002

Post-transcriptional control of chloroplast gene expression. Accumulation of stable psaC mRNA is due to downstream RNA cleavages in the ndhD gene.

Intergenic cleavages, intron splicing, and editing of primary transcripts of the plastid ndhH-D operon produce multiple overlapping RNAs, of which the most abundant by far is the monocistronic 400-nucleotide mRNA of psaC (encoding the PsaC protein of photosystem I), in contrast with the low level of transcripts of the six ndh genes. Like other plastid operons containing genes for functionally unrelated proteins, the contrasting accumulation of ndh and psaC transcripts provides a model to investigate the mechanisms of the post-transcriptional control of gene expression, a feature of chloroplast genetic machinery, with a minimum of interference by transcriptional control. In leek (Allium porrum L), the ndhD transcript (which follows the psaC gene and ends the ndhH-D operon) requires C --> U editing to restore its start codon and may be used as a marker for the processing of psaC and ndhD transcripts. By determining the editing state and 5' end sequences of specific transcripts, we demonstrated that stable monocistronic psaC mRNA results from downstream cleavages in the ndhD sequence, which renders non-functional ndhD transcripts as by-products. Alternative psaC-ndhD intergenic cleavages produce complete mRNAs for both genes, but only take place in precursors containing editing-restored ndhD start codons. Hence, post-transcriptional control acts by promoting the ndhD cleavage alternative, which allows the accumulation of psaC mRNA at the expense of ndhD mRNA levels.

Base Sequence↗
PubMed1990

The MURF3 gene of T. brucei contains multiple domains of extensive editing and is homologous to a subunit of NADH dehydrogenase.

Mitochondrial MURF3 transcripts of T. brucei are extensively edited by the addition and deletion of uridines. The editing creates potential initiation and termination codons and a continuous open reading frame. The predicted amino acid sequence has homology to a subunit of NADH dehydrogenase (ND7). ND7 is independently edited in two distinct domains, suggesting two editing initiation sites. Editing in the two domains is differentially regulated: the 5' domain is edited in both bloodstream and procyclic forms but the 3' domain is completely edited only in the bloodstream form. Two potential guide RNA (gRNA) coding sequences were identified in the same minicircle. One is complementary to edited sequence in the 5' domain, the other to edited sequence in the 3' domain.

Amino Acid Sequence↗
PubMed2005

12th annual congress of the European Society of Gene Therapy.

The 2004 European Society of Gene Therapy (ESGT) meeting took place at Tampere Hall in Finland and highlighted advances in a variety of topics, including cancer, zinc-fingers, stem cells, small interfering RNA (siRNA), microRNA, and recent developments of non-viral and viral vectors. This meeting was attended by 513 participants from 32 countries, and included 106 oral and 224 poster presentations. One of the aims of this meeting was to take a critical look at gene therapy and the prospects for the future. Se-veral presentations reported on RNA-based technologies, such as siRNA, as potential new classes of therapeutics against a wide range of diseases and for use in expression libraries to identify functional genes involved in biological phenotypes. Critical assessments were made of other aspects of gene therapy, such as genome editing and the use of protein transduction domains (PTDs) in gene- and protein-based therapies, where many researchers have failed to reproduce initial findings reported in the literature. Safety issues related to viral vectors were also important areas of discussion, especially following details released by the UK Gene Therapy Advisory Committee of perhaps the first known case of lentiviral vector-associated oncogenesis. Finally, updates were presented on the clinical development of viral vectors in anticancer therapies with evidence of significant improvements in the mean survival of patients.

Clinical Trials as Topic↗
PubMed2000

B cell receptor expression level determines the fate of developing B lymphocytes: receptor editing versus selection.

During B lymphocyte development, antibody genes are assembled by DNA recombination. Successful cell surface expression of IgM promotes developmental progression. However, when antigen receptors bind autoantigen, development is blocked and ongoing antibody gene recombination occurs, which often alters antibody specificity in a process called receptor editing. We demonstrate here a significant role of developmental block and receptor editing in B cell receptor quality control. During development a functional, non-self-reactive receptor undergoes receptor editing if its expression is below a certain threshold. Doubling the receptor gene dose promotes development in the absence of autoantigen, but allows editing when autoantigen is present. Thus, both underexpressed and harmful B cell receptors can undergo correction by receptor editing.

Animals↗