[Why do plants continue editing RNA?].
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Chloroplasts↗
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RNA editing has been lost in the mitochondrial cox3 and rps13 mRNAs in Asparagales.
RNA editing in plant mitochondria alters the RNA sequence by converting C-to-U or U-to-C at a specific site. We investigated the requirement for RNA editing in the complete genomic sequences of the gene encoding the cytochrome oxidase subunit III (cox3) and the ribosomal protein S13 (rps13) in 59 closely related species within the Asparagales and Liliales (monocots). To obtain a comprehensive picture of the degree of variation in editing we explored the non-synonymous RNA editing sites within the newly sequenced cox3 and rps13 genes by a comparative phylogenetic approach. RNA editing is predicted to occur in all the surveyed species, but to different extents. Zero to one non-synonymous editing site is inferred in the cox3 mRNA in species from Amaryllidaceae and Iridaceae. No RNA editing of rps13 mRNAs is required in Amaryllidaceae, because all respective genomic sequences resemble the edited version of the mRNAs of other analysed land plants. The observed absence of cox3 and rps13 RNA editing in Iridaceae and Amaryllidaceae and the striking RNA editing reduction of ccb2 in the latter family is likely generated by recombination and reverse transcription mediated events involving edited mitochondrial transcripts.
Amino Acid Sequence↗
Maxicircle CR1 transcripts of Trypanosoma brucei are edited and developmentally regulated and encode a putative iron-sulfur protein homologous to an NADH dehydrogenase subunit.
The maxicircle of Trypanosoma brucei encodes components of the mitochondrial oxidative phosphorylation system, as do other mitochondrial DNAs, but maxicircle gene identification is complicated by extensive editing of some transcripts. We found that transcripts from the CR1 region were extensively edited, as are other transcripts from maxicircle regions which exhibit strong G versus C strand bias. Editing added 259 uridines and removed 46 uridines to produce an approximately 574-nucleotide mature mRNA. Partially edited cDNAs and potential guide RNAs were also characterized. Initiation and termination codons were created, and they defined an open reading frame encoding a predicted protein of 145 amino acids. This protein contains two iron-sulfur cysteine motifs and is homologous to a subunit of NADH dehydrogenase and to other electron-carrier proteins. Higher levels of both edited and unedited CR1 transcripts accumulated in bloodstream forms of the parasite than in procyclic forms, suggesting developmental regulation of CR1 gene expression.
Amino Acid Sequence↗
Cotranscriptional editing of Physarum mitochondrial RNA requires local features of the native template.
RNAs in the mitochondrion of Physarum polycephalum are edited by the precise cotranscriptional addition of non-encoded nucleotides. Here we describe experiments to address the basis of editing specificity using a series of chimeric templates generated by either rearranging the DNA present in editing-competent mitochondrial transcription elongation complexes (mtTECs) or linking it to exogenous DNA. Notably, run-on transcripts synthesized from rearranged mtTECs are edited at the natural sites, even when different genes are ligated together, yet exogenous, deproteinized DNA does not support editing. Furthermore, the accuracy of nucleotide insertion in chimeric RNAs argues that any cis-acting determinants of cytidine insertion are limited to small regions surrounding editing sites. Taken together, these observations strongly suggest that template-associated factors affect read-out of the mitochondrial genome.
Animals↗
Global effect of PEG-IFN-alpha and ribavirin on gene expression in PBMC in vitro.
Using oligonucleotide microarrays, we have examined the expression of 22,000 genes in peripheral blood cells treated with pegylated interferon-alpha2b (PEG-IFN-alpha) and ribavirin. Treatment with ribavirin had very little effect on gene expression, whereas treatment with PEG-IFN-alpha had a dramatic effect, modulating the expression of approximately 1000 genes (at p < 0.001). In addition to genes previously reported to be induced by type I or type II IFNs, many novel genes were found to be upregulated, including transcription factors, such as ATF3, ATF4, properdin, a key regulator of the complement pathway, a homeobox gene (HESX1), and an RNA editing enzyme (apobec3). Chemokines CXCL10 and CXCL11 were upregulated, whereas CXCL5 was downregulated. Cytokines interleukin-15 (IL-15) and IL-18 were also significantly induced, whereas IL-1alpha and IL-1beta were downregulated. Most other interleukins were not affected. The results of the microarrays were confirmed by kinetic real-time PCR. These data indicate that IFN treatment causes upregulation of genes associated with the stress response, apoptosis, and signaling, and an equal number of genes are downregulated, including those associated with protein synthesis, specific cytokines and chemokines and other biosynthetic functions.
Cells, Cultured↗
A third member of the RNA-specific adenosine deaminase gene family, ADAR3, contains both single- and double-stranded RNA binding domains.
Members of the double-stranded RNA- (dsRNA) specific adenosine deaminase gene family convert adenosine residues into inosines in dsRNA and are involved in A-to-I RNA editing of transcripts of glutamate receptor (GluR) subunits and serotonin receptor subtype 2C (5-HT(2C)R). We have isolated hADAR3, the third member of this class of human enzyme and investigated its editing site selectivity using in vitro RNA editing assay systems. As originally reported for rat ADAR3 or RED2, purified ADAR3 proteins could not edit GluR-B RNA at the "Q/R" site, the "R/G" site, and the intronic "hot spot" site. In addition, ADAR3 did not edit any of five sites discovered recently within the intracellular loop II region of 5-HT(2C)R RNAs, confirming its total lack of editing activity for currently known substrate RNAs. Filter-binding analyses revealed that ADAR3 is capable of binding not only to dsRNA but also to single-stranded RNA (ssRNA). Deletion mutagenesis identified a region rich in arginine residues located in the N-terminus that is responsible for binding of ADAR3 to ssRNA. The presence of this ssRNA-binding domain as well as its expression in restricted brain regions and postmitotic neurons make ADAR3 distinct from the other two ADAR gene family members, editing competent ADAR1 and ADAR2. ADAR3 inhibited in vitro the activities of RNA editing enzymes of the ADAR gene family, raising the possibility of a regulatory role in RNA editing.
Adenosine Deaminase↗
Adenovirus VAI RNA antagonizes the RNA-editing activity of the ADAR adenosine deaminase.
The virus-associated VAI RNA of adenovirus is a small highly structured RNA that is required for the efficient translation of cellular and viral mRNAs at late times after infection. VAI RNA antagonizes the activation of the interferon-inducible RNA-dependent protein kinase, PKR, an important regulator of translation. The RNA-specific adenosine deaminase, ADAR, is an interferon-inducible RNA-editing enzyme that catalyzes the site-selective C-6 deamination of adenosine to inosine. ADAR possesses three copies of the highly conserved RNA-binding motif (dsRBM) that are similar to the two copies found in PKR, the enzyme in which the prototype dsRBM motif was discovered. We have examined the effect of VAI RNA on ADAR function. VAI RNA impairs the activity of ADAR deaminase. This inhibition can be observed in extracts prepared from interferon-treated human cells and from monkey COS cells in which wild-type recombinant ADAR was expressed. Analysis of wild-type and mutant forms of VA RNA suggests that the central domain is important in the antagonism of ADAR activity. These results suggest that VAI RNA may modulate viral and cellular gene expression by modulating RNA editing as well as mRNA translation.
Adenosine Deaminase↗
A CRISPR-Cas9 Toolkit Enabling Tunable Integration and Transient Homologous Recombination Enhancement in Yarrowia lipolytica.
Although the oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory, its application remains constrained by inefficient homology-directed repair (HDR) and a lack of precise genomic integration tools. To address these limitations, we developed a comprehensive genetic toolkit featuring three synergistic advancements. First, we systematically identified 55 neutral integration sites with tunable expression profiles, enabling stable, position-independent gene integration with predictable transcriptional output across a 12.88-fold dynamic range. Second, we established a dual-readout high-throughput screening platform combining colony morphology analysis with hrGFP fluorescence. This approach accurately measures locus-specific homologous recombination (HR) efficiency while eliminating false positives by dominant non-homologous end joining (NHEJ). Third, we engineered a transient HR enhancement system by fusing the Sae2 exonuclease to Cas9 via a flexible (GGGGS)3 linker. This fusion significantly boosts HR efficiency and surpasses the cleavage activity of unmodified Cas9 without introducing permanent genomic modifications or compromising cellular fitness. Finally, HR efficiency for single-gene integration was increased from 46.5% to 77.5% while the dual-locus editing efficiency reached 64.1% when using 500-bp homology arms, and the engineered strains demonstrated improved genetic stability compared to those with constitutive HR enhancement.
Yarrowia↗
New retron systems from environmental bacteria identify triggers of anti-phage defense and expand tools for genome editing.
Retrons are bacterial immune systems that protect a bacterial population against phages by killing infected hosts. Retrons typically comprise a reverse transcriptase (RT), a template noncoding RNA that is partially reverse transcribed into RT-DNA, and a toxic effector. The reverse transcriptase (RT), noncoding RNA, and RT-DNA complex sequester the toxic effector until triggered by phage infection, at which point the toxin is released to induce cell death. Due to their ability to produce single-stranded DNA in vivo, retrons have also been engineered to produce donor templates for genome editing in both prokaryotes and eukaryotes. However, the current repertoire of experimentally characterized retrons is limited, with most retrons sourced from clinical and laboratory strains of bacteria. To better understand retron biology and natural diversity, and to expand the current toolbox of retron-based genome editors, we developed a pipeline to isolate retrons and their bacterial hosts from a variety of environmental samples. Here, we identify seven new retron systems, each isolated from a different host bacterium. We characterize DNA production by these retrons and test their ability to defend against a panel of Escherichia coli phages. We find that two of these retrons are disrupted by other elements, in one case a group II intron and in another a separate defense system, yet both retrons still produce RT-DNA. For two other retrons, we further unravel their mechanism of defense by identifying the phage genes responsible for triggering abortive infection. Finally, we engineer these retrons for genome editing in E. coli, demonstrating their potential use in a biotechnological application.
Gene Editing↗
Editing and translation of ribosomal protein S13 transcripts: unedited translation products are not detectable in maize mitochondria.
Maize mitochondrial transcripts for the ribosomal protein S13 gene (rps13) have six C- to -U editing sites, and each nucleotide conversion causes a change in the amino acid specified by the effected codons. Sequence analysis of 30 cDNA clones indicated that 73% of the cDNAS were edited at all six sites and 3% were completely unedited. Antibodies were produced against synthetic peptides that corresponded to unedited or edited translation products at editing sites V and VI (80% and 83% edited, respectively). Antibody preparations were purified that selectively recognized the edited or unedited forms of the epitope. The antibody preparations were highly sensitive to the amino-acid residue encoded at editing site VI, but relatively insensitive to the residue encoded at editing site V. Immunological analyses demonstrated that the edited translation product accumulated as a ribosomal protein, but that the unedited translation product was not detected in the mitochondrion, in the ribosomal fraction, or in a post-ribosomal supernatant. These results, taken together with other studies which demonstrated that incompletely edited transcripts are incorporated into polyribosomes, suggest that incompletely edited transcripts may be translated, but polypeptides encoded by incompletely edited RNAs may be unstable and, consequently, fail to accumulate.
Amino Acid Sequence↗
The single-stranded DNA-binding protein of Deinococcus radiodurans.
BACKGROUND: Deinococcus radiodurans R1 is one of the most radiation-resistant organisms known and is able to repair an unusually large amount of DNA damage without induced mutation. Single-stranded DNA-binding (SSB) protein is an essential protein in all organisms and is involved in DNA replication, recombination and repair. The published genomic sequence from Deinococcus radiodurans includes a putative single-stranded DNA-binding protein gene (ssb; DR0100) requiring a translational frameshift for synthesis of a complete SSB protein. The apparently tripartite gene has inspired considerable speculation in the literature about potentially novel frameshifting or RNA editing mechanisms. Immediately upstream of the ssb gene is another gene (DR0099) given an ssb-like annotation, but left unexplored. RESULTS: A segment of the Deinococcus radiodurans strain R1 genome encompassing the ssb gene has been re-sequenced, and two errors involving omitted guanine nucleotides have been documented. The corrected sequence incorporates both of the open reading frames designated DR0099 and DR0100 into one contiguous ssb open reading frame (ORF). The corrected gene requires no translational frameshifts and contains two predicted oligonucleotide/oligosaccharide-binding (OB) folds. The protein has been purified and its sequence is closely related to the Thermus thermophilus and Thermus aquaticus SSB proteins. Like the Thermus SSB proteins, the SSBDr functions as a homodimer. The Deinococcus radiodurans SSB homodimer stimulates Deinococcus radiodurans RecA protein and Escherichia coli RecA protein-promoted DNA three-strand exchange reactions with at least the same efficiency as the Escherichia coli SSB homotetramer. CONCLUSIONS: The correct Deinococcus radiodurans ssb gene is a contiguous open reading frame that codes for the largest bacterial SSB monomer identified to date. The Deinococcus radiodurans SSB protein includes two OB folds per monomer and functions as a homodimer. The Deinococcus radiodurans SSB protein efficiently stimulates Deinococcus radiodurans RecA and also Escherichia coli RecA protein-promoted DNA strand exchange reactions. The identification and purification of Deinococcus radiodurans SSB protein not only allows for greater understanding of the SSB protein family but provides an essential yet previously missing player in the current efforts to understand the extraordinary DNA repair capacity of Deinococcus radiodurans.
DNA Repair↗
Complete nucleotide sequence of the chloroplast genome from a leptosporangiate fern, Adiantum capillus-veneris L.
We determined the complete nucleotide sequence of the chloroplast genome of the leptosporangiate fern, Adiantum capillus-veneris L. (Pteridaceae). The circular genome is 150,568 bp, with a large single-copy region (LSC) of 82,282 bp, a small-single copy region (SSC) of 21,392 bp and inverted repeats (IR) of 23,447 bp each. We compared the sequence to other published chloroplast genomes to infer the location of putative genes. When the IR is considered only once, we assigned 118 genes, of which 85 encode proteins, 29 encode tRNAs and 4 encode rRNAs. Four protein-coding genes, all four rRNA genes and six tRNA genes occur in the IR. Most (57) putative protein-coding genes appear to start with an ATG codon, but we also detected five other possible start codons, some of which suggest tRNA editing. We also found 26 apparent stop codons in 18 putative genes, also suggestive of RNA editing. We found all but one of the tRNA genes necessary to encode the complete repertoire required for translation. The missing trnK gene appears to have been disrupted by a large inversion, relative to other published chloroplast genomes. We detected several structural rearrangements that may provide useful information for phylogenetic studies.
Base Sequence↗
The formation of mitochondrial ribonucleoprotein complexes involving guide RNA molecules in Trypanosoma brucei.
Transcripts from mitochondrial (maxicircle) genes of kinetoplastid organisms undergo RNA editing characterized by a series of reactions that insert and delete uridine nucleotides within the sequence of the pre-mRNAs. Guide RNAs, which complement fully edited mRNAs, provide the information for the edited sequence by an unknown mechanism. We report here that guide RNA molecules associate with other mitochondrial components to form four specific, stable ribonucleoprotein complexes. The complexes form very rapidly at a low monovalent cation concentration, and their formation is blocked by heparin or pretreatment of the mitochondrial lysate with SDS. ATP hydrolysis is not required but slightly stimulates complex association up to concentrations of 5 mM. The results are suggestive of a sequential assembly of the ribonucleoprotein complexes, and their possible involvement during the kinetoplastid RNA editing is discussed.
Amino Acid Sequence↗
TALEs, TALENs, and TALE Base Editors: From Plant Pathology to Biotechnology.
TALEs (transcription activator-like effectors) are an excellent example of how studying pathogen-host interactions can lead to significant biotechnology inventions. TALEs are bacterial effectors that are translocated into plant cells via a bacterial type III secretion system. Once inside the host cell, they are imported into the nucleus to bind specific promoters and induce expression of target genes, thereby supporting the bacterial infection. TALEs are found throughout many, but not all, Xanthomonas pathovars, which can be severe pathogens of different crops. The key feature of TALEs is their modular DNA-binding domain, which allows a simple evolutionary adaptation to novel DNA sequences as well as simple cloning of designer TALEs with desired DNA-binding specificity. Accordingly, TALE nucleases started the genome-editing revolution, and TALE base editors are the latest tools to efficiently edit chloroplast and mitochondrial genomes. We review recent advances in Xanthomonas genomics, synthesize current knowledge about naturally occurring TALEs, and highlight current roles of TALEs in genome editing and synthetic biology.
Xanthomonas↗
Neurological dysfunctions in mice expressing different levels of the Q/R site-unedited AMPAR subunit GluR-B.
We generated mouse mutants with targeted AMPA receptor (AMPAR) GluR-B subunit alleles, functionally expressed at different levels and deficient in Q/R-site editing. All mutant lines had increased AMPAR calcium permeabilities in pyramidal neurons, and one showed elevated macroscopic conductances of these channels. The AMPAR-mediated calcium influx induced NMDA-receptor-independent long-term potentiation (LTP) in hippocampal pyramidal cell connections. Calcium-triggered neuronal death was not observed, but mutants had mild to severe neurological dysfunctions, including epilepsy and deficits in dendritic architecture. The seizure-prone phenotype correlated with an increase in the macroscopic conductance, as independently revealed by the effect of a transgene for a Q/R-site-altered GluR-B subunit. Thus, changes in GluR-B gene expression and Q/R site editing can affect critical architectural and functional aspects of excitatory principal neurons.
Alleles↗
Prime Editing for p47phox-Deficient Chronic Granulomatous Disease.
Chronic granulomatous disease (CGD) is a severe monogenic immunodeficiency caused by damaging variants in genes required for microbicidal NADPH oxidase activity. Autosomal recessive p47phox-deficient CGD (p47-CGD) is predominantly caused by a two-nucleotide deletion in exon 2 (delGT) of NCF1. We developed PM359, an autologous CD34+ hematopoietic stem-cell therapy in which prime editing is used to correct delGT. Two participants received PM359 after myeloid conditioning with busulfan: neutrophils and platelets engrafted promptly in both patients. Adverse events were consistent with myeloid conditioning with busulfan. NADPH oxidase activity was observed in neutrophils within 1 month and was maintained for 6 months and 4 months as of the last follow-up visit in Participants 1 and 2, respectively. These results support further investigation of prime editing of CD34+ cells to treat p47-CGD. (Funded by Prime Medicine; ClinicalTrials.gov number, NCT06559176.).
Adolescent↗
B cell receptor editing in tolerance and autoimmunity.
The random assembly of immunoglobulin (Ig) genes often creates a B cell receptor that is self-reactive, and such cells are subjected to negative selection. A primary mechanism to extinguish this self-reactivity is receptor editing, which allows continued recombination of Ig genes and replacement of the self-reactive receptor with a new innocuous receptor. Recent data now suggest that receptor editing may also promote autoimmunity in an autoimmune context. This mechanism has also been implicated in the process of B cell positive selection and maturation. Here we discuss the contribution of receptor editing in B-lymphopoiesis and its importance for B cell tolerance and autoimmunity.
Autoimmunity↗
Lipid disorders and mutations in the APOB gene.
BACKGROUND: Plasma lipoproteins are important determinants of atherosclerosis. Apolipoprotein (apo) B is a large, amphipathic glycoprotein that plays a central role in human lipoprotein metabolism. Two forms of apoB are produced from the APOB gene by a unique posttranscriptional editing process: apoB-48, which is required for chylomicron production in the small intestine, and apoB-100, required for VLDL production in the liver. In addition to being the essential structural component of VLDL, apoB-100 is the ligand for LDL-receptor-mediated endocytosis of LDL particles. CONTENT: The study of monogenic dyslipidemias has revealed important aspects of metabolic pathways. In this review, we discuss the regulation of apoB metabolism and examine how APOB gene defects can lead to both hypo- and hypercholesterolemia. The key clinical, metabolic, and genetic features of familial hypobetalipoproteinemia and familial ligand-defective apoB-100 are described. SUMMARY: Missense mutations in the LDL-receptor-binding domain of apoB cause familial ligand-defective apoB-100, characterized by hypercholesterolemia and premature coronary artery disease. Other mutations in APOB can cause familial hypobetalipoproteinemia, characterized by hypocholesterolemia and resistance to atherosclerosis. These naturally occurring mutations reveal key domains in apoB and demonstrate how monogenic dyslipidemias can provide insight into biologically important mechanisms.
Apolipoproteins B↗