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At least 145 records · Page 8Linked to original sources
PubMed1995

In vivo testing of a tobacco plastid DNA segment for guide RNA function in psbL editing.

A C-to-U RNA editing event creates a functional initiation codon for translation of the psbL mRNA in tobacco plastids. Small trans-acting guide RNAs (gRNAs) have been shown to be involved in editing site selection in kinetoplastid mitochondria. A computer search of the tobacco plastid genome (ptDNA) identified such a putative gRNA, a 14-nucleotide sequence motif that is complementary to the psbL mRNA, including the A nucleotide required to direct the C-to-U change. The critical A nucleotide of the putative gRNA gene was changed to G by plastid transformation. We report here that the introduced mutation did not abolish psbL editing. Since no other region of the plastid genome contains significant complementarity to the psbL editing site we suggest that, if gRNAs serve as trans-acting factors for plastid psbL mRNA editing, they either have only a limited complementarity to the editing site, or are encoded in the nuclear genome.

Base Sequence↗
PubMed1990

RNA editing in transcripts of the mitochondrial genes of the insect trypanosome Crithidia fasciculata.

With the aid of cDNA and RNA sequence analysis, we have determined to what extent transcripts of mitochondrial maxicircle genes of the insect trypanosome Crithidia fasciculata are altered by RNA editing, a novel mechanism of gene expression which operates via the insertion and deletion of uridine residues. Editing of cytochrome c oxidase (cox) subunit II and III transcripts and of maxicircle unidentified reading frame (MURF) 2 RNA is limited to a small section and results in the creation of a potential AUG translational initiation codon (coxIII, MURF2) or the removal of a frameshift (coxII). No differences with the genomic sequences were observed in the remainder of these RNAs. Surprisingly, NADH dehydrogenase subunit I transcripts were completely unedited in the coding region, implying that an AUG translational initiation codon is absent. The partial ribosomal RNA sequences determined also conform to the gene sequences. Together these results lead to the conclusion that the unusual sequences predicted by the protein and rRNA genes must indeed be present in the gene products. Editing also occurred in the poly(A) tail of RNAs from all protein genes, including those that are unedited in the coding region. The tails display a large variation in AU sequence motifs. Finally, some cDNAs contained sequences absent from both the DNA and the edited RNA. Some of these may represent intermediates in the RNA editing process. We argue, however, that long runs of T may be artefacts of cDNA synthesis.

Animals↗
PubMed1995

Glutamate receptor RNA editing in vitro by enzymatic conversion of adenosine to inosine.

RNA encoding the B subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of ionotropic glutamate receptor (GluR-B) undergoes a posttranscriptional modification in which a genomically encoded adenosine is represented as a guanosine in the GluR-B complementary DNA. In vitro editing of GluR-B RNA transcripts with HeLa cell nuclear extracts was found to result from an activity that converts adenosine to inosine in regions of double-stranded RNA by enzymatic base modification. This activity is consistent with that of a double-stranded RNA-specific adenosine deaminase previously described in Xenopus oocytes and widely distributed in mammalian tissues.

Adenosine↗
PubMed1997

Evolution of dnaQ, the gene encoding the editing 3' to 5' exonuclease subunit of DNA polymerase III holoenzyme in Gram-negative bacteria.

The nucleotide sequences of the dnaQ genes from Salmonella typhimurium and Buchnera aphidicola, encoding the epsilon-subunit of the DNA polymerase III holoenzyme, have been determined. The Salmonella typhimurium dnaQ protein consists of 243 amino acid residues with a calculated molecular weight of 27224. The Buchnera aphidicola dnaQ protein contains 233 amino acid residues with a calculated molecular weight of 27170. A multiple sequence alignment of the amino acid sequences of the dnaQ proteins and those of DNA polymerase IIIs from Gram-positive bacteria produced six homologous segments. These homologous segments contain highly conserved amino acid sequence motifs involved in catalytically important metal ion bindings (ligands 1, 2 and 3). However, metal ligand 4 is found to be altered in the 3'-5' exonuclease domain of the family C DNA polymerases and dnaQ proteins in Gram-negative bacteria. From these results, we propose that the last common ancestor of the dnaQ gene of Gram-negative bacteria and the DNA polymerase III gene (pol C gene) of Gram-positive bacteria was a single gene containing both 3'-5' exonuclease and DNA polymerase domains and then the dnaQ gene separated from the polymerase gene in Gram-negative bacteria.

Amino Acid Sequence↗
PubMed1999

Left-handed Z-DNA: structure and function.

Z-DNA is a high energy conformer of B-DNA that forms in vivo during transcription as a result of torsional strain generated by a moving polymerase. An understanding of the biological role of Z-DNA has advanced with the discovery that the RNA editing enzyme double-stranded RNA adenosine deaminase type I (ADAR1) has motifs specific for the Z-DNA conformation. Editing by ADAR1 requires a double-stranded RNA substrate. In the cases known, the substrate is formed by folding an intron back onto the exon that is targeted for modification. The use of introns to direct processing of exons requires that editing occurs before splicing. Recognition of Z-DNA by ADAR1 may allow editing of nascent transcripts to be initiated immediately after transcription, ensuring that editing and splicing are performed in the correct sequence. Structural characterization of the Z-DNA binding domain indicates that it belongs to the winged helix-turn-helix class of proteins and is similar to the globular domain of histone-H5.

Adenosine Deaminase↗
PubMed1993

Selective import of nuclear-encoded tRNAs into mitochondria of the protozoan Leishmania tarentolae.

The trypanosomatid mitochondrial genome does not encode tRNA genes at all and experimental evidence obtained with Leishmania tarentolae shows that tRNAs in mitochondria represent a selected set of imported nuclear-encoded tRNAs. In this paper we present the data showing that tRNAs derived from the clustered genomic tRNA genes are invariably imported into mitochondria, while tRNA from the solitary gene is not. By sequencing a cosmid DNA clone of L. tarentolae genomic DNA, we have identified a 1.5-kb subclone encoding a duplicate set of the closely linked tRNA(Tyr) (GTA) and tRNA(Thr) (AGT) genes. Northern analysis shows that these tRNAs are imported into mitochondria. In contrast, when the tRNA gene [tRNA(Gln) (CUG)] located alone in a 40-kb DNA fragment was examined, the corresponding tRNA was not detected in the mitochondrion. This "loner" tRNA gene is highly unusual since the 3'-flanking putative RNA polymerase III transcription termination signal sequence is characterized by a long string of 8 Ts followed by an A and a stretch of 7 Cs, while all other trypanosomatid tRNA genes whose tRNA transcripts are imported are terminated by a possible transcription termination signal of only 4-6 Ts. Whether the correlation found between the gene organization and tRNA-import characteristics is of general significance needs to be investigated further. A simple computer analysis presented in this paper rules out the possibility that tRNAs found in the trypanosomatid mitochondrion are the products of the U-addition type 'RNA editing' of maxicircle DNA.

Animals↗
PubMed2003

RNA-modifying machines in archaea.

It has been known for nearly half a century that coding and non-coding RNAs (mRNA, and tRNAs and rRNAs respectively) play critical roles in the process of information transfer from DNA to protein. What is both surprising and exciting, are the discoveries in the last decade that cells, particularly eukaryotic cells, contain a plethora of non-coding RNAs and that these RNAs can either possess catalytic activity or can function as integral components of dynamic ribonucleoprotein machines. These machines appear to mediate diverse, complex and essential processes such as intron excision, RNA modification and editing, protein targeting, DNA packaging, etc. Archaea have been shown to possess RNP complexes; some of these are authentic homologues of the eukaryotic complexes that function as machines in the processing, modification and assembly of rRNA into ribosomal subunits. Deciphering how these RNA-containing machines function will require a dissection and analysis of the component parts, an understanding of how the parts fit together and an ability to reassemble the parts into complexes that can function in vitro. This article summarizes our current knowledge about small-non-coding RNAs in Archaea, their roles in ribosome biogenesis and their relationships to the complexes that have been identified in eukaryotic cells.

Amino Acid Sequence↗
PubMed2025

CRISPRoff epigenome editing for programmable gene silencing in human cell lines and primary T cells.

The advent of CRISPR-based technologies has enabled the rapid advancement of programmable gene manipulation in cells, tissues, and whole organisms. An emerging platform for targeted gene perturbation is epigenetic editing, the direct editing of chemical modifications on DNA and histones that ultimately results in repression or activation of the targeted gene. In contrast to CRISPR nucleases, epigenetic editors modulate gene expression without inducing DNA breaks or altering the genomic sequence of host cells. Recently, we developed the CRISPRoff epigenetic editing technology that simultaneously establishes DNA methylation and repressive histone modifications at targeted gene promoters. Transient expression of CRISPRoff and the accompanying single guide RNAs in mammalian cells results in transcriptional repression of targeted genes that is memorized heritably by cells through cell division and differentiation. Here, we describe our protocol for the delivery of CRISPRoff through plasmid DNA transfection, as well as the delivery of CRISPRoff mRNA, into transformed human cell lines and primary immune cells. We also provide guidance on evaluating target gene silencing and highlight key considerations when utilizing CRISPRoff for gene perturbations. Our protocols are broadly applicable to other CRISPR-based epigenetic editing technologies, as programmable genome manipulation tools continue to evolve rapidly.

Humans↗
PubMed2025

Rescue of imprinted genes by epigenome editing in human cellular models of Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a genomic imprinting disorder caused by the loss of function of the paternal chromosome 15q11-13, resulting in a spectrum of symptoms associated with hypothalamic dysfunction. PWS patients lack the expression of paternally expressed genes (PEGs) in the 15q11-13 locus but possess an epigenetically silenced set of these genes in the maternal allele. Thus, activation of these silenced genes can serve as a therapeutic target for PWS. Here, we leverage CRISPR-based epigenome editing system to modulate the DNA methylation status of the PWS imprinting control region (PWS-ICR) in induced pluripotent stem cells (iPSCs) derived from PWS patients. Successful demethylation in the PWS-ICR restores the PEG expression from the maternal allele and reorganizes the methylation patterns in other PWS-associated imprinted regions beyond the PWS-ICR. Remarkably, these corrected epigenomic patterns and PEG expression are maintained following the differentiation of these cells into hypothalamic organoids. Finally, the single-cell transcriptomic analysis of epigenome-edited organoids demonstrates a partial restoration of the transcriptomic dysregulation observed in PWS. This study highlights the utility of epigenome editing technology as a therapeutic approach in addressing PWS and potentially other imprinting disorders.

Prader-Willi Syndrome↗
PubMed2004

Distant structural homology leads to the functional characterization of an archaeal PIN domain as an exonuclease.

Genome sequencing projects have focused attention on the problem of discovering the functions of protein domains that are widely distributed throughout living species but which are, as yet, largely uncharacterized. One such example is the PIN domain, found in eukaryotes, bacteria, and Archaea, and with suggested roles in signaling, RNase editing, and/or nucleotide binding. The first reported crystal structure of a PIN domain (open reading frame PAE2754, derived from the crenarchaeon, Pyrobaculum aerophilum) has been determined to 2.5 A resolution and is presented here. Mapping conserved residues from a multiple sequence alignment onto the structure identifies a putative active site. The discovery of distant structural homology with several exonucleases, including T4 phage RNase H and flap endonuclease (FEN1), further suggests a likely function for PIN domains as Mg2+-dependent exonucleases, a hypothesis that we have confirmed in vitro. The tetrameric structure of PAE2754, with the active sites inside a tunnel, suggests a mechanism for selective cleavage of single-stranded overhangs or flap structures. These results indicate likely DNA or RNA editing roles for prokaryotic PIN domains, which are strikingly numerous in thermophiles, and in organisms such as Mycobacterium tuberculosis. They also support previous hypotheses that eukaryotic PIN domains participate in RNAi and nonsense-mediated RNA degradation.

Amino Acid Sequence↗
PubMed1991

Molecular cloning of a ribonuclease H (RNase HI) gene from an extreme thermophile Thermus thermophilus HB8: a thermostable RNase H can functionally replace the Escherichia coli enzyme in vivo.

A DNA fragment encoding Ribonuclease H (EC 3. 1.26.4) was isolated from an extreme thermophilic bacterium, Thermus thermophilus HB8, by its ability to complement the temperature-sensitive growth of an Escherichia coli rnhA deficient mutant. The primary amino acid sequence showed 56% similarity to that of E. coli RNase HI but little or no homology to E. coli RNase HII. Enzymes derived from thermophilic organisms tend to have fewer cysteines than their bacterial counterparts. However, T. thermophilus RNase H has one more cysteine than its E. coli homologue. Stability of the RNase H in extracts of T. thermophilus to elevated temperatures was the same for the protein expressed in E. coli. T. thermophilus RNase H should, therefore, be a useful tool for editing RNA-DNA hybrid molecules at higher temperatures and may also be stable enough to be used in a cyclical process. It was suggested that regulation of expression of the RNase H may be different from that of E. coli. RNase HI.

Amino Acid Sequence↗
PubMed1987

Specificity and efficiency of editing of mismatches involved in the formation of base-substitution mutations by the 3'----5' exonuclease activity of phage T4 DNA polymerase.

The specificity and efficiency of base-mispair editing by the 3'----5' exonuclease activity of phage T4 DNA polymerase has been measured using a sensitive infectivity assay. A series of oligodeoxynucleotide primer chains was synthesized chemically. These primers, when hybridized to phi X174 single-stranded DNAs containing an amber codon, result in a mispaired nucleotide at the 3'-hydroxyl end of the primer chain within the amber codon. DNA synthesis on these primer X templates without the removal of the mispaired terminal nucleotide results in the formation of heteroduplex molecules that yield viable revertants upon transfection into an amber nonsuppressor host. This method permits determination of the efficiency of editing of a mismatch to 1 in 10(6) mismatches that escape editing and allows all eight mispairs that can yield viable revertants at an amber codon to be studied. The results of experiments with primers hybridizing to phi X174 am16 and am3 codons show that the order of mispair editing by T4 DNA polymerase is Ttemplate X Gprimer less than (A X G, T X C) less than (T X T, G X A, G X G, A X C) less than A X A. The efficiency of editing depends upon the mispair, as well as the neighboring DNA sequence. Under these conditions of synthesis, the 3'----5' exonuclease activity, depending upon the mispair and DNA sequences beyond the nearest neighbors, is estimated to contribute a factor of from 2.3 X 10(3)- to greater than 10(6)-fold to the accuracy of T4 DNA polymerase.

Base Sequence↗
PubMed1996

A mutation in a Saccharomyces cerevisiae gene (RAD3) required for nucleotide excision repair and transcription increases the efficiency of mismatch correction.

RAD3 functions in DNA repair and transcription in Saccharomyces cerevisiae and particular rad3 alleles confer a mutator phenotype, possibly as a consequence of defective mismatch correction. We assessed the potential involvement of the Rad3 protein in mismatch correction by comparing heteroduplex repair in isogenic rad3-1 and wild-type strains. The rad3-1 allele increased the spontaneous mutation rate but did not prevent heteroduplex repair or bias its directionality. Instead, the efficiency of mismatch correction was enhanced in the rad3-1 strain. This surprising result prompted us to examine expression of yeast mismatch repair genes. We determined that MSH2, but not MLH1, is transcriptionally regulated during the cell-cycle like PMS1, and that rad3-1 does not increase the transcript levels for these genes in log phase cells. These observations suggest that the rad3-1 mutation gives rise to an enhanced efficiency of mismatch correction via a process that does not involve transcriptional regulation of mismatch repair. Interestingly, mismatch repair also was more efficient when error-editing by yeast DNA polymerase delta was eliminated. We discuss our results in relation to possible mechanisms that may link the rad3-1 mutation to mismatch correction efficiency.

Adenosine Triphosphatases↗
PubMed2001

Probing DNA polymerase fidelity mechanisms using time-resolved fluorescence anisotropy.

Prior to undergoing postsynthetic 3'-5' editing (proofreading), a defective DNA primer terminus must be transferred from the 5'-3' polymerase active site to a remote 3'-5' exonuclease site. To elucidate the mechanisms by which this occurs, we have used time-resolved fluorescence spectroscopy to study the interaction of dansyl-labeled DNA primer/templates with the Klenow fragment of Escherichia coli DNA polymerase I. The dansyl probe is positioned such that when the DNA substrate occupies the polymerase active site, the probe is solvent-exposed and possesses a short average fluorescence lifetime (4.7 ns) and extensive angular diffusion (42.5 degrees). Conversely, when the DNA substrate occupies the exonuclease active site, the probe becomes buried within the protein, resulting in an increase in the average lifetime (14.1 ns) and a decrease in the degree of angular diffusion (14.4 degrees ). If both polymerase and exonuclease binding modes are populated (lower limit approximately 5%), their markedly different fluorescence properties cause the anisotropy to decay with a characteristic "dip and rise" shape. Nonlinear least-squares analysis of these data recovers the ground-state mole fractions of exposed (x(e)) and buried (x(b)) probes, which are equivalent to the equilibrium proportions of the DNA substrate bound at the polymerase and exonuclease sites, respectively. The distribution between the polymerase and exonuclease binding modes is given by the equilibrium partitioning constant K(pe) (equal to x(b)/x(e)). The important determinants of the proofreading process can therefore be identified by changes made to either the protein or DNA that perturb the partitioning equilibrium and hence alter the magnitude of K(pe).

DNA Polymerase I↗
PubMed1993

DNA polymerase III accessory proteins. V. Theta encoded by holE.

The polIII core subassembly of DNA polymerase III holoenzyme is composed of the alpha (DNA polymerase), epsilon (editing exonuclease), and theta subunits. We have identified holE encoding theta (8.6 kDa) at 40.4 min, expressed and purified 300 mg of theta, and have studied its function by constituting the polIII core from pure alpha, epsilon, and theta subunits. The theta subunit binds the epsilon proofreader tightly, but it does not form a detectable complex with alpha. The epsilon subunit also binds to alpha (Maki, H., and Kornberg, A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 4389-4392). Hence, the subunit arrangement of the polIII core is linear, alpha epsilon theta. Interaction of theta with epsilon slightly stimulated epsilon in excision of a 3' terminal mismatched nucleotide, suggesting a possible role for theta in fidelity.

Amino Acid Sequence↗
PubMed1997

No editing of mitochondrial plasmid transcripts in mitochondria of Physarum that have an RNA-editing system.

Two types of RNA editing have been reported in the mitochondria of Physarum; extensive insertions of nucleotides and single-base substitutions. In the Ng strain of P. polycephalum and its derivatives, mitochondria have a specific plasmid (mF) that promotes fusion of mitochondria. We examined the editing of transcripts derived from the mF plasmid. For analysis, we selected three regions of the plasmid, including a DNA fragment that corresponded to missing conserved domains of the RNA polymerase. In contrast to the mitochondrial DNA (mtDNA), no RNA editing of the transcripts of the mF plasmid was detected. Our results suggest that the mechanism of transcription of the mitochondrial plasmid is independent of that of mtDNA, indicating that the plasmid has a different evolutionary origin from the mtDNA.

Animals↗
PubMed2005

Heterochromatin: on the ADAR radar?

Vigilin proteins, the absence of which is known to cause abnormalities in heterochromatin, have been found to bind edited RNAs. Molecular complexes including vigilin comprise proteins involved with RNA editing and with DNA repair, making connections between these processes and RNA-based silencing mechanisms.

Autoantigens↗
PubMed2026

Chemotactic sensing of extracellular antibiotic resistance genes enables their efficient removal by Stutzerimonas stutzeri.

The dissemination of antibiotic resistance genes (ARGs) in wastewater environments poses a severe threat to public health. Extracellular ARGs (eARGs) persist as free DNA fragments that are refractory to efficient removal by conventional physicochemical treatment technologies. Here, we isolated Stutzerimonas stutzeri CHY07 from municipal sewage and demonstrated that extracellular DNA fragments, including eARGs, can serve as chemoattractants for environmental bacteria. Through genomic mining, molecular docking, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and protein-ligand interaction profiling, we identified the chemoreceptor Mcp16 as the primary sensor of extracellular DNA and revealed that it achieves sequence-independent recognition of the DNA phosphate backbone. We further established the endogenous pentapeptide VRSVR as a methylation substrate for CheR and constructed the engineered strain CHY07-2 (mcp16::VRSVR) using an SSB/CRISPR-Cas9 ribonucleoprotein (RNP) system. This strain exhibited significantly enhanced chemotactic responsiveness, achieving 72-h removal efficiencies of 96.56% and 91.60% for low- and high-molecular-weight eARGs in non-sterile WWTP secondary effluent; conversely, mcp16 deletion markedly attenuated both chemotaxis and removal, whereas in situ complementation restored them. These findings reveal a "chemotaxis-contact-removal" cascade - with a proposed self-reinforcing loop - in eARG-removing bacteria, providing both a theoretical framework and a technical paradigm for enhancing pollutant removal through targeted amplification of microbial chemotaxis.

Chemotaxis↗