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PubMed1994

Mitochondrial RNA editing of cytochrome c oxidase subunit II (coxII) in the primitive vascular plant Psilotum nudum.

A 634-nucleotide tract, including primers, was amplified via the polymerase chain reaction within a mitochondrial coxII gene of the primitive vascular plant Psilotum nudum and sequenced. Alignment with homologous coxII gene sequences from diverse plant species having known RNA editing sites which restore amino acid sequence consensus was used to infer eight sites of C-to-U transitions in Psilotum. In every case, the predicted editing event would confer the selective advantage of conserving the amino acid residue at a site where amino acid sequence divergence has not been observed in other plant species. The plant mitochondrial editing machinery is shown for the first time to extend to one of the deepest branches of vascular plant phylogeny.

Amino Acid Sequence↗
PubMed1999

Disproportionate relationship between APOBEC-1 expression and apolipoprotein B mRNA editing activity.

Apolipoprotein B (apoB) mRNA editing is a site-specific (nucleotide 6666) cytidine to uridine transition catalyzed by a cytidine deaminase, APOBEC-1, in the context of a multiprotein complex referred to as the C/U editosome. This report quantifies for the first time the effect of altering APOBEC-1 protein abundance on the proportion of edited apoB mRNAs using transfected McArdle rat hepatoma cells which had been sorted by flow cytometry into populations expressing different levels of green fluorescent protein-APOBEC-1 chimera, GFP-APOBEC. A correlation was observed in which increased expression of GFP-APOBEC protein resulted in a higher proportion of edited apoB mRNA. The number of enzyme molecules required to increase the proportion of edited apoB RNAs was disproportionately high relative to that which might have been predicted from a typical catalytic relationship. Moreover, editing of apoB mRNA at inappropriate sites (promiscuous editing) occurred in response to overexpressing GFP-APOBEC. The data suggest that experimental manipulation of APOBEC-1 abundance in the absence of other regulatory considerations will always result in some level of promiscuous editing. Coordinate expression of APOBEC-1 and the auxiliary proteins and/or regulation of their interactions may be required to increase editing activity without losing editing-site fidelity.

APOBEC-1 Deaminase↗
PubMed1994

Differential RNA editing efficiency of AMPA receptor subunit GluR-2 in human brain.

RNA editing in rat brain has been found to control a determinant of cation flow in alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid (AMPA)-gated channels. Here we provide the first evidence that this RNA editing phenomenon occurs in human brain and is differentially regulated. Sequence analysis of human genomic DNA revealed a Q codon (CAG) in the putative channel-forming segment of human GluR-2, whereas in the majority of cDNA clones an R codon (CGG) was found. Examination of editing in various brain tissues revealed differences in the efficiency of this process. The hippocampus, cerebellum and temporal cortex harbour 100% edited GluR-2, whereas only 72% of substantia nigra, 89% of corpus striatum and 96% of fetal cDNAs have been found to be edited. This new discovery of differential efficiency of RNA editing has important implications in AMPA receptor channel-mediated calcium influx. AMPA receptors are thought to mediate the majority of the fast excitatory synaptic neurotransmission; the RNA editing process may therefore play a critical role in normal brain function and development. Dysfunction of this RNA editing process may have neuropathological consequences and could be related to certain neurodegenerative diseases.

Animals↗
PubMed2025

Strain-Promoted mRNA Transdermal Delivery by Lipoic Lipid Nanoparticles for Therapeutic Skin Genome Editing.

Lipid nanoparticle (LNP)-mRNA formulations have revolutionized the field of nucleic acid therapeutics, yet their broader clinical application is constrained by inflammatory side effects and oxidative stress, particularly in the context of inflammatory diseases. Herein, we report the rational design and synthesis of a lipoic acid-based ionizable lipid library to address these limitations. By leveraging the antioxidant properties and thiol-mediated uptake potential of lipoic acid, we identified LA-A2B2CD3 as an optimal candidate through a structure-activity relationship study and design of experiment (DOE) optimization. LA-A2B2CD3 LNPs exhibited superior reactive oxygen species scavenging, enhanced mRNA translation, and reduced inflammatory cytokine production in vitro and in vivo. Mechanistic studies revealed that the efficient cellular uptake and the transdermal delivery capacity of LA-A2B2CD3 heavily rely on the reducible disulfide ring of lipoic acid. Application of LA-A2B2CD3 LNPs for the localized transdermal delivery of Cas9 mRNA and CD93 sgRNA in a murine model of psoriasis resulted in effective CD93 genome editing and the inhibition of the CD93-p38 MAPK-AKT-SMAD2/3 pathway, leading to significant therapeutic improvement. This work presents a robust, biocompatible LNP platform with minimized immunogenicity and strong potential for genome-editing therapies in inflammatory conditions, offering a transformative approach for the mRNA-based treatment of skin and other inflammation-related disorders.

Animals↗
PubMed2000

In vitro analysis of the binding of ADAR2 to the pre-mRNA encoding the GluR-B R/G site.

The ADAR family of RNA-editing enzymes deaminates adenosines within RNA that is completely or largely double stranded. In mammals, most of the characterized substrates encode receptors involved in neurotransmission, and these substrates are thought to be targeted by the mammalian enzymes ADAR1 and ADAR2. Although some ADAR substrates are deaminated very promiscuously, mammalian glutamate receptor B (gluR-B) pre-mRNA is deaminated at a few specific adenosines. Like most double-stranded RNA (dsRNA) binding proteins, ADARs bind to many different sequences, but few studies have directly measured and compared binding affinities. We have attempted to determine if ADAR deamination specificity occurs because the enzymes bind to targeted regions with higher affinities. To explore this question we studied binding of rat ADAR2 to a region of rat gluR-B pre-mRNA that contains the R/G editing site, and compared a wild-type molecule with one containing mutations that decreased R/G site editing. Although binding affinity to the two sequences was almost identical, footprinting studies indicate ADAR2 binds to the wild-type RNA at a discrete region surrounding the editing site, whereas binding to the mutant appeared nonspecific.

Adenosine Deaminase↗
PubMed1994

Preferential selection of adenosines for modification by double-stranded RNA adenosine deaminase.

Double-stranded RNA adenosine deaminase (dsRAD), previously called the double-stranded RNA (dsRNA) unwinding/modifying activity, modifies adenosines to inosines within dsRNA. We used ribonuclease U2 and a mutant of ribonuclease T1 to map the sites of modification in several RNA duplexes. We found that dsRAD had a 5' neighbor preference (A = U > C > G) but no apparent 3' neighbor preference. Further, the proximity of the strand termini affected whether an adenosine was modified. Most importantly, dsRAD exhibited selectivity, modifying a minimal number of adenosines in short dsRNAs. Our results suggest that the specific editing of glutamate receptor subunit B mRNA could be performed in vivo by dsRAD without the aid of specificity factors, and support the hypothesis that dsRAD is responsible for hypermutations in certain RNA viruses.

Adenosine↗
PubMed1999

Confirmation of predicted edits and demonstration of unpredicted edits in Acanthamoeba castellanii mitochondrial tRNAs.

In the amoeboid protozoon Acanthamoeba castellanii 13 of the 16 mtDNA-encoded tRNA sequences have mis-matches at one or more of the first three positions in the acceptor stem. A previous study had indicated that these mis-matches are corrected by a form of RNA editing. In the present study, the pattern of editing was further investigated by sequence analysis of both halves of the acceptor stem of 11 mtDNA-encoded tRNAs. The results confirm all of the remaining editing sites predicted on the basis of the secondary structure modelling of A. castellanii mitochondrial tRNAs, and identify two unexpected edits. We also investigated the expression and editing of transcripts of an unusual trnX gene specifying an eight-nucleotide anticodon loop sequence. Although no mature 3'-CCAOH-containing tRNAX products were detected, editing was observed in some circularized tRNAX clones. The implications of these results with respect to the mechanism of editing and the evolutionary origin of this process are discussed.

Acanthamoeba↗
PubMed1996

Hyperediting of multiple cytidines of apolipoprotein B mRNA by APOBEC-1 requires auxiliary protein(s) but not a mooring sequence motif.

An RNA-binding cytidine deaminase (APOBEC-1) and unidentified auxiliary protein(s) are required for apolipoprotein (apo) B mRNA editing. A sequence motif on apoB mRNA ("mooring sequence," nucleotides 6671-6681) is obligatory for the editing of cytidine 6666 (C6666), the only cytidine on apoB mRNA converted to uridine in normal animals. Transgenic animals with hepatic overexpression of APOBEC-1 develop liver tumors, and other non-apoB mRNAs are edited, suggesting a loss of the normally precise specificity. In this study, we examined apoB mRNA from these transgenic animals to determine if cytidines aside from C6666 are edited. Multiple cytidines downstream from C6666 in apoB mRNA were edited extensively by the overexpressed APOBEC-1. This pathophysiological "hyperediting" could be mimicked in vitro by incubating a synthetic apoB RNA substrate with the transgenic mouse liver extracts. Multiple cytidines in the synthetic apoB RNA were edited by recombinant APOBEC-1 but only with supplementation of the auxiliary protein(s). Mutations in the mooring sequence markedly decreased the normal editing of C6666 but, surprisingly, increased the hyperediting of downstream cytidines. Furthermore, cytidines in an apoB RNA substrate lacking the mooring sequence were also edited in vitro. These results indicate that the hyperediting of apoB mRNA by overexpressed APOBEC-1 depends upon auxiliary protein(s) but is independent of the mooring sequence motif. These results suggest that hyperediting may represent the first step in a two-step recognition model for normal apoB mRNA editing.

APOBEC-1 Deaminase↗
PubMed1999

Synthesis and utilization of 13C(8)-enriched purines.

A new and more efficient method is presented for the synthesis of 13C(8)-enriched adenine. In addition, we present for the first time the synthesis of 13C(8)-enriched 2-aminopurine and purine. All three analogues have been converted to the corresponding ribonucleosides. The adenine analogue has been further converted to the 2'-deoxy-nucleoside and incorporated into a synthetic oligonucleotide. Data is presented demonstrating the utility of 13C-enrichment in heteronuclear isotope-edited NMR spectra.

Base Sequence↗
PubMed1993

The 5'-terminal region of the apocytochrome b transcript in Crithidia fasciculata is successively edited by two guide RNAs in the 3' to 5' direction.

We analyzed the chimeric guide RNA (gRNA)-mRNA molecules in Crithidia fasciculata that are predicted to transiently exist in editing of the 5'-terminal domain of apocytochrome b (CYb) mRNA, by polymerase chain reaction amplification and DNA sequencing, and obtained evidence suggesting that among the 14 editing sites numbered from 3' to 5', the sequence in the 3'-half of the sites (3' block) was specified by one guide RNA species (gRNA-I) and that in the remaining half of the sites (5' block) by the other guide RNA species (gRNA-II) and that the direction of editing in each block was 3' to 5'. The predicted transition site of editing by two gRNAs was between the first and second U residues from the 3' end within editing site 7. We found that a stretch of the edited sequence in the 3' block of mRNA could form a stable duplex with a stretch immediately upstream of the guide sequence in gRNA-II. The result leads to a successive editing model that the 3' block of pre-edited mRNA is first edited by gRNA-I, and after completion of editing, the 5' portion of gRNA-II pairs with the edited mRNA for editing of the 5' block.

Animals↗
PubMed2001

Atlas-assisted localization analysis of functional images.

OBJECTIVE: This paper introduces a method for localization analysis of functional images assisted by a brain atlas. The usefulness of the system developed, based on this method, is analyzed for human brain mapping and neuroradiology. MATERIALS AND METHODS: We use an enhanced and extended electronic Talairach-Tournoux brain atlas, containing segmented and labeled subcortical structures, Brodmann's areas, and gyri. The brain atlas serves as a tool for anatomy referencing, segmentation, labeling, registration, and providing 3D anatomical relationships. The process of localization analysis is decomposed into five steps: data loading, feature extraction, data normalization, identification and editing of loci, and getting labels and values. This analysis is supported by near real-time data-to-atlas warping based on the Talairach transformation. Metanalysis is enabled by merging the current and external lists of activation loci. RESULTS: We have designed, developed, tested, and deployed a commercial system for atlas-assisted localization analysis of functional images. This is the first system where an electronic version of the Talairach-Tournoux brain atlas is used interactively for analysis of functional images. This system runs on personal computers and provides functions for a rapid normalization of anatomical and functional volumetric data, data segmentation and labeling, readout of Talairach coordinates, and data display. It also is empowered with several unique features including: interactive warping facilitating fine tuning of the data-to-atlas fit, a backtracking mechanism to compensate for missing landmarks and enhancing the outcome of the overall process of data analysis, navigation on the triplanar formed by the data and the atlas, multiple-images-in-one display with atlas-anatomy-function blending, a fast locus-controlled generation of results, editing of loci, multiple label display, and saving and reading of loci. The system normalizes a single image in near real-time (0.7 s), so analysis of anatomical and functional datasets can be done on-the-fly regardless of the number of slices. The same task performed by the state-of-the-art non-linear registration methods may require up to several days. CONCLUSIONS: The system is a useful tool for atlas-assisted localization analysis and a helpful adjunct to function/location metanalysis in human brain mapping research. It is also a step forward in bringing the atlas and the clinical data together within a practical and powerful solution that is fast and flexible, yet low-cost and affordable.

Anatomy, Artistic↗
PubMed1994

Complete RNA editing of unspliced and dicistronic transcripts of the intron-containing reading frame IRF170 from maize chloroplasts.

The maize plastome harbors within the rps4-rps14 gene cluster the reading frame IRF170, which is interrupted by two introns. Although the function of the encoded peptide of 170 amino acids is not known, the conservation of IRF170 homologs in other plastomes is a strong indication that IRF170 is a functional gene. Amplification and sequence analyses of IRF170 specific cDNAs reveals two C-to-U editing events occurring within each of the first two exons. This situation allows an analysis of the temporal order between editing and splicing of a chloroplast transcript. By using intron-specific primer combinations, cDNAs derived from partially or even unspliced IRF170 transcripts could be amplified which in all cases showed complete editing. Complete editing was also observed with a cDNA derived from a transcript in which the proximal rps4 and the 5' half of IRF170-encoded sequences were still linked. This demonstrates that editing of the IRF170 transcript is an early processing step preceding both splicing and cleavage to monocistronic mRNA.

Amino Acid Sequence↗
PubMed1996

Copper deficiency increases hepatic apolipoprotein B secretion and mRNA editing in rats.

Male weanling Sprague-Dawley rats were assigned to copper-deficient (9.0 mumol Cu/kg diet) or copper-adequate (102 mumol Cu/kg diet) dietary treatments for 6 wk. Pulse-chase studies using freshly isolated rat liver parenchymal cells demonstrated that apolipoprotein B (apoB)-48 and apoB-100 syntheses were not altered, but secretion was increased twofold in hepatocytes derived from copper-deficient rats. Both plasma apoB-48 and apoB-100 levels were increased by copper deficiency, but only the apoB-48 increase was significant. Hepatic apoB mRNA editing, expressed as a ratio of apoB-48 mRNA to apoB-48 plus apoB-100 mRNA, was significantly increased from 60.8% in copper-adequate to 70.2% in copper-deficient rats. Moreover, hepatic apoB mRNA abundance was not significantly altered by copper deficiency. Thus the increased amount of nascent apoB-48 secreted into the medium as well as the enhanced apoB mRNA editing may have contributed to the differential increase in plasma apoB-48 over apoB-100 level in copper-deficient rats.

Animals↗
PubMed1996

Two nucleotides 5'-adjacent to the anticodon of rat cytoplasmic tRNA(Asp) are not edited.

Cytoplasmic tRNA(Asp) of rat liver was purified by the solid-phase hybridization method and its nucleotide sequence was analyzed by Donis-Keller's method. The results suggested that the two nucleotides next to the anticodon were identical to its gene sequence, a finding that is inconsistent with a previous report demonstrating by several methods that C32 and T33 on the tRNA(Asp) gene are post-transcriptionally converted to U32 and C33, respectively (Beier et al (1992) Nucleic Acids Res 20, 2679-2683). Our results indicate that the tRNA hybridized to an oligonucleotide, designed on the basis of the tRNA(Asp) gene sequence, undergoes no editing and possesses C32 and U33 as predicted from the DNA sequence. Analysis of cDNA synthesized from the purified tRNA(Asp) by the RT-PCR method supported the finding that RNA editing is not involved in the maturation process of rat cytoplasmic tRNA(Asp).

Adenosine Triphosphate↗
PubMed2003

Nervous system targets of RNA editing identified by comparative genomics.

An unknown number of precursor messenger RNAs undergo genetic recoding by modification of adenosine to inosine, a reaction catalyzed by the adenosine deaminases acting on RNA (ADARs). Discovery of these edited transcripts has always been serendipitous. Using comparative genomics, we identified a phylogenetic signature of RNA editing. We report the identification and experimental verification of 16 previously unknown ADAR target genes in the fruit fly Drosophila and one in humans-more than the sum total previously reported. All of these genes are involved in rapid electrical and chemical neurotransmission, and many of the edited sites recode conserved and functionally important amino acids. These results point to a pivotal role for RNA editing in nervous system function.

Adenosine↗
PubMed1992

Measuring the determinants of work values for psychiatrists' services in the resource-based relative value scale study.

OBJECTIVE: As part of the Harvard resource-based relative value scale study, the authors investigated how well the codes in the Physician's Current Procedural Terminology, 4th edition, or CPT-4, match psychiatric services to the work involved in evaluating and managing patients and how patient care characteristics affect different levels of psychiatric work. METHOD: A random sample of over 200 psychiatrists and subspecialists was asked to use 68 typical clinical examples or vignettes to evaluate services described by CPT codes. Data were analyzed by multivariate statistical methods. RESULTS: The survey showed that the existing coding system does not adequately describe the work that psychiatrists do. Within a single code (e.g., 90844, individual medical psychotherapy), there was wide (more than twofold) variation in the estimates, from multiple measurements based on different vignettes, of the amount of work represented. Estimates of work values varied significantly according to treatment setting and patient characteristics: psychiatric services in the hospital showed an average work value 25% greater than that for office services; treating new patients involved 18% more effort than treating established patients; and treating patients described as at risk of harming self or others increased the psychiatrists' work effort by 36%. CONCLUSIONS: Revisions in coding evaluation and management services in the new Medicare fee schedule for psychiatric services should be further refined and then implemented. These revisions would bring the coding system into line with psychiatric practice, making it a better way of accounting for the relative work involved in treating patients of varying difficulty.

Fee Schedules↗
PubMedDate not supplied

Sense from nonsense: how the genetic information of chloroplasts is altered by RNA editing.

Plastid transcripts can be subject to an RNA processing mechanism changing the identity of individual nucleotides and thus altering the information content of the mRNA. This processing step was termed RNA editing and adds a novel mechanism to the multitude of RNA maturation events required before mRNAs can serve as faithful templates in plastid protein biosynthesis. RNA editing in chloroplasts proceeds by the conversion of individual cytidine residues to uridine and, in some bryophytes, also by the reverse event, uridine-to-cytidine transitions. The discovery of RNA editing in chloroplasts has provided researchers with a wealth of molecular and evolutionary puzzles, many of which are not yet solved. However, recent work employing chloroplast transformation technologies has shed some light on the molecular mechanisms by which RNA editing sites are recognized with extraordinarily high precision. Also, extensive phylogenetic studies have provided intriguing insights in the evolutionary dynamics with which editing sites may come and go. This review summarizes the state-of-the-art in the field of chloroplast RNA editing, discusses mechanistic and evolutionary aspects of editing and points out some of the important open questions surrounding this enigmatic RNA processing step.

Base Sequence↗
PubMedDate not supplied

Base-language effects on word identification in bilingual speech: evidence from categorical perception experiments.

The categorical perception paradigm was used to investigate whether French-English bilinguals categorize a code-switched word as French or English on the basis of its acoustic-phonetic information alone or whether they are influenced by the base-language context in which the word occurs, that is, by the language in which the majority of words are spoken. Subjects identified stimuli from computer-edited series that ranged from an English to a French word as either the English or the French endpoint. The stimuli were preceded by either an English or a French context sentence. In accord with previous studies (Grosjean, 1988), it was found that the base language had a contrastive effect on the perception of a code-switched word when the endpoints of the between-language series were phonetically marked as English and French, respectively. When the endpoints of the series were phonetically unmarked and thus compatible with either language, however, no effect of the base language was found; in particular, we failed to find the assimilative effect that has been observed with other paradigms (Grosjean, 1988; Soares and Grosjean, 1984; Macnamara and Kushnir, 1971). The current results provide confirming evidence that the perception of a code-switched word is influenced by the base-language context in which it occurs and, moreover, that the nature of the effect depends on the acoustic-phonetic characteristics of the code-switched word. In addition, the finding that a contrastive effect occurs across all paradigms used to date, but that an assimilative effect occurs in only some paradigms, suggests that these two context effects may arise at different stages of processing.

Humans↗