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PubMed2001

RNA editing of the human serotonin 5-HT2C receptor alters receptor-mediated activation of G13 protein.

The recent completion of the human genome predicted the presence of only 30,000 genes, stressing the importance of mechanisms that increase molecular diversity at the post-transcriptional level. One such post-transcriptional event is RNA editing, which generates multiple protein isoforms from a single gene, often with profound functional consequences. The human serotonin 5-HT(2C) receptor undergoes RNA editing that creates multiple receptor isoforms. One consequence of RNA editing of cell surface receptors may be to alter the pattern of activation of heterotrimeric G-proteins and thereby shift preferred intracellular signaling pathways. We examined the ability of the nonedited 5-HT(2C) receptor isoform (INI) and two extensively edited isoforms, VSV and VGV, to interact with various G-protein alpha subunits. Two functional assays were utilized: the cell-based functional assay, Receptor Selection/Amplification Technology(TM), in which the pharmacological consequences of co-expression of 5HT(2C) receptor isoforms with G-protein alpha subunits in fibroblasts were studied, and 5HT(2C) receptor-mediated rearrangements of the actin cytoskeleton in stable cell lines. These studies revealed that the nonedited 5-HT(2C) receptor functionally couples to G(q) and G(13). In contrast, coupling to G(13) was not detected for the extensively edited 5-HT(2C) receptors. Thus, RNA editing represents a novel mechanism for regulating the pattern of activation of heterotrimeric G-proteins, molecular switches that control an enormous variety of biological processes.

3T3 Cells↗

PubMed2003

Water quality and health in the new millennium: the role of the World Health Organization Guidelines for Drinking-Water Quality.

In this report the role of the WHO Guidelines for Drinking Water Quality in promoting safe drinking water for the world's population is briefly described. The guidelines are being revised in a third edition to emphasize an integrated approach to water quality assessment and management from source to consumer. The forthcoming guidelines will: be risk-based and quantitative, emphasize quality protection and prevention of contamination, be proactive and participatory, and address the needs of those in developing countries who have no access to piped community water supplies. The guidelines emphasize the maintenance of microbial quality to prevent waterborne infectious disease as an essential goal. In addition, they address protection from chemical toxicants and other contaminants of public health concern. The forthcoming 3rd edition of the WHO GDWQ intend to be responsive to the under-served in developing countries by inclusion of non-piped supplies and addressing practical systems for their collection, treatment and storage at household level to provide safe water. Beyond the inclusion of these and possibly additional household water collection, treatment and storage systems, what is needed is to achieve their widespread use is an education and dissemination campaign that promotes and explains them and their benefits. Such a communication and marketing campaign is best done by including as many different sectors and stakeholders as possible in the process. It will be important to acknowledge that safe water is one of essential components or needs for healthy living, along with adequate sanitation and proper nutrition. Together, these are the essential health needs to be met in the developing and the developed world. All three contribute to reduced disease and increased health, and the lack of one can degrade the beneficial impact of the others. The importance of safe water, sanitation and nutrition to human health and well-being can be stated no better than it was by United Nations Secretary General Kofi Annan in his statement on "Freedom from Want" in the Millennium Report, 03/04/00. "How can we call human beings free and equal in dignity when over a billion of them are struggling to survive on less than one dollar a day, without safe drinking water, and when half of all humanity lacks adequate sanitation? Some of us are worrying about whether the stock market will crash, or struggling to master our latest computer, while more than half our fellow men and women have much more basic worries, such as where their children's next meal is coming from."

Consumer Product Safety↗

PubMed2006

A-to-I pre-mRNA editing of the serotonin 2C receptor: comparisons among inbred mouse strains.

The serotonin receptor 5HT2CR pre-mRNA is subject to adenosine deamination (RNA editing) at five residues located within a 15 nucleotide stretch of the coding region. Such changes of adenosine to inosine (A-to-I) can produce 32 mRNA variants, encoding 24 different protein isoforms, some of which vary in biochemical and pharmacological properties. Because serotonin mediates diverse neurological processes relevant to behavior and because inbred mouse strains vary in their responses to tests of learning and behavior, we have examined the A-to-I editing patterns of the 5HT2CR mRNA in whole brains from eight mouse strains. By sequencing approximately 100 clones from individual mice, we generated detailed information on levels of editing at each site and patterns of editing that identify a total of 28 mRNA and 20 protein isoforms. Significant differences between individuals from different strains were found in total editing frequency, in the proportion of transcripts with 1 and 4 edited sites, in editing frequency at the A, B, E and D sites, in amino acid frequencies at positions 157 and 161, and in subsets of major protein isoforms. Primer extension assays were used to show that individuals within strains (six C3H.B-+rd1 and four 129SvImrJ) displayed no significant differences in any feature. These findings suggest that genetic background contributes to subtle variation in 5HT2CR mRNA editing patterns which may have consequences for pharmacological treatments and behavioral testing.

Adenosine↗

PubMed1994

RNA editing of tobacco petB mRNAs occurs both in chloroplasts and non-photosynthetic proplastids.

We found an RNA editing site in the protein coding region of tobacco (Nicotiana tabacum) petB transcripts. This editing (CCA to CUA) leads to an amino acid conversion from proline to leucine. It is observed not only in chloroplasts isolated from tobacco leaves but also in non-photosynthetic proplastids isolated from a tobacco cell culture. Also unspliced pre-mRNA shows complete editing. The editing site is the same as that recently observed in the maize petB transcripts which restores the codon for a highly conserved leucine residue, suggesting that RNA editing at this site is critical for the protein (cytochrome b6) function.

Base Sequence↗

PubMed1994

RNA editing and mitochondrial genomic organization in the cryptobiid kinetoplastid protozoan Trypanoplasma borreli.

The bodonids and cryptobiids represent an early diverged sister group to the trypanosomatids among the kinetoplastid protozoa. The trypanosome type of uridine insertion-deletion RNA editing was found to occur in the cryptobiid fish parasite Trypanoplasma borreli. A pan-edited ribosomal protein, S12, and a novel 3'- and 5'-edited cytochrome b, in addition to an unedited cytochrome oxidase III gene and an apparently unedited 12S rRNA gene, were found in a 6-kb fragment of the 80- to 90-kb mitochondrial genome. The gene order differs from that in trypanosomatids, as does the organization of putative guide RNA genes; guide RNA-like molecules are transcribed from tandemly repeated 1-kb sequences organized in 200- and 170-kb molecules instead of minicircles. The presence of pan-editing in this lineage is consistent with an ancient evolutionary origin of this process.

Animals↗

PubMed1999

Synthetic substrate analogs for the RNA-editing adenosine deaminase ADAR-2.

We have synthesized structural analogs of a natural RNA editing substrate and compared editing reactions of these substrates by recombinant ADAR-2, an RNA-editing adenosine deaminase. Deamination rates were shown to be sensitive to structural changes at the 2[prime]-carbon of the edited adenosine. Methylation of the 2[prime]-OH caused a large decrease in deamination rate, whereas 2[prime]-deoxyadenosine and 2[prime]-deoxy-2[prime]-fluoroadenosine were deaminated at a rate similar to adenosine. In addition, a duplex containing as few as 19 bp of the stem structure adjacent to the R/G editing site of the GluR-B pre-mRNA supports deamination of the R/G adenosine by ADAR-2. This identification and initial characterization of synthetic RNA editing substrate analogs further defines structural elements in the RNA that are important for the deamination reaction and sets the stage for additional detailed structural, thermodynamic and kinetic studies of the ADAR-2 reaction.

Adenosine↗

PubMed1997

The mental health of informal caregivers in Ontario: an epidemiological survey.

OBJECTIVES: This study describes the mental health status, disability, physical health, and mental health service utilization of informal care-givers under the age of 65 in the province of Ontario. METHODS: The study analyzed data collected in the 1991 province-wide, population-based mental health supplement to the Ontario Health Survey. Diagnoses from the Diagnostic and Statistical Manual of Mental Disorders, 3rd edition, revised, were generated on the basis of a structured diagnostic interview. Caregivers and noncaregivers are compared here on past-year prevalence of psychiatric disorder, physical illness, disability, and utilization of mental health services. The possible confounding effects of age, sex, employment status, and economic disadvantage are explored. RESULTS: Informal caregivers (n = 1219) constituted 15.0% of the sample. Caregivers had higher rates of affective (6.3% vs 4.2%) and anxiety (17.5% vs 10.9%) disorders than noncaregivers and used health services for mental health problems at nearly twice the rate. CONCLUSIONS: Documentation of the prevalence of caregiving and the increased prevalence of psychiatric disorders, disability, and service utilization among caregivers is of critical importance as governments continue to move toward community-based care. To accomplish this goal, the needs of caregivers must be acknowledged and met by the establishment of appropriate and readily accessible support services.

Adolescent↗

PubMed1999

In vivo lactate editing in single voxel proton spectroscopy and proton spectroscopic imaging by homonuclear polarisation transfer.

Volume-selective lactate editing has been performed successfully in vitro and in vivo in the brain on a clinical scanner using a PRESS-based single voxel 1H spectroscopy and a 1H spectroscopic imaging sequence. The PRESS sequence was made sensitive to homonuclear polarisation by replacing the standard 180 degree refocusing pulses with 90 degree pulses. Two acquisitions were made at a total echo time around 2/J (J is the coupling constant for CH and CH3 spins in lactate approximately 7 Hz) whose individual echo times differed by 5.5 ms. Subtraction of one signal from the other yielded the lactate resonance alone. The technique is an effective method of separating the overlapping signals of lactate and lipids. Furthermore this editing method can be performed without state of the art MRI scanner hardware.

Brain↗

PubMed1994

Disruption of RNA editing in Leishmania tarentolae by the loss of minicircle-encoded guide RNA genes.

RNA editing in kinetoplastids appears to be a labile genetic trait that is affected by prolonged cell culture. The transcripts of the G1-G5 cryptogenes are pan-edited in the recently isolated LEM125 strain of Leishmania tarentolae, but not in the UC strain which has been in culture for 55 years. At least 32 minicircle-encoded guide RNAs (gRNAs) for the editing of G1-G5 transcripts are present in LEM125 and absent in UC. We hypothesize that specific minicircle sequence classes encoding gRNAs for the editing of these transcripts were lost during the long culture history of the UC strain. The protein products, which include components of complex I of the respiratory chain, are probably not required during the culture stage of the Leishmania life cycle.

Amino Acid Sequence↗

PubMed2002

Chloroplast ribosomal S14 protein transcript is edited to create a translation initiation codon in the moss Physcomitrella patens.

The rps14 transcript is edited in the moss Physcomitrella patens chloroplast by a C-to-U transition, to create a translation initiation codon, AUG. The efficiency of RNA editing was low, with approximately 20% of rps14 transcripts edited. This suggests that the translation of rps14 mRNA is strictly regulated by RNA editing. This is the first report of RNA editing in P. patens and the creation of a translation initiation codon in rps14 mRNA in chloroplasts.

Amino Acid Sequence↗

PubMed1998

Cis- and trans-splicing and RNA editing are required for the expression of nad2 in wheat mitochondria.

In wheat mitochondria, the gene coding for subunit 2 of the NADH-ubiquinone oxidoreductase (nad2) is divided into five exons located in two distant genomic regions. The first two exons of the gene, a and b, lie 22 kb downstream of exons c, d, and e, on the same DNA strand. All introns of nad2 are group II introns. A trans-splicing event is required to join exons b and c. It involves base pairing of the two precursor RNAs in the stem of domain IV of the intron. A gene coding for tRNA(Tyr) is located upstream of exon c. In addition to splicing processes, mRNA editing is also required for the correct expression of nad2. The mature mRNA is edited at 36 positions, distributed over its five exons, resulting in 28 codon modifications. Editing increases protein hydrophobicity and conservation.

Amino Acid Sequence↗

PubMed2001

Isolation, characterization and developmental regulation of the human apobec-1 complementation factor (ACF) gene.

Mammalian apolipoprotein B (apo B) mRNA undergoes site-specific C to U deamination which is mediated by a multicomponent enzyme complex containing a minimal core composed of apobec-1 and a complementation factor, ACF. We have isolated and characterized the human ACF gene and examined its tissue-specific and developmental expression. The ACF gene spans approximately 80 kb and contains 15 exons, three of which are non-coding. Multiple alternative splice acceptor sites were found, generating at least nine different transcripts. Of these, the majority (approximately 75-89%) encode functional protein. In order to examine the role of ACF mRNA expression in the regulation of apo B mRNA editing, we examined a panel of fetal intestinal and hepatic mRNAs as well as RNA from an intestinal cell line. A developmental increase in C to U RNA editing has been previously noted in the human intestine. In both instances, the pattern of alternative splicing and overall abundance of ACF mRNA was relatively constant during development in both liver and small intestine. Taken together, the data demonstrate a complex pattern of differential, tissue-specific splicing of ACF mRNA, but suggest that other mechanisms are responsible for the developmental increase noted in intestinal apo B mRNA editing in humans.

APOBEC-1 Deaminase↗

PubMed1993

Hepatic polysomes that contain apoprotein B mRNA have unusual physical properties.

To characterize the association of the mRNA for apoprotein B (apoB) with ribosomes, rat hepatic cytoplasmic extracts were fractionated by density gradient centrifugation. On linear sucrose gradients, the sedimentation velocity of the 14.4-kilobase apoB mRNA was retarded compared to the mRNAs for other hepatic proteins, which were concentrated in fractions containing the bulk of the polysomes. This unusual distribution of apoB mRNA could not be explained by cotranslational association of nascent apoB peptides with lipids, based on experiments using either detergents to delipidate proteins or puromycin to release nascent peptides from polysomes. The results were also not the result of the editing of apoB mRNA, since the sucrose gradient distributions of both edited and nonedited forms were similar. In contrast, the distribution of a 3'-truncated apoB mRNA (apoB-42, 5.8 kilobases) expressed in rat hepatoma cells resembled that of mRNA of a typical hepatic protein. As opposed to the sedimentation velocity results, on equilibrium density gradients most hepatic apoB mRNA was found in the fraction that contained polysomes. Based on these data, the elongation rate of nascent apoB, and the calculated translational yield of apoB mRNA, we conclude that the majority of rat hepatic apoB mRNA must be part of polysomal complexes with unusual physical properties related to the presence of sequence(s) in the 3'-region of the message. These sequences may either be primary determinants of structural features or binding sites for protein factors that effect conformational changes.

Animals↗

PubMed2003

Elevated activity of the large form of ADAR1 in vivo: very efficient RNA editing occurs in the cytoplasm.

Mammalian cells express small and large forms of the RNA editing enzyme ADAR1, referred to as ADAR1-S and ADAR1-L, respectively. Here we observed that ADAR1-L was >70-fold more active than was ADAR1-S when assayed with a substrate that could be edited in either the nucleus or cytoplasm, and was also much more active when assayed with a substrate that was generated in the cytoplasm during viral replication. In contrast, when a substrate that could only be edited within the nucleus was assayed, the activity of ADAR1-S was found to be somewhat higher than that of ADAR1-L. We show here not only that editing could occur in the cytoplasm but also that the process was extremely efficient, occurred rapidly, and could occur in the absence of translation. Consistent with the observation that editing in the cytoplasm can be very efficient, deletion of the nuclear localization signal from ADAR2 resulted in a protein with 15-fold higher activity when tested with a substrate that contained an editing site in the mature message. In addition to its potential role in an antiviral response, we propose that ADAR1-L is the form primarily responsible for editing mRNAs in which the editing site is retained after processing.

Adenosine Deaminase↗

PubMed2001

Mitochondrial atp6 transcript editing during microgametogenesis in male-sterile sorghum.

A marked reduction of mitochondrial atp6 transcript-editing capability in sorghum anthers and pollen has been invoked as a factor in the loss of viability of male gametophytes in lines carrying the IS1112C male-sterile cytoplasm. We initiated a systematic examination of transcript editing of sorghum atp6 during microgametogenesis, from microspores through pollen, in two sets of male-fertile and near-isogenic, male-sterile lines. Transcript editing in cDNA populations from fertile lines indicated an average of about 90% editing among 15 sites examined. Parallel samples from sterile lines demonstrated a detectable reduction in editing efficiency, most evident in late pollen samples. However, editing efficiency in these cells averaged 80%, substantially higher than the 20% previously observed. Thus male-sterile lines retain substantial atp6-editing efficiency in an environment where the sterility and fertility restoration traits are stably expressed. Drastically reduced editing of atp6 transcripts in anthers and pollen of male-sterile sorghum is not demonstrable in a field environment; and thus it is not a factor in the loss of microgametophyte viability in this system.

Base Sequence↗

PubMed1999

RNA editing.

The term RNA editing describes those molecular processes in which the information content is altered in an RNA molecule. To date such changes have been observed in tRNA. rRNA and mRNA molecules of eukaryotes, but not prokaryotes. The demonstration of RNA editing in prokaryotes may only be a matter of time, considering the range of species in which the various RNA editing processes have been found. RNA editing occurs in the nucleus, as well as in mitochondria and plastids, which are thought to have evolved from prokaryotic-like endosymbionts. Most of the RNA editing processes, however, appear to be evolutionarily recent acquisitions that arose independently. The diversity of RNA editing mechanisms includes nucleoside modifications such as C to U and A to I deaminations, as well as non-templated nucleotide additions and insertions. RNA editing in mRNAs effectively alters the amino acid sequence of the encoded protein so that it differs from that predicted by the genomic DNA sequence.

Amino Acid Sequence↗

PubMed2002

Topology correction in brain cortex segmentation using a multiscale, graph-based algorithm.

Reconstructing an accurate and topologically correct representation of the cortical surface of the brain is an important objective in various neuroscience applications. Most cortical surface reconstruction methods either ignore topology or correct it using manual editing or methods that lead to inaccurate reconstructions. Shattuck and Leahy recently reported a fully automatic method that yields a topologically correct representation with little distortion of the underlying segmentation. We provide an alternate approach that has several advantages over their approach, including the use of arbitrary digital connectivities, a flexible morphology-based multiscale approach, and the option of foreground-only or background-only correction. A detailed analysis of the method's performance on 15 magnetic resonance brain images is provided.

Algorithms↗

PubMedDate not supplied

Expanding genome capacity via RNA editing.

RNA editing, which results in the creation of RNA molecules that differ from the template from which they were made, is a highly specific process. Alterations include converting one base to another, removal of one nucleotide and substitution of another, deletion of encoded residues, and insertion of non-templated nucleotides. Such changes have marked effects on gene expression, ranging from defined amino acid changes to the de novo creation of entire open reading frames. Editing can be regulated in a developmental or tissue-specific manner, and is likely to play a role in the etiology of human disease.

Genetic Techniques↗