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Ronald B. Emeson - One of the best experts on this subject based on the ideXlab platform.
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high throughput multiplexed transcript analysis yields enhanced resolution of 5 hydroxytryptamine2c receptor mrna editing profiles
Molecular Pharmacology, 2010Co-Authors: Michael V Morabito, Randi J Ulbricht, Richard T Oneil, David C Airey, Bing Zhang, Lily Wang, Ronald B. EmesonAbstract:RNA editing is a Post-Transcriptional Modification in which adenosine residues are converted to inosine (adenosine-to-inosine editing). Commonly used methodologies to quantify RNA editing levels involve either direct sequencing or pyrosequencing of individual cDNA clones. The limitations of these methods lead to a small number of clones characterized in comparison to the number of mRNA molecules in the original sample, thereby producing significant sampling errors and potentially erroneous conclusions. We have developed an improved method for quantifying RNA editing patterns that increases sequence analysis to an average of more than 800,000 individual cDNAs per sample, substantially increasing accuracy and sensitivity. Our method is based on the serotonin 2C receptor (5-hydroxytryptamine2C; 5HT2C) transcript, an RNA editing substrate in which up to five adenosines are modified. Using a high-throughput multiplexed transcript analysis, we were able to quantify accurately the expression of twenty 5HT2C isoforms, each representing at least 0.25% of the total 5HT2C transcripts. Furthermore, this approach allowed the detection of previously unobserved changes in 5HT2C editing in RNA samples isolated from different inbred mouse strains and dissected brain regions, as well as editing differences in alternatively spliced 5HT2C variants. This approach provides a novel and efficient strategy for large-scale analyses of RNA editing and may prove to be a valuable tool for uncovering new information regarding editing patterns in specific disease states and in response to pharmacological and physiological perturbation, further elucidating the impact of 5HT2C RNA editing on central nervous system function.
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an innovative real time pcr method to measure changes in rna editing of the serotonin 2c receptor 5 ht2cr in brain
Journal of Neuroscience Methods, 2009Co-Authors: Maria Fe Lanfranco, Patricia K Seitz, Michael V Morabito, Elaine Sandersbush, Ronald B. Emeson, Kathryn A CunninghamAbstract:The serotonin 2C receptor (5-HT2CR) plays a significant role in psychiatric disorders (e.g., depression) and is a target for pharmacotherapy. The 5-HT2CR is widely expressed in brain and spinal cord and is the only G-protein coupled receptor currently known to undergo mRNA editing, a Post-Transcriptional Modification that results in translation of distinct, though closely related, protein isoforms. The 5-HT2CR RNA can be edited at five sites to alter up to three amino acids resulting in modulation of receptor:G-protein coupling and constitutive activity. To rapidly quantify changes ex vivo in individual 5-HT2CR isoform levels in response to treatment, we adapted quantitative (real-time) reverse transcription polymerase chain reaction (qRT-PCR) utilizing TaqMan® probes modified with a minor groove binder (MGB). Probes were developed for four 5-HT2CR RNA isoforms and their sensitivity and specificity were validated systematically using standard templates. Relative expression of the four isoforms was measured in cDNAs from whole brain extracted from 129S6 and C57BL/6J mice. Rank order derived from this qRT-PCR analysis matched that derived from DNA sequencing. In mutant mice solely expressing either non-edited or fully edited 5-HT2CR transcripts, only expected transcripts were detected. These data suggest this qRT-PCR method is a precise and rapid means to detect closely related mRNA sequences ex vivo without the necessity of characterizing the entire 5-HT2CR profile. Implementation of this technique will expand and expedite studies of specific brain 5-HT2CR mRNA isoforms in response to pharmacological, behavioral and genetic manipulation, particularly in ex vivo studies which require rapid collection of data on large numbers of samples.
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regulation of serotonin 2c receptor g protein coupling by rna editing
Nature, 1997Co-Authors: Colleen M Burns, Elaine Sandersbush, Susan M Rueter, Linda K Hutchinson, Herve Canton, Ronald B. EmesonAbstract:The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) elicits a wide array of physiological effects by binding to several receptor subtypes. The 5-HT2 family of receptors belongs to a large group of seven-transmembrane-spanning G-protein-coupled receptors and includes three receptor subtypes (5-HT2A, 5-HT2B and 5-HT2C) which are linked to phospholipase C, promoting the hydrolysis of membrane phospholipids and a subsequent increase in the intracellular levels of inositol phosphates and diacylglycerol1. Here we show that transcripts encoding the 2C subtype of serotonin receptor (5-HT2CR) undergo RNA editing events in which genomically encoded adenosine residues are converted to inosines by the action of double-stranded RNA adenosine deaminase(s). Sequence analysis of complementary DNA isolates from dissected brain regions have indicated the tissue-specific expression of seven major 5-HT2C receptor iso-forms encoded by eleven distinct RNA species. Editing of 5-HT2CR messenger RNAs alters the amino-acid coding potential of the predicted second intracellular loop of the receptor and can lead to a 10–15-fold reduction in the efficacy of the interaction between receptors and their G proteins. These observations indicate that RNA editing is a new mechanism for regulating serotonergic signal transduction and suggest that this Post-Transcriptional Modification may be critical for modulating the different cellular functions that are mediated by other members of the G-protein-coupled receptor superfamily.
Remko Offringa - One of the best experts on this subject based on the ideXlab platform.
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pin driven polar auxin transport in plant developmental plasticity a key target for environmental and endogenous signals
New Phytologist, 2014Co-Authors: Myckel E J Habets, Remko OffringaAbstract:Contents 'Summary' 362 I. 'Introduction' 362 II. 'Auxin action' 363 III. 'PIN regulation by a complex network of feedback loops' 366 IV. 'PIN trafficking regulated by environmental signals' 369 V. 'Regulation of PIN proteins by internal signals' 371 VI. 'Conclusions/future perspectives' 372 'Acknowledgements' 373 References 373 Summary Plants master the art of coping with environmental challenges in two ways: on the one hand, through their extensive defense systems, and on the other, by their developmental plasticity. The plant hormone auxin plays an important role in a plant's adaptations to its surroundings, as it specifies organ orientation and positioning by regulating cell growth and division in response to internal and external signals. Important in auxin action is the family of PIN-FORMED (PIN) auxin transport proteins that generate auxin maxima and minima by driving polar cell-to-cell transport of auxin through their asymmetric subcellular distribution. Here, we review how regulatory proteins, the cytoskeleton, and membrane trafficking affect PIN expression and localization. Transcriptional regulation of PIN genes alters protein abundance, provides tissue-specific expression, and enables feedback based on auxin concentrations and crosstalk with other hormones. Post-Transcriptional Modification, for example by PIN phosphorylation or ubiquitination, provides regulation through protein trafficking and degradation, changing the direction and quantity of the auxin flow. Several plant hormones affect PIN abundance, resulting in another means of crosstalk between auxin and these hormones. In conclusion, PIN proteins are instrumental in directing plant developmental responses to environmental and endogenous signals.
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pin driven polar auxin transport in plant developmental plasticity a key target for environmental and endogenous signals
New Phytologist, 2014Co-Authors: Myckel E J Habets, Remko OffringaAbstract:Plants master the art of coping with environmental challenges in two ways: on the one hand, through their extensive defense systems, and on the other, by their developmental plasticity. The plant hormone auxin plays an important role in a plant's adaptations to its surroundings, as it specifies organ orientation and positioning by regulating cell growth and division in response to internal and external signals. Important in auxin action is the family of PIN-FORMED (PIN) auxin transport proteins that generate auxin maxima and minima by driving polar cell-to-cell transport of auxin through their asymmetric subcellular distribution. Here, we review how regulatory proteins, the cytoskeleton, and membrane trafficking affect PIN expression and localization. Transcriptional regulation of PIN genes alters protein abundance, provides tissue-specific expression, and enables feedback based on auxin concentrations and crosstalk with other hormones. Post-Transcriptional Modification, for example by PIN phosphorylation or ubiquitination, provides regulation through protein trafficking and degradation, changing the direction and quantity of the auxin flow. Several plant hormones affect PIN abundance, resulting in another means of crosstalk between auxin and these hormones. In conclusion, PIN proteins are instrumental in directing plant developmental responses to environmental and endogenous signals.
Myckel E J Habets - One of the best experts on this subject based on the ideXlab platform.
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pin driven polar auxin transport in plant developmental plasticity a key target for environmental and endogenous signals
New Phytologist, 2014Co-Authors: Myckel E J Habets, Remko OffringaAbstract:Contents 'Summary' 362 I. 'Introduction' 362 II. 'Auxin action' 363 III. 'PIN regulation by a complex network of feedback loops' 366 IV. 'PIN trafficking regulated by environmental signals' 369 V. 'Regulation of PIN proteins by internal signals' 371 VI. 'Conclusions/future perspectives' 372 'Acknowledgements' 373 References 373 Summary Plants master the art of coping with environmental challenges in two ways: on the one hand, through their extensive defense systems, and on the other, by their developmental plasticity. The plant hormone auxin plays an important role in a plant's adaptations to its surroundings, as it specifies organ orientation and positioning by regulating cell growth and division in response to internal and external signals. Important in auxin action is the family of PIN-FORMED (PIN) auxin transport proteins that generate auxin maxima and minima by driving polar cell-to-cell transport of auxin through their asymmetric subcellular distribution. Here, we review how regulatory proteins, the cytoskeleton, and membrane trafficking affect PIN expression and localization. Transcriptional regulation of PIN genes alters protein abundance, provides tissue-specific expression, and enables feedback based on auxin concentrations and crosstalk with other hormones. Post-Transcriptional Modification, for example by PIN phosphorylation or ubiquitination, provides regulation through protein trafficking and degradation, changing the direction and quantity of the auxin flow. Several plant hormones affect PIN abundance, resulting in another means of crosstalk between auxin and these hormones. In conclusion, PIN proteins are instrumental in directing plant developmental responses to environmental and endogenous signals.
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pin driven polar auxin transport in plant developmental plasticity a key target for environmental and endogenous signals
New Phytologist, 2014Co-Authors: Myckel E J Habets, Remko OffringaAbstract:Plants master the art of coping with environmental challenges in two ways: on the one hand, through their extensive defense systems, and on the other, by their developmental plasticity. The plant hormone auxin plays an important role in a plant's adaptations to its surroundings, as it specifies organ orientation and positioning by regulating cell growth and division in response to internal and external signals. Important in auxin action is the family of PIN-FORMED (PIN) auxin transport proteins that generate auxin maxima and minima by driving polar cell-to-cell transport of auxin through their asymmetric subcellular distribution. Here, we review how regulatory proteins, the cytoskeleton, and membrane trafficking affect PIN expression and localization. Transcriptional regulation of PIN genes alters protein abundance, provides tissue-specific expression, and enables feedback based on auxin concentrations and crosstalk with other hormones. Post-Transcriptional Modification, for example by PIN phosphorylation or ubiquitination, provides regulation through protein trafficking and degradation, changing the direction and quantity of the auxin flow. Several plant hormones affect PIN abundance, resulting in another means of crosstalk between auxin and these hormones. In conclusion, PIN proteins are instrumental in directing plant developmental responses to environmental and endogenous signals.
Alessandro Barbon - One of the best experts on this subject based on the ideXlab platform.
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genome wide analysis of consistently rna edited sites in human blood reveals interactions with mrna processing genes and suggests correlations with cell types and biological variables
BMC Genomics, 2018Co-Authors: Edoardo Giacopuzzi, Massimo Gennarelli, Chiara Sacco, Alice Filippini, Jessica Mingardi, Chiara Magri, Alessandro BarbonAbstract:A-to-I RNA editing is a co−/Post-Transcriptional Modification catalyzed by ADAR enzymes, that deaminates Adenosines (A) into Inosines (I). Most of known editing events are located within inverted ALU repeats, but they also occur in coding sequences and may alter the function of encoded proteins. RNA editing contributes to generate transcriptomic diversity and it is found altered in cancer, autoimmune and neurological disorders. Emerging evidences indicate that editing process could be influenced by genetic variations, biological and environmental variables. We analyzed RNA editing levels in human blood using RNA-seq data from 459 healthy individuals and identified 2079 sites consistently edited in this tissue. As expected, analysis of gene expression revealed that ADAR is the major contributor to editing on these sites, explaining ~ 13% of observed variability. After removing ADAR effect, we found significant associations for 1122 genes, mainly involved in RNA processing. These genes were significantly enriched in genes encoding proteins interacting with ADARs, including 276 potential ADARs interactors and 9 ADARs direct partners. In addition, our analysis revealed several factors potentially influencing RNA editing in blood, including cell composition, age, Body Mass Index, smoke and alcohol consumption. Finally, we identified genetic loci associated with editing levels, including known ADAR eQTLs and a small region on chromosome 7, containing LOC730338, a lincRNA gene that appears to modulate ADARs mRNA expression. Our data provides a detailed picture of the most relevant RNA editing events and their variability in human blood, giving interesting insights on potential mechanisms behind this Post-Transcriptional Modification and its regulation in this tissue.
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genome wide analysis of consistently rna edited sites in human blood reveals interactions with mrna processing genes and suggests correlations with cell types and biological variables
bioRxiv, 2018Co-Authors: Edoardo Giacopuzzi, Massimo Gennarelli, Chiara Sacco, Alice Filippini, Jessica Mingardi, Chiara Magri, Alessandro BarbonAbstract:Abstract Background A-to-I RNA editing is a co-/Post-Transcriptional Modification catalyzed by ADAR enzymes, that deaminates Adenosines (A) into Inosines (I). Most of known editing events are located within inverted ALU repeats, but they also occur in coding sequences and may alter the function of encoded proteins. RNA editing contributes to generate transcriptomic diversity and it is found altered in cancer, autoimmune and neurological disorders. Emerging evidences indicate that editing process could be influenced by genetic variations, biological and environmental variables. Results We analyzed RNA editing levels in human blood using RNA-seq data from 459 healthy individuals and identified 2,079 sites consistently edited in this tissue. As expected, analysis of gene expression revealed that ADAR is the major contributor to editing on these sites, explaining ∼13% of observed variability. After removing ADAR effect, we found significant associations for 1,122 genes, mainly involved in RNA processing. These genes were significantly enriched in genes encoding proteins interacting with ADARs, including 276 potential ADARs interactors and 9 ADARs direct partners. In addition, our analysis revealed several factors potentially influencing RNA editing in blood, including cell composition, age, Body Mass Index, smoke and alcohol consumption. Finally, we identified genetic loci associated with editing levels, including known ADAR eQTLs and a small region on chromosome 7, containing LOC730338, a lincRNA gene that appears to modulate ADARs mRNA expression. Conclusions Our data provides a detailed picture of the most relevant RNA editing events and their variability in human blood, giving interesting insights on potential mechanisms behind this Post-Transcriptional Modification and its regulation in this tissue.
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genome wide analysis of rna sites consistently edited in human blood reveals interactions with mrna processing genes and suggests correlations with biological and drug related variables
bioRxiv, 2018Co-Authors: Edoardo Giacopuzzi, Massimo Gennarelli, Chiara Sacco, Chiara Magri, Alessandro BarbonAbstract:Background: A-to-I RNA editing is a co-/Post-Transcriptional Modification catalyzed by ADAR enzymes, that deaminate Adenosines (A) into Inosines (I). Most of known editing events are located within inverted ALU repeats, but they also occur in coding sequences and may alter the function of encoded proteins. RNA editing contributes to generate transcriptomic diversity and it is found altered in cancer, autoimmune and neurological disorders. However, little is known about how editing process could be influenced by genetic variations, biological and environmental variables. Results: We analyzed RNA editing levels in human blood using RNA-seq data from 459 healthy individuals and identified 2,079 sites consistently edited in this tissue, that we considered the most biologically relevant editing sites. As expected, analysis of gene expression revealed that ADAR is the major contributor to editing on these sites, explaining ~13% of observed variability. After removing ADAR effect, we found significant associations for 1,122 genes, mainly involved in RNA processing. These genes were significantly enriched in genes encoding proteins interacting with ADARs, including 276 potential ADARs interactors and 9 ADARs direct partners. In addition, association analysis on 28 biological and drugs intake variables revealed several factors potentially influencing RNA editing in blood, including sex, age, BMI, drugs and medications. Finally, we identified genetic loci associated to editing levels, including known ADAR eQTLs and a small region on chromosome 7, containing LOC730338 lincRNA gene. Conclusions: Our data provides a detailed picture of the most relevant RNA editing events and their variability in human blood, giving interesting insights on the mechanisms behind this Post-Transcriptional Modification and its regulation in this tissue.
Gideon Rechavi - One of the best experts on this subject based on the ideXlab platform.
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n 6 methyladenosine in mrna disrupts trna selection and translation elongation dynamics
Nature Structural & Molecular Biology, 2016Co-Authors: Junhong Choi, Jin Chen, Arjun Prabhakar, Hasan Demirci, Ka Weng Ieong, Alexey Petrov, Gideon Rechavi, Sean T Oleary, Dan DominissiniAbstract:N(6)-methylation of adenosine (forming m(6)A) is the most abundant Post-Transcriptional Modification within the coding region of mRNA, but its role during translation remains unknown. Here, we used bulk kinetic and single-molecule methods to probe the effect of m(6)A in mRNA decoding. Although m(6)A base-pairs with uridine during decoding, as shown by X-ray crystallographic analyses of Thermus thermophilus ribosomal complexes, our measurements in an Escherichia coli translation system revealed that m(6)A Modification of mRNA acts as a barrier to tRNA accommodation and translation elongation. The interaction between an m(6)A-modified codon and cognate tRNA echoes the interaction between a near-cognate codon and tRNA, because delay in tRNA accommodation depends on the position and context of m(6)A within codons and on the accuracy level of translation. Overall, our results demonstrate that chemical Modification of mRNA can change translational dynamics.
