The Experts below are selected from a list of 129 Experts worldwide ranked by ideXlab platform
Matthew J A Wood - One of the best experts on this subject based on the ideXlab platform.
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silencing of parkinson s disease associated genes with artificial Mirtron mimics of mir 1224
Nucleic Acids Research, 2012Co-Authors: Christopher R. Sibley, Yiqi Seow, Marc S Weinberg, Helen J Curtis, Matthew J A WoodAbstract:Mirtrons are a recently described category of microRNA (miRNA) relying on splicing rather than processing by the microprocessor complex to generate pre-miRNA precursors of the RNA interference (RNAi) pathway. Their discovery and subsequent verification provides important information about a distinct class of miRNA and inherent advantages that could be exploited to silence genes of interest. These include micro-processor-independent biogenesis, pol-II-dependent transcription, accurate species generation and the delivery of multiple artificial Mirtrons as introns within a single host transcript. Here we determined the sequence motifs required for correct processing of the mmu-miR-1224 Mirtron and incorporated these into artificial Mirtrons targeting Parkinson's disease-associated LRRK2 and α-synuclein genes. By incorporating these rules associated with processing and splicing, artificial Mirtrons could be designed and made to silence complementary targets either at the mRNA or protein level. We further demonstrate with a LRRK2 targeting artificial Mirtron that neuronal-specific silencing can be directed under the control of the human synapsin promoter. Finally, multiple Mirtrons were co-delivered within a single host transcript, an eGFP reporter, to allow simultaneous targeting of two or more targets in a combinatorial approach. Thus, the unique characteristics of artificial Mirtrons make this an attractive approach for future RNAi applications. © 2012 The Author(s).
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Artificial Mirtron-mediated gene knockdown: Functional DMPK silencing in mammalian cells
RNA, 2012Co-Authors: Yiqi Seow, Christopher R. Sibley, Matthew J A WoodAbstract:Mirtrons are introns that form pre-miRNA hairpins after splicing to produce RNA interference (RNAi) effectors distinct from Drosha-dependent intronic miRNAs. Here we present a design algorithm for artificial Mirtrons and demonstrate, for the first time, efficient gene knockdown of myotonic dystrophy protein kinase (DMPK) target sequences in Renilla luciferase 3' UTR and subsequently pathogenic DMPK mRNA, causative of Type I myotonic dystrophy, using artificial Mirtrons cloned as eGFP introns. Deep sequencing of artificial Mirtrons suggests that functional mature transcripts corresponding to the designed sequence were produced in high abundance. They were further shown to be splicing-dependent, Drosha-independent, and partially dependent on exportin-5, resulting in the precise generation of pre-miRNAs. In a murine myoblast line containing a pathogenic copy of human DMPK with more than 500 CUG repeats, the DMPK artificial Mirtron corrected DM1-associated splicing abnormalities of the Serca-1 mRNA, demonstrating the therapeutic potential of Mirtron-mediated RNAi. Thus, further development and exploitation of the unique properties of Mirtrons will benefit future research and therapeutic RNAi applications as an alternative to conventional RNAi strategies.
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the biogenesis and characterization of mammalian micrornas of Mirtron origin
Nucleic Acids Research, 2012Co-Authors: Christopher R. Sibley, Yiqi Seow, Sheena Saayman, Krijn K Dijkstra, Samir El Andaloussi, Marc S Weinberg, Matthew J A WoodAbstract:Mirtrons, short hairpin pre-microRNA (miRNA) mimics directly produced by intronic splicing, have recently been identified and experimentally confirmed in invertebrates. While there is evidence to suggest several mammalian miRNAs have Mirtron origins, this has yet to be experimentally demonstrated. Here, we characterize the biogenesis of mammalian Mirtrons by ectopic expression of splicing-dependent Mirtron precursors. The putative Mirtrons hsa-miR-877, hsa-miR-1226 and mmu-miR-1224 were designed as introns within eGFP. Correct splicing and function of these sequences as introns was shown through eGFP fluorescence and RT–PCR, while all Mirtrons suppressed perfectly complementary luciferase reporter targets to levels similar to that of corresponding independently expressed pre-miRNA controls. Splicingdeficient mutants and disruption of key steps in miRNA biogenesis demonstrated that Mirtronmediated gene knockdown was splicing-dependent, Drosha-independent and had variable dependence on RNAi pathway elements following pre-miRNA formation. The silencing effect of hsa-miR-877 was further demonstrated to be mediated by the generation of short anti-sense RNA species expressed with low abundance. Finally, the mammalian Mirtron hsa-miR-877 was shown to reduce mRNA levels of an endogenous transcript containing hsa-miR-877 target sites in neuronal SH-SY5Y cells. This work confirms the Mirtron origins of three mammalian miRNAs and suggests that they are a functional class of splicing-dependent miRNAs which are physiologically active.
Pilar Bustossanmamed - One of the best experts on this subject based on the ideXlab platform.
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the small rna diversity from medicago truncatula roots under biotic interactions evidences the environmental plasticity of the mirnaome
Genome Biology, 2014Co-Authors: Damien Formey, Erika Sallet, Christine Lelandaisbriere, Cecile Ben, Pilar BustossanmamedAbstract:Background: Legume roots show a remarkable plasticity to adapt their architecture to biotic and abiotic constraints, including symbiotic interactions. However, global analysis of miRNA regulation in roots is limited, and a global view of the evolution of miRNA-mediated diversification in different ecotypes is lacking. Results: In the model legume Medicago truncatula, we analyze the small RNA transcriptome of roots submitted to symbiotic and pathogenic interactions. Genome mapping and a computational pipeline identify 416 miRNA candidates, including known and novel variants of 78 miRNA families present in miRBase. Stringent criteria of pre-miRNA prediction yield 52 new mtr-miRNAs, including 27 Mirtrons. Analyzing miRNA precursor polymorphisms in 26 M. truncatula ecotypes identifies higher sequence polymorphism in conserved rather than Medicago-specific miRNA precursors. An average of 19 targets, mainly involved in environmental responses and signalling, is predicted per novel miRNA. We identify miRNAs responsive to bacterial and fungal pathogens or symbionts as well as their related Nod and Myc-LCO symbiotic signals. Network analyses reveal modules of new and conserved co-expressed miRNAs that regulate distinct sets of targets, highlighting potential miRNA-regulated biological pathways relevant to pathogenic and symbiotic interactions. Conclusions: We identify 52 novel genuine miRNAs and large plasticity of the root miRNAome in response to the environment, and also in response to purified Myc/Nod signaling molecules. The new miRNAs identified and their sequence variation across M. truncatula ecotypes may be crucial to understand the adaptation of root growth to the soil environment, notably in the agriculturally important legume crops.
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the small rna diversity from medicago truncatula roots under biotic interactions evidences the environmental plasticity of the mirnaome
Genome Biology, 2014Co-Authors: Damien Formey, Erika Sallet, Christine Lelandaisbriere, Cecile Ben, Pilar BustossanmamedAbstract:Legume roots show a remarkable plasticity to adapt their architecture to biotic and abiotic constraints, including symbiotic interactions. However, global analysis of miRNA regulation in roots is limited, and a global view of the evolution of miRNA-mediated diversification in different ecotypes is lacking. In the model legume Medicago truncatula, we analyze the small RNA transcriptome of roots submitted to symbiotic and pathogenic interactions. Genome mapping and a computational pipeline identify 416 miRNA candidates, including known and novel variants of 78 miRNA families present in miRBase. Stringent criteria of pre-miRNA prediction yield 52 new mtr-miRNAs, including 27 Mirtrons. Analyzing miRNA precursor polymorphisms in 26 M. truncatula ecotypes identifies higher sequence polymorphism in conserved rather than Medicago-specific miRNA precursors. An average of 19 targets, mainly involved in environmental responses and signalling, is predicted per novel miRNA. We identify miRNAs responsive to bacterial and fungal pathogens or symbionts as well as their related Nod and Myc-LCO symbiotic signals. Network analyses reveal modules of new and conserved co-expressed miRNAs that regulate distinct sets of targets, highlighting potential miRNA-regulated biological pathways relevant to pathogenic and symbiotic interactions. We identify 52 novel genuine miRNAs and large plasticity of the root miRNAome in response to the environment, and also in response to purified Myc/Nod signaling molecules. The new miRNAs identified and their sequence variation across M. truncatula ecotypes may be crucial to understand the adaptation of root growth to the soil environment, notably in the agriculturally important legume crops.
Timothy E Saunders - One of the best experts on this subject based on the ideXlab platform.
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the Mirtron mir 1010 functions in concert with its host gene skip to balance elevation of nacrβ2
Scientific Reports, 2020Co-Authors: Christopher Amourda, Timothy E SaundersAbstract:Mirtrons are non-canonical miRNAs arising by splicing and debranching from short introns. A plethora of introns have been inferred by computational analyses as potential Mirtrons. Yet, few have been experimentally validated and their functions, particularly in relation to their host genes, remain poorly understood. Here, we found that Drosophila larvae lacking either the Mirtron miR-1010 or its binding site in the nicotinic acetylcholine receptor β2 (nAcRβ2) 3′UTR fail to grow properly and pupariate. Increase of cortical nAcRβ2 mediated by neural activity elevates the level of intracellular Ca2+, which in turn activates CaMKII and, further downstream, the transcription factor Adf-1. We show that miR-1010 downregulates nAcRβ2. We reveal that Adf-1 initiates the expression of SKIP, the host gene of miR-1010. Preventing synaptic potentials from overshooting their optimal range requires both SKIP to temper synaptic potentials (incoherent feedforward loop) and miR-1010 to reduce nAcRβ2 mRNA levels (negative feedback loop). Our results demonstrate how a Mirtron, in coordination with its host gene, contributes to maintaining appropriate receptor levels, which in turn may play a role in maintaining homeostasis.
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the Mirtron mir 1010 functions in concert with its host gene skip to maintain synaptic homeostasis
Social Science Research Network, 2018Co-Authors: Christopher Amourda, Timothy E SaundersAbstract:Mirtrons are non-canonical miRNAs arising by splicing and debranching from short introns. A plethora of introns have been inferred by computational analyses as potential Mirtrons. Yet, few have been experimentally validated and their functions, particularly in relation to their host genes, remain poorly understood. Here, we found that larvae lacking the Mirtron miR-1010 are unable to grow properly and pupariate. We show that miR-1010 downregulates nAcRβ2. Increase of cortical nAcRb2 mediated by neural activity elevates the level of intracellular Ca2 , which in turn activates CaMKII and, further downstream, the transcription factor Adf-1. We reveal that Adf-1 initiates the expression of SKIP, the host gene of miR-1010. Preventing synaptic potentials from overshooting their optimal range requires both SKIP to temper synaptic potentials (incoherent feedforward loop) and miR-1010 to reduce nAcRβ2 mRNA levels (negative feedback loop). Our results demonstrate how a Mirtron, in coordination with its host gene, contributes to maintaining homeostasis.
Eric C Lai - One of the best experts on this subject based on the ideXlab platform.
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A Drosophila pasha mutant distinguishes the canonical microRNA and Mirtron pathways.
Molecular and cellular biology, 2008Co-Authors: Raquel Martin, Peter Smibert, David M Tyler, Abdullah Yalcin, Ulrich Schafer, Thomas Tuschl, Eric C LaiAbstract:Canonical primary microRNA (miRNA) transcripts and Mirtrons are proposed to transit distinct nuclear pathways en route to generating mature approximately 22 nucleotide regulatory RNAs. We generated a null allele of Drosophila pasha, which encodes a double-stranded RNA-binding protein partner of the RNase III enzyme Drosha. Analysis of this mutant yielded stringent evidence that Pasha is essential for the biogenesis of canonical miRNAs but is dispensable for the processing and function of Mirtron-derived regulatory RNAs. The pasha mutant also provided a unique tool to study the developmental requirements for Drosophila miRNAs. While pasha adult somatic clones are similar in many respects to those of dicer-1 clones, pasha mutant larvae revealed an unexpected requirement for the miRNA pathway in imaginal disc growth. These data suggest limitations to somatic clonal analysis of miRNA pathway components.
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mammalian Mirtron genes
Molecular Cell, 2007Co-Authors: Eugene Berezikov, Wei Jen Chung, Jason Willis, Edwin Cuppen, Eric C LaiAbstract:Mirtrons are alternative precursors for microRNA biogenesis that were recently described in invertebrates. These short hairpin introns use splicing to bypass Drosha cleavage, which is otherwise essential for the generation of canonical animal microRNAs. Using computational and experimental strategies, we now establish that mammals have Mirtrons as well. We identified 3 Mirtrons that are well conserved and expressed in diverse mammals, 16 primate-specific Mirtrons, and 46 candidates supported by limited cloning evidence in primates. As with some fly and worm Mirtrons, the existence of well-conserved mammalian Mirtrons indicates their relatively ancient incorporation into endogenous regulatory pathways. However, as worms, flies, and mammals each have different sets of Mirtrons, we hypothesize that different animals may have independently evolved the capacity for this hybrid small RNA pathway. This notion is supported by our observation of several clade-specific features of mammalian and invertebrate Mirtrons.
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the Mirtron pathway generates microrna class regulatory rnas in drosophila
Cell, 2007Co-Authors: Katsutomo Okamura, Joshua W Hagen, Hong Duan, David M Tyler, Eric C LaiAbstract:Summary The canonical microRNA (miRNA) pathway converts primary hairpin precursor transcripts into ∼22 nucleotide regulatory RNAs via consecutive cleavages by two RNase III enzymes, Drosha and Dicer. In this study, we characterize Drosophila small RNAs that derive from short intronic hairpins termed "Mirtrons." Their nuclear biogenesis appears to bypass Drosha cleavage, which is essential for miRNA biogenesis. Instead, Mirtron hairpins are defined by the action of the splicing machinery and lariat-debranching enzyme, which yield pre-miRNA-like hairpins. The Mirtron pathway merges with the canonical miRNA pathway during hairpin export by Exportin-5, and both types of hairpins are subsequently processed by Dicer-1/loqs. This generates small RNAs that can repress perfectly matched and seed-matched targets, and we provide evidence that they function, at least in part, via the RNA-induced silencing complex effector Ago1. These findings reveal that Mirtrons are an alternate source of miRNA-type regulatory RNAs.
Christopher R. Sibley - One of the best experts on this subject based on the ideXlab platform.
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silencing of parkinson s disease associated genes with artificial Mirtron mimics of mir 1224
Nucleic Acids Research, 2012Co-Authors: Christopher R. Sibley, Yiqi Seow, Marc S Weinberg, Helen J Curtis, Matthew J A WoodAbstract:Mirtrons are a recently described category of microRNA (miRNA) relying on splicing rather than processing by the microprocessor complex to generate pre-miRNA precursors of the RNA interference (RNAi) pathway. Their discovery and subsequent verification provides important information about a distinct class of miRNA and inherent advantages that could be exploited to silence genes of interest. These include micro-processor-independent biogenesis, pol-II-dependent transcription, accurate species generation and the delivery of multiple artificial Mirtrons as introns within a single host transcript. Here we determined the sequence motifs required for correct processing of the mmu-miR-1224 Mirtron and incorporated these into artificial Mirtrons targeting Parkinson's disease-associated LRRK2 and α-synuclein genes. By incorporating these rules associated with processing and splicing, artificial Mirtrons could be designed and made to silence complementary targets either at the mRNA or protein level. We further demonstrate with a LRRK2 targeting artificial Mirtron that neuronal-specific silencing can be directed under the control of the human synapsin promoter. Finally, multiple Mirtrons were co-delivered within a single host transcript, an eGFP reporter, to allow simultaneous targeting of two or more targets in a combinatorial approach. Thus, the unique characteristics of artificial Mirtrons make this an attractive approach for future RNAi applications. © 2012 The Author(s).
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Artificial Mirtron-mediated gene knockdown: Functional DMPK silencing in mammalian cells
RNA, 2012Co-Authors: Yiqi Seow, Christopher R. Sibley, Matthew J A WoodAbstract:Mirtrons are introns that form pre-miRNA hairpins after splicing to produce RNA interference (RNAi) effectors distinct from Drosha-dependent intronic miRNAs. Here we present a design algorithm for artificial Mirtrons and demonstrate, for the first time, efficient gene knockdown of myotonic dystrophy protein kinase (DMPK) target sequences in Renilla luciferase 3' UTR and subsequently pathogenic DMPK mRNA, causative of Type I myotonic dystrophy, using artificial Mirtrons cloned as eGFP introns. Deep sequencing of artificial Mirtrons suggests that functional mature transcripts corresponding to the designed sequence were produced in high abundance. They were further shown to be splicing-dependent, Drosha-independent, and partially dependent on exportin-5, resulting in the precise generation of pre-miRNAs. In a murine myoblast line containing a pathogenic copy of human DMPK with more than 500 CUG repeats, the DMPK artificial Mirtron corrected DM1-associated splicing abnormalities of the Serca-1 mRNA, demonstrating the therapeutic potential of Mirtron-mediated RNAi. Thus, further development and exploitation of the unique properties of Mirtrons will benefit future research and therapeutic RNAi applications as an alternative to conventional RNAi strategies.
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the biogenesis and characterization of mammalian micrornas of Mirtron origin
Nucleic Acids Research, 2012Co-Authors: Christopher R. Sibley, Yiqi Seow, Sheena Saayman, Krijn K Dijkstra, Samir El Andaloussi, Marc S Weinberg, Matthew J A WoodAbstract:Mirtrons, short hairpin pre-microRNA (miRNA) mimics directly produced by intronic splicing, have recently been identified and experimentally confirmed in invertebrates. While there is evidence to suggest several mammalian miRNAs have Mirtron origins, this has yet to be experimentally demonstrated. Here, we characterize the biogenesis of mammalian Mirtrons by ectopic expression of splicing-dependent Mirtron precursors. The putative Mirtrons hsa-miR-877, hsa-miR-1226 and mmu-miR-1224 were designed as introns within eGFP. Correct splicing and function of these sequences as introns was shown through eGFP fluorescence and RT–PCR, while all Mirtrons suppressed perfectly complementary luciferase reporter targets to levels similar to that of corresponding independently expressed pre-miRNA controls. Splicingdeficient mutants and disruption of key steps in miRNA biogenesis demonstrated that Mirtronmediated gene knockdown was splicing-dependent, Drosha-independent and had variable dependence on RNAi pathway elements following pre-miRNA formation. The silencing effect of hsa-miR-877 was further demonstrated to be mediated by the generation of short anti-sense RNA species expressed with low abundance. Finally, the mammalian Mirtron hsa-miR-877 was shown to reduce mRNA levels of an endogenous transcript containing hsa-miR-877 target sites in neuronal SH-SY5Y cells. This work confirms the Mirtron origins of three mammalian miRNAs and suggests that they are a functional class of splicing-dependent miRNAs which are physiologically active.