Noncoding RNA
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DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs

CRISPR genome-editing technology makes it possible to quickly and cheaply delete non-protein-coding regulatory elements. We present a vector system adapted for this purpose called DECKO (Double Excision CRISPR Knockout), which applies a simple two-step cloning to generate lentiviral vectors expressing two guide RNAs (gRNAs) simultaneously. The key feature of DECKO is its use of a single 165 bp starting oligonucleotide carrying the variable sequences of both gRNAs, making it fully scalable from single-locus studies to complex library cloning.

Via Biswapriya Biswavas Misra
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Biswapriya Biswavas Misra's curator insight, October 25, 2015 3:16 PM
Abstract
Background

CRISPR genome-editing technology makes it possible to quickly and cheaply delete non-protein-coding regulatory elements. We present a vector system adapted for this purpose called DECKO (Double Excision CRISPR Knockout), which applies a simple two-step cloning to generate lentiviral vectors expressing two guide RNAs (gRNAs) simultaneously. The key feature of DECKO is its use of a single 165 bp starting oligonucleotide carrying the variable sequences of both gRNAs, making it fully scalable from single-locus studies to complex library cloning.

Results

We apply DECKO to deleting the promoters of one protein-coding gene and two oncogenic lncRNAs, UCA1 and the highly-expressed MALAT1, focus of many previous studies employing RNA interference approaches. DECKO successfully deleted genomic fragments ranging in size from 100 to 3000 bp in four human cell lines. Using a clone-derivation workflow lasting approximately 20 days, we obtained 9 homozygous and 17 heterozygous promoter knockouts in three human cell lines. Frequent target region inversions were observed. These clones have reductions in steady-state MALAT1 RNA levels of up to 98 % and display reduced proliferation rates.

Conclusions

We present a dual CRISPR tool, DECKO, which is cloned using a single starting oligonucleotide, thereby affording simplicity and scalability to CRISPR knockout studies of non-coding genomic elements, including long non-coding RNAs.

Keywords:

CRISPR; Genome editing; DECKO; Long non-coding RNA; lncRNA

 
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Small RNA profiling reveals regulation of Arabidopsis miR168 and heterochromatic siRNA415 in response to fungal elicitors

Small RNA profiling reveals regulation of Arabidopsis miR168 and heterochromatic siRNA415 in response to fungal elicitors | Noncoding RNA | Scoop.it
“ Background Small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), have emerged as important regulators of eukaryotic gene expression. In plants, miRNAs play critical roles in development, nutrient homeostasis and abiotic stress responses. Accumulating evidence also reveals that sRNAs are involved in plant immunity. Most studies on pathogen-regulated sRNAs have been conducted in Arabidopsis plants infected with the bacterial pathogen Pseudomonas syringae, or treated with the flagelin-derived elicitor peptide flg22 from P. syringae. This work investigates sRNAs that are regulated by elicitors from the fungus Fusarium oxysporum in Arabidopsis. Results Microarray analysis revealed alterations on the accumulation of a set of sRNAs in response to elicitor treatment, including miRNAs and small RNA sequences derived from massively parallel signature sequencing. Among the elicitor-regulated miRNAs was miR168 which regulates ARGONAUTE1, the core component of the RNA-induced silencing complex involved in miRNA functioning. Promoter analysis in transgenic Arabidopsis plants revealed transcriptional activation of MIR168 by fungal elicitors. Furthermore, transgenic plants expressing a GFP-miR168 sensor gene confirmed that the elicitor-induced miR168 is active. MiR823, targeting Chromomethylase3 (CMT3) involved in RNA-directed DNA methylation (RdDM) was also found to be regulated by fungal elicitors. In addition to known miRNAs, microarray analysis allowed the identification of an elicitor-inducible small RNA that was incorrectly annotated as a miRNA. Studies on Arabidopsis mutants impaired in small RNA biogenesis demonstrated that this sRNA, is a heterochromatic-siRNA (hc-siRNA) named as siRNA415. Hc-siRNAs are known to be involved in RNA-directed DNA methylation (RdDM). SiRNA415 is detected in several plant species. Conclusion Results here presented support a transcriptional regulatory mechanism underlying MIR168 expression. This finding highlights the importance of miRNA functioning in adaptive processes of Arabidopsis plants to fungal infection. The results of this study also lay a foundation for the involvement of RdDM processes through the activity of siRNA415 and miR823 in mediating regulation of immune responses in Arabidopsis plants.”
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Repression of microRNA biogenesis by silencing of OsDCL1 activates the basal resistance to Magnaporthe oryzae in rice

“ Highlights • OsDCL1 RNAi lines showed enhanced resistance to rice blast. • A negative feedback loop between miR162a and OsDCL1 was identified. • Differentially expressed miRNAs responsive to rice blast infection were identified. • PR and PTI responsive genes were constitutively activated in OsDCL1 RNAi lines. Abstract The RNaseIII enzyme Dicer-like 1 (DCL1) processes the microRNA biogenesis and plays a determinant role in plant development. In this study, we reported the function of OsDCL1 in the immunity to rice blast, the devastating disease caused by the fungal pathogen, Magnaporthe oryzae. Expression profiling demonstrated that different OsDCLs responded dynamically and OsDCL1 reduced its expression upon the challenge of rice blast pathogen. In contrast, miR162a predicted to target OsDCL1 increased its expression, implying a negative feedback loop between OsDCL1 and miR162a in rice. In addition to developmental defects, the OsDCL1-silencing mutants showed enhanced resistance to virulent rice blast strains in a non-race specific manner. Accumulation of hydrogen peroxide and cell death were observed in the contact cells with infectious hyphae, revealing that silencing of OsDCL1 activated cellular defense responses. In OsDCL1 RNAi lines, 12 differentially expressed miRNAs were identified, of which 5 and 7 were down- and up-regulated, respectively, indicating that miRNAs responded dynamically in the interaction between rice and rice blast. Moreover, silencing of OsDCL1 activated the constitutive expression of defense related genes. Taken together, our results indicate that rice is capable of activating basal resistance against rice blast by perturbing OsDCL1-dependent miRNA biogenesis pathway.”
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Elicitation of hypersensitive responses in Nicotiana glutinosa by the suppressor of RNA silencing protein P0 from poleroviruses

Elicitation of hypersensitive responses in Nicotiana glutinosa by the suppressor of RNA silencing protein P0 from poleroviruses | Noncoding RNA | Scoop.it
“ Plant disease resistance (R) proteins that confer resistance to viruses recognize viral gene products with diverse functions, including viral suppressors of RNA silencing (VSRs). The P0 protein from poleroviruses is a VSR that targets the ARGONAUTE1 (AGO1) protein for degradation, thereby disrupting RNA silencing and antiviral defences. Here, we report resistance against poleroviruses in Nicotiana glutinosa directed against Turnip yellows virus (TuYV) and Potato leafroll virus (PLRV). The P0 proteins from TuYV (P0Tu), PLRV (P0PL) and Cucurbit aphid-borne yellows virus (P0CA) were found to elicit a hypersensitive response (HR) in N. glutinosa accession TW59, whereas other accessions recognized P0PL only. Genetic analysis showed that recognition of P0Tu by a resistance gene designated RPO1 (Resistance to POleroviruses 1) is inherited as a dominant allele. Expression of P0 from a Potato virus X (PVX) expression vector transferred recognition to the recombinant virus on plants expressing RPO1, supporting P0 as the unique Polerovirus factor eliciting resistance. The induction of HR required a functional P0 protein, as P0Tu mutants with substitutions in the F-box motif that abolished VSR activity were unable to elicit HR. We surmised that the broad P0 recognition seen in TW59 and the requirement for the F-box protein motif could indicate detection of P0-induced AGO1 degradation and disruption of RNA silencing; however, other viral silencing suppressors, including the PVX P25 that also causes AGO1 degradation, failed to elicit HR in N. glutinosa. Investigation of P0 elicitation of RPO1 could provide insight into P0 activities within the cell that trigger resistance.”
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High-throughput sequencing reveals differential expression of miRNAs in tomato inoculated with Phytophthora infestans - Springer

High-throughput sequencing reveals differential expression of miRNAs in tomato inoculated with Phytophthora infestans - Springer | Noncoding RNA | Scoop.it
Main conclusion
The characterization and compare expression profiling of the miRNA transcriptome lay a solid foundation for unraveling the complex miRNA-mediated regulatory network in tomato resistance mechanisms against LB.
MicroRNAs (miRNAs) are a class of small endogenous non-coding RNAs with 20–24 nt. They have been identified in many plants with their diverse regulatory roles in biotic stresses. The knowledge, that miRNAs regulate late blight (LB), caused by Phytophthora infestans, is rather limited. In this study, we used miRNA-Seq to investigate the miRNA expression difference between the tomatoes treated with and without P. infestans. A total of 42,714,516 raw reads were generated from two small RNA libraries by high-throughput sequencing. Finally, 207 known miRNAs and 67 new miRNAs were obtained. The differential expression profile of miRNAs in tomato was further analyzed with twofold change (P value ≤0.01). A total of 70 miRNAs were manifested to change significantly in samples treated with P. infestans, including 50 down-regulated miRNAs and 20 up-regulated miRNAs. Moreover, a total of 73 target genes were acquired for 28 differentially expressed miRNAs by psRNATarget analysis. By enrichment pathway analysis of target genes, plant–pathogen interaction was the most highly relevant pathway which played an important role in disease defense. In addition, 30 miRNAs were selected for qRT-PCR to validate their expression patterns. The expression patterns for targets of miR6027, miR5300, miR476b, miR159a, miR164a and miRn13 were selectively examined, and the results showed that there was a negative correlation on the expression patterns between miRNAs and their targets. The targets have previously been reported to be related with plant immune and involved in plant–pathogen interaction pathway in this study, suggesting these miRNAs might act as regulators in process of tomato resistance against P. infestans. These discoveries will provide us useful information to explain tomato resistance mechanisms against LB.
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TUT7 controls the fate of precursor microRNAs by using three different uridylation mechanisms. - PubMed - NCBI

TUT7 controls the fate of precursor microRNAs by using three different uridylation mechanisms. - PubMed - NCBI | Noncoding RNA | Scoop.it
EMBO J. 2015 May 15. pii: e201590931. [Epub ahead of print]
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Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis. Using biochemistry, single-molecule, and deep sequencing techniques, we here investigate the mechanism by which human TUT7 (also known as ZCCHC6) recognizes and uridylates precursor miRNAs (pre-miRNAs) in the absence of Lin28. We find that the overhang of a pre-miRNA is the key structural element that is recognized by TUT7 and its paralogues, TUT4 (ZCCHC11) and TUT2 (GLD2/PAPD4). For group II pre-miRNAs, which have a 1-nt 3' overhang, TUT7 restores the canonical end structure (2-nt 3' overhang) through mono-uridylation, thereby promoting miRNA biogenesis. For pre-miRNAs where the 3' end is further recessed into the stem (as in 3' trimmed pre-miRNAs), TUT7 generates an oligo-U tail that leads to degradation. In contrast to Lin28-stimulated oligo-uridylation, which is processive, a distributive mode is employed by TUT7 for both mono- and oligo-uridylation in the absence of Lin28. The overhang length dictates the frequency (but not duration) of the TUT7-RNA interaction, thus explaining how TUT7 differentiates pre-miRNA species with different overhangs. Our study reveals dual roles and mechanisms of uridylation in repair and removal of defective pre-miRNAs.

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microRNA167-directed regulation of the auxin response factors, GmARF8a and GmARF8b, is required for soybean (Glycine max L.) nodulation and lateral root development

“ Legumes root nodules convert atmospheric nitrogen gas (N2) into ammonium through symbiosis with a prokaryotic microsymbiont broadly called 'rhizobia'. Auxin signaling is required for determinant nodule development; however, the molecular mechanism of auxin-mediated nodule formation remains largely unknown. Here we show in soybean (Glycine max L. Merr) that microRNA (miRNA) miR167 acts as a positive regulator of lateral root organs, namely nodules and lateral roots. miR167c expression was up-regulated in the vasculature, pericycle and cortex of soybean roots following inoculation with Bradyrhizobium japonicum strain USDA110 (the microsymbiont). It was found to positively regulate nodule numbers directly by repressing the target genes GmARF8a and GmARF8b (homologous genes of Arabidopsis AtARF8 that encode auxin response factors). Moreover, the expression of miR167 and its targets were up- or down-regulated by auxin, respectively. The miR167-GmARF8 module also positively regulated nodulation efficiency under low microsymbiont density, a condition often associated with environmental stress. The regulatory role of miR167 on nodule initiation was dependent on the Nod factor receptor GmNFR1α, and it acts upstream of the nodulation-associated genes GmNIN, GmNSP1, GmENOD40-1, GmHAP2-1 and GmHAP2-2. miR167 also promoted lateral root numbers. Collectively, our findings establish a key role for the miR167-GmARF8 module in auxin-mediated nodule and lateral root formation in soybean.”
Via Jean-Michel Ané
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Yale lab uncovers new pathway for passing genetic messages between cells

Yale lab uncovers new pathway for passing genetic messages between cells | Noncoding RNA | Scoop.it

A Yale-led research team has described a novel pathway for the delivery of microRNA (miRNA), the tiny RNA molecules that can move between cells to regulate gene expression.


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PNAS: Phytophthora effector targets a novel component of small RNA pathway in plants to promote infection (2015)

PNAS: Phytophthora effector targets a novel component of small RNA pathway in plants to promote infection (2015) | Noncoding RNA | Scoop.it

Phytophthora is a major threat to agriculture. However, the molecular interaction of these severe pathogens with plant hosts is poorly understood. Here, we report that the Phytophthora Suppressor of RNA Silencing 1 (PSR1) effectively promotes infection in Arabidopsis thaliana by directly targeting an essential protein containing a aspartate–glutamate–alanine–histidine-box RNA helicase domain. This PSR1-Interacting Protein 1 (PINP1) is required for the accumulation of distinct classes of endogenous small RNAs and acts as a positive regulator of plant immunity. Silencing of PINP1 impaired the assembly of microRNA-processing complexes in the nucleus, leading to defects in development and immunity. This study revealed a conserved RNA helicase as a regulator of RNA silencing and provides mechanistic insight into Phytophthora pathogenesis.


Via Kamoun Lab @ TSL
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A Non-canonical RNA Silencing Pathway Promotes mRNA Degradation in Basal Fungi

A Non-canonical RNA Silencing Pathway Promotes mRNA Degradation in Basal Fungi | Noncoding RNA | Scoop.it
“ The increasing knowledge on the functional relevance of endogenous small RNAs (esRNAs) as riboregulators has stimulated the identification and characterization of these molecules in numerous eukaryotes. In the basal fungus Mucor circinelloides, an emerging opportunistic human pathogen, esRNAs that regulate the expression of many protein coding genes have been described. These esRNAs share common machinery for their biogenesis consisting of an RNase III endonuclease Dicer, a single Argonaute protein and two RNA-dependent RNA polymerases. We show in this study that, besides participating in this canonical dicer-dependent RNA interference (RNAi) pathway, the rdrp genes are involved in a novel dicer-independent degradation process of endogenous mRNAs. The analysis of esRNAs accumulated in wild type and silencing mutants demonstrates that this new rdrp-dependent dicer-independent regulatory pathway, which does not produce sRNA molecules of discrete sizes, controls the expression of target genes promoting the specific degradation of mRNAs by a previously unknown RNase. This pathway mainly regulates conserved genes involved in metabolism and cellular processes and signaling, such as those required for heme biosynthesis, and controls responses to specific environmental signals. Searching the Mucor genome for candidate RNases to participate in this pathway, and functional analysis of the corresponding knockout mutants, identified a new protein, R3B2. This RNase III-like protein presents unique domain architecture, it is specifically found in basal fungi and, besides its relevant role in the rdrp-dependent dicer-independent pathway, it is also involved in the canonical dicer-dependent RNAi pathway, highlighting its crucial role in the biogenesis and function of regulatory esRNAs. The involvement of RdRPs in RNA degradation could represent the first evolutionary step towards the development of an RNAi mechanism and constitutes a genetic link between mRNA degradation and post-transcriptional gene silencing.”
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Two-Faced RNAs | The Scientist Magazine®

Two-Faced RNAs | The Scientist Magazine® | Noncoding RNA | Scoop.it
The same microRNAs can have opposing roles in cancer.

Via Integrated DNA Technologies
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Transcription Factors and microRNA-Co-Regulated Genes in Gastric Cancer Invasion in Ex Vivo

Transcription Factors and microRNA-Co-Regulated Genes in Gastric Cancer Invasion in  Ex Vivo | Noncoding RNA | Scoop.it
Aberrant miRNA expression abnormally modulates gene expression in cells and can contribute to tumorigenesis in humans.

Via Krishan Maggon
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Krishan Maggon 's curator insight, April 11, 2015 10:39 AM

Citation: Shi Y, Wang J, Xin Z, Duan Z, Wang G, et al. (2015) Transcription Factors and microRNA-Co-Regulated Genes in Gastric Cancer Invasion in Ex Vivo. PLoS ONE 10(4): e0122882. doi:10.1371/journal.pone.0122882

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Suppression of endogenous gene silencing by bidirectional cytoplasmic RNA decay in Arabidopsis

Plant immunity against foreign gene invasion takes advantage of posttranscriptional gene silencing (PTGS). How plants elaborately avert inappropriate PTGS of endogenous coding genes remains unclear. We demonstrate in Arabidopsis that both 5′-3′ and 3′-5′ cytoplasmic RNA decay pathways act as repressors of transgene and endogenous PTGS. Disruption of bidirectional cytoplasmic RNA decay leads to pleiotropic developmental defects and drastic transcriptomic alterations, which are substantially rescued by PTGS mutants. Upon dysfunction of bidirectional RNA decay, a large number of 21- to 22-nucleotide endogenous small interfering RNAs are produced from coding transcripts, including multiple microRNA targets, which could interfere with their cognate gene expression and functions. This study highlights the risk of unwanted PTGS and identifies cytoplasmic RNA decay pathways as safeguards of plant transcriptome and development.

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A Non-canonical RNA Silencing Pathway Promotes mRNA Degradation in Basal Fungi

A Non-canonical RNA Silencing Pathway Promotes mRNA Degradation in Basal Fungi | Noncoding RNA | Scoop.it
The increasing knowledge on the functional relevance of endogenous small RNAs (esRNAs) as riboregulators has stimulated the identification and characterization of these molecules in numerous eukaryotes. In the basal fungus Mucor circinelloides, an emerging opportunistic human pathogen, esRNAs that regulate the expression of many protein coding genes have been described. These esRNAs share common machinery for their biogenesis consisting of an RNase III endonuclease Dicer, a single Argonaute protein and two RNA-dependent RNA polymerases. We show in this study that, besides participating in this canonical dicer-dependent RNA interference (RNAi) pathway, the rdrp genes are involved in a novel dicer-independent degradation process of endogenous mRNAs. The analysis of esRNAs accumulated in wild type and silencing mutants demonstrates that this new rdrp-dependent dicer-independent regulatory pathway, which does not produce sRNA molecules of discrete sizes, controls the expression of target genes promoting the specific degradation of mRNAs by a previously unknown RNase. This pathway mainly regulates conserved genes involved in metabolism and cellular processes and signaling, such as those required for heme biosynthesis, and controls responses to specific environmental signals. Searching the Mucor genome for candidate RNases to participate in this pathway, and functional analysis of the corresponding knockout mutants, identified a new protein, R3B2. This RNase III-like protein presents unique domain architecture, it is specifically found in basal fungi and, besides its relevant role in the rdrp-dependent dicer-independent pathway, it is also involved in the canonical dicer-dependent RNAi pathway, highlighting its crucial role in the biogenesis and function of regulatory esRNAs. The involvement of RdRPs in RNA degradation could represent the first evolutionary step towards the development of an RNAi mechanism and constitutes a genetic link between mRNA degradation and post-transcriptional gene silencing.

Via Francis Martin
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Widespread Polycistronic Transcripts in Fungi Revealed by Single-Molecule mRNA Sequencing

Widespread Polycistronic Transcripts in Fungi Revealed by Single-Molecule mRNA Sequencing | Noncoding RNA | Scoop.it
“ Genes in prokaryotic genomes are often arranged into clusters and co-transcribed into polycistronic RNAs. Isolated examples of polycistronic RNAs were also reported in some higher eukaryotes but their presence was generally considered rare. Here we developed a long-read sequencing strategy to identify polycistronic transcripts in several mushroom forming fungal species including Plicaturopsis crispa, Phanerochaete chrysosporium, Trametes versicolor, and Gloeophyllum trabeum. We found genome-wide prevalence of polycistronic transcription in these Agaricomycetes, involving up to 8% of the transcribed genes. Unlike polycistronic mRNAs in prokaryotes, these co-transcribed genes are also independently transcribed. We show that polycistronic transcription may interfere with expression of the downstream tandem gene. Further comparative genomic analysis indicates that polycistronic transcription is conserved among a wide range of mushroom forming fungi. In summary, our study revealed, for the first time, the genome prevalence of polycistronic transcription in a phylogenetic range of higher fungi. Furthermore, we systematically show that our long-read sequencing approach and combined bioinformatics pipeline is a generic powerful tool for precise characterization of complex transcriptomes that enables identification of mRNA isoforms not recovered via short-read assembly.”
Via Francis Martin
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Frontiers | Elongator and its epigenetic role in plant development and responses to abiotic and biotic stresses | Plant Physiology

Frontiers | Elongator and its epigenetic role in plant development and responses to abiotic and biotic stresses | Plant Physiology | Noncoding RNA | Scoop.it
“ Elongator, a six-subunit protein complex, was initially isolated as an interactor of hyperphosphorylated RNA polymerase II in yeast, and was subsequently identified in animals and plants. Elongator has been implicated in multiple cellular activities or biological processes including tRNA modification, histone modification, DNA demethylation or methylation, tubulin acetylation, and exocytosis. Studies in the model plant Arabidopsis thaliana suggest that the structure of Elongator and its functions are highly conserved between plants and yeast. Disruption of the Elongator complex in plants leads to aberrant growth and development, resistance to abiotic stresses, and susceptibility to plant pathogens. The morphological and physiological phenotypes of Arabidopsis Elongator mutants are associated with decreased histone acetylation and/or altered DNA methylation. This review summarizes recent findings related to the epigenetic function of Elongator in plant development and responses to abiotic and biotic stresses.”
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Cell: A Receptor Pair with an Integrated Decoy Converts Pathogen Disabling of Transcription Factors to Immunity (2015)

Cell: A Receptor Pair with an Integrated Decoy Converts Pathogen Disabling of Transcription Factors to Immunity (2015) | Noncoding RNA | Scoop.it

Microbial pathogens infect host cells by delivering virulence factors (effectors) that interfere with defenses. In plants, intracellular nucleotide-binding/leucine-rich repeat receptors (NLRs) detect specific effector interference and trigger immunity by an unknown mechanism. The Arabidopsis-interacting NLR pair, RRS1-R with RPS4, confers resistance to different pathogens, including Ralstonia solanacearum bacteria expressing the acetyltransferase effector PopP2. We show that PopP2 directly acetylates a key lysine within an additional C-terminal WRKY transcription factor domain of RRS1-R that binds DNA. This disrupts RRS1-R DNA association and activates RPS4-dependent immunity. PopP2 uses the same lysine acetylation strategy to target multiple defense-promoting WRKY transcription factors, causing loss of WRKY-DNA binding and transactivating functions needed for defense gene expression and disease resistance. Thus, RRS1-R integrates an effector target with an NLR complex at the DNA to switch a potent bacterial virulence activity into defense gene activation.

Clémentine Le Roux, Gaëlle Huet, Alain Jauneau, Laurent Camborde, Dominique Trémousaygue, Alexandra Kraut, Binbin Zhou, Marie Levaillant, Hiroaki Adachi, Hirofumi Yoshioka, Sylvain Raffaele, Richard Berthomé, Yohann Couté, Jane E. Parker, Laurent Deslandes


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Identification and functional characterization of soybean root hair microRNAs expressed in response to Bradyrhizobium japonicum infection - Yan - 2015 - Plant Biotechnology Journal - Wiley Online L...

Identification and functional characterization of soybean root hair microRNAs expressed in response to Bradyrhizobium japonicum infection - Yan - 2015 - Plant Biotechnology Journal - Wiley Online L... | Noncoding RNA | Scoop.it
Three soybean [Glycine max (L) Merr.] small RNA libraries were generated and sequenced using the Illumina platform to examine the role of miRNAs during soybean nodulation. The small RNA libraries were derived from root hairs inoculated with Bradyrhizobium japonicum (In_RH) or mock-inoculated with water (Un_RH), as well as from the comparable inoculated stripped root samples (i.e. inoculated roots with the root hairs removed). Sequencing of these libraries identified a total of 114 miRNAs, including 22 novel miRNAs. A comparison of miRNA abundance among the 114 miRNAs identified 66 miRNAs that were differentially expressed between root hairs and stripped roots, and 48 miRNAs that were differentially regulated in infected root hairs in response to B. japonicum when compared to uninfected root hairs (P ≤ 0.05). A parallel analysis of RNA ends (PARE) library was constructed and sequenced to reveal a total of 405 soybean miRNA targets, with most predicted to encode transcription factors or proteins involved in protein modification, protein degradation and hormone pathways. The roles of gma-miR4416 and gma-miR2606b during nodulation were further analysed. Ectopic expression of these two miRNAs in soybean roots resulted in significant changes in nodule numbers. miRNA target information suggested that gma-miR2606b regulates a Mannosyl-oligosaccharide 1, 2-alpha-mannosidase gene, while gma-miR4416 regulates the expression of a rhizobium-induced peroxidase 1 (RIP1)-like peroxidase gene, GmRIP1, during nodulation.

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Yale lab uncovers new pathway for passing genetic messages between cells

Yale lab uncovers new pathway for passing genetic messages between cells | Noncoding RNA | Scoop.it

A Yale-led research team has described a novel pathway for the delivery of microRNA (miRNA), the tiny RNA molecules that can move between cells to regulate gene expression.


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A Stress-Induced Small RNA Modulates Alpha-Rhizobial Cell Cycle Progression

A Stress-Induced Small RNA Modulates Alpha-Rhizobial Cell Cycle Progression | Noncoding RNA | Scoop.it

Mechanisms adjusting replication initiation and cell cycle progression in response to environmental conditions are crucial for microbial survival. Functional characterization of the trans-encoded small non-coding RNA (trans-sRNA) EcpR1 in the plant-symbiotic alpha-proteobacterium Sinorhizobium meliloti revealed a role of this class of riboregulators in modulation of cell cycle regulation. EcpR1 is broadly conserved in at least five families of the Rhizobiales and is predicted to form a stable structure with two defined stem-loop domains. In S. meliloti, this trans-sRNA is encoded downstream of the divK-pleD operon. ecpR1 belongs to the stringent response regulon, and its expression was induced by various stress factors and in stationary phase. Induced EcpR1 overproduction led to cell elongation and increased DNA content, while deletion of ecpR1 resulted in reduced competitiveness. Computationally predicted EcpR1 targets were enriched with cell cycle-related mRNAs. Post-transcriptional repression of the cell cycle key regulatory genes gcrA and dnaA mediated by mRNA base-pairing with the strongly conserved loop 1 of EcpR1 was experimentally confirmed by two-plasmid differential gene expression assays and compensatory changes in sRNA and mRNA. Evidence is presented for EcpR1 promoting RNase E-dependent degradation of the dnaA mRNA. We propose that EcpR1 contributes to modulation of cell cycle regulation under detrimental conditions.

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Computational Biology in microRNA - Li - 2015 - Wiley Interdisciplinary Reviews: RNA - Wiley Online Library

Computational Biology in microRNA - Li - 2015 - Wiley Interdisciplinary Reviews: RNA - Wiley Online Library | Noncoding RNA | Scoop.it
“Computational Biology in microRNA: MicroRNA (miRNA) is a class of small endogenous noncoding RNA species, whic... http://t.co/KZmVsOhOOD”
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Using Antisense Technologies to Modulate Noncoding RNA Function

Using Antisense Technologies to Modulate Noncoding RNA Function | Noncoding RNA | Scoop.it

Subfamilies of RNA are being increasingly recognized as having important roles in gene regulation. Some of these RNAs have similar lengths and splicing structures to mRNA, but do not encode proteins. Already thousands of these noncoding RNAs, which include long intergenic noncoding RNAs (lincRNAs), long noncoding RNAs (lncRNAs), and small nucleolar RNAs (snoRNAs), have been discov­ered, but relatively few have been function­ally characterized. Most noncoding RNAs are localized to the nucleus, limiting the use of RNAi for loss-of-function studies due to the low prevalence of the requisite enzymes in the nucleus [1]. However, RNase H, which is present in the nucleus, can be engaged to mediate RNA degradation, enabling the use of chemically modified antisense DNA oligonucleotides to downregulate targeted nuclear noncoding RNAs.


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Cleavage of INDOLE-3-ACETIC ACID INDUCIBLE28 mRNA by MicroRNA847 Upregulates Auxin Signaling to Modulate Cell Proliferation and Lateral Organ Growth in Arabidopsis

Cleavage of INDOLE-3-ACETIC ACID INDUCIBLE28 mRNA by MicroRNA847 Upregulates Auxin Signaling to Modulate Cell Proliferation and Lateral Organ Growth in Arabidopsis | Noncoding RNA | Scoop.it
“ MicroRNAs function in a range of developmental processes. Here, we demonstrate that miR847 targets the mRNA of the auxin/indole acetic acid (Aux/IAA) repressor-encoding gene IAA28 for cleavage. The rapidly increased accumulation of miR847 in Arabidopsis thaliana coincided with reduced IAA28 mRNA levels upon auxin treatment. This induction of miR847 by auxin was abolished in auxin receptor tir1-1 and auxin-resistant axr1-3 mutants. Further analysis demonstrates that miR847 functions as a positive regulator of auxin-mediated lateral organ development by cleaving IAA28 mRNA. Importantly, the ectopic expression of miR847 increases the expression of cell cycle genes as well as the neoplastic activity of leaf cells, prolonging later-stage rosette leaf growth and producing leaves with serrated margins. Moreover, both miR847 and IAA28 mRNAs are specifically expressed in marginal meristems of rosette leaves and lateral root initiation sites. Our data indicate that auxin-dependent induction of miR847 positively regulates meristematic competence by clearing IAA28 mRNA to upregulate auxin signaling, thereby determining the duration of cell proliferation and lateral organ growth in Arabidopsis. IAA28 mRNA encodes an Aux/IAA repressor protein, which is degraded through the proteasome in response to auxin. Altered signal sensitization to IAA28 mRNA levels, together with targeted IAA28 degradation, ensures a robust signal derepression.”
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Increase of microRNA-210, Decrease of Raptor Gene Expression and Alteration of Mammalian Target of Rapamycin Regulated Proteins following Mithramycin Treatment of Human Erythroid Cells

Increase of microRNA-210, Decrease of Raptor Gene Expression and Alteration of Mammalian Target of Rapamycin Regulated Proteins following Mithramycin Treatment of Human Erythroid Cells | Noncoding RNA | Scoop.it
Expression and regulation of microRNAs is an emerging issue in erythroid differentiation and globin gene expression in hemoglobin disorders.

Via Krishan Maggon
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Krishan Maggon 's curator insight, April 8, 2015 2:26 AM

Citation: Bianchi N, Finotti A, Ferracin M, Lampronti I, Zuccato C, et al. (2015) Increase of microRNA-210, Decrease of Raptor Gene Expression and Alteration of Mammalian Target of Rapamycin Regulated Proteins following Mithramycin Treatment of Human Erythroid Cells. PLoS ONE 10(4): e0121567. doi:10.1371/journal.pone.0121567

Rescooped by zengwp from TAL effector science
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TALEN-engineered human cell lines with microRNA-21 null mutations - RNA & DISEASE

Kurata and Lin 2015

Dysregulation of microRNA-21 (miR-21) is associated with many types of cancer as well as with kidney and cardiovascular diseases. Aberrant expression of miR-21 leads to multiple phenotypic alterations including cellular proliferation, invasiveness, apoptosis, and fibrosis. We recently used transcription activator-like effector nucleases to engineer human cell lines with miR-21 null mutations. As expected, loss of miR-21 resulted in decrease cell proliferation and reduced transforming activity in culture and in xenografts. Besides an increase of apoptotic gene expression, miR-21 knockout cells also had significantly increased expression of genes involved in extracellular matrix interaction. Results from small RNA sequencing suggest that miR-21 deletion changed the microRNA expression profile. These results raise intriguing possibilities that loss of miR-21 expression may influence cellular interactions and that cells with long term miR-21 deficiency may compensate for the loss of this highly expressed microRNA by changing the abundance of alternate microRNAs or the AGO2 protein in order to maintain the microRNA-AGO2 homeostasis. Further characterization and utilization of miR-21 knockout human cells will shed new light on this pathologically important microRNA.


Via dromius
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