[Show abstract][Hide abstract] ABSTRACT: Small RNAs (sRNAs) are loaded into ARGONAUTE (AGO) proteins to induce gene silencing. In plants, the 5'-terminal nucleotide is important for sRNA sorting into different AGOs. Here we show that microRNA (miRNA) duplex structure also contributes to miRNA sorting. Base pairing at the 15th nucleotide of a miRNA duplex is important for miRNA sorting in both Arabidopsis AGO1 and AGO2. AGO2 favours miRNA duplexes with no middle mismatches, whereas AGO1 tolerates, or prefers, duplexes with central mismatches. AGO structure modelling and mutational analyses reveal that the QF-V motif within the conserved PIWI domain contributes to recognition of base pairing at the 15th nucleotide of a duplex, while the DDDE catalytic core of AtAGO2 is important for recognition of the central nucleotides. Finally, we rescued the adaxialized phenotype of ago1-12, which is largely due to miR165 loss-of-function, by changing miR165 duplex structure which we predict redirects it to AGO2.
[Show abstract][Hide abstract] ABSTRACT: High-throughput sequencing is a powerful tool for exploring small RNA populations in plants. The ever-increasing output from an Illumina Sequencing System allows for multiplexing multiple samples while still obtaining sufficient data for small RNA discovery and characterization. Here we describe a protocol for generating multiplexed small RNA libraries for sequencing up to 12 samples in one lane of an Illumina HiSeq System single-end, 50 base pair run. RNA ligases are used to add the 3’ and 5’ adaptors to purified small RNAs; ligation products that lack a small RNA molecule (adaptor-adaptor products) are intentionally depleted. After cDNA synthesis, a linear PCR step amplifies the DNA fragments. The 3’ PCR primers used here include unique 6-nucleotide sequences to allow for multiplexing up to 12 samples.
[Show abstract][Hide abstract] ABSTRACT: The identification of viroid-derived small RNAs (vd-sRNAs) of 21-24 nucleotides (nt) in plants infected by viroids (infectious non-protein-coding RNAs of just 250-400 nt), supports their targeting by dicer-like enzymes, the first host RNA silencing barrier. However, whether viroids, like RNA viruses, are also targeted by the RNA-induced silencing complex (RISC) remains controversial. At the RISC core is one Argonaute (AGO) protein that, guided by endogenous or viral sRNAs, targets complementary RNAs. To examine whether AGO proteins also load vd-sRNAs, leaves of Nicotiana benthamiana infected by potato spindle tuber viroid (PSTVd) were agroinfiltrated with plasmids expressing epitope-tagged versions of AGO1, AGO2, AGO3, AGO4, AGO5, AGO6, AGO7, AGO9 and AGO10 from Arabidopsis thaliana. Immunoprecipitation analyses of the agroinfiltrated halos revealed that all AGOs, except AGO6, AGO7 and AGO10, associated with vd-sRNAs: AGO1, AGO2 and AGO3 preferentially with those of 21 and 22 nt, while AGO4, AGO5 and AGO9 additionally bound those of 24 nt. Deep sequencing analyses showed that sorting of vd-sRNAs into AGO1, AGO2, AGO4 and AGO5 depended essentially on their 5’-terminal nucleotide, with the profiles of the corresponding AGO-loaded vd-sRNAs adopting specific hot spot distributions along the viroid genome. Furthermore, agroexpression of AGO1, AGO2, AGO4 and AGO5 on PSTVd-infected tissue attenuated the level of the genomic RNAs, suggesting that they, or their precursors, are RISC-targeted. In contrast to RNA viruses, PSTVd infection of N. benthamiana did not affect miR168-mediated regulation of the endogenous AGO1, which loaded vd-sRNAs with specificity similar to its A. thaliana counterpart.
Journal of Virology 08/2014; 88(20):11933. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are used for small RNA-based, specific gene silencing or knockdown in plants. Current methods to generate amiRNA or syn-tasiRNA constructs are not well adapted for cost-effective, large-scale production, or for multiplexing to specifically suppress multiple targets. Here we describe simple, fast and cost-effective methods with high-throughput capability to generate amiRNA and multiplexed syn-tasiRNA constructs for efficient gene silencing in Arabidopsis and other plant species. AmiRNA or syn-tasiRNA inserts resulting from the annealing of two overlapping and partially complementary oligonucleotides are ligated directionally into a zero background BsaI/ccdB ('B/c')-based expression vector. B/c vectors for amiRNA and syn-tasiRNA cloning and expression contain a modified version of Arabidopsis MIR390a or TAS1c precursors, respectively, in which a fragment of the endogenous sequence was substituted by a ccdB cassette. Several amiRNA and syn-tasiRNA sequences designed to target one or more endogenous genes were validated in transgenic plants that a) exhibited the expected phenotypes predicted by loss of target gene function, b) accumulated high levels of accurately processed amiRNAs or syn-tasiRNAs, and c) had reduced levels of the corresponding target RNAs.
[Show abstract][Hide abstract] ABSTRACT: The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using Parallel Analysis of RNA Ends (PARE) data is lacking in B. distachyon.
B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million distinct genome-matched sequences that resulted represent an extensive dataset to analyze small RNA-guided cleavage events. Analysis of the PARE and miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner.
B. distachyon miRNAs and target RNAs were experimentally identified and analyzed. Knowledge gained from this study should provide insights into the roles of miRNAs and the regulation of their targets in B. distachyon and related plants.
[Show abstract][Hide abstract] ABSTRACT: In eukaryotes, RNA silencing pathways utilize 20-30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focused on plant and animal RNA silencing systems. Phytophthora species belong to a phylogenetically distinct group of economically important plant pathogens that cause billions of dollars in yield losses annually as well as ecologically devastating outbreaks. We analyzed the small RNA-generating components of the genomes of P. infestans, P. sojae and P. ramorum using bioinformatics, genetic, phylogenetic and high-throughput sequencing-based methods. Each species produces two distinct populations of small RNAs that are predominantly 21- or 25-nucleotides long. The 25-nucleotide small RNAs were primarily derived from loci encoding transposable elements and we propose that these small RNAs define a pathway of short-interfering RNAs that silence repetitive genetic elements. The 21-nucleotide small RNAs were primarily derived from inverted repeats, including a novel microRNA family that is conserved among the three species, and several gene families, including Crinkler effectors and type III fibronectins. The Phytophthora microRNA is predicted to target a family of amino acid/auxin permeases, and we propose that 21-nucleotide small RNAs function at the post-transcriptional level. The functional significance of microRNA-guided regulation of amino acid/auxin permeases and the association of 21-nucleotide small RNAs with Crinkler effectors remains unclear, but this work provides a framework for testing the role of small RNAs in Phytophthora biology and pathogenesis in future work.
PLoS ONE 01/2013; 8(10):e77181. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicer-independent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicer-defective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses.
The Plant Cell 09/2012; 24(9):3613-29. · 9.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In nematodes, plants, and fungi, RNAi is remarkably potent and persistent due to the amplification of initial silencing signals by RNA-dependent RNA polymerases (RdRPs). In Caenorhabditis elegans (C. elegans), the interaction between the RNA-induced silencing complex (RISC) loaded with primary small interfering RNAs (siRNAs) and the target messenger RNA (mRNA) leads to the recruitment of RdRPs and synthesis of secondary siRNAs using the target mRNA as the template. The mechanism and genetic requirements for secondary siRNA accumulation are not well understood.
From a forward genetic screen for C. elegans genes required for RNAi, we identified rde-10, and through proteomic analysis of RDE-10-interacting proteins, we identified a protein complex containing the new RNAi factor RDE-11, the known RNAi factors RSD-2 and ERGO-1, and other candidate RNAi factors. The RNAi defective genes rde-10 and rde-11 encode a novel protein and a RING-type zinc finger domain protein, respectively. Mutations in rde-10 and rde-11 genes cause dosage-sensitive RNAi deficiencies: these mutants are resistant to low dosage but sensitive to high dosage of double-stranded RNAs. We assessed the roles of rde-10, rde-11, and other dosage-sensitive RNAi-defective genes rsd-2, rsd-6, and haf-6 in both exogenous and endogenous small RNA pathways using high-throughput sequencing and qRT-PCR. These genes are required for the accumulation of secondary siRNAs in both exogenous and endogenous RNAi pathways.
The RDE-10/RDE-11 complex is essential for the amplification of RNAi in C. elegans by promoting secondary siRNA accumulation.
Current biology: CB 04/2012; 22(10):881-90. · 10.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests.
Journal of Virology 03/2012; 86(11):6002-9. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endogenous small interfering RNAs (siRNAs) are a class of naturally occuring regulatory RNAs found in fungi, plants, and animals. Some endogenous siRNAs are required to silence transposons or function in chromosome segregation; however, the specific roles of most endogenous siRNAs are unclear. The helicase gene eri-6/7 was identified in the nematode Caenorhabditis elegans by the enhanced response to exogenous double-stranded RNAs (dsRNAs) of the null mutant. eri-6/7 encodes a helicase homologous to small RNA factors Armitage in Drosophila, SDE3 in Arabidopsis, and Mov10 in humans. Here we show that eri-6/7 mutations cause the loss of 26-nucleotide (nt) endogenous siRNAs derived from genes and pseudogenes in oocytes and embryos, as well as deficiencies in somatic 22-nucleotide secondary siRNAs corresponding to the same loci. About 80 genes are eri-6/7 targets that generate the embryonic endogenous siRNAs that silence the corresponding mRNAs. These 80 genes share extensive nucleotide sequence homology and are poorly conserved, suggesting a role for these endogenous siRNAs in silencing of and thereby directing the fate of recently acquired, duplicated genes. Unlike most endogenous siRNAs in C. elegans, eri-6/7-dependent siRNAs require Dicer. We identify that the eri-6/7-dependent siRNAs have a passenger strand that is ∼19 nt and is inset by ∼3-4 nts from both ends of the 26 nt guide siRNA, suggesting non-canonical Dicer processing. Mutations in the Argonaute ERGO-1, which associates with eri-6/7-dependent 26 nt siRNAs, cause passenger strand stabilization, indicating that ERGO-1 is required to separate the siRNA duplex, presumably through endonucleolytic cleavage of the passenger strand. Thus, like several other siRNA-associated Argonautes with a conserved RNaseH motif, ERGO-1 appears to be required for siRNA maturation.
[Show abstract][Hide abstract] ABSTRACT: We report the 207-Mb genome sequence of the North American Arabidopsis lyrata strain MN47 based on 8.3× dideoxy sequence coverage. We predict 32,670 genes in this outcrossing species compared to the 27,025 genes in the selfing species Arabidopsis thaliana. The much smaller 125-Mb genome of A. thaliana, which diverged from A. lyrata 10 million years ago, likely constitutes the derived state for the family. We found evidence for DNA loss from large-scale rearrangements, but most of the difference in genome size can be attributed to hundreds of thousands of small deletions, mostly in noncoding DNA and transposons. Analysis of deletions and insertions still segregating in A. thaliana indicates that the process of DNA loss is ongoing, suggesting pervasive selection for a smaller genome. The high-quality reference genome sequence for A. lyrata will be an important resource for functional, evolutionary and ecological studies in the genus Arabidopsis.
[Show abstract][Hide abstract] ABSTRACT: De novo DNA methylation in Arabidopsis thaliana is catalyzed by the methyltransferase DRM2, a homolog of the mammalian de novo methyltransferase DNMT3. DRM2 is targeted to DNA by small interfering RNAs (siRNAs) in a process known as RNA-directed DNA Methylation (RdDM). While several components of the RdDM pathway are known, a functional understanding of the underlying mechanism is far from complete. We employed both forward and reverse genetic approaches to identify factors involved in de novo methylation. We utilized the FWA transgene, which is methylated and silenced when transformed into wild-type plants, but unmethylated and expressed when transformed into de novo methylation mutants. Expression of FWA is marked by a late flowering phenotype, which is easily scored in mutant versus wild-type plants. By reverse genetics we discovered the requirement for known RdDM effectors AGO6 and NRPE5a for efficient de novo methylation. A forward genetic approach uncovered alleles of several components of the RdDM pathway, including alleles of clsy1, ktf1, and nrpd/e2, which have not been previously shown to be required for the initial establishment of DNA methylation. Mutations were mapped and genes cloned by both traditional and whole genome sequencing approaches. The methodologies and the mutant alleles discovered will be instrumental in further studies of de novo DNA methylation.
Epigenetics: official journal of the DNA Methylation Society 03/2011; 6(3):344-54. · 4.58 Impact Factor