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.
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 siRNAs and the target 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, as well as 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 (dsRNAs). 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.
rde-10; rde-11; RNAi; endogenous siRNAs; RNA silencing protein co-factors
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.
Light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. Here, we used chromatin immunoprecipitation (ChIP) sequencing to uncover direct targets of the Neurospora crassa circadian regulator White Collar Complex (WCC). The WCC is a blue-light receptor and the key transcription factor of the circadian oscillator. It controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to light. We found that in response to light, WCC binds to hundreds of genomic regions, including the promoters of previously identified clock- and light-regulated genes. We show that WCC directly controls the expression of 24 transcription factor genes, including the clock-controlled adv-1 gene, which controls a circadian output pathway required for daily rhythms in development. Our findings provide links between the key circadian activator and effectors in downstream regulatory pathways.
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.
DNA methylation; Arabidopsis; de novo; genetic screen; whole-genome sequencing
RNA interference pathways may involve amplification of secondary siRNAs by RNA-dependent RNA polymerases. In plants, RDR6-dependent secondary siRNAs arise from transcripts targeted by some microRNA (miRNA). Here, Arabidopsis thaliana secondary siRNA from mRNA, and trans-acting siRNA, are shown to be triggered through initial targeting by 22 nt miRNA that associate with AGO1. In contrast to canonical 21 nt miRNA, 22 nt miRNA primarily arise from foldback precursors containing asymmetric bulges. Using artificial miRNA constructs, conversion of asymmetric foldbacks to symmetric foldbacks resulted in production of 21 nt forms of miR173, miR472 and miR828. Both 21 and 22 nt forms associated with AGO1 and guided accurate slicer activity, but only 22 nt miRNA were competent to trigger RDR6-dependent siRNA from target RNA. These data suggest that AGO1 functions differentially with 21 and 22 nt miRNA to engage the RDR6-associated amplification apparatus.
Intercellular transport of viruses through cytoplasmic connections, termed plasmodesmata (PD), is essential for systemic infection in plants by viruses. Previous genetic and ultrastructural data revealed that the potyvirus cyclindrical inclusion (CI) protein is directly involved in cell-to-cell movement, likely through the formation of conical structures anchored to and extended through PD. In this study, we demonstrate that plasmodesmatal localization of CI in N. benthamiana leaf cells is modulated by the recently discovered potyviral protein, P3N-PIPO, in a CI:P3N-PIPO ratio-dependent manner. We show that P3N-PIPO is a PD-located protein that physically interacts with CI in planta. The early secretory pathway, rather than the actomyosin motility system, is required for the delivery of P3N-PIPO and CI to PD. Moreover, CI mutations that disrupt virus cell-to-cell movement compromise PD-localization capacity. These data suggest that the CI and P3N-PIPO complex coordinates the formation of PD-associated structures that facilitate the intercellular movement of potyviruses in infected plants.
Plant viral pathogens cause an estimated US$60 billion loss in crop yields worldwide each year. Potyviruses, accounting for ∼30% of known plant viruses, include many agriculturally important viruses. Despite their importance, the cell-to-cell spread of potyviruses remains poorly understood. Previous studies have shown that at early time points of infection, the virus-encoded CI protein, one of 11 known potyviral proteins, is associated with cone-shaped structures at plasmodesmata (PD) and is involved in viral cell-to-cell movement. In this paper, we show that a newly identified potyviral protein, P3N-PIPO, is a PD-located protein and directs the CI protein to PD, facilitating the deposition of the cone-shaped structures of CI at PD by interacting with CI protein. We demonstrate that the mutant of CI, which impairs potyviral cell-to-cell movement, loses its ability to accumulate at PD. We further reveal that P3N-PIPO utilizes the secretory pathway rather than the actomyosin motility system for trafficking to PD. Taken together, the data presented in this study suggest that CI and P3N-PIPO coordinates the formation of conical structure at PD for potyviral cell-to-cell spread.
Flowering is the primary trait affected by ambient temperature changes. Plant microRNAs (miRNAs) are small non-coding RNAs playing an important regulatory role in plant development. In this study, to elucidate the mechanism of flowering-time regulation by small RNAs, we identified six ambient temperature-responsive miRNAs (miR156, miR163, miR169, miR172, miR398 and miR399) in Arabidopsis via miRNA microarray and northern hybridization analyses. We also determined the expression profile of 120 unique miRNA loci in response to ambient temperature changes by miRNA northern hybridization analysis. The expression of the ambient temperature-responsive miRNAs and their target genes was largely anticorrelated at two different temperatures (16 and 23°C). Interestingly, a lesion in short vegetative phase (SVP), a key regulator within the thermosensory pathway, caused alteration in the expression of miR172 and a subset of its target genes, providing a link between a thermosensory pathway gene and miR172. The miR172-overexpressing plants showed a temperature-independent early flowering phenotype, suggesting that modulation of miR172 expression leads to temperature insensitivity. Taken together, our results suggest a genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs under non-stress temperature conditions.
Short RNAs, and in particular microRNAs, are important regulators of gene expression both within defined regulatory pathways and at the epigenetic scale. We investigated the short RNA (sRNA) population (18-24 nt) of the transcriptome of green leaves from the sequenced Populus trichocarpa using a concatenation strategy in combination with 454 sequencing.
The most abundant size class of sRNAs were 24 nt. Long Terminal Repeats were particularly associated with 24 nt sRNAs. Additionally, some repetitive elements were associated with 22 nt sRNAs. We identified an sRNA hot-spot on chromosome 19, overlapping a region containing both the proposed sex-determining locus and a major cluster of NBS-LRR genes. A number of phased siRNA loci were identified, a subset of which are predicted to target PPR and NBS-LRR disease resistance genes, classes of genes that have been significantly expanded in Populus. Additional loci enriched for sRNA production were identified and characterised. We identified 15 novel predicted microRNAs (miRNAs), including miRNA*sequences, and identified a novel locus that may encode a dual miRNA or a miRNA and short interfering RNAs (siRNAs).
The short RNA population of P. trichocarpa is at least as complex as that of Arabidopsis thaliana. We provide a first genome-wide view of short RNA production for P. trichocarpa and identify new, non-conserved miRNAs.
In genetic hybrids, the silencing of nucleolar rRNA genes inherited from one progenitor is the epigenetic phenomenon known as nucleolar dominance. An RNAi knockdown screen identified the Arabidopsis de novo cytosine methyltransferase, DRM2 and the methylcytosine binding domain proteins, MBD6 and MBD10 as activities required for nucleolar dominance. MBD10 localizes throughout the nucleus, but MBD6 preferentially associates with silenced rRNA genes, and does so in a DRM2-dependent manner. DRM2 methylation is thought to be guided by siRNAs whose biogenesis requires RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Consistent with this hypothesis, knockdown of DCL3 or RDR2 disrupts nucleolar dominance. In genetic hybrids, the silencing of nucleolar rRNA genes inherited from one progenitor is the epigenetic phenomenon known as nucleolar dominance. An RNAi knockdown screen identified the Arabidopsis de novo cytosine methyltransferase, DRM2 and the methylcytosine binding domain proteins, MBD6 and MBD10 as activities required for nucleolar dominance. MBD10 localizes throughout the nucleus, but MBD6 preferentially associates with silenced rRNA genes, and does so in a DRM2-dependent manner. DRM2 methylation is thought to be guided by siRNAs whose biogenesis requires RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Consistent with this hypothesis, knockdown of DCL3 or RDR2 disrupts nucleolar dominance. Collectively, these results indicate that in addition to directing the silencing of retrotransposons and noncoding repeats, siRNAs specify de novo cytosine methylation patterns that are recognized by MBD6 and MBD10 in the large-scale silencing of rRNA gene loci.
In metazoans Piwi-related Argonaute proteins have been linked to germ-line maintenance, and to a class of germ-line enriched small RNAs termed piRNAs. Here we show that an abundant class of 21-nucleotide small RNAs (21U-RNAs) are expressed in the C. elegans germ line, interact with the C. elegans Piwi-family member, PRG-1, and depend on PRG-1 activity for their accumulation. The PRG-1 protein is expressed throughout development and localizes to nuage-like structures called P-granules. Although 21U-RNA loci share a conserved upstream sequence motif, the mature 21U-RNAs are not conserved and, with few exceptions, fail to exhibit complementarity or evidence for direct regulation of other expressed sequences. Our findings demonstrate that 21U-RNAs are the piRNAs of C. elegans and link this class of small RNAs and their associated Piwi Argonaute to the maintenance of temperature-dependent fertility.
Large molecular sequence databases are fundamental resources for modern bioscientists. Whether for project-specific purposes or sharing data with colleagues, it is often advantageous to maintain smaller sequence databases. However, this is usually not an easy task for the average bench scientist.
We present the Personal Sequence Database (PSD), a suite of tools to create and maintain small- to medium-sized web-accessible sequence databases. All interactions with PSD tools occur via the internet with a web browser. Users may define sequence groups within their database that can be maintained privately or published to the web for public use. A sequence group can be downloaded, browsed, searched by keyword or searched for sequence similarities using BLAST. Publishing a sequence group extends these capabilities to colleagues and collaborators. In addition to being able to manage their own sequence databases, users can enroll sequences in BLASTAgent, a BLAST hit tracking system, to monitor NCBI databases for new entries displaying a specified level of nucleotide or amino acid similarity.
The PSD offers a valuable set of resources unavailable elsewhere. In addition to managing sequence data and BLAST search results, it facilitates data sharing with colleagues, collaborators and public users. The PSD is hosted by the authors and is available at .
Development of the Arabidopsis Small RNA Project (ASRP) Database, which provides information and tools for the analysis of microRNA, endogenous siRNA and other small RNA-related features, has been driven by the introduction of high-throughput sequencing technology. To accommodate the demands of increased data, numerous improvements and updates have been made to ASRP, including new ways to access data, more efficient algorithms for handling data, and increased integration with community-wide resources. New search and visualization tools have also been developed to improve access to small RNA classes and their targets. ASRP is publicly available through a web interface at http://asrp.cgrb.oregonstate.edu/db/
Eukaryotes contain a diversified set of small RNA-guided pathways that control genes, repeated sequences, and viruses at the transcriptional and posttranscriptional levels. Genome-wide profiles and analyses of small RNAs, particularly the large class of 24-nucleotide (nt) short interfering RNAs (siRNAs), were done for wild-type Arabidopsis thaliana and silencing pathway mutants with defects in three RNA-dependent RNA polymerase (RDR) and four Dicer-like (DCL) genes. The profiling involved direct analysis using a multiplexed, parallel-sequencing strategy. Small RNA-generating loci, especially those producing predominantly 24-nt siRNAs, were found to be highly correlated with repetitive elements across the genome. These were found to be largely RDR2- and DCL3-dependent, although alternative DCL activities were detected on a widespread level in the absence of DCL3. In contrast, no evidence for RDR2-alternative activities was detected. Analysis of RDR2- and DCL3-dependent small RNA accumulation patterns in and around protein-coding genes revealed that upstream gene regulatory sequences systematically lack siRNA-generating activities. Further, expression profiling suggested that relatively few genes, proximal to abundant 24-nt siRNAs, are regulated directly by RDR2- and DCL3-dependent silencing. We conclude that the widespread accumulation patterns for RDR2- and DCL3-dependent siRNAs throughout the Arabidopsis genome largely reflect mechanisms to silence highly repeated sequences.
The discovery of small RNA molecules that inactivate genes during normal growth and development or on invasion by viruses and other mobile genetic elements has profoundly altered our views about how genes are regulated. We set out to investigate the global patterns of small RNA formation and activity across the entire genome of the plant, Arabidopsis thaliana. We used new technology and bioinformatics to characterize and compare these patterns in normal plants and in plants with defects in various genes involved in RNA silencing. The results show vast populations of small RNAs in plants and reveal how specialized branches of the RNA-silencing pathway have been adopted for inactivation of different types of genome sequences.
There are a lot of small RNAs in
Arabidopis that regulate other genes. These RNAs are produced by a number of enzymes; the authors have used mutants of these enzymes to see what each of them does.
MicroRNAs (miRNAs) are an extensive class of noncoding genes that regulate gene expression through posttranscriptional repression. Given the potential for large viral genomes to encode these transcripts, we examined the human cytomegalovirus AD169 genome for miRNAs using a bioinformatics approach. We identified 406 potential stem-loops, of which 110 were conserved between chimpanzee cytomegalovirus and several strains of human cytomegalovirus. Of these conserved stem-loops, 13 exhibited a significant score using the MiRscan algorithm. Examination of total RNA from human cytomegalovirus-infected cells demonstrated that 5 of the 13 predicted miRNAs were expressed during infection. These studies demonstrate that human cytomegalovirus encodes multiple conserved miRNAs and suggest that human cytomegalovirus may utilize an miRNA strategy to regulate cellular and viral gene function.
Plum pox virus (PPV) is a member of the Potyvirus genus that, in nature, infects trees of the Prunus genus. Although PPV infects systemically several species of the Nicotiana genus, such as N. clevelandii and N. benthamiana, and replicates in the inoculated leaves of N. tabacum, it is unable to infect systemically the last host. The long-distance movement defect of PPV was corrected in transgenic tobacco plants expressing the 5"-terminal region of the genome of tobacco etch virus (TEV), a potyvirus that infects systemically tobacco. The fact that PPV was unable to move to upper noninoculated leaves in tobacco plants transformed with the same TEV transgene, but with a mutation in the HC protein (HC-Pro)-coding sequences, identifies the multifunctional HC-Pro as the complementing factor, and strongly suggests that a defect in an HC-Pro activity is responsible for the long-distance movement defect of PPV in tobacco. Whereas PPV HC-Pro strongly intensifies the symptoms caused by potato virus X (PVX) in the PPV systemic hosts N. clevelandii and N. benthamiana, it has no apparent effect on PVX pathogenicity in tobacco, supporting the hypothesis that long-distance movement and pathogenicity enhancement are related activities of the potyviral HC proteins. The movement defect of PPV in tobacco could also be complemented by cucumber mosaic virus in a mixed infection, demonstrating that at least some components of the long-distance machinery of the potyviruses are not strictly virus specific. A general conclusion of this work is that the HC-Pro might be a relevant factor for controlling the host range of the potyviruses.
The tobacco etch potyvirus (TEV) RNA-dependent RNA polymerase (NIb) has been shown to interact with the proteinase domain of the VPg-proteinase (NIa). To investigate the significance of this interaction, a Saccharomyces cerevisiae two-hybrid assay was used to isolate conditional NIa mutant proteins with temperature-sensitive (ts) defects in interacting with NIb. Thirty-six unique tsNIa mutants with substitutions affecting the proteinase domain were recovered. Most of the mutants coded for proteins with little or no proteolytic activity at permissive and nonpermissive temperatures. However, three mutant proteins retained proteolytic activity at both temperatures and, in two cases (tsNIa-Q384P and tsNIa-N393D), the mutations responsible for the ts interaction phenotype could be mapped to single positions. One of the mutations (N393D) conferred a ts-genome-amplification phenotype when it was placed in a recombinant TEV strain. Suppressor NIb mutants that restored interaction with the tsNIa-N393D protein at the restrictive temperature were recovered by a two-hybrid selection system. Although most of the suppressor mutants failed to stimulate amplification of genomes encoding the tsNIa-N393D protein, two suppressors (NIb-I94T and NIb-C380R) stimulated amplification of virus containing the N393D substitution by approximately sevenfold. These results support the hypothesis that interaction between NIa and NIb is important during TEV genome replication.
The 3′-terminal 350 nucleotides of the tobacco etch potyvirus (TEV) genome span the end of the capsid protein (CP)-coding sequence and the 3′ nontranslated region (NTR). The CP-coding sequence within this region contains a 105-nucleotide cis-active element required for genome replication (S. Mahajan, V. V. Dolja, and J. C. Carrington, J. Virol. 70:4370–4379, 1996). To investigate the sequence and secondary structure requirements within the CP cis-active region and the 3′ NTR, a systematic linker-scanning mutagenesis analysis was done. Forty-six mutations, each with two to six nucleotide substitutions, were introduced at consecutive hexanucleotide positions in the genome of a recombinant TEV strain expressing a reporter protein (β-glucuronidase). Genome amplification activity of each mutant in the protoplast cell culture system was measured. Mutations that severely debilitated genome amplification were identified throughout the CP-coding cis-active sequence and at several distinct locations within the 3′ NTR. However, based on a computer model of RNA folding, mutations that had the most severe effects mapped to regions that were predicted to form base-paired secondary structures. Linker-scanning mutations predicted to affect either strand of a base-paired structure within the CP-coding cis-active sequence, a base-paired structure between two segments of the CP-coding cis-active sequence and a contiguous 14-nucleotide segment of the 3′ NTR, and a base-paired structure near the 3′ terminus of the 3′ NTR inactivated genome amplification. Compensatory mutations that restored base pair interactions in each of these regions restored amplification activity, although to differing levels depending on the structure restored. These data reveal that the 3′ terminus of the TEV genome consists of a series of functionally discrete sequences and secondary structures and that the CP-coding sequence and 3′ NTR are coadapted for genome amplification function through a requirement for base pair interactions.
Tobacco etch virus, a plant potyvirus, expresses its RNA genome as a large polyprotein precursor which undergoes extensive proteolytic processing to yield seven or more mature products. Two of these products, proteins with apparent molecular weights of 49,000 and 54,000 (49K and 54K proteins), aggregate in the form of crystalline inclusions within the nuclei of infected cells. Cell-free translation of synthetic transcripts was used to map the genes for these two products on the viral genome and to express an enzymatically active protein. The 49K protein was determined to be a viral protease responsible for several cleavages of the polyprotein, including its own autocatalytic excision. Analyses of products expressed from the 49K protein genes which were altered by deletion revealed that only the carboxyl-terminal half was required for proteolytic activity.
Multicellular eukaryotes produce small RNA molecules (approximately 21–24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and genome structure. Formation or activity of small RNAs requires factors belonging to gene families that encode DICER (or DICER-LIKE [DCL]) and ARGONAUTE proteins and, in the case of some siRNAs, RNA-dependent RNA polymerase (RDR) proteins. Unlike many animals, plants encode multiple DCL and RDR proteins. Using a series of insertion mutants of Arabidopsis thaliana, unique functions for three DCL proteins in miRNA (DCL1), endogenous siRNA (DCL3), and viral siRNA (DCL2) biogenesis were identified. One RDR protein (RDR2) was required for all endogenous siRNAs analyzed. The loss of endogenous siRNA in dcl3 and rdr2 mutants was associated with loss of heterochromatic marks and increased transcript accumulation at some loci. Defects in siRNA-generation activity in response to turnip crinkle virus in dcl2 mutant plants correlated with increased virus susceptibility. We conclude that proliferation and diversification of DCL and RDR genes during evolution of plants contributed to specialization of small RNA-directed pathways for development, chromatin structure, and defense.
In plants, RNA-mediated silencing pathways have diversified in unique ways. This study elucidates the specific functions of some of the key regulators in development, chromatin structure, and pathogen defense
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.
Endogenous small interfering RNAs (siRNAs) are a class of small RNAs present in fungi, plants, and animals. Small RNAs, including microRNAs, are known to regulate the expression levels of genes, silence invading elements such as transposons, and act in cell division. However, the function of many endogenous siRNAs is unknown. We have found that the ERI-6/7 helicase is required for a subset of endogenous siRNAs present in the nematode Caenorhabditis elegans. The ERI-6/7 helicase acts in a pathway together with the Argonaute protein ERGO-1 to produce two types of siRNAs: a primary class of 26 nucleotides in length present in oocytes and embryos, and a class of 22 nucleotide siRNAs present in later stages of development. These siRNAs correspond to only about one hundred genes. Interestingly, we found that these genes fall into groups of genes that contain nearly identical DNA sequences. The sequences of these genes are not conserved in other organisms, not even in related nematodes. These results point to a potential function of these endogenous siRNAs: silencing of recently acquired, duplicated genes. Our work demonstrates a new role of small RNAs, different from known functions in transposon silencing and regulation of gene expression.
We present the 207 Mb genome sequence of the outcrosser Arabidopsis lyrata, which diverged from the self-fertilizing species A. thaliana about 10 million years ago. It is generally assumed that the much smaller A. thaliana genome, which is only 125 Mb, constitutes the derived state for the family. Apparent genome reduction in this genus can be partially attributed to the loss of DNA from large-scale rearrangements, but the main cause lies in the hundreds of thousands of small deletions found throughout the genome. These occurred primarily in non-coding DNA and transposons, but protein-coding multi-gene families are smaller in A. thaliana as well. 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.
GENE-counter is a complete Perl-based computational pipeline for analyzing RNA-Sequencing (RNA-Seq) data for differential gene expression. In addition to its use in studying transcriptomes of eukaryotic model organisms, GENE-counter is applicable for prokaryotes and non-model organisms without an available genome reference sequence. For alignments, GENE-counter is configured for CASHX, Bowtie, and BWA, but an end user can use any Sequence Alignment/Map (SAM)-compliant program of preference. To analyze data for differential gene expression, GENE-counter can be run with any one of three statistics packages that are based on variations of the negative binomial distribution. The default method is a new and simple statistical test we developed based on an over-parameterized version of the negative binomial distribution. GENE-counter also includes three different methods for assessing differentially expressed features for enriched gene ontology (GO) terms. Results are transparent and data are systematically stored in a MySQL relational database to facilitate additional analyses as well as quality assessment. We used next generation sequencing to generate a small-scale RNA-Seq dataset derived from the heavily studied defense response of Arabidopsis thaliana and used GENE-counter to process the data. Collectively, the support from analysis of microarrays as well as the observed and substantial overlap in results from each of the three statistics packages demonstrates that GENE-counter is well suited for handling the unique characteristics of small sample sizes and high variability in gene counts.
Endogenous small RNAs (endo-siRNAs) interact with Argonaute (AGO) proteins to mediate sequence-specific regulation of diverse biological processes. Here, we combine deep-sequencing and genetic approaches to explore the biogenesis and function of endo-siRNAs in C. elegans. We describe conditional alleles of the dicer-related helicase, drh-3, that abrogate both RNA interference and the biogenesis of endo-siRNAs, called 22G-RNAs. DRH-3 is a core component of RNA-dependent RNA polymerase (RdRP) complexes essential for several distinct 22G-RNA systems. We show that in the germ-line, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germ-line nuage structures called P-granules. WAGO-1 silences certain genes, transposons, pseudogenes and cryptic loci. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one WAGO-mediated surveillance pathway. These findings broaden our understanding of the biogenesis and diversity of 22G-RNAs and suggest novel regulatory functions for small RNAs.
Considerable progress has been made in identifying the targets of plant microRNAs, many of which regulate the stability or translation of mRNAs that encode transcription factors involved in development. In most cases, it is unknown, however, which immediate transcriptional targets mediate downstream effects of the microRNA-regulated transcription factors. We identified a new process controlled by the miR319-regulated clade of TCP (TEOSINTE BRANCHED/CYCLOIDEA/PCF) transcription factor genes. In contrast to other miRNA targets, several of which modulate hormone responses, TCPs control biosynthesis of the hormone jasmonic acid. Furthermore, we demonstrate a previously unrecognized effect of TCPs on leaf senescence, a process in which jasmonic acid has been proposed to be a critical regulator. We propose that miR319-controlled TCP transcription factors coordinate two sequential processes in leaf development: leaf growth, which they negatively regulate, and leaf senescence, which they positively regulate.
Short, single-stranded RNA molecules called microRNAs (miRNAs) regulate gene expression by negatively controlling both the stability and translation of target messenger RNAs that they recognize through sequence complementarity. In plants, miRNAs mostly regulate other regulators, the DNA-binding transcription factors. We investigated the downstream events regulated by five TCP (TEOSINTE BRANCHED/CYCLOIDEA/PCF) transcription factors that are controlled by the microRNA miR319 in Arabidopsis thaliana. The miR319-regulated TCPs were previously known to be important for limiting the growth of leaves. By applying a combination of genome-wide, biochemical, and genetic studies, we identified new TCP targets that include enzymes responsible for the synthesis of the hormone jasmonic acid. Our analysis of leaf extracts from plants with increased activity of miR319 confirms that altered expression of the biosynthetic genes leads to changed jasmonic acid levels. These plants show also an altered senescence behavior that becomes more normal again when the plants are treated with jasmonate. We propose that the miR319-regulated TCP factors thus coordinate different aspects of leaf development and physiology: growth, which they negatively regulate, and aging, which they positively regulate.
A plant microRNA and its targets turn out to regulate both early and late stages of leaf development: early on, they inhibit growth, while later on, they promote the onset of senescence.