Argonaute Genes Research Articles

Duplication and Diversification of Dipteran Argonaute Genes, and the Evolutionary Divergence of Piwi and Aubergine.

Genetic studies of Drosophila melanogaster have provided a paradigm for RNA interference (RNAi) in arthropods, in which the microRNA and antiviral pathways are each mediated by a single Argonaute (Ago1 and Ago2) and germline suppression of transposable elements is mediated by a trio of Piwi-subfamily Argonaute proteins (Ago3, Aub, and Piwi). Without a suitable evolutionary context, deviations from this can be interpreted as derived or idiosyncratic. Here we analyze the evolution of Argonaute genes across the genomes and transcriptomes of 86 Dipteran species, showing that variation in copy number can occur rapidly, and that there is constant flux in some RNAi mechanisms. The lability of the RNAi pathways is illustrated by the divergence of Aub and Piwi (182–156 Ma), independent origins of multiple Piwi-family genes in Aedes mosquitoes (less than 25Ma), and the recent duplications of Ago2 and Ago3 in the tsetse fly Glossina morsitans. In each case the tissue specificity of these genes has altered, suggesting functional divergence or innovation, and consistent with the action of dynamic selection pressures across the Argonaute gene family. We find there are large differences in evolutionary rates and gene turnover between pathways, and that paralogs of Ago2, Ago3, and Piwi/Aub show contrasting rates of evolution after duplication. This suggests that Argonautes undergo frequent evolutionary expansions that facilitate functional divergence.

RNAi pathway genes are resistant to small RNA mediated gene silencing in the protozoan parasite Entamoeba histolytica.

The RNA interference pathway in the protist Entamoeba histolytica plays important roles in permanent gene silencing as well as in the regulation of virulence determinants. Recently, a novel RNA interference (RNAi)-based silencing technique was developed in this parasite that uses a gene endogenously silenced by small RNAs as a “trigger” to induce silencing of other genes that are fused to it. Fusion to a trigger gene induces the production of gene-specific antisense small RNAs, resulting in robust and permanent silencing of the cognate gene. This approach has silenced multiple genes including those involved in virulence and transcriptional regulation. We now demonstrate that all tested genes of the amebic RNAi pathway are unable to be silenced using the trigger approach, including Argonaute genes (Ago2-1, Ago2-2, and Ago2-3), RNaseIII, and RNA-dependent RNA polymerase (RdRP). In all situations (except for RdRP), fusion to a trigger successfully induces production of gene-specific antisense small RNAs to the cognate gene. These small RNAs are capable of silencing a target gene in trans, indicating that they are functional; despite this, however, they cannot silence the RNAi pathway genes. Interestingly, when a trigger is fused to RdRP, small RNA induction to RdRP does not occur, a unique phenotype hinting that either RdRP is highly resistant to being a target of small RNAs or that small RNA generation may be controlled by RdRP. The inability of the small RNA pathway to silence RNAi genes in E. histolytica, despite the generation of functional small RNAs to these loci suggest that epigenetic factors may protect certain genomic loci and thus determine susceptibility to small RNA mediated silencing.

Identification and characterization of Argonaute gene family and meiosis-enriched Argonaute during sporogenesis in maize.

Argonaute (AGO) proteins play a key role in regulation of gene expression through small RNA-directed RNA cleavage and translational repression, and are essential for multiple developmental processes. In the present study, 17 AGO genes of maize (Zea mays L., ZmAGOs) were identified using a Hidden Markov Model and validated by rapid amplification of cDNA ends assay. Subsequently, quantitative PCR revealed that expressions of these genes were higher in reproductive than in vegetative tissues. AGOs presented five temporal and spatial expression patterns, which were likely modulated by DNA methylation, 5'-untranslated exons and microRNA-mediated feedback loops. Intriguingly, ZmAGO18b was highly expressed in tassels during meiosis. Furthermore, in situ hybridization and immunofluorescence showed that ZmAGO18b was enriched in the tapetum and germ cells in meiotic anthers. We hypothesized that ZmAGOs are highly expressed in reproductive tissues, and that ZmAGO18b is a tapetum and germ cell-specific member of the AGO family in maize.

SAT0565 The Expression and Regulation of the Argonaute Protein Family Member PIWIL4 in RA

BackgroundAberrant expression of PIWIL (P-element induced wimpy testis like RNA-mediated gene silencing) 1-4 genes, members of the Argonaute gene family, was reported in cancer. PIWIL proteins build complexes with piwi-interacting...

Identification, Characterization and Expression Profiling of Dicer-Like, Argonaute and RNA-Dependent RNA Polymerase Gene Families in Foxtail Millet

Post-transcriptional control of gene expression is achieved through RNA interference where the activities of Dicer-like (DCL), Argonautes (AGO) and RNA-dependent RNA polymerases (RDRs) are significant. Hence, considering the importance of DCL, AGO and RDRs, a comprehensive genome-wide analysis was performed in foxtail millet. The study identified 8 DCL, 19 AGO and 11 RDR genes. Phylogenetic and domain analysis provided interesting information on the evolutionary and structural aspects of these proteins. The orthologs of Setaria italica DCL (SiDCL), AGO (SiAGO) and RDRs (SiRDRs) were identified in sorghum, maize and rice, and the evolutionary relationships among the orthologous gene pairs were investigated. Promoter analysis of SiDCL, SiAGO and SiRDR genes revealed the presence of unique and common cis-acting elements at the upstream of respective gene sequences, which serves as binding sites for several developmental and stress-related transcription factors. In silico expression profiling using RNA-sequence data showed tissue-specific expression patterns of these genes in foxtail millet. Candidate genes representing each sub-family were chosen for expression analysis through quantitative real-time PCR (qRT-PCR) under salinity, dehydration and hormonal treatments. It revealed the differential expression pattern of candidate genes at different time points of stresses. This is the first report on genome-wide analysis of SiDCL, SiAGO and SiRDR gene families in foxtail millet, which provides basic genomic information and insights into the putative roles of these genes in abiotic stresses. The present study will serve as a base for further functional characterization of these gene families in foxtail millet and related grass species.

Transfection of BmCPV genomic dsRNA in silkmoth-derived Bm5 cells: Stability and interactions with the core RNAi machinery

While several studies have been conducted to investigate the stability of dsRNA in the extracellular medium (hemolymph, gut content, saliva), little is known regarding the persistence of dsRNA once it has been introduced into the cell. Here, we investigate the stability of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) genomic dsRNA fragments after transfection into Bombyx-derived Bm5 cells. Using RT-PCR as a detection method, we found that dsRNA could persist for long periods (up to 8days) in the intracellular environment. While the BmCPV genomic dsRNA was processed by the RNAi machinery, its presence had no effects on other RNAi processes, such as the silencing of a luciferase reporter by dsLuc. We also found that transfection of BmCPV genomic dsRNA could not establish a viral infection in the Bm5 cells, even when co-transfections were carried out with dsRNAs targeting Dicer and Argonaute genes, suggesting that the neutralization by RNAi does not play a role in the establishment of an in vitro culture system. The mechanism of the dsRNA stability in Bm5 cells is discussed, as well as the implications for the establishment for an in vitro culture system for BmCPV.

In silico identification, phylogenetic and bioinformatic analysis of argonaute genes in plants.

Argonaute protein family is the key players in pathways of gene silencing and small regulatory RNAs in different organisms. Argonaute proteins can bind small noncoding RNAs and control protein synthesis, affect messenger RNA stability, and even participate in the production of new forms of small RNAs. The aim of this study was to characterize and perform bioinformatic analysis of Argonaute proteins in 32 plant species that their genome was sequenced. A total of 437 Argonaute genes were identified and were analyzed based on lengths, gene structure, and protein structure. Results showed that Argonaute proteins were highly conserved across plant kingdom. Phylogenic analysis divided plant Argonautes into three classes. Argonaute proteins have three conserved domains PAZ, MID and PIWI. In addition to three conserved domains namely, PAZ, MID, and PIWI, we identified few more domains in AGO of some plant species. Expression profile analysis of Argonaute proteins showed that expression of these genes varies in most of tissues, which means that these proteins are involved in regulation of most pathways of the plant system. Numbers of alternative transcripts of Argonaute genes were highly variable among the plants. A thorough analysis of large number of putative Argonaute genes revealed several interesting aspects associated with this protein and brought novel information with promising usefulness for both basic and biotechnological applications.

Evolution of RNA interference proteins dicer and argonaute in Basidiomycota

RNA interference (RNAi) refers to a mechanism in which cells control gene expression, protect the genome against mobile repetitive DNA sequences, retro elements and transposons, and defend themself against viruses. Two core components, dicer and argonaute, are central in the RNAi machinery. In this study the evolution of argonaute and dicer genes were analyzed with 43 fungal genomes, with the focus on Basidiomycota. Argonaute and dicer genes are widely represented in Basidiomycota as well as in other fungal groups, but the number of copies of them vary. However, in certain lineages, argonaute or dicer is missing. Our results suggest an ancient duplication of dicer and argonaute genes concurrently with early diversification of the Basidiomycota followed by additional species-specific duplications and losses of more recent origin. Several distinct RNAi pathways exist in fungi, based on structural similarity and phylogenetic relationship, our results indicate that quelling possibly exists in most Basidiomycota, while we could not find any evidence for the MSUD (meiotic-silencing) pathway in Basidiomycota. RNAi has been developed to be an important tool for reverse genetics studies. Because both argonaute and dicer are present in almost all Basidiomycota our results indicate that it should be possible to develop RNAi as a tool for functional studies of genes in most Basidiomycota species.

Genome‐wide screening for components of small interfering RNA (siRNA) and micro‐RNA (miRNA) pathways in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)

The brown planthopper (BPH), Nilaparvata lugens, is a major rice pest in Asia, and accumulated evidence indicates that this species is susceptible to RNA interference (RNAi); however, the mechanism underlying RNAi and parental RNAi has not yet been determined. We comprehensively investigated the repertoire of core genes involved in small interfering RNA (siRNA) and micro-RNA (miRNA) pathways in the BPH by comparing its newly assembled transcriptome and genome with those of Drosophila melanogaster, Tribolium castaneum and Caenorhabditis elegans. Our analysis showed that the BPH possesses one drosha and two Dicer (dcr) genes, three dsRNA-binding motif protein genes, two Argonaute (ago) genes, two Eri-1-like genes (eri-1), and a Sid-1-like gene (sid-1). Additionally, we report for first time that parental RNAi might occur in this species, and siRNA pathway and Sid-1 were required for high efficiency of systemic RNAi triggered by exogenous dsRNA. Furthermore, our results also demonstrated that the miRNA pathway was involved in BPH metamorphosis as depletion of the ago1 or dcr1 gene severely impaired ecdysis. The BPH might be a good model system to study the molecular mechanism of systemic RNAi in hemimetabolous insects, and RNAi has potential to be developed to control this pest in agricultural settings.

A Single Argonaute Gene Participates in Exogenous and Endogenous RNAi and Controls Cellular Functions in the Basal Fungus Mucor circinelloides

The mechanism of RNAi is well described in metazoans where it plays a role in diverse cellular functions. However, although different classes of endogenous small RNAs (esRNAs) have been identified in fungi, their biological roles are poorly described due, in part, to the lack of phenotype of mutants affected in the biogenesis of these esRNAs. Argonaute proteins are one of the key components of the RNAi pathways, in which different members of this protein family participate in the biogenesis of a wide repertoire of esRNAs molecules. Here we identified three argonaute genes of the fungus Mucor circinelloides and investigated their participation in exogenous and endogenous RNAi. We found that only one of the ago genes, ago-1, is involved in RNAi during vegetative growth and is required for both transgene-induced RNA silencing and the accumulation of distinct classes of esRNAs derived from exons (ex-siRNAs). Classes I and II ex-siRNAs bind to Ago-1 to control mRNA accumulation of the target protein coding genes. Class III ex-siRNAs do not specifically bind to Ago-1, but requires this protein for their production, revealing the complexity of the biogenesis pathways of ex-siRNAs. We also show that ago-1 is involved in the response to environmental signals, since vegetative development and autolysis induced by nutritional stress are affected in ago-1 − M. circinelloides mutants. Our results demonstrate that a single Ago protein participates in the production of different classes of esRNAs that are generated through different pathways. They also highlight the role of ex-siRNAs in the regulation of endogenous genes in fungi and expand the range of biological functions modulated by RNAi.

Resistance to Germline RNA Interference in a Caenorhabditis elegans Wild Isolate Exhibits Complexity and Nonadditivity

Resolving the genetic complexity of heritable phenotypic variation is fundamental to understanding the mechanisms of evolution and the etiology of human disease. Trait variation among isolates from genetically efficient model organisms offers the opportunity to dissect genetic architectures and identify the molecular mechanisms of causation. Here we present a genetic analysis of loss of sensitivity to gene knockdown via exogenous RNA interference in the germline of a wild isolate of the roundworm Caenorhabditis elegans. We find that the loss of RNA interference sensitivity in the wild isolate CB4856 is recessive to the sensitivity of the lab strain N2. A cross of the strains produced F2 with intermediate sensitivities, and the segregation of the trait among F2s strongly deviated from a single locus recessive allele expectation. Linkage analysis in recombinant inbred lines derived from CB4856 and N2 identified a single significant locus on chromosome I that includes the argonaute gene ppw-1. The alleles for ppw-1 were unable to explain the sensitivity of 18 (12.1%) of the recombinant inbred lines. Complementation tests and F2 segregation analysis of these recombinant inbred lines revealed cases of complex epistatic suppression and enhancement of the effects of ppw-1. We conclude that the variation in RNA interference sensitivity between CB4856 and N2 likely involves the nonadditive interactions of eight or more genes in addition to ppw-1.

Expression pattern of Piwi-like genes in adult Myzostoma cirriferum (Annelida)

Piwi-like genes are a subgroup of Argonaute genes which participate as gene regulators by gene silencing. In most bilaterians, such as mouse, human, insects, and zebrafish, their expression is mostly limited to gonadal stem cells. But there are some striking exceptions to this pattern; flatworms and acoels also express Piwi-like genes in somatic stem cells, due to their unique replacement system. Annelid species like Capitella teleta and Platynereis dumerilii express these genes in cells of the posterior growth zone as well as in gonadal stem cells. To investigate the expression pattern of Piwi-like genes in another annelid, we established in situ hybridization for adult Myzostoma cirriferum. Piwi-like gene transcripts recovered in an mRNA-seq library of pooled adult stages of M. cirriferum were expanded using RACE PCR, cloned and sequenced. ML analysis confirmed the identity of both transcripts as part of the Piwi1-like or Piwi2-like subfamily of Argonaute proteins. The results of in situ hybridization studies show that the expression of both Piwi-like genes, Mc-Piwi1 and Mc-Piwi2, is clearly located only in gonadal stem cells, and as such we did not find any evidence for the existence of a posterior growth zone nor expression in somatic stem cells.

Abstract 1935: Expression of human Piwi-like genes is associated with prognosis for soft tissue sarcoma patients and is different in gender.

Abstract The family of Argonaute genes is essential for stem cell maintenance and RNA interference. Piwi-like genes as a subclass of the Argonaute genes are expressed mainly in the germline. However, these genes may be re-expressed in tumors, and expression of the Piwi-like genes is associated with prognosis in several types of tumors. We analyzed the expression of Piwi-like mRNAs (Piwi-like 2-4) in 125 soft tissue sarcoma (STS) samples by qPCRs. Statistical tests were applied to study the correlation of expression levels with tumor-specific survival for STS patients. In multivariate Cox's regression analyses, we detected that low Piwi-like 2 and Piwi-like 4 mRNA expression were significantly associated with a worse prognosis (RR = 1.87; p = 0.032 and RR = 1.82; p = 0.039). In addition, low expression of both genes was associated with a 2.58-fold increased risk of tumor-related death (p = 0.01). Next we analysed expression of Piwi-like genes gender specific. Piwi-like 4 and combined Piwi-like 2 and 4 mRNA levels correlated significantly with prognosis (RR = 3.53; p = 0.002 and RR = 5.23; p = 0.004) only for female but not for male patients. However, combined low Piwi-like 2 and 3 transcript levels were associated with worse survival (RR = 5.90; p = 0.02) for male patients. In this study, we identified a significant association between the expression of Piwi-like 2 and 4 mRNAs and the tumor-specific survival of soft tissue sarcoma patients. A connection between gender and the impact of Piwi-like mRNA expressions on STS patients’ prognosis was shown for the first time. Citation Format: Thomas Greither, Franziska Koser, Matthias Kappler, Matthias Bache, Christine Lautenschläger, Steffen Goebel, Hans-Jürgen Holzhausen, Sven Wach, Peter Würl, Helge W. Taubert. Expression of human Piwi-like genes is associated with prognosis for soft tissue sarcoma patients and is different in gender. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1935. doi:10.1158/1538-7445.AM2013-1935

Cloning and characterization of two Argonaute genes in wheat (Triticum aestivum L.)

BackgroundArgonaute proteins are key components of RNA interference (RNAi), playing important roles in RNA-directed gene silencing. Various classes of Argonaute genes have been identified from plants and might be involved in developmental regulation. However, little is known about these genes in wheat (Triticum aestivum).ResultsIn this study, two full-length cDNAs of Argonaute were cloned from wheat, designated as TaAGO1b and TaAGO4. The cDNA of TaAGO1b is 3273 bp long and encodes 868 amino acids, with a predicted molecular weight of ~97.78 kDa and pI of 9.29. The 3157-bp TaAGO4 encodes 916 amino acids, with a molecular mass of 102.10 kDa and pI of 9.12. Genomics analysis showed that TaAGO1b and TaAGO4 contain 20 and 18 introns, respectively. Protein structural analysis demonstrated that typical PAZ and PIWI domains were found in both TaAGO1b and TaAGO4. From the highly conserved PIWI domains, we detected conserved Asp-Asp-His (DDH) motifs that function as a catalytic triad and have critical roles during the process of sequence-specific cleavage in the RNAi machinery. Structural modelling indicated that both TaAGOs can fold to a specific α/β structure. Moreover, the three aligned DDH residues are spatially close to each other at the “slicer” site of the PIWI domain. Expression analysis indicated that both genes are ubiquitously expressed in vegetative and reproductive organs, including the root, stem, leaf, anther, ovule, and seed. However, they are differentially expressed in germinating endosperm tissues. We were interested to learn that the two TaAGOs are also differentially expressed in developing wheat plants and that their expression patterns are variously affected by vernalization treatment. Further investigation revealed that they can be induced by cold accumulation during vernalization.ConclusionsTwo putative wheat Argonaute genes, TaAGO1b and TaAGO4, were cloned. Phylogenetic analysis, prediction of conserved domains and catalytic motifs, and modelling of their protein structures suggested that they encode functional Argonaute proteins. Temporal and spatial expression analyses indicated that these genes are potentially involved in developmental regulation of wheat plants.

Genome-wide identification, molecular cloning, expression profiling and posttranscriptional regulation analysis of the Argonaute gene family in Salvia miltiorrhiza, an emerging model medicinal plant

BackgroundArgonaute (AGO) is the core component of RNA-induced silencing complex. The AGO gene family has been analyzed in various plant species; however, there is no report about AGOs in the well-known Traditional Chinese Medicine (TCM) plant, Salvia miltiorrhiza.ResultsThrough a genome-wide analysis, we identified ten SmAGO genes in S. miltiorrhiza. Full-length cDNAs of all SmAGOs were subsequently cloned and sequenced. These SmAGOs were characterized using a comprehensive approach. Sequence features, gene structures and conserved domains were analyzed by the comparison of SmAGOs and AtAGOs. Phylogenetic relationships among AGO proteins from S. miltiorrhiza, Arabidopsis and rice were revealed. The expression levels of SmAGO genes in various tissues of S. miltiorrhiza were investigated. The results implied that some SmAGOs, such as SmAGO1, SmAGO2, SmAGO3, SmAGO7 and SmAGO10, probably played similar roles as their counterparts in Arabidopsis; whereas the others could be more species-specialized. It suggests the conservation and diversity of AGOs in plants. Additionally, we identified a total of 24 hairpin structures, representing six miRNA gene families, to be miRNA precursors. Using the modified 5′-RACE method, we confirmed that SmAGO1 and SmAGO2 were targeted by S. miltiorrhiza miR168a/b and miR403, respectively. It suggests the conservation of AGO1-miR168 and AGO2-miR403 regulatory modules in S. miltiorrhiza and Arabidopsis.ConclusionsThis is the first attempt to explore SmAGOs and miRNAs in S. miltiorrhiza. The results provide useful information for further elucidation of gene silencing pathways in S. miltiorrhiza.

Evidence for small RNAs homologous to effector-encoding genes and transposable elements in the oomycete Phytophthora infestans.

Phytophthora infestans is the oomycete pathogen responsible for the devastating late blight disease on potato and tomato. There is presently an intense research focus on the role(s) of effectors in promoting late blight disease development. However, little is known about how they are regulated, or how diversity in their expression may be generated among different isolates. Here we present data from investigation of RNA silencing processes, characterized by non-coding small RNA molecules (sRNA) of 19–40 nt. From deep sequencing of sRNAs we have identified sRNAs matching numerous RxLR and Crinkler (CRN) effector protein genes in two isolates differing in pathogenicity. Effector gene-derived sRNAs were present in both isolates, but exhibited marked differences in abundance, especially for CRN effectors. Small RNAs in P. infestans grouped into three clear size classes of 21, 25/26 and 32 nt. Small RNAs from all size classes mapped to RxLR effector genes, but notably 21 nt sRNAs were the predominant size class mapping to CRN effector genes. Some effector genes, such as PiAvr3a, to which sRNAs were found, also exhibited differences in transcript accumulation between the two isolates. The P. infestans genome is rich in transposable elements, and the majority of sRNAs of all size classes mapped to these sequences, predominantly to long terminal repeat (LTR) retrotransposons. RNA silencing of Dicer and Argonaute genes provided evidence that generation of 21 nt sRNAs is Dicer-dependent, while accumulation of longer sRNAs was impacted by silencing of Argonaute genes. Additionally, we identified six microRNA (miRNA) candidates from our sequencing data, their precursor sequences from the genome sequence, and target mRNAs. These miRNA candidates have features characteristic of both plant and metazoan miRNAs.

Expression of human Piwi-likegenes is associated with prognosis for soft tissue sarcoma patients

BackgroundArgonaute genes are essential for RNA interference, stem cell maintenance and differentiation. The Piwi-like genes, a subclass of the Argonaute genes, are expressed mainly in the germline. These genes may be re-expressed in tumors, and expression of the Piwi-like genes is associated with prognosis in several types of tumors.MethodsWe measured the expression of Piwi-like mRNAs (Piwi-like 2–4) in 125 soft tissue sarcoma (STS) samples by qPCRs. Statistical tests were applied to study the correlation of expression levels with tumor-specific survival for STS patients.ResultsIn multivariate Cox’s regression analyses, we showed that low Piwi-like 2 and Piwi-like 4 mRNA expression were significantly associated with a worse prognosis (RR = 1.87; p = 0.032 and RR = 1.82; p = 0.039). Low expression of both genes was associated with a 2.58-fold increased risk of tumor-related death (p = 0.01). Piwi-like 4 and combined Piwi-like 2 and 4 mRNA levels correlated significantly with prognosis (RR = 3.53; p = 0.002 and RR = 5.23; p = 0.004) only for female but not for male patients. However, combined low Piwi-like 2 and 3 transcript levels were associated with worse survival (RR = 5.90; p = 0.02) for male patients.ConclusionsIn this study, we identified a significant association between the expression of Piwi-like 2 and 4 mRNAs and the tumor-specific survival of soft tissue sarcoma patients. Furthermore, a connection between sex and the impact of Piwi-like mRNA expressions on STS patients’ prognosis was shown for the first time.

MicroRNA profiles and their control of male gametophyte development in rice

Plant microRNAs (miRNAs) act as negative regulators of gene expression by slicing target transcripts or inhibiting translation. A number of miRNAs play important roles in development. In order to investigate the potential function of miRNAs during male gametogenesis in rice, we obtained both gene and small RNA expression profiles by combining microarray and high-throughput sequencing technologies. From the microarray datasets, 2,925 male gametophyte-specific genes were identified, including 107 transcription factors and three significant Argonaute genes (AGO12, AGO13, and AGO17). From the sRNA-Seq datasets, 104 unique miRNAs (miRus) were identified, including 47 known miRus and 57 novel miRus; interestingly, most of the new miRus are pollen-specific and not conserved among species. Furthermore, an interactive network of miRNA-target was constructed based on the two datasets. By employing enrichment analysis, the miRNA-regulated targets were found to be involved in both the up and down pathways, but predominantly in the down pathways, including 37 GO biological processes and 32 KEGG pathways. These findings indicate that miRNAs play a broad regulatory role during male gametophyte development in rice.

Repression of Germline RNAi Pathways in Somatic Cells by Retinoblastoma Pathway Chromatin Complexes

The retinoblastoma (Rb) tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene.

Genome-wide identification of Dicer-like, Argonaute and RNA-dependent RNA polymerase gene families and their expression analyses in response to viral infection and abiotic stresses in Solanum lycopersicum

Dicer, Argonaute and RNA-dependent RNA polymerase form the core components to trigger RNA silencing. Although tomato (Solanum lycopersicum) is a dicotyledon model plant, no systematic analysis and expression profiling of these genes in tomato has been undertaken previously. In this study, seven Dicer-like (SlDCLs), 15 Argonaute (SlAGOs) and six RNA-dependent RNA polymerase (SlRDRs) genes were identified in tomato. These genes were categorized into four subgroups based on phylogenetic analyses. Comprehensive analyses of gene structure, genomic localization and similarity among these genes were performed. Their expression patterns were investigated by means of expression models in different tissues and organs using online data and semi-quantitative RT-PCR. Many of the candidate genes were up-regulated in response to Tomato yellow leaf curl virus infection and abiotic stresses. The expression models of tandem gene duplications among SlDCL2s indicated the DCL2 family plays an important role in the evolution of tomato.