The RNA interference (RNAi) mediated by homology-dependent degradation of the target mRNA with small RNA molecules plays a key role in controlling transcription and translation processes in a number of eukaryotic organisms. The RNAi machinery is also evolutionarily conserved in a wide variety of fungal species, including pathogenic fungi. To elucidate the physiological functions of the RNAi pathway in Cryptococcus neoformans that causes fungal meningitis, here we performed genetic analyses for genes encoding Argonaute (AGO1 and AGO2), RNA-dependent RNA polymerase (RDP1), and Dicers (DCR1 and DCR2) in both serotype A and D C. neoformans. The present study shows that Ago1, Rdp1, and Dcr2 are the major components of the RNAi process occurring in C. neoformans. However, the RNAi machinery is not involved in regulation of production of two virulence factors (capsule and melanin), sexual differentiation, and diverse stress response. Comparative transcriptome analysis of the serotype A and D RNAi mutants revealed that only modest changes occur in the genome-wide transcriptome profiles when the RNAi process was perturbed. Notably, the serotype D rdp1Δ mutants showed an increase in transcript abundance of active retrotransposons and transposons, such as T2 and T3, the latter of which is a novel serotype D-specific transposon of C. neoformans. In a wild type background both T2 and T3 were found to be weakly active mobile elements, although we found no evidence of Cnl1 retrotransposon mobility. In contrast, all three transposable elements exhibited enhanced mobility in the rdp1Δ mutant strain. In conclusion, the RNAi pathway plays an important role in controlling transposon activity and genome integrity of C. neoformans.
RNA interference; RNA-dependent RNA polymerase; Dicer; Argonaute; Transposon
Yeast and filamentous fungi have been essential model systems for unveiling the secrets of RNA interference (RNAi). Research on these organisms has contributed to identifying general mechanisms and conserved eukaryotic RNAi machinery that can be found from fungi to mammals. The development of deep sequencing technologies has brought on the last wave of studies on RNAi in fungi, which has been focused on the identification of new types of functional small RNAs (sRNAs). These studies have discovered an unexpected diversity of sRNA, biogenesis pathways and new functions that are the focus of this review.
fungi; sRNA; siRNA; masiRNAs; esRNA; ex-siRNA; milRNA; qiRNA
When recognized by the RNA interference (RNAi) pathway, double-stranded RNA (dsRNA) produced in eukaryotic cells results in posttranscriptional gene silencing. In addition, dsRNA can trigger the interferon response as part of the immune response in vertebrates. In this study, we show that dsRNA, but not short interfering RNA (siRNA), induces the expression of qde-2 (an Argonaute gene) and dcl-2 (a Dicer gene), two central components of the RNAi pathway in the filamentous fungus Neurospora crassa. The induction of QDE-2 by dsRNA is required for normal gene silencing, indicating that this is a regulatory mechanism that allows the optimal function of the RNAi pathway. In addition, we demonstrate that Dicer proteins (DCLs) regulate QDE-2 posttranscriptionally, suggesting a role for DCLs or siRNA in QDE-2 accumulation. Finally, a genome-wide search revealed that additional RNAi components and homologs of antiviral and interferon-stimulated genes are also dsRNA-activated genes in Neurospora. Together, our results suggest that the activation of the RNAi components is part of a broad ancient host defense response against viral and transposon infections.
RNA interference (RNAi) pathways are widespread in metaozoans but the genes required show variable occurrence or activity in eukaryotic microbes, including many pathogens. While some Leishmania lack RNAi activity and Argonaute or Dicer genes, we show that Leishmania braziliensis and other species within the Leishmania subgenus Viannia elaborate active RNAi machinery. Strong attenuation of expression from a variety of reporter and endogenous genes was seen. As expected, RNAi knockdowns of the sole Argonaute gene implicated this protein in RNAi. The potential for functional genetics was established by testing RNAi knockdown lines lacking the paraflagellar rod, a key component of the parasite flagellum. This sets the stage for the systematic manipulation of gene expression through RNAi in these predominantly diploid asexual organisms, and may also allow selective RNAi-based chemotherapy. Functional evolutionary surveys of RNAi genes established that RNAi activity was lost after the separation of the Leishmania subgenus Viannia from the remaining Leishmania species, a divergence associated with profound changes in the parasite infectious cycle and virulence. The genus Leishmania therefore offers an accessible system for testing hypothesis about forces that may select for the loss of RNAi during evolution, such as invasion by viruses, changes in genome plasticity mediated by transposable elements and gene amplification (including those mediating drug resistance), and/or alterations in parasite virulence.
RNAi interference pathways play fundamental roles in eukaryotes and provide important methods for the analysis of gene function. Occasionally RNAi has been lost, precluding its use as a tool, as well as raising the question of what forces could lead to loss of such a key pathway. Genomic and functional studies previously showed that within trypanosomatids protozoans RNAi was absent in both Leishmania major and Trypanosoma cruzi. The genome of L. braziliensis, a member of the early diverging Leishmania subgenus Viannia, retained key genes required for RNAi such as an Argonaute. We demonstrated that in fact L. braziliensis shows strong RNAi activity with reporter and endogenous genes affecting flagellar function. These data suggest that RNAi may be productively applied for functional genomic studies in L. braziliensis. We mapped the evolutionary point at which RNAi was lost in lineage leading to Leishmania and Crithidia, and establish that RNAi must have been lost at least twice in the trypanosomatids, once on the lineage leading to T. cruzi and independently following the divergence of the Viannia subgenus from other Leishmania species. Lastly, we discuss hypotheses concerning the forces leading to the loss of RNAi in Leishmania evolution, including viral invasion, increased genome plasticity, and altered virulence.
Double-stranded RNA has been shown to induce gene silencing in diverse eukaryotes and by a variety of pathways. We have examined the taxonomic distribution and the phylogenetic relationship of key components of the RNA interference (RNAi) machinery in members of five eukaryotic supergroups. On the basis of the parsimony principle, our analyses suggest that a relatively complex RNAi machinery was already present in the last common ancestor of eukaryotes and consisted, at a minimum, of one Argonaute-like polypeptide, one Piwi-like protein, one Dicer, and one RNA-dependent RNA polymerase. As proposed before, the ancestral (but non-essential) role of these components may have been in defense responses against genomic parasites such as transposable elements and viruses. From a mechanistic perspective, the RNAi machinery in the eukaryotic ancestor may have been capable of both small-RNA-guided transcript degradation as well as transcriptional repression, most likely through histone modifications. Both roles appear to be widespread among living eukaryotes and this diversification of function could account for the evolutionary conservation of duplicated Argonaute-Piwi proteins. In contrast, additional RNAi-mediated pathways such as RNA-directed DNA methylation, programmed genome rearrangements, meiotic silencing by unpaired DNA, and miRNA-mediated gene regulation may have evolved independently in specific lineages.
RNA interference; Transposon silencing; Heterochromatin; RNAi phylogenetics
Eukaryotic cells express a wide variety of endogenous small regulatory RNAs that regulate heterochromatin formation, developmental timing, defense against parasitic nucleic acids, and genome rearrangement. Many small regulatory RNAs are thought to function in nuclei 1-2. For instance, in plants and fungi siRNAs associate with nascent transcripts and direct chromatin and/or DNA modifications 1-2. To further understand the biological roles of small regulatory RNAs, we conducted a genetic screen to identify factors required for RNA interference (RNAi) in C. elegans nuclei 3. Here we show that nrde-2 encodes an evolutionarily conserved protein that is required for small interfering (si)RNA-mediated silencing in nuclei. NRDE-2 associates with the Argonaute protein NRDE-3 within nuclei and is recruited by NRDE-3/siRNA complexes to nascent transcripts that have been targeted by RNAi. We find that nuclear-localized siRNAs direct a NRDE-2-dependent silencing of pre-mRNAs 3’ to sites of RNAi, a NRDE-2-dependent accumulation of RNA Polymerase (RNAP) II at genomic loci targeted by RNAi, and NRDE-2-dependent decreases in RNAP II occupancy and RNAP II transcriptional activity 3’ to sites of RNAi. These results define NRDE-2 as a component of the nuclear RNAi machinery and demonstrate that metazoan siRNAs can silence nuclear-localized RNAs co-transcriptionally. In addition, these results establish a novel mode of RNAP II regulation; siRNA-directed recruitment of NRDE factors that inhibit RNAP II during the elongation phase of transcription.
Beyond their role in post-transcriptional gene silencing, Dicer and Argonaute, two components of the RNA interference (RNAi) machinery, were shown to be involved in epigenetic regulation of centromeric heterochromatin and transcriptional gene silencing. In particular, RNAi mechanisms appear to play a role in repeat induced silencing and some aspects of Polycomb-mediated gene silencing. However, the functional interplay of RNAi mechanisms and Polycomb group (PcG) pathways at endogenous loci remains to be elucidated.
Here we show that the endogenous Dicer-2/Argonaute-2 RNAi pathway is dispensable for the PcG mediated silencing of the homeotic Bithorax Complex (BX-C). Although Dicer-2 depletion triggers mild transcriptional activation at Polycomb Response Elements (PREs), this does not induce transcriptional changes at PcG-repressed genes. Moreover, Dicer-2 is not needed to maintain global levels of methylation of lysine 27 of histone H3 and does not affect PRE-mediated higher order chromatin structures within the BX-C. Finally bioinformatic analysis, comparing published data sets of PcG targets with Argonaute-2-bound small RNAs reveals no enrichment of these small RNAs at promoter regions associated with PcG proteins.
We conclude that the Dicer-2/Argonaute-2 RNAi pathway, despite its role in pairing sensitive gene silencing of transgenes, does not have a role in PcG dependent silencing of major homeotic gene cluster loci in Drosophila.
In eukaryotes, RNA interference (RNAi) is a major mechanism of defense against viruses and transposable elements as well of regulating translation of endogenous mRNAs. The RNAi systems recognize the target RNA molecules via small guide RNAs that are completely or partially complementary to a region of the target. Key components of the RNAi systems are proteins of the Argonaute-PIWI family some of which function as slicers, the nucleases that cleave the target RNA that is base-paired to a guide RNA. Numerous prokaryotes possess the CRISPR-associated system (CASS) of defense against phages and plasmids that is, in part, mechanistically analogous but not homologous to eukaryotic RNAi systems. Many prokaryotes also encode homologs of Argonaute-PIWI proteins but their functions remain unknown.
We present a detailed analysis of Argonaute-PIWI protein sequences and the genomic neighborhoods of the respective genes in prokaryotes. Whereas eukaryotic Ago/PIWI proteins always contain PAZ (oligonucleotide binding) and PIWI (active or inactivated nuclease) domains, the prokaryotic Argonaute homologs (pAgos) fall into two major groups in which the PAZ domain is either present or absent. The monophyly of each group is supported by a phylogenetic analysis of the conserved PIWI-domains. Almost all pAgos that lack a PAZ domain appear to be inactivated, and the respective genes are associated with a variety of predicted nucleases in putative operons. An additional, uncharacterized domain that is fused to various nucleases appears to be a unique signature of operons encoding the short (lacking PAZ) pAgo form. By contrast, almost all PAZ-domain containing pAgos are predicted to be active nucleases. Some proteins of this group (e.g., that from Aquifex aeolicus) have been experimentally shown to possess nuclease activity, and are not typically associated with genes for other (putative) nucleases. Given these observations, the apparent extensive horizontal transfer of pAgo genes, and their common, statistically significant over-representation in genomic neighborhoods enriched in genes encoding proteins involved in the defense against phages and/or plasmids, we hypothesize that pAgos are key components of a novel class of defense systems. The PAZ-domain containing pAgos are predicted to directly destroy virus or plasmid nucleic acids via their nuclease activity, whereas the apparently inactivated, PAZ-lacking pAgos could be structural subunits of protein complexes that contain, as active moieties, the putative nucleases that we predict to be co-expressed with these pAgos. All these nucleases are predicted to be DNA endonucleases, so it seems most probable that the putative novel phage/plasmid-defense system targets phage DNA rather than mRNAs. Given that in eukaryotic RNAi systems, the PAZ domain binds a guide RNA and positions it on the complementary region of the target, we further speculate that pAgos function on a similar principle (the guide being either DNA or RNA), and that the uncharacterized domain found in putative operons with the short forms of pAgos is a functional substitute for the PAZ domain.
The hypothesis that pAgos are key components of a novel prokaryotic immune system that employs guide RNA or DNA molecules to degrade nucleic acids of invading mobile elements implies a functional analogy with the prokaryotic CASS and a direct evolutionary connection with eukaryotic RNAi. The predictions of the hypothesis including both the activities of pAgos and those of the associated endonucleases are readily amenable to experimental tests.
This article was reviewed by Daniel Haft, Martijn Huynen, and Chris Ponting.
Although RNA-mediated interference (RNAi) is a widely conserved process among eukaryotes, including many fungi, it is absent from the budding yeast Saccharomyces cerevisiae. Three human proteins, Ago2, Dicer and TRBP, are sufficient for reconstituting the RISC complex in vitro. To examine whether the introduction of human RNAi genes can reconstitute RNAi in S. cerevisiae, genes encoding these three human proteins were introduced into S. cerevisiae. We observed both siRNA and siRNA- and RISC-dependent silencing of the target gene GFP. Thus, human Ago2, Dicer and TRBP can functionally reconstitute human RNAi in S. cerevisiae, in vivo, enabling the study and use of the human RNAi pathway in a facile genetic model organism.
Dicer ribonucleases of plants and invertebrate animals including Caenorhabditis elegans recognize and process a viral RNA trigger into virus-derived small interfering RNAs (siRNAs) to guide specific viral immunity by Argonaute-dependent RNA interference (RNAi). C. elegans also encodes three Dicer-related helicase (drh) genes closely related to the RIG-I-like RNA helicase receptors which initiate broad-spectrum innate immunity against RNA viruses in mammals. Here we developed a transgenic C. elegans strain that expressed intense green fluorescence from a chromosomally integrated flock house virus replicon only after knockdown or knockout of a gene required for antiviral RNAi. Use of the reporter nematode strain in a feeding RNAi screen identified drh-1 as an essential component of the antiviral RNAi pathway. However, RNAi induced by either exogenous dsRNA or the viral replicon was enhanced in drh-2 mutant nematodes, whereas exogenous RNAi was essentially unaltered in drh-1 mutant nematodes, indicating that exogenous and antiviral RNAi pathways are genetically distinct. Genetic epistatic analysis shows that drh-1 acts downstream of virus sensing and viral siRNA biogenesis to mediate specific antiviral RNAi. Notably, we found that two members of the substantially expanded subfamily of Argonautes specific to C. elegans control parallel antiviral RNAi pathways. These findings demonstrate both conserved and unique strategies of C. elegans in antiviral defense.
The genome of Caenorhabditis elegans encodes three Dicer-related helicases (DRHs) highly homologous to the DExD/H box helicase domain found in two distinct families of virus sensors, Dicer ribonucleases and RIG-I-like helicases (RLRs). Dicer initiates the specific, RNAi-mediated viral immunity in plants, fungi and invertebrates by producing virus-derived small interfering RNAs (siRNAs). By contrast, mammalian RLRs trigger interferon production and broad-spectrum viral immunity, although one of the three RLRs may act as both a negative and positive regulator of viral immunity. In this study we developed a transgenic C. elegans strain for high-throughput genetic screens and identified 35 genes including drh-1 that are required for RNAi-mediated viral immunity. Genetic epistatic analyses demonstrate that drh-1 mediates RNAi immunity downstream of the production of viral siRNAs. Notably, we found that drh-2 functions as a negative regulator of the viral immunity. Thus, both nematode DRHs and mammalian RLRs participate in antiviral immune responses. Unlike mammalian RLRs, however, nematode DRH-1 employs an RNAi effector mechanism and is unlikely to be involved in direct virus sensing.
A recent study by Massirer et al. in the nematode C. elegans has shown that a family of microRNAs (miRNAs), miR-35-41, regulates the efficiency of RNA interference (RNAi), revealing a new connection between these small RNA pathways. In this commentary, we discuss the potential mechanisms for cross regulation in the miRNA and RNAi pathways and the implications for gene expression. While miRNAs are genetically encoded, the small interfering RNAs (siRNAs) that function in RNAi can originate from processing of exogenous dsRNA (exo-RNAi) or from the production of siRNAs from endogenous transcripts (endo-RNAi). These small RNA pathways involve Dicer and Argonaute proteins and typically use antisense base pairing to target mRNAs for downregulated expression. The discovery that loss of miR-35–41 results in enhanced exo-RNAi sensitivity and reduced endo-RNAi effectiveness suggests that these miRNAs normally help balance the RNAi pathways. The effect of mir-35–41 on RNAi is largely through lin-35, the C. elegans homolog of the tumor suppressor Retinoblastoma (Rb) gene. lin-35/Rb previously has been shown to regulate RNAi sensitivity through unclear mechanisms and the new finding that accumulation of LIN-35/Rb protein is dependent on miR-35–41 adds another layer of complexity to this process. The utilization of miRNAs to control the responsiveness of RNAi exemplifies the cross-regulation embedded in small RNA-directed pathways.
C. elegans; RNAi; lin-35; miR-35-41; miRNA; retinoblastoma (Rb)
An extended dsRBD with a novel zinc-binding motif mediates nuclear retention of fission yeast Dicer
The Dicer ribonuclease Dcr1 plays an important role in the biogenesis of small regulatory RNAs. Surprisingly, RNA binding by the double-stranded RNA binding domain (dsRBD) is dispensable for Dcr1 function, while zinc coordination of the extended dsRBD is required for its nuclear localization and RNA silencing.
Dicer proteins function in RNA interference (RNAi) pathways by generating small RNAs (sRNAs). Here, we report the solution structure of the C-terminal domain of Schizosaccharomyces pombe Dicer (Dcr1). The structure reveals an unusual double-stranded RNA binding domain (dsRBD) fold embedding a novel zinc-binding motif that is conserved among dicers in yeast. Although the C-terminal domain of Dcr1 still binds nucleic acids, this property is dispensable for proper functioning of Dcr1. In contrast, disruption of zinc coordination renders Dcr1 mainly cytoplasmic and leads to remarkable changes in gene expression and loss of heterochromatin assembly. In summary, our results reveal novel insights into the mechanism of nuclear retention of Dcr1 and raise the possibility that this new class of dsRBDs might generally function in nucleocytoplasmic trafficking and not substrate binding. The C-terminal domain of Dcr1 constitutes a novel regulatory module that might represent a potential target for therapeutic intervention with fungal diseases.
dicer; dsRBD; heterochromatin; RNA interference; zinc-binding domain
RNA interference (RNAi) was initially discovered as a post-transcriptional gene silencing mechanism in which short RNAs are used to target complementary RNAs for degradation. During the past several years, it has been demonstrated that RNAi-related processes are also involved in transcriptional gene silencing by directing formation of heterochromatin. The dynamic DNA rearrangement during sexual reproduction of the ciliated protozoan Tetrahymena provides an extreme example of RNAi-directed heterochromatin formation. In this process, small RNAs of ~28-29 nt, which are processed by the Dicer-like protein Dcl1p and are associated with the Argonaute family protein Twi1p, induce heterochromatin formation at complementary genomic sequences by recruiting the histone H3 lysine 9/27 methyltransferase Ezl1p and chromodomain proteins. Eventually these heterochromatinized regions are targeted for DNA elimination. In many eukaryotes, one of the major roles for RNAi-related mechanisms is silencing transposons, and DNA elimination in Tetrahymena is also believed to have evolved as a transposon defense by removing transposon-related sequences from the somatic genome. Because DNA elimination is achieved by the coordinated actions of non-coding RNA transcription, RNA processing, RNA transport, RNA-RNA and RNA-protein interactions, RNA degradation and RNA-directed chromatin modifications, DNA elimination in Tetrahymena is a useful model to study ‘RNA infrastructure’.
DNA elimination; Tetrahymena; ciliate; RNAi; small RNA; siRNA; piRNA; CRISPR; non-coding RNA; epigenetics; heterochromatin; transcriptional silencing; transposon silencing; Argonaute; Piwi; Dicer
RNA interference (RNAi) is a eukaryotic gene-silencing mechanism that functions in antiviral immunity in diverse organisms. To combat RNAi-mediated immunity, viruses encode viral suppressors of RNA silencing (VSRs) that target RNA and protein components in the RNAi machinery. Although the endonuclease Dicer plays key roles in RNAi immunity, little is known about how VSRs target Dicer. Here, we show that the B2 protein from Wuhan nodavirus (WhNV), the counterpart of Flock House virus (FHV), suppresses Drosophila melanogaster RNAi by directly interacting with Dicer-2 (Dcr-2) and sequestering double-stranded RNA (dsRNA) and small interfering RNA (siRNA). Further investigations reveal that WhNV B2 binds to the RNase III and Piwi-Argonaut-Zwille (PAZ) domains of Dcr-2 via its C-terminal region, thereby blocking the activities of Dcr-2 in processing dsRNA and incorporating siRNA into the RNA-induced silencing complex (RISC). Moreover, we uncover an interrelationship among diverse activities of WhNV B2, showing that RNA binding enhances the B2–Dcr-2 interaction by promoting B2 homodimerization. Taken together, our findings establish a model of suppression of Drosophila RNAi by WhNV B2 targeting both Dcr-2 and RNA and provide evidence that an interrelationship exists among diverse activities of VSRs to antagonize RNAi.
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.
Genetic and biochemical analyses of RNA interference (RNAi) and microRNA (miRNA) pathways have revealed proteins such as Argonaute/PIWI and Dicer that process and present small RNAs to their targets. Well validated small RNA pathway cofactors, such as the Argonaute/PIWI proteins show distinctive patterns of conservation or divergence in particular animal, plant, fungal, and protist species. We compared 86 divergent eukaryotic genome sequences to discern sets of proteins that show similar phylogenetic profiles with known small RNA cofactors. A large set of additional candidate small RNA cofactors have emerged from functional genomic screens for defects in miRNA- or siRNA-mediated repression in C. elegans and D. melanogaster1,2 and from proteomic analyses of proteins co-purifying with validated small RNA pathway proteins3,4. The phylogenetic profiles of many of these candidate small RNA pathway proteins are similar to those of known small RNA cofactor proteins. We used a Bayesian approach to integrate the phylogenetic profile analysis with predictions from diverse transcriptional coregulation and proteome interaction datasets to assign a probability for each protein for a role in a small RNA pathway. Testing high-confidence candidates from this analysis for defects in RNAi silencing, we found that about half of the predicted small RNA cofactors are required for RNAi silencing. Many of the newly identified small RNA pathway proteins are orthologues of proteins implicated in RNA splicing. In support of a deep connection between the mechanism of RNA splicing and small RNA-mediated gene silencing, the presence of the Argonaute proteins and other small RNA components in the many species analysed strongly correlates with the number of introns in that species.
Heterochromatin is the tightly packaged dynamic region of the eukaryotic chromosome that plays a vital role in cellular processes such as mitosis and meiotic recombination. Recent experiments in Schizosaccharomyces pombe have revealed the structure of centromeric heterochromatin is affected in RNAi pathway mutants. It has also been shown in fission yeast that the heterochromatin barrier is traversed by RNA Pol II and that the passage of RNA Pol II through heterochromatin is important for heterochromatin structure. Thus, an intricate interaction between the RNAi machinery and RNA Pol II affects heterochromatin structure. However, the role of the RNAi machinery and RNA Pol II on the metazoan heterochromatin landscape is not known. This study analyses the interaction of the small RNA machinery and RNA Pol II on Drosophila heterochromatin structure.
The results in this paper show genetic and biochemical interaction between RNA Pol II (largest and second largest subunit) and small RNA silencing machinery components (dcr-2, ago1, ago2, piwi, Lip [D], aub and hls). Immunofluorescence analysis of polytene chromosomes from trans-heterozygotes of RNA Pol II and different mutations of the small RNA pathways show decreased H3K9me2 and mislocalization of Heterochromatin protein-1. A genetic analysis performed on these mutants showed a strong suppression of white-mottled4h position effect variegation. This was further corroborated by a western blot analysis and chromatin immunoprecipitation, which showed decreased H3K9me2 in trans-heterozygote mutants compared to wild type or single heterozygotes. Co-immunoprecipitation performed using Drosophila embryo extracts showed the RNA Pol II largest subunit interacting with Dcr-2 and dAGO1. Co-localization performed on polytene chromosomes showed RNA Pol II and dAGO1 overlapping at some sites.
Our experiments show a genetic and biochemical interaction between RNA Pol II (largest and second largest subunits) and the small RNA silencing machinery in Drosophila. The interaction has functional aspects in terms of determining H3K9me2 and HP-1 deposition at the chromocentric heterochromatin. Thus, RNA Pol II has an important role in establishing heterochromatin structure in Drosophila.
Small interfering RNAs (siRNAs) and genome-encoded microRNAs (miRNAs) silence genes via complementary interactions with mRNAs. With thousands of miRNA genes identified and genome sequences of diverse eukaryotes available for comparison, the opportunity emerges for insights into origin and evolution of RNA interference (RNAi). The miRNA repertoires of plants and animals appear to have evolved independently. However, conservation of the key proteins involved in RNAi suggests that the last common ancestor of modern eukaryotes possessed siRNA-based mechanisms. Prokaryotes have a RNAi-like defense system that is functionally analogous but not homologous to eukaryotic RNAi. The protein machinery of eukaryotic RNAi seems to have been pieced together from ancestral proteins of archaeal, bacterial and phage origins that are involved in DNA repair and RNA-processing pathways.
The specificity of RNAi pathways is determined by several classes of small RNAs, which include siRNAs, piRNAs, endo-siRNAs, and microRNAs (miRNAs). These small RNAs are invariably incorporated into large Argonaute (Ago)-containing effector complexes known as RNA-induced silencing complexes (RISCs), which they guide to silencing targets. Both genetic and biochemical strategies have yielded conserved molecular components of small RNA biogenesis and effector machineries. However, given the complexity of these pathways, there are likely to be additional components and regulators that remain to be uncovered. We have undertaken a comparative and comprehensive RNAi screen to identify genes that impact three major Ago-dependent small RNA pathways that operate in Drosophila S2 cells. We identify subsets of candidates that act positively or negatively in siRNA, endo-siRNA and miRNA pathways. Our studies indicate that many components are shared among all three Argonaute-dependent silencing pathways, though each is also impacted by discrete sets of genes.
Cytosine methylation of DNA is an important epigenetic gene silencing mechanism in plants, fungi, and animals. In the filamentous fungus Neurospora crassa, nearly all known DNA methylations occur in transposon relics and repetitive sequences, and DNA methylation does not depend on the canonical RNAi pathway. disiRNAs are Dicer-independent small non-coding RNAs that arise from gene-rich part of the Neurospora genome. Here we describe a new type of DNA methylation that is associated with the disiRNA loci. Unlike the known DNA methylation in Neurospora, disiRNA loci DNA methylation (DLDM) is highly dynamic and is regulated by an on/off mechanism. Some disiRNA production appears to rely on pol II directed transcription. Importantly, DLDM is triggered by convergent transcription and enriched in promoter regions. Together, our results establish a new mechanism that triggers DNA methylation.
DNA methylation in eukayrotes refers to the modification of cytidines at 5th position with methyl group (5mC). Though absent in some species, DNA methylation is conserved across fungi, plants and animals and plays a critical role in X chromosome inactivation, genomic imprinting, transposon silencing etc. In addition, DNA methylation also occurs at the promoter sequence to regulate gene expression. Filamentous fungus Neurospora crassa has a well-known mechanism of DNA methylation for genomic defense. During sexual stage repetitive sequences (e.g. transposons) are recognized and point mutations are introduced. During vegetative stage these mutations serve as signals for establishing static DNA methylation to silence all copies of the sequences. In this study, we report a new type of DNA methylation in Neurospora. It is tightly linked to a type of non-coding small RNA termed dicer-independent siRNA (disiRNA) and therefore was termed disiRNA loci DNA methylation (DLDM). DLDM is dynamic regulated and shows an on/off pattern, i.e. most alleles contain no 5mC but some are densely methylated. Interestingly, DLDM can be triggered by convergent transcription and is accumulated at promoter regions. In summary, our findings demonstrate a new type of dynamic DNA methylation.
A variety of small RNAs, including the Dicer-dependent miRNAs and the Dicer-independent Piwi-interacting RNAs, associate with Argonaute family proteins to regulate gene expression in diverse cellular processes. These two species of small RNA have not been found in fungi. Here, by analyzing small RNA associated with the Neurospora Argonaute protein QDE-2, we show that diverse pathways generate miRNA-like small RNAs (milRNAs) and Dicer-independent small interfering RNAs (disiRNAs) in this filamentous fungus. Surprisingly, milRNAs are produced by at least four different mechanisms that use a distinct combination of factors, including Dicers, QDE-2, the exonuclease QIP and an RNAse III domain-containing protein MRPL3. In contrast, disiRNAs originate from loci producing overlapping sense and antisense transcripts, and do not require the known RNAi components for their production. Taken together, these results uncover several pathways for small RNA production in filamentous fungi, shedding light on the diversity and evolutionary origins of eukaryotic small RNAs.
RNA interference (RNAi) uses small RNA molecules to regulate transcriptional and post-transcriptional gene expression. In recent years, a number of structural studies provided insights into the molecular architecture and mechanism of functional modules of RNAi. Mechanisms of nucleic acid recognition and cleavage have been revealed by structural studies of proteins and their nucleic acid complexes involved in RNA biogenesis, for example, Argonaute, PIWI, RNase III, Dicer, Drosha and DGCR8. While quite a few questions remain, an excellent structural and mechanistic overview of RNAi processes has already emerged. In this review, we examine functional modules and their assemblies in RNAi processes.
RNAi is a conserved mechanism in which small interfering RNAs (siRNAs) guide the degradation of cognate RNAs, but also promote heterochromatin assembly at repetitive DNA elements such as centromeric repeats1,2. However, the full extent of RNAi functions and its endogenous targets have not been explored. Here we show that in the fission yeast Schizosaccharomyces pombe, RNAi and heterochromatin factors cooperate to silence diverse loci, including sexual differentiation genes, genes encoding transmembrane proteins, and retrotransposons that are also targeted by the exosome RNA degradation machinery. In the absence of the exosome, transcripts are processed preferentially by the RNAi machinery, revealing siRNA clusters and corresponding increase in heterochromatin modifications across large domains containing genes and retrotransposons. We show that the generation of siRNAs and heterochromatin assembly by RNAi is triggered by a mechanism involving the canonical poly(A) polymerase Pla1 and an associated RNA surveillance factor Red1, which also activate the exosome. Remarkably, siRNA production and heterochromatin modifications at these target loci are regulated by environmental growth conditions, and by developmental signals that induce gene expression during sexual differentiation. Our analyses uncover interplay between RNAi and the exosome that is conserved in higher eukaryotes, and show that differentiation signals modulate RNAi silencing to regulate developmental genes.
Small RNA regulatory pathways (SRRPs) control key aspects of development and anti-viral defense in metazoans. Members of the Argonaute family of catalytic enzymes degrade target RNAs in each of these pathways. SRRPs include the microRNA, small interfering RNA (siRNA) and PIWI-type gene silencing pathways. Mosquitoes generate viral siRNAs when infected with RNA arboviruses. However, in some mosquitoes, arboviruses survive antiviral RNA interference (RNAi) and are transmitted via mosquito bite to a subsequent host. Increased knowledge of these pathways and functional components should increase understanding of the limitations of anti-viral defense in vector mosquitoes. To do this, we compared the genomic structure of SRRP components across three mosquito species and three major small RNA pathways.
The Ae. aegypti, An. gambiae and Cx. pipiens genomes encode putative orthologs for all major components of the miRNA, siRNA, and piRNA pathways. Ae. aegypti and Cx. pipiens have undergone expansion of Argonaute and PIWI subfamily genes. Phylogenetic analyses were performed for these protein families. In addition, sequence pattern recognition algorithms MEME, MDScan and Weeder were used to identify upstream regulatory motifs for all SRRP components. Statistical analyses confirmed enrichment of species-specific and pathway-specific cis-elements over the rest of the genome.
Analysis of Argonaute and PIWI subfamily genes suggests that the small regulatory RNA pathways of the major arbovirus vectors, Ae. aegypti and Cx. pipiens, are evolving faster than those of the malaria vector An. gambiae and D. melanogaster. Further, protein and genomic features suggest functional differences between subclasses of PIWI proteins and provide a basis for future analyses. Common UCR elements among SRRP components indicate that 1) key components from the miRNA, siRNA, and piRNA pathways contain NF-kappaB-related and Broad complex transcription factor binding sites, 2) purifying selection has occurred to maintain common pathway-specific elements across mosquito species and 3) species-specific differences in upstream elements suggest that there may be differences in regulatory control among mosquito species. Implications for arbovirus vector competence in mosquitoes are discussed.
Suppression of viral infection by RNA in a nucleotide sequence homology-dependent manner was first reported in plants in early 1990s. Studies in the past 15 years have established a completely new RNA-based immune system against viruses that is mechanistically Riverside, CA, USA. related to RNA silencing or RNA interference (RNAi). This viral immunity begins with recognition of viral double-stranded or structured RNA by the Dicer nuclease family of host immune receptors. In fungi, plants and invertebrates, the viral RNA trigger is processed into small interfering RNAs (siRNAs) to direct specific silencing of the homologous viral genomic and/or messenger RNAs by an RNaseH-like Argonaute protein. Deep sequencing of virus-derived siRNAs indicates that the immunity against viruses with a positive-strand RNA genome is induced by Dicer recognition of dsRNA formed during the initiation of viral progeny (+)RNA synthesis. The RNA-based immune pathway in these organisms overlaps the canonical dsRNA-siRNA pathway of RNAi and may require amplification of viral siRNAs by host RNA-dependent RNA polymerase in plants and nematodes. Production of virus-derived small RNAs is undetectable in mammalian cells infected with RNA viruses. However, infection of mammals with several nucleus-replicating DNA viruses induces production of virus-derived microRNAs capable of silencing host and viral mRNAs as found for viral siRNAs. Remarkably, recent studies indicate that prokaryotes also produce virus-derived small RNAs known as CRISPR RNAs to guide antiviral defense in a manner that has yet to be defined. In this article, we review the recent progress on the identification and mechanism of the key components including viral sensors, viral triggers, effectors, and amplifiers, of the small RNA-directed viral immunity. We also highlight some of the many unresolved questions.
viral; pattern recognition receptors; RNA silencing