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1.  The ERI-6/7 Helicase Acts at the First Stage of an siRNA Amplification Pathway That Targets Recent Gene Duplications 
PLoS Genetics  2011;7(11):e1002369.
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.
Author Summary
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.
PMCID: PMC3213143  PMID: 22102828
2.  Sequence-dependent base pair stepping dynamics in XPD helicase unwinding 
eLife  2013;2:e00334.
Helicases couple the chemical energy of ATP hydrolysis to directional translocation along nucleic acids and transient duplex separation. Understanding helicase mechanism requires that the basic physicochemical process of base pair separation be understood. This necessitates monitoring helicase activity directly, at high spatio-temporal resolution. Using optical tweezers with single base pair (bp) resolution, we analyzed DNA unwinding by XPD helicase, a Superfamily 2 (SF2) DNA helicase involved in DNA repair and transcription initiation. We show that monomeric XPD unwinds duplex DNA in 1-bp steps, yet exhibits frequent backsteps and undergoes conformational transitions manifested in 5-bp backward and forward steps. Quantifying the sequence dependence of XPD stepping dynamics with near base pair resolution, we provide the strongest and most direct evidence thus far that forward, single-base pair stepping of a helicase utilizes the spontaneous opening of the duplex. The proposed unwinding mechanism may be a universal feature of DNA helicases that move along DNA phosphodiester backbones.
eLife digest
During many cellular processes, the double helix must be transiently unwound so that the enzymes responsible for maintaining the genome can access the two strands. During DNA synthesis, for instance, the two strands of DNA are first separated and then used as templates for the production of new strands. The role of destabilizing, separating and unwinding the double helix falls to enzymes known as DNA helicases.
Helicases are also involved in separating strands of nucleic acids during myriad other cellular processes, including DNA repair, transcription and translation. While the functions of helicases are clear, the precise mechanisms by which they unwind DNA are not.
Here, Qi et al. have investigated the mechanism of a helicase called XPD, which is involved in DNA repair and the initiation of transcription of DNA into RNA. Using optical tweezers—in which a laser beam is used to exert extremely small forces on a single DNA molecule—they followed the activity of individual molecules of XPD as they unwound DNA with base pair resolution.
Qi et al. observed that the helicase unwinds DNA strands 1 base pair at a time, but that it sometimes moves backwards by 1 base pair and at other times makes 5 base pair backward and forward steps. The frequency of these backwards steps depends on the availability of ATP, and the sequence of the DNA. Due to the high resolution of the data, Qi et al. were able to correlate these stepping dynamics with the DNA sequence with base pair level accuracy. While some helicases actively separate the strands, using energy derived from ATP to break the hydrogen bonds between pairs of bases, Qi et al. showed that XPD appears to take advantage of momentary separations that arise spontaneously between base pairs.
As well as providing insights into the role of XPD in DNA repair and transcription, the work of Qi et al. presents a method that could be used to explore the mechanisms of other helicases. Given that the unwinding mechanism described here is likely to be a universal feature of enzymes related to XPD, the current work could shed light on a number of other cellular processes involving XPD-like helicases, such as homologous DNA recombination, inter-strand cross-link repair, and accurate chromosome segregation.
PMCID: PMC3668415  PMID: 23741615
helicase; Xeroderma pigmentosum group D helicase; molecular motor; DNA repair; optical tweezers; single molecule; None
3.  Molecular insights into RNA and DNA helicase evolution from the determinants of specificity for a DEAD-box RNA helicase 
eLife  null;3:e04630.
How different helicase families with a conserved catalytic ‘helicase core’ evolved to function on varied RNA and DNA substrates by diverse mechanisms remains unclear. In this study, we used Mss116, a yeast DEAD-box protein that utilizes ATP to locally unwind dsRNA, to investigate helicase specificity and mechanism. Our results define the molecular basis for the substrate specificity of a DEAD-box protein. Additionally, they show that Mss116 has ambiguous substrate-binding properties and interacts with all four NTPs and both RNA and DNA. The efficiency of unwinding correlates with the stability of the ‘closed-state’ helicase core, a complex with nucleotide and nucleic acid that forms as duplexes are unwound. Crystal structures reveal that core stability is modulated by family-specific interactions that favor certain substrates. This suggests how present-day helicases diversified from an ancestral core with broad specificity by retaining core closure as a common catalytic mechanism while optimizing substrate-binding interactions for different cellular functions.
eLife digest
Living cells store their genetic material as DNA, which can be copied to make another molecule called RNA. DNA consists of two strands that are wound around each other in a double helix. RNA is made in a similar way to DNA, but it is usually present as a single strand that folds into a three-dimensional structure that is held in shape by regions of the molecule interacting with each other.
Before DNA and RNA can perform their essential tasks in cells, enzymes called helicases must separate the interacting strands. A large group of helicases, known as superfamily 1 and 2, are involved in virtually all aspects of the control of RNA and DNA structure. All of these helicases contain a region called the ‘helicase core’, but they work in different ways. For example, some move along the DNA or RNA strand whilst they unwind it, while others can unwind RNA without moving. It remains unclear how these helicases have evolved different ways to unwind DNA and RNA structures using the same helicase core.
Mallam et al. have now analyzed a helicase from yeast called Mss116, which belongs to superfamily 2. It is known from previous work that Mss116 binds to many different RNA molecules and—unlike most other helicases—it does not require any extra proteins to help. This makes it an ideal model to study the properties of a helicase core on its own.
Helicases use the energy released from breaking down molecules called nucleotides to pull apart the bonds that hold DNA and RNA strands together. The experiments found that for Mss116, a nucleotide called ATP is the best for providing the energy needed to unwind RNA but other nucleotides can work less efficiently. The experiments also show that in addition to RNA, Ms116 is able to unwind double-stranded DNA molecules that have a certain shape.
Using a technique called X-ray crystallography, Mallam et al. observed the structure of the Mss116 core when it is bound to RNA and DNA. While there are some shared points of contact between the helicase and the DNA or RNA, there are more points of contact between Mss116 and RNA than between Mss116 and DNA.
Mallam et al. propose that present-day helicases have diversified from enzymes that had broad specificity for RNA and DNA, by optimizing interactions that favor the binding of particular nucleotides and nucleic acids. These changes enabled the helicases to become a versatile set of tools that control the structure of RNA or DNA in different ways.
PMCID: PMC4383044  PMID: 25497230
RNA helicase; enzyme specificity; molecular evolution; RNA unwinding; DEAD-box protein; enzyme mechanism; S. cerevisiae
4.  The Expanding Functions of Cellular Helicases: The Tombusvirus RNA Replication Enhancer Co-opts the Plant eIF4AIII-Like AtRH2 and the DDX5-Like AtRH5 DEAD-Box RNA Helicases to Promote Viral Asymmetric RNA Replication 
PLoS Pathogens  2014;10(4):e1004051.
Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. Several of the co-opted host factors bind to the viral RNA, which plays multiple roles, including mRNA function, as an assembly platform for the viral replicase (VRC), template for RNA synthesis, and encapsidation during infection. It is likely that remodeling of the viral RNAs and RNA-protein complexes during the switch from one step to another requires RNA helicases. In this paper, we have discovered a second group of cellular RNA helicases, including the eIF4AIII-like yeast Fal1p and the DDX5-like Dbp3p and the orthologous plant AtRH2 and AtRH5 DEAD box helicases, which are co-opted by tombusviruses. Unlike the previously characterized DDX3-like AtRH20/Ded1p helicases that bind to the 3′ terminal promoter region in the viral minus-strand (−)RNA, the other class of eIF4AIII-like RNA helicases bind to a different cis-acting element, namely the 5′ proximal RIII(−) replication enhancer (REN) element in the TBSV (−)RNA. We show that the binding of AtRH2 and AtRH5 helicases to the TBSV (−)RNA could unwind the dsRNA structure within the RIII(−) REN. This unique characteristic allows the eIF4AIII-like helicases to perform novel pro-viral functions involving the RIII(−) REN in stimulation of plus-strand (+)RNA synthesis. We also show that AtRH2 and AtRH5 helicases are components of the tombusvirus VRCs based on co-purification experiments. We propose that eIF4AIII-like helicases destabilize dsRNA replication intermediate within the RIII(−) REN that promotes bringing the 5′ and 3′ terminal (−)RNA sequences in close vicinity via long-range RNA-RNA base pairing. This newly formed RNA structure promoted by eIF4AIII helicase together with AtRH20 helicase might facilitate the recycling of the viral replicases for multiple rounds of (+)-strand synthesis, thus resulting in asymmetrical viral replication.
Author Summary
Genome-wide screens for host factors affecting tombusvirus replication in yeast indicated that subverted cellular RNA helicases likely play major roles in virus replication. Tombusviruses do not code for their own helicases and they might recruit host RNA helicases to aid their replication in infected cells. Accordingly, in this paper, the authors show that the yeast eIF4AIII-like Fal1p and Dbp3p and the orthologous plant AtRH2 and AtRH5 DEAD-box helicases are co-opted by Tomato bushy stunt virus (TBSV) to aid viral replication. The authors find that eIF4AIII-like helicases bind to the replication enhancer element (REN) in the viral (−)RNA and they promote (+)-strand TBSV RNA synthesis in vitro. Data show that eIF4AIII-like helicases are present in the viral replicase complex and they bind to the replication proteins. In addition, the authors show synergistic effect between eIF4AIII-like helicases and the previously identified DDX3-like Ded1p/AtRH20 DEAD box helicases, which bind to a different cis-acting region in the viral (−)RNA, on stimulation of plus-strand synthesis. In summary, the authors find that two different groups of cellular helicases promote TBSV replication via selectively enhancing (+)-strand synthesis through different mechanisms.
PMCID: PMC3990711  PMID: 24743583
5.  An RIG-I-Like RNA Helicase Mediates Antiviral RNAi Downstream of Viral siRNA Biogenesis in Caenorhabditis elegans 
PLoS Pathogens  2009;5(2):e1000286.
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.
Author Summary
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.
PMCID: PMC2629121  PMID: 19197349
6.  The miR-35-41 Family of MicroRNAs Regulates RNAi Sensitivity in Caenorhabditis elegans 
PLoS Genetics  2012;8(3):e1002536.
RNA interference (RNAi) utilizes small interfering RNAs (siRNAs) to direct silencing of specific genes through transcriptional and post-transcriptional mechanisms. The siRNA guides can originate from exogenous (exo–RNAi) or natural endogenous (endo–RNAi) sources of double-stranded RNA (dsRNA). In Caenorhabditis elegans, inactivation of genes that function in the endo–RNAi pathway can result in enhanced silencing of genes targeted by siRNAs from exogenous sources, indicating cross-regulation between the pathways. Here we show that members of another small RNA pathway, the mir-35-41 cluster of microRNAs (miRNAs) can regulate RNAi. In worms lacking miR-35-41, there is reduced expression of lin-35/Rb, the C. elegans homolog of the tumor suppressor Retinoblastoma gene, previously shown to regulate RNAi responsiveness. Genome-wide microarray analyses show that targets of endo–siRNAs are up-regulated in mir-35-41 mutants, a phenotype also displayed by lin-35/Rb mutants. Furthermore, overexpression of lin-35/Rb specifically rescues the RNAi hypersensitivity of mir-35-41 mutants. Although the mir-35-41 miRNAs appear to be exclusively expressed in germline and embryos, their effect on RNAi sensitivity is transmitted to multiple tissues and stages of development. Additionally, we demonstrate that maternal contribution of miR-35-41 or lin-35/Rb is sufficient to reduce RNAi effectiveness in progeny worms. Our results reveal that miRNAs can broadly regulate other small RNA pathways and, thus, have far reaching effects on gene expression beyond directly targeting specific mRNAs.
Author Summary
RNA interference (RNAi) has become a widely used approach for silencing genes of interest. This tool is possible because endogenous RNA silencing pathways exist broadly across organisms, including humans, worms, and plants. The general RNAi pathway utilizes small ∼21-nucleotide RNAs to target specific protein-coding genes through base-pairing interactions. Since RNAs from exogenous sources require some of the same factors as endogenous small RNAs to silence gene expression, there can be competition between the pathways. Thus, perturbations in the endogenous RNAi pathway can result in enhanced silencing efficiency by exogenous small RNAs. MicroRNAs (miRNAs) comprise another endogenous small RNA pathway, but their biogenesis and mechanism of gene silencing are distinct in many ways from RNAi pathways. Here we show that a family of miRNAs regulates the effectiveness of RNAi in Caenorhabditis elegans. Loss of mir-35-41 results in enhanced RNAi by exogenous RNAs and reduced silencing of endogenous RNAi targets. The embryonic miR-35-41 miRNAs regulate the sensitivity to RNAi through lin-35/Rb, a homolog of the human Retinoblastoma tumor suppressor gene previously shown to regulate RNAi effectiveness in C. elegans. Additionally, we show that this sensitivity can be passed on to the next generation of worms, demonstrating a far-reaching effect of the miR-35-41 miRNAs on gene regulation by other small RNA pathways.
PMCID: PMC3297572  PMID: 22412382
7.  Functional Specialization of the Small Interfering RNA Pathway in Response to Virus Infection 
PLoS Pathogens  2013;9(8):e1003579.
In Drosophila, post-transcriptional gene silencing occurs when exogenous or endogenous double stranded RNA (dsRNA) is processed into small interfering RNAs (siRNAs) by Dicer-2 (Dcr-2) in association with a dsRNA-binding protein (dsRBP) cofactor called Loquacious (Loqs-PD). siRNAs are then loaded onto Argonaute-2 (Ago2) by the action of Dcr-2 with another dsRBP cofactor called R2D2. Loaded Ago2 executes the destruction of target RNAs that have sequence complementarity to siRNAs. Although Dcr-2, R2D2, and Ago2 are essential for innate antiviral defense, the mechanism of virus-derived siRNA (vsiRNA) biogenesis and viral target inhibition remains unclear. Here, we characterize the response mechanism mediated by siRNAs against two different RNA viruses that infect Drosophila. In both cases, we show that vsiRNAs are generated by Dcr-2 processing of dsRNA formed during viral genome replication and, to a lesser extent, viral transcription. These vsiRNAs seem to preferentially target viral polyadenylated RNA to inhibit viral replication. Loqs-PD is completely dispensable for silencing of the viruses, in contrast to its role in silencing endogenous targets. Biogenesis of vsiRNAs is independent of both Loqs-PD and R2D2. R2D2, however, is required for sorting and loading of vsiRNAs onto Ago2 and inhibition of viral RNA expression. Direct injection of viral RNA into Drosophila results in replication that is also independent of Loqs-PD. This suggests that triggering of the antiviral pathway is not related to viral mode of entry but recognition of intrinsic features of virus RNA. Our results indicate the existence of a vsiRNA pathway that is separate from the endogenous siRNA pathway and is specifically triggered by virus RNA. We speculate that this unique framework might be necessary for a prompt and efficient antiviral response.
Author Summary
The RNA interference (RNAi) pathway utilizes small non-coding RNAs to silence gene expression. In insects, RNAi regulates endogenous genes and functions as an RNA-based immune system against viral infection. Here we have uncovered details of how RNAi is triggered by RNA viruses. Double-stranded RNA (dsRNA) generated as a replication intermediate or from transcription of the RNA virus can be used as substrate for the biogenesis of virus-derived small interfering RNAs (vsiRNAs). Unlike other dsRNAs, virus RNA processing involves Dicer but not its canonical partner protein Loqs-PD. Thus, vsiRNA biogenesis is mechanistically different from biogenesis of endogenous siRNAs or siRNAs derived from other exogenous RNA sources. Our results suggest a specialization of the pathway dedicated to silencing of RNA viruses versus other types of RNAi silencing. The understanding of RNAi mechanisms during viral infection could have implications for the control of insect-borne viruses and the use of siRNAs to treat viral infections in humans.
PMCID: PMC3757037  PMID: 24009507
8.  The DNA/RNA-Dependent RNA Polymerase QDE-1 Generates Aberrant RNA and dsRNA for RNAi in a Process Requiring Replication Protein A and a DNA Helicase 
PLoS Biology  2010;8(10):e1000496.
The Neurospora RNA-dependent RNA polymerase QDE-1 is an RNA polymerase that can use both RNA and DNA as templates, suggesting a new mechanism for small RNA production.
The production of aberrant RNA (aRNA) is the initial step in several RNAi pathways. How aRNA is produced and specifically recognized by RNA-dependent RNA polymerases (RdRPs) to generate double-stranded RNA (dsRNA) is not clear. We previously showed that in the filamentous fungus Neurospora, the RdRP QDE-1 is required for rDNA-specific aRNA production, suggesting that QDE-1 may be important in aRNA synthesis. Here we show that a recombinant QDE-1 is both an RdRP and a DNA-dependent RNA polymerase (DdRP). Its DdRP activity is much more robust than the RdRP activity and occurs on ssDNA but not dsDNA templates. We further show that Replication Protein A (RPA), a single-stranded DNA-binding complex that interacts with QDE-1, is essential for aRNA production and gene silencing. In vitro reconstitution assays demonstrate that QDE-1 can produce dsRNA from ssDNA, a process that is strongly promoted by RPA. Furthermore, the interaction between QDE-1 and RPA requires the RecQ DNA helicase QDE-3, a homolog of the human Werner/Bloom Syndrome proteins. Together, these results suggest a novel small RNA biogenesis pathway in Neurospora and a new mechanism for the production of aRNA and dsRNA in RNAi pathways.
Author Summary
Small RNA molecules (20–30 nucleotides) play important roles in many cellular processes in eukaryotic organisms by silencing gene expression. To generate the many forms of small RNAs, DNA is first transcribed to produce single-stranded RNA (ssRNA), which then is converted to double-stranded RNA (dsRNA) by an RNA-dependent RNA polymerase (RdRP). However, it is not clear how the ssRNA templates are synthesized from DNA and specifically recognized by RdRPs amidst a sea of single-stranded, cellular RNAs. We previously showed that in the filamentous fungus Neurospora the production of one type of small RNA called qiRNA, which is specifically induced after DNA damage, requires the RdRP QDE-1. Here, we investigated the precise contributions of QDE-1 to the synthesis of ssRNA and dsRNA. We show that QDE-1 is surprisingly promiscuous in its template choice in that it is able to synthesize RNA from both ssRNA and single-stranded DNA (ssDNA). These results suggest that QDE-1 first generates ssRNA from a DNA template and then converts the ssRNA into dsRNA; this combination of activities in one protein ensures the specific action by RdRP on aberrant RNA in lieu of other single-stranded cellular RNA. In addition, we identified Replication Protein A, a ssDNA-binding protein that interacts with QDE-1, as an essential factor for small RNA production. Furthermore, we were able to reconstitute synthesis of dsRNA from ssDNA in a test tube using purified QDE-1 and RPA proteins, demonstrating the ability of this relatively simple biosynthetic system to generate the nucleic acid trigger for gene regulation. Together, these results uncover the details of a new and important small RNA production mechanism in cells.
PMCID: PMC2950127  PMID: 20957187
9.  RNA Interference: Biology, Mechanism, and Applications 
Double-stranded RNA-mediated interference (RNAi) is a simple and rapid method of silencing gene expression in a range of organisms. The silencing of a gene is a consequence of degradation of RNA into short RNAs that activate ribonucleases to target homologous mRNA. The resulting phenotypes either are identical to those of genetic null mutants or resemble an allelic series of mutants. Specific gene silencing has been shown to be related to two ancient processes, cosuppression in plants and quelling in fungi, and has also been associated with regulatory processes such as transposon silencing, antiviral defense mechanisms, gene regulation, and chromosomal modification. Extensive genetic and biochemical analysis revealed a two-step mechanism of RNAi-induced gene silencing. The first step involves degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity. In the second step, the siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the cognate mRNA and degrades it. Several key components such as Dicer, RNA-dependent RNA polymerase, helicases, and dsRNA endonucleases have been identified in different organisms for their roles in RNAi. Some of these components also control the development of many organisms by processing many noncoding RNAs, called micro-RNAs. The biogenesis and function of micro-RNAs resemble RNAi activities to a large extent. Recent studies indicate that in the context of RNAi, the genome also undergoes alterations in the form of DNA methylation, heterochromatin formation, and programmed DNA elimination. As a result of these changes, the silencing effect of gene functions is exercised as tightly as possible. Because of its exquisite specificity and efficiency, RNAi is being considered as an important tool not only for functional genomics, but also for gene-specific therapeutic activities that target the mRNAs of disease-related genes.
PMCID: PMC309050  PMID: 14665679
10.  Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone 
PLoS Pathogens  2015;11(7):e1005067.
RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3′-to-5′ unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our understanding of enteroviruses and the two types of RNA remodeling activities.
Author Summary
Enteroviruses contain a large number of closely related human pathogens, including poliovirus, EV71, and coxsackie viruses, and cause ~3 billion infections annually. Among the nonstructural proteins of enteroviruses or picornaviruses, protein 2CATPase is the most conserved and complex but the least understood. On the basis of sequence analyses, this protein has been predicted as a putative superfamily 3 (SF3) helicase that supposedly plays a pivotal role in enteroviral RNA replication. However, attempts to determine the helicase activity associated with 2CATPase have been unsuccessful. We found that eukaryotically expressed EV71 or CAV16 2CATPase does possess an ATP-dependent RNA helicase activity that 3′→5′ unwinds RNA helices like other SF3 helicases; surprisingly, it also functions as an RNA chaperone that remodels RNA structures in an ATP-independent manner. Moreover, we determined the domain requirements for these two RNA remodeling activities associated with 2CATPase and provide both in vitro and cellular evidence of their potential roles during viral RNA replication. Additionally, our study provides the first evidence that RNA helicase and chaperoning activities can be integrated within one protein, thereby introducing an extended view of RNA remodeling proteins.
PMCID: PMC4517893  PMID: 26218680
11.  MicroRNA–Directed siRNA Biogenesis in Caenorhabditis elegans 
PLoS Genetics  2010;6(4):e1000903.
RNA interference (RNAi) is a post-transcriptional silencing process, triggered by double-stranded RNA (dsRNA), leading to the destabilization of homologous mRNAs. A distinction has been made between endogenous RNAi–related pathways and the exogenous RNAi pathway, the latter being essential for the experimental use of RNAi. Previous studies have shown that, in Caenorhabditis elegans, a complex containing the enzymes Dicer and the Argonaute RDE-1 process dsRNA. Dicer is responsible for cleaving dsRNA into short interfering RNAs (siRNAs) while RDE-1 acts as the siRNA acceptor. RDE-1 then guides a multi-protein complex to homologous targets to trigger mRNA destabilization. However, endogenous role(s) for RDE-1, if any, have remained unexplored. We here show that RDE-1 functions as a scavenger protein, taking up small RNA molecules from many different sources, including the microRNA (miRNA) pathway. This is in striking contrast to Argonaute proteins functioning directly in the miRNA pathway, ALG-1 and ALG-2: these proteins exclusively bind miRNAs. While playing no significant role in the biogenesis of the main pool of miRNAs, RDE-1 binds endogenous miRNAs and triggers RdRP activity on at least one perfectly matching, endogenous miRNA target. The resulting secondary siRNAs are taken up by a set of Argonaute proteins known to act as siRNA acceptors in exogenous RNAi, resulting in strong mRNA destabilization. Our results show that RDE-1 in an endogenous setting is actively screening the transcriptome using many different small RNAs, including miRNAs, as a guide, with implications for the evolution of transcripts with a potential to be recognized by Dicer.
Author Summary
Due to its intrinsic characteristics, RNA interference (RNAi) has become one of the most widely used tools in cell biology and has revolutionized approaches to elucidate gene function. The process, also known as RNA silencing, is triggered by dsRNA molecules that are cleaved by Dicer proteins into small interfering RNAs (siRNAs). The rde-1 gene from Caenorhabditis elegans was one of the first genes found in association with this mechanism and encodes the only Argonaute protein in worms, which is by itself essential for the classical RNAi pathway triggered by exogenously introduced dsRNA. However, little is known about endogenous functions of RDE-1. Here we show that RDE-1 binds to many classes of small RNAs, including microRNAs. We show that miR-243 is efficiently bound by RDE-1 and triggers regular RNAi on an endogenous target, implying that many RNA species, including miRNAs, are constantly being screened against the transcriptome using the canonical exogenous RNAi pathway.
PMCID: PMC2851571  PMID: 20386745
12.  mRNA turnover rate limits siRNA and microRNA efficacy 
Based on a simple model of the mRNA life cycle, we predict that mRNAs with high turnover rates in the cell are more difficult to perturb with RNAi.We test this hypothesis using a luciferase reporter system and obtain additional evidence from a variety of large-scale data sets, including microRNA overexpression experiments and RT–qPCR-based efficacy measurements for thousands of siRNAs.Our results suggest that mRNA half-lives will influence how mRNAs are differentially perturbed whenever small RNA levels change in the cell, not only after transfection but also during differentiation, pathogenesis and normal cell physiology.
What determines how strongly an mRNA responds to a microRNA or an siRNA? We know that properties of the sequence match between the small RNA and the mRNA are crucial. However, large-scale validations of siRNA efficacies have shown that certain transcripts remain recalcitrant to perturbation even after repeated redesign of the siRNA (Krueger et al, 2007). Weak response to RNAi may thus be an inherent property of the mRNA, but the underlying factors have proven difficult to uncover.
siRNAs induce degradation by sequence-specific cleavage of their target mRNAs (Elbashir et al, 2001). MicroRNAs, too, induce mRNA degradation, and ∼80% of their effect on protein levels can be explained by changes in transcript abundance (Hendrickson et al, 2009; Guo et al, 2010). Given that multiple factors act simultaneously to degrade individual mRNAs, we here consider whether variable responses to micro/siRNA regulation may, in part, be explained simply by the basic dynamics of mRNA turnover. If a transcript is already under strong destabilizing regulation, it is theoretically possible that the relative change in abundance after the addition of a novel degrading factor would be less pronounced compared with a stable transcript (Figure 1). mRNA turnover is achieved by a multitude of factors, and the influence of such factors on targetability can be explored. However, their combined action, including yet unknown factors, is summarized into a single property: the mRNA decay rate.
First, we explored the theoretical relationship between the pre-existing turnover rate of an mRNA, and its expected susceptibility to perturbation by a small RNA. We assumed a basic model of the mRNA life cycle, in which the rate of transcription is constant and the rate of degradation is described by first-order kinetics. Under this model, the relative change in steady-state expression level will become smaller as the pre-existing decay rate grows larger, independent of the transcription rate. This relationship persists also if we assume various degrees of synergy and antagonism between the pre-existing factors and the external factor, with increasing synergism leading to transcripts being more equally targetable, regardless of their pre-existing decay rate.
We next generated a series of four luciferase reporter constructs with destabilizing AU-rich elements (AREs) of various strengths incorporated into their 3′ UTRs. To evaluate how the different constructs would respond to perturbation, we performed co-transfections with an siRNA targeted at the coding region of the luciferase gene. This reduced the signal of the non-destabilized construct to 26% compared with a control siRNA. In contrast, the most destabilized construct showed 42% remaining reporter activity, and we could observe a dose–response relationship across the series.
The reporter experiment encouraged an investigation of this effect on real-world mRNAs. We analyzed a set of 2622 siRNAs, for which individual efficacies were determined using RT–qPCR 48 h post-transfection in HeLa cells ( Of these, 1778 could be associated with an experimentally determined decay rate (Figure 4A). Although the overall correlation between the two variables was modest (Spearman's rank correlation rs=0.22, P<1e−20), we found that siRNAs directed at high-turnover (t1/2<200 min) and medium-turnover (2001000 min) transcripts (P<8e−11 and 4e−9, respectively, two-tailed KS-test, Figure 4B). While 41.6% (498/1196) of the siRNAs directed at low-turnover transcripts reached 10% remaining expression or better, only 16.7% (31/186) of the siRNAs that targeted high-turnover mRNAs reached this high degree of silencing (Figure 4B). Reduced targetability (25.2%, 100/396) was also seen for transcripts with medium-turnover rate.
Our results based on siRNA data suggested that turnover rates could also influence microRNA targeting. By assembling genome-wide mRNA expression data from 20 published microRNA transfections in HeLa cells, we found that predicted target mRNAs with short and medium half-life were significantly less repressed after transfection than their long-lived counterparts (P<8e−5 and P<0.03, respectively, two-tailed KS-test). Specifically, 10.2% (293/2874) of long-lived targets versus 4.4% (41/942) of short-lived targets were strongly (z-score <−3) repressed. siRNAs are known to cause off-target effects that are mediated, in part, by microRNA-like seed complementarity (Jackson et al, 2006). We analyzed changes in transcript levels after transfection of seven different siRNAs, each with a unique seed region (Jackson et al, 2006). Putative ‘off-targets' were identified by mapping of non-conserved seed matches in 3′ UTRs. We found that low-turnover mRNAs (t1/2 >1000 min) were more affected by seed-mediated off-target silencing than high-turnover mRNAs (t1/2 <200 min), with twice as many long-lived seed-containing transcripts (3.8 versus 1.9%) being strongly (z-score <−3) repressed.
In summary, mRNA turnover rates have an important influence on the changes exerted by small RNAs on mRNA levels. It can be assumed that mRNA half-lives will influence how mRNAs are differentially perturbed whenever small RNA levels change in the cell, not only after transfection but also during differentiation, pathogenesis and normal cell physiology.
The microRNA pathway participates in basic cellular processes and its discovery has enabled the development of si/shRNAs as powerful investigational tools and potential therapeutics. Based on a simple kinetic model of the mRNA life cycle, we hypothesized that mRNAs with high turnover rates may be more resistant to RNAi-mediated silencing. The results of a simple reporter experiment strongly supported this hypothesis. We followed this with a genome-wide scale analysis of a rich corpus of experiments, including RT–qPCR validation data for thousands of siRNAs, siRNA/microRNA overexpression data and mRNA stability data. We find that short-lived transcripts are less affected by microRNA overexpression, suggesting that microRNA target prediction would be improved if mRNA turnover rates were considered. Similarly, short-lived transcripts are more difficult to silence using siRNAs, and our results may explain why certain transcripts are inherently recalcitrant to perturbation by small RNAs.
PMCID: PMC3010119  PMID: 21081925
microRNA; mRNA decay; RNAi; siRNA
13.  Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9) 
Nucleic Acids Research  2013;41(5):3457-3470.
Intensive research interest has focused on small RNA-processing machinery and the RNA-induced silencing complex (RISC), key cellular machines in RNAi pathways. However, the structural mechanism regarding RISC assembly, the primary step linking small RNA processing and RNA-mediated gene silencing, is largely unknown. Human RNA helicase A (DHX9) was reported to function as an RISC-loading factor, and such function is mediated mainly by its dsRNA-binding domains (dsRBDs). Here, we report the crystal structures of human RNA helicase A (RHA) dsRBD1 and dsRBD2 domains in complex with dsRNAs, respectively. Structural analysis not only reveals higher siRNA duplex-binding affinity displayed by dsRBD1, but also identifies a crystallographic dsRBD1 pair of physiological significance in cooperatively recognizing dsRNAs. Structural observations are further validated by isothermal titration calorimetric (ITC) assay. Moreover, co-immunoprecipitation (co-IP) assay coupled with mutagenesis demonstrated that both dsRBDs are required for RISC association, and such association is mediated by dsRNA. Hence, our structural and functional efforts have revealed a potential working model for siRNA recognition by RHA tandem dsRBDs, and together they provide direct structural insights into RISC assembly facilitated by RHA.
PMCID: PMC3597700  PMID: 23361462
14.  The Drosophila Helicase MLE Targets Hairpin Structures in Genomic Transcripts 
PLoS Genetics  2016;12(1):e1005761.
RNA hairpins are a common type of secondary structures that play a role in every aspect of RNA biochemistry including RNA editing, mRNA stability, localization and translation of transcripts, and in the activation of the RNA interference (RNAi) and microRNA (miRNA) pathways. Participation in these functions often requires restructuring the RNA molecules by the association of single-strand (ss) RNA-binding proteins or by the action of helicases. The Drosophila MLE helicase has long been identified as a member of the MSL complex responsible for dosage compensation. The complex includes one of two long non-coding RNAs and MLE was shown to remodel the roX RNA hairpin structures in order to initiate assembly of the complex. Here we report that this function of MLE may apply to the hairpins present in the primary RNA transcripts that generate the small molecules responsible for RNA interference. Using stocks from the Transgenic RNAi Project and the Vienna Drosophila Research Center, we show that MLE specifically targets hairpin RNAs at their site of transcription. The association of MLE at these sites is independent of sequence and chromosome location. We use two functional assays to test the biological relevance of this association and determine that MLE participates in the RNAi pathway.
Author Summary
In virtually all RNA molecules, single stranded regions undergo complementary base-pairing with neighboring regions to form double-stranded structures called stem-loops or hairpins. During the fundamental processes of transcription and translation, these RNA structures are reshaped by helicases—enzymes that separate paired regions of nucleic acids. In addition, small non-coding RNA molecules involved in regulating gene expression rely on sequences that allow them to form hairpin structures. In this paper we present evidence that the Drosophila helicase MLE, well known for its role in dosage compensation, participates in the processing of hairpin RNAs in the pathway that leads to induced RNA interference.
PMCID: PMC4710571  PMID: 26752049
15.  Mutations in Mtr4 Structural Domains Reveal Their Important Role in Regulating tRNAiMet Turnover in Saccharomyces cerevisiae and Mtr4p Enzymatic Activities In Vitro 
PLoS ONE  2016;11(1):e0148090.
RNA processing and turnover play important roles in the maturation, metabolism and quality control of a large variety of RNAs thereby contributing to gene expression and cellular health. The TRAMP complex, composed of Air2p, Trf4p and Mtr4p, stimulates nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae. The Mtr4 protein structure is composed of a helicase core and a novel so-called arch domain, which protrudes from the core. The helicase core contains highly conserved helicase domains RecA-1 and 2, and two structural domains of unclear functions, winged helix domain (WH) and ratchet domain. How the structural domains (arch, WH and ratchet domain) coordinate with the helicase domains and what roles they are playing in regulating Mtr4p helicase activity are unknown. We created a library of Mtr4p structural domain mutants for the first time and screened for those defective in the turnover of TRAMP and exosome substrate, hypomodified tRNAiMet. We found these domains regulate Mtr4p enzymatic activities differently through characterizing the arch domain mutants K700N and P731S, WH mutant K904N, and ratchet domain mutant R1030G. Arch domain mutants greatly reduced Mtr4p RNA binding, which surprisingly did not lead to significant defects on either in vivo tRNAiMet turnover, or in vitro unwinding activities. WH mutant K904N and Ratchet domain mutant R1030G showed decreased tRNAiMet turnover in vivo, as well as reduced RNA binding, ATPase and unwinding activities of Mtr4p in vitro. Particularly, K904 was found to be very important for steady protein levels in vivo. Overall, we conclude that arch domain plays a role in RNA binding but is largely dispensable for Mtr4p enzymatic activities, however the structural domains in the helicase core significantly contribute to Mtr4p ATPase and unwinding activities.
PMCID: PMC4731217  PMID: 26820724
16.  Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito's RNA Interference Pathway 
PLoS Pathogens  2009;5(2):e1000299.
A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV) infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi), is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA), which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs). These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2) infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti.
Author Summary
Dengue viruses, globally the most prevalent arboviruses, are transmitted to humans by persistently infected Aedes aegypti mosquitoes. Understanding the mechanisms mosquitoes use to modulate infections by these agents of serious human diseases should give us critical insights into virus–vector interactions leading to transmission. RNA interference (RNAi) is an innate defense mechanism used by invertebrates to inhibit RNA virus infections; however, little is known about the antiviral role of RNAi in mosquitoes. RNAi is triggered by double-stranded RNA, leading to degradation of RNA with sequence homology to the dsRNA trigger. We show that dengue virus type 2 (DENV2) infection of Ae. aegypti by the natural route generates dsRNA and DENV2-specific small interfering RNAs, hallmarks of the RNAi response; nevertheless, persistent infection of mosquitoes occurs, suggesting that DENV2 circumvents RNAi. We also show that DENV2 infection is modulated by RNAi, since impairment by silencing expression of genes encoding important sensor and effector proteins in the RNAi pathway increases virus replication in the vector and decreases the incubation period before virus transmission. Our findings indicate a significant role for RNAi in determining the mosquito vector's potential for transmitting human diseases.
PMCID: PMC2633610  PMID: 19214215
17.  Putative SF2 helicases of the early-branching eukaryote Giardia lamblia are involved in antigenic variation and parasite differentiation into cysts 
BMC Microbiology  2012;12:284.
Regulation of surface antigenic variation in Giardia lamblia is controlled post-transcriptionally by an RNA-interference (RNAi) pathway that includes a Dicer-like bidentate RNase III (gDicer). This enzyme, however, lacks the RNA helicase domain present in Dicer enzymes from higher eukaryotes. The participation of several RNA helicases in practically all organisms in which RNAi was studied suggests that RNA helicases are potentially involved in antigenic variation, as well as during Giardia differentiation into cysts.
An extensive in silico analysis of the Giardia genome identified 32 putative Super Family 2 RNA helicases that contain almost all the conserved RNA helicase motifs. Phylogenetic studies and sequence analysis separated them into 22 DEAD-box, 6 DEAH-box and 4 Ski2p-box RNA helicases, some of which are homologs of well-characterized helicases from higher organisms. No Giardia putative helicase was found to have significant homology to the RNA helicase domain of Dicer enzymes. Additionally a series of up- and down-regulated putative RNA helicases were found during encystation and antigenic variation by qPCR experiments. Finally, we were able to recognize 14 additional putative helicases from three different families (RecQ family, Swi2/Snf2 and Rad3 family) that could be considered DNA helicases.
This is the first comprehensive analysis of the Super Family 2 helicases from the human intestinal parasite G. lamblia. The relative and variable expression of particular RNA helicases during both antigenic variation and encystation agrees with the proposed participation of these enzymes during both adaptive processes. The putatives RNA and DNA helicases identified in this early-branching eukaryote provide initial information regarding the biological role of these enzymes in cell adaptation and differentiation.
PMCID: PMC3566956  PMID: 23190735
RNA/DNA helicases; Giardia lamblia; Encystation; Antigenic variation; Cell differentiation; Gene expression; RNAi; Dicer
18.  HrpA, an RNA Helicase Involved in RNA Processing, Is Required for Mouse Infectivity and Tick Transmission of the Lyme Disease Spirochete 
PLoS Pathogens  2013;9(12):e1003841.
The Lyme disease spirochete Borrelia burgdorferi must differentially express genes and proteins in order to survive in and transit between its tick vector and vertebrate reservoir. The putative DEAH-box RNA helicase, HrpA, has been recently identified as an addition to the spirochete's global regulatory machinery; using proteomic methods, we demonstrated that HrpA modulates the expression of at least 180 proteins. Although most bacteria encode an HrpA helicase, RNA helicase activity has never been demonstrated for HrpAs and the literature contains little information on the contribution of this protein to bacterial physiology or pathogenicity. In this work, we report that B. burgdorferi HrpA has RNA-stimulated ATPase activity and RNA helicase activity and that this enzyme is essential for both mammalian infectivity by syringe inoculation and tick transmission. Reduced infectivity of strains carrying mutations in the ATPase and RNA binding motif mutants suggests that full virulence expression requires both ATPase and coupled helicase activity. Microarray profiling revealed changes in RNA levels of two-fold, or less in an hrpA mutant versus wild-type, suggesting that the enzyme functions largely or exclusively at the post-transcriptional level. In this regard, northern blot analysis of selected gene products highly regulated by HrpA (bb0603 [p66], bba74, bb0241 [glpK], bb0242 and bb0243 [glpA]) suggests a role for HrpA in the processing and translation of transcripts. In addition to being the first demonstration of RNA helicase activity for a bacterial HrpA, our data indicate that the post-transcriptional regulatory functions of this enzyme are essential for maintenance of the Lyme disease spirochete's enzootic cycle.
Author Summary
The bacterium causing Lyme disease, Borrelia burgdorferi, must differentially express genes and proteins in order to survive in and transit between its tick vector and animals that it infects. RNA helicases, enzymes that unwind double-stranded RNA, have recently emerged as major players in all types of processes involving RNA in higher organisms. But in spite of the ubiquitous presence of RNA helicases in bacteria, little is known regarding their function. The Lyme disease spirochete, which has a complex lifecycle involving ticks and vertebrate animals, encodes a single putative RNA helicase, HrpA. Here we establish that the purified protein indeed displays both ATPase and RNA helicase activity. In addition to being the first demonstration of RNA helicase activity for a bacterial HrpA, our data indicate that HrpA is involved in RNA processing of some genes. Our findings also show that HrpA is essential for both tick transmission and mouse infection and establish the RNA helicase as an important component in both parts of the spirochete's lifecycle.
PMCID: PMC3868530  PMID: 24367266
19.  Antiviral RNA Interference against Orsay Virus Is neither Systemic nor Transgenerational in Caenorhabditis elegans 
Journal of Virology  2015;89(23):12035-12046.
Antiviral RNA-mediated silencing (RNA interference [RNAi]) acts as a powerful innate immunity defense in plants, invertebrates, and mammals. In Caenorhabditis elegans, RNAi is systemic; i.e., RNAi silencing signals can move between cells and tissues. Furthermore, RNAi effects can be inherited transgenerationally and may last for many generations. Neither the biological relevance of systemic RNAi nor transgenerational RNAi is currently understood. Here we examined the role of both pathways in the protection of C. elegans from viral infection. We studied the Orsay virus, a positive-strand RNA virus related to Nodaviridae and the first and only virus known to infect C. elegans. Immunity to Orsay virus infection requires the RNAi pathway. Surprisingly, we found that genes required for systemic or transgenerational RNAi did not have a role in antiviral defense. Furthermore, we found that Orsay virus infection did not elicit a systemic RNAi response even when a target for RNAi was provided by using transgenes. Finally, we show that viral siRNAs, the effectors of RNAi, are not inherited to a level that provides any significant resistance to viral infection in the next generation. We conclude that systemic or transgenerational RNAi does not play a role in the defense against natural Orsay virus infection. Furthermore, our data suggest that there is a qualitative difference between experimental RNAi and antiviral RNAi. Our data are consistent with a model of systemic and transgenerational RNAi that requires a nuclear or germ line component that is lacking in almost all RNA virus infections.
IMPORTANCE Since its discovery in Caenorhabditis elegans, RNAi has proven a valuable scientific tool in many organisms. In C. elegans, exogenous RNAi spreads throughout the organism and can be passed between generations; however, there has been controversy as to the endogenous role(s) that the RNAi pathway plays. One endogenous role for which spreading both within the infected organism and between generations would be advantageous is a role in viral defense. In plants, antiviral RNAi is systemic and the spread of RNAi between cells provides protection against subsequent viral infection. Here we investigated this by using the only naturally occurring virus known to infect C. elegans, Orsay virus, and surprisingly found that, in contrast to the exogenous RNAi pathway, the antiviral RNAi response targeted against this virus does not spread systemically throughout the organism and cannot be passed between generations. These results suggest that there are differences between the two pathways that remain to be discovered.
PMCID: PMC4645334  PMID: 26401037
20.  Suppression of RNAi by dsRNA-Degrading RNaseIII Enzymes of Viruses in Animals and Plants 
PLoS Pathogens  2015;11(3):e1004711.
Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)—mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in unrelated RNA and DNA viruses and can be detected in viral genomes using gene modeling and protein structure prediction programs.
Author Summary
RNA interference (RNAi) is a cellular mechanism activated by double-stranded RNA (dsRNA). Cellular dsRNA-specific RNaseIII enzymes (Dicer) recognize dsRNA and process it into double-stranded small interfering RNAs (ds-siRNAs) of 21–25 nucleotides (nt). siRNAs guide RNAi to degrade also single-stranded RNA homologous to the trigger. RNAi regulates gene expression, controls transposons, and represents an important antiviral defense mechanism. Therefore, viruses encode proteins dedicated to countering RNAi. In this study, the RNaseIII enzymes of a fish DNA virus (PPIV) and a plant RNA virus (SPCSV) were compared for suppression of RNAi in non-host organisms. The fish iridovirus RNaseIII suppressed RNAi in a plant and a nematode. It also enhanced accumulation of an RNAi suppressor deficient virus in plants, and suppressed antiviral RNAi and could rescue multiplication of an unrelated, RNAi suppressor-defective virus in nematodes. In contrast, the plant virus RNaseIII could suppress RNAi only in plants. Our results underscore that the active viral RNaseIII enzymes suppress RNAi. Their activity in suppression of RNAi seems to differ for the spectrum of unrelated organisms. Understanding of this novel mechanism of RNAi suppression may inform means of controlling the diseases and economic losses which the RNaseIII-containing viruses cause in animal and plant production.
PMCID: PMC4352025  PMID: 25747942
21.  Structural Basis of RNA Recognition and Activation by Innate Immune Receptor RIG-I 
Nature  2011;479(7373):423-427.
RIG-I (Retinoic acid Inducible Gene - I) is a cytoplasmic pathogen recognition receptor that recognizes pathogen-associated molecular pattern (PAMP) motifs to differentiate between viral and cellular RNAs. RIG-I is activated by blunt-ended double-stranded (ds) RNA with or without a 5′-triphosphate (ppp), single-stranded (ss) RNA marked by 5′-ppp1 and poly-uridine sequence2,3. Upon binding to such PAMP motifs, RIG-I initiates a signaling cascade that induces innate immune defenses and inflammatory cytokines to establish an antiviral state. The RIG-I pathway is highly regulated and aberrant signaling leads to apoptosis, altered cell differentiation, inflammation, autoimmune diseases, and cancer4,5. The helicase and repressor domain (RD) of RIG-I recognize dsRNA and 5′-ppp RNA to activate the amino-terminal two CAspase Recruitment Domains (CARDs) for signaling. To understand the synergy between helicase and RD for RNA binding and the contribution of ATP hydrolysis to RIG-I activation, we determined the structure of human RIG-I helicase-RD in complex with dsRNA and an ATP-analog. The helicase-RD organizes into a ring around dsRNA, capping one end, while contacting both strands utilizing previously uncharacterized motifs to recognize dsRNA. Small angle X-ray scattering (SAXS), limited proteolysis, and differential scanning fluorimetry (DSF) suggest that RIG-I is in an extended and flexible conformation that compacts upon binding RNA. These results provide a detailed view of the helicase role in dsRNA recognition, the synergy between RD and the helicase for RNA binding, organization of full-length RIG-I bound to dsRNA, and evidence of a conformational change upon RNA binding. The RIG-I helicase-RD structure is consistent with dsRNA translocation without unwinding and cooperative binding to RNA. The structure yields unprecedented insight into innate immunity and has broader impact into other areas of biology, including RNA interference and DNA repair, which utilize homologous helicase domains within Dicer and FANCM.
PMCID: PMC3430514  PMID: 21947008
22.  The RNAi machinery controls distinct responses to environmental signals in the basal fungus Mucor circinelloides 
BMC Genomics  2015;16(1):237.
RNA interference (RNAi) is a conserved mechanism of genome defence that can also have a role in the regulation of endogenous functions through endogenous small RNAs (esRNAs). In fungi, knowledge of the functions regulated by esRNAs has been hampered by lack of clear phenotypes in most mutants affected in the RNAi machinery. Mutants of Mucor circinelloides affected in RNAi genes show defects in physiological and developmental processes, thus making Mucor an outstanding fungal model for studying endogenous functions regulated by RNAi. Some classes of Mucor esRNAs map to exons (ex-siRNAs) and regulate expression of the genes from which they derive. To have a broad picture of genes regulated by the silencing machinery during vegetative growth, we have sequenced and compared the mRNA profiles of mutants in the main RNAi genes by using RNA-seq. In addition, we have achieved a more complete phenotypic characterization of silencing mutants.
Deletion of any main RNAi gene provoked a deep impact in mRNA accumulation at exponential and stationary growth. Genes showing increased mRNA levels, as expected for direct ex-siRNAs targets, but also genes with decreased expression were detected, suggesting that, most probably, the initial ex-siRNA targets regulate the expression of other genes, which can be up- or down-regulated. Expression of 50% of the genes was dependent on more than one RNAi gene in agreement with the existence of several classes of ex-siRNAs produced by different combinations of RNAi proteins. These combinations of proteins have also been involved in the regulation of different cellular processes. Besides genes regulated by the canonical RNAi pathway, this analysis identified processes, such as growth at low pH and sexual interaction that are regulated by a dicer-independent non-canonical RNAi pathway.
This work shows that the RNAi pathways play a relevant role in the regulation of a significant number of endogenous genes in M. circinelloides during exponential and stationary growth phases and opens up an important avenue for in-depth study of genes involved in the regulation of physiological and developmental processes in this fungal model.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1443-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4417260  PMID: 25880254
Asexual sporulation; Sexual interaction; pH regulation; Non-canonical RNAi pathway; esRNAs; mRNA profiling
23.  Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3′-to-5′ Helicase That Unwinds 3′-Tailed RNA Duplexes and RNA:DNA Hybrids 
Journal of Bacteriology  2015;197(19):3057-3065.
Mycobacteria have a large and distinctive ensemble of DNA helicases that function in DNA replication, repair, and recombination. Little is known about the roster of RNA helicases in mycobacteria or their roles in RNA transactions. The 912-amino-acid Mycobacterium smegmatis HelY (MSMEG_3885) protein is a bacterial homolog of the Mtr4 and Ski2 helicases that regulate RNA 3′ processing and turnover by the eukaryal exosome. Here we characterize HelY as an RNA-stimulated ATPase/dATPase and an ATP/dATP-dependent 3′-to-5′ helicase. HelY requires a 3′ single-strand RNA tail (a loading RNA strand) to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The findings that HelY ATPase is unresponsive to a DNA polynucleotide cofactor and that HelY is unable to unwind a 3′-tailed duplex in which the loading strand is DNA distinguish HelY from other mycobacterial nucleoside triphosphatases/helicases characterized previously. The biochemical properties of HelY, which resemble those of Mtr4/Ski2, hint at a role for HelY in mycobacterial RNA catabolism.
IMPORTANCE RNA helicases play crucial roles in transcription, RNA processing, and translation by virtue of their ability to alter RNA secondary structure or remodel RNA-protein interactions. In eukarya, the RNA helicases Mtr4 and Ski2 regulate RNA 3′ resection by the exosome. Mycobacterium smegmatis HelY, a bacterial homolog of Mtr4/Ski2, is characterized here as a unidirectional helicase, powered by RNA-dependent ATP/dATP hydrolysis, that tracks 3′ to 5′ along a loading RNA strand to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The biochemical properties of HelY suggest a role in bacterial RNA transactions. HelY homologs are present in pathogenic mycobacteria (e.g., M. tuberculosis and M. leprae) and are widely prevalent in Actinobacteria and Cyanobacteria but occur sporadically elsewhere in the bacterial domain.
PMCID: PMC4560288  PMID: 26170411
24.  RNAi-Dependent and Independent Control of LINE1 Accumulation and Mobility in Mouse Embryonic Stem Cells 
PLoS Genetics  2013;9(11):e1003791.
In most mouse tissues, long-interspersed elements-1 (L1s) are silenced via methylation of their 5′-untranslated regions (5′-UTR). A gradual loss-of-methylation in pre-implantation embryos coincides with L1 retrotransposition in blastocysts, generating potentially harmful mutations. Here, we show that Dicer- and Ago2-dependent RNAi restricts L1 accumulation and retrotransposition in undifferentiated mouse embryonic stem cells (mESCs), derived from blastocysts. RNAi correlates with production of Dicer-dependent 22-nt small RNAs mapping to overlapping sense/antisense transcripts produced from the L1 5′-UTR. However, RNA-surveillance pathways simultaneously degrade these transcripts and, consequently, confound the anti-L1 RNAi response. In Dicer−/− mESC complementation experiments involving ectopic Dicer expression, L1 silencing was rescued in cells in which microRNAs remained strongly depleted. Furthermore, these cells proliferated and differentiated normally, unlike their non-complemented counterparts. These results shed new light on L1 biology, uncover defensive, in addition to regulatory roles for RNAi, and raise questions on the differentiation defects of Dicer−/− mESCs.
Author Summary
A basal network of gene regulation orchestrates the processes ensuring maintenance of genome integrity. Eukaryotic small RNAs generated by the RNAse-III Dicer have emerged as central players in this network, by mediating gene silencing at the transcriptional or post-transcriptional level via RNA interference (RNAi). To gain insight into their potential developmental functions in mammals, we have characterized small RNA expression profiles during mouse Embryonic Stem Cell (mESCs) differentiation, a model for early mammalian development. Long interspersed elements 1 (L1) are non-long-terminal-repeat retrotransposons that dominate the mouse genomic landscape, and are expressed in germ cells or during early development and mESCs. Based on clear precedents in plants and fission yeast, we investigated a role for RNAi and other RNA-based pathways in the regulation of L1 transcription and mobilization. Our work uncovered the existence of small (s)RNAs that map to active L1 elements. Some have characteristics of cognate siRNA produced by Dicer, while others display strand biases and length heterogeneity that evoke their biogenesis through RNA surveillance pathways, in a Dicer-independent manner. Furthermore, genetic ablation of DICER or of ARGONAUTE proteins has complex and profound consequences on L1 transcription and mobilization, indicating that endogenous RNAi do indeed maintain genomic integrity against L1 proliferation.
PMCID: PMC3820764  PMID: 24244175
25.  From promoting to inhibiting: diverse roles of helicases in HIV-1 Replication 
Retrovirology  2012;9:79.
Helicases hydrolyze nucleotide triphosphates (NTPs) and use the energy to modify the structures of nucleic acids. They are key players in every cellular process involving RNA or DNA. Human immunodeficiency virus type 1 (HIV-1) does not encode a helicase, thus it has to exploit cellular helicases in order to efficiently replicate its RNA genome. Indeed, several helicases have been found to specifically associate with HIV-1 and promote viral replication. However, studies have also revealed a couple of helicases that inhibit HIV-1 replication; these findings suggest that HIV-1 can either benefit from the function of cellular helicases or become curtailed by these enzymes. In this review, we focus on what is known about how a specific helicase associates with HIV-1 and how a distinct step of HIV-1 replication is affected. Despite many helicases having demonstrated roles in HIV-1 replication and dozens of other helicase candidates awaiting to be tested, a deeper appreciation of their involvement in the HIV-1 life cycle is hindered by our limited knowledge at the enzymatic and molecular levels regarding how helicases shape the conformation and structure of viral RNA-protein complexes and how these conformational changes are translated into functional outcomes in the context of viral replication.
PMCID: PMC3484045  PMID: 23020886
HIV-1; helicase; RNP

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