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Phillip D. Zamore - One of the best experts on this subject based on the ideXlab platform.
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the tiny conserved zinc finger protein gtsf1 helps piwi proteins achieve their full catalytic potential preprint
bioRxiv, 2021Co-Authors: Amena Arif, Deniz M Ozata, Cecilia Anderson, Natsuko Izumi, Yukihide Tomari, Phillip D. ZamoreAbstract:Abstract Argonaute proteins use nucleic acid guides to find and bind specific DNA or RNA target sequences. Argonaute proteins can be found in all kingdoms of life, and play diverse biological functions including genome defense, gene regulation, and chromosome partitioning. Many Argonautes retain their ancestral endoribonuclease activity, cleaving the phosphodiester bond between target nucleotides t10 and t11. In animals, a specialized class of Argonautes, the PIWI proteins, use 21–35 nt PIWI-interacting RNAs (piRNAs) to direct transposon silencing, protect the germline genome, and regulate gene expression during gametogenesis1. The piRNA pathway is required for fertility in one or both sexes of nearly all animals. Both piRNA production and function require RNA cleavage catalyzed by PIWI proteins. Spermatogenesis in mice and other placental mammals requires three distinct, developmentally regulated PIWI proteins: MIWI (PIWIL1), MILI (PIWIL2), and MIWI2 (PIWIL4)24; the piRNA-guided endoribonuclease activities of MIWI and MILI are essential to produce functional sperm5,6. piRNA-directed silencing in mice, insects, and worms also requires Gametocyte-Specific Factor 1 (GTSF1), a PIWI-associated protein of unknown function712. Here, we report that GTSF1 potentiates the weak, intrinsic, piRNA-directed RNA cleavage activities of PIWI proteins, transforming them into efficient endoribonucleases. GTSF1 represents the first example of an auxiliary protein that potentiates the catalytic activity of an Argonaute protein.
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to degrade a microrna destroy its Argonaute protein
Molecular Cell, 2021Co-Authors: Phillip D. ZamoreAbstract:Han et al. (2020) and Shi et al. (2020) report that the E3 ubiquitin ligase ZSWIM8 senses when an RNA and an Argonaute protein-bound microRNA are extensively base paired and directs Argonaute destruction by the proteasome. The result is degradation of the microRNA.
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single molecule imaging reveals that Argonaute reshapes the binding properties of its nucleic acid guides
Cell, 2015Co-Authors: William Salomon, Phillip D. Zamore, Samson M Jolly, Melissa J Moore, Victor SerebrovAbstract:SUMMARY Argonaute proteins repress gene expression and defend against foreign nucleic acids using short RNAs or DNAs to specify the correct target RNA or DNA sequence. We have developed single-molecule methods to analyze target binding and cleavage mediated by the Argonaute:guide complex, RISC. We find that both eukaryotic and prokaryotic Argonaute proteins reshape the fundamental properties of RNA:RNA, RNA:DNA, and DNA:DNA hybridization—a small RNA or DNA bound to Argonaute as a guide no longer follows the well-established rules by which oligonucleotides find, bind, and dissociate from complementary nucleic acid sequences. Argonautes distinguish substrates from targets with similar complementarity. Mouse AGO2, for example, bindstighter tomiRNAtargetsthan itsRNAicleavage product, even though the cleaved product contains more base pairs. By re-writing the rules for nucleic acid hybridization, Argonautes allow oligonucleotides to serve as specificity determinants with thermodynamic and kinetic properties more typical of RNA-binding proteins than of RNA or DNA.
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Argonaute divides its rna guide into domains with distinct functions and rna binding properties
Cell, 2012Co-Authors: Fabian C Floresjasso, William Salomon, Phillip D. ZamoreAbstract:Summary MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) guide Argonaute proteins to silence mRNA expression. Argonaute binding alters the properties of an RNA guide, creating functional domains. We show that the domains established by Argonaute—the anchor, seed, central, 3′ supplementary, and tail regions—have distinct biochemical properties that explain the differences between how animal miRNAs and siRNAs bind their targets. Extensive complementarity between an siRNA and its target slows the rate at which fly Argonaute2 (Ago2) binds to and dissociates from the target. Highlighting its role in antiviral defense, fly Ago2 dissociates so slowly from extensively complementary target RNAs that essentially every fully paired target is cleaved. Conversely, mouse AGO2, which mainly mediates miRNA-directed repression, dissociates rapidly and with similar rates for fully paired and seed-matched targets. Our data narrow the range of biochemically reasonable models for how Argonaute-bound siRNAs and miRNAs find, bind, and regulate their targets.
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the 3 to 5 exoribonuclease nibbler shapes the 3 ends of micrornas bound to drosophila Argonaute1
Current Biology, 2011Co-Authors: Bo W Han, Phillip D. Zamore, Juihung Hung, Zhiping Weng, Stefan L AmeresAbstract:Summary Background MicroRNAs (miRNAs) are ∼22 nucleotide (nt) small RNAs that control development, physiology, and pathology in animals and plants. Production of miRNAs involves the sequential processing of primary hairpin-containing RNA polymerase II transcripts by the RNase III enzymes Drosha in the nucleus and Dicer in the cytoplasm. miRNA duplexes then assemble into Argonaute proteins to form the RNA-induced silencing complex (RISC). In mature RISC, a single-stranded miRNA directs the Argonaute protein to bind partially complementary sequences, typically in the 3′ untranslated regions of messenger RNAs, repressing their expression. Results Here, we show that after loading into Argonaute1 (Ago1), more than a quarter of all Drosophila miRNAs undergo 3′ end trimming by the 3′-to-5′ exoribonuclease Nibbler (CG9247). Depletion of Nibbler by RNA interference (RNAi) reveals that miRNAs are frequently produced by Dicer-1 as intermediates that are longer than ∼22 nt. Trimming of miRNA 3′ ends occurs after removal of the miRNA ∗ strand from pre-RISC and may be the final step in RISC assembly, ultimately enhancing target messenger RNA repression. In vivo, depletion of Nibbler by RNAi causes developmental defects. Conclusions We provide a molecular explanation for the previously reported heterogeneity of miRNA 3′ ends and propose a model in which Nibbler converts miRNAs into isoforms that are compatible with the preferred length of Ago1-bound small RNAs.
Alexei A. Aravin - One of the best experts on this subject based on the ideXlab platform.
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a programmable pago nuclease with universal guide and target specificity from the mesophilic bacterium kurthia massiliensis
Nucleic Acids Research, 2021Co-Authors: Ekaterina Kropocheva, Alexei A. Aravin, Daria Esyunina, Anton Kuzmenko, Andrey KulbachinskiyAbstract:Argonaute proteins are programmable nucleases that are found in both eukaryotes and prokaryotes and provide defense against invading genetic elements. Although some prokaryotic Argonautes (pAgos) were shown to recognize RNA targets in vitro, the majority of studied pAgos have strict specificity toward DNA, which limits their practical use in RNA-centric applications. Here, we describe a unique pAgo nuclease, KmAgo, from the mesophilic bacterium Kurthia massiliensis that can be programmed with either DNA or RNA guides and can precisely cleave both DNA and RNA targets. KmAgo binds 16-20 nt long 5'-phosphorylated guide molecules with no strict specificity for their sequence and is active in a wide range of temperatures. In bacterial cells, KmAgo is loaded with small DNAs with no obvious sequence preferences suggesting that it can uniformly target genomic sequences. Mismatches between the guide and target sequences greatly affect the efficiency and precision of target cleavage, depending on the mismatch position and the nature of the reacting nucleic acids. Target RNA cleavage by KmAgo depends on the formation of secondary structure indicating that KmAgo can be used for structural probing of RNA. These properties of KmAgo open the way for its use for highly specific nucleic acid detection and cleavage.
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a programmable pago nuclease with universal guide and target specificity from the mesophilic bacterium kurthia massiliensis
bioRxiv, 2021Co-Authors: Ekaterina Kropocheva, Alexei A. Aravin, Daria Esyunina, Anton Kuzmenko, Andrey KulbachinskiyAbstract:ABSTRACT Argonaute proteins are programmable nucleases that are found in both eukaryotes and prokaryotes and provide defense against invading genetic elements. Although some prokaryotic Argonautes (pAgos) were shown to recognize RNA targets in vitro, the majority of studied pAgos have strict specificity toward DNA, which limits their practical use in RNA-centric applications. Here, we describe a unique KmAgo nuclease from the mesophilic bacterium Kurthia massiliensis that can be programmed with either DNA or RNA guides and can precisely cleave both DNA and RNA targets. KmAgo preferentially binds 16-20 nt long 5′-phosphorylated guide molecules with no strict specificity for their sequence and is active in a wide range of temperatures. In bacterial cells, KmAgo is loaded with small DNAs with no obvious sequence preferences suggesting that it can uniformly target genomic sequences. Target cleavage by KmAgo depends on the formation of secondary structure indicating that KmAgo can be used for structural probing of RNA targets. Mismatches between the guide and target sequences greatly affect the efficiency and precision of target cleavage, depending on the mismatch position and the nature of the reacting nucleic acid. These properties of KmAgo open the way for its use for highly specific nucleic acid detection and cleavage.
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Recognition of double-stranded DNA by the Rhodobacter sphaeroides Argonaute protein.
Biochemical and biophysical research communications, 2020Co-Authors: Lidia Lisitskaya, Alexei A. Aravin, Daria Esyunina, Ivan Petushkov, Andrey KulbachinskiyAbstract:In contrast to eukaryotic Argonaute proteins that act on RNA targets, prokaryotic Argonautes (pAgos) can target DNA, using either small RNA or small DNA guides for its recognition. Since pAgos can recognize only a single strand of DNA and lack a helicase activity, it remains unknown how double-stranded DNA can be bound both in vitro and in vivo. Here, using in vitro reconstitution and footprinting assays we analyze formation of specific complexes with target DNA by a catalytically inactive pAgo, RsAgo from Rhodobacter sphaeroides programmed with small guide RNAs. We showed that RsAgo can recognize a specific site in double-stranded DNA after stepwise reconstitution of the complex from individual oligonucleotides or after prior melting of the DNA target. When bound, RsAgo stabilizes an open DNA bubble corresponding to the length of the guide molecule and protects the target DNA from nuclease cleavage. The results suggest that RsAgo and, possibly, other RNA-guided pAgos cannot directly attack double-stranded DNA and likely require DNA opening by other cellular processes for their action.
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programmable dna cleavage by ago nucleases from mesophilic bacteria clostridium butyricum and limnothrix rosea
Nucleic Acids Research, 2019Co-Authors: Anton Kuzmenko, Alexei A. Aravin, Andrey Kulbachinskiy, Denis Yudin, Sergei RyazanskyAbstract:Argonaute (Ago) proteins are key players in RNA interference in eukaryotes, where they function as RNA-guided RNA endonucleases. Prokaryotic Argonautes (pAgos) are much more diverse than their eukaryotic counterparts but their cellular functions and mechanisms of action remain largely unknown. Some pAgos were shown to use small DNA guides for endonucleolytic cleavage of complementary DNA in vitro. However, previously studied pAgos from thermophilic prokaryotes function at elevated temperatures, which limits their potential use as a tool in genomic applications. Here, we describe two pAgos from mesophilic bacteria, Clostridium butyricum (CbAgo) and Limnothrix rosea (LrAgo), that act as DNA-guided DNA nucleases at physiological temperatures. In comparison with previously studied pAgos, CbAgo and LrAgo do not show strong preferences for the 5'-nucleotide in guide DNA and can use not only 5'-phosphorylated but also 5'-hydroxyl DNA guides. Both CbAgo and LrAgo can tolerate guide/target mismatches in the seed region, but are sensitive to mismatches in the 3'-guide region. Both pAgos can perform programmable endonucleolytic cleavage of double-stranded DNA substrates, showing enhanced activity at AT-rich regions and at elevated temperatures. The biochemical characterization of mesophilic pAgo proteins paves the way for their use for DNA manipulations both in vitro and in vivo.
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bacterial Argonaute samples the transcriptome to identify foreign dna
Molecular Cell, 2013Co-Authors: Ivan Olovnikov, Ravi Sachidanandam, Ken Y Chan, Dianne K Newman, Alexei A. AravinAbstract:Eukaryotic Argonautes bind small RNAs and use them as guides to find complementary RNA targets and induce gene silencing. Though homologs of eukaryotic Argonautes are present in many bacteria and archaea, their small RNA partners and functions are unknown. We found that the Argonaute of Rhodobacter sphaeroides (RsAgo) associates with 15–19 nt RNAs that correspond to the majority of transcripts. RsAgo also binds single-stranded 22–24 nt DNA molecules that are complementary to the small RNAs and enriched in sequences derived from exogenous plasmids as well as genome-encoded foreign nucleic acids such as transposons and phage genes. Expression of RsAgo in the heterologous E. coli system leads to formation of plasmid-derived small RNA and DNA and plasmid degradation. In a R. sphaeroides mutant lacking RsAgo, expression of plasmid-encoded genes is elevated. Our results indicate that RNAi-related processes found in eukaryotes are also conserved in bacteria and target foreign nucleic acids.
Leemor Joshuator - One of the best experts on this subject based on the ideXlab platform.
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analysis of the c elegans Argonaute family reveals that distinct Argonautes act sequentially during rnai
Cell, 2006Co-Authors: Erbay Yigit, Niraj H Tolia, Leemor Joshuator, Martin J Simard, Pedro J Batista, Yanxia Bei, Ka Ming Pang, Chunchieh G Chen, Shohei Mitani, Craig C MelloAbstract:Argonaute (AGO) proteins interact with small RNAs to mediate gene silencing. C. elegans contains 27 AGO genes, raising the question of what roles these genes play in RNAi and related gene-silencing pathways. Here we describe 31 deletion alleles representing all of the previously uncharacterized AGO genes. Analysis of single- and multiple-AGO mutant strains reveals functions in several pathways, including (1) chromosome segregation, (2) fertility, and (3) at least two separate steps in the RNAi pathway. We show that RDE-1 interacts with trigger-derived sense and antisense RNAs to initiate RNAi, while several other AGO proteins interact with amplified siRNAs to mediate downstream silencing. Overexpression of downstream AGOs enhances silencing, suggesting that these proteins are limiting for RNAi. Interestingly, these AGO proteins lack key residues required for mRNA cleavage. Our findings support a two-step model for RNAi, in which functionally and structurally distinct AGOs act sequentially to direct gene silencing.
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purified Argonaute2 and an sirna form recombinant human risc
Nature Structural & Molecular Biology, 2005Co-Authors: Fabiola V Rivas, Niraj H Tolia, Gregory J Hannon, Jijoon Song, Juan P Aragon, Leemor JoshuatorAbstract:Genetic, biochemical and structural studies have implicated Argonaute proteins as the catalytic core of the RNAi effector complex, RISC. Here we show that recombinant, human Argonaute2 can combine with a small interfering RNA (siRNA) to form minimal RISC that accurately cleaves substrate RNAs. Recombinant RISC shows many of the properties of RISC purified from human or Drosophila melanogaster cells but also has surprising features. It shows no stimulation by ATP, suggesting that factors promoting product release are missing from the recombinant enzyme. The active site is made up of a unique Asp-Asp-His (DDH) motif. In the RISC reconstitution system, the siRNA 5′ phosphate is important for the stability and the fidelity of the complex but is not essential for the creation of an active enzyme. These studies demonstrate that Argonaute proteins catalyze mRNA cleavage within RISC and provide a source of recombinant enzyme for detailed biochemical studies of the RNAi effector complex.
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Argonaute2 is the catalytic engine of mammalian rnai
Science, 2004Co-Authors: Jidong Liu, Scott M Hammond, Fabiola V Rivas, Jijoon Song, Leemor Joshuator, Michelle A Carmell, Carolyn Marsden, Michael J Thomson, Gregory J HannonAbstract:Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. Here, we show that the multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity. This protein is essential for mouse development, and cells lacking Argonaute2 are unable to mount an experimental response to siRNAs. Mutations within a cryptic ribonuclease H domain within Argonaute2, as identified by comparison with the structure of an archeal Argonaute protein, inactivate RISC. Thus, our evidence supports a model in which Argonaute contributes “Slicer” activity to RISC, providing the catalytic engine for RNAi.
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crystal structure of Argonaute and its implications for risc slicer activity
Science, 2004Co-Authors: Jijoon Song, Gregory J Hannon, Leemor Joshuator, Stephanie K SmithAbstract:Argonaute proteins and small interfering RNAs (siRNAs) are the known signature components of the RNA interference effector complex RNA-induced silencing complex (RISC). However, the identity of "Slicer," the enzyme that cleaves the messenger RNA (mRNA) as directed by the siRNA, has not been resolved. Here, we report the crystal structure of the Argonaute protein from Pyrococcus furiosus at 2.25 angstrom resolution. The structure reveals a crescent-shaped base made up of the amino-terminal, middle, and PIWI domains. The Piwi Argonaute Zwille (PAZ) domain is held above the base by a "stalk"-like region. The PIWI domain (named for the protein piwi) is similar to ribonuclease H, with a conserved active site aspartate-aspartate-glutamate motif, strongly implicating Argonaute as "Slicer." The architecture of the molecule and the placement of the PAZ and PIWI domains define a groove for substrate binding and suggest a mechanism for siRNA-guided mRNA cleavage.
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the crystal structure of the Argonaute2 paz domain reveals an rna binding motif in rnai effector complexes
Nature Structural & Molecular Biology, 2003Co-Authors: Jijoon Song, Niraj H Tolia, Gregory J Hannon, Robert A Martienssen, Jidong Liu, Stephanie K Smith, Jonathan I Schneiderman, Leemor JoshuatorAbstract:RISC, the RNA-induced silencing complex, uses short interfering RNAs (siRNAs) or micro RNAs (miRNAs) to select its targets in a sequence-dependent manner. Key RISC components are Argonaute proteins, which contain two characteristic domains, PAZ and PIWI. PAZ is highly conserved and is found only in Argonaute proteins and Dicer. We have solved the crystal structure of the PAZ domain of Drosophila Argonaute2. The PAZ domain contains a variant of the OB fold, a module that often binds single-stranded nucleic acids. PAZ domains show low-affinity nucleic acid binding, probably interacting with the 3' ends of single-stranded regions of RNA. PAZ can bind the characteristic two-base 3' overhangs of siRNAs, indicating that although PAZ may not be a primary nucleic acid binding site in Dicer or RISC, it may contribute to the specific and productive incorporation of siRNAs and miRNAs into the RNAi pathway.
Andrey Kulbachinskiy - One of the best experts on this subject based on the ideXlab platform.
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a programmable pago nuclease with universal guide and target specificity from the mesophilic bacterium kurthia massiliensis
Nucleic Acids Research, 2021Co-Authors: Ekaterina Kropocheva, Alexei A. Aravin, Daria Esyunina, Anton Kuzmenko, Andrey KulbachinskiyAbstract:Argonaute proteins are programmable nucleases that are found in both eukaryotes and prokaryotes and provide defense against invading genetic elements. Although some prokaryotic Argonautes (pAgos) were shown to recognize RNA targets in vitro, the majority of studied pAgos have strict specificity toward DNA, which limits their practical use in RNA-centric applications. Here, we describe a unique pAgo nuclease, KmAgo, from the mesophilic bacterium Kurthia massiliensis that can be programmed with either DNA or RNA guides and can precisely cleave both DNA and RNA targets. KmAgo binds 16-20 nt long 5'-phosphorylated guide molecules with no strict specificity for their sequence and is active in a wide range of temperatures. In bacterial cells, KmAgo is loaded with small DNAs with no obvious sequence preferences suggesting that it can uniformly target genomic sequences. Mismatches between the guide and target sequences greatly affect the efficiency and precision of target cleavage, depending on the mismatch position and the nature of the reacting nucleic acids. Target RNA cleavage by KmAgo depends on the formation of secondary structure indicating that KmAgo can be used for structural probing of RNA. These properties of KmAgo open the way for its use for highly specific nucleic acid detection and cleavage.
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a programmable pago nuclease with universal guide and target specificity from the mesophilic bacterium kurthia massiliensis
bioRxiv, 2021Co-Authors: Ekaterina Kropocheva, Alexei A. Aravin, Daria Esyunina, Anton Kuzmenko, Andrey KulbachinskiyAbstract:ABSTRACT Argonaute proteins are programmable nucleases that are found in both eukaryotes and prokaryotes and provide defense against invading genetic elements. Although some prokaryotic Argonautes (pAgos) were shown to recognize RNA targets in vitro, the majority of studied pAgos have strict specificity toward DNA, which limits their practical use in RNA-centric applications. Here, we describe a unique KmAgo nuclease from the mesophilic bacterium Kurthia massiliensis that can be programmed with either DNA or RNA guides and can precisely cleave both DNA and RNA targets. KmAgo preferentially binds 16-20 nt long 5′-phosphorylated guide molecules with no strict specificity for their sequence and is active in a wide range of temperatures. In bacterial cells, KmAgo is loaded with small DNAs with no obvious sequence preferences suggesting that it can uniformly target genomic sequences. Target cleavage by KmAgo depends on the formation of secondary structure indicating that KmAgo can be used for structural probing of RNA targets. Mismatches between the guide and target sequences greatly affect the efficiency and precision of target cleavage, depending on the mismatch position and the nature of the reacting nucleic acid. These properties of KmAgo open the way for its use for highly specific nucleic acid detection and cleavage.
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Recognition of double-stranded DNA by the Rhodobacter sphaeroides Argonaute protein.
Biochemical and biophysical research communications, 2020Co-Authors: Lidia Lisitskaya, Alexei A. Aravin, Daria Esyunina, Ivan Petushkov, Andrey KulbachinskiyAbstract:In contrast to eukaryotic Argonaute proteins that act on RNA targets, prokaryotic Argonautes (pAgos) can target DNA, using either small RNA or small DNA guides for its recognition. Since pAgos can recognize only a single strand of DNA and lack a helicase activity, it remains unknown how double-stranded DNA can be bound both in vitro and in vivo. Here, using in vitro reconstitution and footprinting assays we analyze formation of specific complexes with target DNA by a catalytically inactive pAgo, RsAgo from Rhodobacter sphaeroides programmed with small guide RNAs. We showed that RsAgo can recognize a specific site in double-stranded DNA after stepwise reconstitution of the complex from individual oligonucleotides or after prior melting of the DNA target. When bound, RsAgo stabilizes an open DNA bubble corresponding to the length of the guide molecule and protects the target DNA from nuclease cleavage. The results suggest that RsAgo and, possibly, other RNA-guided pAgos cannot directly attack double-stranded DNA and likely require DNA opening by other cellular processes for their action.
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programmable dna cleavage by ago nucleases from mesophilic bacteria clostridium butyricum and limnothrix rosea
Nucleic Acids Research, 2019Co-Authors: Anton Kuzmenko, Alexei A. Aravin, Andrey Kulbachinskiy, Denis Yudin, Sergei RyazanskyAbstract:Argonaute (Ago) proteins are key players in RNA interference in eukaryotes, where they function as RNA-guided RNA endonucleases. Prokaryotic Argonautes (pAgos) are much more diverse than their eukaryotic counterparts but their cellular functions and mechanisms of action remain largely unknown. Some pAgos were shown to use small DNA guides for endonucleolytic cleavage of complementary DNA in vitro. However, previously studied pAgos from thermophilic prokaryotes function at elevated temperatures, which limits their potential use as a tool in genomic applications. Here, we describe two pAgos from mesophilic bacteria, Clostridium butyricum (CbAgo) and Limnothrix rosea (LrAgo), that act as DNA-guided DNA nucleases at physiological temperatures. In comparison with previously studied pAgos, CbAgo and LrAgo do not show strong preferences for the 5'-nucleotide in guide DNA and can use not only 5'-phosphorylated but also 5'-hydroxyl DNA guides. Both CbAgo and LrAgo can tolerate guide/target mismatches in the seed region, but are sensitive to mismatches in the 3'-guide region. Both pAgos can perform programmable endonucleolytic cleavage of double-stranded DNA substrates, showing enhanced activity at AT-rich regions and at elevated temperatures. The biochemical characterization of mesophilic pAgo proteins paves the way for their use for DNA manipulations both in vitro and in vivo.
Huimin Meng - One of the best experts on this subject based on the ideXlab platform.
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dicer and Argonaute genes involved in rna interference in the entomopathogenic fungus metarhizium robertsii
Applied and Environmental Microbiology, 2017Co-Authors: Huimin Meng, Zhangxun Wang, Yulong Wang, Bo HuangAbstract:RNA interference (RNAi) is a gene silencing mechanism that plays an important role in gene regulation in a number of eukaryotic organisms. Two core components, Dicer and Argonaute, are central in the RNAi machinery. However, the physiological roles of Dicer and Argonaute in the entomopathogenic fungus Metarhizium robertsii remained unclear. Here, the roles of genes encoding Dicers ( Mrdcl1 and Mrdcl2 ) and Argonautes ( Mrago1 and Mrago2 ) in M.robertsii were investigated. The results showed that the Dicer protein MrDCL2 and Argonaute protein MrAGO1 are the major components of the RNAi process occurring in M. robertsii . The Dicer and Argonaute genes were not involved in regulation of growth and diverse abiotic stress response in M. robertsii under tested conditions. Moreover, our results showed that the Dicer and Argonaute genes mutant demonstrated reduced abilities to produce conidia, compared to the wild type and the gene-rescued mutant. Especially, the conidial yields in Δ dcl2 and Δ ago1 were reduced by 55.8% and 59.3% compared with those from the control strains. Subsequently, for WT and Δ dcl2 strains, digital gene expression (DGE) profiling analysis of the stage of mycelium growth and conidiogenesis revealed that modest changes occur in development or metabolism processes, which could explain the reduction in conidiation in Δ dcl2 mutant. In addition, we further applied high-throughput sequencing technology to identify sRNAs that are differentially expressed in the WT and Δ dcl2 , and found that 4 known milRNAs and 8 novel milRNAs were Mrdcl2 -dependent in M. robertsii . IMPORTANCE The identification and characterization of components in RNAi have contributed significantly to our understanding of the mechanism and functions of RNAi in eukaryotes. Here, we found that Dicer and Argonaute genes play an important role in regulating conidiation in M. robertsii . Our study also demonstrates that diverse small RNA pathways exist in M. robertsii . The study provides a theoretical platform for exploration of the functions of Dicer and Argonaute genes involved in RNAi in fungi.
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dicer and Argonaute genes involved in rna interference in the entomopathogenic fungus metarhizium robertsii
Applied and Environmental Microbiology, 2017Co-Authors: Huimin Meng, Zhangxun Wang, Yulong Wang, Hong Zhu, Bo HuangAbstract:RNA interference (RNAi) is a gene-silencing mechanism that plays an important role in gene regulation in a number of eukaryotic organisms. Two core components, Dicer and Argonaute, are central in the RNAi machinery. However, the physiological roles of Dicer and Argonaute in the entomopathogenic fungus Metarhizium robertsii have remained unclear. Here, the roles of genes encoding Dicer (M. robertsiidcl1 [Mrdcl1] and Mrdcl2) and Argonaute (Mrago1 and Mrago2) proteins in M. robertsii were investigated. The results showed that the Dicer-like protein MrDCL2 and Argonaute protein MrAGO1 are the major components of the RNAi process occurring in M. robertsii The Dicer and Argonaute genes were not involved in the regulation of growth and diverse abiotic stress response in M. robertsii under the tested conditions. Moreover, our results showed that the Dicer and Argonaute gene mutants demonstrated reduced abilities to produce conidia, compared to the wild type (WT) and the gene-rescued mutant. In particular, the conidial yields in the Δdcl2 and Δago1 mutants were reduced by 55.8% and 59.3%, respectively, compared with those from the control strains. Subsequently, for the WT and Δdcl2 mutant strains, digital gene expression (DGE) profiling analysis of the stage of mycelium growth and conidiogenesis revealed that modest changes occur in development or metabolism processes, which may explain the reduction in conidiation in the Δdcl2 mutant. In addition, we further applied high-throughput sequencing technology to identify small RNAs (sRNAs) that are differentially expressed in the WT and the Δdcl2 mutant and found that 4 known microRNA-like small RNAs (milRNAs) and 8 novel milRNAs were Mrdcl2 dependent in M. robertsiiIMPORTANCE The identification and characterization of components in RNAi have contributed significantly to our understanding of the mechanism and functions of RNAi in eukaryotes. Here, we found that Dicer and Argonaute genes play an important role in regulating conidiation in M. robertsii Our study also demonstrates that diverse small RNA pathways exist in M. robertsii The study provides a theoretical platform for exploration of the functions of Dicer and Argonaute genes involved in RNAi in fungi.