ABSTRACT
East African cichlid fishes have diversified in an explosive fashion, but the (epi)genetic basis of the phenotypic diversity of these fishes remains largely unknown. Although transposable elements (TEs) have been associated with phenotypic variation in cichlids, little is known about their transcriptional activity and epigenetic silencing. Here, we describe dynamic patterns of TE expression in African cichlid gonads and during early development. Orthology inference revealed an expansion of piwil1 genes in Lake Malawi cichlids, likely driven by PiggyBac TEs. The expanded piwil1 copies have signatures of positive selection and retain amino acid residues essential for catalytic activity. Furthermore, the gonads of African cichlids express a Piwi-interacting RNA (piRNA) pathway that target TEs. We define the genomic sites of piRNA production in African cichlids and find divergence in closely related species, in line with fast evolution of piRNA-producing loci. Our findings suggest dynamic co-evolution of TEs and host silencing pathways in the African cichlid radiations. We propose that this co-evolution has contributed to cichlid genomic diversity.
ABSTRACT
PIWI-interacting RNAs (piRNAs) are responsible for preventing the movement of transposable elements in germ cells and protect the integrity of germline genomes. In this review, we examine the common elements of piRNA-guided silencing as well as the differences observed between species. We have categorized the mechanisms of piRNA biogenesis and function into modules. Individual PIWI proteins combine these modules in various ways to produce unique PIWI-piRNA pathways, which nevertheless possess the ability to perform conserved functions. This modular model incorporates conserved core mechanisms and accommodates variable co-factors. Adaptability is a hallmark of this RNA-based immune system. We believe that considering the differences in germ cell biology and resident transposons in different organisms is essential for placing the variations observed in piRNA biology into context, while still highlighting the conserved themes that underpin this process.
ABSTRACT
The harnessing of the CRISPR-Cas9 system allows for quick and inexpensive genome editing in tissue culture models. Traditional CRISPR-Cas9 genome editing techniques rely on the ability of single progenitor cells to expand into new pools in a process known as clonal expansion. This is a significant technical challenge that is difficult to overcome for nontransformed cell culture models such as Drosophila ovarian somatic sheath cells (OSCs). OSCs are a unique ex vivo model for epigenetic regulation by PIWI-interacting RNAs (piRNAs) that establish restriction of mobile genetic elements in germ cells to protect genome integrity. Here, we provide a protocol to generate endogenously tagged proteins and gene knockouts without the need for clonal selection. We combine CRISPR-Cas genome editing and knockin of antibiotic selection markers to generate edited cell pools. At the example of Drosophila piwi in OSCs, we demonstrate a strategy that relies on the insertion of an artificial intron to accommodate a selection marker with minimal disturbance of the resulting mRNA. In brief, our donor cassette contains a peptide tag and an optimized intron that accommodates a selection marker driven by an independent promoter on the other genomic strand. The selection marker is transcribed as an independent mRNA, and the intron is efficiently removed from the mRNA encoding the endogenously tagged (endo-tagged) piwi gene. The endo-tagged Piwi protein is expressed at wild-type levels and appropriately localizes to the nucleus of OSCs. We also describe strategies for C-terminal tagging and generation of knockout alleles in OSCs and in human embryonic kidney cells, discuss different design strategies, and provide a plasmid toolkit (available at Addgene). Our protocol enables robust genome editing in OSCs for the first time and provides a simple and time-saving alternative for other cell culture systems. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Design and cloning of single-guide RNA plasmids Basic Protocol 2: Design and cloning of donor template plasmids for epitope tagging Alternate Protocol: Design and cloning of donor template plasmids for gene knockout Basic Protocol 3: Transfection and selection of edited cell pools.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Animals , Humans , Gene Editing/methods , CRISPR-Cas Systems/genetics , Epigenesis, Genetic , Drosophila/genetics , RNA, MessengerABSTRACT
PIWI proteins and their associated PIWI-interacting RNAs (piRNAs) constitute a small RNA-based adaptive immune system that restricts the deleterious activity of mobile genetic elements to protect genome integrity. Self/nonself discrimination is at the very core of successful defence and relies on complementary base-pairing in RNA-guided immunity. How the millions of piRNA sequences faithfully discriminate between self and nonself and how they adapt to novel genomic invaders remain key outstanding questions in genome biology. This review aims to introduce principles of piRNA silencing in the context of metazoan small RNA pathways. A distinct feature of piRNAs is their origin from single-stranded instead of double-stranded RNA precursors, and piRNAs require a unique set of processing factors. Novel nucleases, helicases and RNA binding proteins have been identified in piRNA biology, and while we are starting to understand some mechanisms of piRNA biogenesis and function, this diverse and prolific class of small RNAs remains full of surprises.
Subject(s)
RNA, Double-Stranded , RNA-Binding Proteins , Animals , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , DNA Helicases/genetics , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Binding Proteins/metabolismABSTRACT
Embryonic stem cells (ESCs) can adopt lineage-specific gene-expression programs by stepwise exposure to defined factors, resulting in the generation of functional cell types. Bulk and single-cell-based assays were employed to catalog gene expression, histone modifications, chromatin conformation, and accessibility transitions in ESC populations and individual cells acquiring a presomitic mesoderm fate and undergoing further specification toward myogenic and neurogenic lineages. These assays identified cis-regulatory regions and transcription factors presiding over gene-expression programs occurring at defined ESC transitions and revealed the presence of heterogeneous cell populations within discrete ESC developmental stages. The datasets were employed to identify previously unappreciated genomic elements directing the initial activation of Pax7 and myogenic and neurogenic gene-expression programs. This study provides a resource for the discovery of genomic and transcriptional features of pluripotent, mesoderm-induced ESCs and ESC-derived cell lineages.
Subject(s)
Embryonic Stem Cells , Transcriptome , Cell Differentiation/genetics , Embryonic Stem Cells/metabolism , Gene Expression Regulation, Developmental , Mesoderm/metabolism , Regulatory Sequences, Nucleic AcidABSTRACT
PIWI-interacting RNAs (piRNAs) guard germline genomes against the deleterious action of mobile genetic elements. PiRNAs use extensive base-pairing to recognize their targets and variable 3'ends could change the specificity and efficacy of piRNA silencing. Here, we identify conserved rules that ensure the generation of a single major piRNA 3'end in flies and mice. Our data suggest that the PIWI proteins initially define a short interval on pre-piRNAs that grants access to the ZUC-processor complex. Within this Goldilocks zone, the preference to cut in front of Uridine determines the ultimate processing site. We observe a mouse-specific roadblock that relocates the Goldilocks zone and generates an opportunity for consecutive trimming. Our data reveal a conserved hierarchy between length and sequence preferences that controls the piRNA sequence space. The unanticipated precision of 3'end formation bolsters the emerging understanding that the functional piRNA sequence space is tightly controlled to ensure effective defense.
ABSTRACT
The combination of genome-editing and epitope tagging provides a powerful strategy to study proteins with high affinity and specificity while preserving their physiological expression patterns. However, stably modifying endogenous genes in cells that do not allow for clonal selection has been challenging. Here, we present a simple and fast strategy to generate stable, endogenously tagged alleles in a non-transformed cell culture model. At the example of piwi in Drosophila ovarian somatic sheath cells, we show that this strategy enables the generation of an N-terminally tagged protein that emulates the expression level and subcellular localization of the wild type protein and forms functional Piwi-piRNA complexes. We further present a concise workflow to establish endogenously N-terminally and C-terminally tagged proteins, and knockout alleles through rapid selection of cell pools in fly and human models.
Subject(s)
Drosophila Proteins , Drosophila , Animals , Drosophila/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Female , Gene Editing , Genes, Reporter , Humans , Ovary/metabolism , RNA, Small Interfering/metabolismABSTRACT
Defense against genome invaders universally relies on RNA-guided immunity. Prokaryotic CRISPR-Cas and eukaryotic RNA interference pathways recognize targets by complementary base-pairing, which places the sequences of their guide RNAs at the center of self/nonself discrimination. Here, we explore the sequence space of PIWI-interacting RNAs (piRNAs), the genome defense of animals, and establish functional priority among individual sequences. Our results reveal that only the topmost abundant piRNAs are commonly present in every cell, whereas rare sequences generate cell-to-cell diversity in flies and mice. We identify a skewed distribution of sequence abundance as a hallmark of piRNA populations and show that quantitative differences of more than a 1000-fold are established by conserved mechanisms of biogenesis. Finally, our genomics analyses and direct reporter assays reveal that abundance determines function in piRNA-guided genome defense. Taken together, we identify an effective sequence space and untangle two classes of piRNAs that differ in complexity and function. The first class represents the topmost abundant sequences and drives silencing of genomic parasites. The second class sparsely covers an enormous sequence space. These rare piRNAs cannot function in every cell, every individual, or every generation but create diversity with potential for adaptation in the ongoing arms race with genome invaders.
Subject(s)
RNA, Guide, Kinetoplastida , Animals , Mice , RNA, Guide, Kinetoplastida/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolismABSTRACT
Crosslinking and immunoprecipitation (CLIP) methods are powerful techniques to interrogate direct protein-RNA interactions and dissect posttranscriptional gene regulatory networks. One widely used CLIP variant is photoactivatable ribonucleoside enhanced CLIP (PAR-CLIP) that involves in vivo labeling of nascent RNAs with the photoreactive nucleosides 4-thiouridine (4SU) or 6-thioguanosine (6SG), which can efficiently crosslink to interacting proteins using UVA and UVB light. Crosslinking of 4SU or 6SG to interacting amino acids changes their base-pairing properties and results in characteristic mutations in cDNA libraries prepared for high-throughput sequencing, which can be computationally exploited to remove abundant background from non-crosslinked sequences and help pinpoint RNA binding protein binding sites at nucleotide resolution on a transcriptome-wide scale. Here we present a streamlined protocol for fluorescence-based PAR-CLIP (fPAR-CLIP) that eliminates the need to use radioactivity. It is based on direct ligation of a fluorescently labeled adapter to the 3'end of crosslinked RNA on immobilized ribonucleoproteins, followed by isolation of the adapter-ligated RNA and efficient conversion into cDNA without the previously needed size fractionation on denaturing polyacrylamide gels. These improvements cut the experimentation by half to 2 days and increases sensitivity by 10-100-fold.
Subject(s)
DNA, Complementary/genetics , Gene Expression Profiling/methods , High-Throughput Nucleotide Sequencing/methods , RNA-Binding Proteins/metabolism , Ribonucleoproteins/metabolism , Binding Sites , Cell Line , Cross-Linking Reagents/chemistry , Electrophoresis, Polyacrylamide Gel , GTP Phosphohydrolases/chemistry , Gene Library , Humans , Immunoprecipitation , Oligonucleotides , Polymerase Chain Reaction/methods , Protein Binding , RNA/chemistry , Ribonucleoproteins/genetics , Sensitivity and Specificity , Software , Thiouridine/chemistry , Ultraviolet RaysABSTRACT
Key to CRISPR-Cas adaptive immunity is maintaining an ongoing record of invading nucleic acids, a process carried out by the Cas1-Cas2 complex that integrates short segments of foreign genetic material (spacers) into the CRISPR locus. It is hypothesized that Cas1 evolved from casposases, a novel class of transposases. We show here that the Methanosarcina mazei casposase can integrate varied forms of the casposon end in vitro, and recapitulates several properties of CRISPR-Cas integrases including site-specificity. The X-ray structure of the casposase bound to DNA representing the product of integration reveals a tetramer with target DNA bound snugly between two dimers in which single-stranded casposon end binding resembles that of spacer 3'-overhangs. The differences between transposase and CRISPR-Cas integrase are largely architectural, and it appears that evolutionary change involved changes in protein-protein interactions to favor Cas2 binding over tetramerization; this in turn led to preferred integration of single spacers over two transposon ends.
Subject(s)
Archaeal Proteins/chemistry , CRISPR-Associated Proteins/metabolism , CRISPR-Cas Systems , DNA/genetics , Methanosarcina/enzymology , Transposases/chemistry , Transposases/metabolism , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , CRISPR-Associated Proteins/chemistry , Clustered Regularly Interspaced Short Palindromic Repeats , DNA/chemistry , DNA/metabolism , DNA Transposable Elements , DNA, Archaeal/chemistry , DNA, Archaeal/genetics , DNA, Archaeal/metabolism , DNA, Intergenic , Methanosarcina/genetics , Models, Molecular , Nucleic Acid Conformation , Protein Conformation , Protein Multimerization , Transposases/geneticsABSTRACT
PIWI-interacting small RNAs (piRNAs) establish sequence-specific adaptive restriction of resident genomic parasites to guard genome integrity. In this issue of Cell, Yu, Koppetsch, et al. describe an innate piRNA-response that specifically fragments the viral RNA genome in the germline of recently invaded koalas. This first line of defense might ensure survival until adaptive immunity develops.
Subject(s)
Phascolarctidae , Animals , Genome , Germ Cells , RNA, Small InterferingABSTRACT
Germline genes that are aberrantly expressed in nongermline cancer cells have the potential to be ideal targets for diagnosis and therapy due to their restricted physiological expression, their broad reactivation in various cancer types, and their immunogenic properties. Among such cancer/testis genes, components of the PIWI-interacting small RNA (piRNA) pathway are of particular interest, as they control mobile genetic elements (transposons) in germ cells and thus hold great potential to counteract genome instability in cancer. Here, we systematically investigate the potential reactivation of functional piRNA-silencing mechanisms in the aberrant context. While we observe expression of individual piRNA-pathway genes in cancer, we fail to detect the formation of functional piRNA-silencing complexes. Accordingly, the expression of a PIWI protein alone remains inconsequential to the cancer cell transcriptome. Our data provide a framework for the investigation of complex aberrant gene-expression signatures and establish that reactivation of piRNA silencing, if at all, is not a prevalent phenomenon in cancer cells.
Subject(s)
Gene Silencing/physiology , Neoplasms/genetics , RNA, Small Interfering/genetics , Cell Line, Tumor , DNA Transposable Elements/genetics , Gene Expression/genetics , Genomic Instability/genetics , Germ Cells/pathology , Humans , Male , Testis/physiology , Transcriptome/geneticsABSTRACT
PIWI-interacting RNAs (piRNAs) are at the center of a small RNA-based immune system that defends genomes against the deleterious action of mobile genetic elements (transposons). PiRNAs are highly variable in sequence with extensive targeting potential. Their diversity is restricted by their preference to start with a Uridine (U) at the 5' most position (1U-bias), a bias that remains poorly understood. Here we uncover that the 1U-bias of Piwi-piRNAs is established by consecutive discrimination against all nucleotides but U, first during piRNA biogenesis and then upon interaction with Piwi's specificity loop. Sequence preferences during piRNA processing also restrict U across the piRNA body with the potential to directly impact target recognition. Overall, the uncovered signatures could modulate specificity and efficacy of piRNA-mediated transposon restriction, and provide a substrate for purifying selection in the ongoing arms race between genomes and their mobile parasites.
Subject(s)
Argonaute Proteins/genetics , Drosophila Proteins/genetics , RNA, Small Interfering/metabolism , Animals , Animals, Genetically Modified , Argonaute Proteins/metabolism , Drosophila/genetics , Drosophila Proteins/metabolism , Female , Mutation , Ovary/metabolism , Protein Domains , RNA, Small Interfering/genetics , Uracil/metabolism , Uridine/genetics , Uridine/metabolismABSTRACT
In mammals, gene silencing by the RNA-induced silencing complex (RISC) is a well-understood cytoplasmic posttranscriptional gene regulatory mechanism. Here, we show that embryonic stem cells (ESCs) contain high levels of nuclear AGO proteins and that in ESCs nuclear AGO protein activity allows for the onset of differentiation. In the nucleus, AGO proteins interact with core RISC components, including the TNRC6 proteins and the CCR4-NOT deadenylase complex. In contrast to cytoplasmic miRNA-mediated gene silencing that mainly operates on cis-acting elements in mRNA 3' untranslated (UTR) sequences, in the nucleus AGO binding in the coding sequence and potentially introns also contributed to post-transcriptional gene silencing. Thus, nuclear localization of AGO proteins in specific cell types leads to a previously unappreciated expansion of the miRNA-regulated transcriptome.
Subject(s)
Argonaute Proteins/physiology , Gene Silencing/physiology , MicroRNAs/physiology , Animals , Argonaute Proteins/genetics , Cell Differentiation/genetics , Cell Line , Cell Nucleus , Cytoplasm , Embryonic Stem Cells/metabolism , Humans , Mammals , Mice , MicroRNAs/genetics , RNA Interference , RNA Stability , RNA, Messenger , RNA, Small Interfering , RNA-Binding Proteins , RNA-Induced Silencing Complex/genetics , RNA-Induced Silencing Complex/metabolism , Transcription FactorsABSTRACT
Transposons are mobile genetic elements that threaten the survival of species by destabilizing the germline genomes. Limiting the spread of these selfish elements is imperative. Germ cells employ specialized small regulatory RNA pathways to restrain transposon activity. PIWI proteins and Piwi-interacting RNAs (piRNAs) silence transposons at the transcriptional and posttranscriptional level with loss-of-function mutant animals universally exhibiting sterility often associated with germ cell defects. This short review aims to illustrate basic strategies of piRNA-guided defense against transposons. Mechanisms of piRNA silencing are most readily studied in Drosophila melanogaster, which serves as a model to delineate molecular concepts and as a reference for mammalian piRNA systems. PiRNA pathways utilize two major strategies to handle the challenges of transposon control: (1) the hard-wired molecular memory of prior transpositions enables recognition of mobile genetic elements and discriminates transposons from host genes; (2) a feed-forward adaptation mechanism shapes piRNA populations to selectively combat the immediate threat of transposon transcripts. In flies, maternally contributed PIWI-piRNA complexes bolster both of these lines of defense and ensure transgenerational immunity. While recent studies have provided a conceptual framework of what could be viewed as an ancient immune system, we are just beginning to appreciate its many molecular innovations.
ABSTRACT
Oncogenic RAS (H-RAS(V12)) induces premature senescence in primary cells by triggering production of reactive oxygen species (ROS), but the molecular role of ROS in senescence remains elusive. We investigated whether inhibition of protein tyrosine phosphatases by ROS contributed to H-RAS(V12)-induced senescence. We identified protein tyrosine phosphatase 1B (PTP1B) as a major target of H-RAS(V12)-induced ROS. Inactivation of PTP1B was necessary and sufficient to induce premature senescence in H-RAS(V12)-expressing IMR90 fibroblasts. We identified phospho-Tyr 393 of argonaute 2 (AGO2) as a direct substrate of PTP1B. Phosphorylation of AGO2 at Tyr 393 inhibited loading with microRNAs (miRNAs) and thus miRNA-mediated gene silencing, which counteracted the function of H-RAS(V12)-induced oncogenic miRNAs. Overall, our data illustrate that premature senescence in H-RAS(V12)-transformed primary cells is a consequence of oxidative inactivation of PTP1B and inhibition of miRNA-mediated gene silencing.
Subject(s)
Argonaute Proteins/metabolism , Gene Silencing , Protein Tyrosine Phosphatase, Non-Receptor Type 1/physiology , Tyrosine/metabolism , ras Proteins/physiology , Argonaute Proteins/chemistry , Cell Line , Cellular Senescence/genetics , Humans , MicroRNAs/metabolism , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Reactive Oxygen Species/metabolism , Tyrosine/chemistry , ras Proteins/genetics , ras Proteins/metabolismABSTRACT
Argonautes are the central protein component in small RNA silencing pathways. Of the four human Argonautes (hAgo1-hAgo4) only hAgo2 is an active slicer. We determined the structure of hAgo1 bound to endogenous copurified RNAs to 1.75 Å resolution and hAgo1 loaded with let-7 microRNA to 2.1 Å. Both structures are strikingly similar to the structures of hAgo2. A conserved catalytic tetrad within the PIWI domain of hAgo2 is required for its slicing activity. Completion of the tetrad, combined with a mutation on a loop adjacent to the active site of hAgo1, results in slicer activity that is substantially enhanced by swapping in the N domain of hAgo2. hAgo3, with an intact tetrad, becomes an active slicer by swapping the N domain of hAgo2 without additional mutations. Intriguingly, the elements that make Argonaute an active slicer involve a sophisticated interplay between the active site and more distant regions of the enzyme.
Subject(s)
Argonaute Proteins/genetics , Eukaryotic Initiation Factors/genetics , RNA, Small Interfering/genetics , Amino Acid Sequence , Animals , Argonaute Proteins/chemistry , Argonaute Proteins/metabolism , Eukaryotic Initiation Factors/chemistry , Eukaryotic Initiation Factors/metabolism , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Models, Molecular , Molecular Sequence Data , Point Mutation , Protein Structure, Tertiary , RNA, Small Interfering/metabolism , Sf9 CellsABSTRACT
PIWI-family proteins and their associated small RNAs (piRNAs) act in an evolutionarily conserved innate immune mechanism to provide essential protection for germ-cell genomes against the activity of mobile genetic elements. piRNA populations comprise a molecular definition of transposons, which permits them to distinguish transposons from host genes and selectively silence them. piRNAs can be generated in two distinct ways, forming either primary or secondary piRNAs. Primary piRNAs come from discrete genomic loci, termed piRNA clusters, and seem to be derived from long, single-stranded precursors. The biogenesis of primary piRNAs involves at least two nucleolytic steps. An unknown enzyme cleaves piRNA cluster transcripts to generate monophosphorylated piRNA 5' ends. piRNA 3' ends are probably formed by exonucleolytic trimming, after a piRNA precursor is loaded into its PIWI partner. Secondary piRNAs arise during the adaptive 'ping-pong' cycle, with their 5' termini being formed by the activity of PIWIs themselves. A number of proteins have been implicated genetically in primary piRNA biogenesis. One of these, Drosophila melanogaster Zucchini, is a member of the phospholipase-D family of phosphodiesterases, which includes both phospholipases and nucleases. Here we produced a dimeric, soluble fragment of the mouse Zucchini homologue (mZuc; also known as PLD6) and show that it possesses single-strand-specific nuclease activity. A crystal structure of mZuc at 1.75 Å resolution indicates greater architectural similarity to phospholipase-D family nucleases than to phospholipases. Together, our data suggest that the Zucchini proteins act in primary piRNA biogenesis as nucleases, perhaps generating the 5' ends of primary piRNAs.
Subject(s)
Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Phospholipase D/chemistry , Phospholipase D/metabolism , RNA, Small Interfering/metabolism , Animals , Biocatalysis , Catalytic Domain , Crystallography, X-Ray , Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Endoribonucleases/chemistry , Endoribonucleases/metabolism , Mice , Models, Molecular , Protein Conformation , Protein Multimerization , RNA, Small Interfering/biosynthesis , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , Static Electricity , Substrate SpecificityABSTRACT
Argonaute proteins lie at the heart of the RNA-induced silencing complex (RISC), wherein they use small RNA guides to recognize targets. Initial insight into the architecture of Argonautes came from studies of prokaryotic proteins, revealing a crescent-shaped base made up of the amino-terminal, PAZ, middle, and PIWI domains. The recently reported crystal structure of human Argonaute-2 (hAgo2), the "slicer" in RNA interference, in complex with a mixed population of RNAs derived from insect cells provides insight into the architecture of a eukaryotic Argonaute protein with defined biochemical and biological functions. Here, we report the structure of human Ago2 bound to a physiologically relevant microRNA, microRNA-20a, at 2.2 Å resolution. The miRNA is anchored at both ends by the Mid and PAZ domains and makes several kinks and turns along the binding groove. Interestingly, miRNA binding confers remarkable stability on hAgo2, locking this otherwise flexible enzyme into a stable conformation.
Subject(s)
Argonaute Proteins/chemistry , Argonaute Proteins/metabolism , MicroRNAs/chemistry , MicroRNAs/metabolism , Argonaute Proteins/isolation & purification , Crystallography, X-Ray , Humans , Models, Molecular , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
Combining RNAi in cultured cells and analysis of mutant animals, we probed the roles of known Piwi-interacting RNA (piRNA) pathway components in the initiation and effector phases of transposon silencing. Squash associated physically with Piwi, and reductions in its expression led to modest transposon derepression without effects on piRNAs, consistent with an effector role. Alterations in Zucchini or Armitage reduced both Piwi protein and piRNAs, indicating functions in the formation of a stable Piwi RISC (RNA-induced silencing complex). Notably, loss of Zucchini or mutations within its catalytic domain led to accumulation of unprocessed precursor transcripts from flamenco, consistent with a role for this putative nuclease in piRNA biogenesis.