ABSTRACT
The systematic determination of protein function is a key goal of modern biology, but remains challenging with current approaches. Here we present ORFtag, a versatile, cost-effective and highly efficient method for the massively parallel tagging and functional interrogation of proteins at the proteome scale. ORFtag uses retroviral vectors bearing a promoter, peptide tag and splice donor to generate fusions between the tag and endogenous open reading frames (ORFs). We demonstrate the utility of ORFtag through functional screens for transcriptional activators, repressors and posttranscriptional regulators in mouse embryonic stem cells. Each screen recovers known and identifies new regulators, including long ORFs inaccessible by other methods. Among other hits, we find that Zfp574 is a highly selective transcriptional activator and that oncogenic fusions often function as transactivators.
ABSTRACT
Members of the diverse heterochromatin protein 1 (HP1) family play crucial roles in heterochromatin formation and maintenance. Despite the similar affinities of their chromodomains for di- and tri-methylated histone H3 lysine 9 (H3K9me2/3), different HP1 proteins exhibit distinct chromatin-binding patterns, likely due to interactions with various specificity factors. Previously, we showed that the chromatin-binding pattern of the HP1 protein Rhino, a crucial factor of the Drosophila PIWI-interacting RNA (piRNA) pathway, is largely defined by a DNA sequence-specific C2H2 zinc finger protein named Kipferl (Baumgartner et al., 2022). Here, we elucidate the molecular basis of the interaction between Rhino and its guidance factor Kipferl. Through phylogenetic analyses, structure prediction, and in vivo genetics, we identify a single amino acid change within Rhino's chromodomain, G31D, that does not affect H3K9me2/3 binding but disrupts the interaction between Rhino and Kipferl. Flies carrying the rhinoG31D mutation phenocopy kipferl mutant flies, with Rhino redistributing from piRNA clusters to satellite repeats, causing pronounced changes in the ovarian piRNA profile of rhinoG31D flies. Thus, Rhino's chromodomain functions as a dual-specificity module, facilitating interactions with both a histone mark and a DNA-binding protein.
Subject(s)
Chromatin , Chromobox Protein Homolog 5 , Chromosomal Proteins, Non-Histone , Drosophila Proteins , Drosophila melanogaster , Animals , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromatin/metabolism , Chromatin/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Evolution, Molecular , Phylogeny , Protein Binding , RNA, Small Interfering/metabolism , RNA, Small Interfering/genetics , Histones/metabolism , Histones/genetics , DNA/metabolism , DNA/geneticsABSTRACT
Genomic imprinting-the non-equivalence of maternal and paternal genomes-is a critical process that has evolved independently in many plant and mammalian species1,2. According to kinship theory, imprinting is the inevitable consequence of conflictive selective forces acting on differentially expressed parental alleles3,4. Yet, how these epigenetic differences evolve in the first place is poorly understood3,5,6. Here we report the identification and molecular dissection of a parent-of-origin effect on gene expression that might help to clarify this fundamental question. Toxin-antidote elements (TAs) are selfish elements that spread in populations by poisoning non-carrier individuals7-9. In reciprocal crosses between two Caenorhabditis tropicalis wild isolates, we found that the slow-1/grow-1 TA is specifically inactive when paternally inherited. This parent-of-origin effect stems from transcriptional repression of the slow-1 toxin by the PIWI-interacting RNA (piRNA) host defence pathway. The repression requires PIWI Argonaute and SET-32 histone methyltransferase activities and is transgenerationally inherited via small RNAs. Remarkably, when slow-1/grow-1 is maternally inherited, slow-1 repression is halted by a translation-independent role of its maternal mRNA. That is, slow-1 transcripts loaded into eggs-but not SLOW-1 protein-are necessary and sufficient to counteract piRNA-mediated repression. Our findings show that parent-of-origin effects can evolve by co-option of the piRNA pathway and hinder the spread of selfish genes that require sex for their propagation.
Subject(s)
Caenorhabditis , Genomic Imprinting , Piwi-Interacting RNA , Repetitive Sequences, Nucleic Acid , Animals , Female , Male , Alleles , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Caenorhabditis/genetics , Caenorhabditis/metabolism , Crosses, Genetic , Fathers , Genome/genetics , Genomic Imprinting/genetics , Hermaphroditic Organisms/genetics , Histone Methyltransferases/genetics , Histone Methyltransferases/metabolism , Mothers , Oocytes/metabolism , Piwi-Interacting RNA/genetics , Protein Biosynthesis , Repetitive Sequences, Nucleic Acid/genetics , RNA, Messenger/genetics , Toxins, Biological/genetics , Transcription, GeneticABSTRACT
ZNF462 haploinsufficiency is linked to Weiss-Kruszka syndrome, a genetic disorder characterized by neurodevelopmental defects, including autism. Though conserved in vertebrates and essential for embryonic development, the molecular functions of ZNF462 remain unclear. We identified its murine homologue ZFP462 in a screen for mediators of epigenetic gene silencing. Here we show that ZFP462 safeguards neural lineage specification of mouse embryonic stem cells (ESCs) by targeting the H3K9-specific histone methyltransferase complex G9A/GLP to silence meso-endodermal genes. ZFP462 binds to transposable elements that are potential enhancers harbouring pluripotency and meso-endoderm transcription factor binding sites. Recruiting G9A/GLP, ZFP462 seeds heterochromatin, restricting transcription factor binding. Loss of ZFP462 in ESCs results in increased chromatin accessibility at target sites and ectopic expression of meso-endodermal genes. Taken together, ZFP462 confers lineage and locus specificity to the broadly expressed epigenetic regulator G9A/GLP. Our results suggest that aberrant activation of lineage non-specific genes in the neuronal lineage underlies ZNF462-associated neurodevelopmental pathology.
Subject(s)
Heterochromatin , Histone-Lysine N-Methyltransferase , Animals , Mice , Heterochromatin/genetics , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Chromatin , Embryonic Stem Cells , Transcription Factors/genetics , Transcription Factors/metabolism , DNA-Binding Proteins/genetics , Nerve Tissue Proteins/geneticsABSTRACT
RNA interference systems depend on the synthesis of small RNA precursors whose sequences define the target spectrum of these silencing pathways. The Drosophila Heterochromatin Protein 1 (HP1) variant Rhino permits transcription of PIWI-interacting RNA (piRNA) precursors within transposon-rich heterochromatic loci in germline cells. Current models propose that Rhino's specific chromatin occupancy at piRNA source loci is determined by histone marks and maternally inherited piRNAs, but also imply the existence of other, undiscovered specificity cues. Here, we identify a member of the diverse family of zinc finger associated domain (ZAD)-C2H2 zinc finger proteins, Kipferl, as critical Rhino cofactor in ovaries. By binding to guanosine-rich DNA motifs and interacting with the Rhino chromodomain, Kipferl recruits Rhino to specific loci and stabilizes it on chromatin. In kipferl mutant flies, Rhino is lost from most of its target chromatin loci and instead accumulates on pericentromeric Satellite arrays, resulting in decreased levels of transposon targeting piRNAs and impaired fertility. Our findings reveal that DNA sequence, in addition to the H3K9me3 mark, determines the identity of piRNA source loci and provide insight into how Rhino might be caught in the crossfire of genetic conflicts.
The genes within our DNA encode the essentials of our body plan and how each task in the body is achieved. However, our genome also contains many repetitive regions of DNA that do not encode functional genes. Some of these regions are genetic parasites known as transposons that try to multiply and spread around the DNA of their host. To prevent transposon DNA from interfering with the way the body operates, humans and other animals have evolved elaborate defense mechanisms to identify transposons and prevent them from multiplying. In one such mechanism, known as the piRNA pathway, the host makes small molecules known as piRNAs that have sequences complementary to those of transposons, and act as guides to silence the transposons. The instructions to make these piRNAs are stored in the form of transposon fragments in dedicated regions of host DNA called piRNA clusters. These clusters thereby act as genetic memory, allowing the host to recognize and silence specific transposons in other locations within the host's genome. In fruit flies, a protein called Rhino binds to piRNA clusters that are densely packed to allow piRNAs to be made. However, it remained unclear how Rhino is able to identify and bind to piRNA clusters, but not to other similarly densely packed regions of DNA. Baumgartner et al. used a combination of genetic, genomic, and imaging approaches to study how Rhino finds its way in the fruit fly genome. They found that another protein called Kipferl interacts with Rhino and is required for Rhino to bind to nearly all piRNA clusters. Since Kipferl can by itself bind to the sequences that Rhino needs to find, the results suggest that Kipferl acts to recruit and initiate Rhino binding within densely packed piRNA clusters. Further experiments found that, in flies lacking Kipferl, Rhino binds to regions of DNA called Satellite repeats, hinting that these selfish sequences may compete for Rhino for their own benefit. The finding that Kipferl and Rhino work together to define the memory system of the piRNA pathway strongly advances our understanding of how a sequence-specific defense system based on small RNAs can be established.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , Animals , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA Transposable Elements/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Guanosine/metabolism , RNA Precursors/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Zinc FingersABSTRACT
RT-qPCR-based diagnostic tests play important roles in combating virus-caused pandemics such as Covid-19. However, their dependence on sophisticated equipment and the associated costs often limits their widespread use. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative nucleic acid detection method that overcomes these limitations. Here, we present a rapid, robust, and sensitive RT-LAMP-based SARS-CoV-2 detection assay. Our 40-min procedure bypasses the RNA isolation step, is insensitive to carryover contamination, and uses a colorimetric readout that enables robust SARS-CoV-2 detection from various sample types. Based on this assay, we have increased sensitivity and scalability by adding a nucleic acid enrichment step (Bead-LAMP), developed a version for home testing (HomeDip-LAMP), and identified open-source RT-LAMP enzymes that can be produced in any molecular biology laboratory. On a dedicated website, rtlamp.org (DOI: 10.5281/zenodo.6033689), we provide detailed protocols and videos. Our optimized, general-purpose RT-LAMP assay is an important step toward population-scale SARS-CoV-2 testing.
ABSTRACT
Nuclear Argonaute proteins, guided by small RNAs, mediate sequence-specific heterochromatin formation. The molecular principles that link Argonaute-small RNA complexes to cellular heterochromatin effectors on binding to nascent target RNAs are poorly understood. Here, we explain the mechanism by which the PIWI-interacting RNA (piRNA) pathway connects to the heterochromatin machinery in Drosophila. We find that Panoramix, a corepressor required for piRNA-guided heterochromatin formation, is SUMOylated on chromatin in a Piwi-dependent manner. SUMOylation, together with an amphipathic LxxLL motif in Panoramix's intrinsically disordered repressor domain, are necessary and sufficient to recruit Small ovary (Sov), a multi-zinc-finger protein essential for general heterochromatin formation and viability. Structure-guided mutations that eliminate the Panoramix-Sov interaction or that prevent SUMOylation of Panoramix uncouple Sov from the piRNA pathway, resulting in viable but sterile flies in which Piwi-targeted transposons are derepressed. Thus, Piwi engages the heterochromatin machinery specifically at transposon loci by coupling recruitment of a corepressor to nascent transcripts with its SUMOylation.
Subject(s)
Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Heterochromatin/genetics , Heterochromatin/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Amino Acid Motifs , Animals , Animals, Genetically Modified , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Binding Sites/genetics , Chromatin/genetics , Chromatin/metabolism , DNA Transposable Elements , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drosophila Proteins/chemistry , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Female , Gene Silencing , Genes, Insect , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/genetics , Intrinsically Disordered Proteins/metabolism , Models, Molecular , Mutation , Nuclear Proteins/chemistry , Oogonial Stem Cells/metabolism , Protein Interaction Domains and Motifs , RNA-Binding Proteins/chemistry , Sumoylation/genetics , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolismABSTRACT
Argonaute proteins of the PIWI clade complexed with PIWI-interacting RNAs (piRNAs) protect the animal germline genome by silencing transposable elements. One of the leading experimental systems for studying piRNA biology is the Drosophila melanogaster ovary. In addition to classical mutagenesis, transgenic RNA interference (RNAi), which enables tissue-specific silencing of gene expression, plays a central role in piRNA research. Here, we establish a versatile toolkit focused on piRNA biology that combines germline transgenic RNAi, GFP marker lines for key proteins of the piRNA pathway, and reporter transgenes to establish genetic hierarchies. We compare constitutive, pan-germline RNAi with an equally potent transgenic RNAi system that is activated only after germ cell cyst formation. Stage-specific RNAi allows us to investigate the role of genes essential for germline cell survival, for example, nuclear RNA export or the SUMOylation pathway, in piRNA-dependent and independent transposon silencing. Our work forms the basis for an expandable genetic toolkit provided by the Vienna Drosophila Resource Center.
Subject(s)
Drosophila melanogaster , AnimalsABSTRACT
Nuclear Argonaute proteins, guided by their bound small RNAs to nascent target transcripts, mediate cotranscriptional silencing of transposons and repetitive genomic loci through heterochromatin formation. The molecular mechanisms involved in this process are incompletely understood. Here, we show that the SFiNX complex, a silencing mediator downstream from nuclear Piwi-piRNA complexes in Drosophila, facilitates cotranscriptional silencing as a homodimer. The dynein light chain protein Cut up/LC8 mediates SFiNX dimerization, and its function can be bypassed by a heterologous dimerization domain, arguing for a constitutive SFiNX dimer. Dimeric, but not monomeric SFiNX, is capable of forming molecular condensates in a nucleic acid-stimulated manner. Mutations that prevent SFiNX dimerization result in loss of condensate formation in vitro and the inability of Piwi to initiate heterochromatin formation and silence transposons in vivo. We propose that multivalent SFiNX-nucleic acid interactions are critical for heterochromatin establishment at piRNA target loci in a cotranscriptional manner.
Subject(s)
Argonaute Proteins/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Gene Expression Regulation, Developmental/genetics , Gene Silencing/physiology , Multiprotein Complexes/metabolism , Animals , Dimerization , Drosophila Proteins/chemistry , Drosophila melanogaster/metabolism , Dyneins/metabolism , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleocytoplasmic Transport Proteins/chemistry , Nucleocytoplasmic Transport Proteins/genetics , Nucleocytoplasmic Transport Proteins/metabolism , Protein Subunits/genetics , Protein Subunits/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
Nibbler (Nbr) is a 3'-to-5' exoribonuclease whose catalytic 3'-end trimming activity impacts microRNA (miRNA) and PIWI-interacting RNA (piRNA) biogenesis. Here, we report on structural and functional studies to decipher the contributions of Nbr's N-terminal domain (NTD) and exonucleolytic domain (EXO) in miRNA 3'-end trimming. We have solved the crystal structures of the NTD core and EXO domains of Nbr, both in the apo-state. The NTD-core domain of Aedes aegypti Nbr adopts a HEAT-like repeat scaffold with basic patches constituting an RNA-binding surface exhibiting a preference for binding double-strand RNA (dsRNA) over single-strand RNA (ssRNA). Structure-guided functional assays in Drosophila S2 cells confirmed a principal role of the NTD in exonucleolytic miRNA trimming, which depends on basic surface patches. Gain-of-function experiments revealed a potential role of the NTD in recruiting Nbr to Argonaute-bound small RNA substrates. The EXO domain of A. aegypti and Drosophila melanogaster Nbr adopt a mixed α/ß-scaffold with a deep pocket lined by a DEDDy catalytic cleavage motif. We demonstrate that Nbr's EXO domain exhibits Mn2+-dependent ssRNA-specific 3'-to-5' exoribonuclease activity. Modeling of a 3' terminal Uridine into the catalytic pocket of Nbr EXO indicates that 2'-O-methylation of the 3'-U would result in a steric clash with a tryptophan side chain, suggesting that 2'-O-methylation protects small RNAs from Nbr-mediated trimming. Overall, our data establish that Nbr requires its NTD as a substrate recruitment platform to execute exonucleolytic miRNA maturation, catalyzed by the ribonuclease EXO domain.
Subject(s)
3' Flanking Region , Drosophila Proteins/chemistry , Exoribonucleases/chemistry , MicroRNAs/chemistry , MicroRNAs/genetics , RNA Processing, Post-Transcriptional , Structure-Activity Relationship , Animals , Argonaute Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster , Exoribonucleases/metabolism , MicroRNAs/metabolism , Models, Biological , Models, Molecular , Molecular Conformation , Mutation , Protein Binding , Protein Interaction Domains and Motifs , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolismABSTRACT
The export of mRNA from nucleus to cytoplasm requires the conserved and essential transcription and export (TREX) complex (THO-UAP56/DDX39B-ALYREF). TREX selectively binds mRNA maturation marks and licenses mRNA for nuclear export by loading the export factor NXF1-NXT1. How TREX integrates these marks and achieves high selectivity for mature mRNA is poorly understood. Here, we report the cryo-electron microscopy structure of the human THO-UAP56/DDX39B complex at 3.3 Å resolution. The seven-subunit THO-UAP56/DDX39B complex multimerizes into a 28-subunit tetrameric assembly, suggesting that selective recognition of mature mRNA is facilitated by the simultaneous sensing of multiple, spatially distant mRNA regions and maturation marks. Two UAP56/DDX39B RNA helicases are juxtaposed at each end of the tetramer, which would allow one bivalent ALYREF protein to bridge adjacent helicases and regulate the TREX-mRNA interaction. Our structural and biochemical results suggest a conserved model for TREX complex function that depends on multivalent interactions between proteins and mRNA.
The DNA of human and other eukaryotic cells is stored inside a compartment called the nucleus. DNA carries the genetic code and provides a blueprint for all of the cell's proteins. However, protein production occurs outside the nucleus, in the main body of the cell. To transmit genetic information from one compartment to the other, the DNA sequences are first transcribed into another molecule called messenger RNA, or mRNA for short. Once made, mRNA exits the nucleus and enters the cell's main body to encounter the machinery that translates its sequence into a protein. Before mRNA can exit the nucleus, it must first undergo a series of modifications, which result in the mRNA molecule being successively bound to specific proteins. Once mRNA has passed through these steps, it is recognized by the transcription-and-export complex, or TREX for short, which is comprised of several proteins. When TREX binds to mRNA, it adds on a final protein which allows the mRNA molecule to be transported out of the nucleus. However, it remained unclear how TREX selects the completed mRNA-protein complexes that are ready for export while at the same time recognizing the wide variety of mRNA molecules produced by cells. Now, Pühringer and Hohmann et al. have identified the first three-dimensional structure of the core of the human TREX complex using a technique called cryo-electron microscopy. This revealed that the seven proteins of the TREX core assemble into a large complex that has four copies of each protein. The structure suggests that TREX can bind to mRNA and its attached proteins in various ways. These different binding arrangements may help the complex select which mRNA molecules are fully modified and ready to be exported. The structure also sheds light on how mutations in this complex can lead to diseases such as BeaulieuBoycottInnes syndrome (BBIS). This work will help guide future research into the activity of TREX, including how its structure changes when it binds to mRNA and deposits the final transport protein. Identifying these structures will make it easier to design experiments that target specific aspects of TREX activity and provide new insights into how these complexes work.
Subject(s)
Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/ultrastructure , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/ultrastructure , Active Transport, Cell Nucleus/physiology , Cryoelectron Microscopy , Humans , Protein Conformation , RNA Transport/physiology , RNA, Messenger/metabolismABSTRACT
Diverse classes of silencing small (s)RNAs operate via ARGONAUTE-family proteins within RNA-induced-silencing-complexes (RISCs). Here, we have streamlined various embodiments of a Q-sepharose-based RISC-purification method that relies on conserved biochemical properties of all ARGONAUTEs. We show, in multiple benchmarking assays, that the resulting 15-min benchtop extraction procedure allows simultaneous purification of all known classes of RISC-associated sRNAs without prior knowledge of the samples-intrinsic ARGONAUTE repertoires. Optimized under a user-friendly format, the method - coined 'TraPR' for Trans-kingdom, rapid, affordable Purification of RISCs - operates irrespectively of the organism, tissue, cell type or bio-fluid of interest, and scales to minute amounts of input material. The method is highly suited for direct profiling of silencing sRNAs, with TraPR-generated sequencing libraries outperforming those obtained via gold-standard procedures that require immunoprecipitations and/or lengthy polyacrylamide gel-selection. TraPR considerably improves the quality and consistency of silencing sRNA sample preparation including from notoriously difficult-to-handle tissues/bio-fluids such as starchy storage roots or mammalian plasma, and regardless of RNA contaminants or RNA degradation status of samples.
Subject(s)
Argonaute Proteins/metabolism , Chromatography, Liquid/methods , RNA, Small Interfering/isolation & purification , RNA-Induced Silencing Complex/chemistry , Animals , Anion Exchange Resins , Argonaute Proteins/isolation & purification , Cell Line, Tumor , Gene Library , Mice , Mice, Inbred C57BL , Polynucleotide 5'-Hydroxyl-Kinase , RNA, Fungal/isolation & purification , RNA, Helminth/isolation & purification , RNA, Neoplasm/isolation & purification , RNA, Plant/isolation & purification , RNA, Protozoan/isolation & purification , RNA, Small Interfering/blood , RNA, Small Interfering/metabolism , Sepharose , Silicon Dioxide , UltracentrifugationABSTRACT
The PIWI-interacting RNA (piRNA) pathway protects genome integrity in part through establishing repressive heterochromatin at transposon loci. Silencing requires piRNA-guided targeting of nuclear PIWI proteins to nascent transposon transcripts, yet the subsequent molecular events are not understood. Here, we identify SFiNX (silencing factor interacting nuclear export variant), an interdependent protein complex required for Piwi-mediated cotranscriptional silencing in Drosophila. SFiNX consists of Nxf2-Nxt1, a gonad-specific variant of the heterodimeric messenger RNA export receptor Nxf1-Nxt1 and the Piwi-associated protein Panoramix. SFiNX mutant flies are sterile and exhibit transposon derepression because piRNA-loaded Piwi is unable to establish heterochromatin. Within SFiNX, Panoramix recruits heterochromatin effectors, while the RNA binding protein Nxf2 licenses cotranscriptional silencing. Our data reveal how Nxf2 might have evolved from an RNA transport receptor into a cotranscriptional silencing factor. Thus, NXF variants, which are abundant in metazoans, can have diverse molecular functions and might have been coopted for host genome defense more broadly.
Subject(s)
Drosophila Proteins/physiology , Drosophila melanogaster/genetics , Heterochromatin/metabolism , Nuclear Proteins/physiology , Nucleocytoplasmic Transport Proteins/physiology , RNA, Small Interfering/genetics , RNA-Binding Proteins/physiology , Amino Acid Sequence , Animals , Argonaute Proteins/physiology , Binding Sites , Crystallography, X-Ray , DNA Transposable Elements/genetics , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Gene Expression Regulation , Gene Silencing , Genome, Insect , Models, Molecular , Multiprotein Complexes , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nucleocytoplasmic Transport Proteins/chemistry , Nucleocytoplasmic Transport Proteins/genetics , Protein Conformation , Protein Interaction Mapping , Protein Multimerization , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Transcription, GeneticABSTRACT
PIWI-interacting RNAs (piRNAs) guide transposon silencing in animals. The 22-30 nt piRNAs are processed in the cytoplasm from long non-coding RNAs that often lack RNA processing hallmarks of export-competent transcripts. By studying how these transcripts achieve nuclear export, we uncover an RNA export pathway specific for piRNA precursors in the Drosophila germline. This pathway requires Nxf3-Nxt1, a variant of the hetero-dimeric mRNA export receptor Nxf1-Nxt1. Nxf3 interacts with UAP56, a nuclear RNA helicase essential for mRNA export, and CG13741/Bootlegger, which recruits Nxf3-Nxt1 and UAP56 to heterochromatic piRNA source loci. Upon RNA cargo binding, Nxf3 achieves nuclear export via the exportin Crm1 and accumulates together with Bootlegger in peri-nuclear nuage, suggesting that after export, Nxf3-Bootlegger delivers precursor transcripts to the piRNA processing sites. These findings indicate that the piRNA pathway bypasses nuclear RNA surveillance systems to export unprocessed transcripts to the cytoplasm, a strategy also exploited by retroviruses.
Subject(s)
Active Transport, Cell Nucleus/physiology , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Heterochromatin/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , RNA, Small Interfering/metabolism , RNA-Binding Proteins/metabolism , Animals , Animals, Genetically Modified , Argonaute Proteins/metabolism , Cell Line , Cell Nucleus/metabolism , Cytoplasm/metabolism , DEAD-box RNA Helicases/metabolism , DNA Transposable Elements , Gene Silencing , Germ Cells/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Karyopherins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription, Genetic , Exportin 1 ProteinABSTRACT
Nuclear small RNA pathways safeguard genome integrity by establishing transcription-repressing heterochromatin at transposable elements. This inevitably also targets the transposon-rich source loci of the small RNAs themselves. How small RNA source loci are efficiently transcribed while transposon promoters are potently silenced is not understood. Here we show that, in Drosophila, transcription of PIWI-interacting RNA (piRNA) clusters-small RNA source loci in animal gonads-is enforced through RNA polymerase II pre-initiation complex formation within repressive heterochromatin. This is accomplished through Moonshiner, a paralogue of a basal transcription factor IIA (TFIIA) subunit, which is recruited to piRNA clusters via the heterochromatin protein-1 variant Rhino. Moonshiner triggers transcription initiation within piRNA clusters by recruiting the TATA-box binding protein (TBP)-related factor TRF2, an animal TFIID core variant. Thus, transcription of heterochromatic small RNA source loci relies on direct recruitment of the core transcriptional machinery to DNA via histone marks rather than sequence motifs, a concept that we argue is a recurring theme in evolution.
Subject(s)
DNA Transposable Elements/genetics , Drosophila melanogaster/genetics , Heterochromatin/genetics , Heterochromatin/metabolism , RNA Polymerase II/metabolism , RNA, Small Interfering/genetics , Transcription, Genetic , Animals , Chromosomal Proteins, Non-Histone/metabolism , Drosophila Proteins/metabolism , Female , Gene Silencing , Heterochromatin/chemistry , Multigene Family/genetics , Promoter Regions, Genetic/genetics , RNA Polymerase II/chemistry , RNA, Small Interfering/biosynthesis , Telomeric Repeat Binding Protein 2/metabolism , Transcription Factor TFIIA/metabolism , Transcription Initiation, GeneticABSTRACT
Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and therefore have important roles in eukaryotic gene silencing. Of the three small RNA classes, microRNAs and short interfering RNAs are processed from double-stranded precursors into defined 21- to 23-mers by Dicer, an endoribonuclease with intrinsic ruler function. PIWI-interacting RNAs (piRNAs)-the 22-30-nt-long guides for PIWI-clade Ago proteins that silence transposons in animal gonads-are generated independently of Dicer from single-stranded precursors. piRNA 5' ends are defined either by Zucchini, the Drosophila homologue of mitoPLD-a mitochondria-anchored endonuclease, or by piRNA-guided target cleavage. Formation of piRNA 3' ends is poorly understood. Here we report that two genetically and mechanistically distinct pathways generate piRNA 3' ends in Drosophila. The initiating nucleases are either Zucchini or the PIWI-clade proteins Aubergine (Aub) or Ago3. While Zucchini-mediated cleavages directly define mature piRNA 3' ends, Aub/Ago3-mediated cleavages liberate pre-piRNAs that require extensive resection by the 3'-to-5' exoribonuclease Nibbler (Drosophila homologue of Mut-7). The relative activity of these two pathways dictates the extent to which piRNAs are directed to cytoplasmic or nuclear PIWI-clade proteins and thereby sets the balance between post-transcriptional and transcriptional silencing. Notably, loss of both Zucchini and Nibbler reveals a minimal, Argonaute-driven small RNA biogenesis pathway in which piRNA 5' and 3' ends are directly produced by closely spaced Aub/Ago3-mediated cleavage events. Our data reveal a coherent model for piRNA biogenesis, and should aid the mechanistic dissection of the processes that govern piRNA 3'-end formation.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , RNA, Small Interfering/biosynthesis , RNA, Small Interfering/genetics , Animals , Argonaute Proteins/metabolism , Cytoplasm/metabolism , Drosophila Proteins/deficiency , Drosophila melanogaster/enzymology , Drosophila melanogaster/metabolism , Endoribonucleases/deficiency , Endoribonucleases/metabolism , Exoribonucleases/deficiency , Exoribonucleases/metabolism , Female , Nuclear Proteins/metabolism , Peptide Initiation Factors/metabolism , RNA Processing, Post-Transcriptional , RNA, Guide, Kinetoplastida/metabolism , RNA, Small Interfering/chemistry , RNA, Small Interfering/metabolism , Transcription, GeneticABSTRACT
The repression of transposable elements in eukaryotes often involves their transcriptional silencing via targeted chromatin modifications. In animal gonads, nuclear Argonaute proteins of the PIWI clade complexed with small guide RNAs (piRNAs) serve as sequence specificity determinants in this process. How binding of nuclear PIWI-piRNA complexes to nascent transcripts orchestrates heterochromatin formation and transcriptional silencing is unknown. Here, we characterize CG9754/Silencio as an essential piRNA pathway factor that is required for Piwi-mediated transcriptional silencing in Drosophila. Ectopic targeting of Silencio to RNA or DNA is sufficient to elicit silencing independently of Piwi and known piRNA pathway factors. Instead, Silencio requires the H3K9 methyltransferase Eggless/SetDB1 for its silencing ability. In agreement with this, SetDB1, but not Su(var)3-9, is required for Piwi-mediated transcriptional silencing genome-wide. Due to its interaction with the target-engaged Piwi-piRNA complex, we suggest that Silencio acts as linker between the sequence specificity factor Piwi and the cellular heterochromatin machinery.
Subject(s)
Argonaute Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/physiology , Gene Expression Regulation, Developmental , Heterochromatin/metabolism , Nuclear Proteins/metabolism , RNA, Small Interfering/metabolism , Animals , DNA/metabolism , DNA Transposable Elements/genetics , Drosophila melanogaster/genetics , Female , Gene Silencing , Genome, Insect/genetics , Histone-Lysine N-Methyltransferase , Histones/metabolism , Methylation , Ovary/physiology , Protein Binding , RNA/metabolism , RNA-Binding Proteins , Repressor Proteins/metabolismABSTRACT
PIWI clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound â¼23- to 30-nucleotide (nt) PIWI-interacting RNAs (piRNAs) that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endonuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the interdependencies between these piRNA biogenesis pathways in developing Drosophila ovaries. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcriptsincluding those from piRNA clustersas primary piRNA precursors and defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways.
Subject(s)
DNA Transposable Elements/genetics , Drosophila melanogaster/genetics , Gene Silencing , RNA, Small Interfering/biosynthesis , RNA, Small Interfering/metabolism , Animals , Argonaute Proteins/metabolism , Cell Nucleus/metabolism , Cytoplasm/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Female , Gene Expression Regulation, Developmental , Germ Cells/metabolism , Ovary/metabolism , Peptide Initiation Factors/metabolism , RNA Processing, Post-TranscriptionalABSTRACT
In animal gonads, PIWI-clade Argonaute proteins repress transposons sequence-specifically via bound Piwi-interacting RNAs (piRNAs). These are processed from single-stranded precursor RNAs by largely unknown mechanisms. Here we show that primary piRNA biogenesis is a 3'-directed and phased process that, in the Drosophila germ line, is initiated by secondary piRNA-guided transcript cleavage. Phasing results from consecutive endonucleolytic cleavages catalyzed by Zucchini, implying coupled formation of 3' and 5' ends of flanking piRNAs. Unexpectedly, Zucchini also participates in 3' end formation of secondary piRNAs. Its function can, however, be bypassed by downstream piRNA-guided precursor cleavages coupled to exonucleolytic trimming. Our data uncover an evolutionarily conserved piRNA biogenesis mechanism in which Zucchini plays a central role in defining piRNA 5' and 3' ends.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/enzymology , Endoribonucleases/metabolism , RNA Cleavage , RNA, Guide, Kinetoplastida/metabolism , RNA, Small Interfering/metabolism , Transcription, Genetic , Animals , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Endoribonucleases/genetics , Evolution, Molecular , Female , Germ Cells/enzymology , Male , Mice , Ovary/enzymology , RNA, Small Interfering/biosynthesis , RNA-Binding Proteins/genetics , Testis/enzymology , Uridine/metabolismABSTRACT
Huang et al. (2013) recently reported that chromatin immunoprecipitation sequencing (ChIP-seq) reveals the genome-wide sites of occupancy by Piwi, a piRNA-guided Argonaute protein central to transposon silencing in Drosophila. Their study also reported that loss of Piwi causes widespread rewiring of transcriptional patterns, as evidenced by changes in RNA polymerase II occupancy across the genome. Here we reanalyze their data and report that the underlying deep-sequencing dataset does not support the authors' genome-wide conclusions.