RESUMO
In animals, PIWI-interacting RNAs (piRNAs) direct PIWI proteins to silence complementary targets such as transposons. In Drosophila and other species with a maternally specified germline, piRNAs deposited in the egg initiate piRNA biogenesis in the progeny. However, Y chromosome loci cannot participate in such a chain of intergenerational inheritance. How then can the biogenesis of Y-linked piRNAs be initiated? Here, using Suppressor of Stellate (Su(Ste)), a Y-linked Drosophila melanogaster piRNA locus as a model, we show that Su(Ste) piRNAs are made in the early male germline via 5'-to-3' phased piRNA biogenesis initiated by maternally deposited 1360/Hoppel transposon piRNAs. Notably, deposition of Su(Ste) piRNAs from XXY mothers obviates the need for phased piRNA biogenesis in sons. Together, our study uncovers a developmentally programmed, intergenerational mechanism that allows fly mothers to protect their sons using a Y-linked piRNA locus.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , RNA de Interação com Piwi , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Argonautas/genéticaRESUMO
In eukaryotes, small RNA guides, such as small interfering RNAs and microRNAs, direct AGO-clade Argonaute proteins to regulate gene expression and defend the genome against external threats. Only animals make a second clade of Argonaute proteins: PIWI proteins. PIWI proteins use PIWI-interacting RNAs (piRNAs) to repress complementary transposon transcripts1,2. In theory, transposons could evade silencing through target site mutations that reduce piRNA complementarity. Here we report that, unlike AGO proteins, PIWI proteins efficiently cleave transcripts that are only partially paired to their piRNA guides. Examination of target binding and cleavage by mouse and sponge PIWI proteins revealed that PIWI slicing tolerates mismatches to any target nucleotide, including those flanking the scissile phosphate. Even canonical seed pairing is dispensable for PIWI binding or cleavage, unlike plant and animal AGOs, which require uninterrupted target pairing from the seed to the nucleotides past the scissile bond3,4. PIWI proteins are therefore better equipped than AGO proteins to target newly acquired or rapidly diverging endogenous transposons without recourse to new small RNA guides. Conversely, the minimum requirements for PIWI slicing are sufficient to avoid inadvertent silencing of host RNAs. Our results demonstrate the biological advantage of PIWI over AGO proteins in defending the genome against transposons and suggest an explanation for why the piRNA pathway was retained in animal evolution.
Assuntos
Proteínas Argonautas , Elementos de DNA Transponíveis , Inativação Gênica , RNA de Interação com Piwi , Animais , Camundongos , Proteínas Argonautas/classificação , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Elementos de DNA Transponíveis/genética , RNA de Interação com Piwi/genética , RNA de Interação com Piwi/metabolismo , Evolução Molecular , Fosfatos/metabolismo , Especificidade por SubstratoRESUMO
Binding affinity quantitatively describes the strength of a molecular interaction and is reported by the equilibrium dissociation constant (KD). Here, we present a protocol to measure KD of mammalian microRNA-loaded Argonaute2 protein by double filter binding. We describe steps for radiolabeling target RNA, measuring concentration of binding-competent protein, setting up binding reactions, separating protein-bound RNA from protein-unbound RNA, preparing library for Illumina sequencing, and performing data analysis. Our protocol is easily applied to other RNA- or DNA-binding proteins. For complete details on the use and execution of this protocol, please refer to Jouravleva et al.1.
RESUMO
In brief: The testis-specific transcription factor, TCFL5, expressed in pachytene spermatocytes regulates the meiotic gene expression program in collaboration with the transcription factor A-MYB. Abstract: In male mice, the transcription factors STRA8 and MEISON initiate meiosis I. We report that STRA8/MEISON activates the transcription factors A-MYB and TCFL5, which together reprogram gene expression after spermatogonia enter into meiosis. TCFL5 promotes the transcription of genes required for meiosis, mRNA turnover, miR-34/449 production, meiotic exit, and spermiogenesis. This transcriptional architecture is conserved in rhesus macaque, suggesting TCFL5 plays a central role in meiosis and spermiogenesis in placental mammals. Tcfl5em1/em1 mutants are sterile, and spermatogenesis arrests at the mid- or late-pachytene stage of meiosis. Moreover, Tcfl5+/em1 mutants produce fewer motile sperm.
Assuntos
Placenta , Fatores de Transcrição , Animais , Feminino , Masculino , Camundongos , Gravidez , Macaca mulatta/metabolismo , Mamíferos/metabolismo , Meiose , Placenta/metabolismo , Sêmen/metabolismo , Espermatócitos/metabolismo , Espermatogênese/genética , Testículo/metabolismo , Fatores de Transcrição/metabolismoRESUMO
In male mice, the transcription factor A MYB initiates the transcription of pachytene piRNA genes during meiosis. Here, we report that A MYB activates the transcription factor Tcfl5 produced in pachytene spermatocytes. Subsequently, A MYB and TCFL5 reciprocally reinforce their own transcription to establish a positive feedback circuit that triggers pachytene piRNA production. TCFL5 regulates the expression of genes required for piRNA maturation and promotes transcription of evolutionarily young pachytene piRNA genes, whereas A-MYB activates the transcription of older pachytene piRNA genes. Intriguingly, pachytene piRNAs from TCFL5-dependent young loci initiates the production of piRNAs from A-MYB-dependent older loci ensuring the self-propagation of pachytene piRNAs. A MYB and TCFL5 act via a set of incoherent feedforward loops that drive regulation of gene expression by pachytene piRNAs during spermatogenesis. This regulatory architecture is conserved in rhesus macaque, suggesting that it was present in the last common ancestor of placental mammals.
RESUMO
In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding domain (dsRBD) proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM structures of Drosophila Dicer-1 that show how Dicer-1 and its partner LoqsPB cooperate (1) before binding pre-miRNA, (2) after binding and in a catalytically competent state, (3) after nicking one arm of the pre-miRNA, and (4) following complete dicing and initial product release. Our reconstructions suggest that pre-miRNA binds a rare, open conformation of the Dicer1â LoqsPB heterodimer. The Dicer-1 dsRBD and three LoqsPB dsRBDs form a tight belt around the pre-miRNA, distorting the RNA helix to place the scissile phosphodiester bonds in the RNase III active sites. Pre-miRNA cleavage shifts the dsRBDs and partially closes Dicer-1, which may promote product release. Our data suggest a model for how the Dicer1â LoqsPB complex affects a complete cycle of pre-miRNA recognition, stepwise endonuclease cleavage, and product release.
Assuntos
Proteínas de Drosophila , MicroRNAs , Animais , Ribonuclease III/genética , Ribonuclease III/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Ligação a RNA/metabolismo , Drosophila/genética , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. In this study, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified traffic light reporter (TLR) system (TLR-multi-Cas variant 1 [MCV1]) that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knockins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knockins in mammalian cell lines.
Assuntos
DNA de Cadeia Simples , Edição de Genes , Humanos , Sistemas CRISPR-Cas/genética , DNA/metabolismo , DNA de Cadeia Simples/genética , Endonucleases/genética , Edição de Genes/métodos , Células HEK293 , Células K562RESUMO
Argonaute proteins use nucleic acid guides to find and bind specific DNA or RNA target sequences. Argonaute proteins have diverse biological functions and many retain their ancestral endoribonuclease activity, cleaving the phosphodiester bond between target nucleotides t10 and t11. In animals, the PIWI proteins-a specialized class of Argonaute proteins-use 21-35 nucleotide 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 catalysed by PIWI proteins. Spermatogenesis in mice and other placental mammals requires three distinct, developmentally regulated PIWI proteins: MIWI (PIWIL1), MILI (PIWIL2) and MIWI22-4 (PIWIL4). The piRNA-guided endoribonuclease activities of MIWI and MILI are essential for the production of functional sperm5,6. piRNA-directed silencing in mice and insects also requires GTSF1, a PIWI-associated protein of unknown function7-12. Here we report that GTSF1 potentiates the weak, intrinsic, piRNA-directed RNA cleavage activities of PIWI proteins, transforming them into efficient endoribonucleases. GTSF1 is thus an example of an auxiliary protein that potentiates the catalytic activity of an Argonaute protein.
Assuntos
Proteínas Argonautas , Peptídeos e Proteínas de Sinalização Intracelular , Clivagem do RNA , RNA Interferente Pequeno , Animais , Proteínas Argonautas/classificação , Proteínas Argonautas/metabolismo , Biocatálise , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , RNA Interferente Pequeno/metabolismoRESUMO
CRISPR-Cas technology has revolutionized genome editing. Its broad and fast-growing application in biomedical research and therapeutics has led to increased demand for guide RNAs. The synthesis of chemically modified single-guide RNAs (sgRNAs) containing >100 nucleotides remains a bottleneck. Here we report the development of a tetrazine ligation method for the preparation of sgRNAs. A tetrazine moiety on the 3'-end of the crRNA and a norbornene moiety on the 5'-end of the tracrRNA enable successful ligation between crRNA and tracrRNA to form sgRNA under mild conditions. Tetrazine-ligated sgRNAs allow efficient genome editing of reporter and endogenous loci in human cells. High-efficiency editing requires structural optimization of the linker.
Assuntos
Edição de Genes , RNA Guia de Cinetoplastídeos , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Humanos , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genéticaRESUMO
RNA Bind-n-Seq (RBNS) is a cost-effective, high-throughput method capable of identifying the sequence preferences of RNA-binding proteins and of qualitatively defining relative dissociation constants. Although RBNS is often described as an unbiased method, several factors may influence the outcome of the analysis. Here, we discuss these biases and present an analytical strategy to estimate absolute binding affinities from RBNS data, extend RBNS to kinetic studies, and develop a framework to compute relative association and dissociation rate constants. As proof of principle, we measured the equilibrium binding properties of mammalian Argonaute2 (AGO2) guided by eight microRNAs (miRNAs) and kinetic parameters for let-7a. The miRNA-binding site repertoires, dissociation constants, and kinetic parameters calculated from RBNS data using our methods correlate well with values measured by traditional ensemble and single-molecule approaches. Our data provide additional quantitative measurements for Argonaute-bound miRNA binding that should facilitate development of quantitative targeting rules for individual miRNAs.
Assuntos
MicroRNAs , Animais , Cinética , MicroRNAs/química , Sítios de Ligação , Proteínas de Ligação a RNA/genética , Termodinâmica , Mamíferos/genéticaRESUMO
Argonautes are nucleic acid-guided proteins that perform numerous cellular functions across all domains of life. Little is known about how distinct evolutionary pressures have shaped each Argonaute's biophysical properties. We applied high-throughput biochemistry to characterize how Thermus thermophilus Argonaute (TtAgo), a DNA-guided DNA endonuclease, finds, binds, and cleaves its targets. We found that TtAgo uses biophysical adaptations similar to those of eukaryotic Argonautes for rapid association but requires more extensive complementarity to achieve high-affinity target binding. Using these data, we constructed models for TtAgo association rates and equilibrium binding affinities that estimate the nucleic acid- and protein-mediated components of the target interaction energies. Finally, we showed that TtAgo cleavage rates vary widely based on the DNA guide, suggesting that only a subset of guides cleaves targets on physiologically relevant timescales.
Assuntos
Proteínas Argonautas , Thermus thermophilus , Proteínas Argonautas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA/genética , Endonucleases/metabolismo , Thermus thermophilus/genéticaRESUMO
In animals, PIWI-interacting RNAs (piRNAs) silence transposons, fight viral infections, and regulate gene expression. piRNA biogenesis concludes with 3' terminal trimming and 2'-O-methylation. Both trimming and methylation influence piRNA stability. Our biochemical data show that multiple mechanisms destabilize unmethylated mouse piRNAs, depending on whether the piRNA 5' or 3' sequence is complementary to a trigger RNA. Unlike target-directed degradation of microRNAs, complementarity-dependent destabilization of piRNAs in mice and flies is blocked by 3' terminal 2'-O-methylation and does not require base pairing to both the piRNA seed and the 3' sequence. In flies, 2'-O-methylation also protects small interfering RNAs (siRNAs) from complementarity-dependent destruction. By contrast, pre-piRNA trimming protects mouse piRNAs from a degradation pathway unaffected by trigger complementarity. In testis lysate and in vivo, internal or 3' terminal uridine- or guanine-rich tracts accelerate pre-piRNA decay. Loss of both trimming and 2'-O-methylation causes the mouse piRNA pathway to collapse, demonstrating that these modifications collaborate to stabilize piRNAs.
Assuntos
Proteínas Argonautas/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Separação Celular , Drosophila melanogaster , Feminino , Citometria de Fluxo , Expressão Gênica , Inativação Gênica , Técnicas Genéticas , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Processamento de Proteína Pós-Traducional , RNA de Cadeia Dupla , Espermatócitos/metabolismo , Espermatogônias/metabolismo , Testículo/metabolismoRESUMO
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.
Assuntos
MicroRNAs , Ubiquitina , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , MicroRNAs/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
In the male germ cells of placental mammals, 26-30-nt-long PIWI-interacting RNAs (piRNAs) emerge when spermatocytes enter the pachytene phase of meiosis. In mice, pachytene piRNAs derive from ~100 discrete autosomal loci that produce canonical RNA polymerase II transcripts. These piRNA clusters bear 5' caps and 3' poly(A) tails, and often contain introns that are removed before nuclear export and processing into piRNAs. What marks pachytene piRNA clusters to produce piRNAs, and what confines their expression to the germline? We report that an unusually long first exon (≥ 10 kb) or a long, unspliced transcript correlates with germline-specific transcription and piRNA production. Our integrative analysis of transcriptome, piRNA, and epigenome datasets across multiple species reveals that a long first exon is an evolutionarily conserved feature of pachytene piRNA clusters. Furthermore, a highly methylated promoter, often containing a low or intermediate level of CG dinucleotides, correlates with germline expression and somatic silencing of pachytene piRNA clusters. Pachytene piRNA precursor transcripts bind THOC1 and THOC2, THO complex subunits known to promote transcriptional elongation and mRNA nuclear export. Together, these features may explain why the major sources of pachytene piRNA clusters specifically generate these unique small RNAs in the male germline of placental mammals.
Assuntos
Epigênese Genética , Éxons/genética , Mamíferos/genética , Estágio Paquíteno/genética , RNA Interferente Pequeno/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Acetilação , Animais , Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Histonas/metabolismo , Íntrons/genética , Masculino , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas/genética , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/genética , Testículo/metabolismo , Transcrição GênicaRESUMO
In many eukaryotes, Argonaute proteins, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15- to 18-nt DNA guides derived from the chromosomal region where replication terminates and associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of gyrase and TtAgo activity slows growth and produces long sausage-like filaments in which the individual bacteria are linked by DNA. Finally, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication.
Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Bactérias/metabolismo , DNA Girase/metabolismo , Replicação do DNA/genética , DNA/metabolismo , Thermus thermophilus/metabolismo , Proteínas Argonautas/genética , Proteínas de Bactérias/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cromossomos/metabolismo , Ciprofloxacina/farmacologia , DNA/genética , Replicação do DNA/efeitos dos fármacos , Endonucleases/metabolismo , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Proteínas Recombinantes , Recombinação Genética/efeitos dos fármacos , Recombinação Genética/genética , Imagem Individual de Molécula , Espectrometria de Massas em Tandem , Thermus thermophilus/genética , Thermus thermophilus/crescimento & desenvolvimento , Thermus thermophilus/ultraestrutura , Inibidores da Topoisomerase II/farmacologiaRESUMO
Pachytene PIWI-interacting RNAs (piRNAs), which comprise >80% of small RNAs in the adult mouse testis, have been proposed to bind and regulate target RNAs like microRNAs, cleave targets like short interfering RNAs or lack biological function altogether. Although piRNA pathway protein mutants are male sterile, no biological function has been identified for any mammalian piRNA-producing locus. Here, we report that males lacking piRNAs from a conserved mouse pachytene piRNA locus on chromosome 6 (pi6) produce sperm with defects in capacitation and egg fertilization. Moreover, heterozygous embryos sired by pi6-/- fathers show reduced viability in utero. Molecular analyses suggest that pi6 piRNAs repress gene expression by cleaving messenger RNAs encoding proteins required for sperm function. pi6 also participates in a network of piRNA-piRNA precursor interactions that initiate piRNA production from a second piRNA locus on chromosome 10, as well as pi6 itself. Our data establish a direct role for pachytene piRNAs in spermiogenesis and embryo viability.
Assuntos
RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Espermatogênese/genética , Animais , Evolução Biológica , Núcleo Celular , Desenvolvimento Embrionário , Feminino , Fertilidade , Deleção de Genes , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Estágio Paquíteno/genética , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Capacitação Espermática/genética , Capacitação Espermática/fisiologia , Interações Espermatozoide-Óvulo/fisiologiaRESUMO
In the fetal mouse testis, PIWI-interacting RNAs (piRNAs) guide PIWI proteins to silence transposons but, after birth, most post-pubertal pachytene piRNAs map to the genome uniquely and are thought to regulate genes required for male fertility. In the human male, the developmental classes, precise genomic origins and transcriptional regulation of postnatal piRNAs remain undefined. Here, we demarcate the genes and transcripts that produce postnatal piRNAs in human juvenile and adult testes. As in the mouse, human A-MYB drives transcription of both pachytene piRNA precursor transcripts and messenger RNAs encoding piRNA biogenesis factors. Although human piRNA genes are syntenic to those in other placental mammals, their sequences are poorly conserved. In fact, pachytene piRNA loci are rapidly diverging even among modern humans. Our findings suggest that, during mammalian evolution, pachytene piRNA genes are under few selective constraints. We speculate that pachytene piRNA diversity may provide a hitherto unrecognized driver of reproductive isolation.
Assuntos
Genoma , Testículo , Adolescente , Animais , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Gravidez , RNA Mensageiro , RNA Interferente PequenoRESUMO
Short hairpin RNAs that are delivered by recombinant adeno-associated virus (rAAV) have the potential to elicit long-term RNAi therapy for human disease. However, the discovery that short hairpin sequences can cause truncation of the rAAV genome calls into question the efficiency and gene-silencing specificity of this strategy in humans. Here, we report that embedding the guide strand of a small silencing RNA into an artificial microRNA (miRNA) scaffold derived from mouse miRNA-33 ensures rAAV genomic integrity and reduces off-targeting by 10-fold, while maintaining effective in vivo target gene repression in mice.