RESUMO
For a decade, mystery has surrounded the mechanisms by which piRNA biogenesis yields distinct size classes of small RNAs within individual PIWI proteins. In this issue of Cell, two studies shed light on this process, identifying conserved PARN-family exonucleases that trim piRNAs to their mature size in silkworms and C. elegans.
RESUMO
The PIWI-interacting RNA (piRNA) pathway is a conserved small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilization. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use noncanonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and poly(A) tails. mRNA processing is important for general mRNA export mediated by nuclear export factor 1 (Nxf1). Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila nuclear export factor family protein Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We found that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters compensate for a lack of canonical features of mature mRNAs to be specifically exported via Nxf3, ensuring proper piRNA production.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Precursores de RNA/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Elementos de DNA Transponíveis/genética , Drosophila/genética , Proteínas de Drosophila/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Ligação a RNA/genéticaRESUMO
The Piwi-interacting RNA (piRNA) pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. piRNA biogenesis requires a specialized machinery that converts long single-stranded precursors into small RNAs of â¼25-nucleotides in length. This process involves factors that operate in two different subcellular compartments: the nuage/Yb body and mitochondria. How these two sites communicate to achieve accurate substrate selection and efficient processing remains unclear. Here, we investigate a previously uncharacterized piRNA biogenesis factor, Daedalus (Daed), that is located on the outer mitochondrial membrane. Daed is essential for Zucchini-mediated piRNA production and the correct localization of the indispensable piRNA biogenesis factor Armitage (Armi). We found that Gasz and Daed interact with each other and likely provide a mitochondrial "anchoring platform" to ensure that Armi is held in place, proximal to Zucchini, during piRNA processing. Our data suggest that Armi initially identifies piRNA precursors in nuage/Yb bodies in a manner that depends on Piwi and then moves to mitochondria to present precursors to the mitochondrial biogenesis machinery. These results represent a significant step in understanding a critical aspect of transposon silencing; namely, how RNAs are chosen to instruct the piRNA machinery in the nature of its silencing targets.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , RNA Helicases/metabolismo , RNA Interferente Pequeno/biossíntese , Animais , Linhagem Celular , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Técnicas de Silenciamento de Genes , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/metabolismoRESUMO
PIWI-interacting RNAs (piRNAs) and their associated PIWI clade Argonaute proteins constitute the core of the piRNA pathway. In gonadal cells, this conserved pathway is crucial for genome defense, and its main function is to silence transposable elements. This is achieved through posttranscriptional and transcriptional gene silencing. Precursors that give rise to piRNAs require specialized transcription and transport machineries because piRNA biogenesis is a cytoplasmic process. The ping-pong cycle, a posttranscriptional silencing mechanism, combines the cleavage-dependent silencing of transposon RNAs with piRNA production. PIWI proteins also function in the nucleus, where they scan for nascent target transcripts with sequence complementarity, instructing transcriptional silencing and deposition of repressive chromatin marks at transposon loci. Although studies have revealed numerous factors that participate in each branch of the piRNA pathway, the precise molecular roles of these factors often remain unclear. In this review, we summarize our current understanding of the mechanisms involved in piRNA biogenesis and function.
Assuntos
Proteínas Argonautas/genética , Elementos de DNA Transponíveis/genética , RNA Interferente Pequeno/genética , Transcrição Gênica , Animais , Drosophila melanogaster/genética , Inativação Gênica , Gônadas/crescimento & desenvolvimento , RNA Interferente Pequeno/biossínteseRESUMO
The FT gene integrates several external and endogenous cues controlling flowering, including information on day length. A complex of the mobile FT protein and the bZIP transcription factor FD in turn has a central role in activating genes that execute the switch from vegetative to reproductive development. Here we reveal that microRNA156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes not only act downstream of FT/FD, but also define a separate endogenous flowering pathway. High levels of miR156 in young plants prevent precocious flowering. A subsequent day length-independent decline in miR156 abundance provides a permissive environment for flowering and is paralleled by a rise in SPL levels. At the shoot apex, FT/FD and SPLs converge on an overlapping set of targets, with SPLs directly activating flower-promoting MADS box genes, providing a molecular substrate for both the redundant activities and the feed-forward action of the miR156/SPL and FT/FD modules in flowering control.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , MicroRNAs/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Meristema/crescimento & desenvolvimento , Transdução de Sinais , TransativadoresRESUMO
Germline genes often become re-expressed in soma-derived human cancers as "cancer/testis antigens" (CTAs), and piRNA (PIWI-interacting RNA) pathway proteins are found among CTAs. However, whether and how the piRNA pathway contributes to oncogenesis in human neoplasms remain poorly understood. We found that oncogenic Ras combined with loss of the Hippo tumor suppressor pathway reactivates a primary piRNA pathway in Drosophila somatic cells coincident with oncogenic transformation. In these cells, Piwi becomes loaded with piRNAs derived from annotated generative loci, which are normally restricted to either the germline or the somatic follicle cells. Negating the pathway leads to increases in the expression of a wide variety of transposons and also altered expression of some protein-coding genes. This correlates with a reduction in the proliferation of the transformed cells in culture, suggesting that, at least in this context, the piRNA pathway may play a functional role in cancer.
Assuntos
Transformação Celular Neoplásica/patologia , Drosophila/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Linhagem Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Células Cultivadas , Elementos de DNA Transponíveis/genética , Drosophila/citologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Ovário/citologia , Transdução de Sinais/genéticaRESUMO
The Drosophila piRNA pathway provides an RNA-based immune system that defends the germline genome against selfish genetic elements. Two interrelated branches of the piRNA system exist: somatic cells that support oogenesis only employ Piwi, whereas germ cells utilize a more elaborate pathway centered on the three gonad-specific Argonaute proteins (Piwi, Aubergine, and Argonaute 3). While several key factors of each branch have been identified, our current knowledge is insufficient to explain the complex workings of the piRNA machinery. Here, we report a reverse genetic screen spanning the ovarian transcriptome in an attempt to uncover the full repertoire of genes required for piRNA-mediated transposon silencing in the female germline. Our screen reveals key factors of piRNA-mediated transposon silencing, including the piRNA biogenesis factors CG2183 (GASZ) and Deadlock. Our data uncover a previously unanticipated set of factors preferentially required for repression of different transposon types.
Assuntos
Elementos de DNA Transponíveis , Drosophila melanogaster/genética , Perfilação da Expressão Gênica/métodos , Ovário/fisiologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Inativação Gênica , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , RNA Interferente Pequeno/genética , Reprodutibilidade dos TestesRESUMO
The PIWI-interacting RNA (piRNA) pathway is a conserved defense mechanism that protects the genetic information of animal germ cells from the deleterious effects of molecular parasites, such as transposons. Discovered nearly a decade ago, this small RNA silencing system comprises PIWI-clade Argonaute proteins and their associated RNA-binding partners, the piRNAs. In this review, we highlight recent work that has advanced our understanding of how piRNAs preserve genome integrity across generations. We discuss the mechanism of piRNA biogenesis, give an overview of common themes as well as differences in piRNA-mediated silencing between species, and end by highlighting known and emerging functions of piRNAs.
Assuntos
Proteínas Argonautas/genética , Drosophila melanogaster/genética , Inativação Gênica , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Animais , Proteínas Argonautas/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transcrição GênicaRESUMO
Adult stem cells support tissue homeostasis and repair throughout the life of an individual. During ageing, numerous intrinsic and extrinsic changes occur that result in altered stem-cell behaviour and reduced tissue maintenance and regeneration. In the Drosophila testis, ageing results in a marked decrease in the self-renewal factor Unpaired (Upd), leading to a concomitant loss of germline stem cells. Here we demonstrate that IGF-II messenger RNA binding protein (Imp) counteracts endogenous small interfering RNAs to stabilize upd (also known as os) RNA. However, similar to upd, Imp expression decreases in the hub cells of older males, which is due to the targeting of Imp by the heterochronic microRNA let-7. In the absence of Imp, upd mRNA therefore becomes unprotected and susceptible to degradation. Understanding the mechanistic basis for ageing-related changes in stem-cell behaviour will lead to the development of strategies to treat age-onset diseases and facilitate stem-cell-based therapies in older individuals.
Assuntos
Senescência Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , MicroRNAs/genética , Proteínas de Ligação a RNA/metabolismo , Nicho de Células-Tronco/fisiologia , Testículo/citologia , Animais , Proteínas Argonautas/metabolismo , Sequência de Bases , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Masculino , Especificidade de Órgãos , RNA Helicases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/antagonistas & inibidores , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Ribonuclease III/metabolismo , Nicho de Células-Tronco/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Drosophila melanogaster/metabolismo , RNA Interferente Pequeno/biossíntese , Animais , Células Cultivadas , Elementos de DNA Transponíveis/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Feminino , Mutação , RNA Helicases/genética , RNA Helicases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Small RNAs directly or indirectly impact nearly every biological process in eukaryotic cells. To perform their myriad roles, not only must precise small RNA species be generated, but they must also be loaded into specific effector complexes called RNA-induced silencing complexes (RISCs). Argonaute proteins form the core of RISCs and different members of this large family have specific expression patterns, protein binding partners and biochemical capabilities. In this Review, we explore the mechanisms that pair specific small RNA strands with their partner proteins, with an eye towards the substantial progress that has been recently made in understanding the sorting of the major small RNA classes - microRNAs (miRNAs) and small interfering RNAs (siRNAs) - in plants and animals.
Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , MicroRNAs/metabolismo , Animais , Dípteros/metabolismo , Humanos , Plantas/metabolismo , Transporte de RNA , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/metabolismoRESUMO
Drosophila Argonaute-1 and Argonaute-2 differ in function and small RNA content. AGO2 binds to siRNAs, whereas AGO1 is almost exclusively occupied by microRNAs. MicroRNA duplexes are intrinsically asymmetric, with one strand, the miR strand, preferentially entering AGO1 to recognize and regulate the expression of target mRNAs. The other strand, miR*, has been viewed as a byproduct of microRNA biogenesis. Here, we show that miR*s are often loaded as functional species into AGO2. This indicates that each microRNA precursor can potentially produce two mature small RNA strands that are differentially sorted within the RNAi pathway. miR* biogenesis depends upon the canonical microRNA pathway, but loading into AGO2 is mediated by factors traditionally dedicated to siRNAs. By inferring and validating hierarchical rules that predict differential AGO loading, we find that intrinsic determinants, including structural and thermodynamic properties of the processed duplex, regulate the fate of each RNA strand within the RNAi pathway.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Drosophila/metabolismo , MicroRNAs/metabolismo , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Regiões 3' não Traduzidas , Animais , Proteínas de Arabidopsis/genética , Proteínas Argonautas , Pareamento de Bases , Northern Blotting , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Imunoprecipitação , MicroRNAs/química , MicroRNAs/genética , Modelos Biológicos , Conformação de Ácido Nucleico , Ligação Proteica , Interferência de RNA , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Complexo de Inativação Induzido por RNA/genética , TermodinâmicaRESUMO
Eukaryotic cells express several classes of small RNAs that regulate gene expression and ensure genome maintenance. Endogenous siRNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs) mainly control gene and transposon expression in the germline, while microRNAs (miRNAs) generally function in post-transcriptional gene silencing in both somatic and germline cells. To provide an evolutionary and developmental perspective on small RNA pathways in nematodes, we identified and characterized known and novel small RNA classes through gametogenesis and embryo development in the parasitic nematode Ascaris suum and compared them with known small RNAs of Caenorhabditis elegans. piRNAs, Piwi-clade Argonautes, and other proteins associated with the piRNA pathway have been lost in Ascaris. miRNAs are synthesized immediately after fertilization in utero, before pronuclear fusion, and before the first cleavage of the zygote. This is the earliest expression of small RNAs ever described at a developmental stage long thought to be transcriptionally quiescent. A comparison of the two classes of Ascaris endo-siRNAs, 22G-RNAs and 26G-RNAs, to those in C. elegans, suggests great diversification and plasticity in the use of small RNA pathways during spermatogenesis in different nematodes. Our data reveal conserved characteristics of nematode small RNAs as well as features unique to Ascaris that illustrate significant flexibility in the use of small RNAs pathways, some of which are likely an adaptation to Ascaris' life cycle and parasitism. The transcriptome assembly has been submitted to NCBI Transcriptome Shotgun Assembly Sequence Database(http://www.ncbi.nlm.nih.gov/genbank/TSA.html) under accession numbers JI163767JI182837 and JI210738JI257410.
Assuntos
Ascaris/genética , Sequenciamento de Nucleotídeos em Larga Escala , RNA Interferente Pequeno/metabolismo , Análise de Sequência de RNA , Animais , Ascaris/metabolismo , Sequência de Bases , Análise por Conglomerados , Sequência Conservada , Desenvolvimento Embrionário/genética , Gametogênese/genética , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Variação Genética , Sequências Repetidas Invertidas , Dados de Sequência Molecular , RNA Interferente Pequeno/genética , Espermatogênese/genética , Zigoto/metabolismoRESUMO
Existing transgenic RNAi resources in Drosophila melanogaster based on long double-stranded hairpin RNAs are powerful tools for functional studies, but they are ineffective in gene knockdown during oogenesis, an important model system for the study of many biological questions. We show that shRNAs, modeled on an endogenous microRNA, are extremely effective at silencing gene expression during oogenesis. We also describe our progress toward building a genome-wide shRNA resource.
Assuntos
Drosophila melanogaster/genética , Genoma de Inseto , Interferência de RNA , Animais , Animais Geneticamente Modificados , Sequência de Bases , Primers do DNA/genética , Feminino , Técnicas de Silenciamento de Genes , Técnicas Genéticas , Vetores Genéticos , MicroRNAs/genética , Oogênese/genética , RNA Interferente Pequeno/genéticaRESUMO
In animals, the piRNA pathway preserves the integrity of gametic genomes, guarding them against the activity of mobile genetic elements. This innate immune mechanism relies on distinct genomic loci, termed piRNA clusters, to provide a molecular definition of transposons, enabling their discrimination from genes. piRNA clusters give rise to long, single-stranded precursors, which are processed into primary piRNAs through an unknown mechanism. These can engage in an adaptive amplification loop, the ping-pong cycle, to optimize the content of small RNA populations via the generation of secondary piRNAs. Many proteins have been ascribed functions in either primary biogenesis or the ping-pong cycle, though for the most part the molecular functions of proteins implicated in these pathways remain obscure. Here, we link shutdown (shu), a gene previously shown to be required for fertility in Drosophila, to the piRNA pathway. Analysis of knockdown phenotypes in both the germline and somatic compartments of the ovary demonstrate important roles for shutdown in both primary biogenesis and the ping-pong cycle. shutdown is a member of the FKBP family of immunophilins. Shu contains domains implicated in peptidyl-prolyl cis-trans isomerase activity and in the binding of HSP90-family chaperones, though the relevance of these domains to piRNA biogenesis is unknown.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Fertilidade/genética , Inativação Gênica , Ovário/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Feminino , Imunofluorescência , Masculino , Ovário/citologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Retroelementos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
In animals a discrete class of small RNAs, the piwi-interacting RNAs (piRNAs), guard germ cell genomes against the activity of mobile genetic elements. piRNAs are generated, via an unknown mechanism, from apparently single-stranded precursors that arise from discrete genomic loci, termed piRNA clusters. Presently, little is known about the signals that distinguish a locus as a source of piRNAs. It is also unknown how individual piRNAs are selected from long precursor transcripts. To address these questions, we inserted new artificial sequence information into piRNA clusters and introduced these marked clusters as transgenes into heterologous genomic positions in mice and flies. Profiling of piRNA from transgenic animals demonstrated that artificial sequences were incorporated into the piRNA repertoire. Transgenic piRNA clusters are functional in non-native genomic contexts in both mice and flies, indicating that the signals that define piRNA generative loci must lie within the clusters themselves rather than being implicit in their genomic position. Comparison of transgenic animals that carry insertions of the same artificial sequence into different ectopic piRNA-generating loci showed that both local and long-range sequence environments inform the generation of individual piRNAs from precursor transcripts.
Assuntos
Drosophila melanogaster/metabolismo , RNA Interferente Pequeno/biossíntese , Animais , Drosophila melanogaster/genética , Camundongos , Camundongos Transgênicos , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genéticaRESUMO
Drosophila endogenous small RNAs are categorized according to their mechanisms of biogenesis and the Argonaute protein to which they bind. MicroRNAs are a class of ubiquitously expressed RNAs of approximately 22 nucleotides in length, which arise from structured precursors through the action of Drosha-Pasha and Dicer-1-Loquacious complexes. These join Argonaute-1 to regulate gene expression. A second endogenous small RNA class, the Piwi-interacting RNAs, bind Piwi proteins and suppress transposons. Piwi-interacting RNAs are restricted to the gonad, and at least a subset of these arises by Piwi-catalysed cleavage of single-stranded RNAs. Here we show that Drosophila generates a third small RNA class, endogenous small interfering RNAs, in both gonadal and somatic tissues. Production of these RNAs requires Dicer-2, but a subset depends preferentially on Loquacious rather than the canonical Dicer-2 partner, R2D2 (ref. 14). Endogenous small interfering RNAs arise both from convergent transcription units and from structured genomic loci in a tissue-specific fashion. They predominantly join Argonaute-2 and have the capacity, as a class, to target both protein-coding genes and mobile elements. These observations expand the repertoire of small RNAs in Drosophila, adding a class that blurs distinctions based on known biogenesis mechanisms and functional roles.
Assuntos
Drosophila melanogaster/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonautas , Linhagem Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Drosophila melanogaster/metabolismo , Ligação Proteica , RNA Helicases/metabolismo , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Retroelementos/genética , Ribonuclease IIIRESUMO
PIWI-interacting RNAs (piRNAs) are small RNAs required to recognize and silence transposable elements. The 5' ends of mature piRNAs are defined through cleavage of long precursor transcripts, primarily by Zucchini (Zuc). Zuc-dependent cleavage typically occurs immediately upstream of a uridine. However, Zuc lacks sequence preference in vitro, pointing towards additional unknown specificity factors. Here, we examine murine piRNAs and reveal a strong and specific enrichment of three sequences (UAA, UAG, UGA)-corresponding to stop codons-at piRNA 5' ends. Stop codon sequences are also enriched immediately after piRNA processing intermediates, reflecting their Zuc-dependent tail-to-head arrangement. Further analyses reveal that a Zuc in vivo cleavage preference at four sequences (UAA, UAG, UGA, UAC) promotes 5' end stop codons. This observation is conserved across mammals and possibly further. Our work provides new insights into Zuc-dependent cleavage and may point to a previously unrecognized connection between piRNA biogenesis and the translational machinery.
Assuntos
Proteínas de Drosophila , Animais , Códon de Terminação/genética , Proteínas de Drosophila/genética , Endorribonucleases/genética , Mamíferos/genética , Camundongos , RNA Interferente Pequeno/genéticaRESUMO
Drosophila melanogaster expresses three classes of small RNAs, which are classified according to their mechanisms of biogenesis. MicroRNAs are approximately 22-23 nucleotides (nt), ubiquitously expressed small RNAs that are sequentially processed from hairpin-like precursors by Drosha/Pasha and Dcr-1/Loquacious complexes. MicroRNAs usually associate with AGO1 and regulate the expression of protein-coding genes. Piwi-interacting RNAs (piRNAs) of approximately 24-28 nt associate with Piwi-family proteins and can arise from single-stranded precursors. piRNAs function in transposon silencing and are mainly restricted to gonadal tissues. Endo-siRNAs are found in both germline and somatic tissues. These approximately 21-nt RNAs are produced by a distinct Dicer, Dcr-2, and do not depend on Drosha/Pasha complexes. They predominantly bind to AGO2 and target both mobile elements and protein-coding genes. Surprisingly, a subset of endo-siRNAs strongly depend for their production on the dsRNA-binding protein Loquacious (Loqs), thought generally to be a partner for Dcr-1 and a cofactor for miRNA biogenesis. Endo-siRNA production depends on a specific Loqs isoform, Loqs-PD, which is distinct from the one, Loqs-PB, required for the production of microRNAs. Paralleling their roles in the biogenesis of distinct small RNA classes, Loqs-PD and Loqs-PB bind to different Dicer proteins, with Dcr-1/Loqs-PB complexes and Dcr-2/Loqs-PD complexes driving microRNA and endo-siRNA biogenesis, respectively.
Assuntos
Drosophila melanogaster/genética , RNA Interferente Pequeno/metabolismo , Animais , Elementos de DNA Transponíveis , Inativação Gênica , Células GerminativasRESUMO
The nuclear pore complex (NPC) is the principal gateway between nucleus and cytoplasm that enables exchange of macromolecular cargo. Composed of multiple copies of ~30 different nucleoporins (Nups), the NPC acts as a selective portal, interacting with factors which individually license passage of specific cargo classes. Here we show that two Nups of the inner channel, Nup54 and Nup58, are essential for transposon silencing via the PIWI-interacting RNA (piRNA) pathway in the Drosophila ovary. In ovarian follicle cells, loss of Nup54 and Nup58 results in compromised piRNA biogenesis exclusively from the flamenco locus, whereas knockdowns of other NPC subunits have widespread consequences. This provides evidence that some Nups can acquire specialised roles in tissue-specific contexts. Our findings consolidate the idea that the NPC has functions beyond simply constituting a barrier to nuclear/cytoplasmic exchange as genomic loci subjected to strong selective pressure can exploit NPC subunits to facilitate their expression.
Transposons are genetic sequences, which, when active, can move around the genome and insert themselves into new locations. This can potentially disrupt the information required for cells to work properly: in reproductive organs, for example, transposon activity can lead to infertility. Many organisms therefore have cellular systems that keep transposons in check. Animal cells comprise two main compartments: the nucleus, which contains the genetic information, and the cytosol, where most chemical reactions necessary for life take place. Molecules continually move between nucleus and cytosol, much as people go in and out of a busy train station. The connecting 'doors' between the two compartments are called Nuclear Pore Complexes (NPCs), and their job is to ensure that each molecule passing through reaches its correct destination. Recent research shows that the individual proteins making up NPCs (called nucleoporins) may play other roles within the cell. In particular, genetic studies in fruit flies suggested that some nucleoporins help to control transposon activity within the ovary but how they did this was still unclear. Munafò et al. therefore set out to determine if the nucleoporins were indeed actively silencing the transposons, or if this was just a side effect of altered nuclear-cytosolic transport. Experiments using cells grown from fruit fly ovaries revealed that depleting two specific nucleoporins, Nup54 and Nup58, re-activated transposons with minimal effects on most genes or the overall health of the cells. This suggests that Nup54 and Nup58 play a direct role in transposon silencing. Further, detailed analysis of gene expression in Nup54- and Nup58-lacking cells revealed that the product of one gene, flamenco, was indeed affected. Normally, flamenco acts as a 'master switch' to turn off transposons. Without Nup54 and Nup58, the molecule encoded by flamenco could not reach its dedicated location in the cytosol, and thus could not carry out its task. These results show that, far from being mere 'doorkeepers' for the nucleus, nucleoporins play important roles adapted to individual tissues in the body. Further research will help determine if the same is true for other organisms, and if these mechanisms can help understand human diseases.