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1.
Cell Rep ; 42(8): 112859, 2023 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-37505984

RESUMO

Biomolecular condensates have been shown to interact in vivo, yet it is unclear whether these interactions are functionally meaningful. Here, we demonstrate that cooperativity between two distinct condensates-germ granules and P bodies-is required for transgenerational gene silencing in C. elegans. We find that P bodies form a coating around perinuclear germ granules and that P body components CGH-1/DDX6 and CAR-1/LSM14 are required for germ granules to organize into sub-compartments and concentrate small RNA silencing factors. Functionally, while the P body mutant cgh-1 is competent to initially trigger gene silencing, it is unable to propagate the silencing to subsequent generations. Mechanistically, we trace this loss of transgenerational silencing to defects in amplifying secondary small RNAs and the stability of WAGO-4 Argonaute, both known carriers of gene silencing memories. Together, these data reveal that cooperation between condensates results in an emergent capability of germ cells to establish heritable memory.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , RNA Interferente Pequeno/genética , Inativação Gênica , Interferência de RNA , Células Germinativas/metabolismo , RNA Nucleotidiltransferases/genética
2.
Genetics ; 224(4)2023 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-37210214

RESUMO

Metazoans guard their germlines against transposons and other foreign transcripts with PIWI-interacting RNAs (piRNAs). Due to the robust heritability of the silencing initiated by piRNAs in Caenorhabditis elegans (C. elegans), previous screens using C. elegans were strongly biased to uncover members of this pathway in the maintenance process but not in the initiation process. To identify novel piRNA pathway members, we have utilized a sensitized reporter strain which detects defects in initiation, amplification, or regulation of piRNA silencing. Using our reporter, we have identified Integrator complex subunits, nuclear pore components, protein import components, and pre-mRNA splicing factors as essential for piRNA-mediated gene silencing. We found the small nuclear processing cellular machine termed the Integrator complex is required for both type I and type II piRNA production. Notably, we identified a role for nuclear pore and nucleolar components NPP-1/Nup54, NPP-6/Nup160, NPP-7/Nup153, and FIB-1 in promoting the perinuclear localization of anti-silencing CSR-1 Argonaute, as well as a role for Importin factor IMA-3 in nuclear localization of silencing Argonaute HRDE-1. Together, we have shown that piRNA silencing in C. elegans is dependent on evolutionarily ancient RNA processing machinery that has been co-opted to function in the piRNA-mediated genome surveillance pathway.


Assuntos
Proteínas de Caenorhabditis elegans , Proteínas de Drosophila , Animais , RNA de Interação com Piwi , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Drosophila/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Inativação Gênica , Proteínas Argonautas/genética , Processamento Pós-Transcricional do RNA , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo
3.
RNA ; 29(5): 557-569, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36737102

RESUMO

PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposon mRNAs and some endogenous mRNAs in various animals. However, C. elegans piRNAs only trigger gene silencing at select predicted targeting sites, suggesting additional cellular mechanisms regulate piRNA silencing. To gain insight into possible mechanisms, we compared the transcriptome-wide predicted piRNA targeting sites to the in vivo piRNA binding sites. Surprisingly, while sequence-based predicted piRNA targeting sites are enriched in 3' UTRs, we found that C. elegans piRNAs preferentially bind to coding regions (CDS) of target mRNAs, leading to preferential production of secondary silencing small RNAs in the CDS. However, our analyses suggest that this CDS binding preference cannot be explained by the action of antisilencing Argonaute CSR-1. Instead, our analyses imply that CSR-1 protects mRNAs from piRNA silencing through two distinct mechanisms-by inhibiting piRNA binding across the entire CSR-1 targeted transcript, and by inhibiting secondary silencing small RNA production locally at CSR-1 bound sites. Together, our work identifies the CDS as the critical region that is uniquely competent for piRNA binding in C. elegans. We speculate the CDS binding preference may have evolved to allow the piRNA pathway to maintain robust recognition of RNA targets in spite of genetic drift. Together, our analyses revealed that distinct mechanisms are responsible for restricting piRNA binding and silencing to achieve proper transcriptome surveillance.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , RNA de Interação com Piwi , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transcriptoma , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , RNA de Cadeia Dupla/metabolismo , Sítios de Ligação , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo
4.
Nat Commun ; 13(1): 5306, 2022 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-36085149

RESUMO

piRNAs function as guardians of the genome by silencing non-self nucleic acids and transposable elements in animals. Many piRNA factors are enriched in perinuclear germ granules, but whether their localization is required for piRNA biogenesis or function is not known. Here we show that GLH/VASA helicase mutants exhibit defects in forming perinuclear condensates containing PIWI and other small RNA cofactors. These mutant animals produce largely normal levels of piRNA but are defective in triggering piRNA silencing. Strikingly, while many piRNA targets are activated in GLH mutants, we observe that hundreds of endogenous genes are aberrantly silenced by piRNAs. This defect in self versus non-self recognition is also observed in other mutants where perinuclear germ granules are disrupted. Together, our results argue that perinuclear germ granules function critically to promote the fidelity of piRNA-based transcriptome surveillance in C. elegans and preserve self versus non-self distinction.


Assuntos
Caenorhabditis elegans , Transcriptoma , Animais , Caenorhabditis elegans/genética , DNA Helicases/genética , Grânulos de Ribonucleoproteínas de Células Germinativas , Células Germinativas , RNA Interferente Pequeno/genética , Transcriptoma/genética
5.
Noncoding RNA ; 8(1)2022 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-35076587

RESUMO

Non-coding RNAs, such as miRNAs and piRNAs, play critical roles in gene regulation through base-pairing interactions with their target molecules. The recent development of the crosslinking, ligation, and sequencing of hybrids (CLASH) method has allowed scientists to map transcriptome-wide RNA-RNA interactions by identifying chimeric reads consisting of fragments from regulatory RNAs and their targets. However, analyzing CLASH data requires scientists to use advanced bioinformatics, and currently available tools are limited for users with little bioinformatic experience. In addition, many published CLASH studies do not show the full scope of RNA-RNA interactions that were captured, highlighting the importance of reanalyzing published data. Here, we present CLASH Analyst, a web server that can analyze raw CLASH data within a fully customizable and easy-to-use interface. CLASH Analyst accepts raw CLASH data as input and identifies the RNA chimeras containing the regulatory and target RNAs according to the user's interest. Detailed annotation of the captured RNA-RNA interactions is then presented for the user to visualize within the server or download for further analysis. We demonstrate that CLASH Analyst can identify miRNA- and piRNA-targeting sites reported from published CLASH data and should be applicable to analyze other RNA-RNA interactions. CLASH Analyst is freely available for academic use.

6.
Genetics ; 215(2): 421-434, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32245789

RESUMO

P granules are phase-separated liquid droplets that play important roles in the maintenance of germ cell fate in Caenorhabditis elegans Both the localization and formation of P granules are highly dynamic, but mechanisms that regulate such processes remain poorly understood. Here, we show evidence that the VASA-like germline RNA helicase GLH-1 couples distinct steps of its ATPase hydrolysis cycle to control the formation and disassembly of P granules. In addition, we found that the phenylalanine-glycine-glycine repeats in GLH-1 promote its localization at the perinucleus. Proteomic analyses of the GLH-1 complex with a GLH-1 mutation that interferes with P granule disassembly revealed transient interactions of GLH-1 with several Argonautes and RNA-binding proteins. Finally, we found that defects in recruiting the P granule component PRG-1 to perinuclear foci in the adult germline correlate with the fertility defects observed in various GLH-1 mutants. Together, our results highlight the versatile roles of an RNA helicase in controlling the formation of liquid droplets in space and time.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Grânulos Citoplasmáticos/metabolismo , RNA Helicases DEAD-box/metabolismo , Cristais Líquidos/química , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , RNA Helicases DEAD-box/genética , Hidrólise
7.
Nucleic Acids Res ; 47(D1): D181-D187, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30357353

RESUMO

PIWI-interacting RNAs (piRNAs) are a class of small noncoding RNAs that guard animal genomes against mutation by silencing transposons. In addition, recent studies have reported that piRNAs silence various endogenous genes. Tens of thousands of distinct piRNAs made in animals do not pair well to transposons and currently the functions and targets of piRNAs are largely unexplored. piRTarBase provides a user-friendly interface to access both predicted and experimentally identified piRNA targeting sites in Caenorhabditis elegans. The user can input genes of interest and retrieve a list of piRNA targeting sites on the input genes. Alternatively, the user can input a piRNA and retrieve a list of its mRNA targets. Additionally, piRTarBase integrates published mRNA and small RNA sequencing data, which will help users identify biologically relevant targeting events. Importantly, our analyses suggest that the piRNA sites found by both predictive and experimental approaches are more likely to exhibit silencing effects on their targets than each method alone. Taken together, piRTarBase offers an integrative platform that will help users to identify functional piRNA target sites by evaluating various information. piRTarBase is freely available for academic use at http://cosbi6.ee.ncku.edu.tw/piRTarBase/.


Assuntos
Sítios de Ligação , Bases de Dados Genéticas , Regulação da Expressão Gênica , Inativação Gênica , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Software , Navegador , Fluxo de Trabalho
8.
Nucleic Acids Res ; 46(W1): W43-W48, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29897582

RESUMO

pirScan is a web-based tool for identifying C. elegans piRNA-targeting sites within a given mRNA or spliced DNA sequence. The purpose of our tool is to allow C. elegans researchers to predict piRNA targeting sites and to avoid the persistent germline silencing of transgenes that has rendered many constructs unusable. pirScan fulfills this purpose by first enumerating the predicted piRNA-targeting sites present in an input sequence. This prediction can be exported in a tabular or graphical format. Subsequently, pirScan suggests silent mutations that can be introduced to the input sequence that would allow the modified transgene to avoid piRNA targeting. The user can customize the piRNA targeting stringency and the silent mutations that he/she wants to introduce into the sequence. The modified sequences can be re-submitted to be certain that any previously present piRNA-targeting sites are now absent and no new piRNA-targeting sites are accidentally generated. This revised sequence can finally be downloaded as a text file and/or visualized in a graphical format. pirScan is freely available for academic use at http://cosbi4.ee.ncku.edu.tw/pirScan/.


Assuntos
Caenorhabditis elegans/genética , Internet , RNA Interferente Pequeno/genética , Software , Animais , Biologia Computacional/tendências , RNA Interferente Pequeno/química
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