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Haifan Lin - One of the best experts on this subject based on the ideXlab platform.

  • PiwiL1 Promotes Gastric Cancer via a piRNA-Independent Mechanism
    2020
    Co-Authors: Shuo Shi, Zhenzhen Yang, Sanhong Liu, Fan Yang, Haifan Lin
    Abstract:

    ABSTRACT Targeted cancer therapy aims to achieve specific elimination of cancerous but not normal cells. Recently, Piwi Proteins, a subfamily of the PAZ-Piwi Domain (PPD) Protein family, have emerged as promising candidates for targeted cancer therapy. PPD Proteins are essential for small non-coding RNA pathways. The Argonaute subfamily partners with microRNA and small interfering RNA, whereas the Piwi subfamily partners with Piwi-interacting RNA (piRNA). Both Piwi Proteins and piRNA are mostly expressed in the germline and best known for their function in transposon silencing, with no detectable function in mammalian somatic tissues. However, Piwi Proteins become aberrantly expressed in multiple types of somatic cancers, thus gaining interest in targeted therapy. Despite this, little is known about the regulatory mechanism of Piwi Proteins in cancer. Here we report that one of the four Piwi Proteins in humans, PiwiL1, is highly expressed in gastric cancer tissues and cell lines. Knocking out PiwiL1 expression (PiwiL1-KO) drastically reduces gastric cancer cell proliferation, migration, metastasis, and tumorigenesis. RNA deep sequencing of gastric cancer cell line SNU-1 reveals that PiwiL1-KO significantly changes the transcriptome, causing the up-regulation of most of its associated transcripts. Surprisingly, few bona fide piRNAs exist in gastric cancer cells. Furthermore, abolishing the piRNA-binding activity of PiwiL1 does not affect its oncogenic function. Thus, PiwiL1 function in gastric cancer cells is independent of piRNA. This piRNA-independent regulation involves interaction with the UPF1-mediated nonsense-mediated mRNA decay (NMD) mechanism. Altogether, our findings reveal a novel and piRNA-independent function of PiwiL1 in promoting gastric cancer. SIGNIFICANCE Precision medicine aims to cure cancer without affecting normal tissues. Piwi Proteins provide a promising opportunity for precision medicine because they are normally expressed only in the testis for male fertility but gain expression in diverse types of cancers. Thus, inhibiting Piwi expression may stop cancer development (and spermatogenesis) without affecting normal body function. To establish causality between Piwi and cancer, we show here that the expression of PiwiL1, a human Piwi Protein, promotes gastric cancer. Surprisingly, this oncogenic function does not require piRNA, the expected partner of Piwi Proteins, but involves the nonsense-mediated mRNA decay mechanism. These findings reveal a new function and action mechanism of Piwi Proteins in oncogenesis, guiding the identification of Piwi inhibitors to cure cancer.

  • Piwi pirna pathway mediated transposable element repression in hydra somatic stem cells
    bioRxiv, 2019
    Co-Authors: Bryan B Teefy, Haifan Lin, Stefan Siebert, Jack F Cazet, Celina E Juliano
    Abstract:

    Abstract Transposable elements (TEs) can damage genomes, thus organisms employ a variety of mechanisms to repress TE expression. However, these mechanisms often fail over time leading to de-repression of TEs in aging tissues. The Piwi-piRNA pathway is a small RNA pathway that represses TE expression in the germline of animals. Here we explore the function of the pathway in the epithelial stem cells of Hydra, a long-lived freshwater cnidarian. Hydra have three stem cell populations; endodermal and ectodermal epithelial stem cells are strictly somatic, whereas the interstitial stem cells retain germline competence. In our previous study, we found that the Piwi Proteins are expressed in all three Hydra stem cell types. In this study, we focus on the ectodermal and endodermal epithelial stem cells to understand the somatic function of the pathway. We isolated piRNAs from Hydra that lack the interstitial lineage and found that these somatic piRNAs map predominantly to TE transcripts and display the conserved sequence signatures typical of germline piRNAs. Three lines of evidence suggest that the Piwi-piRNA pathway represses TEs in Hydra epithelial stem cells. First, epithelial knockdown of the Hydra Piwi Protein hywi resulted in upregulation of TE expression. Second, degradome sequencing revealed evidence of Piwi-mediated cleavage of TE RNAs in epithelial cells using the ping-pong mechanism. Finally, we demonstrated a direct association between Hywi Protein and TE transcripts in epithelial cells using RNA immunoprecipitation. Interestingly, we found that RNAi knockdown of hywi leads to an upregulation of genes involved in innate immunity, which may be in response to TE upregulation; this is consistent with recent studies on TE expression in mammalian cells. Altogether, this study suggests a function for the Piwi-piRNA pathway in maintaining the long-lived somatic cell lineages of Hydra and may point to a broader role for this pathway in protecting somatic tissue from TE-induced damage.

  • heat shock Protein dnaja1 stabilizes Piwi Proteins to support regeneration and homeostasis of planarian schmidtea mediterranea
    Journal of Biological Chemistry, 2019
    Co-Authors: Haifan Lin, Chen Wang, Zhenzhen Yang, Fanghao Guo, Shuo Shi, Xiaoshuai Han, An Zeng, Qing Jing
    Abstract:

    Piwi Proteins are key regulators of germline and somatic stem cells throughout different evolutionary lineages. However, how Piwi Proteins themselves are regulated remains largely unknown. To identify candidate Proteins that interact with Piwi Proteins and regulate their stability, here we established a yeast two-hybrid (Y2H) assay in the planarian species Schmidtea mediterranea We show that DNAJA1, a heat shock Protein 40 family member, interacts with the Piwi Protein SMEDWI-2, as validated by the Y2H screen and co-immunoprecipitation assays. We found that DNAJA1 is enriched in planarian adult stem cells, the nervous system, and intestinal tissues. DNAJA1-knockdown abolished planarian regeneration and homeostasis, compromised stem cell maintenance and Piwi-interacting RNA (piRNA) biogenesis, and deregulated SMEDWI-1/2 target genes. Mechanistically, we observed that DNAJA1 is required for the stability of SMEDWI-1 and SMEDWI-2 Proteins. Furthermore, we noted that human DNAJA1 binds to Piwi-like RNA-mediated gene silencing 1 (PiwiL1) and is required for PiwiL1 stability in human gastric cancer cells. In summary, our results reveal not only an evolutionarily conserved functional link between Piwi and DNAJA1 that is essential for Piwi Protein stability and piRNA biogenesis, but also an important role of DNAJA1 in the control of Proteins involved in stem cell regulation.

  • Piwi maintains germline stem cells and oogenesis in Drosophila through negative regulation of Polycomb group Proteins
    Nature Genetics, 2016
    Co-Authors: Jamy C. Peng, Na Liu, Anton Valouev, Haifan Lin
    Abstract:

    The Drosophila melanogaster Piwi Protein regulates both niche and intrinsic mechanisms to maintain germline stem cells, but its underlying mechanism remains unclear. Here we report that Piwi interacts with Polycomb group complexes PRC1 and PRC2 in niche and germline cells to regulate ovarian germline stem cells and oogenesis. Piwi physically interacts with the PRC2 subunits Su(z)12 and Esc in the ovary and in vitro. Chromatin coimmunoprecipitation of Piwi, the PRC2 enzymatic subunit E(z), histone H3 trimethylated at lysine 27 (H3K27me3) and RNA polymerase II in wild-type and Piwi mutant ovaries demonstrates that Piwi binds a conserved DNA motif at ∼ 72 genomic sites and inhibits PRC2 binding to many non-Piwi-binding genomic targets and H3K27 trimethylation. Moreover, Piwi influences RNA polymerase II activities in Drosophila ovaries, likely via inhibiting PRC2. We hypothesize that Piwi negatively regulates PRC2 binding by sequestering PRC2 in the nucleoplasm, thus reducing PRC2 binding to many targets and influencing transcription during oogenesis.

  • Piwi Proteins Are Dispensable for Mouse Somatic Development and Reprogramming of Fibroblasts into Pluripotent Stem Cells
    PloS one, 2014
    Co-Authors: Ee-chun Cheng, Dongwan D. Kang, Zhong Wang, Haifan Lin
    Abstract:

    Piwi Proteins play essential and conserved roles in germline development, including germline stem cell maintenance and meiosis. Because germline regulators such as OCT4, NANOG, and SOX2 are known to be potent factors that reprogram differentiated somatic cells into induced pluripotent stem cells (iPSCs), we investigated whether the Piwi Protein family is involved in iPSC production. We find that all three mouse Piwi genes, Miwi, Mili, and Miwi2, are expressed in embryonic stem cells (ESCs) at higher levels than in fibroblasts, with Mili being the highest. However, mice lacking all three Piwi genes are viable and female fertile, and are only male sterile. Furthermore, embryonic fibroblasts derived from Miwi/Mili/Miwi2 triple knockout embryos can be efficiently reprogrammed into iPS cells. These iPS cells expressed pluripotency markers and were capable of differentiating into all three germ layers in teratoma assays. Genome-wide expression profiling reveals that the triple knockout iPS cells are very similar to littermate control iPS cells. These results indicate that Piwi Proteins are dispensable for direct reprogramming of mouse fibroblasts.

Kathleen Collins - One of the best experts on this subject based on the ideXlab platform.

  • Transgenerational function of Tetrahymena Piwi Protein Twi8p at distinctive noncoding RNA loci.
    RNA, 2017
    Co-Authors: Brian M. Farley, Kathleen Collins
    Abstract:

    : Transgenerational transmission of genome-regulatory epigenetic information can determine phenotypes in the progeny of sexual reproduction. Sequence specificity of transgenerational regulation derives from small RNAs assembled into Piwi-Protein complexes. Known targets of transgenerational regulation are primarily transposons and transposon-derived sequences. Here, we extend the scope of Piwi-mediated transgenerational regulation to include unique noncoding RNA loci. Ciliates such as Tetrahymena have a phenotypically silent germline micronucleus and an expressed somatic macronucleus, which is differentiated anew from a germline genome copy in sexual reproduction. We show that the nuclear-localized Tetrahymena Piwi Protein Twi8p shuttles from parental to zygotic macronuclei. Genetic elimination of Twi8p has no phenotype for cells in asexual growth. On the other hand, cells lacking Twi8p arrest in sexual reproduction with zygotic nuclei that retain the germline genome structure, without the DNA elimination and fragmentation required to generate a functional macronucleus. Twi8p-bound small RNAs originate from long-noncoding RNAs with a terminal hairpin, which become detectable in the absence of Twi8p. Curiously, the loci that generate Twi8p-bound small RNAs are essential for asexual cell growth, even though Twi8 RNPs are essential only in sexual reproduction. Our findings suggest the model that Twi8 RNPs act on silent germline chromosomes to permit their conversion to expressed macronuclear chromosomes. Overall this work reveals that a Piwi Protein carrying small RNAs from long-noncoding RNA loci has transgenerational function in establishing zygotic nucleus competence for gene expression.

  • a tetrahymena Piwi bound to mature trna 3 fragments activates the exonuclease xrn2 for rna processing in the nucleus
    Molecular Cell, 2012
    Co-Authors: Mary T. Couvillion, Gergana Bounova, Elizabeth Purdom, Terence P Speed, Kathleen Collins
    Abstract:

    Emerging evidence suggests that Argonaute (Ago)/Piwi Proteins have diverse functions in the nucleus and cytoplasm, but the molecular mechanisms employed in the nucleus remain poorly defined. The Tetrahymena thermophila Ago/Piwi Protein Twi12 is essential for growth and functions in the nucleus. Twi12-bound small RNAs (sRNAs) are 3′ tRNA fragments that contain modified bases and thus are attenuated for base pairing to targets. We show that Twi12 assembles an unexpected complex with the nuclear exonuclease Xrn2. Twi12 functions to stabilize and localize Xrn2, as well as to stimulate its exonuclease activity. Twi12 function depends on sRNA binding, which is required for its nuclear import. Depletion of Twi12 or Xrn2 induces a cellular ribosomal RNA processing defect known to result from limiting Xrn2 activity in other organisms. Our findings suggest a role for an Ago/Piwi Protein and 3′ tRNA fragments in nuclear RNA metabolism.

  • A growth-essential Tetrahymena Piwi Protein carries tRNA fragment cargo
    Genes and Development, 2010
    Co-Authors: Mary T. Couvillion, Ravi Sachidanandam, Kathleen Collins
    Abstract:

    Argonaute/Piwi Proteins associate with small RNAs that typically provide sequence specificity for RNP function in gene and genome regulation. Here we show that Twi12, a Tetrahymena Piwi Protein essential for growth, is loaded with mature tRNA fragments. The tightly bound ~18- to 22-nucleotide tRNA 3' fragments are biochemically distinct from the tRNA halves produced transiently in response to stress. Notably, the end positions of Twi12-bound tRNA 3' fragments precisely match RNAs detected in total small RNA of mouse embryonic stem cells and human cancer cells. Our studies demonstrate unanticipated evolutionary conservation of mature tRNA processing to tRNA fragment small RNAs.

Ravi Sachidanandam - One of the best experts on this subject based on the ideXlab platform.

  • distinct roles of rna helicases mvh and tdrd9 in Piwi slicing triggered mammalian pirna biogenesis and function
    Developmental Cell, 2017
    Co-Authors: Joanna M Wenda, Ravi Sachidanandam, Radha Raman Pandey, Zhaolin Yang, David Homolka, Pietro Spinelli, Ramesh S. Pillai
    Abstract:

    Small RNAs called Piwi-interacting RNAs (piRNAs) act as an immune system to suppress transposable elements in the animal gonads. A poorly understood adaptive pathway links cytoplasmic slicing of target RNA by the Piwi Protein MILI to loading of target-derived piRNAs into nuclear MIWI2. Here we demonstrate that MILI slicing generates a 16-nt by-product that is discarded and a pre-piRNA intermediate that is used for phased piRNA production. The ATPase activity of Mouse Vasa Homolog (MVH) is essential for processing the intermediate into piRNAs, ensuring transposon silencing and male fertility. The ATPase activity controls dissociation of an MVH complex containing Piwi Proteins, piRNAs, and slicer products, allowing safe handover of the intermediate. In contrast, ATPase activity of TDRD9 is dispensable for piRNA biogenesis but is essential for transposon silencing and male fertility. Our work implicates distinct RNA helicases in specific steps along the nuclear piRNA pathway.

  • Piwi slicing and exd1 drive biogenesis of nuclear pirnas from cytosolic targets of the mouse pirna pathway
    Molecular Cell, 2016
    Co-Authors: Zhaolin Yang, Ravi Sachidanandam, Radha Raman Pandey, Andrew A. Mccarthy, Michael Reuter, K M Chen, David Homolka, Bruno Kotska Rodino Janeiro, Marieodile Fauvarque, Ramesh S. Pillai
    Abstract:

    Piwi-interacting RNAs (piRNAs) guide Piwi Proteins to suppress transposons in the cytoplasm and nucleus of animal germ cells, but how silencing in the two compartments is coordinated is not known. Here we demonstrate that endonucleolytic slicing of a transcript by the cytosolic mouse Piwi Protein MILI acts as a trigger to initiate its further 5′→3′ processing into non-overlapping fragments. These fragments accumulate as new piRNAs within both cytosolic MILI and the nuclear MIWI2. We also identify Exonuclease domain-containing 1 (EXD1) as a partner of the MIWI2 piRNA biogenesis factor TDRD12. EXD1 homodimers are inactive as a nuclease but function as an RNA adaptor within a PET (Piwi-EXD1-Tdrd12) complex. Loss of Exd1 reduces sequences generated by MILI slicing, impacts biogenesis of MIWI2 piRNAs, and de-represses LINE1 retrotransposons. Thus, piRNA biogenesis triggered by Piwi slicing, and promoted by EXD1, ensures that the same guides instruct Piwi Proteins in the nucleus and cytoplasm.

  • The MID-Piwi module of Piwi Proteins specifies nucleotide- and strand-biases of piRNAs
    RNA, 2014
    Co-Authors: Elisa Cora, Ravi Sachidanandam, Radha Raman Pandey, Jordi Xiol, Josh Taylor, Andrew A. Mccarthy, Ramesh S. Pillai
    Abstract:

    Piwi-interacting RNAs (piRNAs) guide Piwi Argonautes to suppress transposon activity in animal gonads. Known piRNA populations are extremely complex, with millions of individual sequences present in a single organism. Despite this complexity, specific Piwi Proteins incorporate piRNAs with distinct nucleotide- and transposon strand-biases (antisense or sense) of unknown origin. Here, we examined the contribution of structural domains in Piwi Proteins toward defining these biases. We report the first crystal structure of the MID domain from a Piwi Argonaute and use docking experiments to show its ability to specify recognition of 5′ uridine (1U-bias) of piRNAs. Mutational analyses reveal the importance of 5′ end-recognition within the MID domain for piRNA biogenesis in vivo. Finally, domain-swapping experiments uncover an unexpected role for the MID-Piwi module of a Piwi Protein in dictating the transposon strand-orientation of its bound piRNAs. Our work identifies structural features that allow distinguishing individual Piwi members during piRNA biogenesis.

  • tudor domain containing 12 tdrd12 is essential for secondary Piwi interacting rna biogenesis in mice
    Proceedings of the National Academy of Sciences of the United States of America, 2013
    Co-Authors: Radha Raman Pandey, Ravi Sachidanandam, Zhaolin Yang, Yoshimi Tokuzawa, Eri Hayashi, Tomoko Ichisaka, Shimpei Kajita, Yuka Asano, Tetsuo Kunieda, Shinichiro Chuma
    Abstract:

    Piwi-interacting RNAs (piRNAs) are gonad-specific small RNAs that provide defense against transposable genetic elements called transposons. Our knowledge of piRNA biogenesis is sketchy, partly due to an incomplete inventory of the factors involved. Here, we identify Tudor domain-containing 12 (TDRD12; also known as ECAT8) as a unique piRNA biogenesis factor in mice. TDRD12 is detected in complexes containing Piwi Protein MILI (PiwiL2), its associated primary piRNAs, and TDRD1, all of which are already implicated in secondary piRNA biogenesis. Male mice carrying either a nonsense point mutation (reproductive mutant 23 or repro23 mice) or a targeted deletion in the Tdrd12 locus are infertile and derepress retrotransposons. We find that TDRD12 is dispensable for primary piRNA biogenesis but essential for production of secondary piRNAs that enter Piwi Protein MIWI2 (PiwiL4). Cell-culture studies with the insect ortholog of TDRD12 suggest a role for the multidomain Protein in mediating complex formation with other participants during secondary piRNA biogenesis.

  • A growth-essential Tetrahymena Piwi Protein carries tRNA fragment cargo
    Genes and Development, 2010
    Co-Authors: Mary T. Couvillion, Ravi Sachidanandam, Kathleen Collins
    Abstract:

    Argonaute/Piwi Proteins associate with small RNAs that typically provide sequence specificity for RNP function in gene and genome regulation. Here we show that Twi12, a Tetrahymena Piwi Protein essential for growth, is loaded with mature tRNA fragments. The tightly bound ~18- to 22-nucleotide tRNA 3' fragments are biochemically distinct from the tRNA halves produced transiently in response to stress. Notably, the end positions of Twi12-bound tRNA 3' fragments precisely match RNAs detected in total small RNA of mouse embryonic stem cells and human cancer cells. Our studies demonstrate unanticipated evolutionary conservation of mature tRNA processing to tRNA fragment small RNAs.

Ramesh S. Pillai - One of the best experts on this subject based on the ideXlab platform.

  • distinct roles of rna helicases mvh and tdrd9 in Piwi slicing triggered mammalian pirna biogenesis and function
    Developmental Cell, 2017
    Co-Authors: Joanna M Wenda, Ravi Sachidanandam, Radha Raman Pandey, Zhaolin Yang, David Homolka, Pietro Spinelli, Ramesh S. Pillai
    Abstract:

    Small RNAs called Piwi-interacting RNAs (piRNAs) act as an immune system to suppress transposable elements in the animal gonads. A poorly understood adaptive pathway links cytoplasmic slicing of target RNA by the Piwi Protein MILI to loading of target-derived piRNAs into nuclear MIWI2. Here we demonstrate that MILI slicing generates a 16-nt by-product that is discarded and a pre-piRNA intermediate that is used for phased piRNA production. The ATPase activity of Mouse Vasa Homolog (MVH) is essential for processing the intermediate into piRNAs, ensuring transposon silencing and male fertility. The ATPase activity controls dissociation of an MVH complex containing Piwi Proteins, piRNAs, and slicer products, allowing safe handover of the intermediate. In contrast, ATPase activity of TDRD9 is dispensable for piRNA biogenesis but is essential for transposon silencing and male fertility. Our work implicates distinct RNA helicases in specific steps along the nuclear piRNA pathway.

  • Piwi slicing and exd1 drive biogenesis of nuclear pirnas from cytosolic targets of the mouse pirna pathway
    Molecular Cell, 2016
    Co-Authors: Zhaolin Yang, Ravi Sachidanandam, Radha Raman Pandey, Andrew A. Mccarthy, Michael Reuter, K M Chen, David Homolka, Bruno Kotska Rodino Janeiro, Marieodile Fauvarque, Ramesh S. Pillai
    Abstract:

    Piwi-interacting RNAs (piRNAs) guide Piwi Proteins to suppress transposons in the cytoplasm and nucleus of animal germ cells, but how silencing in the two compartments is coordinated is not known. Here we demonstrate that endonucleolytic slicing of a transcript by the cytosolic mouse Piwi Protein MILI acts as a trigger to initiate its further 5′→3′ processing into non-overlapping fragments. These fragments accumulate as new piRNAs within both cytosolic MILI and the nuclear MIWI2. We also identify Exonuclease domain-containing 1 (EXD1) as a partner of the MIWI2 piRNA biogenesis factor TDRD12. EXD1 homodimers are inactive as a nuclease but function as an RNA adaptor within a PET (Piwi-EXD1-Tdrd12) complex. Loss of Exd1 reduces sequences generated by MILI slicing, impacts biogenesis of MIWI2 piRNAs, and de-represses LINE1 retrotransposons. Thus, piRNA biogenesis triggered by Piwi slicing, and promoted by EXD1, ensures that the same guides instruct Piwi Proteins in the nucleus and cytoplasm.

  • Impact of nuclear Piwi elimination on chromatin state in Drosophila melanogaster ovaries
    Nucleic Acids Research, 2014
    Co-Authors: M S Klenov, Ramesh S. Pillai, E. Y. Yakushev, A. D. Stolyarenko, S. A. Lavrov, Alina P. Korbut, Michael Reuter, V A Gvozdev
    Abstract:

    The Piwi-interacting RNA (piRNA)-interacting Piwi Protein is involved in transcriptional silencing of transposable elements in ovaries of Drosophila melanogaster. Here we characterized the genome-wide effect of nuclear Piwi elimination on the presence of the heterochromatic H3K9me3 mark and HP1a, as well as on the transcription-associated mark H3K4me2. Our results demonstrate that a significant increase in the H3K4me2 level upon nuclear Piwi loss is not accompanied by the alterations in H3K9me3 and HP1a levels for several germline-expressed transposons, suggesting that in this case Piwi prevents transcription by a mechanism distinct from H3K9 methylation. We found that the targets of Piwi-dependent chromatin repression are mainly related to the elements that display a higher level of H3K4me2 modification in the absence of silencing, i.e. most actively transcribed elements. We also show that Piwi-guided silencing does not significantly influence the chromatin state of dual-strand piRNA-producing clusters. In addition, host Protein-coding gene expression is essentially not affected due to the nuclear Piwi elimination, but we noted an increase in small nuclear spliceosomal RNAs abundance and propose Piwi involvement in their post-transcriptional regulation. Our work reveals new aspects of transposon silencing in Drosophila, indicating that transcription of transposons can underpin their Piwi dependent silencing, while canonical heterochromatin marks are not obligatory for their repression.

  • The MID-Piwi module of Piwi Proteins specifies nucleotide- and strand-biases of piRNAs
    RNA, 2014
    Co-Authors: Elisa Cora, Ravi Sachidanandam, Radha Raman Pandey, Jordi Xiol, Josh Taylor, Andrew A. Mccarthy, Ramesh S. Pillai
    Abstract:

    Piwi-interacting RNAs (piRNAs) guide Piwi Argonautes to suppress transposon activity in animal gonads. Known piRNA populations are extremely complex, with millions of individual sequences present in a single organism. Despite this complexity, specific Piwi Proteins incorporate piRNAs with distinct nucleotide- and transposon strand-biases (antisense or sense) of unknown origin. Here, we examined the contribution of structural domains in Piwi Proteins toward defining these biases. We report the first crystal structure of the MID domain from a Piwi Argonaute and use docking experiments to show its ability to specify recognition of 5′ uridine (1U-bias) of piRNAs. Mutational analyses reveal the importance of 5′ end-recognition within the MID domain for piRNA biogenesis in vivo. Finally, domain-swapping experiments uncover an unexpected role for the MID-Piwi module of a Piwi Protein in dictating the transposon strand-orientation of its bound piRNAs. Our work identifies structural features that allow distinguishing individual Piwi members during piRNA biogenesis.

  • analysis of small rna guided endonuclease activity in endogenous Piwi Protein complexes from mouse testes
    Methods of Molecular Biology, 2014
    Co-Authors: Michael Reuter, Ramesh S. Pillai
    Abstract:

    Small RNAs associate with members of the Argonaute family to function in gene regulation, transposon control, and creation of silent chromatin domains. In this partnership, small RNAs act as guides for the bound Argonaute and other associated Proteins. Complementary base pairing of small RNAs to target nucleic acid molecules allow specificity for the small RNA-mediated functions. One key activity of some Argonaute Protein family members is their small RNA-guided endonuclease activity called Slicer action. Here we describe a protocol that can be used to probe slicer activity in endogenous Piwi complexes isolated from mouse testes.

V A Gvozdev - One of the best experts on this subject based on the ideXlab platform.

  • Induction of transposon silencing in the Drosophila germline
    Biochemistry (Moscow), 2017
    Co-Authors: S. S. Ryazansky, M S Klenov, A. D. Stolyarenko, V A Gvozdev
    Abstract:

    In this review we consider the role of the piRNA system in transposable element silencing in the Drosophila melanogaster germline. We focus on new data that demonstrate the mechanisms of initiation of piRNA biogenesis in ovarian germinal cells and the role of Piwi Protein in this process, including our own results.

  • Functions of piRNAs and the Piwi Protein in Drosophila
    Russian Journal of Genetics, 2015
    Co-Authors: V A Gvozdev, A. D. Stolyarenko, M S Klenov
    Abstract:

    Short regulatory RNAs 25–35 nucleotides in length, along with RNA-binding Proteins of the Piwi family, constitute an evolutionarily conserved system that functions mainly in eukaryotic gonads. The system can be regarded as a variation of the RNA interference mechanism, which is based on the recognition of target RNA as a result of complementary interactions with piRNAs. Different variants of this regulatory system function both in germline cells, including stem cells, and somatic cells of the niche, ensuring maintenance of the germline stem cells and their differentiation. One of the most important functions (but not the only one) of this system is the repression of transposons, which guarantees genome stability in germline cells. This review focuses on the works of the authors in the context of outstanding international achievements in the rapidly evolving research area, the biology of piRNA and the functions of the Piwi Protein.

  • Piwi Protein as a nucleolus visitor in Drosophila melanogaster
    Molecular Biology, 2015
    Co-Authors: E A Mikhaleva, M S Klenov, E. Y. Yakushev, A. D. Stolyarenko, Ya. M. Rozovsky, V A Gvozdev
    Abstract:

    The evolutionarily conserved nuclear Piwi Protein of Drosophila melanogaster is the defining member of the Argonaute small RNA-binding Protein family. Guided by piRNAs, Piwi functions in transposon silencing in somatic and germ cells of gonads. We found that in ovarian somatic and germ cells, as well as in the established ovarian somatic cell line, Piwi accumulates predominantly in the nucleolus, the main nuclear compartment which participates not only in rRNA synthesis, but also in various cell stress responses. We have shown the colocalization of Piwi with the nucleolar marker Proteins fibrillarin and Nopp140. The PiwiNt mutation which prevents the transport of Piwi to the nucleus and disrupts transposon silencing led to 6-8 fold upregulation of rRNA gene expression, as assessed by the level of transcripts of transposon insertions in 28S rRNA genes. RNase treatment of live cultured ovarian somatic cells depleted Piwi from the nucleolus. The same effect was observed upon inhibition of the activity of RNA polymerase I, which transcribes rRNA genes, but not RNA polymerase II. In contrast, upon heat shock, Piwi concentrated in the nucleolus and was depleted from the nucleoplasm. These results implicate Piwi in RNA polymerase I activity modulation and stress response in the nucleolus. Possible noncanonical Piwi functions are discussed, which are not related to the role of Piwi in transposon silencing.

  • Piwi Protein as a nucleolus visitor in Drosophila melanogaster
    Molekuliarnaia biologiia, 2015
    Co-Authors: E A Mikhaleva, E Iu Iakushev, A D Stoliarenko, M S Klenov, Ia M Pozovskiĭ, V A Gvozdev
    Abstract:

    The evolutionarily conserved nuclear Piwi Protein of Drosophila melanogaster is a representative of the Argonaute small RNA binding Protein family. Guided by small piRNAs, Piwi functions in transposon silencing in somatic and germ cells of the gonad. We found that in ovarian somatic and germ cells, as well as in the established ovarian somatic cell line, Piwi is concentrated predominantly in the nucleolus--the main nuclear compartment, participating not only in rRNA synthesis, but also in various cell stress responses. We demonstrated the colocalization of Piwi with nucleolar marker Proteins--fibrillarin and Nopp140. A mutation preventing Piwi transport to the nucleus and disturbing transposon silencing (Piwi(Nt)) leads to 6-8-fold upregulation of rRNA genes expression, as evaluated by the level of transcripts of transposon insertions in 28S rRNA genes. RNase treatment of live cultured ovarian somatic cells depletes Piwi from the nucleolus. The same effect is observed upon inhibiting RNA polymerase I which transcribes rRNA, but not RNA polymerase II. In contrast, upon heat shock Piwi is concentrated in the nucleolus and is depleted from the nucleoplasm. These results implicate Piwi in RNA polymerase activity modulation and stress response in the nucleolus. We discuss possible noncanonical Piwi functions along with its canonical role in transposon silencing by piRNAs.

  • Functions of piRNAs and the Piwi Protein in Drosophila
    Genetika, 2015
    Co-Authors: V A Gvozdev, A. D. Stolyarenko, M S Klenov
    Abstract:

    Short (25-35 nucleotides) regulatory piPHK, along with RNA-binding Proteins of the Piwi family, constitute an evolutionarily conserved system that functions mainly in eukaryotic gonads. The system can be regarded as a variant of the mechanism of RNA interference, which is based on the recognition of target RNA as a result of complementary interactions with piRNA. The variants of this regulatory system function in the germline cells, including stem cells and somatic cells of the niche, ensuring maintenance of the germline stem cells and their differentiation. One of the most important functions (but not the only one) of this system is the repression of transposons, which guarantees genome stability in germline cells. This review focuses on the works of the authors of the review in the context of outstanding international achievements in a rapidly evolving re- search area, the biology of piRNA and the function of the Piwi Protein.