Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
Cell ; 167(2): 484-497.e9, 2016 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-27693359

RESUMO

PIWI-clade Argonaute proteins associate with PIWI-interacting RNAs (piRNAs) and silence transposable elements in animal gonads. Here, we report the crystal structure of a silkworm PIWI-clade Argonaute, Siwi, bound to the endogenous piRNA, at 2.4 Å resolution. Siwi adopts a bilobed architecture consisting of N-PAZ and MID-PIWI lobes, in which the 5' and 3' ends of the bound piRNA are anchored by the MID-PIWI and PAZ domains, respectively. A structural comparison of Siwi with AGO-clade Argonautes reveals notable differences in their nucleic-acid-binding channels, likely reflecting the distinct lengths of their guide RNAs and their mechanistic differences in guide RNA loading and cleavage product release. In addition, the structure reveals that Siwi and prokaryotic, but not eukaryotic, AGO-clade Argonautes share unexpected similarities, such as metal-dependent 5'-phosphate recognition and a potential structural transition during the catalytic-tetrad formation. Overall, this study provides a critical starting point toward a mechanistic understanding of piRNA-mediated transposon silencing.


Assuntos
Proteínas Argonautas/química , Bombyx/metabolismo , Proteínas de Insetos/química , RNA Interferente Pequeno/química , Animais , Proteínas Argonautas/isolamento & purificação , Bombyx/química , Bombyx/genética , Linhagem Celular , Cristalografia por Raios X , Elementos de DNA Transponíveis/genética , Inativação Gênica , Humanos , Proteínas de Insetos/isolamento & purificação , Conformação de Ácido Nucleico , RNA Interferente Pequeno/isolamento & purificação
2.
Nucleic Acids Res ; 49(5): 2700-2720, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33590099

RESUMO

In animal gonads, transposable elements are actively repressed to preserve genome integrity through the PIWI-interacting RNA (piRNA) pathway. In mice, piRNAs are abundantly expressed in male germ cells, and form effector complexes with three distinct PIWIs. The depletion of individual Piwi genes causes male-specific sterility with no discernible phenotype in female mice. Unlike mice, most other mammals have four PIWI genes, some of which are expressed in the ovary. Here, purification of PIWI complexes from oocytes of the golden hamster revealed that the size of the PIWIL1-associated piRNAs changed during oocyte maturation. In contrast, PIWIL3, an ovary-specific PIWI in most mammals, associates with short piRNAs only in metaphase II oocytes, which coincides with intense phosphorylation of the protein. An improved high-quality genome assembly and annotation revealed that PIWIL1- and PIWIL3-associated piRNAs appear to share the 5'-ends of common piRNA precursors and are mostly derived from unannotated sequences with a diminished contribution from TE-derived sequences, most of which correspond to endogenous retroviruses. Our findings show the complex and dynamic nature of biogenesis of piRNAs in hamster oocytes, and together with the new genome sequence generated, serve as the foundation for developing useful models to study the piRNA pathway in mammalian oocytes.


Assuntos
Proteínas Argonautas/metabolismo , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonautas/genética , Feminino , Genômica , Masculino , Mesocricetus , Metáfase , Fosforilação , RNA Interferente Pequeno/genética , Testículo/metabolismo
3.
BMC Neurol ; 17(1): 91, 2017 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-28506213

RESUMO

BACKGROUND: To analyze voxel-wise correlation between cerebral blood flow (CBF) measured using ASL-MRI and cognition in patients with Alzheimer's disease (AD). METHODS: Forty-one patients diagnosed with AD or mild cognitive impairment due to AD were recruited for this study. CBF images were obtained using ASL-MRI (n = 41) with a post-labeling delay (PLD) of 1.5 and 2.5 s (PLD1.5 and PLD2.5, respectively) using a 3 T scanner, in addition to brain perfusion SPECT with N-isopropyl-4-[I-123]iodoamphetamine (n = 28). Voxel-based analyses were performed for ASL-MRI and SPECT using Mini-Mental State Examination (MMSE) scores as covariates. Differences in CBF between PLD1.5 and PLD2.5 were assessed using a paired t-test with SPM12. RESULTS: Significant positive correlations were observed between MMSE scores and CBF at PLD1.5 in the right posterior cingulate cortex (PCC), and both temporo-parietal association cortexes. At PLD2.5, significant positive correlations were determined for MMSE scores and CBF in the superior parietal lobule and the right temporo-parietal association cortex. SPECT showed significant positive correlations in the PCC and both temporo-parietal association cortexes (right-side dominant). PLD1.5 showed significantly higher CBF than PLD2.5 in the proximal areas of vascular territories of the anterior, middle, and posterior cerebral arteries. CONCLUSIONS: Significant positive correlations in CBF, measured with both ASL-MRI and SPECT, with cognition were found in the PCC and temporo-parietal association cortexes. PLD1.5 and PLD2.5 showed similar correlations with cognition, although the CBF images had significant differences.


Assuntos
Doença de Alzheimer , Circulação Cerebrovascular/fisiologia , Disfunção Cognitiva , Processamento de Imagem Assistida por Computador/métodos , Imageamento por Ressonância Magnética/métodos , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/fisiopatologia , Disfunção Cognitiva/diagnóstico por imagem , Disfunção Cognitiva/fisiopatologia , Estudos Transversais , Feminino , Humanos , Masculino , Marcadores de Spin
4.
Dev Biol ; 400(1): 43-58, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25624265

RESUMO

Mammalian spermatogenesis has been investigated extensively in rodents and a strictly controlled developmental process has been defined at cellular and molecular levels. In comparison, primate spermatogenesis has been far less well characterized. However, important differences between primate and rodent spermatogenesis are emerging so it is not always accurate to extrapolate findings in rodents to primate systems. Here, we performed an extensive immunofluorescence study of spermatogenesis in neonatal, juvenile, and adult testes in the common marmoset (Callithrix jacchus) to determine primate-specific patterns of gene expression that underpin primate germ cell development. Initially we characterized adult spermatogonia into two main classes; mitotically active C-KIT(+)Ki67(+) cells and mitotically quiescent SALL4(+)PLZF(+)LIN28(+)DPPA4(+) cells. We then explored the expression of a set of markers, including PIWIL1/MARWI, VASA, DAZL, CLGN, RanBPM, SYCP1 and HAPRIN, during germ cell differentiation from early spermatocytes through round and elongating spermatids, and a clear program of gene expression changes was determined as development proceeded. We then examined the juvenile marmoset testis. Markers of gonocytes demonstrated two populations; one that migrates to the basal membrane where they form the SALL4(+) or C-KIT(+) spermatogonia, and another that remains in the lumen of the seminiferous tubule. This later population, historically identified as pre-spermatogonia, expressed meiotic and apoptotic markers and were eliminated because they appear to have failed to correctly migrate. Our findings provide the first platform of gene expression dynamics in adult and developing germ cells of the common marmoset. Although we have characterized a limited number of genes, these results will facilitate primate spermatogenesis research and understanding of human reproduction.


Assuntos
Callithrix/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Espermatogênese/fisiologia , Testículo/crescimento & desenvolvimento , Animais , Apoptose/fisiologia , Western Blotting , Marcadores Genéticos/genética , Masculino , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , Testículo/citologia
5.
RNA ; 20(8): 1223-37, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24914035

RESUMO

Small RNAs mediate gene silencing by binding Argonaute/Piwi proteins to regulate target RNAs. Here, we describe small RNA profiling of the adult testes of Callithrix jacchus, the common marmoset. The most abundant class of small RNAs in the adult testis was piRNAs, although 353 novel miRNAs but few endo-siRNAs were also identified. MARWI, a marmoset homolog of mouse MIWI and a very abundant PIWI in adult testes, associates with piRNAs that show characteristics of mouse pachytene piRNAs. As in other mammals, most marmoset piRNAs are derived from conserved clustered regions in the genome, which are annotated as intergenic regions. However, unlike in mice, marmoset piRNA clusters are also found on the X chromosome, suggesting escape from meiotic sex chromosome inactivation by the X-linked clusters. Some of the piRNA clusters identified contain antisense-orientated pseudogenes, suggesting the possibility that pseudogene-derived piRNAs may regulate parental functional protein-coding genes. More piRNAs map to transposable element (TE) subfamilies when they have copies in piRNA clusters. In addition, the strand bias observed for piRNAs mapped to each TE subfamily correlates with the polarity of copies inserted in clusters. These findings suggest that pachytene piRNA clusters determine the abundance and strand-bias of TE-derived piRNAs, may regulate protein-coding genes via pseudogene-derived piRNAs, and may even play roles in meiosis in the adult marmoset testis.


Assuntos
Callithrix/genética , Perfilação da Expressão Gênica , Família Multigênica , RNA Interferente Pequeno/genética , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sequência de Bases , Elementos de DNA Transponíveis , Expressão Gênica , Genoma , Masculino , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Mapeamento Físico do Cromossomo , Ligação Proteica , Pseudogenes , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Alinhamento de Sequência , Testículo/metabolismo
6.
Cell Rep ; 39(11): 110894, 2022 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-35705038

RESUMO

The germ-cell-specific RNA-binding protein (RBP) NANOS2 plays a pivotal role in male gonocyte differentiation and spermatogonial stem cell maintenance. Although NANOS2 interacts with the CNOT deadenylation complex and Dead end 1 (DND1) to repress target RNAs, the molecular mechanisms underlying target mRNA selection remain unclear because of the limited cell resource in vivo. Here, we demonstrate that exogenous NANOS2-DND1 suppresses target mRNAs in somatic cells. Using this somatic cell system, we find that NANOS2 interacts with RNA-bound DND1 and recruits the CNOT complex to the mRNAs. However, a fusion construct composed of the CNOT1-binding site of NANOS2 (NIM) and DND1 fails to repress the target gene expression. Therefore, NANOS2 is required not only for recruitment of the CNOT complex but also for selecting the target mRNA with DND1. This study reveals that NANOS2 functions as a second-layer RBP for the target recognition and functional adaptation of DND1.


Assuntos
Células Germinativas , Proteínas de Neoplasias , Proteínas de Ligação a RNA , RNA , Diferenciação Celular , Células Germinativas/metabolismo , Humanos , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição
7.
iScience ; 24(8): 102890, 2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34401671

RESUMO

During murine germ cell development, male germ cells enter the mitotically arrested G0 stage, which is an initial step of sexually dimorphic differentiation. The male-specific RNA-binding protein NANOS2 has a key role in suppressing the cell cycle in germ cells. However, the detailed mechanism of how NANOS2 regulates the cell cycle remains unclear. Using single-cell RNA sequencing (scRNA-seq), we extracted the cell cycle state of each germ cell in wild-type and Nanos2-KO testes and revealed that Nanos2 expression starts in mitotic cells and induces mitotic arrest. We identified Rheb, a regulator of mTORC1, and Ptma as possible targets of NANOS2. We propose that repression of the cell cycle is a primary function of NANOS2 and that it is mediated via the suppression of mTORC1 activity through the repression of Rheb in a post-transcriptional manner.

8.
Methods Mol Biol ; 1463: 205-216, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27734358

RESUMO

The mouse testis has served as a popular model system to study a wide range of biological processes, including germ cell development, meiosis, epigenetic changes of chromatin, transposon silencing, and small RNA-mediated epigenetic modifications. PIWI-interacting RNAs (piRNAs) are a class of small RNAs that are almost exclusively expressed in animal gonads. They repress transposons by forming effector complexes with PIWI proteins to maintain genome integrity of the germline. Here we describe detailed procedures of how to produce monoclonal antibodies against a mouse nuclear PIWI protein, MIWI2, which functions in de novo DNA methylation of target transposon loci. We then describe how to use the antibodies to isolate associated complexes and to detect MIWI2 immunohistochemically.


Assuntos
Anticorpos Monoclonais/metabolismo , Proteínas Argonautas/imunologia , RNA Interferente Pequeno/genética , Animais , Epigênese Genética , Redes Reguladoras de Genes , Imunização , Masculino , Camundongos , Células NIH 3T3 , Testículo/imunologia , Testículo/metabolismo
9.
Primates ; 57(1): 129-35, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26530217

RESUMO

Spermatogonia are specialized cells responsible for continuous spermatogenesis and the production of offspring. Because of this biological property, in vitro culture of spermatogonia provides a powerful methodology to advance reproductive biology and engineering. However, methods for culturing primate spermatogonia are poorly established. We have designed a novel method for culturing spermatogonia in the common marmoset (Callithrix jacchus), a small primate. By using our method with a suite of growth factors, adult marmoset testis-derived germ cells could be cultured in the form of a floating sphere for several weeks. Notably, this method could be applied not only to freshly isolated cells but also to cryopreserved cell stocks. The spheres enriched spermatogonia and early spermatocytes, and could be assembled from a C-KIT(+) spermatogonial population. Techniques for culturing spermatogonia could facilitate increased understanding of primate reproduction as well as the preservation of valuable biomaterials from nonhuman primates.


Assuntos
Callithrix/fisiologia , Técnicas de Cultura de Células/métodos , Espermatogênese , Espermatogônias/crescimento & desenvolvimento , Testículo/crescimento & desenvolvimento , Animais , Masculino
10.
Curr Biol ; 25(7): R280-3, 2015 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-25829012

RESUMO

Technologies have been developed in animal germ cells that produce artificial piRNAs from transgenes in piRNA clusters to silence target genes by cleaving their transcripts. A new study provides a simple way to generate artificial piRNAs to direct de novo DNA methylation in mice.


Assuntos
Metilação de DNA , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , RNA Interferente Pequeno , Animais , Humanos , Masculino
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA