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1.
Nat Commun ; 12(1): 4498, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301931

RESUMO

In animal germlines, PIWI proteins and the associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons. Here we report the extensive sequence and quantitative correlations between 2',3'-cyclic phosphate-containing RNAs (cP-RNAs), identified using cP-RNA-seq, and piRNAs in the Bombyx germ cell line and mouse testes. The cP-RNAs containing 5'-phosphate (P-cP-RNAs) identified by P-cP-RNA-seq harbor highly consistent 5'-end positions as the piRNAs and are loaded onto PIWI protein, suggesting their direct utilization as piRNA precursors. We identified Bombyx RNase Kappa (BmRNase κ) as a mitochondria-associated endoribonuclease which produces cP-RNAs during piRNA biogenesis. BmRNase κ-depletion elevated transposon levels and disrupted a piRNA-mediated sex determination in Bombyx embryos, indicating the crucial roles of BmRNase κ in piRNA biogenesis and embryonic development. Our results reveal a BmRNase κ-engaged piRNA biogenesis pathway, in which the generation of cP-RNAs promotes robust piRNA production.


Assuntos
Endorribonucleases/genética , Perfilação da Expressão Gênica/métodos , Proteínas de Insetos/genética , RNA Interferente Pequeno/genética , RNA/genética , Animais , Sequência de Bases , Bombyx , Linhagem Celular , Endorribonucleases/metabolismo , Feminino , Proteínas de Insetos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mutação , Ácidos Fosfatídicos/química , RNA/química , RNA/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA-Seq/métodos , Testículo/metabolismo
2.
PLoS Genet ; 15(11): e1008469, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31721758

RESUMO

RNA molecules generated by ribonuclease cleavage sometimes harbor a 2',3'-cyclic phosphate (cP) at their 3'-ends. Those cP-containing RNAs (cP-RNAs) form a hidden layer of transcriptome because standard RNA-seq cannot capture them as a result of cP's prevention of an adapter ligation reaction. Here we provide genome-wide analyses of short cP-RNA transcriptome across multiple mouse tissues. Using cP-RNA-seq that can exclusively sequence cP-RNAs, we identified numerous novel cP-RNA species which are mainly derived from cytoplasmic tRNAs, mRNAs, and rRNAs. Determination of the processing sites of substrate RNAs for cP-RNA generation revealed highly-specific RNA cleavage events between cytidine and adenosine in cP-RNA biogenesis. cP-RNAs were not evenly derived from the overall region of substrate RNAs but rather from specific sites, implying that cP-RNAs are not from random degradation but are produced through a regulated biogenesis pathway. The identified cP-RNAs were abundantly accumulated in mouse tissues, and the expression levels of cP-RNAs showed age-dependent reduction. These analyses of cP-RNA transcriptome unravel a novel, abundant class of non-coding RNAs whose expression could have physiological roles.


Assuntos
Envelhecimento/genética , Sequência de Bases/genética , RNA/genética , Transcriptoma/genética , Envelhecimento/patologia , Animais , Regulação da Expressão Gênica/genética , Genômica , Humanos , Camundongos , Fosfatos/química , Fosfatos/metabolismo , RNA/química , Clivagem do RNA/genética , RNA Ribossômico/genética , RNA Nucleolar Pequeno/genética , RNA de Transferência/genética , RNA não Traduzido/genética , Análise de Sequência de RNA
3.
Nucleic Acids Res ; 45(15): 9108-9120, 2017 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-28645172

RESUMO

Transfer RNAs (tRNAs) function in translational machinery and further serves as a source of short non-coding RNAs (ncRNAs). tRNA-derived ncRNAs show differential expression profiles and play roles in many biological processes beyond translation. Molecular mechanisms that shape and regulate their expression profiles are largely unknown. Here, we report the mechanism of biogenesis for tRNA-derived Piwi-interacting RNAs (td-piRNAs) expressed in Bombyx BmN4 cells. In the cells, two cytoplasmic tRNA species, tRNAAspGUC and tRNAHisGUG, served as major sources for td-piRNAs, which were derived from the 5'-part of the respective tRNAs. cP-RNA-seq identified the two tRNAs as major substrates for the 5'-tRNA halves as well, suggesting a previously uncharacterized link between 5'-tRNA halves and td-piRNAs. An increase in levels of the 5'-tRNA halves, induced by BmNSun2 knockdown, enhanced the td-piRNA expression levels without quantitative change in mature tRNAs, indicating that 5'-tRNA halves, not mature tRNAs, are the direct precursors for td-piRNAs. For the generation of tRNAHisGUG-derived piRNAs, BmThg1l-mediated nucleotide addition to -1 position of tRNAHisGUG was required, revealing an important function of BmThg1l in piRNA biogenesis. Our study advances the understanding of biogenesis mechanisms and the genesis of specific expression profiles for tRNA-derived ncRNAs.


Assuntos
Proteínas Argonauta/genética , Bombyx/genética , Proteínas de Insetos/genética , RNA Interferente Pequeno/genética , RNA de Transferência de Ácido Aspártico/genética , RNA de Transferência de Histidina/genética , Animais , Proteínas Argonauta/metabolismo , Sequência de Bases , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/crescimento & desenvolvimento , Células Germinativas/metabolismo , Proteínas de Insetos/metabolismo , Conformação de Ácido Nucleico , RNA Interferente Pequeno/metabolismo , RNA de Transferência de Ácido Aspártico/metabolismo , RNA de Transferência de Histidina/metabolismo
4.
Sci Rep ; 7(1): 4110, 2017 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-28646211

RESUMO

Piwi proteins and their bound Piwi-interacting RNAs (piRNAs) are predominantly expressed in the germline and play crucial roles in germline development by silencing transposons and other targets. Bombyx mori BmN4 cells are culturable germ cells that equip the piRNA pathway. Because of the scarcity of piRNA-expressing culturable cells, BmN4 cells are being utilized for the analyses of piRNA biogenesis. We here report that the piRNA biogenesis in BmN4 cells is regulated by cell density. As cell density increased, the abundance of Piwi proteins and piRNA biogenesis factors was commonly upregulated, resulting in an increased number of perinuclear nuage-like granules where Piwi proteins localize. Along with these phenomena, the abundance of mature piRNAs also globally increased, whereas levels of long piRNA precursor and transposons decreased, suggesting that increasing cell density promotes piRNA biogenesis pathway and that the resultant accumulation of mature piRNAs is functionally significant for transposon silencing. Our study reveals a previously uncharacterized link between cell density and piRNA biogenesis, designates cell density as a critical variable in piRNA studies using BmN4 cell system, and suggests the alteration of cell density as a useful tool to monitor piRNA biogenesis and function.


Assuntos
Bombyx/genética , Células Germinativas/metabolismo , RNA Interferente Pequeno/genética , Animais , Contagem de Células , Linhagem Celular , Células Cultivadas , Biologia Computacional , Grânulos Citoplasmáticos/metabolismo , Elementos de DNA Transponíveis , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo
5.
Dev Growth Differ ; 58(8): 641-650, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27585825

RESUMO

In many animals, the germ plasm is sufficient and necessary for primordial germ cell (PGC) formation. It contains germinal granules and abundant mitochondria (germline-Mt). However, the role of germline-Mt in germ cell formation remains poorly understood. In Xenopus, the germ plasm is distributed as many small islands at the vegetal pole, which gradually aggregates to form a single large mass in each of the four vegetal pole cells at the early blastula stage. Polymerized microtubules and the adapter protein kinesin are required for the aggregation of germ plasm. However, it remains unknown whether germline-Mt trafficking is important for the cytoplasmic transport of germinal granules during germ plasm aggregation. In this study, we focused on the mitochondrial small GTPase protein Rhot1 to inhibit mitochondrial trafficking during the germ plasm aggregation. Expression of Rhot1ΔC, which lacks the C-terminal mitochondrial transmembrane domain, inhibited the aggregation of germline-Mt during early development. In Rhot1-inhibited embryos, germinal granule components did not aggregate during cleavage stages, which reduced the number of PGCs on the genital ridge at tail-bud stage. These results suggest that mitochondrial trafficking is involved in the aggregation of germinal granule components, which are essential for the formation of PGCs.


Assuntos
Embrião não Mamífero/embriologia , Células Germinativas/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Proteínas Mitocondriais/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Embrião não Mamífero/citologia , Desenvolvimento Embrionário/fisiologia , Células Germinativas/citologia , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas de Xenopus/genética , Xenopus laevis , Proteínas rho de Ligação ao GTP/genética
6.
Nat Protoc ; 11(3): 476-89, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26866791

RESUMO

RNA digestions catalyzed by many ribonucleases generate RNA fragments that contain a 2',3'-cyclic phosphate (cP) at their 3' termini. However, standard RNA-seq methods are unable to accurately capture cP-containing RNAs because the cP inhibits the adapter ligation reaction. We recently developed a method named cP-RNA-seq that is able to selectively amplify and sequence cP-containing RNAs. Here we describe the cP-RNA-seq protocol in which the 3' termini of all RNAs, except those containing a cP, are cleaved through a periodate treatment after phosphatase treatment; hence, subsequent adapter ligation and cDNA amplification steps are exclusively applied to cP-containing RNAs. cP-RNA-seq takes ∼6 d, excluding the time required for sequencing and bioinformatics analyses, which are not covered in detail in this protocol. Biochemical validation of the existence of cP in the identified RNAs takes ∼3 d. Even though the cP-RNA-seq method was developed to identify angiogenin-generating 5'-tRNA halves as a proof of principle, the method should be applicable to global identification of cP-containing RNA repertoires in various transcriptomes.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Fosfatos/análise , RNA/genética , Análise de Sequência de RNA/métodos , Linhagem Celular Tumoral , DNA Complementar/genética , Humanos , Fosfatos/metabolismo , RNA/química , RNA de Transferência/química , RNA de Transferência/genética , Ribonuclease Pancreático/genética
7.
RNA Biol ; 12(5): 501-8, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25833336

RESUMO

Transfer RNAs (tRNAs) play a central role in translation and also recently appear to have a variety of other functions in biological processes beyond translation. Here we report the development of Four-Leaf clover qRT-PCR (FL-PCR), a convenient PCR-based method, which can specifically quantify individual mature tRNA species. In FL-PCR, T4 RNA ligase 2 specifically ligates a stem-loop adapter to mature tRNAs but not to precursor tRNAs or tRNA fragments. Subsequent TaqMan qRT-PCR amplifies only unmodified regions of the tRNA-adapter ligation products; therefore, FL-PCR quantification is not influenced by tRNA post-transcriptional modifications. FL-PCR has broad applicability for the quantification of various tRNAs in different cell types, and thus provides a much-needed simple method for analyzing tRNA abundance and heterogeneity.


Assuntos
Conformação de Ácido Nucleico , RNA de Transferência/química , RNA de Transferência/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sequência de Bases , Linhagem Celular Tumoral , Humanos , Dados de Sequência Molecular
8.
Zygote ; 22(2): 266-74, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-23186935

RESUMO

The POU family subclass V (POU-V) proteins have important roles in maintaining cells in an undifferentiated state. In Xenopus, expression of the POU-V protein Oct60 was detected in oocytes and was found to decrease in blastula- to gastrula-stage embryos. In addition, Oct60 overexpression inhibits some signals in early embryogenesis, including Activin/Nodal, BMP, and Wnt signalling. In this report, we analysed mechanisms of Oct60 promoter activation and discovered that Oct60 transcription was activated ectopically in somatic nuclei by oocyte extract treatment. Promoter assays demonstrated that Oct60 transcription was activated in oocytes specifically and that this activation was dependent on an Octamer-Sox binding motif. ChIP assays showed that the Oct60 protein binds the motif. These results suggest that Oct60 transcription is regulated by a positive-feedback loop in Xenopus oocytes.


Assuntos
Embrião não Mamífero/fisiologia , Retroalimentação Fisiológica , Regulação da Expressão Gênica no Desenvolvimento , Oócitos/fisiologia , Transcrição Genética/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Sequência de Bases , Blástula/citologia , Blástula/fisiologia , Imunoprecipitação da Cromatina , Embrião não Mamífero/citologia , Feminino , Gástrula/citologia , Gástrula/fisiologia , Luciferases , Dados de Sequência Molecular , Oócitos/citologia , Fatores do Domínio POU , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais , Proteínas de Xenopus/genética , Xenopus laevis/genética
9.
Dev Growth Differ ; 55(2): 217-28, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23278717

RESUMO

Primordial germ cells (PGCs) arise in the early embryo and migrate toward the future gonad through species-specific pathways. They are assumed to change their migration properties dependent on their own genetic program and/or environmental cues, though information concerning the developmental change in PGC motility is limited. First, we re-examined the distribution of PGCs in the endodermal region of Xenopus embryos at various stages by using an antibody against Xenopus Daz-like protein, and found four stages of migration, namely clustering, dispersing, directionally migrating and re-aggregating. Next, we isolated living PGCs at each stage and directly examined their morphology and locomotive activity in cell cultures. PGCs at the clustering stage were round in shape with small blebs and showed little motility. PGCs in both the dispersing and the directionally migrating stages alternated between the locomotive phase with an elongated morphology and the pausing phase with a rugged morphology. The locomotive activity of the elongated PGCs was accompanied by the persistent formation of a large bleb at the leading front. The duration of the locomotive phase was shortened gradually with the transition from the dispersing stage to the directionally migrating stage. At the re-aggregating stage, PGCs became round in shape and showed no motility. Thus, we directly showed that the locomotive activity of PGCs changes dynamically depending upon the migrating stage. We also showed that the locomotion and blebbing of the PGCs required F-actin, myosin II activity and RhoA/Rho-associated protein kinase (ROCK) signaling.


Assuntos
Movimento Celular , Embrião não Mamífero/citologia , Células Germinativas/citologia , Xenopus laevis/embriologia , Actinas/metabolismo , Animais , Forma Celular , Células Cultivadas , Ectoderma/citologia , Ectoderma/embriologia , Ectoderma/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Células Germinativas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Miosina Tipo II/metabolismo , Transdução de Sinais , Fatores de Tempo , Imagem com Lapso de Tempo/métodos , Proteínas de Xenopus/metabolismo , Quinases Associadas a rho/metabolismo
10.
Dev Biol ; 349(2): 462-9, 2011 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-21112323

RESUMO

Mitochondria are accurately transmitted to the next generation through a female germ cell in most animals. Mitochondria produce most ATP, accompanied by the generation of reactive oxygen species (ROS). A specialized mechanism should be necessary for inherited mitochondria to escape from impairments of mtDNA by ROS. Inherited mitochondria are named germ-line mitochondria, in contrast with somatic ones. We hypothesized that germ-line mitochondria are distinct from somatic ones. The protein profiles of germ-line and somatic mitochondria were compared, using oocytes at two different stages in Xenopus laevis. Some subunits of ATP synthase were at a low level in germ-line mitochondria, which was confirmed immunologically. Ultrastructural histochemistry using 3,3'-diaminobenzidine (DAB) showed that cytochrome c oxidase (COX) activity of germ-line mitochondria was also at a low level. Mitochondria in one oocyte were segregated into germ-line mitochondria and somatic mitochondria, during growth from stage I to VI oocytes. Respiratory activity represented by ATP synthase expression and COX activity was shown to be low during most of the long gametogenetic period. We propose that germ-line mitochondria that exhibit suppressed respiration alleviate production of ROS and enable transmission of accurate mtDNA from generation to generation.


Assuntos
Células Germinativas/citologia , Mitocôndrias/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Xenopus laevis/embriologia , 3,3'-Diaminobenzidina , Animais , Western Blotting , Respiração Celular/fisiologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Perfilação da Expressão Gênica , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Oócitos/metabolismo , Xenopus laevis/metabolismo
11.
Dev Growth Differ ; 52(2): 235-44, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20151992

RESUMO

Primordial germ cells (PGCs) in Xenopus embryo are specified in the endodermal cell mass and migrate dorsally toward the future gonads. The role of the signal mediated by Notch and Suppressor of Hairless [Su(H)] was analyzed on the migrating PGCs at the tailbud stage. X-Notch-1 and X-Delta-1 are expressed in the migrating PGCs and surrounding endodermal cells, whereas X-Delta-2 and X-Serrate-1 are expressed preferentially in the PGCs. Suppression and constitutive activation of the Notch/Su(H) signaling in the whole endoderm region or selectively in the PGCs resulted in an increase in ectopic PGCs located in lateral or ventral regions. Knocking down of the Notch ligands by morpholino oligonucleotides revealed that X-Delta-2 was indispensable for the correct PGC migration. The ectopic PGCs seemed to have lost their motility in the Notch/Su(H) signal-manipulated embryos. Our results suggest that a cell-to-cell interaction via the Notch/Su(H) pathway has a significant role in the PGC migration by regulating cell motility.


Assuntos
Movimento Celular , Células Germinativas/citologia , Células Germinativas/metabolismo , Receptores Notch/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Endoderma/citologia , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Receptores Notch/genética , Fatores de Transcrição/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética
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