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1.
Nat Commun ; 12(1): 4802, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376665

RESUMO

During meiosis, gene expression is silenced in aberrantly unsynapsed chromatin and in heterogametic sex chromosomes. Initiation of sex chromosome silencing is disrupted in meiocytes with sex chromosome-autosome translocations. To determine whether this is due to aberrant synapsis or loss of continuity of sex chromosomes, we engineered Caenorhabditis elegans nematodes with non-translocated, bisected X chromosomes. In early meiocytes of mutant males and hermaphrodites, X segments are enriched with euchromatin assembly markers and active RNA polymerase II staining, indicating active transcription. Analysis of RNA-seq data showed that genes from the X chromosome are upregulated in gonads of mutant worms. Contrary to previous models, which predicted that any unsynapsed chromatin is silenced during meiosis, our data indicate that unsynapsed X segments are transcribed. Therefore, our results suggest that sex chromosome chromatin has a unique character that facilitates its meiotic expression when its continuity is lost, regardless of whether or not it is synapsed.


Assuntos
Caenorhabditis elegans/genética , Inativação Gênica , Meiose/genética , Cromossomo X/genética , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Pareamento Cromossômico/genética , Feminino , Células Germinativas/citologia , Células Germinativas/metabolismo , Histonas/metabolismo , Hibridização in Situ Fluorescente , Masculino , Microscopia de Fluorescência , Transcrição Genética
2.
Aging Cell ; 20(6): e13386, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34061407

RESUMO

Oogenesis is one of the first processes to fail during aging. In women, most oocytes cannot successfully complete meiotic divisions already during the fourth decade of life. Studies of the nematode Caenorhabditis elegans have uncovered conserved genetic pathways that control lifespan, but our knowledge regarding reproductive aging in worms and humans is limited. Specifically, little is known about germline internal signals that dictate the oogonial biological clock. Here, we report a thorough characterization of the changes in the worm germline during aging. We found that shortly after ovulation halts, germline proliferation declines, while apoptosis continues, leading to a gradual reduction in germ cell numbers. In late aging stages, we observed that meiotic progression is disturbed and crossover designation and DNA double-strand break repair decrease. In addition, we detected a decline in the quality of mature oocytes during aging, as reflected by decreasing size and elongation of interhomolog distance, a phenotype also observed in human oocytes. Many of these altered processes were previously attributed to MAPK signaling variations in young worms. In support of this, we observed changes in activation dynamics of MPK-1 during aging. We therefore tested the hypothesis that MAPK controls oocyte quality in aged worms using both genetic and pharmacological tools. We found that in mutants with high levels of activated MPK-1, oocyte quality deteriorates more rapidly than in wild-type worms, whereas reduction of MPK-1 levels enhances quality. Thus, our data suggest that MAPK signaling controls germline aging and could be used to attenuate the rate of oogenesis quality decline.

3.
RNA Biol ; 18(3): 435-445, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32892705

RESUMO

Long intergenic non-coding RNAs (lincRNAs) are transcripts longer than 200 nucleotides that are transcribed from non-coding loci yet undergo biosynthesis similar to coding mRNAs. The disproportional number of lincRNAs expressed in testes suggests that lincRNAs are important during gametogenesis, but experimental evidence has implicated very few lincRNAs in this process. We took advantage of the relatively limited number of lincRNAs in the genome of the nematode Caenorhabditis elegans to systematically analyse the functions of lincRNAs during meiosis. We deleted six lincRNA genes that are highly and dynamically expressed in the C. elegans gonad and tested the effects on central meiotic processes. Surprisingly, whereas the lincRNA deletions did not strongly impact fertility, germline apoptosis, crossovers, or synapsis, linc-4 was required for somatic growth. Slower growth was observed in linc-4-deletion mutants and in worms depleted of linc-4 using RNAi, indicating that linc-4 transcripts are required for this post-embryonic process. Unexpectedly, analysis of worms depleted of linc-4 in soma versus germline showed that the somatic role stems from linc-4 expression in germline cells. This unique feature suggests that some lincRNAs, like some small non-coding RNAs, are required for germ-soma interactions.

4.
Genetics ; 212(1): 213-229, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30867196

RESUMO

During meiosis, a series of evolutionarily conserved events allow for reductional chromosome division, which is required for sexual reproduction. Although individual meiotic processes have been extensively studied, we currently know far less about how meiosis is regulated and coordinated. In the Caenorhabditis elegans gonad, mitogen-activated protein kinase (MAPK) signaling drives oogenesis while undergoing spatial activation and deactivation waves. However, it is currently unclear how MAPK activation is governed and how it facilitates the progression of oogenesis. Here, we show that the oocyte and germline-related 2 (ogr-2) gene affects proper progression of oogenesis. Complete deletion of ogr-2 results in delayed meiotic entry and late spatial onset of double-strand break repair. Elevated levels of apoptosis are observed in this mutant, independent of the meiotic canonical checkpoints; however, they are dependent on the MAPK terminal member MPK-1/ERK. MPK-1 activation is elevated in diplotene in ogr-2 mutants and its aberrant spatial activation correlates with stages where meiotic progression defects are evident. Deletion of ogr-2 significantly reduces the expression of lip-1, a phosphatase reported to repress MPK-1, which is consistent with OGR-2 localization at chromatin in germ cells. We suggest that OGR-2 modulates the expression of lip-1 to promote the timely progression of meiosis through MPK-1 spatial deactivation.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas do Ovo/metabolismo , Sistema de Sinalização das MAP Quinases , Meiose , Oogênese , Proteínas Tirosina Fosfatases/metabolismo , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/fisiologia , Proteínas do Ovo/fisiologia , Feminino
5.
Elife ; 72018 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-30403374

RESUMO

Although aging-regulating pathways were discovered a few decades ago, it is not entirely clear how their activities are orchestrated, to govern lifespan and proteostasis at the organismal level. Here, we utilized the nematode Caenorhabditis elegans to examine whether the alteration of aging, by reducing the activity of the Insulin/IGF signaling (IIS) cascade, affects protein SUMOylation. We found that IIS activity promotes the SUMOylation of the germline protein, CAR-1, thereby shortening lifespan and impairing proteostasis. In contrast, the expression of mutated CAR-1, that cannot be SUMOylated at residue 185, extends lifespan and enhances proteostasis. A mechanistic analysis indicated that CAR-1 mediates its aging-altering functions, at least partially, through the notch-like receptor glp-1. Our findings unveil a novel regulatory axis in which SUMOylation is utilized to integrate the aging-controlling functions of the IIS and of the germline and provide new insights into the roles of SUMOylation in the regulation of organismal aging.


Assuntos
Envelhecimento/metabolismo , Caenorhabditis elegans/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Insulina/metabolismo , Proteostase , Transdução de Sinais , Sumoilação , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Técnicas de Silenciamento de Genes , Células Germinativas/metabolismo , Gônadas/metabolismo , Longevidade , Modelos Biológicos , Estresse Fisiológico , Transcrição Genética
6.
Genetics ; 210(2): 587-605, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30093412

RESUMO

Developmental programs are executed by tightly controlled gene regulatory pathways. Here, we combined the unique sample retrieval capacity afforded by laser capture microscopy with analysis of mRNA abundance by CEL-Seq (cell expression by linear amplification and sequencing) to generate a spatiotemporal gene expression map of the Caenorhabditis elegans syncytial germline from adult hermaphrodites and males. We found that over 6000 genes exhibit spatiotemporally dynamic expression patterns throughout the hermaphrodite germline, with two dominant groups of genes exhibiting reciprocal shifts in expression at late pachytene during meiotic prophase I. We found a strong correlation between restricted spatiotemporal expression and known developmental and cellular processes, indicating that these gene expression changes may be an important driver of germ cell progression. Analysis of the male gonad revealed a shift in gene expression at early pachytene and upregulation of subsets of genes following the meiotic divisions, specifically in early and late spermatids, mostly transcribed from the X chromosome. We observed that while the X chromosome is silenced throughout the first half of the gonad, some genes escape this control and are highly expressed throughout the germline. Although we found a strong correlation between the expression of genes corresponding to CSR-1-interacting 22G-RNAs during germ cell progression, we also found that a large fraction of genes may bypass the need for CSR-1-mediated germline licensing. Taken together, these findings suggest the existence of mechanisms that enable a shift in gene expression during prophase I to promote germ cell progression.


Assuntos
Gametogênese , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Células Germinativas/citologia , Meiose , Cromossomo X/genética , Inativação do Cromossomo X
7.
Elife ; 5: e12039, 2016 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-26920220

RESUMO

Asymmetric disassembly of the synaptonemal complex (SC) is crucial for proper meiotic chromosome segregation. However, the signaling mechanisms that directly regulate this process are poorly understood. Here we show that the mammalian Rho GEF homolog, ECT-2, functions through the conserved RAS/ERK MAP kinase signaling pathway in the C. elegans germline to regulate the disassembly of SC proteins. We find that SYP-2, a SC central region component, is a potential target for MPK-1-mediated phosphorylation and that constitutively phosphorylated SYP-2 impairs the disassembly of SC proteins from chromosomal domains referred to as the long arms of the bivalents. Inactivation of MAP kinase at late pachytene is critical for timely disassembly of the SC proteins from the long arms, and is dependent on the crossover (CO) promoting factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1. We propose that the conserved MAP kinase pathway coordinates CO designation with the disassembly of SC proteins to ensure accurate chromosome segregation.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Segregação de Cromossomos , Troca Genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Sistema de Sinalização das MAP Quinases , Meiose , Processamento de Proteína Pós-Traducional , Complexo Sinaptonêmico/metabolismo , Animais , Caenorhabditis elegans , Linhagem Celular , Proteína Quinase 1 Ativada por Mitógeno/metabolismo
8.
Genetics ; 195(3): 1181-5, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23979579

RESUMO

We adapted the CRISPR-Cas9 system for template-mediated repair of targeted double-strand breaks via homologous recombination in Caenorhabditis elegans, enabling customized and efficient genome editing. This system can be used to create specific insertions, deletions, and base pair changes in the germline of C. elegans.


Assuntos
Sistemas CRISPR-Cas , Caenorhabditis elegans/genética , Genoma Helmíntico , Animais , Quebras de DNA de Cadeia Dupla , DNA de Helmintos/genética , Marcação de Genes , Engenharia Genética , Recombinação Homóloga , Mutagênese , Mutagênese Insercional , Edição de RNA/genética
9.
Nat Methods ; 10(8): 741-3, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23817069

RESUMO

We report the use of clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease Cas9 to target genomic sequences in the Caenorhabditis elegans germ line using single-guide RNAs that are expressed from a U6 small nuclear RNA promoter. Our results demonstrate that targeted, heritable genetic alterations can be achieved in C. elegans, providing a convenient and effective approach for generating loss-of-function mutants.


Assuntos
Caenorhabditis elegans/genética , Genoma , Sequências Repetidas Invertidas , RNA Guia/genética , RNA Nuclear Pequeno/genética , Animais , Engenharia Genética/métodos , Regiões Promotoras Genéticas , Proteínas de Peixe-Zebra/genética
10.
PLoS Biol ; 10(8): e1001378, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22927794

RESUMO

Successful execution of the meiotic program depends on the timely establishment and removal of sister chromatid cohesion. LAB-1 has been proposed to act in the latter by preventing the premature removal of the meiosis-specific cohesin REC-8 at metaphase I in C. elegans, yet the mechanism and scope of LAB-1 function remained unknown. Here we identify an unexpected earlier role for LAB-1 in promoting the establishment of sister chromatid cohesion in prophase I. LAB-1 and REC-8 are both required for the chromosomal association of the cohesin complex subunit SMC-3. Depletion of lab-1 results in partial loss of sister chromatid cohesion in rec-8 and coh-4 coh-3 mutants and further enhanced chromatid dissociation in worms where all three kleisins are mutated. Moreover, lab-1 depletion results in increased Aurora B kinase (AIR-2) signals in early prophase I nuclei, coupled with a parallel decrease in signals for the PP1 homolog, GSP-2. Finally, LAB-1 directly interacts with GSP-1 and GSP-2. We propose that LAB-1 targets the PP1 homologs to the chromatin at the onset of meiosis I, thereby antagonizing AIR-2 and cooperating with the cohesin complex to promote sister chromatid association and normal progression of the meiotic program.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Cromátides/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Prófase Meiótica I , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Aurora Quinase B , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Troca Genética , Reparo do DNA , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismo , Técnicas do Sistema de Duplo-Híbrido
12.
Proc Natl Acad Sci U S A ; 103(36): 13397-402, 2006 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-16938876

RESUMO

In Caenorhabditis elegans, the antiapoptotic protein CED-9 is localized at the mitochondria, where it binds the proapoptotic protein CED-4. Induction of apoptosis begins when the proapoptotic protein EGL-1 is expressed and binds CED-9. The binding of EGL-1 to CED-9 releases CED-4 from CED-9 and causes the activation of the caspase CED-3. Upon its release from CED-9, CED-4 rapidly translocates to the nuclear envelope (NE) in a CED-3-independent manner. However, the identity of the NE receptor for CED-4 and its possible role in the execution of apoptosis has remained unknown. Here, we show that the inner nuclear membrane SUN-domain protein matefin/SUN-1 is the NE receptor for CED-4. Our data demonstrate that matefin/SUN-1 binds CED-4 and is specifically required for CED-4 translocation and maintenance at the NE. The role of matefin/SUN-1 in the execution of apoptosis is further suggested by the significant reduction in the number of apoptotic cells in the organism after matefin/SUN-1 down-regulation by RNAi. The finding that matefin/SUN-1 is required for the execution of apoptosis adds an important link between cytoplasmic and nuclear apoptotic events.


Assuntos
Apoptose , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Helminto/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Caenorhabditis elegans/genética , Caspases/metabolismo , Regulação para Baixo , Ativação Enzimática , Mitocôndrias/metabolismo , Membrana Nuclear/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Interferência de RNA , Proteínas Repressoras/metabolismo
13.
Nat Rev Mol Cell Biol ; 7(10): 782-8, 2006 10.
Artigo em Inglês | MEDLINE | ID: mdl-16926857

RESUMO

The novel SUN-domain family of nuclear envelope proteins interacts with various KASH-domain partners to form SUN-domain-dependent 'bridges' across the inner and outer nuclear membranes. These bridges physically connect the nucleus to every major component of the cytoskeleton. SUN-domain proteins have diverse roles in nuclear positioning, centrosome localization, germ-cell development, telomere positioning and apoptosis. By serving both as mechanical adaptors and nuclear envelope receptors, we propose that SUN-domain proteins connect cytoplasmic and nucleoplasmic activities.


Assuntos
Citoesqueleto/fisiologia , Membrana Nuclear/fisiologia , Proteínas Nucleares/fisiologia , Animais , Citoesqueleto/metabolismo , Previsões , Modelos Biológicos , Membrana Nuclear/química , Proteínas Nucleares/metabolismo , Filogenia , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia
14.
J Struct Biol ; 140(1-3): 232-40, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12490171

RESUMO

Nuclear membranes and nuclear pore complexes (NPCs) are conserved in both animals and plants. However, the lamina composition and the dimensions of NPCs vary between plants, yeast, and vertebrates. In this study, we established a protocol that preserves the structure of Caenorhabditis elegans embryonic cells for high-resolution studies with thin-section transmission electron microscopy (TEM). We show that the NPCs are bigger in C. elegans embryos than in yeast, with dimensions similar to those in higher eukaryotes. We also localized the C. elegans nuclear envelope proteins Ce-lamin and Ce-emerin by pre-embedding gold labeling immunoelectron microscopy. Both proteins are present at or near the inner nuclear membrane. A fraction of Ce-lamin, but not Ce-emerin, is present in the nuclear interior. Removing the nuclear membranes leaves both Ce-lamin and Ce-emerin associated with the chromatin. Eliminating the single lamin protein caused cell death as visualized by characteristic changes in nuclear architecture including condensation of chromatin, clustering of NPCs, membrane blebbing, and the presence of vesicles inside the nucleus. Taken together, these results show evolutionarily conserved protein localization, interactions, and functions of the C. elegans nuclear envelope.


Assuntos
Caenorhabditis elegans/ultraestrutura , Núcleo Celular/ultraestrutura , Membrana Nuclear/ultraestrutura , Animais , Caenorhabditis elegans/embriologia , Morte Celular , Núcleo Celular/metabolismo , Imuno-Histoquímica , Laminas/metabolismo , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Microscopia Imunoeletrônica , Lâmina Nuclear , Proteínas Nucleares , Interferência de RNA , Timopoietinas/metabolismo
15.
J Struct Biol ; 137(1-2): 146-53, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12064941

RESUMO

Invertebrates and in Drosophila, lamins and lamin-associated proteins are primary targets for cleavage by caspases. Eliminating mammalian lamins causes apoptosis, whereas expressing mutant lamins that cannot be cleaved by caspase-6 delay apoptosis. Caenorhabditis elegans has a single lamin protein, Ce-lamin, and a caspase, CED-3, that is responsible for most if not all somatic apoptosis. In this study we show that in C. elegans embryos induced to undergo apoptosis Ce-lamin is degraded surprisingly late. In such embryos CED-4 translocated to the nuclear envelope but the cytological localization of Ce-lamin remained similar to that in wild-type embryos. TUNEL labeling indicated that Ce-lamin was degraded only after DNA is fragmented. Ce-lamin, Ce-emerin, or Ce-MAN1 were not cleaved by recombinant CED-3, showing that these lamina proteins are not substrates for CED-3 cleavage. These results suggest that lamin cleavage probably is not essential for apoptosis in C. elegans.


Assuntos
Apoptose , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/fisiologia , Núcleo Celular/patologia , Núcleo Celular/fisiologia , Laminas/fisiologia , Lâmina Nuclear/fisiologia , Animais , Caenorhabditis elegans/metabolismo , Caspases/biossíntese , Técnica Indireta de Fluorescência para Anticorpo , Marcação In Situ das Extremidades Cortadas , Laminas/metabolismo , Proteínas de Membrana/biossíntese , Proteínas Nucleares/biossíntese , Plasmídeos/metabolismo , Ligação Proteica
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