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1.
EMBO J ; 42(11): e105002, 2023 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-37078421

RESUMEN

During meiosis, chromosomes with homologous partners undergo synaptonemal complex (SC)-mediated pairing, while the remaining unpaired chromosomes are heterochromatinized through unpaired silencing. Mechanisms underlying homolog recognition during SC formation are still unclear. Here, we show that the Caenorhabditis elegans Argonaute proteins, CSR-1 and its paralog CSR-2, interacting with 22G-RNAs, are required for synaptonemal complex formation with accurate homology. CSR-1 in nuclei and meiotic cohesin, constituting the SC lateral elements, were associated with nonsimple DNA repeats, including minisatellites and transposons, and weakly associated with coding genes. CSR-1-associated CeRep55 minisatellites were expressing 22G-RNAs and long noncoding (lnc) RNAs that colocalized with synaptonemal complexes on paired chromosomes and with cohesin regions of unpaired chromosomes. CeRep55 multilocus deletions reduced the efficiencies of homologous pairing and unpaired silencing, which were supported by the csr-1 activity. Moreover, CSR-1 and CSR-2 were required for proper heterochromatinization of unpaired chromosomes. These findings suggest that CSR-1 and CSR-2 play crucial roles in homology recognition, achieving accurate SC formation between chromosome pairs and condensing unpaired chromosomes by targeting repeat-derived lncRNAs.


Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , ARN/metabolismo , Cromosomas , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Emparejamiento Cromosómico/genética , Complejo Sinaptonémico/metabolismo , Meiosis/genética
2.
EMBO J ; 26(24): 5007-19, 2007 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-18007599

RESUMEN

In the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) play important roles as intermediates. Primary siRNAs are produced from trigger dsRNAs by an RNaseIII-related enzyme called Dicer; in some organisms, secondary siRNAs are also produced by processes involving RNA-dependent RNA polymerases (RdRPs), which act on target mRNAs. Using a cell-free assay system prepared from Caenorhabditis elegans, we analyzed the production and activity of secondary siRNAs. In this cell-free system, RdRP activity acts on mRNA-derived templates to produce small RNAs. The RRF-1 complex is predominantly responsible for the RdRP activity, and synthesizes secondary-type siRNA molecules in a Dicer-independent manner. Notably, secondary-type siRNAs induce a prominent Slicer activity to cleave target mRNAs far more effectively than primary-type siRNAs. An Argonaute protein, CSR-1, is responsible for the Slicer activity induced by secondary-type siRNAs. Secondary rather than primary siRNAs may play a major role in the destabilization of target transcripts during RNAi in C. elegans.


Asunto(s)
Caenorhabditis elegans/genética , Interferencia de ARN , ARN de Helminto/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sistema Libre de Células , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Complejos Multiproteicos/metabolismo , ARN de Helminto/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleasa III , Transgenes
4.
Curr Biol ; 15(4): 378-83, 2005 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-15723801

RESUMEN

RNA interference (RNAi) is an ancient, highly conserved mechanism in which small RNA molecules (siRNAs) guide the sequence-specific silencing of gene expression . Several silencing machinery protein components have been identified, including helicases, RNase-related proteins, double- and single-stranded RNA binding proteins, and RNA-dependent RNA polymerase-related proteins . Work on these factors has led to the revelation that RNAi mechanisms intersect with cellular pathways required for development and fertility . Despite rapid progress in understanding key steps in the RNAi pathway, it is clear that many factors required for both RNAi and related developmental mechanisms have not yet been identified. Here, we report the characterization of the C. elegans gene rde-3. Genetic analysis of presumptive null alleles indicates that rde-3 is required for siRNA accumulation and for efficient RNAi in all tissues, and it is essential for fertility and viability at high temperatures. RDE-3 contains conserved domains found in the polymerase beta nucleotidyltransferase superfamily, which includes conventional poly(A) polymerases, 2'-5' oligoadenylate synthetase (OAS), and yeast Trf4p . These findings implicate a new enzymatic modality in RNAi and suggest possible models for the role of RDE-3 in the RNAi mechanism.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Modelos Biológicos , Nucleotidiltransferasas/metabolismo , Interferencia de ARN , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Cartilla de ADN , Fertilidad/genética , Componentes del Gen , Datos de Secuencia Molecular , Nucleotidiltransferasas/genética , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN
5.
Nucleic Acids Res ; 33(1): 347-55, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-15653635

RESUMEN

In Caenorhabditis elegans, the activity of transposable elements is repressed in the germline. One of the mechanisms involved in this repression is RNA interference (RNAi), a process in which dsRNA targets cleavage of mRNAs in a sequence-specific manner. The first gene found to be involved in RNAi and transposon silencing in C.elegans is mut-7, a gene encoding a putative exoribonuclease. Here, we show that the MUT-7 protein resides in complexes of approximately 250 kDa in the nucleus and in the cytosol. In addition, we find that upon triggering of RNAi the cytosolic MUT-7 complex increases in size. This increase is independent of the presence of target RNA, but does depend on the presence of RDE-1 and RDE-4, two proteins involved in small interfering RNA (siRNA) production. Finally, using a yeast two-hybrid screen, we identified RDE-2/MUT-8 as one of the other components of this complex. This protein is encoded by the rde-2/mut-8 locus, previously implicated in RNAi and transposon silencing. Using genetic complementation analysis, we show that the interaction between these two proteins is required for efficient RNAi in vivo. Together these data support a role for the MUT-7/RDE-2 complex downstream of siRNA formation, but upstream of siRNA mediated target RNA recognition, possibly indicating a role in the siRNA amplification step.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Interferencia de ARN , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Exorribonucleasas/química , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Biblioteca de Genes , Estructura Terciaria de Proteína , Técnicas del Sistema de Dos Híbridos
6.
Mol Biol Cell ; 14(7): 2972-83, 2003 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-12857879

RESUMEN

Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types. In many cases, this response has a systemic character in that silencing of gene expression is observed in cells distal from the site of dsRNA delivery. The molecular mechanisms underlying the mobile nature of RNA silencing are unknown. For example, although cellular entry of dsRNA is possible, cellular exit of dsRNA from normal animal cells has not been directly observed. We provide evidence that transgenic strains of Caenorhabditis elegans transcribing dsRNA from a tissue-specific promoter do not exhibit comprehensive systemic RNA interference phenotypes. In these same animals, modifications of environmental conditions can result in more robust systemic RNA silencing. Additionally, we find that genetic mutations can influence the systemic character of RNA silencing in C. elegans and can separate mechanisms underlying systemic RNA silencing into tissue-specific components. These data suggest that trafficking of RNA silencing signals in C. elegans is regulated by specific physiological and genetic factors.


Asunto(s)
Caenorhabditis elegans/metabolismo , Interferencia de ARN , Transporte de ARN/fisiología , Animales , Caenorhabditis elegans/genética , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Modelos Biológicos , Mutación , Regiones Promotoras Genéticas
8.
Cell ; 109(7): 861-71, 2002 Jun 28.
Artículo en Inglés | MEDLINE | ID: mdl-12110183

RESUMEN

Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. Here we show that the C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. Our findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Endorribonucleasas/metabolismo , Proteínas del Helminto/metabolismo , ARN Helicasas/química , ARN Helicasas/metabolismo , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Clonación Molecular , Secuencia Conservada , ARN Helicasas DEAD-box , Silenciador del Gen , Humanos , Datos de Secuencia Molecular , Mapeo Peptídico , Unión Proteica , ARN Helicasas/genética , ARN Bicatenario/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Ribonucleasa III , Homología de Secuencia de Aminoácido
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