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ABSTRACT: Argonaute (AGO) proteins interact with small RNAs to mediate gene silencing. C. elegans contains 27 AGO genes, raising the question of what roles these genes play in RNAi and related gene-silencing pathways. Here we describe 31 deletion alleles representing all of the previously uncharacterized AGO genes. Analysis of single- and multiple-AGO mutant strains reveals functions in several pathways, including (1) chromosome segregation, (2) fertility, and (3) at least two separate steps in the RNAi pathway. We show that RDE-1 interacts with trigger-derived sense and antisense RNAs to initiate RNAi, while several other AGO proteins interact with amplified siRNAs to mediate downstream silencing. Overexpression of downstream AGOs enhances silencing, suggesting that these proteins are limiting for RNAi. Interestingly, these AGO proteins lack key residues required for mRNA cleavage. Our findings support a two-step model for RNAi, in which functionally and structurally distinct AGOs act sequentially to direct gene silencing.
Cell 12/2006; 127(4):747-57. · 32.40 Impact Factor
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Thomas F Duchaine,
James A Wohlschlegel,
Scott Kennedy, Yanxia Bei,
Darryl Conte,
Kaming Pang,
Daniel R Brownell,
Sandra Harding,
Shohei Mitani,
Gary Ruvkun,
John R Yates,
Craig C Mello
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ABSTRACT: In plants, animals, and fungi, members of the Dicer family of RNase III-related enzymes process double-stranded RNA (dsRNA) to initiate small-RNA-mediated gene-silencing mechanisms. To learn how C. elegans Dicer, DCR-1, functions in multiple distinct silencing mechanisms, we used a mass-spectrometry-based proteomics approach to identify DCR-1-interacting proteins. We then generated and characterized deletion alleles for the corresponding genes. The interactors are required for production of three species of small RNA, including (1) small interfering RNAs (siRNAs), derived from exogenous dsRNA triggers (exo-siRNAs); (2) siRNAs derived from endogenous triggers (endo-siRNAs); and (3) developmental regulatory microRNAs (miRNAs). One interactor, the conserved RNA-phosphatase homolog PIR-1, is required for the processing of a putative amplified DCR-1 substrate. Interactors required for endo-siRNA production include ERI-1 and RRF-3, whose loss of function enhances RNAi. Our findings provide a first glimpse at the complex biochemical niche of Dicer and suggest that competition exists between DCR-1-mediated small-RNA pathways.
Cell 02/2006; 124(2):343-54. · 32.40 Impact Factor
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ABSTRACT: At the onset of embryogenesis, key developmental regulators called determinants are activated asymmetrically to specify the body axes and tissue layers. In C. elegans, this process is regulated in part by a conserved family of CCCH-type zinc finger proteins that specify the fates of early embryonic cells. The asymmetric localization of these and other determinants is regulated in early embryos through motor-dependent physical translocation as well as selective proteolysis.
We show here that the CCCH-type zinc finger protein OMA-1 serves as a nexus for signals that regulate the transition from oogenesis to embryogenesis. While OMA-1 promotes oocyte maturation during meiosis, destruction of OMA-1 is needed during the first cell division for the initiation of ZIF-1-dependent proteolysis of cell-fate determinants. Mutations in four conserved protein kinase genes-mbk-2/Dyrk, kin-19/CK1alpha, gsk-3, and cdk-1/CDC2-cause stabilization of OMA-1 protein, and their phenotypes are partially suppressed by an oma-1 loss-of-function mutation. OMA-1 proteolysis also depends on Cyclin B3 and on a ZIF-1-independent CUL-2-based E3 ubiquitin ligase complex, as well as the CUL-2-interacting protein ZYG-11 and the Skp1-related proteins SKR-1 and SKR-2.
Our findings suggest that a CDK1/Cyclin B3-dependent activity links OMA-1 proteolysis to completion of the first cell cycle and support a model in which OMA-1 functions to prevent the premature activation of cell-fate determinants until after they are asymmetrically partitioned during the first mitosis.
Current Biology 02/2006; 16(1):47-55. · 9.65 Impact Factor
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ABSTRACT: beta-Catenin regulates cell adhesion and cellular differentiation during development, and misregulation of beta-catenin contributes to numerous forms of cancer in humans. Here we describe Caenorhabditis elegans conditional alleles of mom-2/Wnt, mom-4/Tak1, and wrm-1/beta-catenin. We use these reagents to examine the regulation of WRM-1/beta-catenin during a Wnt-signaling-induced asymmetric cell division. While WRM-1 protein initially accumulates in the nuclei of all cells, signaling promotes the retention of WRM-1 in nuclei of responding cells. We show that both PRY-1/Axin and the nuclear exportin homolog IMB-4/CRM-1 antagonize signaling. These findings reveal how Wnt signals direct the asymmetric localization of beta-catenin during polarized cell division.
Genes & Development 09/2005; 19(15):1749-54. · 11.66 Impact Factor
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ABSTRACT: In early C. elegans embryos, signaling between a posterior blastomere, P2, and a ventral blastomere, EMS, specifies endoderm and orients the division axis of the EMS cell. Although Wnt signaling contributes to this polarizing interaction, no mutants identified to date abolish P2/EMS signaling. Here, we show that two tyrosine kinase-related genes, src-1 and mes-1, are required for the accumulation of phosphotyrosine between P2 and EMS. Moreover, src-1 and mes-1 mutants strongly enhance endoderm and EMS spindle rotation defects associated with Wnt pathway mutants. SRC-1 and MES-1 signal bidirectionally to control cell fate and division orientation in both EMS and P2. Our findings suggest that Wnt and Src signaling function in parallel to control developmental outcomes within a single responding cell.
Developmental Cell 08/2002; 3(1):113-25. · 14.03 Impact Factor
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ABSTRACT: β-Catenin regulates cell adhesion and cellular differentiation during development, and misregulation of β-catenin contributes to numerous forms of cancer in humans. Here we describe Caenorhabditis elegans conditional alleles of mom-2/Wnt , mom-4/Tak1 , and wrm-1 /β-catenin. We use these reagents to examine the regulation of WRM-1/β-catenin during a Wnt-signaling-induced asymmetric cell division. While WRM-1 protein initially accumulates in the nuclei of all cells, signaling promotes the retention of WRM-1 in nuclei of responding cells. We show that both PRY-1/Axin and the nuclear exportin homolog IMB-4/CRM-1 antagonize signaling. These findings reveal how Wnt signals direct the asymmetric localization of β-catenin during polarized cell division.
