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C C Mello

Publications and source records attributed to C C Mello.

At least 19 recordsLinked to original sources
PubMed2001

Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing.

RNAi is a gene-silencing phenomenon triggered by double-stranded (ds) RNA and involves the generation of 21 to 26 nt RNA segments that guide mRNA destruction. In Caenorhabditis elegans, lin-4 and let-7 encode small temporal RNAs (stRNAs) of 22 nt that regulate stage-specific development. Here we show that inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-1), and two homologs of rde-1 (alg-1 and alg-2), cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further we show that dcr-1, alg-1, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7 stRNAs. Our findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation.

Animals↗
PubMed2000

Genetic requirements for inheritance of RNAi in C. elegans.

In Caenorhabditis elegans, the introduction of double-stranded RNA triggers sequence-specific genetic interference (RNAi) that is transmitted to offspring. The inheritance properties associated with this phenomenon were examined. Transmission of the interference effect occurred through a dominant extragenic agent. The wild-type activities of the RNAi pathway genes rde-1 and rde-4 were required for the formation of this interfering agent but were not needed for interference thereafter. In contrast, the rde-2 and mut-7 genes were required downstream for interference. These findings provide evidence for germ line transmission of an extragenic sequence-specific silencing factor and implicate rde-1 and rde-4 in the formation of the inherited agent.

Animals↗
PubMed1999

Structural correlates for enhanced stability in the E2 DNA-binding domain from bovine papillomavirus.

Papillomaviral E2 proteins participate in viral DNA replication and transcriptional regulation. We have solved the solution structure of the DNA-binding domain of the E2 protein from bovine papillomavirus (BPV-1). The structure calculation used 2222 distance and 158 dihedral angle restraints for the homodimer (202 residues in total), which were derived from homonuclear and heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopic data. The root-mean-square deviation for structured regions of the monomer when superimposed to the average is 0.73 +/- 0.10 A for backbone atoms and 1.42 +/- 0.16 A for heavy atoms. The 101 residue construct used in this study (residues 310-410) is about 4.5 kcal/mol more stable than a minimal domain comprising the C-terminal 85 amino acid residues (residues 326-410). The structure of the core domain contained within BPV-1 E2 is similar to the corresponding regions of other papilloma viral E2 proteins. Here, however, the extra N-terminal 16 residues form a flap that covers a cavity at the dimer interface and play a role in DNA binding. Interactions between residues in the N-terminal extension and the core domain correlate with the greater stability of the longer form of the protein relative to the minimal domain.

Animals↗
PubMed1999

The rde-1 gene, RNA interference, and transposon silencing in C. elegans.

Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, we have selected C. elegans mutants resistant to dsRNA-mediated interference (RNAi). Two loci, rde-1 and rde-4, are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. We show that rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. We discuss implications for the mechanism of RNAi and the possibility that one natural function of RNAi is transposon silencing.

Amino Acid Sequence↗
PubMed1999

WRM-1 activates the LIT-1 protein kinase to transduce anterior/posterior polarity signals in C. elegans.

During C. elegans development, Wnt/WG signaling is required for differences in cell fate between sister cells born from anterior/posterior divisions. A beta-catenin-related gene, wrm-1, and the lit-1 gene are effectors of this signaling pathway and appear to downregulate the activity of POP-1, a TCF/LEF-related protein, in posterior daughter cells. We show here that lit-1 encodes a serine/threonine protein kinase homolog related to the Drosophila tissue polarity protein Nemo. We demonstrate that the WRM-1 protein binds to LIT-1 in vivo and that WRM-1 can activate the LIT-1 protein kinase when coexpressed in vertebrate tissue culture cells. This activation leads to phosphorylation of POP-1 and to apparent changes in its subcellular localization. Our findings provide evidence for novel regulatory avenues for an evolutionarily conserved Wnt/WG signaling pathway.

Amino Acid Sequence↗
PubMed1999

MOM-4, a MAP kinase kinase kinase-related protein, activates WRM-1/LIT-1 kinase to transduce anterior/posterior polarity signals in C. elegans.

In C. elegans, a Wnt/WG-like signaling pathway down-regulates the TCF/LEF-related protein, POP-1, to specify posterior cell fates. Effectors of this signaling pathway include a beta-catenin homolog, WRM-1, and a conserved protein kinase, LIT-1. WRM-1 and LIT-1 form a kinase complex that can directly phosphorylate POP-1, but how signaling activates WRM-1/LIT-1 kinase is not yet known. Here we show that mom-4, a genetically defined effector of polarity signaling, encodes a MAP kinase kinase kinase-related protein that stimulates the WRM-1/LIT-1-dependent phosphorylation of POP-1. LIT-1 kinase activity requires a conserved residue analogous to an activating phosphorylation site in other kinases, including MAP kinases. These findings suggest that anterior/posterior polarity signaling in C. elegans may involve a MAP kinase-like signaling mechanism.

Amino Acid Sequence↗
PubMed1999

pos-1 encodes a cytoplasmic zinc-finger protein essential for germline specification in C. elegans.

Germ cells arise during early C. elegans embryogenesis from an invariant sequence of asymmetric divisions that separate germ cell precursors from somatic precursors. We show that maternal-effect lethal mutations in the gene pos-1 cause germ cell precursors to inappropriately adopt somatic cell fates. During early embryogenesis, pos-1 mRNA and POS-1 protein are present predominantly in the germ precursors. POS-1 is a novel protein with two copies of a CCCH finger motif previously described in the germline proteins PIE-1 and MEX-1 in C. elegans, and in the mammalian TIS11/Nup475/TTP protein. However, mutations in pos-1 cause several defects in the development of the germline blastomeres that are distinct from those caused by mutations in pie-1 or mex-1. The earliest defect detected in pos-1 mutants is the failure to express APX-1 protein from maternally provided apx-1 mRNA, suggesting that POS-1 may have an important role in regulating the expression of maternal mRNAs in germline blastomeres.

Amino Acid Sequence↗
PubMed1998

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.

Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.

Animals↗
PubMed1997

Wnt signaling and an APC-related gene specify endoderm in early C. elegans embryos.

In a 4-cell stage C. elegans embryo, signaling by the P2 blastomere induces anterior-posterior polarity in the adjacent EMS blastomere, leading to endoderm formation. We have taken genetic and reverse genetic approaches toward understanding the molecular basis for this induction. These studies have identified a set of genes with sequence similarity to genes that have been shown to be, or are implicated in, Wnt/Wingless signaling pathways in other systems. The C. elegans genes described here are related to wnt/wingless, porcupine, frizzled, beta-catenin/armadillo, and the human adenomatous polyposis coli gene, APC. We present evidence that there may be partially redundant inputs into endoderm specification and that a subset of these genes appear also to function in determining cytoskeletal polarity in certain early blastomeres.

Adenomatous Polyposis Coli Protein↗
PubMed1996

MEX-3 is a KH domain protein that regulates blastomere identity in early C. elegans embryos.

After the first division of the C. elegans embryo, the posterior blastomere can produce numerous muscles while the anterior blastomere cannot. We show here that maternal-effect lethal mutations in the gene mex-3 cause descendants of the anterior blastomere to produce muscles by a pattern of development similar to that of a descendant of the wild-type posterior blastomere. mex-3 encodes a probable RNA-binding protein that is distributed unequally in early embryos and that is a component of germline-specific granules called P granules. We propose that MEX-3 contributes to anterior-posterior asymmetry by regulating one or more mRNAs involved in specifying the fate of the posterior blastomere.

Alleles↗
PubMed1996

The PIE-1 protein and germline specification in C. elegans embryos.

Totipotent germline blastomeres in Caenorhabditis elegans contain, but do not respond to, factors that promote somatic differentiation in other embryonic cells. Mutations in the maternal gene pie-1 result in the germline blastomeres adopting somatic cell fates. Here we show that pie-1 encodes a nuclear protein, PIE-1, that is localized to the germline blastomeres throughout early development. During division of each germline blastomere, PIE-1 initially associates with both centrosomes of the mitotic spindle. However, PIE-1 rapidly disappears from the centrosome destined for the somatic daughter, and persists in the centrosome of the daughter that becomes the next germline blastomere. The PIE-1 protein contains potential zinc-finger motifs also found in the mammalian growth-factor response protein TIS-11/NUP475 (refs 4-7). The localization and genetic properties of pie-1 provide an example of a repressor-based mechanism for preserving pluripotency within a stem cell lineage.

Amino Acid Sequence↗
PubMed1996

Repression of gene expression in the embryonic germ lineage of C. elegans.

The distinction between soma and germline was recognized more than a century ago: somatic cells form the body of an organism, whereas germ cells serve to produce future generations. In Caenorhabditis elegans, the separation of some and germline occurs through a series of asymmetrical divisions, in which embryonic germline blastomeres divide unequally to produce one somatic daughter and one germline daughter. Here we show that after each asymmetrical division, embryonically transcribed RNAs are detected in somatic, but not germline, blastomeres. This asymmetry depends on the activity of the germline specific factor, PIE-1. In the absence of PIE-1, embryonically transcribed RNAs are detected in both somatic and germline blastomeres. Furthermore, ectopic expression of PIE-1 in somatic blastomeres can significantly reduce the accumulation of new transcripts in these cells. Taken together, these results suggest that germ-cell fate depends on an inhibitory mechanism that blocks new gene expression in the early embryonic germ lineage.

Animals↗
PubMed1996

par-6, a gene involved in the establishment of asymmetry in early C. elegans embryos, mediates the asymmetric localization of PAR-3.

The generation of asymmetry in the one-cell embryo of Caenorhabditis elegans is necessary to establish the anterior-posterior axis and to ensure the proper identity of early blastomeres. Maternal-effect lethal mutations with a partitioning defective phenotype (par) have identified several genes involved in this process. We have identified a new gene, par-6, which acts in conjunction with other par genes to properly localize cytoplasmic components in the early embryo. The early phenotypes of par-6 embryos include the generation of equal-sized blastomeres, improper localization of P granules and SKN-1 protein, and abnormal second division cleavage patterns. Overall, this phenotype is very similar to that caused by mutations in a previously described gene, par-3. The probable basis for this similarity is revealed by our genetic and immunolocalization results; par-6 acts through par-3 by localizing or maintaining the PAR-3 protein at the cell periphery. In addition, we find that loss-of-function par-6 mutations act as dominant bypass suppressors of loss-of-function mutations in par-2.

Alleles↗
PubMed1996

An inductive interaction in 4-cell stage C. elegans embryos involves APX-1 expression in the signalling cell.

During the 4-cell stage of C. elegans embryogenesis, the P2 blastomere provides a signal that allows two initially equivalent sister blastomeres, called ABa and ABp, to adopt different fates. Preventing P2 signalling in wild-type embryos results in defects in ABp development that are similar to those caused by mutations in the glp-1 and apx-1 genes, which are homologs of the Drosophila genes Notch and Delta, respectively. Previous studies have shown that GLP-1 protein is expressed in 4-cell stage embryos in both ABa and ABp. In this report, we show that APX-1 protein is expressed in the P2 blastomere and that a temperature-sensitive apx-1 mutant has a temperature-sensitive period between the 4-cell and 8-cell stages. We propose that APX-1 is part or all of the P2 signal that induces ABp to adopt a fate different than ABa.

Animals↗
PubMed1994

par-2, a gene required for blastomere asymmetry in Caenorhabditis elegans, encodes zinc-finger and ATP-binding motifs.

The par-2 gene of Caenorhabditis elegans functions in early embryogenesis to ensure an asymmetric first cleavage and the segregation of cytoplasmic factors. Both processes appear to be required to generate daughter blastomeres with distinct developmental potential. We isolated an allele of par-2 by using a screen for maternal-effect lethal mutations in a strain known for its high frequency of transposition events. A transposable element was found to be linked to this allele. Sequences flanking the site of transposon insertion were cloned and found to rescue the par-2 mutant phenotype. DNA in the par-2 region hybridized to a 2.3-kb germ-line-enriched mRNA. The cDNA corresponding to this germ-line-enriched message was cloned, sequenced, and used to identify the molecular lesions associated with three par-2 alleles. Sequence analysis of the par-2 cDNA revealed that the predicted protein contained two distinct motifs found in other known proteins: an ATP-binding site and a cysteine-rich motif which identifies the par-2 gene product as a member of a growing class of putative zinc-binding proteins.

Adenosine Triphosphate↗
PubMed1994

The maternal genes apx-1 and glp-1 and establishment of dorsal-ventral polarity in the early C. elegans embryo.

The sister blastomeres ABp and ABa are equipotent at the beginning of the 4-cell stage in C. elegans embryos, but soon become committed to different fates. We show that the glp-1 gene, a homolog of the Notch gene of Drosophila, functions in two distinct cell-cell interactions that specify the ABp and ABa fates. These interactions both require maternal expression of glp-1. We show that a second maternal gene, apx-1, functions with glp-1 only in the specification of the ABp fate and that apx-1 can encode a protein homologous to the Delta protein of Drosophila. Our results suggest how interactions mediated by glp-1 and apx-1 contribute to the establishment of the dorsal-ventral axis in the early C. elegans embryo.

Amino Acid Sequence↗