Crystal structures of human DcpS in ligand-free and m7GDP-bound forms suggest a dynamic mechanism for scavenger mRNA decapping

Eukaryotic cells utilize DcpS, a scavenger decapping enzyme, to degrade the residual cap structure following 3'-5' mRNA decay, thereby preventing the premature decapping of the capped long mRNA and misincorporation of methylated nucleotides in nucleic acids. We report the structures of DcpS in ligand-free form and in a complex with m7GDP. apo-DcpS is a symmetric dimer, strikingly different from the asymmetric dimer observed in the structures of DcpS with bound cap analogues. In contrast, and similar to the m7GpppG-DcpS complex, DcpS with bound m7GDP is an asymmetric dimer in which the closed state appears to be the substrate-bound complex, whereas the open state mimics the product-bound complex. Comparisons of these structures revealed conformational changes of both the N-terminal swapped-dimeric domain and the cap-binding pocket upon cap binding. Moreover, Tyr273 in the cap-binding pocket displays remarkable conformational changes upon cap binding. Mutagenesis and biochemical analysis suggest that Tyr273 seems to play an important role in cap binding and product release. Examination of the crystallographic B-factors indicates that the N-terminal domain in apo-DcpS is inherently flexible, and in a dynamic state ready for substrate binding and product release.     

Isolation and characterization of rabbit anti-m3 2,2,7G antibodies.

Antibodies specific for intact 2,2,7-trimethylguanosine (m3 2,2,7G) were induced by immunization of rabbits with a nucleoside-human serum albumen (HSA) conjugate. Competition radioimmunoassay showed that the antibody distinguishes well between intact m3 2,2,7G and its alkali-hydrolysed form (m3 2,2,7G*). Antibody specificity is largely dependent on the presence of all three methyl groups in m3 2,2,7G: none of the less extensively methylated nucleosides m7G, m2G and m2 2,2G is able to compete efficiently with the homologous hapten. Little or no competition was observed with m1G, m1A, m6A, m5U and each of the four unmodified ribonucleosides. Binding studies with nucleoplasmic RNAs from Ehrlich ascites cells suggest that the antibody reacts specifically with the m3 2,2,7G-containing cap structure of the small nuclear U-RNAs (U-snRNAs). Thus the antibody should be a valuable tool for studying the role of the 5'-terminal regions of the U-snRNAs of eucaryotic cells.     

Large-scale induced fit recognition of an m(7)GpppG cap analogue by the human nuclear cap-binding complex

The heterodimeric nuclear cap-binding complex (CBC) binds to the 5' cap structure of RNAs in the nucleus and plays a central role in their diverse maturation steps. We describe the crystal structure at 2.1 A resolution of human CBC bound to an m(7)GpppG cap analogue. Comparison with the structure of uncomplexed CBC shows that cap binding induces co-operative folding around the dinucleotide of some 50 residues from the N- and C-terminal extensions to the central RNP domain of the small subunit CBP20. The cap-bound conformation of CBP20 is stabilized by an intricate network of interactions both to the ligand and within the subunit, as well as new interactions of the CBP20 N-terminal tail with the large subunit CBP80. Although the structure is very different from that of other known cap-binding proteins, such as the cytoplasmic cap-binding protein eIF4E, specificity for the methylated guanosine again is achieved by sandwiching the base between two aromatic residues, in this case two conserved tyrosines. Implications for the transfer of capped mRNAs to eIF4E, required for translation initiation, are discussed.     

Functional analyses of vertebrate TCF proteins in C. elegans embryos

In the canonical Wnt pathway, signaling results in the stabilization and increased levels of β-catenin in responding cells. β-catenin then enters the nucleus, functioning as a coactivator for the Wnt effector, TCF/LEF protein. In the absence of Wnt signaling, TCF is complexed with corepressors, together repressing Wnt target genes. In C. elegans, Wnt signaling specifies the E blastomere to become the endoderm precursor. Activation of endoderm genes in E requires not only an increase in β-catenin level, but a concomitant decrease in the nuclear level of POP-1, the sole C. elegans TCF. A decrease in nuclear POP-1 levels requires Wnt-induced phosphorylation of POP-1 and 14-3-3 protein-mediated nuclear export. Nuclear POP-1 levels remain high in the sister cell of E, MS, where POP-1 represses the expression of endoderm genes. Here we express three vertebrate TCF proteins (human TCF4, mouse LEF1 and Xenopus TCF3) in C. elegans embryos and compare their localization, repression and activation functions to POP-1. All three TCFs are localized to the nucleus in C. elegans embryos, but none undergoes Wnt-induced nuclear export. Although unable to undergo Wnt-induced nuclear export, human TCF4, but not mouse LEF1 or Xenopus TCF3, can repress endoderm genes in MS, in a manner very similar to POP-1. This repressive activity requires that human TCF4 recognizes specific promoter sequences upstream of endoderm genes and interacts with C. elegans corepressors. Domain swapping identified two regions of POP-1 that are sufficient to confer nuclear asymmetry to human TCF4 when swapped with its corresponding domains. Despite undergoing Wnt-induced nuclear export, the human TCF4/POP-1 chimeric protein continues to function as a repressor for endoderm genes in E, a result we attribute to the inability of hTCF4 to bind to C. elegans β-catenin. Our results reveal a higher degree of species specificity among TCF proteins for coactivator interactions than for corepressor interactions, and uncover a basic difference between how POP-1 and human TCF4 steady state nuclear levels are regulated.     

DiS-C3(3) monitoring of in vivo mitochondrial membrane potential in C. elegans

The mitochondrial respiratory chain plays a crucial role in cellular and organismal health. In addition to being the major source of energy for most cells, mitochondrial respiratory chain function regulates or modulates redox and metabolite homeostasis, apoptosis and the generation of reactive oxygen species. In order to measure the relative in vivo mitochondrial membrane potential of different strains of the nematode, Caenorhabditis elegans, we have developed a fluorescence assay using the cationic, lipophilic carbocyanine dye, diS-C(3)(3). We demonstrate that two complex I-deficient mutants have significantly lower mitochondrial membrane potentials in vivo than wild type animals. Our fluorescence assay will enable us to better dissect and understand the complex phenotypic consequences of mitochondrial dysfunction.     

Saccharomyces cerevisiae mRNA populations of different intrinsic stability in unstressed and heat shocked cells display almost constant m7GpppA:m7GpppG 5'-cap structure ratios

The half-lives of mRNAs in yeast vary from about 1 to over 100 min. While mRNA stabilities must strongly influence overall gene expression in this organism, very little is known about how they are determined. Labellings of yeast cells were conducted to investigate whether the 5'-cap structures of yeast mRNAs might influence their stability. Variation of the pulse-labelling period from 7.5 min to 120 min did not have any major influence on the relative labelling of m7GpppA (A cap) and m7GpppG (G cap) in total polyadenylated RNA. Whether an mRNA has the A cap or the G cap does not therefore have a marked effect on its stability. During the heat shock response the relative labelling of A caps to G caps in total polyadenylated RNA also does not fluctuate appreciably. This indicates that cap structure alone does not determine the destabilisation of non-heat shock mRNAs and stabilisation of heat shock mRNAs during this stress response.     

Probing single-cell micromechanics in vivo: the microrheology of C. elegans developing embryos

Cells are not directly accessible in vivo and therefore their mechanical properties cannot be measured by methods that require a direct contact between probe and cell. Here, we introduce a novel in vivo assay based on particle tracking microrheology whereby the extent and time-lag dependence of the mean squared displacements of thermally excited nanoparticles embedded within the cytoplasm of developing embryos reflect local viscoelastic properties. As a proof of principle, we probe local viscoelastic properties of the cytoplasm of developing Caenorhabditis elegans embryos. Our results indicate that unlike differentiated cells, the cytoplasm of these embryos does not exhibit measurable elasticity, but is highly viscous. Furthermore, the viscosity of the cytoplasm does not vary along the anterior-posterior axis of the embryo during the first cell division. These results support the hypothesis that the asymmetric positioning of the mitotic spindle stems from an asymmetric distribution of elementary force generators as opposed to asymmetric viscosity of the cytoplasm.     

The glp-1 locus and cellular interactions in early C. elegans embryos

Interactions between the early blastomeres in a C. elegans embryo are required for the specification of certain cell fates. Blastomeres that produce neurons and skin cells when cultured in isolation are induced to also produce pharyngeal cells in intact embryos. We have identified maternal effect lethal mutations that, on the basis of phenotype and temperature-sensitive period, appear to disrupt this inductive interaction. These mutations are all alleles of glp-1, a gene also involved in the control of germ cell proliferation during postembryonic development of C. elegans.     

Generation of asymmetry and segregation of germ-line granules in early C. elegans embryos

Germ-line granules in C. elegans embryos (P granules) can be visualized by immunofluorescence microscopy using a monoclonal antibody. In mutant zygotes with abnormal spindle orientations and in wild-type zygotes treated with the microtubule inhibitors nocodazole, colcemid, vinblastine, and griseofulvin, both P-granule segregation to the posterior pole and the concomitant pseudocleavage occur apparently normally, but the normally concurrent migration of the pronuclei is inhibited. Conversely, treatment of wild-type embryos with the microfilament inhibitors cytochalasins D and B inhibits P-granule segregation and pseudocleavage, as well as other manifestations of polarity, without preventing pronuclear migration. The results suggest that P-granule segregation does not require either the spindle or cytoplasmic microtubules, but that this process as well as generation of other asymmetries does require cytoskeletal functions that depend on microfilaments.     

A direct interaction between IP(3) receptors and myosin II regulates IP(3) signaling in C. elegans

Molecular and physiological studies of cells implicate interactions between the cytoskeleton and the intracellular calcium signalling machinery as an important mechanism for the regulation of calcium signalling. However, little is known about the functions of such mechanisms in animals. A key component of the calcium signalling network is the intracellular release of calcium in response to the production of the second messenger inositol 1,4,5-trisphosphate (IP(3)), mediated by the IP(3) receptor (IP(3)R). We show that C. elegans IP(3)Rs, encoded by the gene itr-1, interact directly with myosin II. The interactions between two myosin proteins, UNC-54 and MYO-1, and ITR-1 were identified in a yeast two-hybrid screen and subsequently confirmed in vivo and in vitro. We defined the interaction sites on both the IP(3)R and MYO-1. To test the effect of disrupting the interaction in vivo we overexpressed interacting fragments of both proteins in C. elegans. This decreased the animal's ability to upregulate pharyngeal pumping in response to food. This is a known IP(3)-mediated process [15]. Other IP(3)-mediated processes, e.g., defecation, were unaffected. Thus it appears that interactions between IP(3)Rs and myosin are required for maintaining the specificity of IP(3) signalling in C. elegans and probably more generally.     

Cytoplasmic flow and the establishment of polarity in C. elegans 1-cell embryos

Early Caenorhabditis elegans embryos provide an excellent model for the study of developmental processes. Development can be studied by direct observation under the light microscope and can be perturbed using laser manipulations, drug inhibitor treatments, and genetic mutants. The first division of the C. elegans embryo is asymmetric, generating two daughter cells unequal in size and developmental fate. These distinct fates are generated by the partitioning of cytoplasmic determinants during the first mitotic cell cycle. Partitioning of these determinants is thought to be driven by cytoplasmic flow. Recent studies in C. elegans in the past year have identified a number of components necessary for this flow, giving us a clearer picture of the molecular mechanisms underlying developmental asymmetry.     

Identification of genes required for cytoplasmic localization in early C. elegans embryos

We have isolated and analyzed eight strict maternal effect mutations identifying four genes, par-1, par-2, par-3, and par-4, required for cytoplasmic localization in early embryos of the nematode C. elegans. Mutations in these genes lead to defects in cleavage patterns, timing of cleavages, and localization of germ line-specific P granules. Four mutations in par-1 and par-4 are fully expressed maternal effect lethal mutations; all embryos from mothers homozygous for these mutations arrest as amorphous masses of differentiated cells but are specifically lacking intestinal cells. Four mutations in par-2, par-3, and par-4 are incompletely expressed maternal effect lethal mutations and are also grandchildless; some embryos from homozygous mothers survive and grow to become infertile adults due to absence of functional germ cells. We propose that all of these defects result from the failure of a maternally encoded system for intracellular localization in early embryos.     

Maternal effect of low temperature on handedness determination in C. elegans embryos

C. elegans embryos, larvae, and adults exhibit several left-right asymmetries with an invariant dextral handedness, which first becomes evident in the embryo at the 6-cell stage. Reversed (sinistral) handedness was not observed among > 10,000 N2 adults reared at 16°C or 20°C under standard conditions. However, among the progeny of adults reproducing at 10°C, the frequency of animals with sinistral handedness was increased to ∼0.5%. Cold pulse experiments indicated that the critical period for this increase was in early oogenesis, several hours before the first appearance of left-right asymmetry in the embryo. Hermaphrodites reared at 10°C and mated with males reared at 20°C produced sinistral outcross as well as sinistral self-progeny, indicating that the low temperature effect on oocytes was sufficient to cause reversals. Increased frequency of reversal was also observed among animals developed from embryos lacking the egg shell. Possible mechanisms for the control of embryonic handedness are discussed in the context of these results, including the hypothesis that handedness could be dictated by the chirality of a gametic component. © 1996 Wiley-Liss, Inc.     

Two phases of astral microtubule activity during cytokinesis in C. elegans embryos

Microtubules of the mitotic spindle are believed to provide positional cues for the assembly of the actin-based contractile ring and the formation of the subsequent cleavage furrow during cytokinesis. In Caenorhabditis elegans, astral microtubules have been thought to inhibit cortical contraction outside the cleavage furrow. Here, we demonstrate by live imaging and RNA interference (RNAi) that astral microtubules play two distinct roles in initiating cleavage furrow formation. In early anaphase, microtubules are required for contractile ring assembly; in late anaphase, microtubules show different cortical behavior and seem to suppress cortical contraction at the poles, as suggested in previous studies. These two distinct phases of microtubule behavior depend on distinct regulatory pathways, one involving the gamma-tubulin complex and the other requiring aurora-A kinase. We propose that temporal and spatial regulation of two distinct phases of astral microtubule behavior is crucial in specifying the position and timing of furrowing.