Post-embryonic expression of C. elegans microRNAs belonging to the lin-4 and let-7 families in the hypodermis and the reproductive system.

MicroRNAs (miRNAs) are regulatory molecules that negatively control gene expression by binding to complementary sequences on target mRNAs. The most thoroughly characterized miRNAs, lin-4 and let-7, direct cell fate determination during the larval transitions in C. elegans and act as key regulators of temporal gene expression. lin-4 and let-7 are founding members of two distinct families of miRNA genes sharing strong sequence homology primarily in the 5' end of the mature miRNAs. In this report, we characterize the temporal and spatial expression patterns of lin-4 and let-7 family members using northern blot analysis and mir::gfp fusion studies. Our results show that lin-4 and let-7 homologues possess distinct temporal and spatial expression patterns during nematode development and that known heterochronic genes regulate their expression. We find that certain lin-4 and let-7 family members display overlapping expression patterns in the hypodermis and the reproductive system, suggesting that combinations of miRNAs from across families may control common developmental events.     

Reciprocal expression of lin-41 and the microRNAs let-7 and mir-125 during mouse embryogenesis.

In C. elegans, heterochronic genes control the timing of cell fate determination during development. Two heterochronic genes, let-7 and lin-4, encode microRNAs (miRNAs) that down-regulate a third heterochronic gene lin-41 by binding to complementary sites in its 3'UTR. let-7 and lin-4 are conserved in mammals. Here we report the cloning and sequencing of mammalian lin-41 orthologs. We find that mouse and human lin-41 genes contain predicted conserved complementary sites for let-7 and the lin-4 ortholog, mir-125, in their 3'UTRs. Mouse lin-41 (Mlin-41) is temporally expressed in developing mouse embryos, most dramatically in the limb buds. Mlin-41 is down-regulated during mid-embryogenesis at the time when mouse let-7c and mir-125 RNA levels are up-regulated. Our results suggest that mammalian lin-41 is temporally regulated by miRNAs in order to direct key developmental events such as limb formation.     

Regulation of the Drosophila lin-41 homologue dappled by let-7 reveals conservation of a regulatory mechanism within the LIN-41 subclade.

Drosophila Dappled (DPLD) is a member of the RBCC/TRIM superfamily, a protein family involved in numerous diverse processes such as developmental timing and asymmetric cell divisions. DPLD belongs to the LIN-41 subclade, several members of which are micro RNA (miRNA) regulated. We re-examined the LIN-41 subclade members and their relation to other RBCC/TRIMs and dpld paralogs, and identified a new Drosophila muscle specific RBCC/TRIM: Another B-Box Affiliate, ABBA. In silico predictions of candidate miRNA regulators of dpld identified let-7 as the strongest candidate. Overexpression of dpld led to abnormal eye development, indicating that strict regulation of dpld mRNA levels is crucial for normal eye development. This phenotype was sensitive to let-7 dosage, suggesting let-7 regulation of dpld in the eye disc. A cell-based assay verified let-7 miRNA down-regulation of dpld expression by means of its 3'-untranslated region. Thus, dpld seems also to be miRNA regulated, suggesting that miRNAs represent an ancient mechanism of LIN-41 regulation.     

microRNA let-7a-3基因甲基化与糖尿病肾病的关系

目的探讨微RNAlet-7a-3基因启动子甲基化与糖尿病肾病的关系。方法采用生物信息学方法预测let-7a-3基因启动子区域甲基化位点,采用real-time PCR及甲基化特异性PCR检测20例糖尿病肾病(DN)患者、20例糖尿病患者(DM)以及20例正常对照(CON)的let-7a-3表达水平及启动子区域甲基化水平。结果 let-7a-3在DN组呈低表达。同时,DN组的平均甲基化率为96.2%±6.2%,DM组的平均甲基化率为76.6%±18.9%,正常组的平均甲基化率为63.2%±28.4%。DN组的平均甲基化水平较DM组和正常组显著增高(P0.01)。结论 let-7a-3启动子区域高甲基化可能是糖尿病肾病患者let-7a-3低表达的一个重要因素,并参与调控糖尿病肾病的发生与发展。     

Fungicidal monoclonal antibody C7 binds to Candida albicans Als3

Monoclonal antibody (MAb) C7 reacted with a >200-kDa component from the Candida albicans cell wall identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry as Als3. It also bound the recombinant N terminus of Als3. Binding of MAb C7 to Als3 may explain the biological activities exerted by the MAb on C. albicans.     

Cloning and regulation of the vertebrate homologue of lin-41 that functions as a heterochronic gene in Caenorhabditis elegans.

The Caenorhabditis elegans gene lin-41 is the one of the heterochronic genes that regulate the timing of many developmental events. MicroRNA let-7 negatively regulates the expression of lin-41 through RNA-RNA interaction on its 3' untranslated region (UTR). Here, we report the isolation of chick and mouse homologues of lin-41 that encode the RBCC-NHL family protein and their expression patterns. C. elegans lin-41 is one of the RBCC-NHL families and the predicted amino acid sequences of isolated two genes encode the same family proteins. Chick and mouse lin-41 expression was also observed in developing limb buds, branchial arches, and tail buds. The 3'UTRs of the mouse and chick lin-41 genes contain multiple let-7 complementary sites. Using luciferase assay, we showed that lin-41 expression can be regulated through let-7 complementary sites.     

Insights from the worm: The C. elegans model for innate immunity

The nematode worm Caenorhabditis elegans comprises an ancestral immune system. C. elegans recognizes and responds to viral, bacterial, and fungal infections. Components of the RNA interference machinery respond to viral infection, while highly conserved MAPK signaling pathways activate the innate immune response to bacterial infection. C. elegans has been particularly important for exploring the role of innate immunity in organismal stress resistance and the regulation of longevity. Also functions of neuronal sensing of infectious bacteria have recently been uncovered. Studies on nematode immunity can be instructive in exploring innate immune signaling in the absence of specialized immune cells and adaptive immunity.     

fzr-1 and lin-35/Rb function redundantly to control cell proliferation in C. elegans as revealed by a nonbiased synthetic screen

We report here a synthetic-lethal screen in Caenorhabditis elegans that overcomes a number of obstacles associated with the analysis of functionally redundant genes. Using this approach, we have identified mutations that synthetically interact with lin-35/Rb, a SynMuv gene and the sole member of the Rb/pocket protein family in C. elegans. Unlike the original SynMuv screens, our approach is completely nonbiased and can theoretically be applied to any situation in which a mutation fails to produce a detectable phenotype. From this screen we have identified fzr-1, a gene that synthetically interacts with lin-35 to produce global defects in cell proliferation control. fzr-1 encodes the C. elegans homolog of Cdh1/Hct1/FZR, a gene product shown in other systems to regulate the APC cyclosome. We have also uncovered genetic interactions between fzr-1 and a subset of class B SynMuv genes, and between lin-35 and the putative SCF regulator lin-23. We propose that lin-35, fzr-1, and lin-23 function redundantly to control cell cycle progression through the regulation of cyclin levels.     

The DAF-7 TGF-beta signaling pathway regulates chemosensory receptor gene expression in C. elegans

Regulation of chemoreceptor gene expression in response to environmental or developmental cues provides a mechanism by which animals can alter their sensory responses. Here we demonstrate a role for the daf-7 TGF-beta pathway in the regulation of expression of a subset of chemoreceptor genes in Caenorhabditis elegans. We describe a novel role of this pathway in maintaining receptor gene expression in the adult and show that the DAF-4 type II TGF-beta receptor functions cell-autonomously to modulate chemoreceptor expression. We also find that the alteration of receptor gene expression in the ASI chemosensory neurons by environmental signals, such as levels of a constitutively produced pheromone, may be mediated via a DAF-7-independent pathway. Receptor gene expression in the ASI and ASH sensory neurons appears to be regulated via distinct mechanisms. Our results suggest that the expression of individual chemoreceptor genes in C. elegans is subject to multiple modes of regulation, thereby ensuring that animals exhibit the responses most appropriate for their developmental stage and environmental conditions.     

Bovine conglutinin binds to an oligosaccharide determinant presented by iC3b, but not by C3, C3b or C3c.

Bovine conglutinin is a serum lectin that agglutinates erythrocytes preincubated with antibodies and complement. This agglutination occurs through the binding of conglutinin to iC3b, a fragment of the complement component C3. It was reported that conglutinin binds fluid-phase C3b and C3c as well as iC3b. We re-investigated the reactivity of conglutinin towards fluid-phase C3 degradation products. ELISA wells were coated with conglutinin and reacted with C3 split products generated in normal human serum, in factor I-deficient serum, or in factor I-depleted serum. Conglutinin-bound C3 fragments were detected with anti-C3c and anti-C3d antibodies. An increased signal was observed during the activation of complement in normal human serum with the peak response after 1-2 hr, following which the signal decreased, reaching background level after 72 hr. The oligosaccharides on C3c, generated in serum, are thus not recognized by conglutinin. No signal was observed when factor I-deficient serum or factor I-depleted serum was used instead of normal serum. Reconstitution with purified factor I re-established the normal pattern. Examination of the conglutinin-bound C3 molecules by SDS-PAGE and Western blotting with anti-C3c and anti-C3d antibodies revealed bands characteristic for iC3b, and no bands corresponding to C3b or C3c. Reduction of the disulphide bonds prior to the incubation of the activated serum with the conglutinin-coated wells revealed a band of 63,000 MW, characteristic of the N-terminal fragment of the alpha-chain of iC3b. We also investigated the binding to the solid-phase conglutinin of purified C3 and degradation products generated with enzymes. In this case, C3 as well as C3b and C3c were bound, suggesting conformational changes in C3 during purification. In conclusion, when C3 conversion takes place at near physiological conditions, conglutinin interacts specifically with the oligosaccharide on the alpha-chain of iC3b.     

Dietary restriction in C. elegans: from rate-of-living effects to nutrient sensing pathways

The nematode Caenorhabditis elegans has been subjected to dietary restriction (DR) by a number of means, with varying results in terms of fecundity and lifespan. Two possible mechanisms by which DR increases lifespan are reduction of metabolic rate and reduction of insulin/IGF-1 signalling. Experimental tests have not supported either possibility. However, interaction studies suggest that DR and insulin/IGF-1 signalling may act in parallel on common regulated processes. In this review, we discuss recent developments in C. elegans DR research, including new discoveries about the biology of nutrient uptake in the gut, and the importance of invasion by the bacterial food source as a determinant of lifespan. The evidence that the effect of DR on lifespan in C. elegans is mediated by the TOR pathway is discussed. We conclude that the effect of DR on lifespan is likely to involve multiple mechanisms, which may differ according to the DR regimen used and the organism under study.     

Start me up: cell signaling and the journey from oocyte to embryo in C. elegans.

Intercellular communication plays a pivotal role in regulating and coordinating oocyte meiosis and fertilization, key triggers for embryonic development. The nematode Caenorhabaditis elegans has emerged as an important experimental paradigm for exploring these fundamental reproductive processes and their regulation. The oocytes of most animal species arrest during meiotic prophase and complete meiosis in response to intercellular signaling in the process of meiotic maturation. Oocyte meiotic maturation is defined by the transition between diakinesis and metaphase of meiosis I and is accompanied by nuclear envelope breakdown and meiotic spindle assembly. As such, the meiotic maturation process is essential for completing meiosis and a prerequisite for successful fertilization. In C. elegans, the processes of meiotic maturation, ovulation, and fertilization are temporally coupled: sperm utilize the major sperm protein as a hormone to trigger oocyte meiotic maturation, and, in turn, the maturing oocyte signals its own ovulation, leading to fertilization. The powerful genetic screens possible in C. elegans have led to the identification of several sperm cell surface proteins that are required for the interaction and fusion of gametes at fertilization. The study of these proteins provides fundamental insights into fertilization mechanisms, their role in speciation, and their potential conservation across phyla. Signaling processes sparked by fertilization are required for meiotic chromosome segregation and initiating the embryonic program. Here we review recent advances in understanding how signaling mechanisms contribute to the oocyte-to-embryo transition in C. elegans.     

Direct interactions between C. elegans RAB-3 and Rim provide a mechanism to target vesicles to the presynaptic density

Rim is a multi-domain, active zone protein that regulates exocytosis and is implicated in vesicle priming and presynaptic plasticity. We recently demonstrated that synaptic defects associated with loss of Caenorhabditis elegans Rim (termed UNC-10) are accompanied by a reduction in docked vesicles adjacent to the presynaptic density. Since Rim is known to interact with the vesicle-associated GTPase Rab3A, here we asked whether UNC-10-dependent recruitment of synaptic vesicles to the presynaptic density was through an UNC-10/Rab-3 interaction. We first established that C. elegans Rab3 (termed RAB-3) in its GTP but not GDP-bound state interacts with UNC-10. We then demonstrated by EM analysis that rab-3 mutant synapses exhibit the same vesicle-targeting defect as unc-10 mutants. Furthermore, unc-10;rab-3 double mutants phenocopy the targeting defects of the single mutants, suggesting UNC-10 and RAB-3 act in the same pathway to target vesicles at the presynaptic density. Endogenous release of unc-10;rab-3 double mutants was similar to that of unc-10 single mutants, but more severe than rab-3 mutants, suggesting the common targeting defects are reflected by the milder rab-3 release defect. Rim has recently been shown to positively regulate calcium influx through direct interactions with calcium channels. Consistent with this notion we found UNC-10 colocalized with the calcium channel, UNC-2 at C. elegans presynaptic densities and synaptic release in unc-10 and rab-3 mutants exhibit reduced calcium-sensitivity. Together these results suggest that vesicles targeted to the presynaptic density by RAB-3/UNC-10 interactions are ideally positioned for efficient calcium-dependent release.     

C. elegans VAB-8 and UNC-73 regulate the SAX-3 receptor to direct cell and growth-cone migrations

During nervous system development, a small number of conserved guidance cues and receptors regulate many axon trajectories. How could a limited number of cues and receptors regulate such complex projection patterns? One way is to modulate receptor function. Here we show that the Caenorhabditis elegans kinesin-related protein VAB-8L, which is necessary and sufficient for posterior cell and growth-cone migrations, directs these migrations by regulating the levels of the guidance receptor SAX-3 (also known as robo). Genetic experiments indicate that VAB-8L and the Rac guanine nucleotide exchange factor activity of UNC-73 (trio) increase the ability of the SLT-1 (slit) and UNC-6 (netrin) guidance pathways to promote posterior guidance. The observations of higher SAX-3 receptor abundance in animals with increasing amounts of VAB-8L, and of physical interactions between UNC-73 and both VAB-8L and the intracellular domain of the SAX-3, support a model whereby VAB-8L directs cell and growth-cone migrations by promoting localization of guidance receptors to the cell surface.     

A phenylalanine hydroxylase gene from the nematode C. elegans is expressed in the hypodermis

We have identified an aromatic amino acid hydroxylase gene from the nematode C. elegans that likely encodes the worm phenylalanine hydroxylase (PheH). The predicted amino acid sequence is most similar to that of other PheH and TrpH proteins. Reporter gene fusions and staining with an antibody to mammalian PheH indicate the gene is expressed in hypodermal cells. A fusion protein expressed in bacteria can convert phenylalanine to tyrosine, and, to a lesser extent, tryptophan to 5-hydroxytryptophan. We hypothesize that the protein is necessary to produce additional tyrosine for protein cross-linking in the nematode cuticle.