ADBP-1 regulates an ADAR RNA-editing enzyme to antagonize RNA-interference-mediated gene silencing in Caenorhabditis elegans

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) mediate gene silencing through evolutionarily conserved pathways. In Caenorhabditis elegans, the siRNA/miRNA pathways are also known to affect transgene expression. To identify genes that regulate the efficiencies of the siRNA/miRNA pathways, we used the expression level of a transgene as an indicator of gene silencing and isolated a transgene-silencing mutant, adbp-1 (ADR-2 binding protein). The adbp-1 mutation caused transgene silencing in hypodermal and intestinal cells in a cell-autonomous manner, depending on the RNA interference (RNAi) machinery. The adbp-1 gene encodes a protein with no conserved domains that is localized in the nucleus. Yeast two-hybrid screening and co-immunoprecipitation analysis demonstrated that ADBP-1 physically interacts with ADR-2, an RNA-editing enzyme from the ADAR (adenosine deaminase acting on dsRNA) family. In the adbp-1 mutant, as previously shown in adr-2 mutants, A-to-I RNA editing was not detected, suggesting that ADBP-1 is required for the RNA-editing activity of ADR-2. We found that ADBP-1 facilitates the nuclear localization of ADR-2. ADBP-1 may regulate ADR-2 activity and the consequent RNA editing and thereby antagonize RNAi-mediated transgene silencing in C. elegans.     

Caenorhabditis elegans ubiquinone biosynthesis genes

Ubiquinone (coenzyme Q, Q) is an essential lipid electron carrier in the mitochondria respiratory chain, and also functions as antioxidant and participates as a cofactor of mitochondrial uncoupling proteins. Caernorhabditis elegans synthesize Q9, but both dietary Q8 intake and endogenous Q9 biosynthesis determine Q balance. Thus, it is of current interest to know the regulatory mechanisms of Q9 biosynthesis in this nematode. Here we review results that leaded to identification of genes involved in Q9 biosynthesis in this nematode using the RNA interference technology. C. elegans coq genes were silenced and depletion of Q content was observed, indicating that the genes related here participate in Q9 biosynthesis. Silenced populations showed an extension of adult life span, probably by the decrease of endogenous oxidative stress produced in mitochondria. We also report the heterologous complementation of C. elegans coq-5 and coq-7 genes in their homologue yeast coq null mutants, leading to restore its ability to growth in non-fermentable sugars. These complemented yeast strains accumulated Q6 but also the intermediate demethoxy-Q6. These findings support the conservative functional homology of these genes.     

Evaluation and identification of reliable reference genes for toxicological study in Caenorhabditis elegans

To identify reliable reference genes for toxicological studies, 16 commonly-used reference genes were selected as candidates to evaluate their expression stabilities under experimental conditions in Caenorhabditis elegans. Sixteen candidates were composed of 12 protein-coding genes and 4 non-coding RNAs, they were act-2, ama-1, arp-6, cdc-42, csq-1, eif-3.C, idhg-1, mdh, pmp-3, rbd-1, tba-1, Y45F10D.4, 18S rRNA, Ce234, U18, and U6. Larval stage 1 synchronized hermaphrodites were exposed to benzo-α-pyrene (BαP), chlorpyrifos, diazinon, gossypol, zinc oxide nanoparticles, and the vehicle control DMSO for 30 h, respectively. Expression stabilities of candidate genes were analyzed using 4 independent evaluating approaches (BestKeeper, the delta Ct approach, geNorm, and NormFinder) followed by a comprehensive method. Results showed that there were slight differences in ranking order between evaluation methods due to their different assumptions and computations. The results also showed that responses of candidate genes to different chemicals were distinct, 18S rRNA was the best for BαP and chlorpyrifos, tba-1 was the most stable gene for diazinon and gossypol treatments, while pmp-3 was more stable for zinc oxide exposure. Additionally, results demonstrated that combinations of multiple genes were more reliable than individual gene, suggesting selecting two or more candidates as reference genes may generate more reliable results for toxicological studies.     

Expression of chimeric genes in Caenorhabditis elegans

We have shown the expression of transformed genes in the nematode Caenorhabditis elegans using a new gene fusion system. Vectors consisting of the flanking regions of a collagen gene (col-1) or a major sperm protein gene of C. elegans fused to the Escherichia coli uidA gene, encoding beta-glucuronidase, were microinjected into worms and found to be propagated as high-copy extrachromosomal tandem arrays. We have detected beta-glucuronidase activity in transformed lines, and have shown that the activity is dependent upon the correct reading frame of the construction and on the presence of the worm sequences. The enzyme activity was shown to be encoded by the chimeric beta-glucuronidase gene by co-segregation analysis and by inactivation with specific antisera. Expression is at a very low level, and seems to be constitutive. We have used histochemical techniques to visualize the enzyme activity in embryos.     

Systematic screen for genes involved in the regulation of oxidative stress in the nematode Caenorhabditis elegans

Oxygen is essential for animals, but high concentrations of oxygen are toxic to them probably because of an increase in reactive oxygen species (ROS). Many genes are involved in the regulation of ROS, but they largely remain to be identified. To identify these genes, we employed the nematode Caenorhabditis elegans as a model organism, and systematically screened for genes that, when down-regulated by RNAi, lead to an increased sensitivity to ROS. We examined approximately 2400 genes on linkage group I and found that knock-down of 9 genes which participate in various cellular functions led to an increased sensitivity to ROS. This finding suggests an implication of a variety of cellular processes in the regulation of oxidative stress.     

Cuticle collagen genes. Expression in Caenorhabditis elegans

Collagen is a structural protein used in the generation of a wide variety of animal extracellular matrices. The exoskeleton of the free-living nematode, Caenorhabditis elegans, is a complex collagen matrix that is tractable to genetic research. Mutations in individual cuticle collagen genes can cause exoskeletal defects that alter the shape of the animal. The complete sequence of the C. elegans genome indicates upwards of 150 distinct collagen genes that probably contribute to this structure. During the synthesis of this matrix, individual collagen genes are expressed in distinct temporal periods, which might facilitate the formation of specific interactions between distinct collagens.     

Wild-type and mutant actin genes in Caenorhabditis elegans

We have sequenced the four actin genes of Caenorhabditis elegans. These four genes encode typical invertebrate actins and are highly homologous, differing from each other by, at most, three amino acid residues. As a first step toward an understanding of the developmental regulation of this gene set we have also sequenced mutant actin genes. The mutant genes were cloned from two independent revertants of a single dominant actin mutant. For both revertants, reversion was accompanied by an actin gene rearrangement. The accumulation of actin mRNA during development in these two revertants is different from that of wild-type animals. We present here a correlation between actin gene structure and expression in wild-type and mutant animals. The results, suggest that co-ordinate regulation of actin genes is not essential for wild-type muscle function. In addition, it appears that changes in the 3' region of at least one of the actin mRNA may affect its steady-state regulation during development.     

Longevity determined by developmental arrest genes in Caenorhabditis elegans

The antagonistic pleiotropy theory of aging proposes that aging takes place because natural selection favors genes that confer benefit early on life at the cost of deterioration later in life. This theory predicts that genes that impact development would play a key role in shaping adult lifespan. To better understand the link between development and adult lifespan, we examined the genes previously known to be essential for development. From a pool of 57 genes that cause developmental arrest after inhibition using RNA interference, we have identified 24 genes that extend lifespan in Caenorhabditis elegans when inactivated during adulthood. Many of these genes are involved in regulation of mRNA translation and mitochondrial functions. Genetic epistasis experiments indicate that the mechanisms of lifespan extension by inactivating the identified genes may be different from those of the insulin/insulin-like growth factor 1 (IGF-1) and dietary restriction pathways. Inhibition of many of these genes also results in increased stress resistance and decreased fecundity, suggesting that they may mediate the trade-offs between somatic maintenance and reproduction. We have isolated novel lifespan-extension genes, which may help understand the intrinsic link between organism development and adult lifespan.     

Trehalose metabolism genes in Caenorhabditis elegans and filarial nematodes

The sugar trehalose is claimed to be important in the physiology of nematodes where it may function in sugar transport, energy storage and protection against environmental stresses. In this study we investigated the role of trehalose metabolism in nematodes, using Caenorhabditis elegans as a model, and also identified complementary DNA clones putatively encoding genes involved in trehalose pathways in filarial nematodes. In C. elegans two putative trehalose-6-phosphate synthase (tps) genes encode the enzymes that catalyse trehalose synthesis and five putative trehalase (tre) genes encode enzymes catalysing hydrolysis of the sugar. We showed by RT-PCR or Northern analysis that each of these genes is expressed as mRNA at all stages of the C. elegans life cycle. Database searches and sequencing of expressed sequence tag clones revealed that at least one tps gene and two tre genes are expressed in the filarial nematode Brugia malayi, while one tps gene and at least one tre gene were identified for Onchocerca volvulus. We used the feeding method of RNA interference in C. elegans to knock down temporarily the expression of each of the tps and tre genes. Semiquantitative RT-PCR analysis confirmed that expression of each gene was silenced by RNA interference. We did not observe an obvious phenotype for any of the genes silenced individually but gas-chromatographic analysis showed >90% decline in trehalose levels when both tps genes were targeted simultaneously. This decline in trehalose content did not affect viability or development of the nematodes.     

Caenorhabditis elegans mutant allele identification by whole-genome sequencing

Identification of the molecular lesion in Caenorhabditis elegans mutants isolated through forward genetic screens usually involves time-consuming genetic mapping. We used Illumina deep sequencing technology to sequence a complete, mutant C. elegans genome and thus pinpointed a single-nucleotide mutation in the genome that affects a neuronal cell fate decision. This constitutes a proof-of-principle for using whole-genome sequencing to analyze C. elegans mutants.     

Number and organization of collagen genes in Caenorhabditis elegans.

We analyzed the number and organization of collagen genes in the nematode Caenorhabditis elegans. Genomic Southern blot hybridization experiments and recombinant phage library screenings indicated that C. elegans has between 40 and 150 distinct collagen genes. A large number of recombinant phages containing collagen genes were isolated from C. elegans DNA libraries. Physical mapping studies indicated that most phage contained a single small collagen gene less than 3 kilobases in size. A few phage contained multiple collagen hybridizing regions and may contain a larger collagen gene or several tightly linked small collagen genes. No overlaps were observed between phages containing different collagen genes, implying that the genes are dispersed in the C. elegans genome. Consistent with the small size of most collagen genes, we found that the predominant class of collagen mRNA in C. elegans is 1.2 to 1.4 kilobases in length. Genomic Southern blot experiments under stringent hybridization conditions revealed considerable sequence diversity among collagen genes. Our data suggest that most collagen genes are unique or are present in only a few copies.     

Functional characterization of SR and SR-related genes in Caenorhabditis elegans

The SR proteins constitute a family of nuclear phosphoproteins, which are required for constitutive splicing and also influence alternative splicing regulation. Initially, it was suggested that SR proteins were functionally redundant in constitutive splicing. However, differences have been observed in alternative splicing regulation, suggesting unique functions for individual SR proteins. Homology searches of the Caenorhabditis elegans genome identified seven genes encoding putative orthologues of the human factors SF2/ASF, SRp20, SC35, SRp40, SRp75 and p54, and also several SR-related genes. To address the issue of functional redundancy, we used dsRNA interference (RNAi) to inhibit specific SR protein function during C.elegans development. RNAi with CeSF2/ASF caused late embryonic lethality, suggesting that this gene has an essential function during C.elegans development. RNAi with other SR genes resulted in no obvious phenotype, which is indicative of gene redundancy. Simultaneous interference of two or more SR proteins in certain combinations caused lethality or other developmental defects. RNAi with CeSRPK, an SR protein kinase, resulted in early embryonic lethality, suggesting an essential role for SR protein phosphorylation during development.