Genome-wide RNAi

In many species, double-stranded RNA can specifically and effectively silence genes. This newly discovered biological phenomenon, called RNA interference (RNAi), has practical implications for functional genomics. As shown by two recent reports, RNAi provides a rapid method to test the function of genes in the nematode Caenorhabditis elegans; most of the genes on C. elegans chromosome I and III have now been tested for RNAi phenotypes. The results validate RNAi as a powerful functional genomics tool for C. elegans, and point the way for similar large-scale studies in other species.     

The AP-3 clathrin-associated complex is essential for embryonic and larval development in Caenorhabditis elegans

The adaptor protein (AP) complexes are involved in membrane transport of many proteins. There are 3 AP complexes in C. elegans unlike mammals that have four. To study the biological functions of the AP-3 complexes of C. elegans, we sought homologues of the mouse and human genes that encode subunits of the AP-3 complexes by screening C. elegans genomic and EST sequences. We identified single copies of homologues of the m3, s3, b3 and d genes. The medium chain of AP-3 is encoded by a single gene in C. elegans but two different genes in mammals. Since there are no known mutations in these genes in C. elegans, we performed RNAi to assess their functions in development. RNAi of each of the genes caused embryonic and larval lethal phenotypes. APM-3 is expressed in most cells, particularly strongly in spermatheca and vulva. We conclude that the products of the C. elegans m3, s3, b3 and d genes are essential for embryogenesis and larval development.     

Innexins in C. elegans

Innexins are functionally analogous to the vertebrate connexins, and the innexin family of gap junction proteins has been identified in many invertebrates, including Drosophila and C. elegans. The genome sequencing project has identified 25 innexins in C. elegans. We are particularly interested in the roles that gap junctions may play in embryonic development and in wiring of the nervous system. To identify the particular C. elegans innexins that are involved in these processes, we are examining their expression patterns using specific antibodies and translational GFP fusions. In addition we are investigating mutant, RNAi and overexpression phenotypes for many of these genes. To date, we have generated specific antibodies to the non-conserved carboxyl termini of 5 innexins. We have constructed GFP translational fusions for 17 innexins and observed expression patterns for 13 of these genes. In total we have characterized expression patterns representing 14 innexins. Mutations have been identified in 5 of these genes, and at least 3 others have RNAi mutant phenotypes. Generalities emerging from our studies include: 1) most tissues and many individual cells express more than one innexin, 2) some innexins are expressed widely, while others are expressed in only a few cells, and 3) there is a potential for functional pairing of innexins.     

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.     

Unique and redundant functions of SR proteins, a conserved family of splicing factors, in Caenorhabditis elegans development

Serine/arginine-rich proteins (SR proteins) constitute a family of RNA-binding proteins conserved throughout metazoans. The SR proteins are essential for constitutive pre-mRNA splicing and also affect regulated pre-mRNA splicing. We identified five putative genes encoding SR proteins (referred to as srp genes) in Caenorhabditis elegans, examined their expression using the gfp gene as a reporter, and suppressed their functions by double-stranded RNA-mediated interference (RNAi). The srp::gfp fusion genes were expressed in the nuclei of most somatic cells and showed no obvious tissue- or stage-specific expression. Simultaneous RNAi of the five srp genes resulted in embryonic lethality, whereas RNAi of individual srp genes caused no obvious morphological abnormality in the F1 progeny, indicating functional redundancy of the SR proteins. However, RNAi of several combinations of srp genes caused various developmental abnormalities, such as abnormal somatic gonad structures, delayed shift of the germ cell sexual differentiation, and abnormal spermatogenesis. Our results suggest that individual SR proteins have unique but somewhat redundant functions in C. elegans development.     

Comparative genomics and adaptive selection of the ATP-binding-cassette gene family in caenorhabditis species

ABC transporters constitute one of the largest gene families in all species. They are mostly involved in transport of substrates across membranes. We have previously demonstrated that the Caenorhabditis elegans ABC family shows poor one-to-one gene orthology with other distant model organisms. To address the evolution dynamics of this gene family among closely related species, we carried out a comparative analysis of the ABC family among the three nematode species C. elegans, C. briggsae, and C. remanei. In contrast to the previous observations, the majority of ABC genes in the three species were found in orthologous trios, including many tandemly duplicated ABC genes, indicating that the gene duplication took place before speciation. Species-specific expansions of ABC members are rare and mostly observed in subfamilies A and B. C. briggsae and C. remanei orthologous ABC genes tend to cluster on trees, with those of C. elegans as an outgroup, consistent with their proposed species phylogeny. Comparison of intron/exon structures of the highly conserved ABCE subfamily members also indicates a closer relationship between C. briggsae and C. remanei than between either of these species and C. elegans. A comparison between insect and mammalian species indicates lineage-specific duplications or deletions of ABC genes, while the family size remains relatively constant. Sites undergoing positive selection within subfamily D, which are implicated in very-long-chain fatty acid transport, were identified. The evolution of these sites might be driven by the changes in food source with time.     

Identification of Heterodera glycines (soybean cyst nematode [SCN]) cDNA sequences with high identity to those of Caenorhabditis elegans having lethal mutant or RNAi phenotypes

The soybean cyst nematode (SCN; Heterodera glycines) is a devastating obligate parasite of Glycine max (soybean) causing one billion dollars in losses to the US economy per year and over ten billion dollars in losses worldwide. While much is understood about the pathology of H. glycines, its genome sequence is not well characterized or fully sequenced. We sought to create bioinformatic tools to mine the H. glycines nucleotide database. One way is to use a comparative genomics approach by anchoring our analysis with an organism, like the free-living nematode Caenorhabditis elegans. Unlike H. glycines, the C. elegans genome is fully sequenced and is well characterized with a number of lethal genes identified through experimental methods. We compared an EST database of H. glycines with the C. elegans genome. Our goal was identifying genes that may be essential for H. glycines survival and would serve as an automated pipeline for RNAi studies to both study and control H. glycines. Our analysis yielded a total of nearly 8334 conserved genes between H. glycines and C. elegans. Of these, 1508 have lethal phenotypes/phenocopies in C. elegans. RNAi of a conserved ribosomal gene from H. glycines (Hg-rps-23) yielded dead and dying worms as shown by positive Sytox fluorescence. Endogenous Hg-rps-23 exhibited typical RNA silencing as shown by RT-PCR. However, an unrelated gene Hg-unc-87 did not exhibit RNA silencing in the Hg-rps-23 dsRNA-treated worms, demonstrating the specificity of the silencing.     

The Ref/Aly proteins are dispensable for mRNA export and development in Caenorhabditis elegans

The mRNA export pathway is highly conserved throughout evolution. We have used RNA interference (RNAi) to functionally characterize bona fide RNA export factors and components of the exon-exon junction complex (EJC) in Caenorhabditis elegans. RNAi of CeNXT1/p15, the binding partner of CeNXF1/TAP, caused early embryonic lethality, demonstrating an essential function of this gene during C. elegans development. Moreover, depletion of this protein resulted in nuclear accumulation of poly(A)(+) RNAs, supporting a direct role of NXT1/p15 in mRNA export in C. elegans. Previously, we have shown that RNAi of CeSRm160, a protein of the EJC complex, resulted in wild-type phenotype; in the present study, we demonstrate that RNAi of CeY14, another component of this complex, results in embryonic lethality. In contrast, depletion of the EJC component CeRNPS1 results in no discernible phenotype. Proteins of the REF/Aly family act as adaptor proteins mediating the recruitment of the mRNA export factor, NXF1/TAP, to mRNAs. The C. elegans genome encodes three members of the REF/Aly family. RNAi of individual Ref genes, or codepletion of two Ref genes in different combinations, resulted in wild-type phenotype. Simultaneous suppression of all three Ref genes did not compromise viability or progression through developmental stages in the affected progeny, and only caused a minor defect in larval mobility. Furthermore, no defects in mRNA export were observed upon simultaneous depletion of all three REF proteins. These results suggest the existence of multiple adaptor proteins that mediate mRNA export in C. elegans.     

Expression profiles of the essential intermediate filament (IF) protein A2 and the IF protein C2 in the nematode Caenorhabditis elegans

The multigene family of intermediate filament (IF) proteins in Caenorhabditis elegans covers 11 members of which four (A1-3, B1) are essential for development. Suppression of a fifth gene (C2) results in a dumpy phenotype. Expression patterns of three essential genes (A1, A3, B1) were already reported. To begin to analyze the two remaining RNAi phenotypes we followed the expression of the A2 and C2 proteins. Expression of A2 mRNA starts in larval stage L1 and continues in the adult. Transgenic A2 promoter/gfp larvae strongly display GFP in the main body hypodermis but not in seam cells. This pattern and the muscle displacement/paralysis induced by RNAi silencing are consistent with the role of this protein in keeping the correct hypodermis/muscle relationship during development. IF protein C2 occurs in the cytoplasm and desmosomes of intestinal cells and in pharynx desmosomes. Expression of C2 starts in the late embryo and persists in all further stages.     

Mining predicted essential genes of Brugia malayi for nematode drug targets

We report results from the first genome-wide application of a rational drug target selection methodology to a metazoan pathogen genome, the completed draft sequence of Brugia malayi, a parasitic nematode responsible for human lymphatic filariasis. More than 1.5 billion people worldwide are at risk of contracting lymphatic filariasis and onchocerciasis, a related filarial disease. Drug treatments for filariasis have not changed significantly in over 20 years, and with the risk of resistance rising, there is an urgent need for the development of new anti-filarial drug therapies. The recent publication of the draft genomic sequence for B. malayi enables a genome-wide search for new drug targets. However, there is no functional genomics data in B. malayi to guide the selection of potential drug targets. To circumvent this problem, we have utilized the free-living model nematode Caenorhabditis elegans as a surrogate for B. malayi. Sequence comparisons between the two genomes allow us to map C. elegans orthologs to B. malayi genes. Using these orthology mappings and by incorporating the extensive genomic and functional genomic data, including genome-wide RNAi screens, that already exist for C. elegans, we identify potentially essential genes in B. malayi. Further incorporation of human host genome sequence data and a custom algorithm for prioritization enables us to collect and rank nearly 600 drug target candidates. Previously identified potential drug targets cluster near the top of our prioritized list, lending credibility to our methodology. Over-represented Gene Ontology terms, predicted InterPro domains, and RNAi phenotypes of C. elegans orthologs associated with the potential target pool are identified. By virtue of the selection procedure, the potential B. malayi drug targets highlight components of key processes in nematode biology such as central metabolism, molting and regulation of gene expression.     

Evolutionarily conserved nuclear migration genes required for early embryonic development in Caenorhabditis elegans

The nudF and nudC genes of the fungus Aspergillus nidulans encode proteins that are members of two evolutionarily conserved families. In A. nidulans these proteins mediate nuclear migration along the hyphae. The human ortholog of nudF is Lis1, a gene essential for neuronal migration in the developing cerebral cortex. The mammalian ortholog of nudC encodes a protein that interacts with Lis1. We have identified orthologs of nudC and Lis1 from the nematode Caenorhabditis elegans. Heterologous expression of the C. elegans nudC ortholog, nud-1, complements the A. nidulans nudC3 mutant, demonstrating evolutionary conservation of function. A C. elegans nud-1::GFP fusion produces sustained fluorescence in sensory neurons and embryos, and transient fluorescence in the gonad, gut, vulva, ventral cord, and hypodermal seam cells. Fusion of GFP to C. elegans lis-1 revealed expression in all major neuronal processes of the animal as well as the multinucleate spermathecal valves and adult seam cells. Phenotypic analysis of either nud-1 and lis-1 by RNA interference yielded similar phenotypes, including embryonic lethality, sterility, altered vulval morphology, and uncoordinated movement. Digital time-lapse video microscopy was used to determine that RNAi-treated embryos exhibited nuclear positioning defects in early embryonic cell division similar to those reported for dynein/dynactin depletion. These results demonstrate that the LIS-1/NUDC-like proteins of C. elegans represent a link between nuclear positioning, cell division, and neuronal function.     

Functional genomic approaches using the nematode Caenorhabditis elegans as a model system

Since the completion of the genome project of the nematode C. elegans in 1998, functional genomic approaches have been applied to elucidate the gene and protein networks in this model organism. The recent completion of the whole genome of C. briggsae, a close sister species of C. elegans, now makes it possible to employ the comparative genomic approaches for identifying regulatory mechanisms that are conserved in these species and to make more precise annotation of the predicted genes. RNA interference (RNAi) screenings in C. elegans have been performed to screen the whole genome for the genes whose mutations give rise to specific phenotypes of interest. RNAi screens can also be used to identify genes that act genetically together with a gene of interest. Microarray experiments have been very useful in identifying genes that exhibit co-regulated expression profiles in given genetic or environmental conditions. Proteomic approaches also can be applied to the nematode, just as in other species whose genomes are known. With all these functional genomic tools, genetics will still remain an important tool for gene function studies in the post genome era. New breakthroughs in C. elegans biology, such as establishing a feasible gene knockout method, immortalized cell lines, or identifying viruses that can be used as vectors for introducing exogenous gene constructs into the worms, will augment the usage of this small organism for genome-wide biology.     

The sequence and associated null phenotype of a C. elegans neurocalcin-like gene

The neuronal calcium sensor (NCS) proteins belong to a subfamily of the EF-hand calcium binding proteins. These proteins are primarily expressed in the nervous system and currently include more than 20 members across species [Nakayama et al., J Mol Evol 34:416-448, 1992]. Two homologues of the ncs genes, Ce-ncs-1 and Ce-ncs-2, have recently been identified in the nematode C. elegans. Here we report the cDNA sequence of a third C. elegans ncs homologue, Ce-ncs-3. We demonstrate that a null mutation in this gene caused by a large deletion in the locus does not confer a visible phenotype in C. elegans. This, in addition to the strong homology between Ce-NCS-3 and the other C. elegans NCS proteins, may indicate functional redundancy between the three genes.     

Most genes encoding cytoplasmic intermediate filament (IF) proteins of the nematode Caenorhabditis elegans are required in late embryogenesis

Intestinal cells of C. elegans show an unexpectedly high complexity of cytoplasmic intermediate filament (IF) proteins. Of the 11 known IF genes six are coexpressed in the intestine, i.e. genes B2, C1, C2, D1, D2, and E1. Specific antibodies and GFP-promoter constructs show that genes B2, D1, D2, and E1 are exclusively expressed in intestinal cells. Using RNA interference (RNAi) by microinjection at 25 degrees C rather than at 20 degrees C we observe for the first time lethal phenotypes for C1 and D2. RNAi at 25 degrees C also shows that the known A1 phenotype occurs already in the late embryo after microinjection and is also observed by feeding which was not the case at 20 degrees C. Thus, RNAi at 25 degrees C may also be useful for the future analysis of other nematode genes. Finally, we show that triple RNAi at 20 degrees C is necessary for the combinations B2, D1, E1 and B2, D1, D2 to obtain a phenotype. Together with earlier results on genes A1, A2, A3, B1, and C2 RNAi phenotypes are now established for all 11IF genes except for the A4 gene. RNAi phenotypes except for A2 (early larval lethality) and C2 (adult phenotype) relate to the late embryo. We conclude that in C. elegans cytoplasmic IFs are required for tissue integrity including late embryonic stages. This is in strong contrast to the mouse, where ablation of IF genes apparently does not affect the embryo proper.     

RNA interference in Caenorhabditis elegans: uptake, mechanism, and regulation

RNA interference (RNAi) is a powerful research tool that has enabled molecular insights into gene activity, pathway analysis, partial loss-of-function phenotypes, and large-scale genomic discovery of gene function. While RNAi works extremely well in the non-parasitic nematode C. elegans, it is also especially useful in organisms that lack facile genetic analysis. Extensive genetic analysis of the mechanisms, delivery and regulation of RNAi in C. elegans has provided mechanistic and phenomenological insights into why RNAi is so effective in this species. These insights are useful for the testing and development of RNAi in other nematodes, including parasitic nematodes where more effective RNAi would be extremely useful. Here, we review the current advances in C. elegans for RNA delivery methods, regulation of cell autonomous and systemic RNAi phenomena, and implications of enhanced RNAi mutants. These discussions, with a focus on mechanism and cross-species application, provide new perspectives for optimizing RNAi in other species.     

Multiple members of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family are essential for viability in Drosophila

Mucin-type O-glycosylation represents a major form of post-translational modification that is conserved across most eukaryotic species. This type of glycosylation is initiated by a family of enzymes (GalNAc-Ts in mammals and PGANTs in Drosophila) whose members are expressed in distinct spatial and temporal patterns during development. Previous work from our group demonstrated that one member of this family is essential for viability and another member modulates extracellular matrix composition and integrin-mediated cell adhesion during development. To investigate whether other members of this family are essential, we employed RNA interference (RNAi) to each gene in vivo. Using this approach, we identified 4 additional pgant genes that are required for viability. Ubiquitous RNAi to pgant4, pgant5, pgant7, or the putative glycosyltransferase CG30463 resulted in lethality. Tissue-specific RNAi was also used to define the specific organ systems and tissues in which each essential family member is required. Interestingly, each essential pgant had a unique complement of tissues in which it was required. Additionally, certain tissues (mesoderm, digestive system, and tracheal system) required more than one pgant, suggesting unique functions for specific enzymes in these tissues. Expanding upon our RNAi results, we found that conventional mutations in pgant5 resulted in lethality and specific defects in specialized cells of the digestive tract, resulting in loss of proper digestive system acidification. In summary, our results highlight essential roles for O-glycosylation and specific members of the pgant family in many aspects of development and organogenesis.