Systematic functional analysis of the Caenorhabditis elegans genome using RNAi

G3: Genes|Genomes|Genetics, 2018

Using combined genetic mapping, Illumina sequencing, bioinformatics analyses, and experimental validation, we identified 60 essential genes from 104 lethal mutations in two genomic regions of Caenorhabditis elegans totaling ∼14 Mb on chromosome III(mid) and chromosome V(left). Five of the 60 genes had not previously been shown to have lethal phenotypes by RNA interference depletion. By analyzing the regions around the lethal missense mutations, we identified four putative new protein functional domains. Furthermore, functional characterization of the identified essential genes shows that most are enzymes, including helicases, tRNA synthetases, and kinases in addition to ribosomal proteins. Gene Ontology analysis indicated that essential genes often encode for enzymes that conduct nucleic acid binding activities during fundamental processes, such as intracellular DNA replication, transcription, and translation. Analysis of essential gene shows that they have fewer paralogs, encode pr...

Science, 2010

Science, 2000

Until now, genome-wide transcriptional profiling has been limited to single-cell organisms. The nematode Caenorhabditis elegans is a well-characterized metazoan in which the expression of all genes can be monitored by oligonucleotide arrays. We used such arrays to quantitate the expression of C. elegans genes throughout the development of this organism. The results provide an estimate of the number of expressed genes in the nematode, reveal relations between gene function and gene expression that can guide analysis of uncharacterized worm genes, and demonstrate a shift in expression from evolutionarily conserved genes to worm-specific genes over the course of development.

Science, 2012

Lab team. Undergraduate research team and graduate student teaching assistant working on a C. elegans project.

BMC Genomics

Background: Essential genes are required for an organism's viability and their functions can vary greatly, spreading across many pathways. Due to the importance of essential genes, large scale efforts have been undertaken to identify the complete set of essential genes and to understand their function. Studies of genome architecture and organization have found that genes are not randomly disturbed in the genome. Results: Using combined genetic mapping, Illumina sequencing, and bioinformatics analyses, we successfully identified 44 essential genes with 130 lethal mutations in genomic regions of C. elegans of around 7.3 Mb from Chromosome I (left). Of the 44 essential genes, six of which were genes not characterized previously by mutant alleles, let-633/let-638 (B0261.1), let-128 (C53H9.2), let-511 (W09C3.4), let-162 (Y47G6A.18), let-510 (Y47G6A.19), and let-131 (Y71G12B.6). Examine essential genes with Hi-C data shows that essential genes tend to cluster within TAD units rather near TAD boundaries. We have also shown that essential genes in the left half of chromosome I in C. elegans function in enzyme and nucleic acid binding activities during fundamental processes, such as DNA replication, transcription, and translation. From protein-protein interaction networks, essential genes exhibit more protein connectivity than nonessential genes in the genome. Also, many of the essential genes show strong expression in embryos or early larvae stages, indicating that they are important to early development. Conclusions: Our results confirmed that this work provided a more comprehensive picture of the essential gene and their functional characterization. These genetic resources will offer important tools for further heath and disease research.

G3: Genes|Genomes|Genetics, 2012

Transcriptional regulation, a primary mechanism for controlling the development of multicellular organisms, is carried out by transcription factors (TFs) that recognize and bind to their cognate binding sites. In Caenorhabditis elegans, our knowledge of which genes are regulated by which TFs, through binding to specific sites, is still very limited. To expand our knowledge about the C. elegans regulatory network, we performed a comprehensive analysis of the C. elegans, Caenorhabditis briggsae, and Caenorhabditis remanei genomes to identify regulatory elements that are conserved in all genomes. Our analysis identified 4959 elements that are significantly conserved across the genomes and that each occur multiple times within each genome, both hallmarks of functional regulatory sites. Our motifs show significant matches to known core promoter elements, TF binding sites, splice sites, and poly-A signals as well as many putative regulatory sites. Many of the motifs are significantly corr...

Nature Reviews Drug Discovery, 2006

In the second half of the twentieth century, Sydney Brenner introduced the soil nematode Caenorhabditis elegans as a model to study development and neurobiology 1 . Today, C. elegans is used to study a much larger variety of biological processes including apoptosis, cell signalling, cell cycle, cell polarity, gene regulation, metabolism, ageing and sex determination 2 . Many key discoveries, both in basic biology and medically relevant areas, were first made in the worm (BOX 1). Together, these studies revealed a surprisingly strong conservation in molecular and cellular pathways between worms and mammals. Indeed, subsequent comparison of the human and C. elegans genomes confirmed that the majority of human disease genes and disease pathways are present in C. elegans 3 . C. elegans has a number of features that make it a powerful tool for the pharmaceutical industry. First, it is easy to culture: although the animal normally grows in the soil and feeds on various bacteria, it can readily be raised in the laboratory on a diet of Escherichia coli. Second, it reproduces rapidly and prolifically: within 3 days it develops from egg to an adult worm of 1.3 mm in length. Short generation time and about 300 progenies per self-fertilizing hermaphrodite enable the large-scale production of several million animals per day. Third, because of its small size, most assays can be carried out in microtitre plates either on agar or in liquid using more than one hundred animals in a single well of a 96-well plate. Fourth, the worm is transparent and, with the use of in vivo fluorescence markers, processes such as axon growth, embryogenesis and fat metabolism can easily be studied in the living animal. Fifth, it is a sophisticated multicellular animal: although the adult hermaphrodite has only 959 somatic cells, these form many different organs and tissues including muscle, hypodermis (skin), intestine, reproductive system, glands, and a nervous system containing 302 neurons 2,4 . Relevance of the C. elegans system Although C. elegans has provided many insights into the underlying mechanisms of human diseases, many still question whether C. elegans can really be used as disease model and, if so, how relevant such a model can be. Does C. elegans demonstrate behaviours that could be characterized as mood disorders? Can worms be depressed? In most cases, there will not be a direct correspondence between human pathology and C. elegans phenotypes. Given that even mammalian models are often not reliably predictive of drug action in humans, it is -from a preclinical model perspective -unrealistic to expect an invertebrate system to give enough confidence to predict drug action and safety in humans. Non-mammalian model organisms will be typically used in early research and should deliver fast answers to a discovery problem, such as the function of a gene, or pioneer medical research to define novel therapeutic entry points. Of the animal models, C. elegans is certainly the fastest and most amenable to cost-effective medium/high-throughput technologies. C. elegans is a valuable disease model if the disease can be defined on a molecular basis. For example, if the underlying cause of depression is a defect in serotonergic signalling, a C. elegans model can be developed to study serotonergic signalling in detail. Such models

2008

The discovery of RNA interference in C. elegans in 1998 has established this nematode as a key model to study RNAi mechanisms. Meanwhile, RNAi has been extensively applied to study a variety of basic biological questions in worms. In C. elegans, the dsRNA required to trigger RNAi can be administered by injection, by soaking, and importantly, by feeding. The delivery of dsRNA through food and the existence of two RNAi feeding libraries allow for large scale RNAi approaches. Thus, RNAi libraries have been used to screen for genes related with processes as diverse as embryonic development, longevity, fat accumulation, DNA damage response, axon guidance or synapse structure, among others. Moreover, since RNAi facilitates the inactivation of more than one gene at the time in the same animal, RNAi assays are being applied to uncover genetics interactions among genes and therefore there is a significant and ongoing progress in identifying components of genetics pathways and networks. Here we review successful applications of RNAi as tool in C. elegans research, and discuss trends in using such tool to shed light into several biological research fields. Applications of RNAi mechanisms nuances Since 1998, when Andy Fire and collaborators published in Nature a "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans" [2], scientists are squeezing all the fresh information published on RNAi mechanisms to get an optimal efficiency and expand the applicability of this revolutionary experimental tool. As examples, C. elegans researchers are now able to conduct RNAi assays in a tissue-specific manner or in strains with hypersensitivity to RNAi. Fire and co-workers observed how injection of dsRNA into worm gonads induced a sequence specific silencing of the corresponding gene. With the help of valuable research in C. elegans, plants and Drosophila, details of RNAi mechanisms have been revealed in the past few years [5, 6]. Once dsRNA is inside the cell, a protein called Dicer slices it to produce small dsRNA molecules of 21-22 nucleotides known as siRNAs. Each siRNA has a "sense" strand (sequence identical to the target gene) that is eliminated and an "antisense" strand (sequence complementary to the target gene) that guides the protein complex RISC to degrade the mRNA transcribed from the targeted gene. RNAi silencing effect was initially thought to happen only through mRNA degradation but two more mechanisms were uncovered acting by blocking transcription [7, 8] and by inhibiting translation [9, 10]. Interestingly, siRNAs are defensive weapons that the eukaryotic cell uses to fight dsRNA viruses and other harmful influences for its genome such as transposons (mobile elements) and repetitive sequences (as transgenes). Thus, worms carrying mutations in genes related with RNAi process are often unable to silence transposons or transgenes in germline tissues [8, 11]. Rather than digging into details of RNAi mechanisms, in this section we intend to list and comment on RNAi mechanism nuances that allow us to profit from RNAi as technique. A remarkable feature of RNAi in C. elegans is the systemic spread of the induced silencing. In other words, application of dsRNA in one part of the animal can produce an RNAi effect in distant tissues. Scientists soon exploited this phenomenon to trigger a systemic RNAi response by soaking worms in dsRNA solution and by feeding worms with bacteria expressing dsRNA [12, 13]. Administration of dsRNA by soaking is more expensive and less reliable than feeding (Fernandez and Piano, personal communication). Moreover, there are two complementary RNAi feeding libraries, commercially available and widely distributed, containing feeding clones to inactivate approximately 86% of the C. elegans genes [14, 15]. These libraries were generated in Julie Ahringer and Marc Vidal labs using as template to synthesize dsRNA genomic DNA and a cDNA library respectively. As consequence, dsRNA by feeding is a technique performed in most of the C. elegans labs routinely. Conveniently RNAi by feeding allows for modulation or titration of the RNAi effect by diluting the bacteria [16, 17]. Interestingly, although the RNA machinery is capable of being saturated, combinatorial RNAi (simultaneous targeting of two different genes) have effectively been developed [16]. The phenomenon of systemic RNAi, or RNAi spreading, has been studied by identifying mutants in which systemic RNAi is impaired [18, 19]. Detailed analysis of these mutations will clarify the spreading process. One of the genes affected for these mutations is sid-1, which encodes a transmembrane protein required for the uptake of dsRNA. In the absence of SID-1 protein, dsRNA can still trigger RNAi in cells where is injected or endogenously synthesized, but it is not effective in other neighboring or distant cells.

Current Opinion in Genetics & Development, 1996

The sequencing of the 100 Mb Caenorhabditis elegans genome-containing-14 000 genes-is-50% complete. One of its most interesting features is its compactness; introns and intergenic distances are unusually small and, surprisingly,-25% of genes are contained in polycistronic transcription units (operons) with only-100 bp between genes.

Genetics, 2020

The emergence of large gene expression datasets has revealed the need for improved tools to identify enriched gene categories and visualize enrichment patterns. While gene ontogeny (GO) provides a valuable tool for gene set enrichment analysis, it has several limitations. First, it is difficult to graph multiple GO analyses for comparison. Second, genes from some model systems are not well represented. For example, 30% of Caenorhabditis elegans genes are missing from the analysis in commonly used databases. To allow categorization and visualization of enriched C. elegans gene sets in different types of genome-scale data, we developed WormCat, a web-based tool that uses a near-complete annotation of the C. elegans genome to identify coexpressed gene sets and scaled heat map for enrichment visualization. We tested the performance of WormCat using a variety of published transcriptomic datasets, and show that it reproduces major categories identified by GO. Importantly, we also found previously unidentified categories that are informative for interpreting phenotypes or predicting biological function. For example, we analyzed published RNA-seq data from C. elegans treated with combinations of lifespan-extending drugs, where one combination paradoxically shortened lifespan. Using WormCat, we identified sterol metabolism as a category that was not enriched in the single or double combinations, but emerged in a triple combination along with the lifespan shortening. Thus, WormCat identified a gene set with potential. phenotypic relevance not found with previous GO analysis. In conclusion, WormCat provides a powerful tool for the analysis and visualization of gene set enrichment in different types of C. elegans datasets. KEYWORDS C. elegans; gene set enrichment analysis; RNA sequencing visualization R NA-SEQ is an indispensable tool for understanding how gene expression changes during development or upon environmental perturbations. As this technology has become less expensive and more robust, it has become more common to generate data from multiple conditions, enabling comparisons of gene expression profiles across biological contexts. The most commonly used method to derive information on the biological function of coexpressed genes is gene ontology (GO) (The Gene Ontology Consortium 2019) (Ashburner et al. 2000), where annotation for each gene follows three major classifications: Biological Process, Molecular Function, or Cellular Component. For example, the Biological Process class refers to genes included in a process that an organism is programmed to execute, and that occurs through specific regulated molecular events. Molecular Function denotes protein activities, and Cellular Component maps the location of activity. Within each of these classifications, functions are broken down in parent-child relationships with increasing functional