Elegans Gene Research Articles

Genome-Wide Temporal Expression Profiling in Caenorhabditis elegans Identifies a Core Gene Set Related to Long-Term Memory.

The identification of genes related to encoding, storage, and retrieval of memories is a major interest in neuroscience. In the current study, we analyzed the temporal gene expression changes in a neuronal mRNA pool during an olfactory long-term associative memory (LTAM) in Caenorhabditis elegans hermaphrodites. Here, we identified a core set of 712 (538 upregulated and 174 downregulated) genes that follows three distinct temporal peaks demonstrating multiple gene regulation waves in LTAM. Compared with the previously published positive LTAM gene set (Lakhina et al., 2015), 50% of the identified upregulated genes here overlap with the previous dataset, possibly representing stimulus-independent memory-related genes. On the other hand, the remaining genes were not previously identified in positive associative memory and may specifically regulate aversive LTAM. Our results suggest a multistep gene activation process during the formation and retrieval of long-term memory and define general memory-implicated genes as well as conditioning-type-dependent gene sets.SIGNIFICANCE STATEMENT The identification of genes regulating different steps of memory is of major interest in neuroscience. Identification of common memory genes across different learning paradigms and the temporal activation of the genes are poorly studied. Here, we investigated the temporal aspects of Caenorhabditis elegans gene expression changes using aversive olfactory associative long-term memory (LTAM) and identified three major gene activation waves. Like in previous studies, aversive LTAM is also CREB dependent, and CREB activity is necessary immediately after training. Finally, we define a list of memory paradigm-independent core gene sets as well as conditioning-dependent genes.

Locomotion Behavior Is Affected by the GαS Pathway and the Two-Pore-Domain K+ Channel TWK-7 Interacting in GABAergic Motor Neurons in Caenorhabditis elegans.

Adjusting the efficiency of movement in response to environmental cues is an essential integrative characteristic of adaptive locomotion behavior across species. However, the modulatory molecules and the pathways involved are largely unknown. Recently, we demonstrated that in Caenorhabditis elegans, a loss-of-function of the two-pore-domain potassium (K2P) channel TWK-7 causes a fast, coordinated, and persistent forward crawling behavior in which five central aspects of stimulated locomotion-velocity, direction, wave parameters, duration, and straightness-are affected. Here, we isolated the reduction-of-function allele cau1 of the C. elegans gene kin-2 in a forward genetic screen and showed that it phenocopies the locomotor activity and locomotion behavior of twk-7(null) animals. Kin-2 encodes the negative regulatory subunit of protein kinase A (KIN-1/PKA). Consistently, we found that other gain-of-function mutants of the GαS-KIN-1/PKA pathway resemble kin-2(cau1) and twk-7(null) in locomotion phenotype. Using the powerful genetics of the C. elegans system in combination with cell type-specific approaches and detailed locomotion analyses, we identified TWK-7 as a putative downstream target of the GαS-KIN-1/PKA pathway at the level of the γ-aminobutyric acid (GABA)ergic D-type motor neurons. Due to this epistatic interaction, we suggest that KIN-1/PKA and TWK-7 may share a common pathway that is probably involved in the modulation of both locomotor activity and locomotion behavior during forward crawling.

An evolutionarily conserved transcriptional response to viral infection in Caenorhabditis nematodes

BackgroundCaenorhabditis elegans is a powerful model organism for probing many biological processes including host-pathogen interactions with bacteria and fungi. The recent identification of nematode viruses that naturally infect C. elegans and Caenorhabditis briggsae provides a unique opportunity to define host-virus interactions in these model hosts.ResultsWe analyzed the transcriptional response of pathogen infected C. elegans and C. briggsae by RNA-seq. We identified a total of 320 differentially expressed genes (DEGs) in C. elegans following Orsay virus infection. The DEGs of known function were enriched for ubiquitin ligase related genes; however, the majority of the genes were of unknown function. Interestingly, many DEGs that responded to Orsay virus infection were similar to those induced by Nematocida parisii infection, which is a natural microsporidia pathogen of C. elegans that like Orsay virus infects intestinal cells. Furthermore, comparison of the Orsay virus DEGs in C. elegans to Santeuil virus DEGs in C. briggsae identified 58 C. elegans genes whose orthologs were likewise differentially expressed in C. briggsae, thereby defining an evolutionarily conserved response to viral infection.ConclusionsThe two different species C. elegans and C. briggsae, which diverged ~18 million years ago, share a common set of transcriptionally responsive genes to viral infection. Furthermore, a subset of these genes were also differentially expressed following infection by a eukaryotic pathogen, N. parisii, suggesting that these genes may constitute a broader pan-microbial response to infection.

Caenorhabditis elegans orthologs of human genes differentially expressed with age are enriched for determinants of longevity.

SummaryWe report a systematic RNAi longevity screen of 82 Caenorhabditis elegans genes selected based on orthology to human genes differentially expressed with age. We find substantial enrichment in genes for which knockdown increased lifespan. This enrichment is markedly higher than published genomewide longevity screens in C. elegans and similar to screens that preselected candidates based on longevity‐correlated metrics (e.g., stress resistance). Of the 50 genes that affected lifespan, 46 were previously unreported. The five genes with the greatest impact on lifespan (>20% extension) encode the enzyme kynureninase (kynu‐1), a neuronal leucine‐rich repeat protein (iglr‐1), a tetraspanin (tsp‐3), a regulator of calcineurin (rcan‐1), and a voltage‐gated calcium channel subunit (unc‐36). Knockdown of each gene extended healthspan without impairing reproduction. kynu‐1(RNAi) alone delayed pathology in C. elegans models of Alzheimer's disease and Huntington's disease. Each gene displayed a distinct pattern of interaction with known aging pathways. In the context of published work, kynu‐1, tsp‐3, and rcan‐1 are of particular interest for immediate follow‐up. kynu‐1 is an understudied member of the kynurenine metabolic pathway with a mechanistically distinct impact on lifespan. Our data suggest that tsp‐3 is a novel modulator of hypoxic signaling and rcan‐1 is a context‐specific calcineurin regulator. Our results validate C. elegans as a comparative tool for prioritizing human candidate aging genes, confirm age‐associated gene expression data as valuable source of novel longevity determinants, and prioritize select genes for mechanistic follow‐up.

Laurencia majuscula var. elegans (Rhodophyta, Rhodomelaceae) is reinstated to specific rank as L. elegans

SUMMARYLaurencia majuscula has recently been determined to be conspecific with Laurencia dendroidea. To determine the correct taxonomic placement of its only non‐typical variety, L. majuscula var. elegans, mitochondrial COI‐5P and chloroplast rbcL gene sequences were analyzed and its phylogenetic position was inferred. Laurencia majuscula var. elegans consistently formed a well‐supported clade distinctly separate from the L. dendroidea clade. Molecular analyses revealed that L. majuscula var. elegans generally shows two distinct forms; a long‐branched form, and a compact form. Often the compact form has been identified as a different species based on morphology alone. Included in these analyses were topotype sequences of two species with which L. majuscula var. elegans is most often misidentified; Laurencia nidifica and Laurencia mcdermidiae. Laurencia nidifica formed a well‐supported clade separate from L. majuscula var. elegans. Laurencia mcdermidiae, however, formed a well‐defined clade sister to the L. majuscula var. elegans clade. Morphological comparisons were also made. It is concluded that L. majuscula var. elegans is morphologically distinct from L. dendroidea, L. nidifica, and L. mcdermidiae. It is proposed that L. majuscula var. elegans be reinstated to species level as L. elegans.

Kollector: transcript-informed, targeted de novo assembly of gene loci.

MotivationDespite considerable advancements in sequencing and computing technologies, de novo assembly of whole eukaryotic genomes is still a time-consuming task that requires a significant amount of computational resources and expertise. A targeted assembly approach to perform local assembly of sequences of interest remains a valuable option for some applications. This is especially true for gene-centric assemblies, whose resulting sequence can be readily utilized for more focused biological research. Here we describe Kollector, an alignment-free targeted assembly pipeline that uses thousands of transcript sequences concurrently to inform the localized assembly of corresponding gene loci. Kollector robustly reconstructs introns and novel sequences within these loci, and scales well to large genomes—properties that makes it especially useful for researchers working on non-model eukaryotic organisms.ResultsWe demonstrate the performance of Kollector for assembling complete or near-complete Caenorhabditis elegans and Homo sapiens gene loci from their respective, input transcripts. In a time- and memory-efficient manner, the Kollector pipeline successfully reconstructs respectively 99% and 80% (compared to 86% and 73% with standard de novo assembly techniques) of C.elegans and H.sapiens transcript targets in their corresponding genomic space using whole genome shotgun sequencing reads. We also show that Kollector outperforms both established and recently released targeted assembly tools. Finally, we demonstrate three use cases for Kollector, including comparative and cancer genomics applications.Availability and ImplementationKollector is implemented as a bash script, and is available at https://github.com/bcgsc/kollectorSupplementary information Supplementary data are available at Bioinformatics online.

Plant-parasitic nematodes: towards understanding molecular players in stress responses.

Background Plant–parasitic nematode interactions occur within a vast molecular plant immunity network. Following initial contact with the host plant roots, plant-parasitic nematodes (PPNs) activate basal immune responses. Defence priming involves the release in the apoplast of toxic molecules derived from reactive species or secondary metabolism. In turn, PPNs must overcome the poisonous and stressful environment at the plant–nematode interface. The ability of PPNs to escape this first line of plant immunity is crucial and will determine its virulence. Scope Nematodes trigger crucial regulatory cytoprotective mechanisms, including antioxidant and detoxification pathways. Knowledge of the upstream regulatory components that contribute to both of these pathways in PPNs remains elusive. In this review, we discuss how PPNs probably orchestrate cytoprotection to resist plant immune responses, postulating that it may be derived from ancient molecular mechanisms. The review focuses on two transcription factors, DAF-16 and SKN-1, which are conserved in the animal kingdom and are central regulators of cell homeostasis and immune function. Both regulate the unfolding protein response and the antioxidant and detoxification pathways. DAF-16 and SKN-1 target a broad spectrum of Caenorhabditis elegans genes coding for numerous protein families present in the secretome of PPNs. Moreover, some regulatory elements of DAF-16 and SKN-1 from C. elegans have already been identified as important genes for PPN infection. Conclusion DAF-16 and SKN-1 genes may play a pivotal role in PPNs during parasitism. In the context of their hub status and mode of regulation, we suggest alternative strategies for control of PPNs through RNAi approaches.

Changes emerging with calcineurin inhibition in Caenorhabditis

Background: It is known that calcineurin inhibition prolongs life in individuals with Caenorhabditis elegans ( C. elegans ). An important part of C. elegans genes shows great similarity to human genes. Aim : This study aims to evaluate the effects of a calcineurin inhibitor on vital functions in C. elegans individuals. Study Design: Animal experimentation Methods: In this study, solutions of cyclosporine A which is a calcineurin inhibitor 1 μM (1.dose), 0.1 μM (2.dose), 0.01 μM (3.dose) and 0.001 μM (4.dose) were prepared and 1 ml from each solution was taken and added to petri dishes, containing 10 ml Nematode Growth Media (NGM). The control group was fed the medium which was not added cyclosporine. The indicated doses of cyclosporine whose synchronization has been completed were transferred to petri dishes in order that each would have 20 pieces. C. elegans were observed using bottom light stereo microscop that provided 6X-50X magnification. Live animals at the same time every day were counted and compared with the control group until all C. elegans in all petri dishes died. Results: The results have showed that there was a statistically significant increase in life span periods of C. elegans exposed to cyclosporin depending on dose increase (p 0,05). Conclusion: Calcineurin inhibition slows down the developmental process in C. elegans and prolongs survival, and may produce positive effects on aging.

Tribbles ortholog NIPI-3 and bZIP transcription factor CEBP-1 regulate a Caenorhabditis elegans intestinal immune surveillance pathway.

BackgroundMany pathogens secrete toxins that target key host processes resulting in the activation of immune pathways. The secreted Pseudomonas aeruginosa toxin Exotoxin A (ToxA) disrupts intestinal protein synthesis, which triggers the induction of a subset of P. aeruginosa-response genes in the nematode Caenorhabditis elegans.ResultsWe show here that one ToxA-induced C. elegans gene, the Tribbles pseudokinase ortholog nipi-3, is essential for host survival following exposure to P. aeruginosa or ToxA. We find that NIPI-3 mediates the post-developmental expression of intestinal immune genes and proteins and primarily functions in parallel to known immune pathways, including p38 MAPK signaling. Through mutagenesis screening, we identify mutants of the bZIP C/EBP transcription factor cebp-1 that suppress the hypersusceptibility defects of nipi-3 mutants.ConclusionsNIPI-3 is a negative regulator of CEBP-1, which in turn negatively regulates protective immune mechanisms. This pathway represents a previously unknown innate immune signaling pathway in intestinal epithelial cells that is involved in the surveillance of cellular homeostasis. Because NIPI-3 and CEBP-1 are also essential for C. elegans development, NIPI-3 is analogous to other key innate immune signaling molecules such as the Toll receptors in Drosophila that have an independent role during development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0334-6) contains supplementary material, which is available to authorized users.

GExplore 1.4: An expanded web interface for queries on Caenorhabditis elegans protein and gene function.

Genetic high-throughput experiments often result in hundreds or thousands of genes satisfying certain experimental conditions. Grouping and prioritizing a large number of genes for further analysis can be a time-consuming challenge. In 2009 we developed a web-based user interface, GExplore, to assist with large-scale data-mining related to gene function in Caenorhabditis elegans. The underlying database contained information about Caenorhabditis elegans genes and proteins including domain organization of the proteins, phenotypic descriptions, expression data and Gene Ontology Consortium annotations. These data enable users to quickly obtain an overview of biological and biochemical functions of a large number of genes at once. Since its inception the underlying database has been updated and expanded significantly. Here we describe the current version of GExplore 1.4, documenting the changes since the original release. GExplore 1.4 now contains information about the domain organization of the proteomes of 9 nematode species, can display the location of Caenorhabditis elegans mutations with respect to the domain organization of the proteins, and includes stage-specific RNAseq gene expression data generated by the modENCODE project. The underlying database has been reorganized to facilitate independent updates of the different parts of the database and to allow the addition of novel data sets in the future. The web interface is available under http://genome.sfu.ca/gexplore.

XRN2 Autoregulation and Control of Polycistronic Gene Expresssion in Caenorhabditis elegans.

XRN2 is a conserved 5’→3’ exoribonuclease that complexes with proteins that contain XRN2-binding domains (XTBDs). In Caenorhabditis elegans (C. elegans), the XTBD-protein PAXT-1 stabilizes XRN2 to retain its activity. XRN2 activity is also promoted by 3'(2'),5'-bisphosphate nucleotidase 1 (BPNT1) through hydrolysis of an endogenous XRN inhibitor 3’-phosphoadenosine-5'-phosphate (PAP). Here, we find through unbiased screening that loss of bpnt-1 function suppresses lethality caused by paxt-1 deletion. This unexpected finding is explained by XRN2 autoregulation, which occurs through repression of a cryptic promoter activity and destabilization of the xrn-2 transcript. De-repression appears to be triggered such that more robust XRN2 perturbation, by elimination of both PAXT-1 and BPNT1, is less detrimental to worm viability than absence of PAXT-1 alone. Indeed, we find that two distinct XRN2 repression mechanisms are alleviated at different thresholds of XRN2 inactivation. Like more than 15% of C. elegans genes, xrn-2 occurs in an operon, and we identify additional operons under its control, consistent with a broader function of XRN2 in polycistronic gene regulation. Regulation occurs through intercistronic regions that link genes in an operon, but a part of the mechanisms may allow XRN2 to operate on monocistronic genes in organisms lacking operons.

Nematode Peptides with Host-Directed Anti-inflammatory Activity Rescue Caenorhabditis elegans from a Burkholderia pseudomallei Infection.

Burkholderia pseudomallei, the causative agent of melioidosis, is among a growing number of bacterial pathogens that are increasingly antibiotic resistant. Antimicrobial peptides (AMPs) have been investigated as an alternative approach to treat microbial infections, as generally, there is a lower likelihood that a pathogen will develop resistance to AMPs. In this study, 36 candidate Caenorhabditis elegans genes that encode secreted peptides of <150 amino acids and previously shown to be overexpressed during infection by B. pseudomallei were identified from the expression profile of infected nematodes. RNA interference (RNAi)-based knockdown of 12/34 peptide-encoding genes resulted in enhanced nematode susceptibility to B. pseudomallei without affecting worm fitness. A microdilution test demonstrated that two peptides, NLP-31 and Y43C5A.3, exhibited anti-B. pseudomallei activity in a dose dependent manner on different pathogens. Time kill analysis proposed that these peptides were bacteriostatic against B. pseudomallei at concentrations up to 8× MIC90. The SYTOX green assay demonstrated that NLP-31 and Y43C5A.3 did not disrupt the B. pseudomallei membrane. Instead, gel retardation assays revealed that both peptides were able to bind to DNA and interfere with bacterial viability. In parallel, microscopic examination showed induction of cellular filamentation, a hallmark of DNA synthesis inhibition, of NLP-31 and Y43C5A.3 treated cells. In addition, the peptides also regulated the expression of inflammatory cytokines in B. pseudomallei infected macrophage cells. Collectively, these findings demonstrate the potential of NLP-31 and Y43C5A.3 as anti-B. pseudomallei peptides based on their function as immune modulators.

Identification and functional characterization of a putative IDE, C28F5.4 (ceIDE-1), in Caenorhabditis elegans: Implications for Alzheimer's disease

Insulin-degrading enzyme (IDE) is a zinc metalloprotease, known to degrade insulin peptide and amyloid-beta (Aβ); the key protein involved in Alzheimer's disease (AD). Considering the important role played by IDE in disease progression of AD and type 2 diabetes mellitus (T2DM), we endeavored to identify the Caenorhabditis elegans (C. elegans) IDE orthologous genes and test them for their role in AD related outcomes. We employed bioinformatics, reverse genetics and molecular biology approaches towards identification and functional characterization of putative IDE candidates in C. elegans. Using in-silico analysis we have identified seven C. elegans genes that possess HXXEH motif, an identifying marker of IDE. We further carried out functional analysis of the identified genes in Aβ expressing C. elegans strain CL4176 [myo-3/Aβ1–42 long 3′-UTR] via studying effect on Aβ induced toxicity, cholinergic neuroanatomy, content of acetylcholine/acetylcholine-esterase, extent of reactive oxygen species and expression of FOXO transcription factor DAF-16. Our findings reveal that amongst the identified putative IDE orthologs, a functionally uncharacterized gene C28F5.4 had a profound effect on the tested endpoints. Knocking down C28F5.4 modulated the AD associated conditions by decreasing Aβ induced toxicity, severely compromising cholinergic neuroanatomy, reducing expression of acetylcholine-transporter, decreasing acetylcholine content, elevating ROS, with no effect on DAF-16 stress-response protein. These studies provide crucial insight into the structural/functional orthology of IDEs across human and nematode species and further our understanding of the involvement of these proteins and insulin pathway in AD. Further studies could aid in identifying novel drug-targets and in understanding the common modulating factors between AD and T2DM.

Gamete interactions require transmembranous immunoglobulin-like proteins with conserved roles during evolution.

C. elegans spe-9 class genes are male germline-enriched in their expression and indispensable during sperm-oocyte fusion. Identification of mammalian orthologs that exhibit similar functions to these C. elegans genes has been a challenge. The mouse Izumo1 gene encodes a sperm-specific, immunoglobulin (Ig)-like transmembrane (TM) protein that is required for gamete fusion. We recently identified the C. elegans spe-45 gene, which shows male germline-enriched expression and encodes an Ig-like TM protein. spe-45 mutant worms produced otherwise normal spermatozoa that cannot fuse with oocytes, causing essentially the same phenotype as that seen in the Izumo1-knockout mice. By counting the number of self-sperm in the spermatheca of spe-45 hermaphrodites, it was found that this gene might be involved in sperm guidance from the uterus into the spermatheca, as well as gamete fusion. Moreover, we discovered that SPE-45 and IZUMO1 share certain functions for gamete fusion, which are presumably related to binding with cis- and/or trans-partners. Intriguingly, various organisms have Ig-like TM proteins that act during gamete interactions, indicating the wide-spread utility of Ig-like domains during fertilization.

English

Screening of Caenorhabditis elegans genes possibly involved in the mitochondrial genome maintenance was performed using our previous validated method of RNAi combined with ethidium bromide. This was to knock down C. elegans genes homologous to yeast genes known to be involved in mtDNA stability but of unknown molecular function or to identify transient components that could play important role on the stability of mtDNA in a temporal and/or spatial manner. C. elegans homologs for 11 genes among 27 yeast genes for which deletion leads to a rho0 state were found, however, only 5 genes were present in the RNAi library. Out of these 5 genes, 1 gene (homolog of GEM1) gave a clear L3 arrest on RNAi and ethidium bromide indicating its involvement on mtDNA stability. Four other genes homologs of MTG2, YER087W, AVL9 and RRG3 did not lead to L3 arrest even though their deletion in Saccharomyces cerevisiae leads to rho0 state. Although MTG2 has been reported to be important in the function and structure on mtDNA stability in yeast, our results did not support those findings in C. elegans. The human homolog of this gene (MIRO1) can be considered as a candidate gene involved in mtDNA stability and sequenced in patients with mtDNA depletion diseases. Key words: mtDNA, Caenorhabditis elegans, nucleoid, RNAi, candidate genes, homolog, MIRO1.

Antimicrobial effectors in the nematode Caenorhabditis elegans: an outgroup to the Arthropoda.

Nematodes and arthropods likely form the taxon Ecdysozoa. Information on antimicrobial effectors from the model nematode Caenorhabditis elegans may thus shed light on the evolutionary origin of these defences in arthropods. This nematode species possesses an extensive armory of putative antimicrobial effector proteins, such as lysozymes, caenopores (or saposin-like proteins), defensin-like peptides, caenacins and neuropeptide-like proteins, in addition to the production of reactive oxygen species and autophagy. As C. elegans is a bacterivore that lives in microbe-rich environments, some of its effector peptides and proteins likely function in both digestion of bacterial food and pathogen elimination. In this review, we provide an overview of C. elegans immune effector proteins and mechanisms. We summarize the experimental evidence of their antimicrobial function and involvement in the response to pathogen infection. We further evaluate the microbe-induced expression of effector genes using WormExp, a recently established database for C. elegans gene expression analysis. We emphasize the need for further analysis at the protein level to demonstrate an antimicrobial activity of these molecules both in vitro and in vivoThis article is part of the themed issue 'Evolutionary ecology of arthropod antimicrobial peptides'.

Localization of RNAPII and 3′ end formation factor CstF subunits on C. elegans genes and operons

ABSTRACTTranscription termination is mechanistically coupled to pre-mRNA 3′ end formation to prevent transcription much beyond the gene 3′ end. C. elegans, however, engages in polycistronic transcription of operons in which 3′ end formation between genes is not accompanied by termination. We have performed RNA polymerase II (RNAPII) and CstF ChIP-seq experiments to investigate at a genome-wide level how RNAPII can transcribe through multiple poly-A signals without causing termination. Our data shows that transcription proceeds in some ways as if operons were composed of multiple adjacent single genes. Total RNAPII shows a small peak at the promoter of the gene cluster and a much larger peak at 3′ ends. These 3′ peaks coincide with maximal phosphorylation of Ser2 within the C-terminal domain (CTD) of RNAPII and maximal localization of the 3′ end formation factor CstF. This pattern occurs at all 3′ ends including those at internal sites in operons where termination does not occur. Thus the normal mechanism of 3′ end formation does not always result in transcription termination. Furthermore, reduction of CstF50 by RNAi did not substantially alter the pattern of CstF64, total RNAPII, or Ser2 phosphorylation at either internal or terminal 3′ ends. However, CstF50 RNAi did result in a subtle reduction of CstF64 binding upstream of the site of 3′ cleavage, suggesting that the CstF50/CTD interaction may facilitate bringing the 3′ end machinery to the transcription complex.

Functional Interplay of Two Paralogs Encoding SWI/SNF Chromatin-Remodeling Accessory Subunits During Caenorhabditis elegans Development.

SWI/SNF ATP-dependent chromatin-remodeling complexes have been related to several cellular processes such as transcription, regulation of chromosomal stability, and DNA repair. The Caenorhabditis elegans gene ham-3 (also known as swsn-2.1) and its paralog swsn-2.2 encode accessory subunits of SWI/SNF complexes. Using RNA interference (RNAi) assays and diverse alleles we investigated whether ham-3 and swsn-2.2 have different functions during C. elegans development since they encode proteins that are probably mutually exclusive in a given SWI/SNF complex. We found that ham-3 and swsn-2.2 display similar functions in vulva specification, germline development, and intestinal cell proliferation, but have distinct roles in embryonic development. Accordingly, we detected functional redundancy in some developmental processes and demonstrated by RNA sequencing of RNAi-treated L4 animals that ham-3 and swsn-2.2 regulate the expression of a common subset of genes but also have specific targets. Cell lineage analyses in the embryo revealed hyper-proliferation of intestinal cells in ham-3 null mutants whereas swsn-2.2 is required for proper cell divisions. Using a proteomic approach, we identified SWSN-2.2-interacting proteins needed for early cell divisions, such as SAO-1 and ATX-2, and also nuclear envelope proteins such as MEL-28. swsn-2.2 mutants phenocopy mel-28 loss-of-function, and we observed that SWSN-2.2 and MEL-28 colocalize in mitotic and meiotic chromosomes. Moreover, we demonstrated that SWSN-2.2 is required for correct chromosome segregation and nuclear reassembly after mitosis including recruitment of MEL-28 to the nuclear periphery.

Caenorhabditis elegans glp-4 Encodes a Valyl Aminoacyl tRNA Synthetase.

Germline stem cell proliferation is necessary to populate the germline with sufficient numbers of cells for gametogenesis and for signaling the soma to control organismal properties such as aging. The Caenorhabditis elegans gene glp-4 was identified by the temperature-sensitive allele bn2 where mutants raised at the restrictive temperature produce adults that are essentially germ cell deficient, containing only a small number of stem cells arrested in the mitotic cycle but otherwise have a morphologically normal soma. We determined that glp-4 encodes a valyl aminoacyl transfer RNA synthetase (VARS-2) and that the probable null phenotype is early larval lethality. Phenotypic analysis indicates glp-4(bn2ts) is partial loss of function in the soma. Structural modeling suggests that bn2 Gly296Asp results in partial loss of function by a novel mechanism: aspartate 296 in the editing pocket induces inappropriate deacylation of correctly charged Val-tRNAval. Intragenic suppressor mutations are predicted to displace aspartate 296 so that it is less able to catalyze inappropriate deacylation. Thus glp-4(bn2ts) likely causes reduced protein translation due to decreased levels of Val-tRNAval. The germline, as a reproductive preservation mechanism during unfavorable conditions, signals the soma for organismal aging, stress and pathogen resistance. glp-4(bn2ts) mutants are widely used to generate germline deficient mutants for organismal studies, under the assumption that the soma is unaffected. As reduced translation has also been demonstrated to alter organismal properties, it is unclear whether changes in aging, stress resistance, etc. observed in glp-4(bn2ts) mutants are the result of germline deficiency or reduced translation.

Novel Alleles of gon-2, a C. elegans Ortholog of Mammalian TRPM6 and TRPM7, Obtained by Genetic Reversion Screens.

TRP (Transient Receptor Potential) cation channels of the TRPM subfamily have been found to be critically important for the regulation of Mg2+ homeostasis in both protostomes (e.g., the nematode, C. elegans, and the insect, D. melanogaster) and deuterostomes (e.g., humans). Although significant progress has been made toward understanding how the activities of these channels are regulated, there are still major gaps in our understanding of the potential regulatory roles of extensive, evolutionarily conserved, regions of these proteins. The C. elegans genes, gon-2, gtl-1 and gtl-2, encode paralogous TRP cation channel proteins that are similar in sequence and function to human TRPM6 and TRPM7. We isolated fourteen revertants of the missense mutant, gon-2(q338), and these mutations affect nine different residues within GON-2. Since eight of the nine affected residues are situated within regions that have high similarity to human TRPM1,3,6 and 7, these mutations identify sections of these channels that are potentially critical for channel regulation. We also isolated a single mutant allele of gon-2 during a screen for revertants of the Mg2+-hypersensitive phenotype of gtl-2(-) mutants. This allele of gon-2 converts a serine to phenylalanine within the highly conserved TRP domain, and is antimorphic against both gon-2(+) and gtl-1(+). Interestingly, others have reported that mutation of the corresponding residue in TRPM7 to glutamate results in deregulated channel activity.