Frolows, Natalya et al. (2019) International Worm Meeting "Biooorthogonal RNA labelling in Caenorhabditis elegans."

Ashe, Alyson, Frolows, Natalya

[International Worm Meeting, 2019]

Small RNAs have been implicated as a major mechanism of epigenetic inheritance, but the identity and interactions of these involved RNA molecules has previously been hard to elucidate. A key to studying these heritable RNA molecules is the ability to selectively isolate RNA from particular cell types and time points of interest. Recently a method of bioorthogonally tagging nascent RNA molecules has been developed which uses the bacterial enzyme uracil phosphoribosyl transferase (UPRT) to insert a chemically tagged uracil analogue, 5-ethynyluracil (5EU) into RNA, allowing for temporal and cell-type control of RNA labelling. This method also allows for bioconjugation via click-chemistry and can be used for biotinylation and streptavidin extraction of the specific RNA of interest. This method was developed in immortalised cell lines and has so far only been adapted to mice and Drosophila melanogaster, but the technique shows great promise as a means to search for heritable RNA molecules within our Caenorhabditis elegans model of transgenerational epigenetic inheritance. Transgenic C. elegans strains that express UPRT under the control of targeted promoters have been created. These strains will be used to optimise the 5EU labelling method to the C. elegans model and then to isolate and identify RNA molecules involved in the TEI mechanism.

Monteiro, Dhruv et al. (2019) International Worm Meeting "The role of set-9 and set-26 in transgenerational epigenetic inheritance in Caenorhabditis elegans."

Mackay, Joel, Monteiro, Dhruv, Ashe, Alyson, Woodhouse, Rachel

[International Worm Meeting, 2019]

Although the inheritance of genetic information has long been established, in recent years, it has become apparent that epigenetic marks can also be transmitted from one generation to the next. This phenomenon, where gene expression patterns are transmitted from one generation to the next with no change to the DNA sequence, is referred to as transgenerational epigenetic inheritance (TEI). It has been observed in a wide range of species, including Mus musculus, Drosophila melanogaster, and Caenorhabditis elegans and has implications for natural selection, viral defence, and germline immortality. The research presented here utilizes C. elegans in an RNA interference (RNAi) assay to assess the inheritance of gene silencing, involving silencing of a gfp transgene by exposure of parent animals to an RNAi trigger and assessing following generations for inheritance of this silencing in the absence of this trigger. Previous work has distinguished three distinct parts to this transgenerational silencing process, including initiation of gene silencing, the establishment of silencing inheritance within the parental germline, and the maintenance of this silencing signal within the progeny. A number of genes have been implicated as involved in one or more of these processes, including histone methyltransferases and Argonaute proteins. This study examines the highly homologous predicted histone methyltransferases set-9 and set-26, to determine in which stage of TEI, if any, they act. The results of the study implicate set-9 as being involved in establishment of a silencing signal and set-26 and as being involved in both establishment of a silencing signal and its maintenance in subsequent generations. Information regarding the structure and related function of SET-9 and SET-26 was elucidated through open source bioinformatics tools in an attempt to understand their differential roles in TEI. Our analysis revealed that both proteins lacked the residues currently understood as necessary for enzymatic activity. Further research is underway to validate these predictions and more closely analyse their protein function.

Woodhouse, Rachel et al. (2021) International Worm Meeting "Disruption of mitochondrial factor SDHA-2 affects sperm motility and male fertility"

Hawdon, Azelle, Woodhouse, Rachel, Wong, Edmund Hen King, Ashe, Alyson

[International Worm Meeting, 2021]

Sperm quality is essential to male fertility. Mitochondrial health contributes to sperm quality, but the precise role of mitochondria in sperm function is not fully understood. SDHA is a component of the succinate dehydrogenase (SDH) complex and plays a critical role in mitochondria in both the citric acid cycle and mitochondrial respiration. In the citric acid cycle SDHA converts succinate to fumarate. Additionally, this reaction contributes electrons to the electron transport chain, responsible for driving ATP synthesis. C. elegans have two orthologues of SDHA, SDHA-1 and SDHA-2. Here, we show that mutation in sdha-2 results in dramatically reduced male fertility due to defective sperm activation. We found that C. elegans harbouring a sdha-2 SNP produce a significantly diminished brood size, one fifth of that of wild-type animals. In vitro sperm activation assays reveal that most mutant sperm do not activate from spermatids to spermatozoa, failing to grow the pseudopod required for motility. As a result, mutant sperm fail to localise to the spermatheca in hermaphrodites, the site of oocyte fertilisation. Instead, sperm display aberrant localisation throughout the uterus. We repaired the sdha-2 SNP in the endogenous locus to wild-type sequence using CRISPR-Cas9 and demonstrated complete rescue of brood size and sperm activation. We identified similar sperm motility defects in sdha-1 mutant animals, further implicating succinate dehydrogenase function, and in icl-1 mutant animals. ICL-1 catalyzes the cleavage of isocitrate to succinate and glyoxylate in the glyoxylate cycle. This suggests that an imbalance in succinate may underlie the dramatic brood size defect, a hypothesis that we have tested by performing metabolomics on these strains. Our results demonstrate a role for SDHA-2 in sperm motility and male reproductive health. In humans, SDH activity is positively correlated with sperm quality, and mutations in SDHA are associated with Leigh Syndrome. This strain may provide a new animal model of human SDH deficiency-associated infertility and other pathologies.

Woodhouse, Rachel et al. (2019) International Worm Meeting "Transgenerational epigenetic inheritance is revealed as a multi-step process by studies of the SET-domain proteins SET-25 and SET-32."

Ashe, Alyson, Low, Jason, Larance, Mark, Woodhouse, Rachel, Buchmann, Gabriele, Boag, Peter, Harney, Dylan, Hoe, Matthew

[International Worm Meeting, 2019]

It has recently become clear that in some circumstances Lamarck may have been right. There are a growing number of examples where a clear case can be made for the inheritance from parent to offspring of environmentally acquired gene expression changes. We have developed a sensor in C. elegans in which RNAi-induced silencing of a GFP transgene is robustly inherited for multiple generations in the absence of the initial RNAi trigger. The visible nature of this phenotype provides an exquisitely sensitive system, whereby we can separate individual animals according to their silencing status and measure effects in these distinct groups. Using this approach, we show that the histone H3 lysine 9 (H3K9) trimethyltransferase SET-25 and the putative histone methyltransferase SET-32 are required in the RNAi-exposed generation for effective transmission of transgene silencing, but are dispensable for silencing inheritance in subsequent generations. This indicates that SET-25 and SET-32 are required for the establishment of a transgenerational silencing signal but not for long-term maintenance of this signal, suggesting that transgenerational epigenetic inheritance is a multi-step process with distinct genetic requirements for establishment and maintenance of heritable silencing. We further characterize SET-25 and SET-32 to gain insights into the nature of the silencing establishment signal. We show that SET-25 is expressed in nuclei in the mitotic zone and embryos, whilst SET-32 is expressed in both the cytoplasm and nuclei throughout the entire germline. Additionally, immunofluorescence and quantitative mass spectrometry reveal aberrant H3K9me3 in set-25 and set-32 mutants. Previous studies have implicated secondary siRNAs as the effector molecules of heritable silencing. We separated animals according to their inherited silencing status (GFP-silenced or GFP-expressing) and sequenced small RNA populations in set-25 and set-32 mutants, and in mutants of the nuclear RNAi inheritance machinery. We found that the abundance of secondary siRNAs does not correlate well with silencing phenotypes in the sequenced generation or in the next generation, suggesting that the current mechanistic models of epigenetic inheritance are incomplete.

Le Pen, Jeremie et al. (2013) International Worm Meeting "A deletion polymorphism in the C. elegans RIG-I homolog disables antiviral siRNA formation and immunity."

Le Pen, Jeremie, Lehrbach, Nicolas J., Miska, Eric A., Felix, Marie-Anne, Cording, Amy, Belicard, Tony, Sarkies, Peter, Ashe, Alyson

[International Worm Meeting, 2013]

RNA interference is an important effector of antiviral immunity in plants and animals. Yet, how Dicer can sense the foreign genomes of RNA viruses is still poorly understood. We address this question using the nematode Caenorhabditis elegans and its natural pathogen, the RNA virus Orsay. Genomic studies on C. elegans wild isolates permitted us to identify a common 159 base-pair deletion in the drh-1 gene as a major determinant of viral sensitivity. We show that DRH-1, a known Dicer interactor in C. elegans and the homologue of the human RIG-I receptor, is required for the generation of viral-derived siRNAs (viRNAs) by Dicer. It is well established in humans that RIG-I can sense foreign nucleic acids and trigger an interferon-based innate immunity response upon infection by RNA viruses such as Influenza. Our work with nematodes demonstrates that the conserved RNA binding domain of RIG-I has an ancient role in viral recognition outside of mammals. We therefore propose that RIG-I/DRH-1 acts as modular viral receptor that can couple viral sensing to different effector pathways including RNAi and interferon responses.

Miska, Eric A. et al. (2011) International Worm Meeting "Modulators of small RNA pathways determine immunity against a natural virus of C. elegans."

Wu, G., Ashe, Alyson, Sanroman, M., Zhao, G., Nuez, I., Miska, Eric A., Franz, C.J., Piffaretti, J., Belicard, T., Goldstein, Leonard D., Wang, D., Jiang, Y., Felix, M.A.

[International Worm Meeting, 2011]

From wild isolates of C. elegans and C. briggsae with unusual morphological phenotypes in intestinal cells, we identified two novel RNA viruses distantly related to known nodaviruses, one infecting specifically C. elegans (Orsay virus), the other C. briggsae (Santeuil virus). Infected animals continuously maintained viral infection for 6 mo (~50 generations), demonstrating that natural cycles of horizontal virus transmission were faithfully recapitulated in laboratory culture. In addition to infecting the natural C. elegans isolate (JU1580), Orsay virus readily infected laboratory C. elegans mutants defective in RNAi and yielded higher levels of viral RNA and infection symptoms as compared to infection of the corresponding wild-type N2 strain. These results demonstrated a clear role for RNAi in the defense against this virus. Furthermore, different wild C. elegans isolates displayed differential susceptibility to infection by Orsay virus, thereby affording genetic approaches to defining antiviral loci. This discovery establishes a bona fide viral infection system to explore the natural ecology of nematodes, host-pathogen co-evolution, the evolution of small RNA responses, and innate antiviral mechanisms. More recently we have been able to demonstrate that the difference in viral immunity between the laboratory reference N2 and the wild isolate JU1580 is due, at least in part, to a defect in the antiviral small RNA response. Sequencing of small RNA populations in N2, rde-1 mutants and JU1580 after infection with the Orsay virus suggests that this defect is downstream of the generation of Dicer-dependent primary siRNAs, but upstream of the generation of antiviral secondary siRNAs. We have used a candidate screen and QTL mapping to identify genes required for an effective antiviral small RNA response. In addition, we have analysed mRNA expression profiles of N2, rde-1 mutants and JU1580 before and after infection with the Orsay virus. We find that viral infection results in the upregulation of a specific set of innate immunity genes. Finally, we find that small RNA pathways not only raise an antiviral response, but also modulate the expression of innate immunity genes.

Frezal, Lise et al. (2013) International Worm Meeting "Microevolution and coexistence of Santeuil and Le Blanc viruses in Caenorhabditis briggsae. ."

Frezal, Lise, Felix, Marie-Anne

[International Worm Meeting, 2013]

We recently found three viruses, Orsay, Santeuil and Le Blanc, which naturally infect Caenorhabditis nematodes (1,2). These ss(+)RNA viruses cause intestinal cell symptoms and are horizontally transmitted. Whereas C. elegans can so far only be infected by the Orsay virus, European C. briggsae genotypes are susceptible to both Santeuil and Le Blanc viruses, and both viruses have been found in the same locations. This vulnerability of C. briggsae to two viruses enables studies of in vivo viral competition and of the mechanisms driving their short-term evolution, as well as the impact of their competition on worm fitness. RNA viruses may evolve rapidly through both high mutation rates and recombination events. The impact of recombination widely varies from one viral species to another but in all cases, for recombination to occur, different virus types have to infect the same host cell. The first step is thus to assess whether different virus species can co-infect the same worm population, the same animal and the same cell. By using quantitative RT-PCR, we demonstrate that the Le Blanc and Santeuil viruses can coexist in a worm population, even when originally introduced at widely different concentrations. The two viruses are jointly maintained over 10 worm generations. We presently investigate the co-infection at the whole organism and single cell levels by tracking the viral RNAs in co-infected worms using Fluorescent In Situ Hybridization. 1- Felix, Ashe, Piffaretti et al. 2011 PloS biology. 2- Franz et al. 2012 Journal of virology.

Frezal, Lise et al. (2015) International Worm Meeting "Evolutionary variation in heat-triggered multigenerational onset of sterility in C. elegans."

Miska, Eric, Felix, Marie-Anne, Frezal, Lise

[International Worm Meeting, 2015]

We serendipitously discovered that many C. elegans wild isolates display a strong Mortal Germ Line (Mrt) phenotype (1) at high temperature, whereby upon chronic exposure to temperatures such as 25degC, they progressively become sterile after several generations. Here we will report on our systematic studies of this multigenerational onset of sterility in wild isolates of C. elegans. We assayed a reference panel of 97 genetically distinct isogenic C. elegans wild isolates (2) for the number of generations at 25degC after which full sterility occurred. Reproducible variation was found among these wild isolates showing that C. elegans displays natural variation in the Mrt phenotype, likely of genetic nature. The N2 strain is among the most resistant ones. In order to genetically map this natural variation in the multigenerational onset of sterility, we generated and investigated 120 recombinant inbred lines derived from parental isolate pairs with contrasted Mrt phenotypes. Previous studies (e.g. 3, 4) strongly suggest that the observed temperature-sensitive Mrt phenotype could be regulated through small RNAs and/or chromatin state modifications that are transmitted across generations. Therefore, we are investigating the evolution of small RNA contents over several generations in naturally Mrt and non-Mrt C. elegans isolates. The aim of this work is to test whether and how epigenetic inheritance systems are modulated by natural genetic variation. We are particularly intrigued by the evolutionary and ecological contexts that allow the evolution of conditional sterility defects.(1) Ahmed & Hodgkin. 2000. Nature. (2) Andersen et al. 2012 Nature Genetics (3) Ashe et al. 2012 Cell (4) Buckley et al. 2012 Nature..

TANGUY, Melanie et al. (2015) International Worm Meeting "A STAT homologue is a key transcription factor in Caenorhabditis elegans antiviral immunity."

Miska, Eric, Sarkies, Peter, Tanguy, Melanie

[International Worm Meeting, 2015]

Innate immunity represents the capacity of any cell types to mount a reaction against any potential danger and is conserved in all domains of life. The recent discovery of a virus able to infect C. elegans in the wild offers the opportunity for characterization of its antiviral immunity mechanisms. We previously showed that the RNAi pathway is a major component of antiviral innate immunity, and is initiated by DRH-1 (1), a member of the family of RIG-I like receptors conserved in mammals (2). Surprisingly, despite the absence of interferon in C. elegans, DRH-1 also acts upstream of a transcriptional response to viral infection (3). Through further analysis of the antiviral transcriptional response, we identified the STAT family transcription factor STA-1 as a main effector of innate immunity. Intriguingly, just as in mammals, STA-1 acts downstream of viral sensing by DRH-1. However, unlike in mammals, STA-1 acts as a constitutive repressor of viral response genes, with DRH-1 acting to release repression as a result of viral infection.Altogether, we propose that DRH-1 has a conserved role in a STAT signaling pathway during viral infection in addition to its initiator of the RNAi response. The respective contribution of RNA interference and interferon pathways across species or even within an organism is a current challenge of antiviral research. In this study, we take advantage of Orsay virus infection in C. elegans and the dual role of its RIG-I like receptor to better understand how organism can shape an efficient response to pathogen.1. Ashe A et al. Elife. 2(0):e00994. (2013)2. Yoneyama M et al. Curr. Opin. Immunol. 32C:48. (2015)3. Sarkies P et al. Genome Res. 23(8):1258. (2013).

Frezal, Lise et al. (2015) International Worm Meeting "Caenorhabditis briggsae and its two natural viruses, coevolution in a 'menage a trois'."

BRESARD, Gautier, Felix, Marie-Anne, Frezal, Lise

[International Worm Meeting, 2015]

We study the natural coevolution between Caenorhabditis briggsae and its two recently described RNA viruses called Santeuil and Le Blanc (1, 2). The main advantage of this system is to combine the access to wild host and virus populations with powerful molecular tools and experimental evolution designs. We characterized the incidence of the two C. briggsae viruses in France and found that they are found in sympatry. By monitoring the viral RNAs in wild-caught C. briggsae isolates using Fluorescent In Situ Hybridization, we demonstrated that the Le Blanc and Santeuil viruses could coexist in one host population, one animal and one intestinal cell. Molecular variation of the wild-caught viruses was assessed by sequencing their two RNA molecules. While both viruses' diversities are geographically structured, we detected balancing selection on the RNA-dependent RNA polymerase (RdRp) locus in one local Santeuil population. Despite the frequent incidence of coinfection in the wild, we found no evidence for genetic exchange (recombination or RNA reassortment) between the Santeuil and Le Blanc viruses. However, we found clear evidence for RNA reassortment between different Santeuil virus variants. Finally, we investigated natural variation in C. briggsae resistance to each virus. We tested a set of wild isolates -representative of C. briggsae worldwide diversity- for their sensitivity to the Santeuil and Le Blanc viruses. While temperate C. briggsae genotypes are generally susceptible to both viruses, the tested tropical C. briggsae genotypes are resistant to both viruses. Most interestingly, two Japanese C. briggsae genotypes show specific resistance to the Le Blanc virus. To understand the genetic basis of the general and virus-specific resistances of C. briggsae, we carried out a QTL-mapping approach using recombinant inbred lines between AF16 and HK104 (3) and identified a main QTL region on chromosome IV responsible for the variation in resistance to Santeuil virus infection.(1) Felix, Ashe, Piffaretti et al. 2011 PloS Biology. (2) Franz et al. 2012 Journal of Virology. (3) Ross et al. 2011 PLoS Genetics..