Conine, Colin et al. (2009) International Worm Meeting "Small RNA Pathways Required to Produce Thermo-tolerant Sperm in C. elegans."

Mello, Craig, Tsai, Hsin-Yue, Batista, Pedro, Conine, Colin

[International Worm Meeting, 2009]

Argonaute-mediated small RNA pathways are involved in a diversity of cellular processes in numerous organisms. In C. elegans null mutations are available for the entire family of over 24 Argonaute genes, and at least 5 different combinations of these mutants result in lethal or sterile phenotypes. Here, we focus on the role of two members of the AGO clade of Argonautes, T22B3.2 and ZK757.3 that are essential for functional sperm at elevated temperatures. The predicted products of these two AGOs are 96% identical at the amino acid level, and while either single mutant is viable and fertile the double mutant (TZK) is temperature sensitive sterile. TZK worms have a reduced brood size at 20 deg C, and are completely sterile at 25 deg C. This sterility appears to reflect a defect in sperm function at high temperature, as, even males reared at 15 deg C fail to produce cross progeny when shifted to 25 deg C. Furthermore, wild-type males can rescue sterile TZK hermaphrodites suggesting that the defect is specific to the male germ line. Consistent with this phenotype we found that a rescuing GFP::T22B3.2 transgene was expressed exclusively in germ-cells undergoing spermatogenesis and in mature spermatozoa. The TZK sterile phenotype was very similar to male-specific infertility observed in a null mutant in the rde-8-related gene C29F5.3. In addition to the rde-8 motif, C29F5.3 contains a cytidine-deaminase domain. RDE-8 is a novel protein with defects in both the exo- and endo-RNAi pathways (see abstract by Tsai et al.). RDE-8 domains have no known function but are found in organisms ranging from bacteria to humans. Surprisingly, certain classes of small RNAs that are absent in rde-8 mutants appear to be restored in rde-8, C29F5.3 double mutant animals. These findings suggest that the loss of these small RNAs may result from an ectopic function of C29F5.3 that is acquired when RDE-8 protein is absent. Gametogenesis appears to be an inherently thermosensitive process both in C. elegans and in many other metazoa. In humans the core body temperature is lethal to sperm, and external male gametogenesis is likely to represent an adaptation that was basal to the evolution of endothermia in the vertebrate lineage. Understanding how TZK argonautes and the novel protein C29F5.3 function to facilitate sperm function may shed light on the inherently ts-process that underlies spermatogenesis. We hope to gain insight into these questions by sequencing the small RNAs that engage T22B3.2, and by systematically applying transcriptomics to identify cytidine-deaminated RNAs expressed in sperm.

Conine, Colin et al. (2013) International Worm Meeting "ALG-3/4 acts through the CSR-1 pathway to promote spermiogenic gene expression and to provide a paternally inherited memory of past gene expression."

Conine, Colin, Moresco, James, Mello, Craig, Yates, John

[International Worm Meeting, 2013]

During each generation germline cells undergo truly dramatic alterations in gene expression and cellular morphology that are required to produce the male and female gametes, and do so while preserving both the genetic and epigenetic information required for pluripotency. In many animals the production of functional male gametes is particularly sensitive to temperature. We have been exploring the question of how small-RNA pathways contribute to the production of functional thermotolerant sperm in C. elegans. Here we show that ALG-3/4-associated 26G-RNAs act upstream of the argonaute CSR-1. csr-1 mutant males are identical phenotypically to alg-3/4 males and like alg-3/4 mutants, exhibit temperature sensitive infertility correlated with reduced expression of spermiogenic mRNAs. CSR-1 protein associates with peripheral spermatogenic chromatin and also localizes prominently in mature spermatozoa. Interestingly, alg-3/4 and csr-1 heterozygous males are normally fertile at all temperatures, however heterozygous progeny produced after 3 to 5 generations of paternal homozygosity exhibit the same thermo-intolerant sterile phenotype observed for homozygous males. These findings are consistent with a role for the ALG-3/4, CSR-1 pathway in maintaining an epigenetic program for thermotolerant spermiogenesis. As expected CSR-1 engages small-RNAs targeting spermiogenic RNAs in males. However, we were surprised to find that CSR-1 also engages small RNAs antisense to female-specific germline mRNAs that are not expressed in males. Taken together our findings suggest that the ALG-3/4 CSR-1 pathway promotes theromtolerance by preserving robust spermiogenic gene expression during meiosis, and that CSR-1 functions epigenetically to transmit a memory of both male- and female-specific gene expression to successive generations.

Conine, Colin et al. (2011) International Worm Meeting "The Ago-class Argonautes ALG-3 and ALG-4 and 26G-RNAs regulate spermatogenic gene expression to promote thermotolerant male fertility in C. elegans."

Mello, Craig, Yates III, John, Conine, Colin, Moresco, James

[International Worm Meeting, 2011]

Gametogenesis is an inherently thermosensitive process in numerous metazoa ranging from worms to man. In C. elegans a variety of germ-line nuage- (P-granule) -associated RNA-binding proteins have been implicated in temperature-dependent fertility. We have previously shown that two AGO-class paralogs, T22B3.2 (ALG-3) and ZK757.3 (ALG-4) are required for male fertility at elevated temperatures. alg-3/4 mutants are completely sterile at 25o and lack a subgroup of small RNAs, named 26G-RNAs that target hundreds of spermatogenesis-expressed mRNAs. alg-3/4 sterility can be rescued by mating with wild type males, suggesting that the infertility results from defects specific to the male germline. A rescuing GFP::ALG-3 transgene is localized in P-granules beginning at the late pachytene stage of male gametogenesis. ALG-3/4 mutant spermatids are defective in spermiogenesis and fail to form motile pseudopods. In order to understand how a molecular deficit in a small-RNA pathway leads to such striking morphological defects in sperm development, we have undertaken several parallel investigations. First we have screened available mutants with similar ts-sterile phenotypes for loss of 26G-RNAs. We have shown that the RdRP involved in the ALG-3/4 pathway, rrf-3, is allelic to fer-15 and that the un-cloned fer genes, fer-2,3,4 and 6, all exhibit small RNA defects identical to those of alg-3/4 mutants. Transmission Electron Microscopy on alg-3/4 mutants and previous EM studies on the fer mutants reveal defective Fibrous Body-Membranous Organelles (FB-MOs), which leads to aberrant arrangement of Major Sperm Proteins (MSPs). Whole-sperm proteomic analysis performed with the Yates lab, along with our transcriptomic analysis reveals striking ALG-3/4-dependent regulation of specific targets at the level of both mRNA and protein accumulation. For example, five non-canonical MSP proteins including the essential gene dct-9 are upregulated more than 20 fold in ALG-3/4 mutants. We are now exploring the possibility that misregulation of specific targets in alg-3/4 mutants underlies the temperature-dependent abnormalities in sperm morphology and function.

Crocker, Olivia et al. (2021) International Worm Meeting "Interrogating the role of paternally contributed tRNA fragments in C. elegans fertilization and development"

Conine, Colin, Crocker, Olivia

[International Worm Meeting, 2021]

Across organisms, males experiencing environmental stressors contribute non-genetic information to future generations. While sperm chromatin retains a small percentage of histones, the overwhelming exchange of histones for protamines in sperm paired with the erasure of DNA methylation after fertilization challenges the traditional epigenetic inheritance paradigms as mechanisms of intergenerational transmission. Prior work has demonstrated that modulating the diet of mammals has effects on the metabolism of offspring by altering the abundance of small RNAs in sperm, particularly the levels of highly abundant ~28-34 nt tRNA fragments (tRFs). After fertilization, the altered abundance of tRFs correlates with modified embryonic gene expression and development. While the potential role of tRFs as a carrier of epigenetic information in sperm is exciting, little is known about the molecular functions of tRFs. In mammals there are greater than 10 RnaseA and RnaseT2 enzymes with the potential to generate tRFs. Thus, redundancy of murine endonucleases makes it difficult to establish a tractable genetic model of tRF function. Recently, we have identified abundant tRFs in C. elegans males which are specifically enriched in sperm. Fortunately, the C. elegans genome encodes only one RnaseT2 (rnst-2) and no RnaseA homologs, allowing for simple genetic manipulation of tRF abundance. We have generated several loss-of-function and catalytically dead alleles of rnst-2 which all exhibit developmental and male fertility defects. Additionally, by endogenously tagging rnst-2 we have found that it is specifically expressed in sperm and in the developing embryo. Finally, rnst-2 regulates the levels of tRFs in male worms, establishing a model to determine if tRFs in sperm can transmit epigenetic information to progeny in C. elegans. To support these studies, we have developed a method for single embryo RNA-Sequencing in worms, permitting the quantitation of gene expression changes across the transcriptome from 2- and 8-cell embryos to determine the first molecular effects of modulating the levels of tRFs in sperm and early development.

de Albuquerque BF et al. (2013) Dev Cell "Is this mine? small RNAs help to decide."

Ketting RF, de Albuquerque BF

[Dev Cell, 2013]

Genomes are constantly challenged by invaders, so determining what belongs is crucial. Small RNAs silence alien DNA, but Conine etal. (2013), Seth etal. (2013), and Wedeles etal. (2013) now report in Cell and Developmental Cell that these tiny transcripts can also license trusted DNA for expression.

Bacon, Grace P. et al. (2015) International Worm Meeting "RNA Helicase A May Function to Upregulate Genes in the ALG-3/ALG-4 26G RNA Interference Pathway."

Bacon, Grace P., Walstrom, Katherine M.

[International Worm Meeting, 2015]

Argonaute proteins have been well established as key players in RNA interference. The RNA interference process generally inhibits gene expression, but in recent years, argonautes were shown to play a role in the upregulation of particular gene targets. In C. elegans, spermatogenesis is a thermosensitive process. The argonaute proteins ALG-3 and ALG-4 have been shown to upregulate certain spermatogenesis genes at elevated temperatures, allowing for the production of functional sperm at these temperatures (Conine et al. 2010). Deletion mutants in RNA Helicase A (rha-1) have similar phenotypes as alg-3/4 mutants, including the production of sterile worms at elevated temperatures. This project sought to determine whether a connection may exist between RHA-1 and ALG-3/4. This was achieved using immunofluorescence assays of spermatocyte features previously identified as changed in male alg-3/4 mutants (Conine et al. 2013). Between the control strain (him-8) and the two mutant strains (alg-3/4 and rha-1), both features (MSP expression and H3K4 dimethylation) were consistently changed. The changes in MSP expression were complicated and not statistically significant. H3K4 dimethylation was statistically significantly (alpha<0.05) lower in both alg-3/4 and rha-1 mutant spermatocytes compared to him-8 spermatocytes. These results were replicable across two procedures of immunofluorescence. H3K4 dimethylation is a common activating histone modification. Our results are consistent with those of Conine et al. and suggest that the increased presence of H3K4 dimethylation in the control samples aided in the upregulation of sperm genes by ALG-3/4 and possibly by RHA-1. Given the wide range of similarities between the effects of these proteins on the sperm of C. elegans, it is likely that RHA-1 plays a role in the ALG-3/4 RNAi interference pathway.Conine, C. C. et al. (2010) PNAS 107, 3588.Conine, C. C. et al. (2013) Cell 155, 1532.

Arda, H Efsun et al. (2009) International Worm Meeting "An Intricate Network of Nuclear Hormone Receptors Regulates Energy Balance in the Nematode C. elegans."

Arda, H Efsun, Walhout, Albertha JM, Taubert, Stefan, Doucette-Stamm, Lynn, Yamamoto, Keith, van Gilst, Marc, Sequerra, Reynaldo, Conine, Colin

[International Worm Meeting, 2009]

Obesity is a growing epidemic and results from an imbalanced energy homeostasis. Most metabolic genes are transcriptionally regulated, therefore deciphering the complex regulatory networks that govern energy homeostasis is crucial to understand the mechanisms underlying obesity. The nematode C. elegans is a powerful model organism to study metabolic transcriptional regulatory networks. Already ~400 genes have been identified that alter the body fat of the worm when inactivated, and an additional ~25 genes were shown to give transcriptional response upon fasting. In order to elucidate the transcriptional regulation of these "fat" and "fasting" genes, we mapped a transcription regulatory network using high-throughput, gene-centered yeast one-hybrid assays. This network contains numerous nuclear hormone receptor (NHR) type transcription factors, which are involved in fat metabolism throughout the Metazoa. We found that most of these previously uncharacterized NHRs partition into different modules based on the targets they share in the network. We show that several of them are necessary to maintain normal levels of body fat. Finally, we also found that many of the transcription factors identified in the fat network can interact with a mediator subunit, MDT-15, which is required for the regulation of fat metabolism both in C. elegans and mammalian systems. Taken together, our results provide an integrated network of transcriptional regulation of energy balance of C. elegans.

Tsai, Hsin-Yue et al. (2009) International Worm Meeting "RDE-8 encodes a novel protein with a conserved domain required for RNAi."

Gu, Weifeng, Mello, Craig, Tsai, Hsin-Yue, Yates III, John R., Chen, Chun-Chieh G., Moresco, James J.

[International Worm Meeting, 2009]

Several classes of small RNAs have been identified in mammals, zebrafish, Drosophila, and the nematode C. elegans. These include Dicer-dependent products (primary siRNAs and miRNAs) as well as secondary siRNAs produced by RNA-dependent RNA polymerase (RdRP). We have recently identified a new RNAi pathway component, RDE-8, which is essential for the accumulation of RdRP-derived small RNAs in both the exo- and endo- RNAi pathways. RDE-8 contains a novel protein motif that is highly conserved throughout all three kingdoms of life. Interestingly, in other organisms the majority of proteins containing the RDE-8 motif also contain RNA binding domains, most often of the CCCH zinc finger or KH domain families. Although RDE-8 itself is not highly conserved, one C. elegans RDE-8 family member (C30F12.1) has a human homolog in which both the amino acid composition and organization of its CCCH and RDE-8 motifs are well conserved. A third C. elegans RDE-8-motif protein also contains a Cytidine deaminase motif and is required for male fertility (See abstract by Colin Conine). These findings suggest that proteins containing the RDE-8 motif function in RNA binding and modification. To explore the role of RDE-8 in small-RNA pathways, we have utilized mass-spectrometry-based proteomics to identify protein interactors, and small-RNA deep sequencing to identify endogenous small RNAs whose biogenesis/stability depend on RDE-8. Our data suggests that RDE-8 is required for several distinct small-RNA pathways and resides in at least two protein complexes. RDE-8 interacts with the sap-domain exonuclease ERI-1b, and is required for the accumulation of ERI-pathway small RNAs. Interestingly, RDE-8 not only co-purifies with ERI-1b, but is required for a ssRNA trimming activity associated with the native ERI-1b/RDE-8 complex. We speculate that this trimming activity is necessary for the processing of substrates (or products) of the RdRP RRF-3. RDE-8 appears to form a second complex with components of the RNAi and transposon silencing small RNA pathway, including RDE-3, MUT-15, and MUT-16. We have not yet identified specific biochemical activities associated with this complex. However, it may also be involved in recruiting RdRPs: in this case, RRF-1 and EGO-1, which function to amplify silencing signals in the exo- and endo-RNAi pathways. We are currently investigating the biochemical properties of the RDE-8 complexes, and are exploring the activity of the RDE-8 motif.

EK Round et al. (1999) International C. elegans Meeting "Cloning and characterization of aex-2 and aex-4, two genes required for defecation"

EK Round, JH Thomas

[International C. elegans Meeting, 1999]

C. elegans follows a stereotyped series of muscle contractions during defecation: first a posterior body-muscle contraction (pBoc), followed by an anterior body-muscle contraction (aBoc) and finally contraction of the enteric muscles which results in expulsion of the gut contents (Exp). In the presence of food, this defecation motor program occurs every 45 seconds. The neurotransmitter GABA specifically activates the expulsion since mutants lacking GABA have enteric muscle contractions in only 15% of defecation cycles and have no defect in aBoc or pBoc. In contrast, mutations in seven defined genes cause defects in both the aBoc and Exp steps of the defecation motor program (Aex phenotype)(1). Colin Thacker and Ann Rose have shown that aex-5 is a proprotein convertase in the kexin/subtilisin family(2), suggesting a role for peptide signaling in the process of defecation. With the hope of identifying molecules that act in the same pathway as aex-5 , we are mapping and cloning two other Aex genes. From a large number of recombinants, we obtained a precise map position for aex-2 between the anchor genes unc-110 and unc-115 on the X chromosome. These data were used to choose cosmids for transgenic rescue experiments. We have rescued the aex-2 phenotype with a pool of two cosmids and are currently working to identify the exact gene responsible for this rescue. We have mapped aex-4 between unc-78 and lin-18 on the X chromosome and are currently mapping aex-4 relative to polymorphisms in this region. 1. J.H. Thomas, 1990. Genetics 124 : 855-872. 2. C. Thacker and A.M. Rose, West Coast Worm Meeting Abstract 1998, #29.

Colin T. Dolphin et al. (2006) European Worm Meeting "C. elegans Reporter Fusion Genes Generated by Seamless Modification of Large Genomic DNA Clones"

Colin T. Dolphin, Ian A. Hope

[European Worm Meeting, 2006]

Colin T. Dolphin1 & Ian A. Hope2. By determining spatial-temporal expression patterns, reporter constructs can provide significant insight into the function of C. elegans genes. An improved reporter gene fusion would contain significant stretches of the 5 and 3 sequence flanking the protein-coding region to maximize the chances of including all associated regulatory elements such that the resulting expression pattern would be highly likely to recapitulate that of the endogenous gene. Furthermore, it would also be desirable, for various reasons, to be able to insert the reporter sequence, seamlessly and in-frame, at any specific position within the target gene. Lastly, the construction method should not be overly complicated and be potentially adaptable for high-throughput construct generation. We have developed such a method.. Reporter genes were inserted precisely, by homologous recombination in E.coli, into C. elegans genes cloned in fosmids. The homologous recombination, commonly referred to as recombineering, is mediated by the bacteriophage lambda Red system. This system is coupled with a simple and robust two-step counter-selection protocol; first a cassette with both positive and negative selectable marker genes is inserted at the target location and the cassette is then replaced with the reporter gene. This procedure allows straightforward, flexible and precise construction of translational reporter gene fusions directly from C. elegans fosmid clones with minimal alterations to the target gene.. We have used the approach to insert either gfp or cfp precisely at the C-termini of three C. elegans target genes, each located centrally within the approx. 35-40kb inserts of different fosmid clones and examined previously using conventional reporter approaches. Recombineered fosmids were used in transformation of C. elegans by microinjection. Resulting transgenic lines revealed reporter expression consistent with previously published data for the tagged genes and also provided additional information including subcellular distributions. This simple and straightforward method generates reporter gene fusions highly likely to recapitulate endogenous gene expression and thus represents an important addition to the functional genomics toolbox. Its flexibility means the procedure will have many applications.