Krzysztof Drabikowski et al. (2005) International Worm Meeting "Global approach to sumoylation in C. elegans."

Jacqueline Ferralli, Ruth Chiquet-Ehrismann, Krzysztof Drabikowski, Ralf Baumeister

[International Worm Meeting, 2005]

Small ubiquitin-related modifier (SUMO) proteins are covalently and reversibly coupled to several intracellular protein targets modulating protein-protein and protein-DNA interactions. Despite rapid progress, key questions about the regulation mechanisms remain to be answered. Is there a SUMO receptor? Where does sumoylation specificity come from and what are the signaling events regulating the timing of SUMO ligation and de-sumoylation? Several SUMO targets belong to signaling pathways involved in cancer and neurodegenerative diseases. SUMO attachment to target proteins affects their function and stability and might be used as a therapeutic target. For example, arsenite is used as a treatment for a certain type of promyelotic leukemia and is acting by specific induction of sumoylation of PML and PMLretinoic acid receptor fusion protein. Enhanced sumoylation of these proteins leads to apoptosis of malignant cells. In the Drosophila model of Huntington disease it was shown that SUMO competes with ubiquitin for the same attachment site. Attachment of SUMO to the pathogenic fragment of huntingtin protein protects it from ubiquitination and degradation and thereby enhancing neurodegeneration. We decided to study the sumoylation process at the level of the entire organism using C. elegans as a model system. Establishing the complete list of SUMO targets is the first step to decipher the pathways regulated by SUMO. Our estimates suggest that the expected number of proteins undergoing sumoylation is 200 in yeast and worms and 600 proteins in humans and mice. Identification of SUMO targets in C. elegans is performed by purification of sumoylated proteins from transgenic worms expressing a tagged form of SUMO. The purified proteins are identified by mass spectrometric analysis. It has been proposed that part of the sumoylation specificity and dynamics is regulated by different de-sumoylating enzymes. In the C. elegans genome there are 6 genes, encoding proteins containing the SUMO protease domain. We performed knock-down experiments of these genes using RNA interference. We have identified the protease responsible for maturation of SUMO by cleaving of the C-terminus. Five other proteases show important differences in the global sumoylation pattern in the worm upon treatment. Manipulating protein activity by affecting their sumoylation state might be a target for therapeutic intervention and studies in C. elegans can help to establish the basic knowledge of sumoylation processes as well as to screen for potential drugs.

Krzysztof Drabikowski et al. (2002) European Worm Meeting "Ten-1 is involved in sperm development in C. elegans."

Krzysztof Drabikowski, Ruth Chiquet-Ehrismann

[European Worm Meeting, 2002]

The ten-m gene was identified by Baumgartner et al. [1994] in Drosophila. It is the first pair rule gene that is not a transcription factor but a cell surface protein. In addition to the early embryonic expression it is also expressed in the developing nervous system of the fly. Members of the Ten- m protein family were also identified in vertebrates and have been named "teneurins" [Minet et al., 1999]. Vertebrate teneurins are highly expressed in the nervous system. They are type II transmembrane proteins. The Nterminal, intracellular part is followed by a single transmembrane domain. The extracellular domain is around 2500 aa and contains EGF-like repeats. The C. elegans ten-1 is located on chromosome III and is mapped to the cosmid R13F6. By 5'-RACE analysis we identified an alternative transcription start located 8 kb upstream of the predicted one. Both ten-1 forms are expressed early in C. elegans morphogenesis. In post-embryonic development the protein from the upstream promoter is expressed in the developing somatic gonad, the pharynx and a subset of neurons. The protein is also expressed in the germ line, both in developing oocytes and sperm. The protein from the downstream promoter in adult worms has a broad neuronal expression. RNAi aimed against ten-1 transcripts results in a low penetrance phenotype affecting worm morphogenesis in various stages of development. To further analyze the function of teneurin in C. elegans we screened a mutant library for deletions in the ten-1 gene. No mutant carrying a deletion of the downstream transcription start that would result in a complete null could be rescued. However, a mutant carrying a deletion of the upstream transcription start including the four first exons was recovered. We conclude that this results in a null mutation of the longer form of ten-1. Heterozygote worms show variable embryonic and larval lethality. Adult worms show defects in gonad migration. Worms have little or no sperm. Sperm is often pushed to the uterus by ovulating oocytes. Ovulation is defective resulting in ruptured oocytes and abnormally small embryos. Endomitotic oocytes form in the uterus. Worms are constipated. No -/- embryos develop suggesting that Ten-1 is involved in oocyte and/or sperm development. +/- males are fertile and have +/- progeny suggesting that at least in males Ten-1- sperm develop. This phenotype is reminiscent of spe-8, spe-12, spe-27 and spe-29 mutants that are defective in spermatid maturation. We are testing the hypothesis of Ten-1 involvement in spermatozoon development.

Krzysztof Drabikowski et al. (2003) International Worm Meeting "Intracellular domain of Ten-1 is released from the membrane and signals directly to the nucleus."

Krzysztof Drabikowski, Ruth Chiquet-Ehrismann, Jacqueline Ferralli

[International Worm Meeting, 2003]

The ten-m gene was identified in Drosophila as the first pair rule gene that is not a transcription factor but a cell surface protein. In addition to the early embryonic expression it is also expressed in the developing nervous system of the fly. Members of the Ten-m protein family were also identified in vertebrates and have been named teneurins. Vertebrate teneurins are highly expressed in developing tissues especially in the nervous system. Members of the Ten family are type II transmembrane proteins. The N-terminal, intracellular part is followed by a single transmembrane domain. The extracellular domain is around 2500 aa and contains EGF-like repeats. The C. elegans ten-1 is located on chromosome III and is mapped to the cosmid R13F6. By 5'-RACE analysis we identified an alternative transcription start located 8 kb upstream of the predicted one. Both ten-1 forms are expressed early in C. elegans morphogenesis. In post-embryonic development the protein from the upstream promoter is expressed in the developing somatic gonad, the pharynx, vulva and body wall muscles and a subset of neurons. It is also expressed in the germ line, both in developing oocytes and sperm. The protein from the downstream promoter in adult worms has a broad neuronal expression. RNAi aimed against ten-1 transcripts affects worm morphogenesis in various stages of development. Embryos die because of elongation defects. Variably abnormal larvae develop. Adult worms show defects in gonad migration. Worms have little or no sperm. Sperm is often pushed to the uterus by ovulating oocytes. Ovulation is defective resulting in ruptured oocytes and abnormally small embryos. Endomitotic oocytes form in the uterus. Worms are constipated. We have developed antibodies against the N- and C-terminal peptides of the Ten-1 protein. The anti C-terminus stains the cell membranes but the anti N-terminus antibody stains cell membranes as well as the nuclei. This suggests the release of the intracellular domain from the membrane and translocation to the nucleus. The Ten-1 nuclear staining is reminiscent of the PML nuclear bodies in vertebrates. We postulate that Ten-1 is a receptor for a morphogenetic cue and its intracellular domain is a membrane bound transcription regulator that signals directly from the membrane to the nucleus.

Krzysztof Drabikowski et al. (2001) International C. elegans Meeting "Ten-1, the C. elegans homologue of the Drosophila pair rule gene ten-m is involved in somatic gonad formation."

Krzysztof Drabikowski, Ruth Chiquet-Ehrismann

[International C. elegans Meeting, 2001]

The ten-m gene was identified by Baumgartner et al. [1994] in Drosophila . It is the first pair rule gene which is not a transcription factor but a cell surface protein. In addition to the early embryonic expression it is also expressed in the developing nervous system of the fly. Members of the Ten-m protein family were also identified in vertebrates and have been named "teneurins" [Minet et al., 1999]. Vertebrate teneurins are highly expressed in the nervous system. They are type II transmembrane proteins. The N-terminal intracellular part is followed by a single transmembrane domain. The extracellular domain is around 2500 aa and contains EGF-like repeats. The C. elegans teneurin is located on chromosome III and is mapped to the cosmid R13F6. By 5'-RACE analysis we identified an alternative transcription start located 8 kb upstream of the predicted one. Both teneurin forms are expressed early in C. elegans morphogenesis. In post-embryonic development the protein from the upstream promoter is expressed in the developing somatic gonad, the pharynx and a subset of neurons. The protein from the downstream promoter in adult worms has a broad neuronal expression. RNAi aimed against teneurin transcripts results in a low penetrance phenotype affecting worm morphogenesis in various stages of development. Embryos arrest at the two- or three-fold stage. Larvae burst during hatching and the survivors often burst at later stages of development. Adult hermaphrodites have very little or no sperm. The gonad is often displaced. In order to further analyse the function of teneurin in C.elegans , we screened a mutant library for deletions in the ten-1 gene. No mutant carrying a deletion of the downstream transcription start that would result in a complete null could be rescued. However, a mutant carrying a deletion of the upstream transcription start including the three first exons was recovered. We conclude that this results in a null mutation of the longer form of teneurin. This mutation affects various aspects of the somatic gonad development. The distal tip cell migrates along the gonad or the gut and occasionally makes an extra turn. It may penetrate the gut or the uterus. The width of the gonad arms is uneven along their length. The vulva-uterine connection is often not established. The worms have less sperm and endomitotic oocytes are formed. In addition to the malformation of the gonad, ten-1 mutants show neuronal migration defects and are constipated. We postulate that teneurin is important for cell-cell interactions and that it is a receptor for morphogenetic cues and regulates cell migration and cell shape.

Liudkovska, Vladyslava et al. (2021) International Worm Meeting "Cytoplasmic polyadenylation by TENT-5 regulates the innate immune response in worms"

Cysewski, Dominik, Dziembowski, Andrzej, Mroczek, Seweryn, Krawczyk, Pawel S., Drabikowski, Krzysztof, Liudkovska, Vladyslava, Ewbank, Jonathan J.

[International Worm Meeting, 2021]

Non-canonical poly(A) polymerases (ncPAPs) belong to the family of the terminal nucleotidyltransferases (TENTs) and are implicated in a range of physiological processes which they regulate on the post-transcriptional level. In Caenorhabditis elegans, cytoplasmic polyadenylation has been mostly studied in the context of gametogenesis, in which ncPAP GLD-2 elongates the poly(A) tails of many germline mRNAs, thereby promoting their stability. However, cytoplasmic polyadenylation may play a much broader role than previously anticipated. In worms, innate immunity is the main mechanism of host defense against pathogens, and hence it is tightly regulated at both the transcriptional and post-transcriptional levels. Although the role of post-transcriptional regulation of gene expression in innate immunity is well-appreciated, the particular role of cytoplasmic polyadenylation in this process has never been addressed. We show that C. elegans protein TENT-5 (PQN-44) is an active cytoplasmic ncPAP. Global transcriptome and proteome analyses revealed that TENT-5 is involved in the regulation of genes influencing the interaction between C. elegans and bacteria, including many genes encoding secreted proteins with antimicrobial activity. The poly(A) tail profiling by direct RNA sequencing on Nanopores showed that TENT-5 polyadenylates and stabilizes mRNAs with signal peptide-encoding sequences, that are translated at the endoplasmic reticulum. This aligns with TENT-5 localization in the intestine, which in worms serves as one of the major surfaces of host-pathogen interaction, and TENT-5 enrichment at the endoplasmic reticulum. Furthermore, loss of tent-5 leads to higher susceptibility of mutant worms to infection with pathogenic bacteria. We propose that TENT-5 regulates the stability and modulates the expression of mRNAs that encode secreted proteins that ensures an energy-effective and rapid response to infection. Importantly, the role of TENT-5 in innate immunity is evolutionarily conserved since murine macrophages devoid of TENT5A and TENT5C ncPAPs also exhibit defects in polyadenylation of numerous mRNAs some of which are of genes orthologous to C. elegans TENT-5 targets. Taken together, our data reveal that cytoplasmic polyadenylation of mRNA encoding innate immunity effector proteins by TENT5 is a previously unknown and essential component of the post-transcriptional regulation of innate immunity.

Topf, Ulrike et al. (2009) International Worm Meeting "High throughput screen for genes interacting with ten-1."

Topf, Ulrike, Chiquet-Ehrismann, Ruth

[International Worm Meeting, 2009]

TEN-1 belongs to the teneurin protein family. Homologues were described in fly, zebra fish, chicken, mouse and man. All teneurin genes encode type II transmembrane proteins consisting of an intracellular domain, a single transmembrane domain and a large extracellular domain which is the most conserved part among the orthologs. Ten-1 is expressed throughout all developmental stages in C. elegans, predominantly in developing gonad and neuronal cells. Ten-1 deletion mutants show severe morphological defects causing lethality, sterility and worms bursting through the vulva. Approximately 40% of mutants develop to viable, fertile worms. By RNAi knock-down, ten-1 was found to be important for germ cell development, epidermal morphogenesis, gonad migration and neuronal pathfinding (Drabikowski et al., 2005). We have recently shown that ten-1 is important for basement membrane integrity. Genetic interactions of ten-1 with basement membrane genes, ina-1, dgn-1 and epi-1 result in synthetic lethal phenotype suggesting that ten-1 acts together with these genes (Trzebiatowska et al., 2008). However, the mechanism of Ten-1 action remains to be discovered. Interestingly, the intracellular domain of TEN-1 was shown to translocate to the nucleus (Drabikowski et al., 2005). Thus, TEN-1 is suggested to function as modulator of signaling events resulting in transcriptional regulation. Little is known, however, about the cleavage mechanism and possible target genes. To discover genes acting upstream or downstream of ten-1 we performed a RNAi -based genome-wide screen. We used an RNAi feeding approach to compare the effect of ten-1 knock down in wild type versus ten-1 deletion animals. We identified 37 genes that may genetically interact with ten-1. Among them, depletion of 4 genes resulted in suppression of the ten-1 mutant phenotype and knock down of 18 genes enhanced the ten-1 phenotype. For 15 other genes only wild type animals were affected and no change of the ten-1 phenotype could be observed. Genes required in neuronal or reproductive systems are enriched in the ten-1 interaction screen. The genome-wide RNAi screen provides a basis for dissecting the ten-1 function and placing it in genetic and developmental pathways. Confirmation of genetic interactions will be performed by crossing the ten-1 mutant into genetic deletion mutants of the candidates identified.

Agnieszka Trzebiatowska et al. (2006) European Worm Meeting "TEN-1 is required for the Formation of the Somatic Gonad in Caenorhabditis elegans"

Krzysztof Drabikowski, Agnieszka Trzebiatowska, Ruth Chiquet-Ehrismann

[European Worm Meeting, 2006]

Agnieszka Trzebiatowska1, Krzysztof Drabikowski2, Ruth Chiquet-Ehrismann1. TEN-1 is a member of a novel family of transmembrane proteins named teneurins that have been characterized in Drosophila, zebrafish, chicken, mouse and man. Teneurins were shown to be mainly expressed in the developing and adult nervous system and to play a role in morphogenesis, cell migration and at sites of pattern formation. C. elegans TEN-1 was proposed to act as a receptor that signals directly to the nucleus. Its intracellular domain is cleaved and translocates to the nucleus where it may influence gene expression. Little is known, however, about the cleavage mechanism and possible target genes. C. elegans has a single teneurin orthologue, which is under control of alternative promoters. The upstream promoter is active in the somatic gonad, some muscle cells, the gut and in a number of neurons. The expression from the downstream promoter is mainly detected in the nervous system. TEN-1 is a type II transmembrane protein consisting of a short intracellular domain followed by a transmembrane domain and a long highly conserved extracellular part containing eight EGF-like repeats, a cysteine rich region and YD repeats.. In order to determine the function of TEN-1 in C.elegans, we investigated two loss-of-function mutants: ten-1(ok641) and ten-1(tm651). The former carries an in frame deletion of four EGF-like repeats and a part of the cysteine rich region; the latter has a deletion that removes the transmembrane domain and introduces a frameshift mutation resulting in the loss of the entire protein except for the first 194 aminoacids. In both mutants transcripts for the predicted truncated proteins could be detected on the level comparable to the wild type form. Both ten-1 mutants show a pleiotropic phenotype, similar to the phenotype observed after reduction of ten-1 expression by RNAi. Mutant worms are sterile due to various defects in the somatic gonad and ectopic germline formation in the proximity of the vulva. Time course analysis of germline development showed that these abnormalities may result from an early defect in the somatic gonad formation. We were able to partially rescue the phenotype of ten-1(ok641) mutant worms by injecting the cosmid F36A3 carrying the entire genomic region of the ten-1 gene. Since such an extrachromosomal array is likely to be silenced in the germline, gonadal defects appear to be the result of impaired signaling in the somatic gonad.