Guo, Yanwu et al. (2017) International Worm Meeting "clk-2 is a novel player in the C. elegans nonsense-mediated mRNA decay."

Ciosk, Rafal, Guo, Yanwu

[International Worm Meeting, 2017]

Surveillance of aberrant RNAs is important for development and disease. To identify novel factors monitoring mRNA processing, we created a strain expressing a GFP reporter under the control of an aberrant 3'UTR. The reporter mRNA is degraded in wild-type animals. Following mutagenesis, we isolated mutants permitting expression of this reporter and found that most of them correspond to known components of the C. elegans nonsense-mediated mRNA decay pathway (NMD; encoded by the smg genes). The SMG proteins are conserved players in the NMD pathway, which is used to degrade aberrant mRNAs containing premature termination codons but also affects the stability many "normal" mRNAs. In worms, a previous study by Quinn et al reported NMD-mediated processing of pseudogenes. However, based on RNA sequencing datasets from different eukaryotes, the connection between NMD and pseudogenes appears to be nematode-specific. In addition to known NMD factors, our screen uncovered clk-2, previously implicated in DNA damage, as a novel player in the nematode NMD. CLK-2/TEL2 is a conserved protein, best known in humans for a function in telomere biology. Nevertheless, TEL2 was previously linked to NMD and our results suggest that CLK-2/TEL2 is a conserved player in the NMD pathway. The molecular mechanism of CLK-2 in NMD is currently under investigation.

Briglin A et al. (1998) East Coast Worm Meeting "The role of PAR-1 in establishing polarity in the early C. elegans embryo."

Briglin A, Kemphues KJ

[East Coast Worm Meeting, 1998]

PAR-1 protein is required for establishing polarity during early embryogenesis in the nematode C.elegans. Mutants in par-1 have an abnormal first cell division and patterning defects. PAR-1 protein is localized asymmetrically, to the posterior periphery of the one cell embryo. The conserved C-terminal domain of PAR-1 was found to interact specifically, both in vivo and in vitro, with NMY-2, a non-muscle myosin type II heavy chain (Guo and Kemphues 1996). RNA interference experiments suggest that removing NMY-2 from the early embryo results in a mislocalization of the PAR-1 protein. At present, the role of the interaction between PAR-1 and NMY-2 in establishing polarity in the early embryo is still unclear. To understand better the role of this interaction in vivo, I will generate mutant PAR-1 proteins that disrupt binding to NMY-2 in vitro and test these proteins for rescue with germline transformation. PAR-1 is predicted to be a serine/threonine protein kinase (Guo and Kemphues 1995). Two par-1 alleles contain mutations in conserved residues of the kinase domain. These mutants suggest that PAR-1 kinase activity is also required to correctly establish polarity. In order to understand the role that PAR-1 plays in the early cell divisions, it will be necessary to identify the substrates for this kinase activity in vivo. To do this, I am conducting a screen for PAR-1 substrates. Guo, S. and K.J. Kemphues. 1995. Cell 81:611-620. Guo, S. and K.J. Kemphues. 1996. Nature 382:455-258.

Dyczkowska, Aneta et al. (2021) International Worm Meeting "Characterizing the roles of ETS-4 transcription factor in fat metabolism"

Guo, Yanwu, Ciosk, Rafal, Dyczkowska, Aneta, Chabowska-Kita, Agnieszka

[International Worm Meeting, 2021]

Obesity is a global health problem associated with many comorbidities, sparking interest in molecular mechanisms controlling body fat. Previously, we found that fat accumulation in C. elegans requires the endoribonuclease REGE-1/Regnase-1, which inhibits the expression of transcription factor ETS-4. Analyzing gene expression dependent on ETS-4, we noticed increased expression of a peptide transporter, PEPT-1, previously implicated in fat regulation. Currently, we investigate the potential link between ETS-4 and PEPT-1-mediated fat loss, and will present our results so far.

Pekec, Tina et al. (2021) International Worm Meeting "Cold survival driven by ferritin-mediated iron regulation"

Komur, Alicja, Ciosk, Rafal, Sobanska, Daria, Guo, Yanwu, Pekec, Tina, Frankowski, Marcin

[International Worm Meeting, 2021]

Hibernation is employed by animals to withstand periods of low energy supply associated with cold temperatures. When adapted to cold, C. elegans can survive near-freezing temperatures for several days. Among factors promoting C. elegans cold survival is the conserved ribonuclease REGE-1/Regnase-1. The main target of REGE-1 is mRNA encoding a transcription factor, ETS-4, previously implicated in insulin signaling and the regulation of body fat. We found that the abnormal accumulation of ETS-4 is also responsible for the reduced cold resistance of rege-1 mutants. Conversely, ets-4 mutants survive cold much better than wild type. Through genetics and functional genomics, we found that, in the cold, the loss of ETS-4 leads to the activation of two transcription factors, DAF-16/FOXO and PQM-1. In contrast to standard cultivation temperatures, where these transcription factors play antagonistic functions, our analysis suggests that, in the cold, these transcription factors cooperate to induce transcription of specific genes. We show that one of their targets, FTN-1/ferritin, facilitates cold survival, and propose that it does so by detoxifying harmful iron species.

Habacher, Cornelia et al. (2017) International Worm Meeting "Ribonuclease mediated control of body fat."

Guo, Yanwu, Habacher, Cornelia, Gaidazis, Dimos, Neagu, Anca, Venz, Richard, Gut, Heinz, Ciosk, Rafal, Kumari, Pooja

[International Worm Meeting, 2017]

A continuous increase in obesity and obesity-related diseases, such as metabolic syndrome and type-II diabetes, has led to a global health crisis. Therefore, the understanding of molecular mechanism controlling fat metabolism is crucial to identify potential new therapeutic targets. Key transcription factors involved in lipid metabolism, such as SBP-1/SREBP, LPD-2/C/EBP and MDT-15, are conserved from nematodes to mammals, and C. elegans has proven to be a powerful model for obesity and metabolic research. The C. elegans RNase REGE-1 is closely related to the mammalian Regnase-1/MCPIP1/Zc3h12a, an extensively studied regulator of innate immunity. The loss of REGE-1 causes a dramatic decrease in overall body fat, accompanied by increased expression of lipid metabolic and innate immunity genes. Using Exon-Intron Split Analysis (EISA), we found that REGE-1 controls fat by targeting mRNA encoding a fat loss-promoting transcription factor, ETS-4, previously implicated in ageing. While REGE-1 degrades ets-4 mRNA via an endonucleolytic cleavage within its 3UTR, ETS-4, in turn, promotes the transcription of rege-1. Thus, ETS-4 and REGE-1 form an auto- regulatory module (ERM), affecting fat by controlling the transcriptional output of ETS-4. The ERM is a novel mechanistic paradigm in fat regulation. Posttranscriptional mechanisms are well suited to a rapid and reversible control of gene expression. Similarly, REGE-1-mediated mRNA degradation might be a way to rapidly re-wire lipid metabolism in response to environmental changes, including change of diet, temperature or exposure to pathogens.

Michael L Stitzel et al. (2004) East Coast Worm Meeting "How are cytoplasmic asymmetries achieved? In vivo studies of germ plasm localization dynamics during early embryogenesis"

Geraldine Seydoux, Denis Wirtz, Michael L Stitzel

[East Coast Worm Meeting, 2004]

The CCCH finger proteins PIE-1, POS-1, and MEX-1 are maternally-encoded germ cell fate regulators that segregate with the germ lineage during early embryogenesis. These proteins, initially distributed throughout the zygote, become enriched at the posterior pole prior to mitosis and are maintained there through the first cell division. Genetic studies have indicated that asymmetric localization of cytoplasmic determinants depends on PAR-1 on the posterior cortex (Guo and Kemphues, 1995), and on MEX-5 and MEX-6, two homologous CCCH finger proteins that segregate opposite PIE-1, POS-1, and MEX-1 in the cytoplasm (Schubert et al., 2000). The molecular mechanisms that lead to cytoplasmic asymmetries in the zygote are not known. To begin to characterize these mechanisms, we are analyzing the in vivo localization dynamics of GFP fusions in wild type and mutant embryos using quantitative time-lapse microscopy and fluorescence recovery after photobleaching (FRAP). FRAP analysis of GFP:PIE-1 indicates that there is a larger immobile fraction of the fusion protein in the posterior compared to the anterior (35% vs. 0%) during mitosis, suggesting that PIE-1 may be tethered in the posterior region of the zygote. We are currently assessing the kinetics of recovery in par-1 and mex-5/-6 (RNAi) mutants to determine what roles they play in enrichment dynamics. References: 1. Guo, S. and Kemphues, KJ. 1995. Cell. 81(4): 611-620. 2. Schubert, C.M., et al., 2000. Mol. Cell. 5(4): 671-682.

Cha, S. et al. (2015) International Worm Meeting "An annotated genome of the root-knot nematode, Meloidogyne chitwoodi."

Cha, S., Bird, D.

[International Worm Meeting, 2015]

Plant-parasitic nematodes are responsible for annual crop losses in excess of USD123 billion worldwide. Most important are the root-knot nematodes (RKN: Meloidogyne spp.), which establish an intimate association with their host. As a genus, Meloidogyne has a host-range that spans the tracheophyta, although individual isolates are more restricted. In cool climates, M. hapla and M. chitwoodi predominate and are a significant problem on potato in Europe and the US. Because of its genetic tractability, we selected M. hapla for sequencing, and an annotated public release (HapPep1) was made in 2008 (Opperman et al., 2008). Since then we have undertaken on-going curation, and the current release (HapPep5) includes ~2xE9 RNA-Seq reads (Guo et al., 2014). We have also sequenced two additional strains of M. hapla (VW8 and LM) but they have not yet been released. Because M. hapla and M. chitwoodi are sympatric, they presumably have similar gene compliments. To test this, we sequenced the M. chitwoodi genome.Genomic DNA was isolated from nematodes collected in a potato field in Washington State, and confirmed by Axel Elling to be M. chitwoodi. Using an Illumina MiSeq II we obtained 20,079,197 short (~300 bp) paired-end reads. The assembly parameters were empirically optimized, and 4,735 contigs assembled; N50 is 82 kbp. The longest is ~758 kbp with coverage of 33 reads per bp. Average coverage genome-wide is 289 reads. At the protein level, CEGMA identified 245 (98.79%) of the core proteins, pointing to near 100% genome coverage. When CEGMA proteins as a query were blasted against the assembled contigs as a database, it was observed that one protein had hits with more than two contigs. Using CEGMA (and other) proteins, as well as M. chitwoodi ESTs, we trained AUGUSTUS for gene prediction. GO categorization was performed using InterProScan. Analysis of these data is in progress, with an emphasis on genes encoding replicas of plant peptide hormones (xenomones). Because of a potential role in speciation of the closely-related parasites, we have, in collaboration with David Lunt (University of Hull), examined the numbers and distribution of transposable elements: not surprising, M. chitwoodi is much more similar to M. hapla than it is to M. incognita.Operman et al., 2008. Proc Nat Acad Sci 105: 14802 -14807.Guo et al. 2014. Worm 3:e29158.

Adrian Cuenca et al. (2004) East Coast Worm Meeting "Structure/function studies of the polarity regulator PAR-1 in C. elegans."

Adrian Cuenca, Geraldine Seydoux

[East Coast Worm Meeting, 2004]

Polarization of the C. elegans zygote along the anterior-posterior axis depends on a signal from the sperm asters, which causes the cortically-enriched PAR proteins to relocalize to distinct anterior and posterior domains. The sperm asters exclude the PAR-3/PAR-6/PKC3 complex from the nearby cortex, allowing PAR-1 and PAR-2 to accumulate there (Goldstein and Hird, 1996; O'Connell et al., 2000; Wallenfang and Seydoux, 2000; Cuenca et al., 2003). We are investigating how the serine-threonine kinase PAR-1 localizes to the cortex. A GFP:PAR-1 fusion accumulates around the sperm-derived centrosomes and on the nearby posterior cortex during polarization. We have identified a domain at the carboxy-terminal end of PAR-1 necessary and sufficient for both of these localizations. This domain is conserved in PAR-1 kinases from other organisms and includes a region that binds to the non-muscle myosin NMY-2 (Guo and Kemphues, 1996). Consistent with earlier results (Boyd et al., 1996), analysis of GFP fused to the localization domain in zygotes depleted for PAR-2 or PAR-3 suggest that PAR-1 can associate with the cortex in the absence of other PAR proteins, but requires PAR-2 and PAR-3 to accumulate specifically on the posterior cortex. We are currently constructing PAR-1 transgenes lacking the localization domain to investigate its relevance to PAR-1 polarity function in vivo.

Jean-Claude Labbe et al. (2001) International C. elegans Meeting "Assessing the role of microtubules in the establishment of the first asymmetric cell division of the C. elegans embryo"

Paul S Maddox, Bob Goldstein, Jean-Claude Labbe, E D Salmon

[International C. elegans Meeting, 2001]

We are using the C. elegans 1-cell embryo as a model to understand how cells divide asymmetrically. The first embryonic division of C. elegans gives rise to a larger, anterior cell, termed AB, and a smaller, posterior cell, P 1 . This asymmetry is the result of the position of the first mitotic spindle, which moves from the center to the posterior of the 1-cell embryo during metaphase. Several mutations, including alleles of the par genes, disrupt embryonic polarity and affect the posterior displacement of the spindle, suggesting that components localized at the cell cortex can influence spindle positioning (1). Recent experiments by Grill et al . (2) demonstrated that severing the spindle at the start of anaphase B results in both the anterior and posterior centrosomes migrating toward their respective poles at a faster rate than normal, with the posterior centrosome moving faster than the anterior one. In order to address the role of astral microtubules in spindle positioning, we ablated either the anterior or the posterior centrosome at various phases of the cell cycle. During anaphase B, ablation of the anterior centrosome caused posterior movement of the posterior centrosome and ablation of the posterior centrosome caused anterior movement of the anterior one; these results are similar to those obtained by Grill et al . However, during prophase (before pronuclear envelope breakdown), ablation of the anterior centrosome resulted in posterior displacement of the posterior centrosome, while ablating the posterior centrosome at this time did not cause anterior movement of the anterior centrosome. The anterior centrosome did not move until later, posibly at anaphase onset. This result suggests that a pulling force acts on the posterior aster before anaphase. One posible mechanism for this could be that components of the posterior cortex might modulate the dynamics of microtubules in this region of the embryo, which could asymmetrically modulate pulling forces. PAR-1, a protein with similarity to MARK kinases that localizes to the posterior cortex of the embryo (3), is a good candidate to have a local effect on posterior microtubules. MARK kinases have been implicated in the phosphorylation of microtubule-associated proteins (MAPs) and in the destabilization of microtubules in vivo and in vitro (4). We monitored dynamics of individual microtubules by imaging embryos expressing an alpha-tubulin:gfp fusion (kindly provided by Karen Oogema) with a spinning-disk confocal microscope. Thus far, preliminary quantitative data analysis suggests a similar stability between anterior and posterior astral micrutubules during the time that the spindle becomes asymmetric in wild-type embryos. We will further address this issue by monitoring microtubule dynamics in par mutants. Furthermore, we are developing an in vitro assay to test whether PAR-1 can directly affect microtubules to modulate their stability. To this end, we have isolated C. elegans MAPs and will determine if they can play a role in this process. Progress toward these goals will be presented. 1. Guo, S. and Kemphues, K.J. (1996). Curr. Opin. Genet. Dev. 6, 408-415 2. Grill, S.W. et al. (2001). Nature 409, 630-633 3. Guo, S. and Kemphues, K.J. (1995). Cell 81, 611-620 4. Drewes, G. et al. (1997) Cell 89, 297-308

CA Shelton et al. (1999) International C. elegans Meeting "The Nonmuscle Myosin Regulatory Light Chain Gene mlc-4 is Required for Cytokinesis, Anterior-Posterior Polarity, and Body Morphology during C. elegans Embryogenesis"

BA Bowerman, CA Shelton, JC Carter

[International C. elegans Meeting, 1999]

Using RNA-mediated genetic interference (RNAi) in a phenotypic screen, we identified the sole conserved nonmuscle myosin II regulatory light chain (nmRLC) gene in Caenorhabditis elegans , which we name mlc-4 . Maternally-supplied mlc-4 function is required for cytokinesis during both meiosis and mitosis, and for establishment of anterior-posterior (a-p) asymmetries after fertilization. Reducing the function of mlc-4 or nmy-2 , a nonmuscle myosin II gene (1), also leads to a loss of polarized cytoplasmic streaming in the C. elegans zygote, supporting models in which cytoplasmic flow may be required to establish a-p differences (2). However, germline P-granules are normally localized to the posterior despite these defects. Because P-granule localization requires an intact actin cytoskeleton (3), these results suggest that an actin-dependent mechanism other than cytoplasmic flow or mlc-4/nmy-2 activity generates some anterior-posterior asymmetries in the C. elegans zygote. To determine if mlc-4 has essential zygotic functions, we isolated a deletion allele. We show that removing zygotic mlc-4 function results in a partial elongation phenotype during embryogenesis. Moreover, A mlc-4::GFP transgene is expressed in lateral rows of hypodermal cells, and these cells fail to properly change shape in mlc-4 mutant animals during elongation. This phenotype is similar to that obtained from let-502 mutations and we suggest that the previously described let-502/mel-11 regulatory pathway (4) may interact with MLC-4 to generate contractile forces required for proper elongation of the embryo. 1. Guo, S. & Kemphues, K.J. Nature 382:455-458 (1996). 2. Goldstein, B. & Hird, S.N. Development 122:1467-1474 (1996). 3. Strome, S. & Wood, W.B. Cell 35:15-25 (1983). 4. Wissmann, A., Ingles, J., McGhee, J.D. & Mains, P.E. Genes Dev 11: 409-422 (1997).