Posner, Rachel et al. (2017) International Worm Meeting "Gene regulation via non-cell autonomous acting small RNAs in C. elegans."

Azmon, Eran, Star, Ekaterina, Anava, Sarit, Rechavi, Oded, Posner, Rachel, Hobert, Oliver, Gingold, Hila, Bracha, Shahar

[International Worm Meeting, 2017]

Exposure of C. elegans to artificial dsRNA can trigger exogenous small RNA-mediated silencing that transmits from somatic cells to the germline, and persists for multiple generations. However, it is unknown whether endogenous types of small RNAs (miRNAs, piRNAs and endo-siRNAs) produce systematic responses and what type of effect they may have on gene regulation. Our recent results suggest that endogenous siRNAs act in a non-cell autonomous manner, orchestrating gene silencing between tissues. We will present a dissection of the major components of the RNAi pathway required for non-cell autonomous gene regulation by endo-siRNAs and provide evidence possibly linking the regulation of physiological phenotypes to small RNAs mobilizing between tissues.

L Saccomanno et al. (1999) International C. elegans Meeting "The role of the STAR family in tra-2 3'UTR translational regulation"

EB Goodwin, L Saccomanno

[International C. elegans Meeting, 1999]

The C. elegans sex determination gene, tra-2 , encodes a large transmembrane protein that is required for female sexual development. Repression of tra-2 translation is necessary for male cell fate development in both somatic and germ line tissues. Two elements located in the 3'UTR of the tra-2 mRNA, called TGEs, are required for tra-2 translational regulation. GLD-1 is a member of the STAR family of RNA binding proteins and regulates tra-2 translation in the hermaphrodite germline to permit spermatogenesis. However, translational regulation of tra-2 also occurs in the soma. The STAR family contains an approximately 200 amino acid domain (STAR domain) that is highly conserved, raising the possibility that another STAR protein represses tra-2 in the soma. The C. elegans sequencing project has identified at least five predicted open reading frames that have strong similarity to GLD-1. We have preliminary evidence that suggests at least one of these five STAR proteins, T21G5.5, can regulate translation through the tra-2 3'UTR. T21G5.5 is 67% identical to GLD-1 at the amino acid level within the STAR domain. I have performed RNA-mediated interference (RNAi) of T21G5.5 in transgenic C. elegans animals that carry a reporter transgene encoding the lacZ gene with the wild type tra-2 3'UTR. The F1 progeny following RNAi of T21G5.5 had an increased level of b -gal staining in the soma compared to wild type transgenic animals. 22% of the RNAi animals had b -gal activity in intestinal cells compared to 12.5% of non-injected. In addition, animals that stained for b -gal after injection with T21G5.5 also showed an increase in the number of b -gal stained intestinal cells (2-8 cells) compared to wild type (1-2 cells). Disruption of T21G5.5 activity also resulted in animals with interesting morphological phenotypes. F1 animals had a dumpy phenotype and an altered tail morphology. In addition, the gonads of these animals were sexually transformed. These preliminary data suggest that T21G5.5 is a likely candidate for a TGE-dependent translational repressor. However, these results do not eliminate the possibility that other STAR proteins are also 3'UTR-dependent translational regulators. We are currently testing this possibility.

Garett M Padilla et al. (2004) Mid-west Worm Meeting "Investigating the Role of a Putative STAR Domain Protein in Caenorhabditis elegans"

Garett M Padilla, Elizabeth B Goodwin

[Mid-west Worm Meeting, 2004]

TRA-2 protein is necessary to promote female sexual development. Male development, at least in part, requires translational repression of tra-2 by two elements called TGEs ( tra-2 and GLI elements) located in the 3! (inverted exclamation mark)- (macron) UTR. tra-2 gain of function mutants that disrupt the TGEs feminize hermaphrodites. These mutations also feminize male animals, resulting in yolk production in the intestines and oocyte development in the germline. Therefore, TGE control regulates tra-2 activity in the germline and soma. GLD-1 is a germline specific protein that contains an RNA binding Maxi-KH/STAR domain that regulates tra-2 translation; however, a tra-2 somatic regulator has yet to be identified. We have identified a STAR domain containing protein in C. elegans , T21G5.5. This gene has two putative transcripts which differ in a 3! (inverted exclamation mark)- (macron) exon and is predicted to bind the same mRNAs as GLD-1 (Ryder et al , 2004). T21G5.5 are 63% identical throughout the STAR domain and 74% identical in the KH RNA binding domain. Both transcripts have been cloned and we are currently investigating their biochemical functions. Western analysis of a deletion strain of T21G5.5 demonstrates increased levels of TRA-2 protein relative to wildtype worms. In addition, RNA interference against T21G5.5a results in some in embryonic lethality and a decrease in fecundity. However, unlike GLD-1 gross germline phenotypes are not observed. Together these results indicate that T21G5.5 may function in the soma as a translational repressor in a role equivalent to GLD-1. Given slight germline defects observed, T21G5.5 may also function in the germline as well. Our future experiments include investigating the tissue specific and subcellular localization of T21G5.5, phenotypic analysis of the deletion strain, and determination of biochemical function. Ryder SP, Frater LA, Abramovitz DL, Goodwin EB, and Williamson JR. RNA target specificity of the STAR/GSG domain post-transcriptional regulatory protein GLD- 1. Nature Structural and Molecular Biology 11 , 20-28 (2004).

Jan E et al. (1998) Midwest Worm Meeting "Searching for tra-2 3'UTR TGE regulators: We've struck GLD-1!"

Graves LE, Goodwin EB, Motzny CK, Jan E

[Midwest Worm Meeting, 1998]

The two first authors contributed equally to this study. The C. elegans sex determination gene, tra-2, is translationally regulated by elements in the 3' untranslated region (3'UTR) called TGEs. tra-2 activity is required for female development. Male development in both somatic and germ line tissues requires that tra-2 translation is repressed via the TGEs. The TGE control is a conserved mechanism present in invertebrates and vertebrates that controls the translation of other mRNAs. A factor, called DRF, in crude worm extract binds the TGEs and may be a repressor of translation. Our working model is that binding of DRF to the TGEs represses tra-2 translation. Using the yeast three hybrid screen, we have identified a protein, GLD-1, that binds specifically to the TGEs and which may be a repressor of tra-2 translation. GLD-1 is a cytoplasmic germ line specific protein and is a member of the STAR family of RNA-binding proteins. The RNA targets of the STAR proteins have not been previously identified. GLD-1 is essential for oogenesis, and is also necessary for spermatogenesis and inhibition of germ cell proliferation. Our results suggest that GLD-1 promotes spermatogenesis by repressing the translation of tra-2 through the TGEs. Six lines of evidence support this conclusion. First, GLD-1 represses the translation of reporter RNAs via the TGEs both in vitro and in vivo. Second, GLD-1 is required to maintain low TRA-2 protein levels in the adult hermaphrodite gonad. Third, ectopic expression of GLD-1 in the soma can repress the development of female cell fates, causing masculinization of somatic structures in hermaphrodite animals. Fourth, genetic analysis indicates that GLD-1 acts upstream of the TGE control. Fifth, antibody experiments show that endogenous GLD-1 is a component of DRF. Sixth, purified bacterially expressed GLD-1 fusion protein binds specifically to the TGEs. These results are consistent with the idea that GLD-1 represses tra-2 translation by acting through the TGEs and that it may be a component of DRF. The conservation of both the TGE control and the STAR family raises the possibility that a subset of STAR proteins may control RNA activity by repressing translation via TGEs.

Ferreira, D.W. et al. (2015) International Worm Meeting "Germline defects in mitochondrial cholesterol transporter C. elegans mutants following bisphenol A or low cholesterol exposures."

Allard, P., Ferreira, D.W.

[International Worm Meeting, 2015]

Bisphenol A (BPA) treatment results in abnormal oocyte development in mammalian species as well as in the nematode C. elegans. In C. elegans, these defects are prevented by exposing the worms to cholesterol. We have therefore begun to investigate whether there is an interaction between BPA and normal cholesterol homeostasis in the germline. Previously, we have shown that mammalian homologs to cholesterol transporters (Steroid Acute Regulator Protein; StAR, 18kDa translocator protein; TSPO, and StAR related lipid transfer protein 3; StarD3) mimic germline phenotypes found in BPA-treated worms. Here we show that developing germ cells of these mutants have increases in germ cell nuclei apoptosis and reduced fertility when exposed to no or low cholesterol conditions. Protruding vulva, as well as missing or underdeveloped gonads, are also frequently seen in strl-1 (homologous to StAR) worms. To investigate a possible interaction between BPA and mitochondrial cholesterol transport, wild type and strl-1 worms were treated with BPA. Diakinetic analysis of the -1, -2 and -3 oocytes from control-treated worms reveals an increased incidence of abnormal chromatin arrangement in strl-1 mutant worms compared to wild type (29, 49 and 78 versus 0, 5 and 25 percent). Interestingly, the occurrence among wild type and strl-1 worms is similar following BPA treatment (6, 34 and 71 percent in wild type versus 7, 30 and 73 in strl-1). Additionally, BPA treatment reduced the incidence of protruding vulva (4 vs 24% in controls) and other severe gonad defects (6 vs 31% in controls) in strl-1 mutants. Together, the data suggest that BPA can at least partially rescue the sensitive germline phenotype in strl-1 worms. .

Punkay EM et al. (1998) Midwest Worm Meeting "All in the Family: Mouse QKI-6, a homologue of GLD-1, preferentially binds to conserved TGEs"

Loushin CL, Goodwin EB, Artzt K, Punkay EM

[Midwest Worm Meeting, 1998]

The sex determination gene, tra-2, promotes female cell fate. Male development, in part, requires translational control of tra-2 by regulatory elements, called TGEs, located in the 3'UTR. Regulation by TGEs is a highly conserved mechanism in both invertebrates and vertebrates. Using the TGEs as bait in a yeast three-hybrid screen, a potential regulator of tra-2 translation was identified. This protein, C. elegans GLD-1, a member of the STAR family, can preferentially bind to the TGEs. In addition, it can also regulate translation via the TGEs both in vitro and in vivo. STAR family proteins contain a single KH RNA binding domain. Their RNA targets have not been previously identified. Mouse QKI proteins, also members of the STAR family, are close homologues of GLD-1 and thus good candidates for translational regulators in vertebrates. QKI proteins are believed to be regulators of myelination and embryonic development. The mutant viable phenotype is characterized by gross dysmyelination, primarily in the CNS, causing the mouse to shudder or "quake". There are also embryonic lethal mutations. qkI has three alternatively spliced forms of 5, 6 and 7 kb. The proteins they encode differ only in their carboxy tails and in their intracellular localization. QKI-5 is confined to the nucleus, while QKI-6 and QKI-7 are found only in the cytoplasm. Since GLD-1 is a cytoplasmic protein, we focused on QKI-6 to test if it possesses the same RNA binding characteristics as GLD-1. Through gel shift assays, it was confirmed that QKI-6 preferentially binds to the TGEs of the 3'UTRs of C. elegans and C. briggsae tra-2, thus mimicking the behavior of GLD-1. This evidence points to a possible role for QKI-6 as the translational regulator of an as yet unidentified gene in mouse.

E Jan et al. (1999) International C. elegans Meeting "GLD-1 represses tra-2 translation through a poly (A) tail-dependent mechanism"

EB Goodwin, E Jan

[International C. elegans Meeting, 1999]

The C. elegans sex determination gene, tra-2 , is translationally regulated by elements in the 3'untranslated region (3'UTR) called TGEs. tra-2 activity is required for female development. Male development in both somatic and germline tissues requires that tra-2 translation is repressed via the TGEs. The TGE control is a conserved mechanism present in invertebrates and vertebrates that controls the translation of other mRNAs. Recently, we have demonstrated that the C. elegans GLD-1 protein binds specifically to the TGEs and represses translation in a TGE-dependent manner in vitro and in vivo . GLD-1 is a cytoplasmic germline specific protein and is a member of the STAR family of RNA binding proteins. GLD-1 is essential for oogenesis and is also necessary for inhibiting premeiotic proliferation and promoting hermaphrodite spermatogenesis. In general, the mechanism of 3'UTR regulation and the function of STAR proteins are not well understood. Polysome analysis suggest the TGEs repress translation by inhibiting ribosome initiation in C. elegans . To better understand the mechanism of GLD-1 repression, we developed a yeast in vitro translation assay. We find that addition of GLD-1 protein to yeast extracts represses translation of reporter RNAs in a TGE-dependent manner. We also find that this repression occurs only when reporter RNAs carry a poly (A) tail, suggesting that GLD-1 represses translation through a poly (A) tail-dependent mechanism. Furthermore, deadenylation is not necesssary for GLD-1 repression, since GLD-1 can inhibit translation of RNAs carrying a stretch of 30 nucleotides 3' to the poly (A) tail which blocks deadenylation. Lastly, the 5'cap of an mRNA is not required for GLD-1 repression suggesting that the mechanism of TGE control is not acting through the cap-binding protein, eIF4E. Our results suggest a model by which binding of GLD-1 to the TGEs represses translation by influencing the effect of the poly (A) tail on ribosome initiation.

Kalchhauser, Irene et al. (2011) International Worm Meeting "FBF-mediated repression of the Cip/Kip cell cycle inhibitor CKI-2 promotes self-renewal of C. elegans germline stem cells."

Ryder, Sean, Farley, Brian, Kalchhauser, Irene, Pauli, Sandra, Ciosk, Rafal

[International Worm Meeting, 2011]

The transition of stem cells from self-renewal to differentiation has been linked to modifications of the cell cycle. However, the relation between key stem cell regulators and cell cycle changes remains poorly understood. RNA-binding proteins of the PUF family regulate stem cells in diverse animals. For example, Caenorhabditis elegans FBF maintains self-renewal of stem cells in the germ line. Here, we report that FBF promotes self-renewal by repressing the mRNA encoding CKI-2Cip/Kip, a Cyclin E/CDK2 inhibitor. We have previously shown that repression of cye-1 (cyclin E) mRNA by the STAR-domain protein GLD-1 inhibits CYE-1/CDK-2 activity, promoting germ cell differentiation. Based on these and current findings, we propose that a post-transcriptional regulatory circuit mediated by FBF and GLD-1 controls the self-renewal versus differentiation decision in the germ line by promoting high CYE-1/CDK-2 activity in stem cells, and repressing CYE-1/CDK-2 activity in differentiating cells.

Scheckel, Claudia et al. (2011) International Worm Meeting "GLD-1 binding marks specific mRNA targets for accumulation in oocytes."

Gaidatzis, Dimos, Scheckel, Claudia, Ciosk, Rafal, Wright, Jane E.

[International Worm Meeting, 2011]

Maternal mRNAs loaded into transcriptionally quiescent oocytes are stored in an inactive but stable form, in case of human oocytes for several decades, to support the oocyte-to-embryo transition (OET). These mRNAs are thought to be stable by 'default', due to a global repression of mRNA decay pathways. However, we find that a large group of mRNAs encoding factors driving OET is specifically stabilized in the C. elegans germ line by the combined function of two conserved RNA regulators. One of them, the sequence-specific RNA binding protein of the STAR family, GLD-1, represses translation of associated mRNAs. This appears to mark them for stabilization that depends on CGH-1, a DDX6-like RNA helicase, which is a component of germline RNA/protein granules and somatic processing (P) bodies. Our findings suggest that the GLD-1 and CGH-1-dependent pathway for mRNA storage ensures efficient accumulation, and consequently function, of OET regulators.

Clark, Laura et al. (2015) International Worm Meeting "Return to the death star: Coryneform bacteria as pathogens of C. elegans."

Burkovski, Andreas, Mattos-Guaraldi, Ana Luiza, Azevedo Antunes, Camila, Hodgkin, Jonathan, Clark, Laura

[International Worm Meeting, 2015]

Two novel nematopathogenic Leucobacter were isolated from nature and display remarkable modes of pathogenicity1, some of which can be recapitulated using members of the human-pathogenic genus Corynebacterium.A bacterium, CBX151, was isolated from nematodes living on a rotting banana trunk in the Cabo Verde islands and identified as a novel subspecies of Leucobacter celer. When introduced to Caenorhabditis elegans N2, CBX151 adhered to the nematode cuticle in a thick "fur coat" and impaired worm movement on solid medium, but did not affect lifespan. However, when infected worms were added to aqueous medium they immediately aggregated by their tails into a rosette-like formation, or "worm-star", from which it was almost impossible to escape. Worms trapped in stars died within 24 hours and CBX151 proliferated using the worm carcases as a nutrient source. This novel phenomenon relies on exceedingly robust adhesion between the bacteria and the worm cuticle.A second bacterium, CBX152, was isolated as a co-infection with CBX151 but identified as a distinct novel species of Leucobacter. CBX152 does not adhere to the worm cuticle but causes a distinctive rectal swelling and is usually lethal within 24 hours of infection. Infection with CBX152 induces a strong immune response in the worms. Surprisingly, C. elegans mutations which confer resistance to CBX152 frequently induce hypersusceptibility to CBX151.Recent work using the human pathogens Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis, and the non-pathogen Corynebacterium glutamicum shows that these bacterial species can also induce star formation slowly in wild-type worms, as well as severe tail swelling. Characterisation of the tail-swelling response using immunocompromised worms reveals the involvement of daf-16 and the ERK map kinase pathway.The fascinating worm-trapping strategy exhibited by CBX151 and pathogenic Corynebacteria opens new avenues of inquiry into the mechanisms by which pathogens exploit host resources. Moreover, interactions between coryneform bacteria and soil nematodes are likely to be ecologically important and diverse, in view of the abundance of these bacteria in soil.1. Hodgkin et al 2013. Curr Biol 23, 2157-2161.