Spaulding EL et al. (2022) Nat Commun "RG/RGG repeats in the C. elegans homologs of Nucleolin and GAR1 contribute to sub-nucleolar phase separation."

Updike DL, Spaulding EL, Feidler AM, Cook LA

[Nat Commun, 2022]

The intrinsically disordered RG/RGG repeat domain is found in several nucleolar and P-granule proteins, but how it influences their phase separation into biomolecular condensates is unclear. We survey all RG/RGG repeats in C. elegans and uncover nucleolar and P-granule-specific RG/RGG motifs. An uncharacterized protein, K07H8.10, contains the longest nucleolar-like RG/RGG domain in C. elegans. Domain and sequence similarity, as well as nucleolar localization, reveals K07H8.10 (NUCL-1) to be the homolog of Nucleolin, a protein conserved across animals, plants, and fungi, but previously thought to be absent in nematodes. Deleting the RG/RGG repeats within endogenous NUCL-1 and a second nucleolar protein, GARR-1 (GAR1), demonstrates these domains are dispensable for nucleolar accumulation. Instead, their RG/RGG repeats contribute to the phase separation of proteins into nucleolar sub-compartments. Despite this common RG/RGG repeat function, only removal of the GARR-1 RG/RGG domain affects worm fertility and development, decoupling precise sub-nucleolar structure from nucleolar function.

Wang J et al. (2023) Carbohydr Polym "A ginseng-derived rhamnogalacturonan I (RG-I) pectin promotes longevity via TOR signalling in Caenorhabditis elegans."

Wang Y, Li X, Yu Y, Xue D, Wang J, Zhou Y

[Carbohydr Polym, 2023]

Panax ginseng C. A. Meyer (ginseng), a traditional Chinese herb, is usually used to improve health and increase anti-aging activity for human. Polysaccharides are bioactive components of ginseng. Herein, using Caenorhabditis elegans as a model, we discovered a ginseng-derived rhamnogalacturonan I (RG-I) pectin WGPA-1-RG promoted longevity via TOR signalling pathway with transcription factors FOXO/DAF-16 and Nrf2/SKN-1 accumulated in the nucleus, where they activated target genes. And the WGPA-1-RG-mediated lifespan extension was dependent on endocytosis, rather than a bacterial metabolic process. Glycosidic linkage analyses combined with arabinose- and galactose-releasing enzyme hydrolyses identified the RG-I backbone of WGPA-1-RG was primarily substituted with α-1,5-linked arabinan, β-1,4-linked galactan and arabinogalactan II (AG-II) side chains. Feeding worms with the WGPA-1-RG-derived fractions which lost distinct structural elements by enzymatic digestions, we found the arabinan side chains prominently contributed to the longevity-promoting activity of WGPA-1-RG. These findings provide a novel ginseng-derived nutrient that potentially increases human longevity.

Spaulding, Emily et al. (2021) International Worm Meeting "The C. elegans homolog of Nucleolin, NUCL-1, contributes to nucleolar organization through its intrinsically disordered RG/RGG repeat domain"

Updike, Dustin, Spaulding, Emily, Feidler, Alexis

[International Worm Meeting, 2021]

Intrinsically disordered domains are found in 30-40% of human proteins, many of which undergo liquid-liquid phase separation (LLPS). How these domains influence LLPS-mediated membraneless organelle (MLO) formation and organization in vivo is unclear. One such domain, consisting of Arginine and Glycine (RG/RGG) repeats, is critical for both P-granule and nucleolar function in C. elegans. We have identified 551 proteins with 3 or more regularly spaced RG/RGG repeats in C. elegans. Gene Ontology analysis reveals that these RG/RGG repeat-containing proteins are enriched in MLOs, including the nucleolus and P-granules. MEME motif discovery was used to identify a phenylalanine-rich RG/RGG motif typical of nucleolar proteins and a tyrosine-rich RG/RGG motif typical of P-granule proteins. These motifs were then used to predict the MLO localization of a highly abundant but uncharacterized protein, K07H8.10. The 176 amino acid-long RG/RGG repeat domain of K07H8.10 is the longest in C. elegans and is interspersed with phenylalanine, predicting nucleolar localization. In addition to its N terminal RG/RGG repeat domain, K07H8.10 contains a coiled-coil acidic domain and two C terminal RNA recognition motifs. Both the HHpred and the MARRVEL bioinformatics toolkits predict homology to Nucleolin, which contains these same three domains, although configured in a different arrangement in nematodes. We have fluorescently tagged K07H8.10 (now named NUCL-1) in the C. elegans germline and confirmed its nucleolar localization. Deleting the N terminal RG/RGG repeat domain of NUCL-1 results in fertile worms and does not impair NUCL-1 localization to the nucleolus. However, super resolution imaging of NUCL-1 in living worms reveals that sub-nucleolar compartmentalization of both NUCL-1 and Fibrillarin (FIB-1) are disrupted. Our results indicate that the NUCL-1 RG/RGG repeat domain is dispensable for localization to the nucleolus but is crucial for overall nucleolar organization.

Kocherscheidt L et al. (2010) Pediatr Allergy Immunol "Cytokine and chemokine responses in adults, newborns and children exposed to Entamoeba histolytica/dispar, Onchocerca volvulus and Plasmodium falciparum."

Agossou A, Hamm DM, Soboslay PT, Gantin RG, Banla M, Kocherscheidt L

[Pediatr Allergy Immunol, 2010]

Cytokine and chemokine response profiles were studied in newborns, 10-yr-old children and post partum mothers. All study groups were repeatedly exposed to Entamoeba histolytica, Onchocerca volvulus and Plasmodium falciparum infections as indicated by their Immunoglobulin (IgG) responses to parasite-specific antigens. As key indicators for regulatory and pro-inflammatory cytokine and chemokine responses, Interferon (IFN)gamma and regulatory IL-10 were investigated, along with the chemokines MIP-1 alpha/CCL3, MIP-1 beta/CCL4, MDC/CCL22 and TARC/CCL17. Entamoeba histolytica antigens (EhAg) strongly activated pro-inflammatory MIP-1 alpha/CCL3 and MIP-1 beta/CCL4 responses of similar magnitude in mothers, children and neonates alike. Plasmodium falciparum antigens (PfAg) enhanced MIP-1 alpha/CCL3, MIP-1 beta/CCL4 and MDC/CCL22 production in neonates, but did not trigger these chemokines in mothers or 10-yr-old children. Onchocerca volvulus antigens (OvAg) activated IFN-gamma and TARC/CCL17 production in mothers but not in neonates and children. Crude IL-10 production [i.e., without subtracting spontaneous cellular release (baseline)] was highest in mothers and somewhat lower in neonates, while the lowest IL-10 amounts of all were released by peripheral blood mononuclear cells from 10-yr-old children. In summary, strong inflammatory chemokine responses to plasmodia and ameba antigens in newborns and 10-yr-old children suggest that adequately balanced immune regulatory mechanisms may not have developed yet in these age groups and that repeated exposure to parasite infections and immune maturation during childhood is required to generate similar cytokine and chemokine profiles as in adults.

Wallis, Dylan et al. (2021) International Worm Meeting "Regulation of C. elegans Argonaute proteins by Arginine Dimethylation"

An Nguyen, Dieu, Phillips, Carolyn, Wallis, Dylan

[International Worm Meeting, 2021]

RNA silencing is a critically important mechanism through which cells regulate gene expression and protect the genome against aberrant RNAs, transposons, and viruses. This suppression of aberrant transcripts is carried out by the evolutionarily conserved small RNA pathway. Small RNAs are loaded onto Argonaute proteins to induce silencing through sequence recognition of specific transcripts through a variety of different silencing mechanisms including post-transcriptional or co-transcriptional silencing. In recent studies, it has been shown that some Argonaute proteins are modified with dimethylarginine within arginine/glycine-rich regions (RG/RGG motifs). In D. melanogaster, dimethylation of the Argonaute protein Aub is required for assembly of the ping-pong complex and ultimately for piRNA mediated transposon silencing. However, it remains unclear whether there are any general principles underlying the function of Argonaute modification by dimethylation. First, to determine if any of the 27 C. elegans Argonaute proteins are dimethylated, we identified Argonaute proteins containing RG/RGG motifs. The proteins with the highest incidence of these motifs are the cytoplasmic Argonautes ERGO-1, CSR-1, ALG-3 and PRG-1, and the nuclear Argonaute HRDE-1. These five Argonaute proteins were tagged using CRISPR and immunoprecipitated. Following mass spectrometry analysis, we identified multiple dimethylation sites within each of these Argonaute proteins. Next, to understand the physiological role of these dimethylarginine modifications, we used CRISPR to generate methylation-defective mutant Argonaute proteins. We are currently focusing on the PRG-1 RG-motif mutant, and assessing how mRNA and small RNA expression change compared to wild-type and a prg-1 null mutant. To further probe PRG-1 function in the absence of dimethylarginine, we will next determine whether the PRG-1 RG-motif mutant activates a sensor for piRNA silencing activity and whether it has transgenerational fertility defects. This work will define the functional relevance of Argonaute protein methylation in the C. elegans germline.

Korbmacher F et al. (2018) Parasite Epidemiol Control "Mansonella perstans, Onchocerca volvulus and Strongyloides stercoralis infections in rural populations in central and southern Togo."

Padjoudoum K, Karabou P, Korbmacher F, Soboslay PT, Gantin RG, Poutouli WP, Kohler C, Komlan K

[Parasite Epidemiol Control, 2018]

Background: are important especially under the aspect of MDA of ivermectin which is performed since decades. Methods: =924) were collected from rural populations in the Regions Central and Plateaux in Togo, and analyzed by parasite-specific real-time PCR and ELISA techniques. Results: were 9.9%. Conclusions: may be ongoing. The degree of positive test results in the examined rural communities advocate for the continuation of MDA with ivermectin and albendazole, and further investigations should address the intensity of transmission of these parasites.

Komlan K et al. (2018) PLoS Negl Trop Dis "Onchocerca volvulus infection and serological prevalence, ocular onchocerciasis and parasite transmission in northern and central Togo after decades of Simulium damnosum s.l. vector control and mass drug administration of ivermectin."

Soboslay PT, Vossberg PS, Solim T, Korbmacher F, Kohler C, Gantin RG, Komlan K, Banla M, Karabou P, Padjoudoum K

[PLoS Negl Trop Dis, 2018]

BACKGROUND: Mass drug administration (MDA) of ivermectin has become the main intervention to control onchocerciasis or "river blindness". In Togo, after many years of MDA, Onchocerca volvulus infection has declined dramatically, and elimination appears achievable, but in certain river basins the current situation remains unknown. We have conducted parasitological, serological, ophthalmological, and entomological assessments in northern and central Togo within the river basins of Oti, Keran and Mo. METHODOLOGY/PRINCIPAL FINDINGS: Examinations were completed in 1,455 participants from 11 onchocerciasis sentinel villages, and O. volvulus transmission by Simulium damnosum sensu lato (s.l.) was evaluated. In children (aged 1-10 years), the prevalence of microfilariae (Mf) was 2.3% and in adults it ranged from 5.1 to 13.3%. Positive IgG4 responses to O. volvulus adult (crude) worm antigen (OvAg) and the recombinant Ov16 antigen were in all-ages 48.7% and 34.4%, and 29.1% and 14.9% in children, respectively. In the river basin villages of Keran, Mo and Oti, the IgG4 seroprevalences to OvAg in children were 51.7%, 23.5% and 12.7%, respectively, and to the Ov16 antigen 33.3% (Keran) and 5.2% (Oti). Onchocerciasis ocular lesions (punctate keratitis, evolving iridocyclitis and chorioretinitis) were observed in children and young adults. O. volvulus-specific DNA (Ov150) was detected by poolscreen in vector samples collected from Tchitchira/Keran(22.8%), Bouzalo/Mo(11.3%), Baghan/Mo(2.9%) and Pancerys/Oti(4.9%); prevalences of O. volvulus infection in S. damnosum s.l. were, respectively, 1%, 0.5%, 0.1% and 0.2%. CONCLUSIONS/SIGNIFICANCE: In the northern and central river basins in Togo, interruption of O. volvulus transmission has not yet been attained. Patent O. volvulus infections, positive antibody responses, progressive ocular onchocerciasis were diagnosed, and parasite transmission by S. damnosum s.l. occurred close to the survey locations. Future interventions may require approaches selectively targeted to non-complying endemic populations, to the seasonality of parasite transmission and national onchocerciasis control programs should harmonize cross-border MDA as a coordinated intervention.

Soboslay PT et al. (2010) Wien Klin Wochenschr "Applied field research for comprehensive helminth infection control."

Schulz-Key H, Stingl P, Djassoa G, Karabou PK, Hamm DM, Douti JK, Heuschkel C, Agossou A, Gantin RG, Soboslay PT, Banla M

[Wien Klin Wochenschr, 2010]

The Institute for Tropical Medicine at University of Tubingen has established 30 years ago in Togo a Research Centre and Onchocerciasis Reference Laboratory (ORL). Onchocerca volvulus infection control and of other neglected tropical diseases has been the focus of activities, and those were performed together with the National Institute of Hygiene in Togo, the Medical Faculty at University of Lome, national disease control programs and district and regional hospitals. The ORL contributed significantly to the assessment of ivermectin as the prime choice for onchocerciasis treatment, and 24 years of repeated annual treatment with ivermectin has progressively reduced disease prevalence and notably the level of ocular and dermal manifestations of onchocerciasis in the endemic population. The ORL has shown that large parts of the rural population in Togo is concurrently infected with intestinal and intravascular protozoan and helminth parasites, notably school children. The application of repeated treatments with albendazole and praziquantel against Schistosoma spp. and instestinal helminthes for several years has reduced infection intensities by more than 80%. Longitudinal investigations of the cellular immune responses in adults and children have found that parasite co-infections will generate prominent pro-inflammatory responses, and a single or few interventions will not suffice to eliminate co-infections and not establish an appropriately balanced immunity.

KM Walstrom et al. (1999) International C. elegans Meeting "RHA-1: A conserved RNA helicase in C. elegans important for development"

KA Swan, KM Walstrom

[International C. elegans Meeting, 1999]

RNA helicases are important proteins involved in RNA metabolic reactions, such as splicing and transcription. Last year we discovered an RNA helicase (RHA-1) required for development in C. elegans . This protein is homologous to Drosophila maleless and human RNA helicase A (hRHA). Each of these proteins interact with DNA binding proteins and are proposed to regulate transcription. Specifically, hRHA is capable of mediating an association between CREB binding protein (CBP) and RNA polymerase II. This suggests that hRHA may play a role in mediating the effects of CBP on transcription (Nakajima et al. (1997) Cell 90 , 1107). RHA is also required for proper gastrulation in mice (Lee et al. (1998) PNAS 95 , 13709). RNA interference (RNAi) was used to deplete C. elegans embryos of RHA-1. We injected 800 bp of double-stranded RNA transcribed from DNA encoding the N-terminus of RHA-1 and observed a range of phenotypes culminating in dead embryos. Some dead embryos exhibited muscle development and most lacked gut granules. This suggests that endodermal development was compromised. Less severely affected progeny reached adulthood but were sterile or laid mainly oocytes. Others have shown that performing RNAi with RNA transcribed from C. elegans cbp-1 results in embryos lacking mesodermal, endodermal, and hypodermal cells (Shi & Mello (1998) Genes and Develop. 12 , 943-955). Thus one explanation of our rha-1 RNAi results is a partial block of CBP function. Alternately, RHA-1 may be required for the function of other transcription factors, or it may be generally required for transcription or translation. Our current work involves biochemical experiments on the arginine-glycine-rich (RG) RNA binding region of RHA-1. This RG region binds specifically to single-stranded RNAs; therefore, it may have sequence specific RNA binding. The RG region of RHA-1 was subcloned from a previously isolated cDNA clone (from Yuji Kohara). This protein has been overexpressed in E. coli as a His-tagged polypeptide. RNA binding results will be discussed.

Nguyen, D.A. et al. (2019) International Worm Meeting "Differential Expression and Localization of CSR-1 Isoforms Allows for Regulation of Distinct Target Genes."

Nguyen, D.A., Phillips, C.M.

[International Worm Meeting, 2019]

Small RNA silencing is a mechanism by which the cell suppresses harmful exogenous and endogenous RNAs. The silencing complex consists of an Argonaute protein and its bound small RNAs, which are typically 18-30nt. In C. elegans, CSR-1 is an essential Argonaute protein that binds to a subclass of 22G-RNA to regulate most germline genes. CSR-1 is quite unique because it protects rather than silences its target mRNAs. Knockdown of CSR-1 results in sterility and embryonic lethality. This Argonaute protein has two differentially expressed isoforms - both sharing complete sequence and structural identity except for an additional exon included at the 5' end of the longer isoform. This exon distinguishes the long isoform, or CSR-1A, from the shorter counterpart, CSR-1B. Unlike CSR-1B which is ubiquitously expressed throughout the germline across development, CSR-1A expression is restricted to the spermatogenesis region of L4 hermaphrodites and males. Furthermore, CSR-1A perfectly colocalizes at P granules with the spermatogenesis-specific Argonaute ALG-3, and an uncharacterized Argonaute WAGO-10. Analysis of small RNA libraries shows that CSR-1A and CSR-1B bind to distinct sets of 22G-RNAs. More specifically, CSR-1A-bound siRNAs are more enriched for spermatogenic genes and surprisingly, mutator-class genes, which consist of peudogenes, repetitive elements, and transposons. The function of CSR-1A targeting this subset of 22G-RNA remains unclear. A hint may be found in the sequence of CSR-1A's unique exon. This exon contains 15 Arginine/Glycine (RG/RGG) motifs in a 158 amino-acid stretch, which are putative target for Protein Arginine Methyltransferase. By mass spectrometry analysis, we can detect at least six dimethylargnine modifications within this exon. Using CRISPR/Cas9, we have mutated all 15 RG/RGG motifs to remove the methylation sites. We are currently determining how the dimethylarginine modification contributes to the differential regulatory mechanism between the two CSR-1 isoforms.