Amrit, Francis RG et al. (2021) International Worm Meeting "TCER-1-regulated alternative splicing promotes stress resilience"

Ghazi, Arjumand, Amrit, Francis RG, KUROYANAGI, Hidehito, Loose, Julia, Pfenning, Andreas

[International Worm Meeting, 2021]

Alternative RNA splicing augments proteomic biodiversity in eukaryotes, and has an emerging role in regulating longevity. This process has been shown to have various roles based on physiological demands including those in response to external stimuli. Here, we show that in the nematode Caenorhabiditis elegans, exposure to the Gram-negative pathogen, Pseudomonas aeruginosa, induces a shift in alternative splicing. In vivo analysis of transgenic reporters of splicing fidelity showed significant splicing changes in multiple tissues. Based on our recent discovery that TCER-1, worm homolog of the human transcription elongation and splicing factor, TCERG1, represses innate immunity in worms, we examined the role of TCER-1 in the modulation of splicing upon pathogen exposure. tcer-1 mutants exhibited aberrant splicing regulation in multiple tissues, and transcriptomic analysis identified 54 exon-skipping and 468 intron retention events upon tcer-1 inactivation. In vivo analyses of these events revealed a TCER-1 dependent splicing pattern that correlated with our stress resilience phenotypes. Further, CRISPR-mediated recreation of alternative splicing observed in tcer-1 mutants enhanced immunoresistance and motor function. Together our findings bring to the forefront the important role of alternative splicing in stress resilience and healthspan, and reveal TCER-1- to be a major regulator of this process.

Amrit, Francis R.G. et al. (2010) Worm Breeder's Gazette "Differential expression of daf-16 splice variants"

May, Robin C., Amrit, Francis R.G.

[Worm Breeder's Gazette, 2010]

Amrit, Francis RG et al. (2015) International Worm Meeting "Molecular Jekyll and Hyde: The Opposing Roles of TCER-1/TCERG1 in Regulating Lifespan and Stress Resilience."

Amrit, Francis RG, Steenberge, Laura, Yanowitz, Judith, McClendon, Brooke, Ghazi, Arjumand

[International Worm Meeting, 2015]

Longevity and stress resistance are intimately linked. In worms, and in many other species, animals that exhibit increased lifespan almost inevitably also display enhanced resistance to various stressors. In most cases, genes that promote longevity also contribute to improved stress resilience. For instance, glp-1 mutants that are long-lived due to elimination of the proliferating germine-stem cells (GSCs) are also highly stress-resistant, and both the long life and stress-resilience of these animals is dependent on the longevity determinant, DAF-16/FOXO. Previously, we identified a conserved transcription elongation and splicing factor, TCER-1/TCERG1, that cooperates with DAF-16/FOXO to selectively augment the longevity of germline-depleted animals.Here, we demonstrate that, unlike DAF-16/FOXO, TCER-1 does not contribute to stress-resistance, instead it represses the animals' ability to combat adversity. tcer-1 reduction-of-function confers resistance to a multitude of biotic and abiotic stressors, both in fertile animals as well as sterile mutants. Using site of action studies and molecular genetic analysis, we have explored the basis of this phenotypic uncoupling. Our experiments reveal a unique role for TCER-1/TCERG1 in balancing length of life, reproductive health and stress resistance during adult life. These data will be discussed.

Amrit, Francis RG et al. (2015) International Worm Meeting "Transcription Factors that Coordinate Lipid Synthesis and Breakdown to Ensure Metabolic Homeostasis and Longevity."

Steenkiste, Elizabeth, Ratnappan, Ramesh, Amrit, Francis RG, Olsen, Carissa P, Yanowitz, Judith L, McClendon, T Brooke, Chen, Shaw-Wen

[International Worm Meeting, 2015]

The coordination of lipid production and degradation is essential for animals' health - lipid imbalances are characteristic of obesity and a feature of many reproductive pathologies and age-related diseases. But how these processes are balanced in multicellular organisms is poorly understood. In C. elegans, removing the germline, using mutations in genes such as glp-1, extends lifespan. Germline loss is a major metabolic challenge that compels the animal to stop fat deposition into eggs and remodel its lipid reserves. This phenomenon provides a unique platform to understand how complex metazoans retain metabolic homeostasis when challenged with alterations in fertility and age. Recent studies, including ours, have shown that germline loss activates conserved transcription factors such as DAF-16/FOXO and TCER-1/TCERG1 in the intestine, the worms' main fat depot. We recently demonstrated that the nuclear hormone receptor, NHR-49/PPARalpha, under partial regulation by DAF-16/FOXO and TCER-1/TCERG1, promotes glp-1 mutants' longevity by elevating fatty-acid beta-oxidation and desaturation- pathways involved in fat buildup and breakdown, respectively. We have now discovered that both lipid synthesis and degradation are widely and concurrently enhanced in response to germline loss. Using transcriptomics, molecular-genetic analyses and biochemical approaches, we demonstrate that these transcription factors activate multiple lipid anabolic (de novo lipid synthesis, fatty-acid desaturation and elongation and triglyceride production) and catabolic (triglyceride hydrolysis, fatty-acid oxidation) pathways in glp-1 mutants. Our data suggest that the coordinated enhancement of lipid synthesis and breakdown, by NHR-49, DAF-16 and TCER-1, facilitates the adaptation to germline loss by ensuring lipid homeostasis, and mediates longevity.

RG Amrit, Francis et al. (2013) International Worm Meeting "The Dual Roles of TCER-1/TCERG1 in Balancing Reproductive Fitness and Longevity."

Ghazi, Arjumand, RG Amrit, Francis, Benos, Takis, Arora, Arshi

[International Worm Meeting, 2013]

The debilitating effects of advacing age on fertility are well-known, but recent studies have shown that reproductive status reciprocally alters aging. We describe a role for TCER-1, the worm homolog of human transcription elongation factor, TCERG1, in establishing the balance between reproductive efficiency and somatic maintenance based on the procreative status of the animal. In C. elegans, removal of germline-stem cells (GSCs) extends lifespan. TCER-1/TCERG1 promotes longevity specifically upon GSC loss, likely by influencing the activity of the conserved longevity determinant, DAF-16/FOXO. We used massively parallel RNA-Sequencing to map the contribution of TCER-1/TCERG1 and DAF-16/FOXO to the transcriptional profile of GSC(-) worms. We found that these proteins share about a third of their transcriptomes and jointly up-regulate a spectrum of genes that alter lipid metabolism and xenobiotic-stress resistance. These targets improve cellular homeostasis and are essential for the longevity of the sterile adult. Strikingly, TCER-1/TCERG1 represses more genes than it activates in GSC(-) worms, and these genes are highly enriched for reproductive roles. We found that tcer-1 mutants have reduced fecundity under normal conditions, including diminished brood size, poor egg viability and premature unfertilized-oocyte production. tcer-1 is expressed in the germline and its mutant phenotypes can be attributed to both defective sperm and eggs. Accordingly, many genes repressed by TCER-1/TCERG1 in GSC(-) worms are important for reproduction and reducing their functions impairs fertility. Our results reveal that TCER-1/TCERG1 plays a dynamic function depending on the reproductive status of the worm. In fertile adults, it promotes reproductive fitness. Upon GSC loss, it represses the expression of reproduction genes, and (in combination with DAF-16/FOXO) elevates the expression of pro-longevity genes. This molecular dexterity of TCER-1/TCERG1 facilitates communication between the germline and the soma, and orchestrates the balance between reproductive vigor and somatic preservation.

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.

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.

Lee M-H et al. (1997) International C. elegans Meeting "TOWARD THE IDENTIFICATION OF IN VIVO RNA TARGETS OF GLD-1."

Lee M-H, Schedl TB

[International C. elegans Meeting, 1997]

gld-1 is a female germ cell specific tumor suppressor gene that is essential for normal oocyte differentiation and meiotic prophase progression (Francis et al., 1995a,b). GLD-1 is a member of a small family of proteins, including the mouse/human Sam68 and Quaking proteins, that are highly related over an ~ 200 amino acid region which contains a 70 to 100 amino acid RNA binding domain called the KH motif (Jones and Schedl, 1995). Extensive mutational analysis of gld-1 has demonstrated the importance of conserved sequences for its in vivo function. GLD-1 is a cytoplasmic protein and therefore is likely to control mRNA translation or RNA stability in the cytoplasm (Jones et al., 1996). Currently, no obvious RNA targets have been identified from genetic analysis. We have employed a biochemical approach to identify in vivo RNA targets of GLD-1. The strategy is based on the ability of anti GLD-1 antibodies to immunoprecipitate (IP) GLD-1 from a worm lysate and the strong likelihood that GLD-1 present in the lysate is functional and bound to RNAs. GLD-1 was IPed with affinity purified polyclonal antibodies from a cytosol extract of adult hermaphrodites or with rabbit IgG as a control. RNAs co-IP with GLD-1, as well as with rabbit IgG, were converted into cDNAs. Non-specifically trapped RNAs from the GLD-1 IP were eliminated by subtracting cDNAs from the GLD-1 IP with cDNAs from the control IP. The difference product after 4 rounds of subtraction (DP4) was used to screen a Lambda ZAP II cDNA library constructed with the cDNAs from the GLD-1 IP. Duplicate filters were also screened with a probe made from control IP cDNA. Clones that are positive only with the DP4 probe are currently being characterized. Francis et al., 1995a Genetics 139, 579-606; Francis et al., 1995b Genetics 139, 607-630; Jones and Schedl, 1995 Genes Dev. 9, 1491-1504; Jones et al., 1996 Dev. Biol. 180, 165-183. This work was supported by NIH Grant HD25614.