Wang, Chris et al. (2011) International Worm Meeting "Identifying TEG-1 interacting proteins that are involved in germline development."

Hansen, Dave, Wang, Chris

[International Worm Meeting, 2011]

In the C. elegans germline, mitotically dividing cells are found at the distal end of each gonad arm, while the more proximal cells enter meiosis and differentiate into gametes. The GLP-1/Notch signaling pathway is the major player in promoting distal germ cell proliferation by suppressing two redundant pathways; one containing GLD-1 and the other GLD-2, which each promote meiotic entry. Genetic enhancer screens using a glp-1 weak gain-of-function background had identified teg-1 (tumours enhancer of glp-1(gf)) as a gene in maintaining the balance between proliferation and meiotic entry. We have identified UAF-1 (the large subunit of the U2 small nuclear ribonucleoparitcle auxiliary factor) and VIG-1 (Drosophila Vasa Intronic Gene ortholog) as potential TEG-1 binding proteins through proteomics. Results from co-immunoprecipitation and bacterial pull-down experiments suggest that TEG-1 directly interacts with UAF-1. Moreover, a complex containing CD2BP2 (human TEG-1 homolog) and U2AF65 human UAF-1 homolog) is detected in HeLa extracts. The human CD2BP2 also physically binds to U2AF65 in vitro. These data, together, indicate that the direct interaction between TEG-1 and UAF-1 is conserved from worms to humans in RNA independent manner. Antibodies raised against VIG-1 detect the protein in the cytoplasm of the germ cells by indirect immunofluorescence. Like teg-1 mutations, vig-1 null mutants also enhance the over-proliferation phenotype in a weak glp-1 gain-of-function background. In addition, vig-1; teg-1 double mutants show pleiotropic germline defective phenotypes. As VIG-1 has been shown to be a component of the miRNA-induced silencing complex, we are currently investigating possible genetic interactions between genes in the miRNA pathway and genes in the genetic pathway controlling the balance between proliferation and differentiation. In addition, we are determining the genetic epistasis of vig-1 in the proliferation/differentiation pathway. A series of biochemical experiments are being performed to verify the interaction between VIG-1 and TEG-1. Results from both genetic and biochemical experiments would provide a better understand on the mechanism of VIG-1in regulating the proliferation vs. meiotic entry decision in the germ line.

Wang, Chris et al. (2013) International Worm Meeting "TEG-1 regulates the stability of miRISC components and the levels of microRNAs."

Hansen, Dave, Wang, Chris

[International Worm Meeting, 2013]

TEG-1 (Tumorous Enhancer of Glp-1(gf))-related proteins are found in organisms from yeast to humans. We have previously reported roles for TEG-1 in germline development, including regulating the mitosis/meiosis decision and germline sex determination (Wang et. al. 2012. Dev. Dyn. 241:505-521). Recently, we found that teg-1 mutants display heterochronic defect phenotypes, including: (1) reiteration of the L2 stage resulting in an increased number of seam cells; (2) defective seam cell fusion at the L4 molt; and (3) retarded alae formation at the L4-adult transition. Since developmental timing defects are often controlled by microRNAs, we sought to determine if TEG-1 also plays a role in microRNA function. We found that mature let-7, mir-58, and mir-62 levels are significantly reduced in teg-1 mutants. To gain insight into the cause of reduced microRNA levels in teg-1 mutants, we performed immunoprecipitation (IP) and found that TEG-1 interacts with VIG-1, a Drosophila Vasa Intronic Gene ortholog, whose activity is required for proper let-7 function and is believed to be a component of the microRNA RNA-induced silencing complex (RISC) (Caudy et. al. 2003. Nature 425:411-414; Chan et. al. 2008. RNA 14:2104-2114). This interaction was confirmed by co-IP experiments using wild-type extracts. Interestingly, we also found that TEG-1 levels are lowered in vig-1 mutants, and both VIG-1 and ALG-1 (an Argonaute component of the RISC complex) levels are reduced in teg-1 mutants. This finding led us to propose that VIG-1 and ALG-1 levels are regulated post-translationally. Consistent with this hypothesis, we found that the mRNA levels of vig-1 and alg-1 remain unchanged in teg-1 mutants, whereas elevated VIG-1 and ALG-1 protein levels are detected in teg-1 mutants and wild-type animals treated with lactacystin, a specific inhibitor of the 26S proteasome. Together, these data suggest that TEG-1 regulates a broad range of microRNAs through stabilizing the microRISC components, potentially by protecting them from proteasomal degradation.

Wang, Chris et al. (2015) International Worm Meeting "TEG-1 CD2BP2 regulates miRNA levels via maintaining miRISC stability in C. elegans and human cells."

Bosse, Gabriel, Wang, Xin, Wang, Chris, Gupta, Pratyush, Simard, Martin, Hansen, Dave

[International Worm Meeting, 2015]

Although the recruitment of miRNA-induced silencing complex (miRISC) by miRNAs to the targeted mRNAs to regulate post-transcriptional gene expression is well documented, the mechanisms by which miRISC components are maintained at appropriate levels for proper function remain largely unknown. Here, we report the C. elegans TEG-1 (Tumorous Enhancer of glp-1(gf)) regulates the stability of two miRISC effectors, VIG-1 and ALG-1, which in turn affects the abundance of various miRNA families. Loss of teg-1 in vivo results in heterochronic defects, including excessive seam cell division and defective seam cell fusion. Since temporal developmental events are often controlled by miRNAs in C. elegans, we sought to determine if TEG-1 plays a role in miRNA function by identifying TEG-1-assoicated proteins. We found that TEG-1 physically interacts with VIG-1, and complexes with mature let-7 miRNA. Moreover, molecular and genetic analyses demonstrate that the levels of lsy-6 expression and mature let-7, miR-58, and miR-62 are significantly reduced in teg-1 mutants, suggesting mutations in teg-1 affect the levels of a broad range of miRNA members. We also found that both VIG-1 and ALG-1 protein levels are reduced in teg-1 mutants, although their mRNA levels remain unchanged. Together, these findings suggest that the association of TEG-1 with miRISC is necessary for miRNAs to function properly during development. We further demonstrate that the human orthologs of TEG-1, VIG-1 and ALG-1 (CD2BP2, SERBP1/PAI-RBP1, and AGO2) complex in HeLa cells, and knockdown of CD2BP2 results in reduced let-7a miRNA levels. Therefore, TEG-1's role in affecting miRNA levels and function is likely conserved from nematodes to humans. We propose that TEG-1 CD2BP2 stabilizes miRISC and mature miRNAs, maintaining them at levels necessary to properly regulate target gene expression.

Infarinato N (2019) J Cell Biol "Xiaochen Wang: Building up our understanding of breaking down."

Infarinato N

[J Cell Biol, 2019]

Wang studies lysosomal degradation pathways using <i>C. elegans</i> as a model system.

Wang JT et al. (2014) Curr Biol "P granules."

Seydoux G, Wang JT

[Curr Biol, 2014]

Wang and Seydoux discuss the functional importance of P granules - the germline-specific RNA granules of C. elegans.

Blumenthal TE et al. (1993) Worm Breeder's Gazette "The C. elegans Cleavage and Polyadenylation Signal"

Fields CA, White O, Blumenthal TE

[Worm Breeder's Gazette, 1993]

THE C. ELEGANS CLEAVAGE AND POLYADENYLATION SlGNAL Tom Blumenthal, Department of Biology, Indiana University. Bloomington, IN 47405; Owen White and Chris Fields, Institute For Genomic Research, Gaithersburg, MD, 20878

Zhang, Dan et al. (2021) International Worm Meeting "The Role of the RNA-Induced Silencing Complex (RISC) Component VIG-1 in C. elegans Germline Stem Cell Regulation"

Hansen, Dave, Wang, Chris, Zhang, Dan

[International Worm Meeting, 2021]

Stem cells are undifferentiated cells that can give rise to a wide variety of specialized cell types. In development and tissue maintenance, these cells play crucial roles in maintaining homeostasis and reproductive fitness. The C. elegans germline is set up in an assembly line-like format, in which mitotically dividing proliferative cells, or stem cells, reside in a distal niche. As cells move away from this niche, they gradually progress through meiosis until ultimately becoming fully differentiated sperm or oocytes. Notch signaling in the distal end of the germline provides the proliferative signal, while downstream post-transcriptional regulatory pathways promote differentiation. Although this main pathway has been well characterized, there exist various components that serve to fine-tune this balance in subtle but important ways. Previous work has identified small RNA molecules, such as miRNAs, as having a role in germline stem cell regulation. Here, the role of vig-1, a gene involved in the RNA-induced silencing complex (RISC) pathway, in C. elegans germline stem cell regulation is investigated. Although vig-1 performs multiple functions in a variety of tissues, it was initially implicated in the germline through its physical protein interaction with another RISC component, teg-1, that was known to modulate germline stem cell balance. We show that vig-1 likely functions to repress the activity of Notch signaling. Genetic null mutants for vig-1 and GLD-1/GLD-2 pathway components do not cause any changes to germline phenotype, suggesting that vig-1 does not function downstream of Notch. However, in worms that lack vig-1 activity, Notch signaling is seen to be enhanced, manifesting through ectopic mitotic activity and over-proliferation of stem cells. In addition, vig-1 can partially suppress Notch loss-of-function mutant phenotypes. In addition, we show that vig-1 may act directly on Notch signaling through increasing the expression of its direct transcriptional targets, LST-1 and SYGL-1. Finally, we propose that vig-1 acts through modulating miRNA levels, as germline miRNA levels are reduced in vig-1 mutants.

Ray C et al. (1990) Worm Breeder's Gazette "CORRECTION Isolation of a C. elegans Cysteine Protease Gene"

McKerrow JH, Ray C

[Worm Breeder's Gazette, 1990]

In the previous issue of the WBG (Vol. 11, #3, page 20) we reported the isolation of a cysteine protease clone from a mixed-stage C. elegans cDNA library. This library was originally obtained from Chris Martin and not from Cynthia Kenyon as was reported in the article. Our apologies for any misunderstanding caused by this oversight.

Vanden Broek, Kara et al. (2021) International Worm Meeting "RACK-1 is required for proper GLD-1 sub-cellular localization and function"

Wang, Chris, Vanden Broek, Kara, Hansen, Dave

[International Worm Meeting, 2021]

Stem cells are central to the development of multi-cellular organisms, including C. elegans and humans. Key to their function is their ability to differentiate into specialized cells or proliferate to maintain their population for future use. We are focusing on identifying the molecular mechanisms that regulate the proliferation/differentiation decision of stem cells using the C. elegans germ line as a model. C. elegans' germline stem cells (GSCs) proliferate to maintain the stem cell pool and differentiate to produce gametes. The conserved STAR family, RNA binding protein, GLD-1/Quaking, promotes differentiation and is required to maintain a proper balance between proliferation and differentiation. When gld-1 activity is lost, along with gld-2 activity, which functions redundantly with gld-1 to promote differentiation, a germline tumour of proliferating stem cells is formed. Previous research has shown that the pattern of GLD-1 germline accumulation is key to controlling its activity. We have found that GLD-1 subcellular localization is likely also involved in regulating GLD-1's activity. We identified rack-1 as a regulator of GLD-1 subcellular localization. rack-1 mutants have a severe disruption in GLD-1's subcellular localization; while wild-type GLD-1 is cytoplasmic, in rack-1 mutants GLD-1 localizes to germ granules. This disruption in localization appears to impact GLD-1's activity, as a loss of rack-1 phenocopies a reduction, or loss, of gld-1 activity in various genetic backgrounds. Our research is revealing a novel mechanism where the activity of GLD-1 is regulated by its subcellular localization. This provides an additional layer of regulation in the proliferation/differentiation decision of C. elegans GSCs.

Munro E (2017) Dev Cell "Protein Clustering Shapes Polarity Protein Gradients."

Munro E

[Dev Cell, 2017]

In this issue of Developmental Cell, Dickinson etal. (2017) and Rodriguez etal. (2017), along with Wang etal. (2017) in Nature Cell Biology, show how PAR protein oligomerization can dynamically couple protein diffusion and transport by cortical flow to control kinase activity gradients and polarity in the C.elegans zygote.