Karen Bennett et al. (2004) Mid-west Worm Meeting "Cross-species RNAi: several dsRNAs from Ascaris sterilize Caenorhabditis"

Ge Gao, Karen Bennett, Sheela Raikar

[Mid-west Worm Meeting, 2004]

The parasitic nematode Ascaris lumbricoides is the most ubiquitous human parasite, infecting ~1/4 th of the world's population, while a similar proportion of Missouri swine are infected with the closely-related species A. suum . Ascaris, worms lay 200,000 eggs/day! Because parasitic nematodes are rapidly developing multi-drug resistance and most anti-helminthic drugs eliminate the adults but have no effect on the eggs, we reasoned that RNA interference (RNAi) might be applicable as a novel anti-parasitic agent. Since ascarid worms cannot be maintained in the laboratory for extended periods, we have begun by testing Ascaris genes in Caenorhabditis . Several Ascaris cDNAs with stretches of identity to C. elegans of >21nts and with an overall match of >80% for ~200nts were amplified from Ascaris ovarian RNA. Thus far, after injection into C. elegans , two A. suum genes have been successful in sterilizing C. elegans . In our first tests ~90% of the offspring were either dead embryos or sterile F1 adult worms. Although both genes are highly conserved, neither has a mammalian counterpart with a match of 21nts. We have recently begun to test two other candidates that are nematode-specific and plan to expand our studies to the more-applicable methods of dsRNA delivery, including feeding and soaking. We plan to test isolated Ascaris suum adults in the laboratory to assure ourselves that RNAi works in this species of nematode before moving on to the daunting task of considering delivery in swine. This work was supported by a pilot award from the MU PPAID (Program Project in Animal Infectious Diseases), funded by the USDA. SR was supported by a University of Missouri LS UROP (Life Science Undergraduate Research Opportunities Program) fellowship.

Karen L Bennett et al. (2002) Mid-west Worm Meeting "Two GLH interactors, critical for meiosis, are not necessary for P granule assembly."

Ruth A Montgomery, April M Orsborn, Pliny A Smith, Karen L Bennett

[Mid-west Worm Meeting, 2002]

GLH-interacting proteins. Their physical interactions with GLH proteins have also been shown by pull-down assays. For two of these, CSN-5 and KGB-1, loss of function results in complete sterility. CSN-5 encodes the subunit 5 of the COP-9 signalosome, a protein complex conserved from plants to man that has been implicated in many cellular functions, including protein degradation. KGB-1, a kinase that GLHs bind, is a predicted MAP serine/threonine kinase in the JNK sub-family. With the loss of either CSN-5 or KBG-1 there is a lack of mature oocytes, fertilized eggs and embryos, with sperm produced. Their phenotypes also differ. csn-5(RNAi) results in little or no oogenesis, severe reductions in mitotic germ cells and tiny gonads, a phenotype essentially identical to that of the combined loss of glh-1 and glh-4 (Kuznicki et al. Development 2000 127:2907-16). For the kgb-1(um3) deletion strain, a presumed null, the sterility is temperature sensitive; at 25-26C oocytes undergo endomitosis, an Emo phenotype, while mitotic germ cells and pachytene nuclei are not reduced and gonads are of normal size. Anti-CSN-5 antibodies reveal CSN-5 protein is found in all cells of the worm, in the cytoplasm and concentrated in the nucleus; thus it may be surprising that csn-5(RNAi) appears to affect only germline function. Like the loss of any of the glhs, when either csn-5 or kgb-1 is reduced or missing, P granules still assemble; however, P granules are missing (and therefore unable to carry out their unknown function) in the proximal gonad. The genetic relationships between the GLH proteins and these binding partners are being pursued with combinatorial RNAi and with the construction of double mutant strains.

McEwen, Tamara Jean et al. (2009) International Worm Meeting "Activating interactions between Dicer, microRNAs and GLH-1 in the C. elegans germline."

McEwen, Tamara Jean, Bennett, Karen L, Beshore, Erica, Shah, Shalin

[International Worm Meeting, 2009]

In Caenorhabditis elegans, maintenance of a productive germline is dependent on the germline RNA helicase, GLH-1, an integral component of the germline-specific P granules that surround the nuclei of germline progenitor cells, germline stem cells, and developing oocytes and sperm. Our laboratory has discovered that GLH-1 and the ribo-endonuclease Dicer-1 (DCR-1) are interdependent both by genetic and biochemical analyses. These two proteins bind each other and are colocalized to P granules at the pachytene stage of oogenesis when many maternal RNAs are processed and stored. When either GLH-1 or DCR-1 is missing from the germline, levels of the other are substantially decreased. MicroRNAs (miRNAs), non-coding RNAs found in plants and animals, are processed by Dicer and are involved in development, apoptosis, and disease progression. Most reports have found miRNAs repress the translation of their target genes; however, evidence indicates miRNAs can also activate their targets. Two miRNAs, miR-67 and miR-83, are predicted to bind overlapping sites in the small, 140-nt, 3''-UTR of glh-1 mRNA. This 3''-UTR also contains multiple sites, specifically nanos response element (NRE) and adenylation control element (ACE) motifs, predicted to bind regulatory proteins. Microarray analysis by the Bass laboratory (U Utah) indicated that glh-1 and many other germline-enriched mRNAs are down-regulated in C. elegans dcr-1 null mutants. We have verified, through quantitative real time PCR, that glh-1 mRNA levels are reduced an average of 2-fold when Dicer is missing. Therefore, we hypothesize that miR-83 and miR-67 may function to up-regulate the translation of glh-1; thus, GLH-1 levels would decrease when these miRNAs are absent. We obtained miR-67 and miR-83 deletion strains and generated the miR-67; miR-83 double mutant. Our preliminary results indicate that GLH-1 protein levels are decreased in miR-67 and miR-83 mutants and in the double, when compared to wildtype animals. In addition, these miR mutants exhibit abnormally-shaped gonads, with some animals displaying the rarely-reported phenotype of a bifurcated distal gonad similar to that occasionally seen with loss of glh-1 or glh-4. Based on our findings to date, we propose these two miRNAs, perhaps in conjunction with yet-to-be-defined regulatory proteins, bind the glh-1 3''UTR and activate glh-1 mRNA translation, resulting in increased GLH-1 protein levels. To determine whether this effect is direct or indirect, we intend to verify that these miRNAs bind glh-1 and hope to create transgenic worms with mutated glh-1 3''-UTRs. We propose the possibility that many mRNAs in the C. elegans germline may be regulated by similar, novel miRNA-mediated activation.

KA Kuznicki et al. (1999) International C. elegans Meeting "Unwinding the P granules: evidence from RNAi and glh mutants"

KL Bennett, JW Kirchner, PA Smith, KA Kuznicki

[International C. elegans Meeting, 1999]

The Bennett laboratory studies the four g erm l ine RNA h elicases (GLHs) , components of the C. elegans P granules. The GLHs are members of the DEAD-box family of RNA helicases and contain retroviral-like CCHC zinc fingers and uncharged glycine-rich repeats (Roussell and Bennett 1993 PNAS 90 :9300-9304, Gruidl et al . 1996 PNAS 93 :13837-13842). GLH-3 and GLH-4, more recently discovered helicases, differ from GLH-1 and GLH-2; GLH-3 lacks the uncharged glycine-rich repeats of the others, while GLH-4 is the most unique. RNAi implies that each GLH has a different role. RNAi of glh-1 or glh-2 produces sterility in a small percentage of the F 1 generation at the restrictive temperature. By contrast, glh-3 RNAi causes smaller brood sizes in the injected worm at the restrictive temperature. Using a combination of the helicase regions of glh-1 , glh-2 , and glh-3 results in a more dramatic temperature sensitive phenotype. Approximately 75% of the F 1 generation is infertile. Other combinatorial glh RNAi injections will further determine if the glhs function synergistically. A glh-3 - strain with a 1.7 kb deletion was identified in our laboratory from a TMP/UV library we generated. Preliminary analysis of the glh-3 - strain indicates that it has a mild temperature sensitive fertility defect, with brood size approximately 60-70% of that seen with wild type worms at 25-26degC. This result is consistent with that seen in the glh-3 RNAi experiments. Ongoing experiments include glh-4 RNAi and screening for additional glh mutants.

Chen, Wenjun et al. (2019) International Worm Meeting "Germline RNA helicases drive the phase separation and perinuclear anchoring of germ granules to promote piRNA-mediated genome surveillance."

Zhang, Donglei, Lee, Heng-Chi, Tu, Shikui, Weng, Zhiping, Chen, Wenjun, Munro, Ed, Lang, Charles, Bennett, Karen, Brown, Jordan

[International Worm Meeting, 2019]

piRNAs function as a guardian of the genome through the silencing of foreign or selfish nucleic acids. However, it remains unknown how piRNAs effectively identify foreign/selfish RNAs among many endogenous mRNAs. Intriguingly, many piRNA pathway factors are enriched in germ granules, the major sites of RNA export in germ cells, implying their localization may facilitate the detection and silencing of foreign nucleic acids. Here we showed that two germline helicases, GLH-1 and GLH-4, play a redundant role in germ granule formation. In glh-1;glh-4 double mutants, many germ factors, including PGL-1 and PRG-1, become completely dispersed into the cytoplasm. To investigate the roles of germline helicases in controlling germ granule formation, we created gene-edited worms that express various mutated GLH-1 helicases. We found that GLH-1 controls the formation and disassembly of germ granules through their binding and release of RNAs, respectively, which are coupled to distinct steps of its ATP hydrolysis cycle. Microscopic and proteomic analyses support a model that RNA binding by GLH-1 allows for the assembly of germ granule factors on RNAs, which promote the phase separation of germ granules. In addition, the FG repeats of GLH-1 is required for the anchoring of germ granules to perinuclear foci. Importantly, piRNA reporter analyses and small RNA cloning data suggest that normal germ granule dynamics and proper nuclear membrane association are both critical for the production of secondary small RNAs and gene silencing. Furthermore, we showed that these glh-1 mutants with abnormal germ granules also exhibit defects in transgenerational gene silencing. Together, our research suggests that the localization and dynamics of germ granules are both controlled by germline helicases and their RNA-bound complexes drive the phase separation of germ granules. In addition, our data support a model that germ granules function as a checkpoint, where small RNAs survey mRNA transcripts as they exit nuclei to identify RNA targets for gene silencing.

Kreutzer MA et al. (1995) International C. elegans Meeting "CHARACTERIZATION OF THE CAENORHABDITIS ELEGANS CYCLIN GENES"

Richards JP, Bennett KL, Kreutzer MA

[International C. elegans Meeting, 1995]

The Bennett laboratory has identified genes that may be essential for nematode germline determination. Drosophila cyclin B RNA has been shown to localize to the Drosophila polar granules, germ granules that many be equivalent to the nematode P granules. Therefore, cyclins may play a role in the development of the germline. We have cloned cDNAs for the C. elegans cyclins A1, B and B3 (Kreutzer, et al. in press). Cyclins A1 and B are most closely related to either A- or B- type cyclins of other species while cyclin B3 is a distant ancestral cyclin gene. While cyclin A1 is a member of a large A-type multigene family that corresponds to a single transcript on northern blots, cyclin B3 appears to be a single gene recognizing a single transcript. In contrast, cyclin B recognizes three transcripts with one cyclin B transcript specific for the paternal germline. Our results suggest that the largest and smallest cyclin B transcripts use different polyadenylation sites. By northern analyses, the two other cyclin B as well as the cyclin A1 and cyclin B3 transcripts are most abundant in the maternal germline. In addition, two types of 3'UTR translational regulatory elements, ACEs and NREs, are found in the 3' untranslated region of each of these genes. We are currently using probes to the cyclin genes for in situ hybridization to wild type and germline defective adult worms and embryos to determine the localization of these RNAs during germline proliferation and embryogenesis.

Te-Wen Lo et al. (2007) International Worm Meeting "C. elegans FGF Receptor Signaling Can Occur via Direct Binding of the SEM-5/Grb2 Adaptor Protein."

Michael Stern, Te-Wen Lo

[International Worm Meeting, 2007]

The components of receptor tyrosine kinase(RTK)signaling complexes help to define the specificity of the effects of their activation. The C. elegans fibroblast growth factor receptor (FGFR), EGL-15, regulates a number of processes, including sex myoblast (SM) migration guidance and fluid homeostasis, both of which require a Grb2/Sos/Ras cassette of signaling components. Here we show that SEM-5/Grb2 can bind directly to EGL-15 to mediate SM chemoattraction. A yeast two-hybrid screen identified SEM-5 as able to interact with the carboxy-terminal domain (CTD) of EGL-15, a domain that is specifically required for SM chemoattraction. This interaction requires the SEM-5 SH2 binding motifs present in the CTD (Y1009 and Y1087), and these sites are required for the CTD role of EGL-15 in SM chemoattraction. Although SEM-5 is required for the fluid homeostasis function of EGL-15, these sites are not required for that function, indicating that SEM-5 can link to EGL-15 through an alternative mechanism. The multisubstrate adaptor protein FRS2 serves to link vertebrate FGFRs to GRB2. In C. elegans, an FRS2-like gene, rog-1, functions upstream of a Ras/MAPK pathway for oocyte maturation1 but is not required for EGL-15 function2 . Thus, unlike the vertebrate FGFRs, which require the multisubstrate adaptor FRS2 to recruit Grb2, EGL-15 can recruit SEM-5/Grb2 directly. 1Matusbara et al., 2007. Genes to Cells; 12(3): 407-420. 2see D. Bennett abstract.

Chen LS et al. (1997) International C. elegans Meeting "MOLECULAR AND GENETIC ANALYSIS OF C. ELEGANS NEF-1, A MEMBER OF THE IG/FNIII SUPERFAMILY OF CELL ADHESION MOLECULES"

Bennett V, Chen LS

[International C. elegans Meeting, 1997]

A cell's identity, its interactions with other cells, and its ablity to adapt to its environment are heavily dependent on the proteins associated with its plasma membrane. Ankyrin, an intracellular protein that couples several integral membrane proteins to the spectrin cytoskeleton, is believed to be instrumental in organizing many membrane proteins into specialized domains. Recently, a family of cell adhesion molecules belonging to the Ig/FnIII superfamily was shown to bind ankyrin; these proteins include neurofascin, L1, NrCam and NgCam in vertebrates and neuroglian in Drosophila (1-6). This family of ankyrin binding CAMs is believed to be involved in neurite outgrowth, axonal fasciculation and targeting, cell migration and synaptogenesis during embryonic and postnatal vertebrate development (1-4). A member of the family has been identified in C. elegans. The gene encoding the homologue has been designated nef-1. In addition to the Ig and FnIII domains, it contains a highly conserved ankyrin binding domain. We are interested in elucidating the role of nef-1 in C. elegans development and its interactions with the C. elegans ankyrin homologue, UNC-44. Interestingly, mutations in unc-44 causes defects in axon guidance (7). 1. Sonderegger and Rathgen, 1992. J.Cell Biol. 119:1387-1394. 2. Rathgen and Jessel, 1991. Sem. of Neurosci. 3:297-307. 3. Grumet, 1991. Curr. Opin. Neurobiol. 1:370-379. 4. Hortsch and Goodman, 1991. Ann. Rev. Cell Biol. 7:505-557. 5. Davis et al., 1993. J. Biol. Chem.232:121-133 6. Davis and Bennett, 1994. J. Biol. Chem.267:18955-18972. 7. Otsuka et al., 1995. J.Cell Biol. 129: 1089-1092.

Karla Opperman et al. (2007) International Worm Meeting "A structure-function analysis of the C. elegans L1CAMs."

Lihsia Chen, Xuelin Wang, Karla Opperman, Shan Zhou

[International Worm Meeting, 2007]

Cell adhesion molecules play important roles during development and in maintaining tissue integrity. L1CAMs are a family of immunoglobulin (Ig)-like cell adhesion molecules that are important in vertebrate nervous system development. L1CAMs are conserved in C. elegans, and are encoded by the lad-1/sax-7 and lad-2 genes. SAX-7 and LAD-2 have distinct functions in the nervous system despite overlapping neuronal expression. SAX-7 is required to maintain neuronal positioning while LAD-2 participates in axon pathfinding. We are performing a structure-function analysis to determine how both proteins mediate their functions. Because the cytoplasmic tails between both proteins are distinct, we are focusing our analysis on how the cytoplasmic tail regulates L1CAM functions. Indeed, while the extracellular domains of SAX-7 and LAD-2 are conserved, the LAD-2 cytoplasmic tail is completely divergent and does not contain any of motifs conserved in SAX-7 and vertebrate L1CAMs. These motifs include the ankyrin-binding motif, which links L1CAMs to the spectrin-actin cytoskeleton, the PDZ-binding motif, and conserved tyrosine residues that are phosphorylated. Our preliminary results reveal that both conserved motifs, as well as phosphorylation one of the tyrosine residues, which regulates ankyrin binding, all contribute to the function of SAX-7 in neuronal position maintenance. (1) Chen L, Ong B, Bennett V. J Cell Biol. 2001 Aug 20;154(4):841-55. (2) Wang X, Kweon J, Larson S, Chen L. Dev Biol. 2005 Aug 15;284(2):273-91. (3) Sasakura H, Inada H, Kuhara A, Fusaoka E, Takemoto D, Takeuchi K, Mori I. EMBO J. 2005 Apr 6;24(7):1477-88. Epub 2005 Mar 17.

Smith PA et al. (1996) Midwest Worm Meeting "C. elegans germline RNA helicases: are they all components of the P granules?"

McCrone JS, Smith PA, Bennett KL, Kuznicki KA, Kirchner JW, Strome S, Gruidl ME

[Midwest Worm Meeting, 1996]

The glh genes are a family of putative germline RNA helicases in C. elegans. glh-1 has been characterized as a germline-specific transcript coding for a predicted protein with eight conserved RNA helicase motifs, glycine-rich imperfect repeats in the N terminus, and four retroviral-like CCHC zinc fingers (Roussell and Bennett, 1993, PNAS USA 90:9300). Antibodies against two regions of GLH-1 fusion proteins recognize the germline-specific P granules of C. elegans throughout all stages of worm development. GLH-1 protein is uniformly distributed in the cytoplasm of the oocyte and segregates with the germline precursor cell from the first embryonic cell division. By Western analysis a 78kD worm protein, the predicted size for GLH-1, is seen. The closely-related glh-2 gene is also germline-specific, maps near glh-1 and codes for a less abundant, larger mRNA. The in situ hybridization patterns of glh-1 and glh-2 differ. The presence of glh-1 RNA is associated with regions of the hermaphrodite gonad where germ cell proliferation, oogenesis, and spermatogenesis occur. glh-2 mRNA is largely confined to the region where oogenesis occurs; however, glh-2 RNA is not associated with spermatogenesis. glh-2 mRNA is also detected in young C. elegans embryos. The predicted GLH-2 protein differs from GLH-1 in having six zinc fingers and different glycine repeats. Preliminary studies with GLH-2 antibodies indicate GLH-2 is present in oocytes and in P granules. The Genome Sequencing Center has uncovered an additional region on LG1 that closely matches glh-1 and glh-2. This potential GLH-3 contains two somewhat divergent CCHC fingers, but lacks the glycine repeats. Questions remain. Are all GLH proteins localized to the P granules? Do their roles differ in the germline?