Aoki, Scott Takeo et al. (2015) International Worm Meeting "The P-granule assembly protein, PGL-1, is a base-specific RNA nuclease."

Kimble, Judith, Bingman, Craig, Aoki, Scott Takeo, Wickens, Marvin, Kershner, Aaron M.

[International Worm Meeting, 2015]

P-granules are RNA-protein assemblies required for germ cell development. Loss of P-granules leads to adult germline deterioration (reviewed in 1-2) and differentiation of germ cells into somatic cell types (3). Granule formation requires PGL-1 and PGL-3 (PGLs), protein paralogs capable of granule self-assembly (4-5). We set out to structurally characterize the PGLs to better understand their molecular function in P-granules. Our biochemical analyses of recombinant PGL-1 identified a dimerization domain in the central region of the protein, referred to as "PGL-1 DD." We then determined the 1.6 A and 3.6 A crystal structures of C. remanei and C. elegans PGL-1 DD, respectively. PGL-1 DD has a novel, alpha helical fold. The proposed dimer forms a positively charged channel with the appropriate dimensions to fit single stranded RNA. Unexpectedly, incubation of PGL-1 DD with oligonucleotides resulted in RNA cleavage. Additional in vitro results demonstrate PGL-1 DD to be a guanosine-specific, single-stranded RNase. The crystal structure allowed us to identify a site in the dimer channel that brought together conserved glutamine and lysine side chains. We postulated these side chains might contribute to PGL's RNase activity. Mutation of this glutamine to alanine abrogated RNA cleavage without affecting either dimerization or RNA binding. We have now created the glutamine to alanine RNase mutants in the endogenous pgl-1 and pgl-3 genes using CRISPR-Cas methods, and we are assessing the role of PGL's enzymatic function in germ cell development. Our results support the notion that PGL-1 may have a dual function in P-granules as an assembly protein and as an RNase. Our results also raise the question of whether other germ granule scaffold proteins, like Drosophila Oskar and zebrafish Bucky Ball, may also have enzymatic activities affecting RNA regulation.1. Updike, D., Strome, S. J Androl 2010; 31(1):53-60.2. Voronina E, Seydoux G, Sassone-Corsi P, Nagamori I. Cold Spring Harb Perspect Biol. 2011 Dec 1;3(12):a002774.3. Updike DL, Knutson AK, Egelhofer TA, Campbell AC, Strome S. Curr Biol. 2014 May 5;24(9):970-5.4. Updike DL, Hachey SJ, Kreher J, Strome S. J Cell Biol. 2011 Mar 21;192(6):939-48.5. Hanazawa M, Yonetani M, Sugimoto A. J Cell Biol. 2011 Mar 21;192(6):929-37.

Carrick, Brian et al. (2017) International Worm Meeting "Genomic analyses of interactions that govern an RNA regulatory network in stem cells."

Guthrie, Timothy, Wickens, Marvin, Carrick, Brian, Kimble, Judith, Kroll-Conner, Peggy, Prasad, Aman, Porter, Douglas

[International Worm Meeting, 2017]

mRNA regulation governs when, where, and how much protein an mRNA produces and thereby determines cell identity, function and behavior. A single mRNA regulatory protein can bind to and control hundreds to thousands of mRNAs, forming a "network" that enables control of a biological outcome. This work focuses on C. elegans FBF-1 and FBF-2 (collectively termed FBF), which maintain germline stem cells (GSCs). FBF is a member of the PUF (Pumilio and FBF) family of RNA-binding proteins (RBPs), which bind mRNAs via sequence-specific elements in the 3' untranslated region (3'UTR) and regulate expression. We have now identified the RNAs bound by FBF in vivo in both oogenic and spermatogenic germlines using the technique of iCLIP (individual-nucleotide resolution CrossLinking and ImmunoPreciptation). The goal of this work is to learn FBF targets that function independently of gender, as well as gamete-specific targets and to thereby gain insight into FBF sub-networks. Like other PUF proteins, FBF acts with protein partners which can modulate binding specificity in vitro. The challenge now is to understand how these partners affect FBF regulatory networks in vivo. Currently, we are delineating protein - protein interfaces of FBF with its partners and generating CRISPR-induced mutants that abrogate those partnerships. Ultimately we will analyze how the FBF subnetworks fluctuate in response to changing FBF partnerships.

Motzny CK et al. (1997) International C. elegans Meeting "IDENTIFYING TRANSLATIONAL REGULATORS OF THE SEX DETERMINING GENE, TRA-2"

Motzny CK, Goodwin EB

[International C. elegans Meeting, 1997]

The sex determination gene, tra-2, is required for female development. Over-production of Tra-2 leads to feminization of both XX and XO animals. Previous studies have identified two Direct Repeat Elements (DREs) in the 3' untranslated region (3' UTR) of tra-2 that regulate translation of the RNA. A factor called DRF binds to the DREs and is proposed to suppress translation of tra-2. Here we report on progress to molecularly identify DRF. We have utilized the yeast three-hybrid system designed and provided by Beilin Zhang and Marvin Wickens to clone DRF. In brief, a vector that transcibes a hybrid RNA containing the viral coat protein MS2 RNA binding site and the DREs was introduced into his- yeast carrying the reporter genes his+ and lacZ downstream of LexA binding sites. The strain also expresses a DNA binding protein composed of a fusion of the LexA DNA binding domain and the MS2 coat protein that anchors the hybrid RNA through the MS2 coat protein and MS2 RNA binding site to the reporter gene promoter. Detectable expression of the reporter genes occurs when a fusion protein composed of the Gal4 activation domain and C. elegans cDNA binds to the DREs bringing the DNA binding domain in proximity to the DNA activation domain. We have identified several candidates for DRF and will present the molecular characterization of these clones. We have also devised a genetic screen to identify translational regulators of tra-2. A GFP transgene expressed in the gut and controlled by the tra-2 3' UTR is utilized in a mutagenesis screen to identify F2 embryos and L1s in which translation of the transgene is disrupted. We will report progress on the screen.

Kyung Won Kim et al. (2005) International Worm Meeting "RNP-8 interacts with the GLD-2 cytoplasmic poly(A) polymerase and regulates its polyadenylation activity"

Kyung Won Kim, Judith Kimble, Liaoteng Wang

[International Worm Meeting, 2005]

Polyadenylation is critical for mRNA stability and translational activation. During oocyte maturation and early embryonic development, cytoplasmic polyadenylation of preexisting mRNAs promotes their translation. The C. elegans gld-2 gene is required for multiple steps in germline development, including the mitosis/meiosis decision. GLD-2 is a cytoplasmic poly(A) polymerase (PAP) that lacks an RNA recognition motif (1); similar PAPs have been identified in virtually all eukaryotic organisms (2). A yeast two-hybrid screen using GLD-2 as bait isolated GLD-3, RNP-8 and LARP-1 (1; L. Wang and J. Kimble, unpublished). GLD-2 has little PAP activity on its own, but it is stimulated in vitro by GLD-3 (1). We have now focused on RNP-8 to ask whether this GLD-2 interactor also stimulates GLD-2 activity and to determine whether RNP-8 has a major role in germline development. Preliminary data reveal that rnp-8(RNAi) and gld-2 mutants have similar defects during oogenesis. Both gld-2 and rnp-8 mRNAs are abundant in embryos, L4 larvae, and adults, and rnp-8 mRNA is enriched in the germ line. To test whether RNP-8 stimulates the GLD-2 PAP activity, we performed assays for PAP activity in vitro. Preliminary data suggest that RNP-8 can indeed stimulate GLD-2 PAP activity. Taken together, we suggest that RNP-8 stimulates GLD-2 activity to control germline development.1. Wang, L., Eckmann, C.R., Kadyk, L.C., Wickens, M., and Kimble J. (2002), A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans. Nature 419: 312-316 2. Kwak, J.E., Wang, L., Ballantyne, S., Kimble J. and Wickens, M. (2004), Mammalian GLD-2 homologs are poly(A) polymerases. PNAS 101: 4407-4412

Kyung Won Kim et al. (2007) International Worm Meeting "RNP-8, a newly identified co-activator of GLD-2 cytoplasmic poly(A) polymerase, and its control of germline development."

Kyung Won Kim, Liaoteng Wang, Judith Kimble

[International Worm Meeting, 2007]

Polyadenylation is critical for mRNA stability and translation. In general, poly(A) tail elongation can stabilize and translationally activate mRNAs, while poly(A) tail removal can trigger mRNA degradation and repress translation. One key regulator of polyadenylation is GLD-2, a widely-conserved cytoplasmic poly(A) polymerase (PAP) (1, 2). GLD-2 proteins are catalytic subunits but have no recognizable RNA-binding domain (1). Our work focuses on GLD-2 in the nematode C. elegans and its control of germline development. Our current model is that a nematode GLD-2 is recruited to specific mRNAs by specific RNA-binding partners. GLD-2 has low PAP activity on its own, but its activity is stimulated by interactions with GLD-3/Bicaudal-C, a conserved RNA-binding protein (1). To identify other GLD-2 partners, we conducted a yeast two-hybrid screen and identified RNP-8, a novel RRM protein that is predicted to bind RNA. Consistent with our model, we find that RNP-8 stimulates GLD-2 PAP activity in vitro. Both rnp-8 mRNA and RNP-8 protein are germline-enriched and abundant in the cytoplasm of germ cells in the pachytene stage of meiotic prophase and in maturing oocytes. An rnp-8 deletion mutant exhibits low percentage of the Mog (for masculinization of germline) phenotype, suggesting that RNP-8 promotes oogenesis. Importantly, its minor phenotypic defects are enhanced by removal of other RNA regulators controlling germline development. 1. Wang, L., Eckmann, C.R., Kadyk, L.C., Wickens, M. and Kimble, J. (2002) A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans. Nature 419: 312-316. 2. Kwak, J.E., Wang, L., Ballantyne, S., Kimble, J., and Wickens, M. (2004) Mammalian GLD-2 homologs are poly(A) polymerases. Proc Natl Acad Sci USA 101, 4407-4412.

Beth E Thompson et al. (2003) International Worm Meeting "Genetic control of early germline proliferation in C. elegans"

Beth E Thompson, Judith Kimble, Andrei G Petcherski

[International Worm Meeting, 2003]

During larval development, germ cells divide from the two present at hatching to about a thousand in adults by a variable pattern of divisions to generate. These germline divisions are controlled by a combination of Notch signaling and RNA regulation. The fbf genes, fbf-1 and fbf-2, encode members of the Puf family of RNA binding proteins (Wickens et al 2002). fbf-1 fbf-2 double mutants proliferate normally during early larval stages, but all germ cells enter into meiosis during L4 and differentiate as sperm (Crittenden et al 2002). We have found that two other genes, fog-1 and fog-3, can be critical for germline divisions during L1 and L2. FOG-1 is a CPEB homologue required for the sperm fate (Barton and Kimble 1991; Jin et al, 2001), and FOG-3 is a Tob family protein also required the sperm fate (Ellis and Kimble, 1995; Chen et al 2000). The germline proliferates normally in both fog-1 and fog-3 single mutants, but they make no sperm and instead make oocytes continuously. However, in an fbf-1 fbf-2 double mutant background, a reduction of either fog-1 or fog-3 by RNAi results in a germline with a reduced number of germ cells compared to the fbf-1 fbf-2 double mutant. Most commonly there appear to be only 4-16 cells; these cells appear to be oocytes; no cell death has been observed. We are currently building the fog-1; fbf-1 fbf-2 triple mutant to characterize these defects in more depth. Progress will be reported at the meeting. Wickens et al (2002) Trends in Genetics 18(3), 150-157; Crittenden et al (2002) Nature 417, 660-663; Barton and Kimble (1991) Genetics 125, 29-39; Jin et al (2001) Developmental Biology 229, 537-553; Ellis and Kimble (1995) Genetics 139, 561-577; Chen et al (2000) Developmental Biology 217, 77-90

Morgan, Dyan et al. (2011) International Worm Meeting "FOG-1 activates fog-3 expression."

Kimble, Judith, Morgan, Dyan, Jeong, Johan, Noble, Daniel

[International Worm Meeting, 2011]

FOG-1 promotes the sperm fate at the expense of oogenesis in both sexes: XO fog-1 mutants are somatically male but make only oocytes; similarly XX fog-1 mutants are somatically hermaphrodite but make only oocytes (1). FOG-1 is a divergent Cytoplasmic Polyadenylation Element Binding (CPEB) protein (2, 3). Xenopus CPEB binds CPEB binding elements (CPEs) in the 3'untranslated region (3'UTR) of target mRNAs to regulate translation via polyadenylation or deadenylation depending on associated cofactors. Genetically, FOG-1 acts at the end of the germline sex determination pathway as does another key regulator of sperm fate specification, FOG-3 (4). Our work provides three lines of evidence suggesting that FOG-1 controls the fog-3 mRNA directly. First, purified recombinant FOG-1 binds the fog-3 3'UTR in a CPE-dependent manner in vitro. Second, FOG-1 antibodies immunoprecipitate fog-3 mRNA from L3 hermaphrodite extracts, whereas control IPs do not. Third, the abundance of an epitope-tagged and rescuing FOG-3 protein drops precipitously in fog-1 mutants, while fog-3 mRNA is not reduced. We suggest that FOG-1 promotes sperm fate specification by translationally activating fog-3 expression. Experiments are in progress to test this idea with transgenes carrying either a wild-type fog-3 3'UTR or a mutant fog-3 3'UTR that lacks CPEs. 1. Barton MK & Kimble J (1990) fog-1, a regulatory gene required for specification of spermatogenesis in the germ line of Caenorhabditis elegans. Genetics 125:29-39. 2. Jin S-W, Kimble J, & Ellis RE (2001) Regulation of cell fate in Caenorhabditis elegans by a novel cytoplasmic polyadenylation element binding protein. Dev. Biol. 229(2):537-553. 3. Luitjens C, Gallegos M, Kraemer B, Kimble J, & Wickens M (2000) CPEB proteins control two key steps in spermatogenesis in C. elegans. Genes Dev. 14(20):2596-2609. 4. Ellis RE & Kimble J (1995) The fog-3 gene and regulation of cell fate in the germ line of Caenorhabditis elegans. Genetics 139:561-577.

Li, Y. et al. (2017) International Worm Meeting "Investigating the role of poly(U) polymerases in germline development."

Li, Y., Maine, E., Snyder, M.

[International Worm Meeting, 2017]

Poly(U) polymerases (PUPs) add uridine to the 3'end of RNA, a common modification that correlates with reduced RNA stability in many organisms. Three PUP proteins have been identified in Caenorhabditis elegans (Kwak & Wickens, 2007). PUP-1/CDE-1 (co-suppression defective) is known to target a subset of siRNAs and is expressed in the germline (van Wolfswinkel et al., 2009). PUP-2 activity is known to regulate a subset of miRNAs that act in the soma (Lehrbach et al., 2009; Ha & Kim, 2014). Our genetic studies reveal a redundant role for PUP-1 and PUP-2 in germline survival and chromatin regulation. Under temperature stress, pup-1/-2(0) hermaphrodites produce progressively fewer progeny over successive generations and become sterile by the third generation. The majority of those sterile adults contain no germ cells due to germ cell death during mid-larval development. Chromatin regulation, particularly the H3K9me2 modification, is abnormal in meiosis in survivors (generations 1-2) if unpaired chromosomes are present. In addition, pup-1/-2(0) sperm are fertilization defective as endomitotic oocytes develop in hermaphrodites and males fail the mating assay. Our molecular studies indicate that PUP-1 and PUP-2 localize to different cellular compartments. PUP-1 localizes to P granules throughout the mitotic and meiotic germline. PUP-2 is detected as cytoplasmic foci in meiotic germ cells beginning at L4 stage. Thus, both proteins are expressed during the stage when H3K9me2 misregulation is first detected and, in later generations, when the germline dies. The expression patterns are consistent with PUP-1 and PUP-2 acting at different points in the RNA turnover process. To better understand the role of uridylation in germline development, we abolished the activities of all three PUPs. Preliminary results suggest the loss of PUP-3 function can restore germline survival in the absence of PUP-1 and PUP-2 function. We hypothesize PUP activities modulate the balance of RNA levels. Absence of any single PUP increases the abundance of its targets, which perturbs the stoichiometric relationships of gene products. However, loss of all three PUPs elevates the gross RNA level, which restores the balance of gene products such that germline viability is restored. Taken together, our findings reveal critical functions for PUPs in germline development.

Julia Kaye et al. (2007) International Worm Meeting "TRANSLATIONAL REGULATION MEDIATES THE ADAPTATION RESPONSE WITH IN THE AWC SENSORY NEURONS."

Noelle L'Etoile, Natalie Rose, Julia Kaye, Brett Goldsworthy

[International Worm Meeting, 2007]

An animals behavior results from the interaction between its genes and its experiences. Within the adult functioning nervous system, neuronal excitation is thought to regulate both transcription and translation in order to modify the organisms response to its environment as a function of experience. Translational changes, in particular, have been shown to contribute to many of the forms of synaptic plasticity thought to underlie memory formation1. In fact, olfactory conditioning in flies has been shown to depend on new protein synthesis and the 3UTR binding translational repressor, Pumilio2. In C. elegans, neuronal plasticity is critical for regulation of the chemotaxis response to attractive odors. This attraction can be modulated by experience such that prior exposure to one odor down regulates the worm''s response to that odor. We call this plasticity olfactory adaptation and is dependent on the cell autonomous function of EGL-43. In C. elegans, olfactory adaptation is both odor-specific and enduring. We present evidence that a new type of Pumilio binding Nanos response element (NR/FBE) within the egl-4 3UTR determines how C. elegans will respond to specific odors in its environment. The Pumilio/FBF protein (PUF), FBF-1, has been previously characterized to regulate protein expression during developmental processes4,6, however, here we show that FBF-1 is required in the adult animal only at the time of odor exposure to adapt the worms response to a single odor in its environment. In addition, we show that FBF-1 binds to the egl-4 NR/FBE. We also employ the fluorescence-based reporter, kaede5, to examine how both FBF-1 and the 3UTR of egl-4 affects translation. PUF proteins and the NREs they bind are uniformly described as negative regulators of translation6; conversely, we demonstrate by use of this kaede reporter that the egl-4 NR/FBE actually increases message translation in the olfactory sensory neuron of the intact adult worm. Thus, classical translational repressors may behave as activators in the context of the adult functioning neuron and their activity can determine specific aspects of the animals behavior. (1) Martin, K. C. (2004). Curr Opin Neurobiol 14, 305-10. (2) Dubnau et al., (2003). Curr Biol 13, 286-96. (3) L''Etoile et al., (2002). Neuron 36,1079-89.(4) Crittenden et al., (2002). Nature 417, 660-3. (5) Ando et al., (2002). Proc Natl Acad Sci U S A 99, 12651-6. (6) Wickens et al., (2002). Trends Genet 18, 150-7.