mirnas Collaborate with a Conserved RNA Binding Protein to Ensure Development and Stress Response in C. elegans

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1 University of Colorado, Boulder CU Scholar Molecular, Cellular, and Developmental Biology Graduate Theses & Dissertations Molecular, Cellular, and Developmental Biology Spring mirnas Collaborate with a Conserved RNA Binding Protein to Ensure Development and Stress Response in C. elegans Rebecca A. Zabinsky University of Colorado Boulder, rzabinsky@gmail.com Follow this and additional works at: Part of the Developmental Biology Commons, Genetics Commons, and the Molecular Biology Commons Recommended Citation Zabinsky, Rebecca A., "mirnas Collaborate with a Conserved RNA Binding Protein to Ensure Development and Stress Response in C. elegans" (2015). Molecular, Cellular, and Developmental Biology Graduate Theses & Dissertations This Dissertation is brought to you for free and open access by Molecular, Cellular, and Developmental Biology at CU Scholar. It has been accepted for inclusion in Molecular, Cellular, and Developmental Biology Graduate Theses & Dissertations by an authorized administrator of CU Scholar. For more information, please contact cuscholaradmin@colorado.edu.

2 mirnas collaborate with a conserved RNA binding protein to ensure development and stress response in C. elegans by Rebecca A. Zabinsky B.A., Scripps College, 2009 A thesis submitted to the Faculty of the Graduate School of the University of Colorado Boulder in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular, Cellular, and Developmental Biology 2015

3 ii This thesis entitled: mirnas collaborate with a conserved RNA binding protein to ensure development and stress response in C. elegans written by Rebecca Ann Zabinsky has been approved for the Department of Molecular, Cellular, and Developmental Biology Min Han, Ph.D., Advisor Tin Tin Su, Ph.D., Committee Chair Date: The final copy of this thesis has been examined by the signatories, and we Find that both the content and the form meet acceptable presentation standards Of scholar work in the above mentioned discipline.

4 Abstract iii Zabinsky, Rebecca Ann (Ph.D., Molecular, Cellular, and Developmental Biology) mirnas collaborate with a conserved RNA binding protein to ensure development and stress response in C. elegans Thesis directed by Professor Min Han mirnas play critical roles in development and other cellular processes in C. elegans even though most individual mirnas are not essential for development or viability (Miska et al., 2007). Extensive studies in the field have suggested that most mirna functions are executed through complex mirna-target interaction networks. Furthermore, such networks may also function semi-redundantly with other regulatory systems to shape gene expression dynamics for proper physiological functions. Hypothesizing that mirnas function in stress conditions, I collaborated with a postdoctoral fellow to investigate the role of a specific mirna mir-71 in starvation induced L1 diapause. Hypothesizing that mirnas collaborate with other gene regulation mechanisms to maintain C. elegans developmental robustness, I, along with another postdoctoral fellow, carried out a genome-wide RNAi screen for genes that interact with the mirna induced silencing complex (mirisc). We found that when the mirisc is compromised by knocking out one of the two partially redundant GW182 proteins, many genes become essential for development (Weaver et al., 2014). Further analysis of the CED-3 caspase and its interaction with the let-7 family of mirnas revealed a widely used but previously unknown role in protein degradation, which contrasts the previously known activity of CED-3 in apoptosis. I further focused on a conserved RNA binding protein Vigilin and its interaction with mir-52, which revealed previously unknown roles of Vigilin and mir-52 in larval development. We hypothesize that Vigilin may function with mir-52 and other specific mirnas to repress a large

5 iv number of target mrnas to regulate development. Our results may shed light on the mechanism of how mirnas work with RNA binding proteins to regulate gene expression for robust development.

6 Acknowledgments v I thank my advisor Min Han for all the opportunities he has provided for me as well as continuous guidance and support. I thank all members of the Han lab for feedback, discussion, advice, and camaraderie. I thank Xiaochang Zhang, Cindy Teng, and Mingxue Cui for collaboration on the L1-induced starvation work. I thank Ben Weaver and Yi Weaver for collaboration on the genome wide RNAi screen and for endless advice, answers, and guidance. I thank Brett Weum for collaboration on the Vigilin project and daily feedback and discussion. I thank the Xue, Blumenthal, Yi, Pace, and Espinosa laboratories for use of reagents and/or equipment. I thank outside collaborators H. Robert Horvitz, Victor Ambros, Eric Moss, Keith Blackwell, Andrew Fire, Shohei Mitani, NBRP, KO Consortium, CGC for C. elegans strains and reagents. This work was supported by NIH Signaling and Cellular Regulation Training Grant 2T32GM (Rebecca Zabinsky), HHMI (Min Han), ACS (Benjamin Weaver), as well as BURST and UROP grants (Brett Weum).

7 Table of Contents Chapter I: Introduction and Background... 1 Discovery of mirnas... 1 Discovery of GW182 proteins... 2 Discovery of Vigilin... 5 Chapter II: mirnas play critical roles in the survival and recovery of C. elegans from starvation-induced L1 diapause... 8 Introduction... 8 Materials and Methods... 9 C. elegans methods... 9 L1 Starvation Survival Assay... 9 Dual color reporter assay and other developmental assays... 9 Results ain-1 mutants display reduced survival rate during L1 diapause mir-71 mutants display reduced survival rate during L1 diapause mir-71 functions to repress age-1 and unc-31 expression mir-71 is not required for L1 arrest but functions in starvation recovery Discussion Chapter III: C. elegans GW182 protein AIN-1 interacts with polya binding protein PAB Introduction Materials and Methods In vitro protein binding assay Immunoprecipitation from worm lysate Results AIN-1 and PAB-1 directly interact in vitro AIN-1 and PAB-1 immunoprecipitate from C. elegans lysate The SLWI motif is not totally required for the AIN-1 interaction with PAB-1 in vivo and possibly also not totally essential for function Discussion Chapter IV: Uncovering developmental functions of mirnas and other regulators by a genetic enhancer screen Introduction Materials and Methods Genome wide RNAi Screen Results Screen hits Example 1: ceh Example 2: ced Example 3: vgln Discussion Chapter V: Several mirnas collaborate with vgln-1 to promote development Introduction Materials and Methods Cloning and CRISPR-Cas Genome integration by bombardment Gas Chromatography vi

8 Crosslinking and Immunoprecipitation (CLIP) Results Vigilin knockout mutant exhibits a weak L1 arrest phenotype that is dramatically enhanced by compromising mirisc Characterization of phenotypes associated with loss of vgln-1 and ain Vigilin binds to RNA and is broadly expressed throughout C. elegans development Identification of specific mirnas that act with VIGN-1 to regulate postembryonic development Predicted mirna target skn-1 is upregulated in vgln-1(lf) Discussion Chapter VI: Concluding Comments and Future Directions References Appendicies Appendix 1: C. elegans strains Appendix 2: Plasmids vii

9 List of Figures viii Figure 1. Annotated conserved domains in GW182 protein homologs Figure 2. ain-1 mutants display a reduced survival rate Figure 3. Dual color reporters reveal age-1 and hbl-1 are regulated by mir Figure 4. mir-71 is not required for entering L1 arrest but is required for resuming development after arrest Figure 5. Model of mir-71 function in IIS and parallel pathways Figure 6. AIN-1 interacts with PAB Figure 7. The SLWI motif is not required for AIN-1 function or interaction with PAB-1 in worm lysate Figure 8. Screen design and results Figure 9. Loss of both ain-1 and ceh-18 results in fewer oocytes Figure 10. CED-3 cooperation with mirnas reveals a non-apoptotic function Figure 11. VGLN-1 is essential for development when the mirisc is compromised Figure 12. Characterization of vgln-1(lf) phenotypes Figure 13. vgln-1(lf) is sensitive to heat at the L1 stage but resistant at the adult stage Figure 14. VGLN-1 binds to RNA is broadly expressed throughout development Figure 15. Specific mirnas cooperate with VGLN-1 to regulate development Figure 16. Model of VGLN-1 function

10 1 Chapter I: Introduction and Background Discovery of mirnas MicroRNAs (mirnas) are small pieces of RNA that make up one of many different mechanisms of gene expression regulation by degrading or preventing translation of target messenger RNAs (mrnas). microrna (mirna)-mediated gene silencing is a gene expression regulatory mechanism crucial for all aspects of cellular processes including animal development, metabolism, stress responses and neuronal behaviors (Ambros, 2004). Misregulation of mirnas has also been linked to numerous human diseases such as cancer (Orellana and Kasinski, 2015; Shenouda and Alahari, 2009). Mutations in the gene for the first mirna discovered, lin-4, was identified in a screen for C. elegans developmental timing defects (Horvitz and Sulston, 1980). lin-4 was later cloned and identified as a tiny 20 nucleotide RNA (Lee et al., 1993). Genetically, the lin-14 mutant phenotype is epistatic to that of lin-4. Specifically, a loss-of-function mutation (lf) in lin-4 results in retarded larval development, while lin-14(lf) results in precocious larval development. The lin-4(lf);lin-14(lf) double mutants display the same phenotype as lin-14(lf). Furthermore, a gain-of-function (gf) allele of lin-14, due to a deletion in the lin-4 targeting site in the lin-14 3 UTR, has the same phenotype as lin-4(lf) (Wightman et al., 1991). These genetic data are consistent with the model that lin-4 represses lin-14 to control developmental timing. The realization of complementarity between the mirna lin-4 and the 3 UTR of the target lin-14 led to the first description of a mirna regulating expression of a target mrna (Lee et al., 1993; Wightman et al., 1993). Although it was suggested that there could be more small RNAs with similar regulatory functions (Wickens and Takayama, 1994), the next mirna found, let-7, was not identified

11 2 until 2000 (Reinhart et al., 2000). Although lin-4 was not recognized as a conserved RNA, let-7 and its target lin-41 were immediately recognized as conserved molecules in Drosophila, humans, and many other animals (Pasquinelli et al., 2000; Slack et al., 2000). This finding sparked a search for more mirnas resulting in the current rich mirna field of research. Although lin-4 and let-7 are two of the most well understood mirnas (Vella and Slack, 2005), they are unusual in that they produce a robust developmental phenotype when the gene activity is disrupted. The vast majority of mirnas and mirna families are not essential for development or viability (Alvarez-Saavedra and Horvitz, 2010; Miska et al., 2007). Due to redundancy and complex interactions between multiple mirnas and multiple targets, classical genetic methods are not effective to elucidate hidden roles of mirnas. Our lab and others have hypothesized that mirnas function in stress resistance when organisms encounter non-ideal growth conditions, and function redundantly with other gene regulatory mechanisms to ensure developmental robustness (Kudlow et al., 2012; Leung and Sharp, 2010, 2007; Vidigal and Ventura, 2014; Weaver et al., 2014; Zhang et al., 2011b). To address these hypotheses, we tested whether mirnas are required for starvation survival (Chapter 2) and whether nonessential genes become essential for development when overall mirna function is compromised (Chapter 4). Discovery of GW182 proteins GW182 was first discovered using autoimmune serum to stain puncta in mammalian cells (Eystathioy et al., 2002; Fritzler and Chan, 2013). The protein contained glycine tryptophan GW repeats and was 182kDa, leading to the name GW182.

12 3 The essential role of GW182 family proteins in mirna-induced silencing complex (mirisc) in any organisms was first identified by former graduate student Lei Ding in the Han lab (Ding et al., 2005). The Han lab became interested in studying mirnas when Lei Ding isolated mutations in a C. elegans GW182 homolog in a genetic screen and his subsequent genetic and biochemical analysis determined its role in mirna regulation of gene expression during development. In particular, after positional cloning of this gene, he showed that the protein encoded by the gene directly interacts with Argonaute ALG-1 and a large number of mirnas (Ding et al., 2005). He named the gene ain-1 (Argonaute- INteracting protein-1). Later, Lei and another graduate student in lab, Liang Zhang, demonstrated that C. elegans GW182 proteins, specifically the partially redundant homologs AIN-1 and AIN-2, are essential for mirna-mediated gene silencing but not mirna biogenesis (Zhang et al., 2007a). These two proteins are both expressed ubiquitously and can suppress the phenotype of the other suggesting that they function partially redundantly (Ding et al., 2005; Zhang et al., 2007b). Shortly after Lei Ding s initial report, several studies, including biochemical analysis in mammalian tissue cultured cells and functional analysis in Drosophila, revealed that the mammalian and Drosophila GW182 proteins also function in the mirisc (Jakymiw et al., 2005; Liu et al., 2005; Meister et al., 2005). In order to further study the role of GW182 proteins and mirnas, members of the Han lab developed systematic immunoprecipitations of the mirisc utilizing AIN-1 and AIN-2 to identify mirna targets (Zhang et al., 2007a). By analyzing mirna::mrna interactions at different stages of C. elegans development, thousands of mirna targets were identified supporting the hypothesis that mirnas orchestrate developmental programs (Zhang et al., 2009). By analyzing mirna::mrna interactions in different tissues of C.

13 elegans, Brian Kudlow discovered that mirna activity in the gut is largely dedicated to 4 repressing the pathogen response (Kudlow et al., 2012) and Minh Than uncovered a role for neuronal mirnas in preventing inappropriate entrance into the stress-induced dauer larval stage (Than et al., 2013). The mechanism of mirna-mediated gene silencing has been best characterized by studying the mirisc proteins in vitro. Several conserved domains within Drosophila and mammalian GW182 proteins have been annotated and thoroughly described (Figure 1) (Braun et al., 2013). Some of these domains are important for interacting with translation regulation factors such as PolyA binding protein (PABP) and decapping complexes, and these interactions are essential for mirna-mediated silencing in vitro (Braun et al., 2013). However, the C. elegans GW182 proteins do not appear to contain the conserved domains required for silencing, or a domain known to bind PABP, leading me to test the interaction between the C. elegans GW182 protein AIN-1 and PABP PAB-1 (Chapter 1).

14 Figure 1. Annotated conserved domains in GW182 protein homologs. 5 The PAM2 motif binds PABP and is shown in purple. Figure from Braun et al., Discovery of Vigilin The Vigilin protein was first cloned from Chicken in 1992, and named because of the VIG repeats in the protein sequence (Grünweller et al., 1997; Henkel et al., 1992; Schmidt et al., 1992). The yeast homolog Scp160 was cloned in 1995 and several studies have illuminated some of the various functions of this protein. Disruption of Scp160 results in cell division defects, decreased viability, and increased DNA content (Weber et al., 1997; Wintersberger et al., 1995). The localization of Scp160 at the ER is dependent on microtubules and dissociates upon stress (Frey et al., 2001; Mitchell et al., 2013). Scp160 exhibits a synthetic lethal phenotype with EAP1, which functions in translation by binding eif4e (Mendelsohn et al., 2003). The Parker lab elegantly characterized RNA binding proteins in yeast, including Scp160, by pulling down mrnas and defining specific mrnas bound to the identified RNA binding proteins (Mitchell et al., 2013). Scp160 is known to interact with Bfr1 and P body components such as the decapping protein Dcp2 and has recently been shown to be required for proper P body formation under normal growth conditions (Weidner et al., 2014). One example of a specific function for Scp160 was described in response to the yeast mating pheromone. Scp160 mediates trafficking of SRO7 mrna to the shmoo tip in a MYO4- dependent manner (Gelin-Licht et al., 2012). Another specific example of an mrna regulated by Xenopus Vigilin is vitellogenin. In response to estrogen, Vigilin binds the 3 UTR of vitellogenin mrna and prevents cleavage by PMR-1 (Cunningham et al., 2000; Dodson and Shapiro, 1997; Nielsen and Shapiro, 1990).

15 6 The Drosophila Vigilin homolog DDP1 is somewhat controversial. This protein was isolated with centromeric dodeca satellite DNA in 1999 (Cortés et al., 1999). Although the protein has been observed to localize mostly in the cytoplasm, it has also been proposed to function in heterochromatin formation and various mutants exhibit different levels of position effect variation (PEV) (Batlle et al., 2011; Huertas et al., 2004; Wang et al., 2005; Zhou et al., 2008). The human Vigilin homolog was originally named HDL binding protein (HDLBP) because it was found to bind HDL and was upregulated by intracellular cholesterol levels (Graham and Oram, 1987). Mutations in this protein have been found to be associated with human cancers (Molyneux et al., 2014) and overexpression of Vigilin was shown to be required for tumor growth in one particular study (Yang et al., 2014). In another study, Vigilin was shown to bind the 3 UTR of c-fms mrna to decrease its stability and inhibit protein expression, thereby suppressing invasion of breast cancer cells (Woo et al., 2011). Most recently human Vigilin was proposed to interact with the CCCTC-binding factor (CTCF) (Liu et al., 2014). Multiple functions of this protein could explain the many different phenotypes and functions observed in different organisms, yet there are still many unanswered questions surrounding this protein. For example, what is the precise role of Vigilin in the mechanism of translational regulation and heterochromatin formation? What function of Vigilin is required for cell growth and division? What other proteins or RNAs does Vigilin function with and regulate? The C. elegans Vigilin homolog C08H9.2 was previously found in a screen for enhancers of the precocious COL-19::GFP expression in animals expressing let-7 at an early

16 developmental stage (Hayes and Ruvkun, 2006). We identified this gene in our screen for 7 genes that become essential for development when the mirisc is compromised (Weaver et al., 2014). Based on homology to Vigilin proteins, we named the gene vgln-1 (ViGiLiN). The surprising interaction between Vigilin and the mirisc exposed additional questions regarding Vigilin: Does Vigilin directly function with mirnas or proteins in the mirisc to regulate translation? Does Vigilin regulate translation of mirna target mrnas and can that finding be used to illuminate the mechanism of Vigilin function in translational regulation? Does Vigilin play an essential role in animal development? To begin to answer these questions, I investigated how the RNA binding protein Vigilin collaborates with mirnas to regulate C. elegans development (Chapter 5).

17 Chapter II: mirnas play critical roles in the survival and recovery of C. elegans from starvation-induced L1 diapause 8 Introduction mirnas have been shown to play important roles in aging and stress conditions (Leung and Sharp, 2010). The worm has proven to be an excellent model for studying stress responses and our lab has studied several stresses such as starvation and pathogen response (Cui et al., 2013; Kudlow et al., 2012; Leung and Sharp, 2010). Starvation is a unique stress to a worm. In a lab with unlimited E. coli as a food source, C. elegans can complete their entire life cycle within three days. However in the wild, C. elegans are likely to experience starvation and thus are required to have the ability to enter a developmental arrest or diapause state at different points during their life cycle. If worms are hatched in the absence of food, they will arrest at the first larval stage and enter L1 diapause (Baugh, 2013). When food becomes absent at later larval stages or the worm encounters crowded conditions and pheromones, they will enter the dauer stage and exit normal development, while adult worms encountering starvation will enter an adult reproductive diapause (Ogawa and Sommer, 2009). In each condition, the worm can resume normal development when food is supplied. We asked whether mirnas affect the worm s ability to survive and recover from starvation-induced L1 diapause. We found that compromising overall mirna function impairs starvation survival. Specifically, mirna mir-71 plays a pivotal role in starvation survival by repressing the expression of age-1, unc-31 and likely other factors in the insulin/insulin-like growth factor signaling (IIS) pathway, as well as genes acting in parallel to the IIS pathway. mir-71 is also required for proper recovery of developmental programs in animals that experienced starvation-induced L1 diapause.

18 Materials and Methods 9 C. elegans methods The wild type (WT) strain used was N2 Bristol and all strains used in this thesis are listed in Appendix 1. Unless otherwise stated, worm strains were maintained and manipulated as described (Stiernagle, 2006) on NGM agar with Escherichia coli OP50 as a food source at 20 o C. L1 Starvation Survival Assay Starvation assays were performed as described (Cui et al., 2013; Zhang et al., 2011b). Worms were well fed for at least two generations, then gravid adults were bleached and synchronized eggs were collected by hypochlorite and sodium hydroxide treatment. About 200 eggs were transferred to plates with HB101 bacteria and adults were bleached again three days later. Those eggs were allowed to hatch overnight in S-basal or M9 buffer without cholesterol in 15ml tubes rocking at 20 o C hours later, the hatched L1 worms were counted and the density was adjusted to 3-5 worms per microliter S-basal. 20µl aliquots of worms were placed on OP50 plates and the number of L1 worms were recorded (Np). Three days later the number of surviving worms were recorded (Ns). The survival rate was Ns/Np, representing an estimate of the whole population, and this was repeated every three days in order to create a survival curve measuring the ability for long-term survival under starvation. Dual color reporter assay and other developmental assays Candidate 3'UTRs were cloned into the modified ppd vector previously used in our lab (Zhang et al., 2009). Plasmids were co-injected according to standard protocols into unc-119(ed3) worms at these concentrations: GFP reporter (4ng/µl), RFP control (4ng/µl), unc-119 rescue plasmid (20ng/µl), pbiiks(+) (72ng/µl). Some plasmids were injected into wild type worms with a different reporter at these concentrations: GFP

19 reporter (4ng/µl), RFP control (4ng/µl), rol-6 marker plasmid (100ng/µl). Images were 10 pseudo colored by Xiaochang Zhang in Photoshop CS3 (Adobe). Results ain-1 mutants display reduced survival rate during L1 diapause We asked whether compromising overall mirna function by knocking out one of the GW182 proteins, AIN-1 and AIN-2, affects L1 starvation survival. Survival rate curves reveal that ain-2(lf) worms survive comparable to WT but ain-1(lf) mutant worms have a reduced survival rate (Figure 2A). We then asked whether the phenotype could be suppressed by expressing AIN-2 in specific tissues. I found that the ain-1(lf) phenotype can be suppressed by expressing AIN-2 in the intestine using the ges-1 promoter but not by expressing AIN-2 in muscle using the myo-3 promoter (Figure 2B), suggesting that ain-1 and ain-2 are functionally redundant and intestinal mirnas are largely sufficient for the role that mirnas play in starvation survival.

20 100% A 100% B WT Percent'Survival' 80% ain/1 ain/2 60% daf/16 40% Percent'Survival' 80% 60% 40% WT ain/1 11 ain/1; ges/1p::ain/2::gf P ain/1; myo/3p::ain/2::g FP 20% 20% 0% Days'of'Starva3on' 0% Days'of'Starva3on' Figure 2. ain-1 mutants display a reduced survival rate. A. Although ain-2 mutants survive comparable to WT, ain-1 mutants display a reduced survival rate. B. Expression of AIN-2 in the intestine, driven by the ges-1 promoter, suppressed the reduced survival rate of ain-1. Each data point represents of sum of three replicates. Error bars represent standard error. During normal development, the IIS pathway represses the FOXO transcription factor daf-16 (Baugh and Sternberg, 2006; Lee and Ashrafi, 2008). However, starvation conditions repress the ocr-2 ion channel expressed in sensory cilia, which usually activates unc-31, a calcium-dependent activator protein for secretion (CAPS). Under growth conditions, unc-31 would activate the insulin receptor daf-2, which in turn activates the signaling cascade including the PI3 kinase age-1, leading to repression of FOXO transcription factor DAF-16. When DAF-16 is activated under starvation conditions, it localizes to the nucleus and turns on stress response genes, leading to arrest of the cell cycle and L1 diapause. daf-16 mutants are unable to survive starvation because they cannot enter L1 diapause. In order to test whether the ain-1(lf) phenotype was dependent on the IIS pathway, a postdoc in the lab, Xiaochang Zhang, knocked out the PI3 kinase age-1 and

21 12 tested starvation survival of the double mutant ain-1(lf);age-1(lf) (Zhang et al., 2011b). We found that the reduced survival rate of ain-1(lf) worms was suppressed by disrupting age-1 and partially suppressed by disrupting unc-31, suggesting that age-1 is a potential downstream target of the mirnas involved in starvation survival. Since unc-31 functions upstream of age-1 in the IIS pathway, these genetic results could be explained by mirnas targeting multiple components of the pathway. mir-71 mutants display reduced survival rate during L1 diapause Xiaochang then screened all available mirna mutants and found that mir-71 mutant worms (among others) display reduced starvation survival rate. This phenotype can also be suppressed by reducing age-1 function, and partially suppressed by knocking out unc-31 (Zhang et al., 2011b). Independently, two different labs showed mir-71 functions in ageing although neither reported direct targets of mir-71 for this function (Boulias and Horvitz, 2012; Smith-Vikos et al., 2014). mir-71 functions to repress age-1 and unc-31 expression The 3'UTRs of multiple components of the IIS pathway have predicted mir-71 target sites based on predictions by Targetscan, PicTar, mirwip, and Lencastre et al., In order to test whether the expression of these predicted target genes is regulated by mir-71, we used a dual color 3'UTR reporter assay previously described by our lab (Zhang et al., 2009). In this case we compared expression of nuclear-localized GFP driven by a ubiquitous promoter with the candidate 3'UTR to the expression of nuclear-localized RFP driven by a ubiquitous promoter with a let-858 3'UTR, which is not regulated by mirnas (Figure 3A). The plasmids with these reporters were injected into wild type and mir-71 mutant worms by a technician in the lab. Xiaochang and I scored expression of the

22 reporters. 13 When using a reporter with the age-1 3'UTR, we observed an obvious increase in GFP in the mir-71(-) mutant worms but not in the wild type worms (Figure 3B). We scored the GFP intensity relative to the RFP intensity in over 100 worms from three different wild type transgenic lines and 4 mir-71(-) transgenic lines (Figure 3B). When GFP was bright and comparable to the RFP, it was scored as (+). If there was weak GFP expression compared to RFP, it was scored as (+/-). If there was no GFP observed but RFP was still visible, it was scored as (-). We observed similar regulation of the unc-31 3'UTR from 2 wild type transgenic lines and 2 mir-71(-) transgenic lines (Zhang et al., 2011b).

23 14 A B C Figure 3. Dual color reporters reveal age-1 and hbl-1 are regulated by mir-71. A. The dual color reporter system. Figure from Zhang et al., 2011b. B. The age- 1 reporter is repressed by mir- 71 and derepressed in mir- 71(- ) mutants. I scored expression of the reporter. Xiaochang analyzed the data graphed on the right by comparing the proportions of GFP(+) worms in mir- 71(+) and mir- 71(- ) mutants; n = 128 and 71, respectively. Error bars represent standard deviation and *** represents p > by Chi squared test. Figure modified from Zhang et al., 2011b. C. The hbl- 1 reporter is repressed by mir- 71. Red arrows point to representative intestinal nuclei. I scored expression of the reporter. Xiaochang analyzed the data graphed on the right by comparing the proportions of GFP(+) worms in mir- 71(+) and mir- 71(- ) mutants; n = 60 each. Error bars represent standard deviation and ** represents p > by Chi squared test. Figure from Zhang et al., 2011b.

24 mir-71 is not required for L1 arrest but functions in starvation recovery We next asked whether mir-71 is required for worms to enter L1 arrest similar to daf-16. Xiaochang used expression of cki-1 and lin-4 promoters to show that cki-1 and lin-4 expressions are up- and down- regulated, respectively, in mir-71(-) mutants, which are indicative of arrest (Zhang et al., 2011b). Normal division of seam cells in the hypodermis was also observed to be arrested in mir-71(-) mutants. The M cell lineage is another effective marker for larval growth progression as the M cell continues to divide and migrate after the first larval stage. I used an M cell lineage marker, P hlh-8 ::GFP (Baugh and Sternberg, 2006; Harfe et al., 1998) to further confirm that mir-71(-) worms arrested at the same stage of development as wild type worms under starvation conditions. By counting the number of worms with a divided M cell, we determined that all WT and mir-71(-) mutants successfully arrested development at the L1 stage (Figure 4A). These four lines of evidence suggest that mir-71 is not required to enter L1 arrest, and therefore mir-71 function is distinct from daf-16 function. 15

25 A B C 16 Figure 4. mir-71 is not required for entering L1 arrest but is required for resuming development after arrest. A. One M cell is visible in arrested L1 worms. The M cell lineage does not divide in wild type or mir-71 mutant worms after 11 days of starvation. Figure from Zhang et al., 2011b. B. VPC division timing is impaired in mir-71 mutants. Worms were staged based on the gonad pictured on the right. Figure from Zhang et al. 2011b. C. Deletion of daf-16 enhances the VPC division timing defect of mir-71(-) mutants. ** represents p <0.005 by Chi squared test. Figure from Zhang et al., 2011b.

26 We hypothesized that if the worms could enter L1 arrest but could not survive 17 starvation, perhaps they were unable to exit arrest and resume normal development. Xiaochang observed that many of the mir-71(-) worms that survived L1 starvation displayed defects in vulval development although well fed mutants did not display these defects. To test the hypothesis that mir-71 was required for vulval cell division in worms recovering from L1 diapause, we scored the percent of worms with normal vulval precursor cell (VPC) division timing. During normal development, the VPC cells divide twice during the L3 larval stage. The L3/L4 stage worms can be identified by observing the gonad size and shape. At this stage of development we counted the VPC daughter cells present in wild type and mir-71(-) mutant worms (Figure 4B). Almost all wild type worms had VPC greatgranddaughter cells present (81/82) but most of the mir-71(-) mutant worms only had the VPC daughter cells present (83/89) after four days of starvation (Figure 4B). The severity of the defect correlates with the duration of L1 starvation and the defect is partially suppressed by reducing age-1 or unc-31 (Zhang et al., 2011b). We reasoned that if the defect was only due to increased daf-16 expression, then knocking out daf-16 should suppress the defect. However, the mir-71(-); daf-16(-) double mutant had a more severe defect (Figure 4C). We concluded that mir-71 regulates vulval developmental timing through a parallel pathway as well as through the IIS pathway. hbl-1 and lin-42 are two heterochronic genes known to regulate developmental timing during L3 larval development, and they contain predicted mir-71 target sites in their 3'UTRs. By using our dual color 3'UTR reporter assay, we confirmed that both these genes are also regulated by mir-71 (Figure 2C, Zhang et al., 2011b), which likely contribute significantly to the impact of mir-71 on larval development after starvation.

27 Discussion 18 By using GW182 protein mutants, we were able to observe the function of mirnas in L1 starvation survival. Further genetic screening and analysis lead to the finding that mirna mir-71 plays a pivotal function in L1 starvation survival and developmental recovery. We showed that mir-71 executes this function by repressing multiple targets in multiple regulatory pathways: age-1 and unc-31 of the IIS pathway, and hbl-1 and lin-42 of the developmental timing regulatory pathway. Clearly mir-71 functions in a complex interaction network as illustrated in our model (Figure 5). Our data further indicated that other mirnas also play roles in starvation survival, suggesting a more complex network of multiple mirnas functioning to regulate many genes in starvation survival. These results reinforce the idea that most mirnas do not regulate specific physiological functions through a robust regulatory interaction between one mirna and one target. Instead, they typically impact specific physiological functions though a mirna-target network involving multiple mirnas and a large number of their targets.

28 19 Starva3on' OCR/2 HBL/1 LIN/42 Andothers mir-71 and other mirnas UNC/31 DAF/2 AGE/1 AKT/2 SGK/1 vulvaldifferenraron DAF/16 survival growth Figure 5. Model of mir-71 function in IIS and parallel pathways.

29 Chapter III: C. elegans GW182 protein AIN-1 interacts with polya binding protein PAB-1 Introduction Argonaute and GW182 proteins are essential components of the mirisc and are required for mirna function. While mirna biogenesis was well understood (Krol et al., 2010; Macfarlane and Murphy, 2010), mechanisms of translational repression and the essential role of GW182 within the mirisc were not when I started to work on my thesis in 2010 (Filipowicz et al., 2008). My research attempted to elucidate the biochemical role of the C. elegans GW182 protein, AIN-1, in mirna-mediated gene silencing. In vitro systems had provided a convenient model to test the essential factors involved in mirna-mediated gene silencing. In mammalian and Drosophila homologs, the N-terminus of GW182 family members, which consists of several GW repeats, binds to Argonaute, while the C- terminus is essential for silencing and binds to polya binding protein (PABP) (Eulalio et al., 2009a, 2009b; Zekri et al., 2009). The interaction between GW182 proteins and PABPs had been shown to be essential for silencing, and occurs through the PAM2 motif within GW182 proteins (Tritschler et al., 2010). The protein sequence of the C. elegans GW182 proteins AIN-1 and AIN-2 varies greatly from the mammalian and Drosophila homologs, as illustrated by Braun et al., 2013 (Figure 1). However, previous studies from our lab showed that these proteins accomplish the same function as the other homologs that contain many conserved domains that are not present in AIN-1 and AIN-2 (Ding et al., 2005; Zhang et al., 2007a). One of these domains is the PAM2 motif, which is known to interact with PABP, and is essential for carrying out the function of mirnamediated gene silencing. I attempted to determine whether the AIN-1 protein could still interact with C. elegans PABP PAB-1 despite the lack of a conserved PAM2 motif. I hypothesized that these proteins may interact through a different motif that had not been characterized. I found that 20

30 C. elegans AIN-1 directly interacts with C. elegans PAB-1 in vitro and that AIN-1 21 immunoprecipitates with PAB-1 from lysate suggesting an in vivo interaction. By disrupting four amino acids within AIN-1, I found that the physical interaction between AIN-1 and PAB-1 is mostly disrupted in vitro, but this mutant AIN-1 protein, when overexpressed, is still functional in the worm possibly due to residual interaction between the mutant AIN-1 and PAB-1. Materials and Methods In vitro protein binding assay The protein coding sequences of ALG-1 and PAB-1 were previously cloned by Lei Ding before a GST tag in the plasmid pgex-2t for expression in BL21 bacteria for use in in vitro binding assays (Table 2 describes all plasmids used). After the expression of PAB-1 and ALG-1 in bacteria was induced with IPTG, the bacterial cells were collected and lysed by sonification in Phosphate buffered saline (PBS) solution with protease inhibitors. The tagged proteins were pulled down by Glutathione Sepharose beads (GE). Once the GST tagged proteins were collected, radiolabeled AIN-1 protein was added and allowed to interact for one hour at 4 o C in binding buffer (1XPBS with 0.1% Triton X-100), then washed with binding buffer. The protein coding sequence of AIN-1 was previously cloned into the plasmid pet- 22b(+) by Lei Ding and I modified parts of the sequence for in vitro translation kits and assays. A Transcription and Translation kit consisting of rabbit reticulocyte (Promega) was used to produce radiolabeled AIN-1 protein according to the vendor s protocol. After the binding assay, proteins bound to beads were analyzed by SDS-PAGE, exposure to a phosphor screen, and documented with a phosphorimager.

31 Immunoprecipitation from worm lysate Worm strains with FLAG tagged PAB-1 were created through genome integration by Jim Mapes (Mapes et al., 2010). I lysed a frozen pellet of worms in liquid nitrogen and performed immunoprecipitations (IPs) as described previously by our lab (Zhang et al., 2009). AIN-1 was immunoprecipitated with our AIN-1 antibody (Ding et al., 2005) and PAB-1::FLAG was immunoprecipitated with a FLAG tag antibody (Sigma). Results were analyzed by SDS-PAGE and western blots with the same antibodies. 22 The IP experiment described in Figure 7 was performed with strains made by crossing ain-1(ku322) to the integrated 3XFLAG::PAB-1 strain CU7916 and injecting the full length AIN-1::GFP or mutant AIN-1(SLWI) constructs. Table 1 lists all the strains used. These injections were performed by myself, a technician, and an undergraduate. Three separately injected AIN-1(SLWI) lines were tested for the IP and two of those were used for the functional assay described in Figure 7. Results AIN-1 and PAB-1 directly interact in vitro If the proposed mechanism of mirna-mediated silencing is conserved in C. elegans, I would expect that C. elegans proteins AIN-1 and PAB-1 interact directly. I used an in vitro binding assay to show that radio-labeled AIN-1 binds to recombinant GST::PAB-1 expressed in bacteria and bound to glutathione sepharose beads. As controls, AIN-1 is shown to bind to C. elegans Argonaute GST::ALG-1 but not to GST alone. By testing different segments of the AIN-1 protein, I found that the AIN-1 N-terminus is not sufficient or required to bind GST::ALG-1 or GST::PAB-1 (Figure 6A). When I mutated four specific amino acids referred to as the SLWI motif, the mutant AIN-1(SLWI) protein no longer bound to GST::PAB-1 but still weakly bound to GST::ALG-1 (Figure 6A).

32 23 A AIN/1 AIN/1N/terminus AIN/1minusN/terminus AIN/1C/terminus AIN/1SLWI390AAAA B IPsample: conservedregion **** 1/10Input GST GST::ALG/1 GST::PAB/1 WB: anr/ain/1 anr/flag Exposure: 1min 10sec Figure 6. AIN-1 interacts with PAB-1. A. AIN-1 C-terminus binds PAB-1 in vitro. Disrupting the SLWI motif prevents binding of AIN-1 to PAB-1 in vitro. B. AIN-1 immunoprecipitates with PAB-1::FLAG and PAB-1::FLAG immunoprecipitates with AIN-1 from C. elegans lysate.

33 AIN-1 and PAB-1 immunoprecipitate from C. elegans lysate To confirm that AIN-1 and PAB-1 are interacting in C. elegans lysate, I 24 immunoprecipitated both proteins and used a western blot to confirm that both proteins pulled down the other (Figure 6B). Tagged PAB-1::FLAG immunoprecipitates with AIN-1 and AIN-1 immunoprecipitates with PAB-1::FLAG from worm lysates. This result supports an in vivo interaction, directly or indirectly, between AIN-1 and PAB-1. Together with the above result from the in vitro binding assay, it supports the hypothesis that AIN-1 and PAB-1 interact directly. The SLWI motif is not totally required for the AIN-1 interaction with PAB-1 in vivo and possibly also not totally essential for function Although several in vitro studies have highlighted the importance of the binding between GW182 proteins and PABP in mirna-mediated gene silencing (Tritschler et al., 2010), the interaction has not been tested in a whole organism. I attempted to address this question by asking if the SLWI motif in AIN-1 was required for the interaction with PAB-1 in worm lysate and function of AIN-1 in the worm. With assistance from a technician and an undergraduate in the lab in microinjection, I introduced the mutant AIN-1(SLWI) protein with a mutated SLWI motif into worms. I found that this mutant protein still immunoprecipitated with PAB-1 suggesting that the SLWI motif was not absolutely required for forming the complex with PAB-1 (Figure 7A). Since the binding efficiency of the mutant protein to PAB-1 was drastically decreased in the in vitro assay (Figure 6A), the proteins might still interact indirectly in vivo (Figure 7A).

34 A Anti- AIN-1 WB Anti- Actin WB B Lysate Anti-FLAG IP Anti-AIN-1 IP Lysate Propor3on'of'worms'alive'on'ain72(RNAi)'normalized' to'propor3on'of'worms'alive'on'mock'rnai' Samples: 1 - Precision Plus Protein ladder 2 - [AIN-1::GFP A] 3 - PAB-1::FLAG 4 - PAB-1::FLAG; [AIN-1(SLWI)::GFP A] 5 - PAB-1::FLAG, [AIN-1(SLWI)::GFP B] 6 - PAB-1::FLAG, [AIN-1(SLWI)::GFP C] 7 - PAB-1::FLAG, [AIN-1:GFP A] 25 Figure 7. The SLWI motif is not required for AIN-1 function or interaction with PAB-1 in worm lysate. A. Western blots were probed with an AIN-1 antibody or Actin antibody. All strains described in the legend were in an ain-1(-) mutant background, therefore lane 3 represents a negative

35 control where no AIN-1 protein was present and lane 2 represents a negative control where no PAB-1::FLAG was present. The anti-flag IP gel shows that PAB-1::FLAG was present in all samples except the negative control when AIN-1or the AIN-1(SLWI) mutant was pulled down. The bottom band present in the two IP gels is the 50kDa heavy chain of the antibody. B. Some of the same strains used in the IP experiment were scored for survival on ain-2(rnai). The ain-1(-) mutant is lethal on ain-2(rnai) but some proportion of all strains with AIN-1 or AIN-1(SLWI) survived. 26 Furthermore the mutant AIN-1(SLWI) protein partially suppressed the ain-1(lf) mutant phenotype suggesting that is still functional (Figure 7B). This result may suggest that the SLWI motif is also not totally required for AIN-1 function. However, since the mutant protein was expressed from a transgene with multiple copies, the activity of the mutant protein could be significant reduced yet still functional overall due to overexpression. Since the protein expressed from the transgene still interacted with PAB-1 in worm lysate, we were not able to directly test the question of whether the interaction between AIN-1 and PAB-1 is essential for mirna-mediated silencing in C. elegans. Discussion Recently the interaction between GW182 proteins and decapping enzymes has been shown to be essential for mirna-mediated gene silencing (reviewed by Jonas and Izaurralde, 2015). It is clear from in vitro assays that interactions between GW182 proteins and PABP and the decapping complex are important for silencing (Jonas and Izaurralde, 2015), but the importance of these interactions have yet to be firmly determined in a whole organism. By using an in vitro binding assay I found that the C. elegans GW182 protein AIN-1 directly interacts with PABP PAB-1. The Izzauralde lab confirmed my results by publishing that the C. elegans AIN-1 protein directly interacts with PABP through an in vitro assay (Kuzuoglu- Öztürk et al., 2012). Furthermore I found that AIN-1 and PAB-1 immunoprecipitate with each

36 27 other from C. elegans lysate. These results confirmed our hypothesis that although the AIN-1 protein sequence is very different from the mammalian and Drosophila homologs, the protein still directly binds to PABP. To determine the importance of the interaction between AIN-1 and PAB-1 in a whole animal, I mutated four amino acids within the AIN-1 protein termed the SLWI motif and found the mutant AIN-1(SLWI) protein was no longer able to prominently bind to PAB-1 in vitro. However, when I used transgenic animals to test the binding between AIN-1(SLWI) and PAB-1 in C. elegans lysate, I observed that the proteins still immunoprecipitated together. This could suggest that the SLWI motif is not absolutely required for binding under natural conditions in worm lysate, or that the interaction in lysate is largely due to an indirect interaction. I found that the AIN-1(SLWI) mutant protein was also sufficient to partially suppress the lethality of ain-1(lf) on ain-2(rnai), suggesting that the protein is still functional and the SLWI motif is not totally required for AIN-1 protein function. Since the transgene was present in multiple copies, it is possible that the binding and functional effect of the mutant AIN-1(SLWI) is significantly due to overexpression of the mutant proteins that overcame the decrease in binding efficiency and function. Therefore, to reach a firm conclusion, single gene insertion, best achieved through recently developed CRISPR-Cas9 techniques, would be necessary to examine the issue further. I decided in 2012 to terminate the project after seeing the publication of the in vitro binding between AIN-1 and PAB-1 by the Izzauralde lab in 2012 (Kuzuoglu-Öztürk et al., 2012). The functional significance of the interaction in live animals remains to be investigated.

37 Chapter IV: Uncovering developmental functions of mirnas and other regulators by a genetic enhancer screen 28 Introduction Development robustness ensures that all organisms complete development regardless of genetic or environmental perturbation. The biologist C. H. Waddington compared developmental robustness to a ball rolling down a hill (Waddington, 1959), since the ball will always end up in the same place, at the bottom of the valley. One way that this robustness is created is through genetic redundancy (Ebert and Sharp, 2012; Félix and Wagner, 2008). Even if one genetic pathway is perturbed, another pathway that serves the same function can substitute for it. In this way organisms will still exhibit developmental robustness despite genetic perturbation. mirnas provide several examples of genetic redundancy and robustness. In Chapter 2 we described an example of one specific mirna, mir-71, that functions to regulate multiple genes in the insulin/insulin-like growth factor signaling (IIS) pathway and in parallel pathways also involved in development after starvation-induced L1 arrest. We also suggested that mir-71 was not the only mirna functioning in the worm s response to starvation, illustrating many levels of regulation by mirnas. In Chapter 3, I attempted to investigate the mechanism of GW182 protein AIN-1 which functions partially redundantly with AIN-2 to ensure mirna-mediated gene silencing. These two stories provide examples of redundancy at the protein level and the mirna target level. We next proposed to study the redundancy or converging activities that exist at the level of pathways such as other mechanisms of gene expression regulation. We hypothesized that C. elegans mirnas collaborate with other gene regulatory mechanisms to ensure developmental robustness. To explore this question, I performed a

38 29 synthetic screen in collaboration with two postdocs in the lab Ben Weaver and Yi Weaver. Our goals were to discover developmental functions of mirnas that might otherwise be masked by genetic redundancy or pleiotropism, and to expose unknown gene regulation mechanisms that collaborate with mirnas. Materials and Methods Genome wide RNAi Screen We screened ain-1 and ain-2 mutants on the entire ORFeome RNAi library (Reboul et al., 2003) using liquid culture similar to a previously published method (Lehner et al., 2006). Our method of screening and confirmation is described in detail by Weaver et al., Results Screen hits Although most C. elegans mirna families are not essential for development or viability (Alvarez-Saavedra and Horvitz, 2010; Miska et al., 2007), one previous screen used sensitized mirna genetic backgrounds to reveal mirna mutant phenotypes (Abbott, 2011; Brenner et al., 2010). Our goal was not only to reveal mirna functions, but also to reveal other gene regulation mechanisms that collaborate with mirnas. In order to compromise overall mirna function, we utilized the GW182 proteins, known to be essential for mirna-mediated gene silencing. Although loss of both ain-1 and ain-2 is lethal, loss of only one compromises mirna function without drastically disrupting development. We screened ain-1 and ain-2 mutants with RNAi of individual genes in the genome from the ORF RNAi library (Reboul et al., 2003), and scored for synthetic developmental phenotypes as sketched in Figure 8A. We found that many genes become essential for development when the mirisc is compromised (Weaver et al., 2014). We confirmed some of these interactions genetically and observed several different

39 phenotypes due to different genes that function redundantly with the mirisc. 30 After screening the entire ORF library and repeating all our hits in quadruplicate, we confirmed 126 final interactors. Gene ontology analysis revealed that the 126 interactors belong to a broad range of functional categories (Figure 8B). Ben Weaver and Yi Weaver performed extensive analysis to better understand the types of genes revealed by our screen (Weaver et al., 2014). A B ain-1 or ain-2 (lf) Dev Phenotype Other Body Structure Apoptosis Metabolism RNA Processing Stress Responsive Cell Mig and Org Gene Reg Protein Stability Unknown RNAi GENE X Figure 8. Screen design and results. A. Strategy to screen ain-1 and ain-2 worms with the ORF RNAi library. Figure from Weaver, Zabinsky et al., B. Gene ontology analysis of screen hits performed by Ben Weaver and Yi Weaver. wt No Phenotype Number of Genes

40 Example 1: ceh-18 One example of a hit from our screen is ceh-18. CEH-18 is a POU domain 31 transcription factor known to be expressed in the distal tip cell and gonadal sheath cells. CEH-18 functions in sheath cell differentiation and signaling to oocytes (Greenstein et al., 1994). We found that in combination with ain-1(lf), animals treated with ceh-18(rnai) were sluggish, small in brood size, and egg laying defective. Specifically, I found that there were fewer oocytes in a single gonad arm of the double mutants compared to WT and single mutants (Figure 9). This enhancer phenotype was particular interesting because our lab previously identified ceh-18 in a synthetic screen with a mutation in the PTEN tumor suppressor daf-18 (Suzuki and Han, 2006). That study found that both daf-18 and ceh-18 function to repress MAPK activity in the oocyte and when both genes are disrupted, MAPK activity drastically increases to inhibit oogenesis. Therefore, mirnas are potentially a third factor that affects MAPK signaling in the oocyte and the combination of compromising mirna regulation and disrupting ceh-18 lead to a similar phenotype. It would be interesting to further investigate this by testing if ain-1(lf) on daf-18(rnai) causes the same phenotype as ain-1(lf) on ceh-18(rnai) or daf-18(lf) on ceh-18(rnai).

41 A D ceh$18 ' B ain$1' C Numberofoocytesin gonadarm ceh$18;ain$1 Figure 9. Loss of both ain-1 and ceh-18 results in fewer oocytes. A. ceh-18 mutants contain an average of 6 oocytes in each gonad arm. B. ain-1 mutants contain an average of 6 oocytes in each gonad arm. C. ceh-18;ain-1 double mutants contain an average of 2 oocytes in each gonad arm, as well as exhibiting other defects such as egg laying defects and a small brood size. D. Average oocytes examined in 20 gonad arms of each genotype. Error bars represent standard error.

42 Example 2: ced-3 Perhaps the most surprising hit from our screen was the well-known cell death 33 caspase ced-3. Without CED-3, all cell death within the worm is abolished but the animals display no obvious developmental defects (Conradt and Xue, 2005), which led to the belief that ced-3 functions quite specifically in apoptosis. The observed severe developmental defects in ain-1(lf); ced-3(lf) double mutants alone suggested to us that the phenotype is the consequence of a non-apoptotic function of ced-3. To confirm this provocative finding, we utilized several established assays. Ben Weaver confirmed that ain-1(lf) does not enhance the ced-3(rf) cell death phenotype by using two genetic interaction assays (Reddien et al., 2007; Weaver et al., 2014). I confirmed that ain-1(lf) does not inhibit the ced-3(lf) cell death phenotype by counting cell corpses in the head of L1 larvae (Figure 10A,B). This assay was performed in a ced-1(lf) mutant background to facilitate the counting by preventing engulfment of cell corpses (Ledwich et al., 2000). Together these assays confirmed that the phenotypes we were analyzing were not related to the cell death function of ced-3, but the consequence of disrupting previously unexplored non-canonical functions of the CED-3 caspase. In order to better characterize the phenotype, Ben Weaver and I both scored multiple developmental phenotypes such as egg laying defective (Egl), Ruptured vulva (Rup), Sluggish movement (Slu) and Dead adults, larvae, and embryos (Dead). I tested whether expression of AIN-1 and AIN-2 in different tissues could suppress the double mutant phenotype, and found that recovering the mirisc activity in the hypodermis and the intestine could both partially suppress the phenotype, suggesting mirnas and CED-3 likely function predominantly in these two tissues (Figure 10C). Lethality associated with

43 expressing ced-3 from a transgene prevented us from testing directly the sufficiency of ced- 3 expression in specific tissues. 34 Ben Weaver and Yi Weaver went on to show that CED-3 collaborates with specific mirnas of the let-7 family and functions to repress the expression of LIN-28 and likely also LIN-14 and DISL-2 (Weaver et al., 2014). All three of these proteins are critical factors in the developmental timing pathway that is also known to regulate differentiation of stem cells both in C. elegans and mammals. This study, which blossomed from our synthetic screen, uncovered an important new function for the well-known cell death caspase CED-3 in developmental timing and stem cell pluripotency.

44 A B C '''''''WT' ' ' ' ' ced73(7)' 35 Figure 10. CED-3 cooperation with mirnas reveals a non-apoptotic function. A. Cell corpses are present in a WT L1 head but not a ced-3(lf) worm head. Figure from Bob Horvitz (Heemels and Editor, 2012). B. Quantification of cell corpses observed in mutants in a ced-1(lf) background. The numbers of each genotype scored are represented above the bar graph. Error bars represent standard deviation. Figure from Weaver et al., C. The pleiotropic developmental defects of ced-3(lf);ain-1(lf) can be partially suppressed by expressing AIN-1 in the hypodermis and gut. The numbers of each strain I scored are represented above the graph. Data was analyzed by Yi Weaver. Error bars represent standard deviation. * represents p < by Fisher s Exact test comparing normal and abnormal animals. Figure from Weaver et al., 2014.

45 Example 3: vgln-1 36 Another interesting hit identified in our screen was an unknown and unnamed RNA binding protein with 14 KH domains, which are domains known to bind RNA. Based on homology to mammalian HDLBP also known as Vigilin, we named the gene vgln-1 (ViGiLiN). Although our initial screen revealed that RNAi of the gene caused egg laying and vulval developmental defects in ain-1 and ain-2 mutants, I observed that after two generations on RNAi, ain-2 mutants arrested at the first L1 larval stage. Further analysis of this synthetic phenotype and the study of the function of VGLN-1 are described in Chapter V. Discussion The goal of our synthetic screen was to discover developmental functions of mirnas otherwise masked by genetic redundancy and to identify unknown regulators that collaborate with mirnas. The ceh-18 example illustrates that mirnas may collaborate with two known mechanisms of MAPK inhibition in the C. elegans gonad. However, the ced-3 example illustrates how our screen was able to reveal an unknown regulatory function of CED-3. Although CED-3 is known as the C. elegans cell death caspase, its role in development was not previously observed because of genetic redundancy. We were only able to reveal this role of CED-3 by using a sensitized genetic background that compromised overall mirna function. Based on further investigation by Ben Weaver, we propose that just like mirnas, CED-3 also functions broadly to repress gene expression for a wide range of functions (Weaver et al., 2014). The identification of vgln-1 in the screen provides another example of genes that were not previously known to function broadly to repress gene expression. By looking for specific mirnas that exhibit a synthetic phenotype with ced-3 and vgln-1 (Weaver et al., 2014, Chapter 5), we also have uncovered new roles for mirnas in development that were otherwise hidden by genetic redundancy. Therefore

46 37 both of the goals of our screen have been realized. The hits I have described exemplify the strength of our screen and suggest that many of our other unstudied hits will likely provide further insights about mirnas and other regulatory mechanisms that collaborate with mirnas to ensure C. elegans robust development.

47 Chapter V: Several mirnas collaborate with vgln-1 to promote development 38 Introduction After identifying more than 100 genes that interact with miriscs from our screen described in Chapter IV, I decided to focus my further in-depth analysis on Vigilin. Although this C. elegans gene, C08H9.2, was unnamed, it is highly conserved and homologous to human HDL Binding Protein, also known as Vigilin. In Chapter 1, I summarized a few elegant studies describing distinct roles for Vigilin in trafficking, protecting, and repressing specific mrnas in yeast, Xenopus, and human cells, suggesting that broad aspects of function are likely associated with this protein. However, there are still outstanding questions regarding the functions and mechanism of functions of this protein, particularly regarding its role in animal development and its relationship with mirna regulation. Based on the synthetic phenotype we observed between ain-2 and vgln-1, I hypothesized that Vigilin functions to repress some of the same target genes repressed by mirnas during larval development. Knocking out both ain-2 and vgln-1 may result in high expression of a group of common target genes that arrests development at the first larval stage. As described in Chapter 3 and illustrated in Figure 1, C. elegans GW182 proteins lack many conserved motifs annotated in Drosophila and mammalian homologs. One of those conserved motifs is an RNA recognition motif (RRM), although the function of this motif remains unknown. I thus hypothesized that AIN-1 and AIN-2 may be interacting with a different protein that contains an RNA binding domain to more efficiently mediate mirna-mediated gene silencing. Vigilin could be an ideal candidate since it contains 15KH domains and Vigilin homologs have been shown to bind and regulate expression of mrnas. Finally, the function of the conserved RRM in mammalian GW182 proteins is still

48 39 not clear. If we can better understand how the C. elegans GW182 proteins function with an RNA binding protein, we may be able to make an important contribution to the understanding of how mammalian GW182 proteins and mirnas function. Several RNA binding proteins have been identified and proposed to affect mirisc activity by enhancing or inhibiting mirna-mediated gene silencing (Leung and Sharp, 2010). Some of those examples include PUF-9, (Nolde et al., 2007), Dnd1 (Kedde et al., 2007), NHL-2, (Hammell et al., 2009), and GLD-1 (Akay et al., 2013). Interactions observed between mirnas and RNA-binding proteins in development have been reviewed (Kedde and Agami, 2008). We hypothesize that Vigilin may be directly regulating mirna targets which could suggest a specific mechanism of mirna-mediated gene silencing. I attempted to test this hypothesis by studying the genetic interaction of vgln-1 and ain-2. Materials and Methods Cloning and CRISPR-Cas9 RNAi constructs were made by amplifying parts from the 3 end of vgln-1 and the 3 UTR of vgln-1, cloning them into the L4440 vector and transforming that into HT115 bacteria. Plasmids for CRISPR-Cas9 were obtained from Addgene (Table 2). I designed a short guide RNA to target Cas9 to the endogenous vgln-1 locus and cloned it into the vector pdd162. I designed a homologous recombination template to replace the vgln-1 gene with unc-119 and cloned it into the vector pbiisk(+). According to the protocol published by Dickinson et al., 2013, unc-119 worms were injected with the homologous recombination plasmid, a positive selection plasmid, and a negative selection plasmid (Table 2). After several generations, two plates were found with wild type worms indicating that they had the unc-119 rescuing

49 40 plasmid. After heat shock to induce the negative selection marker, only one of these plates still had live wild type worms indicating that the unc-119 had been integrated into the genome and the extrachromosomal array had been lost. Genome integration by bombardment The transcriptional reporter construct consists of the endogenous vgln-1 promoter driving GFP with the endogenous vgln-1 3 UTR P vgln-1 ::gfp (kuis103). The fusion reporter construct, P vgln-1 ::vgln-1::gfp (kuis102), consists of the endogenous vgln-1 promoter driving the endogenous vgln-1 cdna fused to GFP with the endogenous vgln-1 3 UTR. These constructs were cloned into the pcfj350 vector with a rescuing unc-119 gene from C. Briggsae (Table 2) and microparticle bombardment (Seydoux lab protocol, Schweinsberg and Grant, 2013) was used to introduce this construct into the genome of unc-119 mutant worms. After several generations, worms that retained the bombarded DNA in their genome no longer exhibited the unc-119 phenotype, and were singled to confirm integration of the reporters. Three out of four lines obtained for each reporter contained high levels of GFP expression. The detailed protocol is available from the Seydoux lab website adapted from Shai Shaham and Praitis et al., Gas Chromatography Worms were washed off NGM plates with water and rinsed three times with M9 buffer. Lipid extraction was performed as previously done by our lab and others (Kniazeva et al., 2008; Miquel and Browse, 1992). Briefly, the worm pellet was resuspended in 1ml hexane, then 100µl ice cold Methanol and Potassium hydroxide were added and the sample was vortexed. After centrifugation, the upper organic phase was transferred to an appropriate vial for analysis. Gas Chromatography (GC) was performed on a HP6890N (Agilent) apparatus.

50 Crosslinking and Immunoprecipitation (CLIP) 41 Mixed stage worms were grown on NGM plates with HB101 bacteria, collected by washing with M9 buffer, and protein was crosslinked to RNA by UV (Broughton and Pasquinelli, 2013) before worms were stored at -70 o C. Worms were lysed by grinding in liquid nitrogen and immediately resuspended in lysis buffer (10mM Tris/Cl ph 7.5, 150 mm NaCl, 0.5mM EDTA, 0.5% NP-40, complete mini Roche protease inhibitor pills, 0.1U/µl Thermo Fisher RNaseOut, 100mM PMSF). Immunoprecipitation was performed with GFP- Trap A fusion proteins coupled to agarose beads according to the vendor s protocol (Chromotek). RNA was digested with dilutions of RNase A (Thermo Fisher), RNA was dephosphorylated with Alkaline phosphatase (Roche), and RNA was radiolabeled with T4 PNK (NEB) and P32 gamma-atp (Perkin Elmer). The protein-rna complex was run on Novex NuPAGE gels and transferred to a nitrocellulose membrane according to XCell II Blott Module manufacturer s directions. Results Vigilin knockout mutant exhibits a weak L1 arrest phenotype that is dramatically enhanced by compromising mirisc Since no null mutant allele of vgln-1 existed, I designed multiple RNAi constructs against different parts of the gene to test if the phenotype was due to off-target RNAi affects. I found that RNAi constructs targeting the 5' end of the gene, the 3' end of the gene, and the 3'UTR all confirmed the synthetic phenotype with ain-2 suggesting that the phenotype is caused by disrupting the vgln-1 gene and not caused by off-target affects (Figure 11A). Based on the RNAi results described in Figure 11A, knocking down Vigilin alone exhibits some proportion of larval lethality. To confirm that this was due to the vgln-1 gene and not the downstream gene in the operon, we made a knockout using the CRISPR-Cas9

51 42 system. This system has been described by several labs for applications in C. elegans to add GFP or change specific endogenous DNA loci. I designed a homologous recombination plasmid to knock out the entire endogenous vgln-1 locus and replace it with a marker gene, unc-119, based on the method described by Dickenson et al., 2013 (Figure 11B). PCR amplification of the locus confirmed that the endogenous vgln-1 gene was no longer there but the unc-119 gene was in its place (Figure 11C). The mutant strain vgln-1(kuis104) was then scored for L1 larval arrest. 20% of vgln-1(lf) progeny arrest at the L1 larval stage and the worms exhibit some developmental delay. This larval arrest phenotype of vgln-1(lf) is dramatically enhanced by reducing mirisc function, as double mutants vgln-1(lf);ain-2(lf) worms were nearly 100% lethal confirming the RNAi phenotype (Figure 11A and D). These results indicate that the conserved RNA binding protein Vigilin functions in early larval development and is essential when the mirisc is compromised. To confirm the vgln-1(lf) defect was due to knocking out vgln-1 and not another gene at another locus in the genome, we tested whether the integrated P vgln-1 ::vgln-1::gfp (kuis102), fusion transgene could revert the phenotype. The L1 arrest phenotype of the vgln- 1(lf) single mutant was suppressed by expression of the P vgln-1 ::vgln-1::gfp (kuis102) translational fusion reporter construct but not by expressing GFP alone by the P vgln-1 ::gfp (kuis103) transcriptional reporter (Figure 11D).

52 A 1.0' B 0.8' vgln31'gene'model' 0.6' ProporJon'L1'arrest' 0.0' rrf33(lf)' ain32(lf);rrf33(lf)' Genotype' C D 0.4' 0.2' vgl-1 C08H9.3 P1 P2 P3 P4 P5 P unc-119 C08H9.3 mock'rnai' vigilin'orf'rnai' vigilin'3'end'rnai' vigilin'3'utr'rnai' WT 1 = P1+P2 WT 2 = P1+P2 WT 3 = P1+P2 vgln-1(-) 4 = P1+P2 vgln-1(-) 5 = P3+P4 WT 6 = P3+P4 WT 7 = P3+P4 vgln-1(-) 8 = P3+P4 vgln-1(-) 9 = P5+P6 WT 10 = P5+P6 WT 11 = P5+P6 vgln-1(-) 12 = P5+P6 vgln-1(-) vgl-1 (-) PercentL1arrest vgl31 ' ' ' ' ' ' ' ''''''C08H9.3' Cas9' sgrna' ''unc3119 ' ' ' ' '' ''unc3119 ' ' ' ' ' ''''''C08H9.3' 100% 80% 60% 40% 20% 0% WT' ' ' vgl31'(3)' ' 43 Genotype Figure 11. VGLN-1 is essential for development when the mirisc is compromised. A. Multiple RNAi constructs targeted against the vgln-1 gene replicate the synthetic L1 arrest. Different constructs are color-coded and error bars represent standard error. B. The CRISPR knockout was designed to replace the endogenous vgln-1 gene with unc-119. C. The vgln-1 null allele was confirmed by PCR. D. vgln-1(lf) exhibits 20% L1 arrest and in combination with ain- 2(lf) the double mutant exhibits nearly 100% L1 arrest.

53 Characterization of phenotypes associated with loss of vgln-1 and ain-2 In order to determine more precisely the time period when the developmental arrest occurs in vgln-1(lf);ain-2(lf) double mutants, we observed the pattern of an AJM-1::GFP reporter (Knight et al., 2002). This reporter marks adherens junctions between seam cells. During postembryonic development, seam cells divide in a characteristic pattern. The pattern observed in arrested vgln-1(rnai) larvae corresponds to 3 hours after hatching, similar to that observed in larvae under starvation-induced diapause (Figure 12A). 44 Other mutants known to arrest development at this stage include animals hatched into a food-less environment (L1 diapause, Baugh, 2013; Baugh and Sternberg, 2006), animals deficient for monomethyl branched-chain fatty acids (mmbcfa), or animals deficient for sphingolipid biosynthesis (Kniazeva et al., 2008; Zhang et al., 2011a). The lipid deficient animals also exhibit defects in intestine apical membrane polarity (Zhang et al., 2012; Zhu et al., 2015). These papers suggested that the polarity defect is connected to polarity-induced L1 arrest. Therefore we asked whether the intestine apical membrane polarity was also disrupted in vgln-1(rnai) animals. Localization of ERM-1::GFP was found to be normal (Figure 12B), suggesting that apical membrane polarity does not play a critical role in vgln-1 L1 arrest. However this does not rule out the possibility that another defect in the intestine prevents nutrients from being absorbed and signaling the worm to grow past the L1 larval stage. We also asked whether vgln-1(lf);ain-2(lf) affect fatty acid composition. I employed gas chromatography (GC) analysis to observe the fatty acid profile during the L1 arrest. ain- 2(lf);rrf-3(lf) worms grown on vgln-1(rnai) were plated on NGM plates and hatched L1s were collected for GC analysis. The fatty acid profile of both control and mutant worms

54 45 were relatively normal, suggesting that there are no major defects in fatty acid synthesis or degradation (Figure 12C). However this does not rule out a defect in overall fat storage. A (starved'in'm9)' B DIC' (vgln31'rnai)' GFP' elt32'rnai'' (40/40'extra'lumen'phenotype)' Mock'RNAi' (50/50'normal)' C17iso F ID 1A, F ront S ignal (1_15_13\1_15_ \009F 0901.D ) 18 C16:0 C Vigilin'RNAi'' (50/50'normal)' ain-2;rrf-3 on mock RNAi replicate F ID 1A, F ront S ignal (1_15_13\1_15_ \010F 1001.D ) 22.5 ain-2;rrf-3 on mock RNAi replicate F ID 1A, F ro n t S ig n a l (1 _ 1 5 _ 1 3 \1 _ 1 5 _ \0 1 1 F D ) 1 8 ain-2;rrf-3 on vgln-1 RNAi replicate F ID 1A, F ront S ignal (1_15_13\1_15_ \012F 1201.D ) ain-2;rrf-3 on vgln-1 RNAi replicate Deple'ng$ainN2$and$C08H9.2$does$not$affect$lipid$biosynthesis/degrada'on$ Figure 12. Characterization of vgln-1 phenotypes. A. vgln-1(lf) arrests development within 3 hours of hatching. Localization of ERM-1::GFP. C. Depleting ain-2 and vgln-1 does not affect lipid biosynthesis/ Figure 12.B.Characterization of vgln-1(lf) phenotypes. degradation. A. vgln-1(lf) arrests development within 3 hours of hatching. B. Localization of ERM-1::GFP is normal in worms on vgln-1(rnai). C. Depleting ain-2 and vgln-1 does not affect lipid biosynthesis and/or degradation.

55 Since a daf-2(rf) mutant with reduced insulin receptor function are reported to 46 exhibit 10% L1 arrest, similar to vgln-1(lf), and are sensitive to heat (Baugh and Sternberg, 2006), I hypothesized that vgln-1(kuis104) might also be sensitive to heat. In order to test this hypothesis, eggs were allowed to grow at 20 o C, 25 o C, or 30 o C. We observed that the L1 arrest phenotype of vgln-1(lf) was dramatically enhanced at a high temperature (Figure 13A), similar to daf-2(rf). However, when adult worms were tested for resistance to heat at 34 o C, vgln-1(lf) mutants survived better than wild type after 6 hours treatment (Figure 13B). This heat resistance phenotype is also observed in daf-2(rf) mutants, in which case it is dependent on daf-16 (Baugh and Sternberg, 2006). We further observed that vgln-1(lf) is extremely sensitive to starvation but vgln-1(lf) is not sensitive to osmotic stress (Figure 13C).

56 A 1 B ProporRonarrestedL1s C FracRonAlive WT daf/2 vgln/1 ain/2 mir/52 lsy/ [NaCl]mM ProporRonaliveadults a`er6hrsat34 o C WT ain/2 vgl/1 daf/ WT WTprecondiRoned ain/2precondironed vgl/1precondironed daf/2precondironed daf/2 vgln/1 ain/2 mir/52 lsy/6 47 Figure 13. vgln-1(lf) is sensitive to heat at the L1 stage but resistant at the adult stage. A. vgln-1(lf) is sensitive to heat at the L1 larval stage. Error bars represent standard deviation from two replicates. B. vgln-1(lf) is resistant to heat at the adult stage. Error bars represent standard deviation from two replicates. C. vgln-1(lf) is not sensitive to osmotic stress. Worms were grown on 20mM or 200mM (preconditioned) NaCl plates before the experiment. Error bars represent standard deviation from two replicates.

57 48 Vigilin binds to RNA and is broadly expressed throughout C. elegans development Based on homology and the presence of 15KH domains, VGLN-1 is predicted to function as an RNA binding protein. To test if the protein binds RNA, the protein and RNA were crosslinked by UV and immunoprecipitated. RNA was radiolabeled and the RNA-protein complex was found to be sensitive to RNase treatment confirming that the protein binds RNA (Figure 14A). A B C VGL31:GFP:'''+'''''+'''''+'''''+'''''''''''''''''3''''''3''''''3''''''3' GFP:''''''''''''''''3''''''3''''''3'''''3'''''''''''''''''+'''''+'''''+'''''+'''' Rnase'A:' '''''''''''''''''+'''''+'''''''3''''''3'' '''''''''''''''''3''''''3''''''+'''''+'' 250kD' 150' 100' 75' ' 50' 37' 25' Radiolabeled'NucleoZdes Stage:'''''''Comma'stage ' CuJcle ' ' 'L132' ' ' ' ' ' ' ' ' ' 'L233' ' ' ' ''' ' ' ' ' ''L43adult ' 'anz3gfp'wb' '' '''''Muscle' '' Figure 14. VGLN-1 binds to RNA is broadly expressed throughout development. A. Cross linking and immunoprecipitation reveals that VGLN-1 binds to RNA. B. VGLNFigure is 14.expressed Vigilin binds to RNA is broadly expressed throughout development. A. Cross and 1::GFP in many tissues throughout development. C. VGLN-1::GFP islinking excluded immunoprecipitation reveals that VGLN-1 binds to RNA. B. VGLN-1::GFP is expressed in many tissues. C. from muscle cytoplasm. VGLN-1::GFP is excluded from muscle cytoplasm.

58 We also made two reporter constructs to observe spacial and temporal expression of Vigilin. The transcriptional reporter construct P vgln-1 ::gfp (kuis103) is expressed throughout 49 development in most tissues (Figure 14B). The translational fusion reporter construct P vgln- 1::vgln-1::gfp (kuis102) is similarly expressed. Expression was especially high in the spermatheca and vulva. While the transcriptional reporter GFP is present in both the cytoplasm and nucleus of muscle and cuticle cells, the translational fusion reporter GFP seems to be excluded from muscle nuclei (Figure 14C). This suggests that VGLN-1 functions mainly in the cytoplasm or that VGLN-1 nuclear import and export is highly regulated since the protein contains conserved nuclear import and export signals. Identification of specific mirnas that act with VIGN-1 to regulate postembryonic development To identify specific mirnas that collaborate with Vigilin to promote post-embryonic development, we screened all of the available C. elegans mirna deletion strains for prominent enhancement of the L1 arrest phenotype using vgln-1(rnai) (Weum, 2015). If Vigilin functions with only a few specific mirnas and targets only a few mrnas involved in L1 development and arrest, we would expect that the vgln-1(lf) mutant would only exhibit a synthetic phenotype with a few mirnas that target the same mrnas important for L1 arrest. However, based on the fact that most mirnas function within a network and the fact that yeast Vigilin Scp160 is known to bind many specific mrnas (Li et al., 2003; Mitchell et al., 2013), it is likely that VGLN-1 collaborates with many mirnas to regulate many mrna targets. Our screen identified several mirna mutants that exhibited developmental phenotypes on vgln-1(rnai) supporting our hypothesis that Vigilin functions broadly. Since several mirna deletions are known to be sensitive to RNAi (Massirer and Pasquinelli, 2013; Massirer et al., 2012), we constructed vgln- 1(lf); mirna(lf) double mutants to verify the results of our RNAi analysis. Specifically, I

59 50 confirmed that deletion mutations of mir-52, mir-83, lsy-6, and mir-265 prominently enhanced the L1 arrest phenotype of vgln-1(lf) (Figure 15A). We also observed that the vgln-1(lf);mir- 52(lf) mutants appear morphologically long. mir-52 is part of the mir-51 family in C. elegans and belongs to the conserved mir- 99/100 family known in other organisms (Brenner et al., 2012). The mir-51 family is one of the few mirna families that displays a severe developmental defect when all members are knocked out; animals with any of the mir-51 family members are embryonic lethal (Alvarez-Saavedra and Horvitz, 2010; Brenner et al., 2012; Shaw et al., 2010). mir-52 is known to be highly expressed at the L1 stage, and is expressed significantly higher than other mir-51 members (Lau et al., 2001; Lee and Ambros, 2001). The expression difference might explain why loss of mir- 52 but not closely related family members enhances the phenotype of vgln-1(lf). Deletion mutations in the other four mirnas do not display obvious developmental defects by themselves (Figure 15A). These results are highly significant because our genetic analysis uncovered previously unknown functions in early larval growth and development associated with these four mirnas. In other words, animals lacking vgln-1 permitted the discovery of important roles of mirnas that were masked by genetic redundancy. In addition, our results reinforce the concept that mirnas often function in mirna::mrna networks involving multiple mirnas and a large number of their targets.

60 1' A B FracMon'of'L1'arrest' 0.8' 0.6' 0.4' 0.2' 0' vgln51:'''''+''''5'''''+'''''5'''''+'''''5'''''+'''''5'''''+'''''5'''''+'''''5''''+'''''5'''''+'''''5''' ''''''''''WT''''''''ain52''''mir552'''mir583'''''lsy56'''''mir5265''mir570'''mir5254' FracJon'of'worms'with'visible' SKN31:GFP''in'ASI'neurons' 1' 0.8' 0.6' 0.4' 0.2' 0' Genotype' Day'2'vgln%1'(36/40)' C D WT' vgln31' 1' 2' 3' Days'of'starvaJon' Day'2'WT'(28/37)' Adult'SKN51::GFP'reporter' Example'of'2'intesMnal'nuclei' 51 response'to'heat'shock'dependent'on'vg Adult'vgln,1(,);'SKN51::GFP'' Example'of'5'intesMnal'nuclei' Number'of'intesMne' nuclei'with'skn51::gfp'' 4.5' 4' 3.5' 3' 2.5' 2' 1.5' 1' 0.5' 0' SKN51::GFP' vgln51;' SKN51::GFP' Figure 15. Specific mirnas cooperate with VGLN-1 to regulate development. A. Multiple mirnas enhance the L1 arrest phenotype of vgln-1(lf). B. Venn diagram of predicted mirna targets constructed by Venny ( C. vgln-1 is required for repression of SKN-1::GFP during L1 starvation. D. SKN-1::GFP localizes to more intestine nuclei in vgln- 1(lf) adults compared to wild type after heat shock.