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  • Thumbnail for The role of RNA-binding proteins in dendrite morphogenesis
    The role of RNA-binding proteins in dendrite morphogenesis by Kellogg, Leah

    Neurons have highly asymmetric cellular morphologies and polarized cellular functions that are necessary for establishing neural circuitry and for proper functioning of the nervous system. Specialized processes, called dendrites, are used by neurons for reception of stimuli, while axons function in the transmission of signals. In neurons, mRNA localization and translational repression are used to change the protein composition of various regions of the cell, allowing for distinct axonal and dendritic morphologies and environments that are equipped for their various cellular tasks. A significant portion of the eukaryotic genome encodes for RNA-binding proteins (RBPs), which play important roles in localizing and translationally regulating RNAs. Since studies have shown that a large number of mRNAs are localized within dendrites, this suggests that the RBPs contribute broadly to neuronal development and function by localizing and regulating mRNAs. Based on a previous screen of RBP-encoding genes that affect dendrite morphogenesis in dendritic arborization neurons (da neurons) in Drosophila that identified 89 genes (Olesnicky, Killian, and Gavis; in preparation), I extended this screen to determine if any of these evolutionarily conserved RBP genes are important for dendrite morphogenesis in C. elegans PVD neurons as well. A significant decrease in dendritic arborization was found in dcr-1 mutants and preliminary results suggest that sup-26 and mtr-4 mutants may have decreased 3rd and 4th order dendritic branching. In addition, several other candidate genes are currently being investigated. Thus far, the results suggest that DCR-1/Dicer, an RBP involved in the microRNA pathway, SUP-26/Alan Shepard, an RBP implicated in translational control of mRNAs, and MTR-4/L(2)35Df, a component of the eukaryotic RNA exosome play an evolutionarily conserved role in dendrite development in flies and worms.

  • Thumbnail for ZK858.4 causes embryonic lethality but does not directly impact MEX-3 localization in an RNAi induced knockdown of E3 ubiquitin ligases in Caenorhabditis elegans
    ZK858.4 causes embryonic lethality but does not directly impact MEX-3 localization in an RNAi induced knockdown of E3 ubiquitin ligases in Caenorhabditis elegans by Reichard, Kathryn L.

    The ubiquitin proteosome system (UPS) functions in the cell to mark specific proteins for degradation. E3 ubiquitin ligases act as recognition factors and increase the specificity of the UPS. MEX-3 is an RNA binding protein in Caenorhabditis elegans that inhibits the translation of PAL-1, a posterior specifying protein, and contributes to development of the anterior of the embryo. MEX-3 is present throughout the oocyte, 1-cell, and 2-cell embryo. However, MEX-3 is then depleted in the posterior after the second cell division, and PAL-1 is then expressed in the two posterior blastomeres of the 4-cell embryo. MEX-3 is rapidly depleted from the entire embryo after the 8-cell stage. This degradation is location and time specific, and thus hypothesized to be caused by the UPS. MEX-3 is hypothesized to be targeted for degradation by a specific E3 ubiquitin ligase, and knockout of this protein should result in increase in universal MEX-3 expression in the early embryo. This study sought to determine the MEX-3 specific E3 ubiquitin ligase(s). Putative E3 ubiquitin ligases expressed during early embryonic development were knocked out in C. elegans, and phenotypes were determined. Of the knocked-out ligase genes, only one, ZK858.4 caused embryonic lethality at both 15˚ and 24˚ C. However, fluorescence microcopy of GFP::MEX-3 demonstrated that ZK858.4 knockout did not appear to increase global MEX-3 concentrations. Determining which protein targets MEX-3 degradation will provide more insight into the molecular mechanisms of determining anterior/posterior patterning in C. elegans early embryonic development.

  • Thumbnail for RNAi induced knockout of zif-1 and of elc-1 E3 ubiquitin ligases affects MEX-3 protein localization during embryo development of Caenorhabditis elegans
    RNAi induced knockout of zif-1 and of elc-1 E3 ubiquitin ligases affects MEX-3 protein localization during embryo development of Caenorhabditis elegans by Heng, Peter

    PAL-1 is a protein that regulates posterior development of Caenorhabditis elegans embryos. Although pal-1 mRNA is present throughout the entire embryo, the PAL-1 protein is only transcribed in the posterior end of the nematode worm. MEX-3, a RNA binding protein, binds to the pal-1 mRNA, preventing its translation in the anterior section of the embryo. The MEX-3 protein is essential to maintaining embryo polarity and ensuring that posterior features develop only in the posterior end of the worm. During development, MEX-3 is present throughout the 1-cell and 2-cell embryo stage. MEX-3 is then degraded in the posterior end during the 4-cell stage, allowing the expression of PAL-1 in the two posterior blastomeres. By the 8-cell stage, MEX-3 is depleted from the entire embryo with remnants remaining in germline cells. The ubiquitination pathway is hypothesized to mark MEX-3 for degradation, localizing the protein at various stages of embryo development. This study screened various E3 ubiquitin ligases to determine which ligases are specifically used to mark MEX-3 for degradation during embryo development. Double-stranded RNA was created for selected E3 ubiquitin ligases and then injected into adult worms. This invoked RNA interference (RNAi) of these ubiquitin ligases in the embryos of the adult worms. Knockout of genes D2089.2, F46A9.5, F59B2.6, and Y82E9BR.15 resulted in embryonic lethality. Fluorescence microscopy of GFP::MEX-3 (green fluorescent protein labeled MEX-3) revealed that only F59B2.6 (zif-1 gene) and Y82E9BR.15 (elc-1 gene) knockouts affected MEX-3 localization. Double knockouts of zif-1 and another developmental gene, mex-5, support the hypothesis that zif-1 acts after other regulatory events in MEX-3 localization.

  • Thumbnail for Screening for ubiquitin ligases involved in Caenorhabditis elegans early embryonic development
    Screening for ubiquitin ligases involved in Caenorhabditis elegans early embryonic development by Lombroso, Adam Paul

    The protein ubiquitination system is a targeted protein degradation pathway that is an essential component of cell cycle progression in mitosis and meiosis. Recent evidence indicates that the ubiquitin system is required for the degradation of zinc finger proteins that play important roles in embryogenesis. It is possible that the ubiquitin system regulates other proteins involved in early embryonic development by controlling which proteins are degraded, and thereby influencing cell fates. In Caenorhabditis elegans (C. elegans), there is a single ubiquitin activating enzyme, which has a well-understood function. The twenty-two ubiquitin conjugating enzymes in C. elegans have been researched to a moderate extent. Finally, there are believed to be about six hundred ubiquitin-protein ligases. Most of these ubiquitin ligases’ exact functions, the proteins they target, remain unknown. Ubiquitin ligases are perhaps the most interesting enzymes in the ubiquitin system because they determine which proteins are targeted for degradation. Two important proteins involved in the embryonic development of C. elegans are posterior alae defective 1 (PAL-1) and muscle excess 3 (MEX-3). PAL-1 is a homeodomain transcription factor protein that is required to specify posterior cell fates. MEX-3 is an RNA-binding protein that binds to pal-1 mRNA in the anterior cells and restricts the translation of PAL-1 to the posterior cells of the embryo, and thereby influences anterior cell fates. Both pal-1 mRNA and MEX-3 protein are present throughout newly fertilized embryos, but by the four-cell stage MEX-3 is depleted in posterior cells and can only bind to pal-1 mRNA in anterior cells, preventing the translation of PAL-1 in these cells. It is thought that MEX-3 depletion in the posterior cells is due to it being targeted by unknown ubiquitin ligases and degraded by the 26S proteasome. Research shows that two homologous mRNA binding proteins (MEX-5 and MEX-6) protect MEX-3 from inactivation and degradation in the anterior, allowing for the repression of PAL-1 translation. One major unanswered question is the identity of the ubiquitin ligase(s) that targets MEX-3 for degradation in the posterior of the embryo. This study attempts to answer that question. RNA interference screening of ubiquitin ligases that are expressed during embryonic development has permitted the identification of 20 ubiquitin ligases that do not target MEX-3 for degradation. Screening of additional ubiquitin ligases may lead to a better understanding of the regulation of many key proteins. By understanding more about the interactions between MEX-3 protein and pal-1 mRNA and how they are regulated, we will learn more about how embryonic development unfolds and what can potentially go wrong.

  • Thumbnail for A novel mutation in the daf-19 gene affects ciliated neuron development in C. elegans
    A novel mutation in the daf-19 gene affects ciliated neuron development in C. elegans by Wells, Kristen Lynn

    The Caenorhabditis elegans male and hermaphrodite nervous systems display sexually dimorphic development characterized, in part, by the presence of 8 hermaphrodite-specific neurons and 89 male-specific neurons. We are interested in identifying the genes and molecular mechanisms that govern sex-specific neural development in C. elegans. Through a mutagenesis screen using a pkd-2::GFP reporter to label male-specific neurons, we recovered several mutants that display defects in sex-specific neural development. Males carrying the sm129 mutation lack pkd-2::GFP expression in the male-specific CEM neurons that are involved in mate finding. Genetic epistasis experiments suggest that CEM neurons are improperly specified or differentiated. We cloned the sm129 mutation and determined that it is an allele daf-19 based on three pieces of evidence: (1) RNAi of daf-19 phenocopies sm129, (2) sm129 fails to complement a daf-19 null mutation, and (3) we found a mutation in daf-19 that likely affects splicing. We are also testing to see if sm129 mutants can be rescued by adding a wild type copy of daf-19. daf-19 encodes an RFX transcription factor that activates genes required for sensory cilia function in ciliated neurons such as the CEMs. daf-19 null mutants lack all sensory cilia, have sensory defects, and display a constitutive dauer phenotype (worms enter an alternative part of the lifecycle associated with starvation survival). We are currently investigating how this mutation affects ciliated neurons such as CEMs but does not affect dauer formation.