Honors Capstone Project
Date of Submission
Arts and Science
Capstone Prize Winner
Won Capstone Funding
Sciences and Engineering
Germline stem cell proliferation in C. elegans is regulated by the distal tip cell (DTC) located at the distal end of each gonad arm. The DTC signals germ cells, through a Notch-type signaling pathway, to proliferate. As germ cells move farther away from the DTC, they transition into meiosis and produce gametes. There are two Notch-type receptors in C. elegans, GLP-1 and LIN-12, which play a role in regulating cell fate decisions. Whenever GLP-1 function is lost, all the germ cells exit mitosis and enter meiosis. If this happens early in development, then the animal is sterile because it does not have a large enough germ cell population to maintain gamete production. On the other hand, if there is a glp-1 gain-of-function (gf) mutation, then the animal develops a distal tumor because the germ cells over-proliferate. In normal development, a healthy balance between mitosis and meiosis is needed for proper germline function. Defects in Notch signaling have been implicated in human developmental disorders and cancers underlining the importance of better understanding how cells regulate this pathway.
Our work identified UBR-5, a HECT-type E3 ubiquitin ligase, as a negative regulator of glp-1 signaling.1 E3 ligases are enzymes involved in attaching ubiquitin to target proteins, marking them for degradation. UBR-5 was shown to negatively regulate GLP-1/Notch signaling in the adult gonad.1 Furthermore, knocking down expression of ubr-5 in a weak, temperature-sensitive, glp-1(ar202ts) gain-of-function background enhanced the gain-of-function phenotype. ubr-5(RNAi) in a rrf-1(0);glp-1(ar202ts) background, where RNAi is defective in the soma, also enhanced the gain-of-function phenotype. This suggests that ubr-5 acts in the germ line to regulate turnover of the Notch receptor rather than in the signaling cell to regulate turnover of the ligand. Redundancy tests with a second E3-ligase, SEL-10, provided evidence that these two E3 ligases act synergistically in the early embryo.
Currently we are aiming to identify specific targets of UBR-5 in the GLP-1/Notch signaling pathway. Evidence suggests that SEL-10, which negatively regulates LIN-12/Notch signaling in C. elegans, is involved in proteasomal degradation of the Notch intracellular domain (ICD)2. We hypothesize that UBR-5 is regulating GLP-1 signaling in an analogous manner by binding to the ICD. Preliminary protein blot data with 3xFLAG-tagged GLP-1 ICD supports this hypothesis. In addition, we are attempting to tag UBR-5 with the 3xFLAG epitope using the CRISPR/Cas9 genome editing system. This reagent will be used for immunoprecipitation and mass spectrometry analysis of UBR-5 binding partners on a more global scale.
Gu, Anniya, "UBR-5, a conserved HECT-Type E3 ubiquitin ligase, negatively regulates notch-type signaling in Caenorhabditis elegans" (2017). Syracuse University Honors Program Capstone Projects. 1053.
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