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RNAi suppressor screen to identify novel genetic interactors with the sydn-1/pfs-2 neurodevelopmental pathway of Caenorhabditis elegans and construction of plasmid vectors for yeast-two-hybrid and in vivo analyses
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Master of Science (MS)
Rose, Jacqueline K.
Proper development of neuronal circuits are crucial for nervous system functioning. A novel pathway regulating axon and synapse development in Caenorhabditis elegans through nuclear 3'-end polyadenylation of nascent mRNA has recently been uncovered (Van Epps et al., 2010). In this pathway, the protein product of the gene synaptic defective enhancer (sydn-1) negatively regulates polyadenylation factor subunit homolog (PFS-2), an evolutionarily conserved scaffolding protein in a multi-protein complex involved in mRNA 3' -end processing. Although 3'-end processing of mRNA has a regulatory role in many cellular processes, regulation of synapse and axon development via this cellular mechanism has not been characterized. An RNAi screen was performed using C. elegans to identify genetic suppressors in this regulatory pathway. This screen was conducted in a sensitized genetic background that allowed for targeted gene silencing and visual screening for suppression of a neuronal phenotype resulting from SYDN-1 mediated misregulation of PFS-2. We identified seven novel genetic interactors. One of these genes, ELAV-type RNA binding protein family (etr-1), encodes a highly conserved protein that is involved in 3'-end alternative splicing during muscle development. The human homolog of ETR-1, CUGBP, elav-like family member (CELF1), is implicated in the development of the genetic disease myotonic dystrophy. Vectors were produced to further study the function of these proteins in the context of the SYDN-1/PFS-2 pathway. One vector contains etr-1 cDNA fused to a gene encoding GFP, and the other contains CELF1 cDNA fused to the yeast-two-hybrid GAL4 DNA-binding domain, to be used for in vivo localization and yeast-two-hybrid assays, respectively.
Western Washington University
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Lee, Mitchell, "RNAi suppressor screen to identify novel genetic interactors with the sydn-1/pfs-2 neurodevelopmental pathway of Caenorhabditis elegans and construction of plasmid vectors for yeast-two-hybrid and in vivo analyses" (2012). WWU Masters Thesis Collection. 243.