Sequence and Target Specificity of the C. elegans Cell Fate Specification Factor POS-1: A Dissertation
AuthorsFarley, Brian M.
Faculty AdvisorSean P. Ryder, Ph.D.
Academic ProgramBiochemistry and Molecular Pharmacology
UMass Chan AffiliationsBiochemistry and Molecular Pharmacology
Document TypeDoctoral Dissertation
KeywordsCaenorhabditis elegans Proteins
3' Untranslated Regions
Amino Acids, Peptides, and Proteins
Animal Experimentation and Research
Biochemistry, Biophysics, and Structural Biology
Cell and Developmental Biology
Nucleic Acids, Nucleotides, and Nucleosides
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AbstractIn most metazoans, early embryogenesis is controlled by the translational regulation of maternally supplied mRNA. Sequence-specific RNA-binding proteins play an important role in regulating early embryogenesis, yet their specificities and regulatory targets are largely unknown. To understand how these RNA-binding proteins select their targets, my research focused on the C. elegans CCCH-type tandem zinc finger protein POS-1. Embryos lacking maternally supplied POS-1 die prior to gastrulation, and exhibit defects in the specification of pharyngeal, intestinal, and germline precursor cells. To identify the regulatory targets that contribute to the POS-1 mutant phenotype, we set out to determine the sequence specificity of POS-1 in vitro, and then use this information to identify regulatory targets in vivo. Using a candidate-based search, we identified a twelve-nucleotide fragment of the mex-3 3' untranslated region (3' UTR) to which POS-1 binds with high affinity. Using quantitative fluorescent electrophoretic mobility shift assays, I determined the affinity of the RNA-binding domain of POS-1 for a panel of single nucleotide mutations of this sequence, and then defined a consensus binding element based on this dataset. POS-1 recognizes the degenerate element UAU 2-3 RDN 1-3 G, where R is any purine (adenosine or guanine), and D is any base except cytosine. A bioinformatics analysis revealed the presence of this element in approximately 40% of C. elegans 3' UTRs, suggesting that POS-1 is capable of binding to and perhaps regulating many transcripts in vivo. POS-1 binding sites alone are not sufficient to pattern the expression of a reporter, suggesting that other factors may contribute to POS-1 specificity. To address the mechanism of POS-1-mediated translational regulation, I investigated the translational regulation of the C. elegans Notch homolog glp-1. Previous work demonstrated that glp-1 translation is repressed in the early embryo in a POS-1-dependent fashion, though it was not clear if this regulation was direct. The glp-1 3' UTR contains two POS-1 binding sites within five nucleotides of each other, and these sites are within a thirty nucleotide region of the 3' UTR required for proper spatiotemporal translation of glp-1. The POS-1 sites overlap with a negative regulatory element that is recognized by GLD-1, and a positive regulatory element recognized by an unknown factor. Both POS-1 and GLD-1 bind to an RNA containing these sites in vitro, and POS-1 competes with GLD-1 for binding. Both proteins are required for translational repression of a glp-1 3' UTR reporter in embryos. Furthermore, only one of the two POS-1 binding sites is required for repression, and the required site is wholly contained within a previously characterized positive regulatory element. Based on this, we propose that POS-1 does not regulate its targets by recruiting regulatory machinery, but instead by competing with factors that do. Thus, sites of POS-1 regulation are highly context dependent, which may contribute to POS-1 specificity.
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/31978
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