RNA Recognition by the Caenorhabditis elegans Embryonic Determinants MEX-5 and MEX-3: A Dissertation

Abstract

Post-transcriptional regulation of gene expression is a mechanism that governs developmental and cellular events in metazoans. In early embryogenesis, transcriptionally quiescent cells depend upon maternally supplied factors such as RNA binding proteins and RNA that control key decisions. Morphogen gradients form and in turn pattern the early embryo generating different cell types and spatial order. In the nematode Caenorhabditis elegans, the early embryo relies upon several RNA binding proteins that control mRNA stability, translation efficiency, and/or mRNA localization of cell fate determinants essential for proper development. MEX-5 and MEX-3 are two conserved RNA-binding proteins required to pattern the anterior/posterior axis and early embryo. Mutation of either gene results in a maternal effect lethal phenotype with proliferating posterior muscle into the anterior blastomeres (Muscle EXcess). Several cell-fate determinants are aberrantly expressed in mex-5 and mex-3 embryos. Both proteins are thought to interact with cis-regulatory elements present in 3’-UTRs of target RNAs controlling their metabolism. However, previous studies failed to demonstrate that these proteins regulate maternal transcripts directly. This dissertation presents a thorough assessment of the RNA binding properties of MEX-5 and MEX-3. Quantitative biochemical approaches were used to determine the RNA binding specificity of both proteins. MEX-5 has a relaxed specificity, binding with high affinity to linear RNA containing a tract of six or more uridines within an eight-nucleotide window. This is very different from its mammalian homologs Tristetraprolin (TTP) and ERF-2. I was able to identify two amino acids present within the MEX-5 RNA binding domain that are required for the differential RNA recognition observed between MEX-5 and TTP. MEX-3 on the other hand is a specific RNA binding protein, recognizing a bipartite element with flexible spacing between two four-nucleotide half-sites. I demonstrate that this element is required for MEX-3 dependent regulation in vivo. Previous studies only identify a small number of candidate regulatory targets of MEX-5 and MEX-3. The defined sequence specificity of both proteins is used to predict new putative targets that may be regulated by either protein. Collectively, this study examines the RNA binding properties of MEX-5 and MEX-3 to clarify their role as post-transcriptional regulators in nematode development.