RNA-binding proteins (RBPs) are important for a wide variety of biological processes involved in gene regulation. However, the structural and dynamic contributions to their biological activity are poorly understood. The tristetraprolin (TTP) family of RBPs, including TTP, TIS11b and TIS11d, regulate the stability of mRNA transcripts encoding for key cancer-related proteins, such as tumor necrosis factor- and vascular endothelial growth factor. Biophysical studies have shown that the RNA binding domain, consisting of two CCCH zinc fingers (ZFs), is folded in the absence of RNA in TIS11d and TIS11b. In TTP, however, only ZF1 adopts a stable fold, while RNA is required to completely fold the tandem zinc finger (TZF). The focus of this research was to understand the origin and biological significance of the structural differences observed for the TZF domains of TTP and TIS11d. Three residues were shown to control the affinity for the structural Zn2+ and determine the folding of ZF2 in the absence of RNA. The partially-folded TZF domain of TTP has greater selectivity for RNA sequences than the fully folded TZF domain of TIS11d. The mRNA destabilizing activity of TTP was increased when the partially disordered RBD of TTP was replaced with the fully structured TZF domain of TIS11d. Disruption of the structure and/or dynamics of the TZF domain observed in the disease-associated mutations of TIS11d, P190L and D219E, results in aberrant cytoplasmic localization. This work demonstrates that the extent of RBD folding in the TTP family is important for differential RNA recognition, mRNA turnover, and protein localization in vivo.

Organisms modulate their response to changing environmental conditions through changes in gene expression, and extensive variations in gene expression are prevalent among individuals even within a population. This widespread plasticity and variability of gene expression is thought to play roles in adaptation and drive novel phenotypes in species. Understanding the mechanisms that contribute to such variations requires the analysis of interactions between the genome and its environment and sequence variations within the genome. This work consists of two projects investigating the plasticity and variation of gene expression during post-embryonic development in the nematode C. elegans. In the first study, I examined the response to changes in population density in developmentally arrested L1 larvae. I systematically characterized arrested L1 larvae from low to high densities using single-worm RNA-seq and uncovered that the density of resuspended L1 larvae regulates the expression of hundreds of mRNAs. Further analysis revealed that the physiological response to changes in density is rapid and signaled by a non-canonical daf-22 ascaroside independent pathway. In the second study, I investigated the evolution of gene expression within species using two genetically divergent C. elegans strains (N2 and CB4856). I carried out RNA-seq and allele-specific analysis across six different conditions and four developmental stages, and we examined gene expression divergence using the homozygous parent and F1 hybrid system. This work provides a new experimental model for studying the evolution of gene expression and a comprehensive view of gene expression variation during development in C. elegans.

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博士(工学) / Doctor of Philosophy in Engineering / 同志社大学 / Doshisha University

The neocortex is considered the seat of higher cognitive function in humans. It develops from a sheet of neural progenitor cells, most of which eventually give rise to neurons. This process of cell fate determination is controlled by precise temporal and spatial gene expression patterns that in turn are affected by epigenetic mechanisms including Polycomb group (PcG) regulation. PcG proteins assemble in multiprotein complexes and catalyze repressive posttranslational histone modifications. Their association with neurodevelopmental disease and various types of cancer of the central nervous system, as well as observations in mouse models, has implicated these epigenetic modifiers in controlling various stages of cortex development. The precise mechanisms conveying PcG-associated transcriptional repression remain incompletely understood and are an active field of research. PcG activity appears to be highly context-specific, raising the question of species-specific differences in the regulation of neural stem and progenitor regulation. In this review, we will discuss our growing understanding of how PcG regulation affects human cortex development, based on studies in murine model systems, but focusing mostly on findings obtained from examining impaired PcG activity in the context of human neurodevelopmental disorders and cancer. Furthermore, we will highlight relevant experimental approaches for functional investigations of PcG regulation in human cortex development.

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