Posttranscriptional Gene Regulation: From Global Models to Functional Mechanisms

<p>Precise regulation of the complex process of gene expression is essential for all aspects of life, and a large degree of this precision is mediated at the posttranscriptional level. The global and individual mechanisms by which posttranscriptional control is coordinated to maintain or alter levels of gene expression as necessary are not fully understood. Identification of the mRNA target sets of individual RNA binding proteins (RBPs) and characterization of the mechanisms by which RBPs regulate the expression of individual mRNAs provide some insight into the global structure of the posttranscriptional environment. However, few studies have integrated these findings into a global model of posttranscriptional control. We have explored the structure and function of the posttranscriptional regulatory system through a combination of global modeling approaches, global studies of mRNA translation and decay, and mechanistic studies of the function of individual RBPs, specifically HuR and Pum1. </p><p>By combining RBP-mRNA association data and transcription factor (TF) target data from separate global studies in yeast, we developed an integrated model of gene expression regulation. Evaluation of this model indicates that posttranscriptional regulation may be responsible for substantially greater contributions to the overall gene expression program than transcriptional regulation. Further, we identified a self-regulatory feature of the posttranscriptional network that suggests a `regulators of regulators' structure may be a defining feature of the posttranscriptional control of gene expression.</p><p>Additionally, we explored the mechanisms and functional consequences of the dynamic association between an RBP, HuR, and its target RNAs through a combination of modeling and experimental approaches, including polysome profile analysis and global measurement of RNA stability. Our model indicates that changes in total mRNA abundance are insufficient to fully explain the dynamics of association between HuR and its targets, suggesting a role for competition and cooperation with other RBPs. Our findings also indicate that HuR may play a role in inhibition of translation in a dynamic immunological system (T cell activation). </p><p>Finally, we performed a mechanistic analysis of the function of the Pum1 RBP and characterized the role of this protein in the translational regulation of several important target mRNAs through the use of luciferase reporter assays. We also provided the first in vivo evidence of a role for specific regions of the Pum1 protein in the mediation of gene expression. However, we were unable to verify previous in vitro reports of a role for Pum1 in the control of translation elongation of verified in vivo mRNA targets, suggesting that Pum1's regulatory function may be context dependent.</p><p>Ultimately, the approaches and findings in this study will provide a framework for the development of a global integrated model of posttranscriptional control. Through the iterative development of models and experimentation, hypotheses can be generated and, tested in the laboratory, and the results of these experiments will then further improve the development of the models. An integrated approach of this type will be necessary to fully understand the highly complex and interconnected nature of the gene expression regulatory system.</p>Dissertatio