My thesis work is aimed at integrating high-throughput biochemical readouts to understand the effect that particular RNA-binding proteins have on their targets' metabolism. I studied several RNA-binding proteins in diverse model organisms. Along the way I have gained insight into the characteristics of target sites for two families of proteins: Argonaute and RBFOX proteins and learned about novel mechanisms they use to control their targets' fate.
In this dissertation, I will present three of my articles that summarize the methods and content of my work. In the first chapter I give an overview of methods I used to quantify the ideal depth of coverage for sequencing experiments in order to sufficiently capture a desired level of complexity. This included the development of novel computational approaches to quantify RNA abundance and splicing with sequencing tools. This was applied to a human model of prostate cancer, LNCaP cells upon stimulation with an androgen compound.
To closely examine mechanisms of miRNA regulation, we generated genome wide maps of the Argonaute protein ALG-1's binding in C. elegans. We found that there exists a large potential for non-canonical associations of ALG-1/miRNA complexes with their targets. Among the surprises we encountered, ALG-1 binds in coding exons but does not seem to repress gene expression when bound there, and we also found an auto-regulatory repression by ALG-1 on miRNA pathway components.
Finally, I turned my attention to the RBFOX family of proteins that is known for their role in RNA splicing regulation. In the work presented in chapter 4, we elucidated a new molecular mechanism whereby RBFOX proteins can regulate RNA splicing from very distant sites. These RNA-bridges, as we have called them because they link RNA regulators with regulated sites via RNA structures, appear to be a common feature of alternatively spliced exons and the regulation of RNA structures like this may be important for dictating splicing outcomes. The application of this knowledge that distant binding sites are functional and that they are mediated by RNA structures is immediately relevant to the design of novel therapeutics for diseases that arise from defects in RNA splicing.
To put the goals of my thesis broadly, I approach two questions: what are the mechanisms of RNA-binding protein targeting, and what are the effects of RNA-binding proteins on their direct targets?