Dissecting the Mechanisms of Quaking-Regulated RNA Processing

Abstract: The overall goal of this research program is to determine how the Quaking-5 isoform (QKI5) of the QKI family of RNA binding proteins regulates gene expression. To achieve this goal, we will determine how QKI5 is regulated, how it executes regulatory functions, and what the consequences of these activities are on the overall gene expression program. Pursuing this goal is the primary mission of my laboratory, and will be an engaging topic upon which I will develop my lab and train post-docs and graduate students. QKI is a conserved regulator of development and tissue-specific gene expression, yet the molecular mechanisms through which its functions are executed remain understudied, particularly for the QKI5 isoform. Its importance is underscored by multiple observations implicating dysregulated QKI function in heart disease, cancer, schizophrenia, and other serious conditions, and our recent discovery that it regulates lineage-specific gene expression. Therefore, this RBP may represent a therapeutic vulnerability for rational, RNA-based drug design, and inform us as to how the earliest cell fate decisions are made. Despite this possibility, there are significant gaps in understanding that must be filled prior to this becoming a reality. This requires us to illuminate the precise molecular mechanisms through which individual target RNAs and the entire transcriptome are regulated. Hence, a major knowledge gap exists in understanding the fundamental mechanisms used by QKI5 to execute its pre-mRNA splicing, transcript localization, and stability/decay functions in RNA processing. This research program will leverage our molecular genetics, biochemical, and genomics expertise to address key gaps in knowledge pertaining to this topic: 1) how upstream regulation of QKI5 impacts its ability to regulate pre-mRNA splicing, and 2) how QKI5 directly and indirectly regulates RNA splicing. Addressing these key gaps will tackle the upstream, intermediate, and downstream impacts on, of, and by QKI regulation. We will use human and mouse cells, and cell extracts to conduct these studies. We propose that QKI5 binding (and thus splicing activity) is influenced by a) competition between pre-mRNA substrates and lncRNAs, and b) protein-protein interactions. Moreover, we propose that regulated splicing by QKI5 is coupled to transcript localization and stability, and that QKI5 influences the core spliceosome machinery. These studies will yield fundamental data that will inform the broader scientific community as to how RBPs can be regulated and regulate gene expression. Overall, these findings will show how complex, multi-step RNA processing is integrated into the overall flow of molecular information in order to maintain cellular homeostasis.