Defining the Universe of Functional RNA-Protein Interactions

Citation

Guo, Jimmy Kang (2024) Defining the Universe of Functional RNA-Protein Interactions. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/wbvq-bz46. https://resolver.caltech.edu/CaltechTHESIS:05012024-121056794

Abstract

RNA has been proposed to mediate many central mechanisms of cell biology, including protein recruitment to chromatin, genome structure organization, and gene expression. In most cases, these critical functions have been widely attributed to the proteins to which RNAs bind. One paradigm example of this is the Xist long non-coding RNA, which complexes with many distinct proteins to orchestrate X-chromosome inactivation. Beyond Xist, there are many critical non-coding RNAs (ncRNAs) that are not yet functionally characterized because we lack information on what proteins they bind to. In this thesis, Chapter 1 discusses the growing gap between the vast potential of ncRNA functions and what has been demonstrated to be functionally meaningful. We highlight critical discrepancies between biochemical evidence supporting specific RNA-protein interactions and genetic evidence demonstrating the same interactions are often dispensable for function. Chapter 2 explores previously reported RNA-protein interactions for many chromatin proteins (i.e., PRC2, CTCF, etc.), demonstrating that they do not represent bona fide interactions in cells. We present Covalent Linkage Affinity Purification (CLAP), a method that employs denaturing purification of RNA-protein complexes, showing that CLAP accurately removes false signals that do not occur in vivo, while retaining known RNA-protein interactions. Chapter 3 details a highly multiplexed method of mapping RBPs and their in vivo binding sites across dozens to hundreds of targets within a single experiment. We present Split and Pool Identification of RBP targets (SPIDR), which enables the rapid, de novo discovery of RNA-protein interactions at an unprecedented scale and separates bona fide RBPs from non-RBPs. Using SPIDR, we uncover a previously unknown LARP1 binding site on the 18S ribosomal RNA that is directly adjacent to the mRNA entry channel, which may explain how LARP1 achieves translational control of sequence-specific mRNAs. Finally, Chapter 4 proposes new experimental and analytical approaches to evaluate the potentially wide universe of ncRNA-protein functions at scale. Together, these results provide a comprehensive framework for evaluating RNA-protein interactions and underscore the growing importance of RNA-mediated functions in cell biology.

Thesis Files