2026 Theses Doctoral

Investigating the protein-protein interactions of RNA Binding Proteins throughout the mRNA life cycle

Street, Lena A.

Post-transcriptional regulation of gene expression is orchestrated by RNA-binding proteins (RBPs), which modulate all aspects of the RNA life cycle including splicing, localization, translation, and decay. Although early research has often studied RBPs as individual regulators, mainly due to technical limitations, it is becoming clear that RNA molecules are generally bound by several RBPs whose coordination directs their fate. These combinatorial interactions produce complex, context-dependent post-transcriptional regulatory networks (PTRNs) whose outcomes are difficult to predict as RBPs may switch function in response to cell conditions, subcellular localization, or post-translational modification, further complicating the understanding of RNA regulation. To begin to address one element of these PTRNs - specifically the protein-protein interactions (PPIs) of RBPs - this dissertation presents a large-scale map of the PPIs of RBPs throughout the mRNA life cycle and a novel high-throughput method to capture PPIs by immunopurification followed by mass spectrometry (IP-MS) that combines pooled IP-MS with sparse signal reconstruction.

In Chapter 1, we review new technological advances transforming our ability to map and interpret PTRNs. Novel multiplexed methods allow parallel profiling of the RNA binding patterns of several RBPs in parallel, whereas more efficient interaction proteomics studies reveal protein–protein interactions and changes of those in distinct biological settings. Complementary RNA-targeting pulldown and single-molecule imaging strategies enable real-time and single-cell-resolution visualization of RNP assembly and dynamics. Overall, these approaches set the stage for future decryption of the spatiotemporal structure of PTRNs and reveal how RBP interactions coordinate sets of RNAs (RNA modules or operons) to collectively regulate them in response to physiological demands. In addition to describing these systems-level approaches to study RBP interaction dynamics, we also outline the next analytical and experimental innovations that could shape our knowledge about RBP function. We believe that a systems-level understanding of RBPs as dynamic, integrated components of multiscale regulatory regimes will be required to unlock the full complexity of gene expression control and its disruption in disease.

Chapter 2 presents an RNA-aware, RBP-centric PPI map across the mRNA life cycle in human cells by IP-MS of ∼100 endogenous RBPs with and without RNase, augmented by size exclusion chromatography-mass spectrometry (SEC-MS). We identify 29,544 interactions between 1,125 proteins and determine that 73% of the IP-MS-identified interactions are RNA-regulated. Our interactome links many proteins, some with unknown functions, to specific mRNA life cycle stages, with nearly half associated with multiple stages. We demonstrate the value of this resource by characterizing novel mRNA regulatory functions of enhancer of rudimentary homolog (ERH) and small nuclear ribonucleoprotein U5 subunit 200 (SNRNP200).

In Chapter 3, we present SPRINT (SParse Reconstruction of INTeractions) integrated experimental and computational platform to accelerate the discovery of PPIs that combines an innovative antibody or lysate pooling scheme with a novel sparse signal reconstruction algorithm, enabling pooled PPI capture experiments. This approach significantly boosts throughput by an order of magnitude, while reducing sample input requirements and maintaining the same amount of hands-on time as standard IP-MS experiments. We demonstrate that the method is comparable to standard individual IP-MS experiments for pooling of both antibodies and lysates.

Chapter 4 discusses IP-MS experimental and analytical considerations, and presents an optimized framework for performing IP-MS experiments.

Chapter 5 provides an outlook on open biological questions and types of methodological approaches that could be applied in future to further our understanding of ribonucleoprotein (RNP) regulation and its importance in post-transcriptional gene expression regulation.

Overall, our findings contribute to the evolving picture of how RBPs mediate post-transcriptional regulation, and provide a novel method to build PPI networks at scale which can be applied to enhance our understanding of how RBPs function in health and disease.