Development and Application of Proteomic and Genomic Methods in RNA Biology

Citation

Honson, Drew D. (2025) Development and Application of Proteomic and Genomic Methods in RNA Biology. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/08dq-1q63. https://resolver.caltech.edu/CaltechTHESIS:12092024-181948039

Abstract

This thesis contains three interrelated projects. Chapter 1 describes the development of a novel RNA-proteomics method: RNA-antisense purification followed by mass spectrometry (RAP-MS 2.0). It contains results of a RAP-MS 2.0 study profiling the protein partners of eight RNAs (7SL, 7SK, RMRP, U1, U2, U6, U7, and Xist) as well as a detailed, step-by-step protocol for the new method. Chapter 2 describes a quality control method for Split and Pool Identification of RBP targets (SPIDR). It identifies an underappreciated failure point in SPIDR experiments (the equal loading of antibody-IDs onto beads), and describes a method for monitoring and resolving this issue. Chapter 3 describes the application of SPIDR to ribosome-associated proteins in human cells. The study both validates existing structures and identifies novel interactions between nucleolar proteins and immature ribosomal RNA, and between protein trafficking factors and the large ribosomal subunit.

Thesis Files