Unveiling the RNA Modification Landscape Through Bioanalytical and Biochemical Analyses

Abstract

Cells face the daunting task to synthesize the correct number of proteins with high fidelity. One mechanism to facilitate this is through the chemical modifications of proteins and nucleic acids. These moieties expand the topologies and chemistries available to biomolecules by altering their biogenesis, localization, function, and stability. Specifically, RNA is post-transcriptionally modified with over 150 different chemical moieties that range from simple methylations and isomerizations to RNA – small molecule conjugates. While modifications were believed to be primarily limited to non-coding (ncRNAs); recent advancements in next-generation RNA sequencing (NGS) and liquid chromatography coupled to tandem mass spectrometry technologies (LC-MS/MS) expanded the RNA modification landscape to protein encoding messenger RNAs (mRNAs). Post-transcriptional modifications of all RNA subclasses affect nearly every stage of the RNA lifecycle, where they play roles in RNA structure and dynamics, stability, RNA – protein interactions, anticodon:codon recognition, and ribosome reading frame maintenance. Thus, it is unsurprising that the dysregulation of RNA modifications or the RNA modifying enzymes has been linked to a host of diseases.

Today, the RNA modification landscape diversity continues to be further unveiled; however, current bioanalytical methods struggle to obtain a comprehensive view of the RNA modification abundance, location, and molecular level consequence in vivo. Over the last 15 years, LC-MS/MS technologies have provided superficial insights into the RNA modifications landscape and its association with cellular stress and disease but continue to struggle to robustly analyze low abundance modifications and sequence complex RNA mixtures. Here, I sought to advance these current LC-MS/MS methodologies to ease the comprehensive interrogation of the RNA modification landscape.

My thesis work primarily consists of the development and implementation of two LC-MS/MS methods that provide separate (but similarly important) information: 1) quantitative LC-MS/MS for global ribonucleoside modification profiling (GRMP) and 2) bottom-up RNA sequencing for complex RNA mixtures. We implemented GRMP to detect four previously unreported modifications in S. cerevisiae mRNA (Chapter 3) and further identified how removing the RNA modification landscape alters the efficacy of hygromycin B to inhibit translation in E. coli and S. cerevisiae (Chapter 4).

Further, we adapted and advanced current bottom-up RNA sequencing pipeline to facilitate the identification of modified sites within complex RNA mixtures. Specifically, adaptations to current RNase digestion schemes enabled the sequencing of highly modified therapeutic siRNAs (Chapter 5) and increased achievable sequence coverages for total tRNA using solely commercially available enzymes (Chapter 7). We further utilized GRMP and bottom-up RNA sequencing to confirm the simplistic RNA modification landscape for the MS2 bacteriophage and provided the first direct comparison between direct nanopore sequencing (DRS) and LC-MS/ to robustly detect modified sites (Chapter 6).

Since the discovery of RNA modifications over 50 years ago, we have gained significant insights into the roles of RNA modifications in vivo. However, our ability to comprehensively interrogate their molecular level consequences has been hindered by the lack of direct bioanalytical methodologies capable of quantifying the abundance and identifying the location of RNA modifications. We sought to fill these technological gaps through the development of quantitative and direct LC-MS/MS methods for global ribonucleoside modification profiling and bottom-up RNA sequencing of complex RNA mixtures. When implemented in tandem with NGS and DRS, these methodologies provide a comprehensive bioanalytical and biochemical pipeline to undertake novel interrogations into the molecular level consequences and dynamics of the RNA modification landscape.