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Title: Rediscovery and Investigation of an Old RNA Modification - Dihydrouridine - with New Chemical Biology Tools
Authors: Dai, Wei
Advisors: Kleiner, Ralph R.K.
Contributors: Chemistry Department
Keywords: dihydrouridine
RNA modification
RNA sequencing
RNA-modifying enzymes
Subjects: Chemistry
Molecular biology
Issue Date: 2023
Publisher: Princeton, NJ : Princeton University
Abstract: Analogous to DNA and proteins, RNAs can be post-synthetically modified with a plethora of chemicalmodifications. RNA modifications have been known for decades, and the initial research endeavors have focused on the more abundant non-coding RNAs, including ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs). Thanks to recent breakthroughs in mass-spectrometry and high-throughput sequencing based methods, investigations of post-transcriptional modifications on messenger RNAs (mRNAs) became possible. Recent studies on N6-methyladenosine (m6A) have revolutionized our understanding of RNA modifications, transforming their perception from static entities with basic structural and conformational roles to dynamic entities with critical regulatory functions. Growing efforts have been made to decipher the epitranscriptomic codes beyond m6A. Here, we describe new strategies to profile RNA-modifying enzymes and map modification sites throughout the transcriptome. First, we developed an approach to systematically identify mRNA-specific modifying enzymes with the activity-based probe 5-fluorocytidine (5-FCyd), named RNA-mediated activity-based protein profiling (RNABPP). We successfully profiled known 5-methylcytidine (m5C) and 5-methyluridine (m5U) methyltransferases. Additionally, a putative dihydrouridine synthase (DUS) – dihydrouridine synthase 3-like (DUS3L) – was also identified. We established the enzymatic activity of DUS3L as a human DUS enzyme using quantitative nucleoside liquid chromatography-mass spectrometry. Furthermore, we mapped its modification sites using individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP)-based sequencing method. Global translation and cell viability assays have also been performed as preliminary examinations of the biological functions of dihydrouridine (DHU) and DUS3L. The second half of the thesis focuses on DHU and its writer enzymes. Chemical-based sequencing methods have been developed to sequence DHU transcriptome-wide using reverse transcription (RT) stop and RT mutation signatures. Differential expression analysis of tRNAs and proteins in DUS1L/DUS2L/DUS3L-KO cells has been conducted to better understand their impacts on gene expression. We also performed dual luciferase reporter assay and HiBiT complementation experiments to further interrogate the roles of DHU and DUS enzymes in protein translation. Taken together, our research provides novel tools for systematic identification of RNA-modifying enzymes and transcriptome-wide mapping of DHU. Our findings have also advanced the understanding of the biological functions of DHU and DUS enzymes, which will facilitate the decryption of epitranscriptomic codes.
Type of Material: Academic dissertations (Ph.D.)
Language: en
Appears in Collections:Chemistry

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