UC Riverside

Chemical modifications on RNA play critical roles in post-transcriptional gene regulation. Like DNA epigenetic modifications, e.g., 5-methyl-2’-deoxycytidine (5-mdC) and 5-hydroxymethyl-2’-deoxycytidine (5-hmdC), they provide an additional layer of regulation by suppressing or enhancing gene expression. The various types of RNA, including ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA), contain over 100 chemical modifications that make up the epitranscriptome. As more modified ribonucleosides are identified, so are the demands for sensitive and accurate methods for evaluating the epitranscriptome and its relationship with disease pathology. In chapter 2, I developed a highly sensitive method for profiling post-transcriptional modifications in RNA by using nano-liquid chromatography-multi-stage mass spectrometry (nLC-MS/MS/MS). This method enabled simultaneous profiling of 31 modified ribonucleosides. To achieve a high-throughput scheduled selected-reaction monitoring, we assigned normalized retention time (iRT) values for each modified ribonucleoside with respect to the canonical ribonucleosides. We utilized the method for the identification of 20 modified ribonucleosides with minimal RNA input. In chapter 3, we identified a RNA 5-methylcytidine (m5C) reader protein, YTHDF2 based on quantitative proteomics. Even though m5C is abundant in all RNA types, the exact role of m5C remains elusive. YTHDF2 uses the same binding pocket to bind N6-methyladenosine (m6A) and m5C. In addition, global m5C levels are altered in HEK293T cells upon CRISPR-mediated knockout of YTHDF2 gene. In chapter 4, I developed a method to identify and quantify a novel RNA modification, 5-hydroxymethyluridine (5-hmrU) by using LC-MS/MS. I quantified 5-hmrU in total RNA and mRNA samples isolated from mouse tissues, cultured cells, and Drosphila melangaster. In addition, we identified ten-eleven translocation (Tet) enzymes, known oxidizers of 5-methylcytosine (m5C) to 5-hydroxymethylcytosine (5-hmC) in DNA and RNA, to also mediate the oxidation of 5-methyluridine (m5U) to 5-hmrU. In chapters 5 and 6, we employ the epitranscriptome global profiling method to evaluate the role of the epitranscriptome on environmental toxin exposure and disease. Chapter 5 focuses on evaluating how levels of ribonucleosides are affected by air pollution and tobacco smoke. Total RNA samples isolated from peripheral blood from truck drivers and office workers were taken for LC-MS/MS/MS analysis. I found that smoking in men was associated with a decrease in global m6A level compared to non-smokers. By contrast, black carbon exposure from air pollution resulted in an increase in m6A level. In chapter 6, I evaluated the role of m6A and its regulatory proteins upon infection with HIV-1 and SARS-CoV-2. I observed similarities in modified ribonucleoside profile in viral RNA genomes of HIV-1 and SARS-CoV-2. In addition, the two viruses may be acting on similar epitranscriptomic pathways to mediate N6,2’-O-dimethyladenosine (m6Am) in mRNA of host cells.