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Abstract
The central dogma describes the flow of genetic information from DNA to RNA to protein. As the intermediary, RNA is a versatile regulator of gene expression. Chemical modifications on RNA, such as N6-methyladenosine (m6A) and pseudouridine (Ψ), profoundly influence RNA fate and function. Dysregulated RNA modifications are frequently observed in cancer, yet their mechanistic contributions to tumorigenesis remain incompletely understood. My PhD research focused on developing technologies and uncovering mechanisms by which RNA modifications regulate cancer biology and may be leveraged for therapeutic development and cancer detection. 1. Development of BIHIND for Ψ detection Ψ occurs in rRNA, tRNA, snRNA, mRNA, and lncRNA. While classical CMC-based Ψ mapping relies on RT-stop analysis, it often causes RNA degradation and requires large RNA input, followed by labor-intensive precipitation steps. To overcome these limitations, I developed BIHIND (Bisulfite Incorporation Hindered ligation-based detection), enabling quantitative and high-throughput Ψ detection without reverse transcription. BIHIND-qPCR quantifies Ψ stoichiometry at single-nucleotide resolution, and BIHIND-seq profiles Ψ transcriptome-wide. We validated BIHIND by monitoring dynamic Ψ changes upon pseudouridine synthase depletion. 2. Mechanistic role of PUS7-mediated Ψ in transcription and CRC apoptosis Although Ψ is known to regulate RNA splicing and translation, its role in transcription was unclear. I discovered that PUS7 catalyzes pseudouridylation of 7SK snRNA, a regulator of RNA polymerase II pausing. Loss of PUS7 reduces Ψ on 7SK, leading to release of P-TEFb, increased Pol II Ser2 phosphorylation, and enhanced transcription elongation. In colorectal cancer (CRC) cells, modulation of 7SK pseudouridylation—either by PUS7 depletion or dCas13b-guided site-specific Ψ editing—induces KLF6/DDIT3-mediated apoptosis and sensitizes cells to 5-FU. 3. Targeted degradation of FTO for AML therapy m6A is the most abundant mRNA modification and is essential for AML stem cell maintenance. We designed FP54, a potent degrader of the m6A eraser FTO based on the inhibitor FB23-2. FP54 selectively degrades FTO and suppresses AML progression in xenograft models more effectively than FB23-2. Mechanistically, FTO loss disrupts ribosome biogenesis and global translation, with especially strong effects on DNA-replication-related mRNAs. These findings support selective degradation of m6A regulators as a therapeutic strategy for AML.