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Abstract
RNA regulation is governed by a multilayered network of RNA-binding proteins (RBPs), chemical modifications, and structural features that together shape transcript stability, translation, and cellular function. Despite the expanding list of RNA modifications and RBPs, technologies for mapping and manipulating these features with precision remain limited. In this dissertation, a suite of next-generation protein engineering and sequencing tools was developed and applied to decode RNA regulation at high resolution. A synthetic, MS2-guided RBP tethering system was first established to modulate mRNA stability and translation in mammalian cells, enabling orthogonal interrogation of RBP functions on RNA transcripts. Next, a TadA-based RNA deamination platform was engineered to map RBP–RNA interactions and m6A sites in vivo with improved specificity and reduced off-target activity compared to APOBEC-based tools. Building on this foundation, eTAM-seq, a quantitative, antibody-free method for transcriptome-wide m6A profiling, was optimized and expanded to achieving enhanced deamination efficiency, reduced sequence-context bias, and ultra–low-input compatibility. Application of this upgraded platform reveals high-resolution methylation landscapes in diverse human cell lines and mouse embryonic tissues. eTAM-seq was further adopted for low-throughput, site-specific quantification of m6A installation and removal, enabling precise measurements of methylation dynamics in targeted perturbation experiments. Finally, computational protein design was incorporated to evolve TadA variants with improved thermostability and activity for future sequencing applications. Together, these methods advance the toolkit for studying RNA modifications and RBP biology, offering quantitative and high-resolution strategies to interrogate the epitranscriptome and its regulatory factors.