This thesis explores the intricate regulatory mechanisms involving embedded tRNA genes within the introns and 3' untranslated regions (UTRs) of protein-coding genes during neural development. Transfer RNAs (tRNAs), essential non-coding RNAs integral to mRNA translation, have recently been found to significantly regulate neighboring protein-coding genes. This study focuses on four tRNAGly(GCC) genes embedded within the introns of VAC14 and the tRNA-Arg-TCT gene within the 3'UTR of HES7, elucidating their regulatory functions. The first chapter employs computational and evolutionary methods to investigate the regulatory role of tRNAGly(GCC) genes within VAC14 introns. Using published DNase-seq, RNA-seq, and ChIP-seq data, I mapped chromatin accessibility changes and transcription factor binding during mouse neuronal differentiation. Functional and comparative genomic analyses revealed that the tRNA genes in the first intron appear to function as enhancer elements, and Zic transcription factors play a previously unrecognized suppressive regulatory role in VAC14 gene transcription during neuronal maturation. The second chapter provides an experimental analysis of the transcriptional regulation of VAC14 by its intronic tRNA-Gly-GCC genes. Utilizing CRISPR/Cas9-mediated homology-directed recombination (HDR) in HEK293T cells, I deleted and mutated the tRNA genes to assess their influence on VAC14 expression. ChIP-seq analysis and western blotting demonstrated that these tRNA genes impact VAC14 expression through transcriptional interference, revealing an interaction between Pol II and Pol III. These findings offer new insights into the transcriptional control mechanisms involved in regulating VAC14. The third chapter examines the regulatory potential of the tRNA-Arg-TCT gene within the HES7 3'UTR, hypothesizing its influence on HES7 expression through chromatin architecture modulation and transcriptional interference. Luciferase reporter assays, along with ChIP-seq and RNA-seq data, demonstrated that the UTR-overlapping tRNA-Arg-TCT gene significantly modulates HES7 expression, shedding light on broader regulatory networks essential for neural development and potential impacts on developmental disorders. This thesis provides novel insights into the complex regulatory networks involving tRNA genes and their impact on host protein-coding genes. Understanding these regulatory mechanisms sheds light on pol3-driven RNA gene regulation of key protein-coding genes during neural development, offering potential therapeutic targets for neurodevelopmental disorders.

Event Host: Qiuxia Tang, Ph.D Candidate, Biomolecular Engineering & Bioinformatics

Advisor: Todd Lowe