The function and regulation of mRNA 5' nucleotide expansion - PROJECT SUMMARY A major mechanism of gene regulation involves processing mRNAs through splicing and polyadenylation, which diversify transcript structures and influence stability, localization, and translation. These well- characterized co- and post-transcriptional modifications allow a single gene to produce multiple functional mRNA isoforms, expanding regulatory and proteomic complexity. Unlike the 3’ ends of mRNAs, which undergo modifications such as polyadenylation, 5' ends have been thought to be exact genomic copies, determined solely by the site of transcription initiation. My research challenges this view with the discovery of mRNA 5’ nucleotide expansion, a previously unrecognized transcriptional phenomenon in which additional A and U nucleotides are incorporated near transcript 5’ ends in a precise and regulated manner. I found that 5’ expansions occur preferentially in specific mRNAs and are influenced by nutrient availability and metabolic states, particularly UTP metabolism. My findings suggest that 5’ expansion fine-tunes cap-proximal RNA- binding protein (RBP) interactions and modulates the translation of Terminal Oligopyrimidine (TOP) mRNAs, a critical subset of transcripts regulated by the mTOR pathway and linked to cell growth and cancer. Despite its evolutionary conservation and regulatory potential, the molecular mechanisms and functional consequences of 5’ expansion are largely unknown. This proposal aims to define the molecular basis, regulatory mechanisms, and physiological impact of mRNA 5’ expansion. In the K99 phase, I will focus on elucidating how mRNA 5’ expansion alters RBP interactions using TurboID-tDeg proximity labeling, focusing on LARP1 and its role in translational repression (Aim 1). I will also investigate how UTP metabolism regulates 5’ expansion and its impact on translation, integrating nucleotide mass spectrometry, transcriptome-wide 5’ mapping, and ribosome profiling to determine how nucleotide availability dictates translational control (Aim 2). In the R00 phase, I will employ the first mRNA 5’ end-focused CRISPR-based screen to identify the genetic regulators of 5’ expansion (Aim 3). To support my transition to independence, I will receive advanced training in transcriptional regulation, nucleotide metabolism, and functional genomics under the mentorship of Dr. Samie Jaffrey and an interdisciplinary advisory committee. I will gain expertise in proximity labeling mass spectrometry, CRISPR screen, and metabolic assays, while refining professional skills in grant writing, scientific communication, and laboratory leadership. This advanced training in transcriptional regulation, nucleotide metabolism, and high- throughput genomics, combined with my research expertise, will position me as a strong candidate for a tenure-track faculty position investigating the molecular mechanisms of gene regulation.
