The Functional Characterization of tRNA-derived Fragment tRF-3021a in Tumor Progression

Small noncoding RNAs are increasingly recognized as key regulators of tumor programs, yet the functional contributions of tRNA-derived fragments (tRFs) in tumor progression, particularly in gliomas, remain poorly defined. This dissertation focuses on tRF-3021a, a tRNA-Ala–derived small RNA. In an initial analysis of TCGA small RNA-seq data, we found that higher expression of tRF-3021a, tRF-3009a or tRF-3030a, are associated with poorer prognosis in low-grade glioma patients. The overall goal of this work is to define the biological function of tRF-3021a and elucidate the cellular pathways through which it supports GBM aggressiveness.

Using gain-of-function and loss-of-function studies in GBM cell lines, we show that depletion of tRF-3021a suppresses tumor cell growth and invasive behavior and is associated with enhanced apoptosis and increased DNA damage. Importantly, tRF-3021a knockdown markedly reduces global protein synthesis, as measured by puromycin labeling. This occurs earlier than the apoptotic changes and is associated with activation of stress-response pathways. Transcriptomic profiling (RNA-seq) reveals broad remodeling of gene-regulatory programs after tRF-3021a depletion, including signatures consistent with impaired translation being the early and apoptosis the late response. Functional rescue experiments further support this model. Restoring tRF-3021a activity reverses key phenotypes, indicating that tRF-3021a specifically helps maintain a pro-survival, pro-growth state in glioma cells.

Finally, we report an unexpected observation that parental tRNAs (including the tRNA source of tRF-3021a) appear increased in RT-based assays after tRF-3021a depletion, raising the possibility that altered tRNA modification or charging states influence tRNA detection and/or translational output. Together, these findings identify tRF-3021a as an oncogenic regulatory small RNA in GBM that supports invasion and proliferation, in part by sustaining efficient protein synthesis and limiting stress-activated, pro-apoptotic programs, and they establish a framework for future mechanistic studies aimed at pinpointing the direct molecular partners and minimal functional determinants of this tRF.