Studies on Endogenous Transfer RNA Fragments as Guides for Messenger RNA Targeting by Ribonuclease P
Degree
Doctor of Philosophy (PhD)
Department
Department of Biological Sciences
Document Type
Dissertation
Abstract
Transfer RNA-derived fragments (tRFs) represent a diverse class of small noncoding RNAs generated through the regulated cleavage of tRNAs. Although tRFs have been implicated in translational control, cellular stress responses, and post-transcriptional gene regulation, the molecular mechanisms underlying many of these functions remain incompletely understood. This dissertation investigates a previously unrecognized role for 3′ tRNA halves as endogenous guides for ribonuclease P (RNase P)-mediated cleavage of messenger RNA.
Nuclear RNase P removes the 5′ leader sequence from precursor tRNAs. We hypothesized that 3′ tRNA halves function as endogenous external guide sequences; by base pairing with complementary mRNA sequences to form tRNA-like hybrid structures that can be recognized and cleaved by RNase P. To test this hypothesis, we generated a custom polyclonal antibody against the essential RNase P-specific subunit Rpp21 and coupled crosslinked immunoprecipitation with deep sequencing (CLIP-seq) to isolate RNAs directly bound by RNase P.
Subsequent chimeric read analysis of CLIP libraries via a custom bioinformatic pipeline identified numerous candidate 3′ tRNA half:mRNA interactions in vivo. Representative synthetic tRNA:mRNA hybrids reconstructed from these candidates were recognized and cleaved by RNase P in vitro at positions analogous to canonical pre-tRNA processing sites. Structural and enzymatic analyses demonstrated that successful cleavage depends on the formation of a structurally competent tRNA-like substrate. Cellular assays further showed that depletion of the RNase P subunit Rpp21 increased the abundance of candidate target transcripts, whereas glutamine deprivation, a condition associated with stress-induced tRNA fragmentation, reduced target mRNA levels. Additionally, we performed deep sequencing analysis of RNA with 5’ phosphate ends from total RNA extracted from cells transfected with either a control oligo or a synthetic 3’ tRNA half to identify in vivo tRF target sites in support of our hypothesis.
These findings support a model in which endogenous 3′ tRNA halves direct site-specific RNase P-mediated cleavage of mRNA through the formation of tRNA-like hybrid substrates. This work expands our understanding of the functional repertoire of tRNA-derived fragments and provides support for a mechanistic link between stress-induced tRNA fragmentation and selective regulation of mRNA abundance as a previously unrecognized mode of post-transcriptional gene regulation in eukaryotic cells.
Date
7-17-2026
Recommended Citation
Eckhardt, Allison V., "Studies on Endogenous Transfer RNA Fragments as Guides for Messenger RNA Targeting by Ribonuclease P" (2026). LSU Doctoral Dissertations. 7136.
https://repository.lsu.edu/gradschool_dissertations/7136
Committee Chair
Vourekas, Anastasios
LSU Acknowledgement
1
LSU Accessibility Acknowledgment
1