A Stress-Induced TRNA Depletion Response Mediates Codon-Based Translational Repression and Growth Suppression

Author

Doowon Huh

Date of Award

2019

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Tavazoie Laboratory

Abstract

Eukaryotic transfer RNAs (tRNAs) can become fragmented upon various cellular stresses, generating tRNA-derived RNA fragments (tRFs). Though this process has been observed for numerous cellular stresses and in many species ranging from plant cells to yeast and human cells, it is still poorly characterized and understood. Such tRNA fragmentation has previously been thought to affect a small fraction of the tRNA pool and was thus presumed to not affect the role of tRNAs in translation. We report that in human cells, oxidative stress can rapidly generate tRFs derived from tyrosyl tRNAGUA—resulting in a significant depletion of the precursor tRNA molecule and mature tRNA while also leading to elevated levels of the tRF. Proteomic and ribosomal profiling of tyrosyl tRNAGUA-depleted cells revealed impaired expression of proteins enriched in its cognate tyrosine codons, comprising growth and metabolic genes. Consistent with these affected pathways, depletion of tyrosyl tRNAGUA or its downstream targets, EPCAM, SCD, or USP3, repressed growth—revealing a tRNA-dependent growth suppressive pathway for oxidative stress response. A synthetic mimetic of the tRF induced upon oxidative stress was used to identify interactions with RNA binding proteins through mass spectrometry. High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) of hnRNPA1 and SSB confirmed the mass spectrometry results and identified endogenous reciprocal interactions between the protein and tRF. Binding of this tRF to hnRNPA1 inhibits destabilization of endogenous targets of this RNA binding protein, leading to increased mRNA expression of DNA damage response and cell cycle regulatory genes. Thus, tRNA fragmentation can both deplete a precursor tRNA molecule with codon-dependent regulatory consequences and also generate small-RNAs that can interact with and regulate RNA binding proteins.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Recommended Citation

Huh, Doowon, "A Stress-Induced TRNA Depletion Response Mediates Codon-Based Translational Repression and Growth Suppression" (2019). Student Theses and Dissertations. 513.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/513