# tRNA processing and nuclear-cytoplasmic dynamics

> **NIH NIH R01** · OHIO STATE UNIVERSITY · 2024 · $20,390

## Abstract

Project Summary
The equipment supplement funds will facilitate parent grant GM122448 (A.K. Hopper, PI) by supporting purchase
of an Integra Mini 96-channel pipetter which will greatly facilitate original proposed work in Aims 2 and 3.
GM122448 focuses on tRNA biology and its subcellular trafficking. tRNAs are small noncoding RNAs that are
essential for decoding the genome by delivering amino acids to translating ribosomes according to codon
directions in mRNAs. Defects in tRNA biology cause numerous human disorders from metabolic diseases, to
neuromuscular diseases, and to cancer. tRNA biology requires a complex set of conserved gene products for
post-transcriptional processing, subcellular traffic, and intron turnover. The research program impacts upon
multiple facets of gene expression, quality control, and issues important to human health. We employ budding
yeast and in vivo technologies to discover unknown important aspects of tRNA biology. In Aim2 of GM122884
we study trafficking of tRNAs between the nucleus and the cytoplasm. Although for decades it was thought that
tRNA movement is unidirectional, nucleus to cytoplasm, we co-discovered that tRNAs move bi-directionally
between the nucleus and the cytoplasm (Shaheen & Hopper 2005) and that the dynamics are conserved
between yeast and vertebrate cells (Shaheen et al. 2007). We developed a new methodology, the HCl/aniline
assay (Nostramo et al. 2020), that reports tRNA retrograde nuclear import and re-export to the cytoplasm. We
are employing this methodology in a genome-wide screen of ~6000 yeast genes to identify and characterize the
proteins functioning in the tRNA retrograde pathway. Aim 3 of GM122884 investigates tRNA introns. Possession
of tRNA introns in subsets of tRNA genes has been conserved from Archaea to humans. We recently made the
exciting discovery (Nostramo et al., Mol. Cell, in revision) that introns spliced from pre-tRNAs provide a novel
complementary-dependent mechanism to fine-tune basal mRNA levels and to assist cellular responses to stress.
We also learned that under particular stresses, subsets of tRNA introns accumulate to high levels due to
increased stability. We discovered one mechanism for tRNA intron turnover (Wu & Hopper 2014); however, there
are at least four additional unknown mechanisms to destroy tRNA introns. None of the yeast annotated RNases
appear to function in the unknown turnover pathways. Thus, we are conducting a genome-wide screen for
mutants that accumulate each of the 8 tRNA intron families whose turnover remains unknown. Although we are
able to grow yeast mutants in the deep 96-well plates, we are unable to extract RNAs in this format due to the
opaqueness of the microtiter plates; therefore, we must conduct the biochemical steps in labor-intensive
analyses of single mutants. The Integra Mini 96-channel pipettes will allow us to conduct all steps preceding
northern gel analyses for 96 strains simultaneously and thereby greatly reduce the time and...

## Key facts

- **NIH application ID:** 11133486
- **Project number:** 3R01GM122884-08S1
- **Recipient organization:** OHIO STATE UNIVERSITY
- **Principal Investigator:** Anita K Hopper
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $20,390
- **Award type:** 3
- **Project period:** 2017-05-01 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11133486

## Citation

> US National Institutes of Health, RePORTER application 11133486, tRNA processing and nuclear-cytoplasmic dynamics (3R01GM122884-08S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/11133486. Licensed CC0.

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