Abstract
Stress is known to induce retrograde tRNA translocation from the cytoplasm to the nucleus but translocation kinetics and tRNA-spatial distribution have not been characterized previously. We microinject fluorescently-labeled tRNA into living cells and use confocal microscopy to image tRNA spatial distribution in single cells at various levels of starvation and to determine translocation rate constants. Retrograde tRNA translocation occurs reversibly, within minutes after nutrition depletion of the extracellular medium. Such nutritional starvation leads to down-regulation of tRNA nuclear import and nearly complete curtailment of its nuclear export. Nuclear tRNA accumulation is suppressed in cells treated with the translation inhibitor puromycin, but is enhanced in cells treated with the microtubule inhibitor nocodazole. tRNA in the cytoplasm exhibits distinct spatial distribution inconsistent with diffusion, implying that such distribution is actively maintained. We propose that tRNA biological complexes and/or cytoplasmic electric fields are the likely regulators of cytoplasmic tRNA spatial distribution.
Highlights
Transfer ribonucleic acid is a 76–93 nucleotidelong adaptor molecule that links a given amino acid to a specific messenger RNA codon and transfers this amino acid to a nascent polypeptide chain at the ribosomal site of protein synthesis [1]
For the first time, the kinetics of Transfer ribonucleic acid (tRNA) nuclear import and export in an experimental system in which tRNA subcellular dynamics can be isolated from tRNA transcription, and which is amenable to measurements in live cells
We monitor in real time the kinetics of tRNA trafficking between the cytoplasm and the nucleus following controlled injection of fluorescent rhd-tRNA into the cytoplasm of single live cells
Summary
Transfer ribonucleic acid (tRNA) is a 76–93 nucleotidelong adaptor molecule that links a given amino acid to a specific messenger RNA (mRNA) codon and transfers this amino acid to a nascent polypeptide chain at the ribosomal site of protein synthesis [1]. It was believed that mature tRNA molecules, following their transcription in the nucleus, are exported to the cytoplasm to undergo aminoacylation and participate in translation [2]. Recently has it been recognized that mature tRNA undergoes retrotranslocation from the cytosol into the nucleus, a reversible process having important roles in cell biology [3]. This phenomenon has been most extensively studied in Saccharomyces cerevisiae [2,4,5,6,7]. Translation in the nucleus has been suggested [18], but this suggestion remains controversial [19]
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