Dynamics of intracellular stress-induced tRNA trafficking

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.

A novel assay provides insight into tRNAPhe retrograde nuclear import and re-export inS. cerevisiae

“…Many stress conditions are known to result in the accumulation of tRNAs in the nucleus, such as amino acid deprivation ( 18 ). A recent study examining the kinetics of tRNA import and re-export upon nutrient deprivation in mouse embryonic fibroblasts microinjected with fluorescently labeled tRNA, found that tRNAs accumulate in the nucleus upon nutrient deprivation and the rate constants of tRNA import and re-export both decrease as nutrient levels decrease ( 43 ). However, the rate of re-export decreases to a greater extent than the rate of import, accounting for the nuclear accumulation of tRNAs.…”

Hijacking tRNAs From Translation: Regulatory Functions of tRNAs in Mammalian Cell Physiology

“…Cytoplasmic tRNAs rapidly translocate into the nucleus under a number of stress conditions, including nutrient starvation (Shaheen and Hopper, 2005;Takano et al, 2005;Hurto et al, 2007;Shaheen et al, 2007;Whitney et al, 2007;Dhakal et al, 2019), heat shock (Miyagawa et al, 2012;Watanabe et al, 2013), viral infection (Zaitseva et al, 2006), and oxidative stress (Schwenzer et al, 2019). This translocation not only directly depletes tRNAs from protein synthesis but also modulates stress response by activation of the GCN2 kinase (Castilho et al, 2014).…”

tRNA Processing and Subcellular Trafficking Proteins Multitask in Pathways for Other RNAs

“…tRNAs move dynamically between the nucleus and the cytoplasm in yeast, protozoa, and vertebrate cells (Shaheen and Hopper, 2005; Takano et al., 2005; Shaheen et al., 2007; Huynh et al., 2010; Barhoom et al., 2011; Ohira and Suzuki, 2011; Watanabe et al., 2013; Dhakal et al., 2018; Kessler et al., 2018). Even though only a subset of eukaryotic tRNA-encoding genes contains introns, we focus on this category of tRNAs.…”

“…Employing nuclear import assays with permeabilized HeLa cells, the Fassati group demonstrated that tRNA nuclear import occurs in vertebrate cells and their studies also showed that tRNA retrograde traffic provides one mechanism by which the retrotranscribed HIV genome can access the nuclear interior in nondividing cells (Zaitseva et al., 2006). Subsequent RNA FISH studies in protozoa, brine shrimp, and vertebrate cells in culture, and, most recently, tagged-tRNAs injected into vertebrate live cells, demonstrate widespread conservation of nuclear import of cytoplasmic tRNAs, especially in response to nutrient and/or heat stress [(Huynh et al., 2010; Barhoom et al., 2011; Miyagawa et al., 2012; Watanabe et al., 2013; Chen et al., 2016; Dhakal et al., 2018) Review: (Huang and Hopper, 2016)].…”