Retrograde nuclear accumulation of cytoplasmic tRNA in rat hepatoma cells in response to amino acid deprivation

Abstract

Until recently, transport of tRNA was presumed to be unidirectional, from the nucleus to the cytoplasm. Our published findings, however, revealed that cytoplasmic tRNAs move retrograde to the nucleus in Saccharomyces cerevisiae and that nuclear accumulation of cytoplasmic tRNAs occurs when cells are nutrient deprived. The findings led us to examine whether retrograde nuclear accumulation of cytoplasmic tRNAs occurs in higher eukaryotes. Using RNA FISH and Northern and Western analyses we show that tRNAs accumulate in nuclei of a hepatoma cell line in response to amino acid deprivation. To discern whether tRNA nuclear accumulation results from nuclear import of cytoplasmic tRNAs, transcription of new RNAs was inhibited, and the location of ''old'' tRNAs in response to nutrient stress was determined. Even in the absence of new RNA synthesis, there were significant tRNA nuclear pools after amino acid depletion, providing strong evidence that retrograde traffic is responsible for the tRNA nuclear pools. Further analyses showed that retrograde tRNA nuclear accumulation in hepatoma cells is a reversible and energy-dependent process. The data provide evidence for retrograde tRNA nuclear accumulation in intact mammalian cells and support the hypothesis that nuclear accumulation of cytoplasmic tRNA and tRNA re-export to the cytoplasm may constitute a universal mechanism for posttranscriptional regulation of global gene expression in response to nutrient availability.nucleus ͉ nutrient deprivation ͉ retrograde traffic I n eukaryotes the nuclear envelope separates mRNA transcription from cytoplasmic translation. The nuclear and cytoplasmic compartments interface at the nuclear pores, which regulate transport of molecules in and out of the nucleus. The majority of nuclear/cytoplasmic exchange occurs via a process that requires the small GTPase Ran and members of the ␤-importin protein family.With the exception of small nuclear RNA, the transport of RNA transcripts across the nuclear envelope was presumed to be unidirectional, from the nucleus to the cytoplasm (for review see ref. 1). This dogma has now been challenged. Yoshihisa and coworkers (2) showed that tRNA splicing in yeast occurs in the cytoplasm. Yet spliced tRNA can accumulate in the nucleus. Accumulation of spliced tRNA in the nucleus prompted examination of whether cytoplasmic tRNA is imported into the nucleus. Our work (3) and the work of Takano et al. (4) showed that tRNA moves retrograde from the cytoplasm to the nucleus in Saccharomyces cerevisiae.Retrograde tRNA nuclear import might serve to proofread tRNAs after splicing to separate improperly spliced tRNAs from the translation machinery. If so, then the retrograde process could be restricted to organisms with cytoplasmic pre-tRNA splicing. According to this scenario, vertebrate cells that splice pre-tRNA in the nucleus (5-7) may not import cytoplasmic tRNAs into their nuclei. However, because some tRNA modifications occur in the cytoplasm (for review see ref. 8) and the long-lived tRNAs could be damaged ...

Transfer RNA Post-Transcriptional Processing, Turnover, and Subcellular Dynamics in the YeastSaccharomyces cerevisiae

“…It is now known that tRNAs can be aminoacylated in the nucleus (Lund and Dahlberg 1998;Sarkar et al 1999;Grosshans et al 2000a), that tRNAs can traffic from the cytoplasm to the nucleus via the tRNA retrograde process (Shaheen and Hopper 2005;Takano et al 2005;Zaitseva et al 2006), and that tRNAs imported into the nucleus can be reexported to the cytoplasm (Whitney et al 2007;Eswara et al 2009) (Figure 3). tRNA subcellular traffic is conserved (Zaitseva et al 2006;Shaheen et al 2007;Barhoom et al 2011), responsive to nutrient availability (Shaheen and Hopper 2005;Hurto et al 2007;Shaheen et al 2007;Whitney et al 2007;Murthi et al 2010), and, in yeast, is coordinated with the formation of P-bodies in the cytoplasm (Hurto and Hopper 2011). In addition to the trafficking of tRNAs between the nucleus and the cytoplasm, some tRNAs encoded by the nucleus are imported into mitochondria (reviewed in Rubio and Hopper 2011;Schneider 2011).…”

Linking tRNA localization with activation of nutritional stress responses

“…The underlying reason for the latter is yet unknown. [26][27][28] To determine whether tRNA retention in the nuclei of HFF would influence the autophagic process similarly to the deletion of yeast LOS1 (Fig. S1), we chose to knock-down the karyopherin Xpo-t by nucleofection with XPOT-specific siRNA.…”

Alzheimer’s disease: amino acid levels and brain metabolic status

“…Under such limiting conditions, amino acids could be playing a signalling role in affected brains, conveying regulatory messages to the transcriptional machinery and affecting the turnover of specific mRNA and proteins. For instance, limitation of several amino acids greatly increases the expression of the gene encoding growth factors, binding proteins, enzymes, amino acids transport systems, retrograde tRNA transport [35,36]. Another possibility is that this deficit could be the result of an ineffective attempt by brain tissues in repairing or gathering energy.…”