tRNAs traffic between the nucleus and the cytoplasm in response to nutrient availability. Using a new assay to track tRNA within cells, we show that tRNA nuclear import is constitutive, whereas tRNA reexport to the cytoplasm is regulated. Msn5 functions only in tRNA re-export, whereas Los1 functions in both the primary and reexport steps.
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 ...
Appropriate nuclear membrane structure is important for all eukaryotic organisms as evidenced by the numerous human diseases and alterations in gene expression caused by inappropriate targeting of proteins to the inner nuclear membrane (INM). We report here the first genome-wide screen to identify proteins functioning in INM targeting. We transformed to near completion the 4850 members of the Saccharomyces cerevisiae deletion collection of unessential genes in the 96-well format with a plasmid encoding a reporter protein, Trm1-II-GFP, which normally resides at the INM. We found that deletion of genes encoding subunits of the N-terminal acetyltransferase, NatC, cause mislocation of Trm1-II-GFP from the INM to the nucleoplasm. Mass spectroscopic analysis indicates that Trm1-II-GFP is N-acetylated. N-terminal mutations of Trm1-II-GFP predicted to ablate N-acetylation cause nucleoplasmic location, whereas a variant with an N-terminal alteration predicted to allow N-acetylation by NatC is located at the INM, providing genetic support that Trm1p-II N-acetylation is necessary for its subnuclear INM location. However, because N-acetylation appears not to be sufficient for INM targeting, it may provide a necessary role for INM targeting by affecting Trm1-II-GFP structure and exposure of cis-acting INM targeting motifs. We also discovered that YIL090W/Ice2p, an integral membrane protein located in the endoplasmic reticulum, is necessary for efficient targeting of Trm1-II-GFP to the INM. YIL090W/Ice2p may serve as a tether for INM proteins or as a regulator of INM tethers. Our methodology can be extrapolated to obtain genome-wide perspectives of mechanisms necessary to achieve appropriate subcellular and/or suborganellar location for any resident protein.T HE nucleus is separated from the cytosol by a doupartments from one another, their structure, biogeneble membrane. Exchange of macromolecules besis, and maintenance likely result from poorly described tween the nuclear interior and the cytoplasm occurs macromolecular interactions. Here we investigate the through nuclear pores, proteinaceous aqueous chanmechanisms that target/tether proteins to the INM. nels that connect the membranes. Appropriate nuclear The nuclear membrane is highly dynamic and has a membrane structure is very important for all organisms complex structure. In higher eukaryotes it is disassemas evidenced by the numerous human diseases, such bled and reassembled each cell division, but in organas Emery-Dreifuss muscular dystrophy and Hutchisonisms such as budding yeast the nuclear membrane reGilford Progeria syndrome (reviewed in Gruenbaum et mains intact during mitosis. This difference perhaps al. 2003), and by numerous alterations in gene expreseliminates some of the dynamics that render analyses sion in yeast (reviewed in Taddei et al. 2004) caused by of nuclear membrane organization particularly difficult inappropriate targeting of proteins that normally reside in higher eukaryotes. The INM is composed of numerat the inner nuclear membrane (I...
Hfq proteins in Gram-negative bacteria play important roles in bacterial physiology and virulence, mediated by binding of the Hfq hexamer to small RNAs and/or mRNAs to post-transcriptionally regulate gene expression. However, the physiological role of Hfqs in Gram-positive bacteria is less clear. Bacillus anthracis, the causative agent of anthrax, uniquely expresses three distinct Hfq proteins, two from the chromosome (Hfq1, Hfq2) and one from its pXO1 virulence plasmid (Hfq3). The protein sequences of Hfq1 and 3 are evolutionarily distinct from those of Hfq2 and of Hfqs found in other Bacilli. Here, the quaternary structure of each B. anthracis Hfq protein, as produced heterologously in Escherichia coli, was characterized. While Hfq2 adopts the expected hexamer structure, Hfq1 does not form similarly stable hexamers in vitro. The impact on the monomer-hexamer equilibrium of varying Hfq C-terminal tail length and other sequence differences among the Hfqs was examined, and a sequence region of the Hfq proteins that was involved in hexamer formation was identified. It was found that, in addition to the distinct higher-order structures of the Hfq homologs, they give rise to different phenotypes. Hfq1 has a disruptive effect on the function of E. coli Hfq in vivo, while Hfq3 expression at high levels is toxic to E. coli but also partially complements Hfq function in E. coli. These results set the stage for future studies of the roles of these proteins in B. anthracis physiology and for the identification of sequence determinants of phenotypic complementation.
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