Crystal structure of Cex1p reveals the mechanism of tRNA trafficking between nucleus and cytoplasm

In all eukaryotes, transcribed precursor tRNAs are maturated by processing and modification processes in nucleus and are transported to the cytoplasm. The cytoplasmic export protein (Cex1p) captures mature tRNAs from the nuclear export receptor (Los1p) on the cytoplasmic side of the nuclear pore complex, and it delivers them to eukaryotic elongation factor 1α. This conserved Cex1p function is essential for the quality control of mature tRNAs to ensure accurate translation. However, the structural basis of how Cex1p recognizes tRNAs and shuttles them to the translational apparatus remains unclear. Here, we solved the 2.2 Å resolution crystal structure of Saccharomyces cerevisiae Cex1p with C-terminal 197 disordered residues truncated. Cex1p adopts an elongated architecture, consisting of N-terminal kinase-like and a C-terminal α-helical HEAT repeat domains. Structure-based biochemical analyses suggested that Cex1p binds tRNAs on its inner side, using the positively charged HEAT repeat surface and the C-terminal disordered region. The N-terminal kinase-like domain acts as a scaffold to interact with the Ran-exportin (Los1p·Gsp1p) machinery. These results provide the structural basis of Los1p·Gsp1p·Cex1p·tRNA complex formation, thus clarifying the dynamic mechanism of tRNA shuttling from exportin to the translational apparatus.     

Cex1p facilitates Rna1p-mediated dissociation of the Los1p-tRNA-Gsp1p-GTP export complex

Nuclear tRNA export plays an essential role in key cellular processes such as regulation of protein synthesis, cell cycle progression, response to nutrient availability and DNA damage and development. Like other nuclear export processes, assembly of the nuclear tRNA export complex in the nucleus is dependent on Ran-GTP/Gsp1p-GTP, and dissociation of the export receptor-tRNA-Ran-GTP/Gsp1p-GTP complex in the cytoplasm requires RanBP1/Yrb1p and RanGAP/Rna1p to activate the GTPase activity of Ran-GTP/Gsp1p-GTP. The Saccharomyces cerevisiae Cex1p and Human Scyl1 have also been proposed to participate in unloading of the tRNA export receptors at the cytoplasmic face of the nuclear pore complex (NPC). Here, we provide evidence suggesting that Cex1p is required for activation of the GTPase activity of Gsp1p and dissociation of the receptor-tRNA-Gsp1p export complex in S. cerevisiae. The data suggest that Cex1p recruits Rna1p from the cytoplasm to the NPC and facilitates Rna1p activation of the GTPase activity of Gsp1p by enabling Rna1p to gain access to Gsp1p-GTP bound to the export receptor tRNA complex. It is possible that this tRNA unloading mechanism is conserved in evolutionarily diverse organisms and that other Gsp1p-GTP-dependent export processes use a pathway-specific component to recruit Rna1p to the NPC.     

Utp8p is a nucleolar tRNA-binding protein that forms a complex with components of the nuclear tRNA export machinery in Saccharomyces cerevisiae

Utp8p is an essential nucleolar component of the nuclear tRNA export machinery in Saccharomyces cerevisiae. It is thought to act at a step between tRNA maturation/aminoacylation and translocation of the tRNA across the nuclear pore complex. To understand the function of Utp8p in nuclear tRNA export, a comprehensive affinity purification analysis was conducted to identify proteins that interact with Utp8p in vivo. In addition to finding proteins that have been shown previously to copurify with Utp8p, a number of new interactions were identified. These interactions include aminoacyl-tRNA synthetases, the RanGTPase Gsp1p, and nuclear tRNA export receptors such as Los1p and Msn5p. Characterization of the interaction of Utp8p with a subset of the newly identified proteins suggests that Utp8p most likely transfer tRNAs to the nuclear tRNA export receptors by using a channeling mechanism.     

Scyl1 Facilitates Nuclear tRNA Export in Mammalian Cells by Acting at the Nuclear Pore Complex

Scyl1 is an evolutionarily conserved N-terminal protein kinase-like domain protein that plays a role in COP1-mediated retrograde protein trafficking in mammalian cells. Furthermore, loss of Scyl1 function has been shown to result in neurodegenerative disorders in mice. Here, we report that Scyl1 is also a cytoplasmic component of the mammalian nuclear tRNA export machinery. Like exportin-t, overexpression of Scyl1 restored export of a nuclear export-defective serine amber suppressor tRNA mutant in COS-7 cells. Scyl1 binds tRNA saturably, and associates with the nuclear pore complex by interacting, in part, with Nup98. Scyl1 copurifies with the nuclear tRNA export receptors exportin-t and exportin-5, the RanGTPase, and the eukaryotic elongation factor eEF-1A, which transports aminoacyl-tRNAs to the ribosomes. Scyl1 interacts directly with exportin-t and RanGTP but not with eEF-1A or RanGDP in vitro. Moreover, exportin-t containing tRNA, Scyl1, and RanGTP form a quaternary complex in vitro. Biochemical characterization also suggests that the nuclear aminoacylation-dependent pathway is primarily responsible for tRNA export in mammalian cells. These findings together suggest that Scyl1 participates in the nuclear aminoacylation-dependent tRNA export pathway and may unload aminoacyl-tRNAs from the nuclear tRNA export receptor at the cytoplasmic side of the nuclear pore complex and channels them to eEF-1A.     

Utp9p Facilitates Msn5p-mediated Nuclear Reexport of Retrograded tRNAs in Saccharomyces cerevisiae

Utp9p is a nucleolar protein that is part of a subcomplex containing several U3 snoRNA-associated proteins including Utp8p, which is a protein that shuttles aminoacyl-tRNAs from the nucleolus to the nuclear tRNA export receptors Los1p and Msn5p in Saccharomyces cerevisiae. Here we show that Utp9p is also an intranuclear component of the Msn5p-mediated nuclear tRNA export pathway. Depletion of Utp9p caused nuclear accumulation of mature tRNAs derived from intron-containing precursors, but not tRNAs made from intronless pre-tRNAs. Utp9p binds tRNA directly and saturably, and copurifies with Utp8p, Gsp1p, and Msn5p, but not with Los1p or aminoacyl-tRNA synthetases. Utp9p interacts directly with Utp8p, Gsp1p, and Msn5p in vitro. Furthermore, Gsp1p forms a complex with Msn5p and Utp9p in a tRNA-dependent manner. However, Utp9p does not shuttle between the nucleus and the cytoplasm. Because tRNA splicing occurs in the cytoplasm and the spliced tRNAs are retrograded back to the nucleus, we propose that Utp9p facilitates nuclear reexport of retrograded tRNAs. Moreover, the data suggest that Utp9p together with Utp8p translocate aminoacyl-tRNAs from the nucleolus to Msn5p and assist with formation of the Msn5p-tRNA-Gsp1p-GTP export complex.     

Utp8p is an essential intranuclear component of the nuclear tRNA export machinery of Saccharomyces cerevisiae

A yeast tRNA three-hybrid interaction approach and an in vivo nuclear tRNA export assay based on amber suppression was used to identify proteins that participate in the nuclear tRNA export process in Saccharomyces cerevisiae. One of the proteins identified by this strategy is Utp8p, an essential 80-kDa nucleolar protein that has been implicated in 18 S ribosomal RNA biogenesis. Our characterization indicated that the major function of Utp8p is in nuclear tRNA export. Like the S. cerevisiae Los1p and the mammalian exportin-t, which are proteins known to facilitate nuclear tRNA export, overexpression of Utp8p restored export of tRNAamTyr mutants defective in nuclear export. Furthermore, depletion of Utp8p blocked nuclear export of mature tRNAs derived from both intronless and intron-containing pre-tRNAs but did not affect tRNA and rRNA maturation, nuclear export of mRNA and ribosomes, or nuclear tRNA aminoacylation. Overexpression of Utp8p also alleviated nuclear retention of non-aminoacylated tRNATyr in a tyrosyl-tRNA synthetase mutant strain. Utp8p binds tRNA directly and saturably, indicating that it has a tRNA-binding site. Utp8p does not appear to function as a tRNA export receptor, because it does not shuttle between the nucleus and the cytoplasm. Taken together, the results suggest that Utp8p is an essential intranuclear component of the nuclear tRNA export machinery, which may channel tRNA to the various tRNA export pathways operating in S. cerevisiae.     

Nutrient stress does not cause retrograde transport of cytoplasmic tRNA to the nucleus in evolutionarily diverse organisms

Intracellular trafficking of tRNA was long thought to be a one-way trip from the site of biogenesis in the nucleus to the translation machinery in the cytoplasm. This view has recently been challenged, however, by the discovery that tRNA can move retrograde from the cytoplasm back to the nucleus in Saccharomyces cerevisiae and rat hepatoma H4IIE cells during nutrient stress and in S. cerevisiae after intron-containing pre-tRNAs are spliced in the cytoplasm. Contrary to studies reported, we present data suggesting that nutrient stress does not cause retrograde transport of cytoplasmic tRNAs to the nucleus in rat hepatoma H4IIE cells, human HeLa and HEK293 cells, and the yeasts Kluyveromyces lactis and S. cerevisiae. However, the efficiency of nuclear re-export of retrograded spliced tRNA was severely affected in S. cerevisiae and two other Saccharomyces species deprived of nutrient. Collectively, the data suggest that nutrient stress does not cause nuclear import of cytoplasmic tRNA; instead, nutrient stress specifically regulates nuclear re-export of retrograded spliced tRNAs but not nuclear export of tRNAs made from intronless pre-tRNAs in Saccharomyces species. Furthermore, we provide evidence suggesting that Mtr10p and the Gsp1pGTP/Gsp1pGDP cycle are not involved in nuclear tRNA import in S. cerevisiae during nutrient stress.     

Role of nuclear pools of aminoacyl-tRNA synthetases in tRNA nuclear export

Reports of nuclear tRNA aminoacylation and its role in tRNA nuclear export (Lund and Dahlberg, 1998; Sarkar et al., 1999; Grosshans et al., 20001) have led to the prediction that there should be nuclear pools of aminoacyl-tRNA synthetases. We report that in budding yeast there are nuclear pools of tyrosyl-tRNA synthetase, Tys1p. By sequence alignments we predicted a Tys1p nuclear localization sequence and showed it to be sufficient for nuclear location of a passenger protein. Mutations of this nuclear localization sequence in endogenous Tys1p reduce nuclear Tys1p pools, indicating that the motif is also important for nucleus location. The mutations do not significantly affect catalytic activity, but they do cause defects in export of tRNAs to the cytosol. Despite export defects, the cells are viable, indicating that nuclear tRNA aminoacylation is not required for all tRNA nuclear export paths. Because the tRNA nuclear exportin, Los1p, is also unessential, we tested whether tRNA aminoacylation and Los1p operate in alternative tRNA nuclear export paths. No genetic interactions between aminoacyl-tRNA synthetases and Los1p were detected, indicating that tRNA nuclear aminoacylation and Los1p operate in the same export pathway or there are more than two pathways for tRNA nuclear export.     

tRNA Nuclear Export in Saccharomyces cerevisiae: In Situ Hybridization Analysis

To understand the factors specifically affecting tRNA nuclear export, we adapted in situ hybridization procedures to locate endogenous levels of individual tRNA families in wild-type and mutant yeast cells. Our studies of tRNAs encoded by genes lacking introns show that nucleoporin Nup116p affects both poly(A) RNA and tRNA export, whereas Nup159p affects only poly(A) RNA export. Los1p is similar to exportin-t, which facilitates vertebrate tRNA export. A los1 deletion mutation affects tRNA but not poly(A) RNA export. The data support the notion that Los1p and exportin-t are functional homologues. Because LOS1 is nonessential, tRNA export in vertebrate and yeast cells likely involves factors in addition to exportin-t. Mutation of RNA1, which encodes RanGAP, causes nuclear accumulation of tRNAs and poly(A) RNA. Many yeast mutants, including those with the rna1-1 mutation, affect both pre-tRNA splicing and RNA export. Our studies of the location of intron-containing pre-tRNAs in the rna1-1 mutant rule out the possibility that this results from tRNA export occurring before splicing. Our results also argue against inappropriate subnuclear compartmentalization causing defects in pre-tRNA splicing. Rather, the data support “feedback” of nucleus/cytosol exchange to the pre-tRNA splicing machinery.     

The Saccharomyces cerevisiae Pus2 protein encoded by YGL063w ORF is a mitochondrial tRNA:Psi27/28-synthase

The RNA:pseudouridine (Psi)-synthase family is one of the most complex families of RNA modification enzymes. Ten genes encoding putative RNA:Psi-synthases have been identified in S. cerevisiae. Most of the encoded enzymes have been characterized experimentally. Only the putative RNA:Psi-synthase Pus2p (encoded by the YGL063w ORF) had no identified substrate. Here, we analyzed Psi residues in cytoplasmic and mitochondrial tRNAs extracted from S. cerevisiae strains, carrying disruptions in the PUS1 and/or PUS2 ORFs. Our results demonstrate that Pus2p is a mitochondrial-specific tRNA:Psi-synthase acting at positions 27 and 28 in tRNAs. The importance of the Asp56 residue in the conserved ARTD motif of the Pus2p catalytic site is demonstrated in vivo. Interestingly, in spite of the absence of a characteristic N-terminal targeting signal, our data strongly suggest an efficient and rapid targeting of Pus2p in yeast mitochondria. In contradiction with the commonly held idea that a unique nuclear gene encodes the enzyme required for both cytoplasmic and mitochondrial tRNA modifications, here we show the existence of an enzyme specifically dedicated to mitochondrial tRNA modification (Pus2p), the corresponding modification in cytoplasmic tRNAs being catalyzed by another protein (Pus1p).     

Inorganic phosphate deprivation causes tRNA nuclear accumulation via retrograde transport in Saccharomyces cerevisiae

Nuclear export of tRNA is an essential eukaryotic function, yet the one known yeast tRNA nuclear exporter, Los1, is nonessential. Moreover recent studies have shown that tRNAs can move retrograde from the cytosol to the nucleus by an undefined process. Therefore, additional gene products involved in tRNA nucleus-cytosol dynamics have yet to be identified. Synthetic genetic array (SGA) analysis was employed to identify proteins involved in Los1-independent tRNA transport and in regulating tRNA nucleus-cytosol distribution. These studies uncovered synthetic interactions between los1Delta and pho88Delta involved in inorganic phopsphate uptake. Further analysis revealed that inorganic phosphate deprivation causes transient, temperature-dependent nuclear accumulation of mature cytoplasmic tRNA within nuclei via a Mtr10- and retrograde-dependent pathway, providing a novel connection between tRNA subcellular dynamics and phosphate availability.     

Protein Kinase A Is Part of a Mechanism That Regulates Nuclear Reimport of the Nuclear tRNA Export Receptors Los1p and Msn5p

The two main signal transduction mechanisms that allow eukaryotes to sense and respond to changes in glucose availability in the environment are the cyclic AMP (cAMP)/protein kinase A (PKA) and AMP-activated protein kinase (AMPK)/Snf1 kinase-dependent pathways. Previous studies have shown that the nuclear tRNA export process is inhibited in Saccharomyces cerevisiae deprived of glucose. However, the signal transduction pathway involved and the mechanism by which glucose availability regulates nuclear-cytoplasmic tRNA trafficking are not understood. Here, we show that inhibition of nuclear tRNA export is caused by a block in nuclear reimport of the tRNA export receptors during glucose deprivation. Cytoplasmic accumulation of the tRNA export receptors during glucose deprivation is not caused by activation of Snf1p. Evidence obtained suggests that PKA is part of the mechanism that regulates nuclear reimport of the tRNA export receptors in response to glucose availability. This mechanism does not appear to involve phosphorylation of the nuclear tRNA export receptors by PKA. The block in nuclear reimport of the tRNA export receptors appears to be caused by activation of an unidentified mechanism when PKA is turned off during glucose deprivation. Taken together, the data suggest that PKA facilitates return of the tRNA export receptors to the nucleus by inhibiting an unidentified activity that facilitates cytoplasmic accumulation of the tRNA export receptors when glucose in the environment is limiting. A PKA-independent mechanism was also found to regulate nuclear tRNA export in response to glucose availability. This mechanism, however, does not regulate nuclear reimport of the tRNA export receptors.     

Yeast Los1p Has Properties of an Exportin-Like Nucleocytoplasmic Transport Factor for tRNA

Saccharomyces cerevisiae Los1p, which is genetically linked to the nuclear pore protein Nsp1p and several tRNA biogenesis factors, was recently grouped into the family of importin/karyopherin-β-like proteins on the basis of its sequence similarity. In a two-hybrid screen, we identified Nup2p as a nucleoporin interacting with Los1p. Subsequent purification of Los1p from yeast demonstrates its physical association not only with Nup2p but also with Nsp1p. By the use of the Gsp1p-G21V mutant, Los1p was shown to preferentially bind to the GTP-bound form of yeast Ran. Furthermore, overexpression of full-length or N-terminally truncated Los1p was shown to have dominant-negative effects on cell growth and different nuclear export pathways. Finally, Los1p could interact with Gsp1p-GTP, but only in the presence of tRNA, as revealed in an indirect in vitro binding assay. These data confirm the homology between Los1p and the recently identified human exportin for tRNA and reinforce the possibility of a role for Los1p in nuclear export of tRNA in yeast.     

Regulation of tRNA Bidirectional Nuclear-Cytoplasmic Trafficking in Saccharomyces cerevisiae

tRNAs in yeast and vertebrate cells move bidirectionally and reversibly between the nucleus and the cytoplasm. We investigated roles of members of the β-importin family in tRNA subcellular dynamics. Retrograde import of tRNA into the nucleus is dependent, directly or indirectly, upon Mtr10. tRNA nuclear export utilizes at least two members of the β-importin family. The β-importins involved in nuclear export have shared and exclusive functions. Los1 functions in both the tRNA primary export and the tRNA reexport processes. Msn5 is unable to export tRNAs in the primary round of export if the tRNAs are encoded by intron-containing genes, and for these tRNAs Msn5 functions primarily in their reexport to the cytoplasm. The data support a model in which tRNA retrograde import to the nucleus is a constitutive process; in contrast, reexport of the imported tRNAs back to the cytoplasm is regulated by the availability of nutrients to cells and by tRNA aminoacylation in the nucleus. Finally, we implicate Tef1, the yeast orthologue of translation elongation factor eEF1A, in the tRNA reexport process and show that its subcellular distribution between the nucleus and cytoplasm is dependent upon Mtr10 and Msn5.     

The nuclear export receptor Xpo1p forms distinct complexes with NES transport substrates and the yeast Ran binding protein 1 (Yrb1p)

Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich nuclear export signal (NES). Xpo1p, the NES-containing protein, and GTP-bound Ran form a complex in the nucleus that translocates across the nuclear pore. We have identified Yrb1p as the major Xpo1p-binding protein in Saccharomyces cerevisiae extracts in the presence of GTP-bound Gsp1p (yeast Ran). Yrb1p is cytoplasmic at steady-state but shuttles continuously between the cytoplasm and the nucleus. Nuclear import of Yrb1p is mediated by two separate nuclear targeting signals. Export from the nucleus requires Xpo1p, but Yrb1p does not contain a leucine-rich NES. Instead, the interaction of Yrb1p with Xpo1p is mediated by Gsp1p-GTP. This novel type of export complex requires the acidic C-terminus of Gsp1p, which is dispensable for the binding to importin beta-like transport receptors. A similar complex with Xpo1p and Gsp1p-GTP can be formed by Yrb2p, a relative of Yrb1p predominantly located in the nucleus. Yrb1p also functions as a disassembly factor for NES/Xpo1p/Gsp1p-GTP complexes by displacing the NES protein from Xpo1p/Gsp1p. This Yrb1p/Xpo1p/Gsp1p complex is then completely dissociated after GTP hydrolysis catalyzed by the cytoplasmic GTPase activating protein Rna1p.     

Pus1p-dependent tRNA pseudouridinylation becomes essential when tRNA biogenesis is compromised in yeast

Yeast Pus1p catalyzes the formation of pseudouridine (psi) at specific sites of several tRNAs, but its function is not essential for cell viability. We show here that Pus1p becomes essential when another tRNA:pseudouridine synthase, Pus4p, or the essential minor tRNA for glutamine are mutated. Strikingly, this mutant tRNA, which carries a mismatch in the T psi C arm, displays a nuclear export defect. Furthermore, nuclear export of at least one wild-type tRNA species becomes defective in the absence of Pus1p. Our data, thus, show that the modifications formed by Pus1p are essential when other aspects of tRNA biogenesis or function are compromised and suggest that impairment of nuclear tRNA export in the absence of Pus1p might contribute to this phenotype.     

Review: Transport of tRNA out of the Nucleus—Direct Channeling to the Ribosome?

Although tRNA was the first substrate whose export from the nuclei of eukaryotic cells had been shown to be carrier-mediated and active, it has only been in the last 2 years that the first mechanistic details of this nucleocytoplasmic transport pathway have begun to emerge. A member of the importin/karyopherin β superfamily, Los1p in yeast and Xpo-t in vertebrates, has been shown to export tRNA in cooperation with the small GTPase Ran (Gsp1p) from the nucleus into the cytoplasm, where tRNA becomes available for translation. However, Los1p is not essential for viability in yeast cells, suggesting that alternative tRNA export pathways exist. Recent results show that aminoacylation and a translation factor are also required for efficient nuclear tRNA export. Thus, protein translation and nuclear export of tRNA appear to be coupled processes.     

Nuclear export of tRNA

Summary Exit of tRNA from the nucleus was shown, long time ago, to be a saturable and carrier-mediated process. Nevertheless, only recently, progress in the field of nucleocytoplasmic transport gave first insight into the mechanism of tRNA nuclear export. A nuclear export receptor for tRNA (Los1p/Xpo-t), belonging to the importin (karyopherin) family, has been characterized in yeast and mammalian cells. Mature tRNA molecules can associate with Los1p/ Xpo-t and the GTP-bound form of the small GTPase Ran to form an export complex in the nucleus. This complex translocates through the nuclear-pore complexes and dissociates upon GTP hydrolysis in the cytoplasm. Genetic studies in yeast have, however, shown thatLOS1 is not essential, unless additional steps in the tRNA biogenesis pathway are impaired, suggesting the existence of additional tRNA nuclear export routes. Furthermore, modification and aminoacylation of tRNA may also be important for efficient transport of tRNA into the cytoplasm.