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Previous evidence suggested that transfer RNAs (tRNAs) crossthe nuclear envelope to the cytosol only once after maturingin the nucleus. We now present evidence for nuclear import oftRNAs in yeast. Several export mutants accumulate mature tRNAsin the nucleus even in the absence of transcription. Importrequires energy but not the Ran cycle. These results indicatethat tRNAs shuttle between the nucleus and cytosol.
1 Department of Chemistry, Graduate School of Science, Japan Science and Technology Corporation, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan. 2 Research Center for Materials Science, Japan Science and Technology Corporation, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan. 3 Institute for Advanced Research, Japan Science and Technology Corporation, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan. 4 Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.
* To whom all correspondence should be addressed. E-mail: tyoshihi{at}biochem.chem.nagoya-u.ac.jp
Nuclear-encoded tRNAs are transcribed, processed in the nucleus,and exported to the cytosol to facilitate translation (1). Moreover,a nuclear pool of mature tRNAs also exists. In Saccharomycescerevisiae, certain mutants defective in nuclear transport andtRNA processing accumulate mature tRNAs in the nucleus, whichsuggests that nuclear mature tRNAs are intermediates waitingfor tRNA export (2–5). Before export, aminoacyl-tRNA synthetasesmay monitor their maturation (6). However, we recently foundthat tRNA-splicing endonuclease localizes to the mitochondrialsurface in yeast and that an endonuclease mutant accumulatesunspliced pre-tRNAs in the cytosol, which indicates that pre-tRNAsare exported to the cytosol and then spliced (7).
Fig. 1. Heterokaryon assay of nuclear import of tRNAs. (A) Heterokaryons formed from MATa cells with sup3+ and MATa cells were incubated in yeast extract, peptone, and dextrose (YPD) for the indicated times and subjected to FISH using probes against mature SptRNA-Meti (mature) and pre-SptRNA-SerUGA (pre). The nucleus and cell outlines are shown in the 4',6'-diamidino-2-phenylindole (DAPI) and Nomarski images, respectively. Target nuclei are marked by arrows. (B) Heterokaryon assays similar to (A) were performed with los1 haploids.
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To understand the origin of nuclear mature tRNAs, we examinedwhether tRNAs are imported into the nucleus from the cytosol,using heterokaryon assays (8). In S. cerevisiae, a heterokaryonwith two different nuclei sharing the same cytosol is formedfrom karyogamy-deficient MATa and MATa cells. To visualize tRNAstranscribed from one nucleus, a sup3+ gene (SptRNA-SerUGA::SptRNA-Meti)from Schizosaccharomyces pombe was introduced into the MATastrain. sup3+ is transcribed and processed to mature SptRNA-SerUGAand SptRNA-Meti in S. cerevisiae (9). Hetero-karyons betweenthe MATa cells (target) and the MATa cells with sup3+ (donor)were subjected to fluorescence in situ hybridization (FISH).Pre-SptRNA-SerUGA was detected in only one nucleus in a heterokaryon(Fig. 1A, pre, and fig. S2). In a wild-type background for tRNAexport, mature SptRNA-Meti was excluded from target nuclei likeendogenous tRNAs (Fig. 1A and fig. S4B). los1 cells, lackingyeast exportin-t (10, 11), exhibit a moderate defect in nuclearexport of mature tRNAs (fig. S4B) (2). In los1 heterokaryons,we observed more SptRNA-Meti in target nuclei (Fig. 1B). Thesedata indicate that cytosolic mature tRNAs enter the nucleus.
Fig. 2. Treatment with thiolutin causes accumulation of mature tRNA-ProUGG in los1msn5 cells. (A) los1msn5 cells treated with the indicated concentrations of thiolutin were subjected to Northern hybridization with an anti-pre-tRNA-ProUGG probe. White triangle, primary transcripts; black triangle, end-matured pre-tRNAs. (B) The cells treated with 5 µg/ml thiolutin for the indicated times were subjected to FISH with probes against tRNA-ProUGG and tRNA-TyrGUA.
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Next, we investigated nuclear import of endogenous tRNAs incells growing vegetatively. If all nuclear mature tRNAs arenewly transcribed, transcription arrest should reduce the nuclearpool of tRNAs through their export. If mature tRNAs are suppliedfrom the cytosol, this should not necessarily be true. A complicatingfactor is that the signal intensity of mature tRNAs in the wild-typenucleus is lower than that in the cytosol. To circumvent thisproblem, we used a los1msn5 double mutant. Msn5p is a homologof mammalian exportin-5, which exports the tRNA-eEF1A complexand pre-miRNAs (microRNAs) (12–14). msn5 by itself didnot alter tRNA localization, but the los1msn5 mutant showeda strong synthetic defect in mature tRNA export (fig. S4), whichsuggests that Msn5p contributes to this export. Using this mutant,we analyzed the pool of nuclear tRNAs when transcription wasblocked by thiolutin, an RNA polymerase inhibitor (15). In thepresence of thiolutin, pre-tRNA-ProUGG disappeared within 1hour (Northern hybridization, Fig. 2A; FISH, Fig. 2B, pre),whereas mature tRNA-ProUGG and tRNA-TyrGUA accumulated in thenucleus (Fig. 2B, mature). Because no detectable transcriptionoccurred under these conditions, nuclear mature tRNAs must havebeen supplied from the preexisting cytosolic pool.
To determine whether nuclear import of mature tRNAs requiresenergy, los1msn5 cells were incubated with NaN3 and 2-deoxyglucose(2-dG). Under these conditions, both the primary transcriptof tRNA-ProUGG and the gradient of mature tRNA-ProUGG acrossthe nuclear envelope (NE) were no longer detected (Fig. 3A andFig. 3B, e). Reappearance of the primary transcript 10 min afterNaN3 and 2-dG removal indicates rapid replenishment of intracellularadenosine triphosphate (Fig. 3A, –thiolutin). Nuclearaccumulation of the mature tRNA was also reestablished afterremoval of NaN3 and 2-dG, even in the presence of thiolutin(Fig. 3B, h and k). We observed similar results with tRNA-IleAAUencoded by intronless genes (Fig. 3B, i and l). These resultsindicate that the import of both intron-containing and intronlesstRNAs requires energy.
Fig. 3. Nuclear import of tRNAs is energy dependent. (A) los1msn5 cells grown in YPD (lane 1) were incubated with NaN3 and 2-dG (YPAdG) for 1 hour (lane 2). The cells were chased without (lanes 3 to 8) or with (lanes 9 to 14) thiolutin for the indicated times. Pre-tRNA-ProUGG was detected by Northern hybridization. White triangle, primary transcripts; black triangle, end-matured pre-tRNAs. (B) los1msn5 cells were treated as in (A) and were harvested before the energy poison treatment (YPD), after the treatment (YPAdG), and after a 2-hour chase without (–thio.) or with (+thio.) thiolutin. Mature tRNA-ProUGG and tRNA-IleAAU were visualized with FISH. (C) los1msn5 cells grown in YPD (lane 1) were incubated in YPAdG (lane 2) and chased with thiolutin for 1 hour (lane 3) or 2 hours (lane 4). Aminoacylation states of tRNA-ProUGG and tRNA-TyrGUA were analyzed with acidic urea-PAGE. Lane 5 contains deacylated tRNA. White and black triangles represent aminoacylated and deacylated tRNAs, respectively.
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Fig. 4. Various tRNA species are imported into the nucleus, and import is Ran independent. (A) cca1-1 cells grown at 23°C were incubated at 37°C for 3 hours, treated with NaN3 and 2-dG for 3 hours at 37°C (YPAdG), and then chased without (–thio.) or with (+thio.) thiolutin for 1.5 hours. Samples were analyzed using FISH (top) and Northern hybridization (bottom) with indicated probes. White, black, and gray triangles represent aminoacylated, deacylated, and 3'-truncated tRNAs, respectively. (B) rna1-1 cells grown at 23°C were incubated at 37°C for 1.5 hours to induce defects and then processed as in (A).
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We analyzed aminoacylation states of mature tRNAs imported intothe nucleus using acidic urea–polyacrylamide gel electrophoresis(urea-PAGE). Mature tRNA-ProUGG and tRNA-TyrGUA in los1msn5cells were present primarily as slower migrating forms evenafter the chase with thiolutin (Fig. 3C, lanes 1, 3, and 4).These forms were converted to faster migrating forms by basetreatment (lane 5), which indicates that the slower migratingtRNAs are aminoacylated. Therefore, imported tRNAs exist mainlyin aminoacylated forms in the nucleus.
We asked whether 3'-truncated tRNAs are imported. cca1-1 cellsare deficient in both de novo formation and repair of 3'-terminalCCA ends of tRNAs (16), and also in tRNA export (4). We thereforetreated cca1-1 cells with NaN3 and 2-dG at a restrictive temperatureto abolish the mature tRNA gradient across the NE and chasedthem with thiolutin. tRNA-TyrGUA and tRNA-IleAAU reaccumulatedin the nucleus to similar levels (Fig. 4A, q to t), althoughthe proportions of the 3'-truncated forms of these tRNAs weredifferent (Fig. 4A, bottom). Taken together with the fact thattRNAs in los1msn5 cells were full-length tRNAs, these resultsindicate that various forms of tRNAs are imported.
To determine the contribution of Ran guanosine triphosphatase(GTPase) (17), we examined tRNA import in rna1-1 cells defectivein RanGAP (Ran GTPase activating protein). The rna1-1 cellsaccumulated mature tRNA-TyrGUA in the nucleus at 37°C (2),and this tRNA gradient disappeared upon treatment with NaN3and 2-dG (Fig. 4B, 37°C, YPAdG). When the cells were transferredto medium with thiolutin, mature tRNA-TyrGUA was reaccumulated(Fig. 4B, +thio.). Because protein import ceased at 37°Cin rna1-1 cells (fig. S5), these results suggest that tRNA importis Ran independent.
Our results indicate that nuclear mature tRNAs are suppliedfrom the cytosol and that tRNAs shuttle between these two compartments.Shuttling may contribute to tRNA quality control, because tRNAshave long lifetimes and may run the risk of inappropriate modifications(18). A quality-control system in the nucleus may repair orfilter out inactive tRNAs from those shuttling through the nucleusand provide only active tRNAs back to the cytosol. Another controversialpossibility would be to supply tRNAs for nuclear translation(19).
References and Notes
1. A. K. Hopper, E. M. Phizicky, Genes Dev.17, 162 (2003).[Free Full Text]
20. We thank A. K. Hopper, K. Weis, P. A. Silver, Y. Ohya, and J. L. Brodsky for experimental materials and critical reading of our manuscript. We appreciate support from our lab members, especially T. Makio and S. Nishikawa. This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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los1 cells, lacking yeast exportin-t (10, 11), exhibit a moderate defect in nuclear export of mature tRNAs (fig. S4B) (2). In 
