DTD (D-Tyr-tRNATyr deacylase) is known to be able to deacylate D-aminoacyl-tRNAs into free D-amino acids and tRNAs and therefore contributes to cellular resistance against D-amino acids in Escherichia coli and yeast. We have found that h-DTD (human DTD) is enriched in the nuclear envelope region of mammalian cells. Treatment of HeLa cells with D-Tyr resulted in nuclear accumulation of tRNATyr. D-Tyr treatment and h-DTD silencing caused tRNATyr downregulation. Furthermore, inhibition of protein synthesis by D-Tyr treatment and h-DTD silencing were also observed. D-Tyr, D-Asp and D-Ser treatment inhibited mammalian cell viability in a dose-dependent manner; overexpression of h-DTD decreased the inhibition rate, while h-DTD-silenced cells became more sensitive to the D-amino acid treatment. Our results suggest that h-DTD may play an important role in cellular resistance against D-amino acids by deacylating D-aminoacyl tRNAs at the nuclear pore. We have also found that m-DTD (mouse DTD) is specifically enriched in central nervous system neurons, its nuclear envelope localization indicates that D-aminoacyl-tRNA editing may be vital for the survival of neurons under high concentration of D-amino acids.

INTRODUCTION

L-amino acids predominantly exist in organisms and are incorporated into proteins. Although most D-amino acids cannot be incorporated into proteins, D-amino acids still have toxic effects in both prokaryotes and eukaryotes [13]. DTD (D-Tyr-tRNATyr deacylase) is a well-conserved protein able to hydrolyse D-Tyr-tRNATyr into free tRNATyr and D-Tyr. It was reported that inactivation of DTD caused increased toxicity of D-Tyr in Escherichia coli and yeast [46], and the toxicity of D-Tyr in E. coli was demonstrated by charging of tRNATyr and starvation of free tRNATyr and L-Tyr-tRNATyr in DTD-deficient E. coli [7]. Besides D-Tyr, DTD was also considered able to act on D-Asp, D-Trp, D-Ser, D-Leu, D-Gln, D-Phe and D-Gly aminoacylated tRNAs. Toxicities of D-Trp, D-Asp, D-Ser and D-Gln were also increased by inactivation of DTD in E. coli [5]. The conserved DTD was found in prokaryotic and eukaryotic species with conserved structures for deacylation [4,6,8]. In Archaea, a novel DTD deacylase was discovered [9]; however, the conserved DTD motif was also found within threonyl-tRNA synthetase as an editing domain for protein synthesis quality control [10]. The h-DTD (human DTD) gene was isolated from a fetal brain cDNA library [11], encoding a protein involved in the assembly of the DNA replication complex [12], and its structure and activity for deacylation and DNA binding was also elucidated [13]. The N-terminal DTD domain of h-DTD is responsible for deacylation and the C-terminal additional domain is responsible for h-DTD's DNA-binding activity. Whether h-DTD carries out its initial function in mammalian cells is not clear yet. In this study, we found that h-DTD is localized around the nuclear pores and may contribute to the D-amino acid resistance in mammalian cells by deacylating D-aminoacyl-tRNAs during tRNA export.

MATERIALS AND METHODS

Antibody

h-DTD polyclonal antibody was raised in rabbit against purified 6-His-tagged h-DTD protein expressed in E. coli, and the antibody was antigen affinity purified (AbMART, China). Antibody specificity was examined (Supplementary Figure S1, at http://www.BiochemJ.org/bj/417/bj4170085add.htm).

Western blot

The whole cell and tissue extracts were prepared and separated on SDS/PAGE (12% gel), and then transferred to a nitrocellulose membrane (Amersham). In addition to rabbit polyclonal anti-h-DTD antibody (1:1000), mouse monoclonal anti-v5 (1:5000, Sigma, St Louis, MO, U.S.A.), rabbit polyclonal anti-FLAG (1:1000, Sigma), rabbit polyclonal anti-Myc (1:1000, MBL, Japan) and mouse monoclonal anti-β-actin (1:5000, Sigma) antibodies were used for detection of the proteins. The HRP (horseradish peroxidase)-conjugated anti-mouse IgG and anti-rabbit IgG antibodies (1:4000, Santa Cruz Biotechnology, U.S.A.) were used as secondary antibodies and signal was visualized using the ECL® (enhanced chemiluminescence; Amersham) system. Cells were donated by the Department of Anatomy, Peking Union Medical College.

Immunofluorescence

Plasmids coding full-length and the C-terminal deletion of h-DTD with both the C-terminal Myc tag and the N-terminal FLAG tag were constructed. HeLa human cervical carcinoma cells (1×104) were plated in each well of 12-well plates and incubated for 1 day under a humidified atmosphere of 5% CO2 and 95% air at 37 °C. HeLa cells were transfected with h-DTD plasmids and fixed with 4% paraformaldehyde at room temperature (25 °C), and permeabilized with 0.1% Triton X-100 and 0.1% sodium citrate for 5 min. Cells were then blocked with 3% BSA in PBS at 37 °C for 30 min and immunostained with rabbit polyclonal anti-FLAG (1:200, Sigma), rabbit polyclonal anti-Myc (1:200, MBL) and NPC (nuclear pore complex) marker (1:300, Abcam ab24609, U.S.A.). Cells were then thoroughly washed three times with PBS. The TRITC-conjugated anti-mouse IgG and FITC-conjugated anti-mouse IgG (1:150, Santa Cruz Biotechnology) were used as secondary antibodies. Cells were viewed with a Leica TCS confocal microscope.

HeLa cells (1×104) were plated in each well of 12-well plates, incubated for 1 day under a humidified atmosphere of 5% CO2 and 95% air at 37 °C, then synchronized with the double-thymidine block method. Briefly, cells were cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% FCS and 10 mM thymidine for 12 h, followed by 9 h in fresh DMEM containing 10% FCS, and then for 12 h in the thymidine-containing medium. Following the last thymidine incubation, the cells were cultured in fresh DMEM containing 10% FCS. Cells synchronized in the G1-phase were fixed as described above immediately. Cells synchronized in the S-phase were fixed between 4 and 6 h. Cells synchronized in the G2-phase were fixed after 8 h. After 6 h of release, nocodazole was added to a final concentration of 40 ng/ml. The culture was continued for another 6 h and round mitotic cells were fixed. Fixed cells were immuno-stained with h-DTD polyclonal antibody (1:200) and NPC marker (1:300).

Immunohistochemistry

Tissues were fixed in 4% PFA in PBS overnight at 4 °C and embedded in paraffin wax using routine histological procedures. Approx. 8 μm thick sections were cut from paraffin blocks and mounted onto polylysine-coated slides (VWR). Slides were dried at 60 °C for 1 h and then deparaffinized in xylene for 4, 3 and 3 min, followed by rehydration in ethanol concentrations declining from 99% to 70% ethanol by several steps lasting 1 min, terminating by running tap water. Sections were rinsed in two shifts of distilled water prior to epitope retrieval. Then sections were rinsed in PBS, treated with 1% hydrogen peroxide for 15 min (to quench endogenous peroxidase reactivity) and thoroughly washed three times with PBS. After pre-blocking with a solution of 1% bovine serum albumin, 5% normal goat serum and 0.2% Triton in PBS, these sections were incubated overnight at 4 °C in the h-DTD polyclonal antibody (1:300), MAP-2 (microtubule-associated protein-2) monoclonal antibody (1:100, Chemicon, U.S.A.) and GFAP (glial fibrillary acidic protein) monoclonal antibody (1:75, Santa Cruz Biotechnology, U.S.A). Control sections were incubated without antibody or with antibody that had been pre-absorbed with 6-His tagged h-DTD protein (0.05 mg/ml). After repeated washings with PBS, sections were incubated for 1 h with anti-rabbit IgG and anti-mouse IgG. Sections were then reacted for 1 h using the ABC (avidin–biotin complex). DAB (3, 3′-diaminobenzidine tetrahydrochloride dihydrate) was used as the chromogen. Immunoreacted sections were dehydrated in alcohol and coverslipped with Permount.

Immunoelectron microscopy

Untreated HeLa cells were harvested, washed twice with PBS, prefixed with 4% paraformaldehyde in PBS for 1 h followed by 2% glutaraldehyde in PBS for 1 h, then embedded in 2% low melting temperature agarose. Cells were further postfixed with 1% OsO4 in PBS for 40 min. The fixed HeLa cells were washed twice with PBS, dehydrated with 50% ethanol for 10 min, 70% ethanol and 2% uranyl acetate for 1 h, 90% ethanol for 10 min and 100% ethanol for 10 min. The samples were then infiltrated with 2:1 mixture of L.R. White resin (London Resin Company, U.K.) and 100% ethanol for 1 h, pure L.R. White resin for 1 h, followed by pure L.R. White resin overnight, and finally in pure L.R. White resin for 1 h. The 1.5 ml microfuge tubes were filled with fresh pure L.R. White resin and polymerized overnight at 60 °C. Thin sections were cut on an MT2-B Ultracut microtome (Sorvall, Japan) using a diamond knife and collected on nickel EM grids with carbon support film. Blocking was carried out with normal goat serum in PBS for 30 min. After a 3 min wash with PBS, grids were incubated for 1 h with 1:2000 h-DTD polyclonal antibody in PBS at room temperature and overnight at 4 °C. Grids were washed five times in PBS for 3 min, and then incubated in 1:40 10 nm gold-labelled goat anti-rabbit IgG secondary antibody (Bios, China) in PBS at room temperature for 30 min. After five washes with PBS for 3 min, the grids were stained with 2% uranyl acetate for 1 h and 2% lead citrate for 5 min. Electronmicrographs were taken with a JEM-1010 transmission electron microscope (JOEL, Japan) operated at an acceleration voltage of 80 kV.

FISH (fluorescence in situ hybridization) and Northern blotting

HeLa cells (1×104) were plated in each well of 12-well plates, incubated for 1 day under a humidified atmosphere of 5% CO2 and 95% air at 37 °C, and then transfected with 100 nM non-silencing RNA or h-DTD-siRNA (h-DTD-small interfering RNA) (5′-AAGCACUGGUCGAAGAGUGUG-3′) [12] (GeneChem, China) using Lipofectamine™ 2000 reagent (Invitrogen) following the manufacturer's instructions. Cells were incubated 6 h after transfection with media containing 0 mM or 7.5 mM D-Tyr for 24 h. A 59 base DNA probe specific for human mature tRNATyr labelled with Alexa 555 (5′-CCGGAATCGAACCAGCGACCTAAGGATCTANAGTCCTCCGCTCTACCNGCTGAGCTATC-3′) was used to visualize tRNATyr by FISH assay as described [14].

HeLa cells (1×106) were plated in each 100 mm dish. Cells were treated as described above. All subsequent steps were performed at 4 °C. The transfected and D-Tyr treated HeLa cells were washed with AMC buffer [14], and lysed in 700 μl AMS buffer with 0.1% NP-40 (Nonidet P-40) for 1 h. Nuclear pellets were washed and resuspended in 700 μl AMS. Nuclear RNA was extracted as described [14] and deacylated tRNA marker was prepared by incubating nuclear RNA in 50 mM Tris/HCl, pH 9.5, at 37 °C for 1 h. RNA from the nuclear fractions was separated by electrophoresis on an 8% polyacrylamide gel containing 8 M urea at 4 °C using 100 mM sodium acetate buffer, pH 5.0 [14].

HeLa cells (2×105) were plated in each well of 6-well plates and treated as described above. Total RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's instructions and separated by electrophoresis on a 15% denaturing polyacrylamide gel.

RNA was transferred to Hybond N+ membrane (Amersham) [14]. A 5′ digoxin tagged probe with the same sequence as above was used to visualize the tRNA bands by Northern blotting.

Protein synthesis and cell viability assays

[3H]Leucine incorporation and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay were carried out according to standard protocols. HeLa cells (6×103) were plated in each well of 96-well plates and transfected with non-silencing or h-DTD-siRNA. D-Tyr (0 mM or 7.5 mM) and 1 μCi/ml [3H]leucine were added to the media, and cells were treated for 24 h. The [3H]leucine incorporation assay was used to measure protein synthesis rate, and the control cell cultures were considered 100% viable. HeLa cells (6×103) were plated in each well of 96-well plates and transfected with pcDNA3.1v5 empty vector plasmid (control)/h-DTD full-length pcDNA3.1v5 plasmid (overexpression of h-DTD) or transfected with non-silencing/h-DTD siRNA as described above. After treatment with 0–7.5 mM D-Tyr, D-Asp and D-Ser for 24 h, cell viability was measured using the MTT assay. MTT solution (10 μl; 5 mg/ml) was added to each well and mixed gently, and incubated at 37 °C for 3 h. The medium was then removed and 200 μl of DMSO added to each well to dissolve the formazan by pipetting. The absorbance was measured at 570 nm. The control cells were considered 100% viable. Inhibition rate is defined as [1−(cell viability of treated cells/control cell viability)]×100%.

Flow cytometry

HeLa cells (4×105) were plated in each 60-mm-diameter dish. Cells were transfected with non-silencing and h-DTD siRNA, and treated with 0 mM or 7.5 mM D-Tyr as described above. Cells were harvested after 24 h and fixed with 70% ethanol at 4 °C for 12 h. Cells were washed twice with PBS and resuspended in 0.5 ml PBS. PI (propidium iodide) and RNase A (50 μg/ml, final concentration) were added and the mixture was incubated at 37 °C for 30 min. Flow cytometry was performed according to standard protocol.

RESULTS

Mammalian DTD protein is highly expressed in CNS (central nervous system) neurons

Mammalian DTD was first found in rabbit reticulocytes and rat liver extracts [15]. In subsequent investigations, Northern blotting analysis showed that h-DTD is widely expressed [11], especially in adult brain, testis, ovary, spleen and fetal brain. We have developed a rabbit polyclonal antibody specific for DTD (Supplementary Figure S1) and examined the multi-tissue expression pattern of h-DTD protein by Western blotting. h-DTD is widely expressed and the highest h-DTD protein level was observed in tissue samples from the CNS, including hippocampus, cerebrum and cerebellum (Figure 1a). There is some ambiguity relating to the expression pattern between h-DTD mRNA and protein, but it is clear that h-DTD is highly expressed in the CNS. We also performed an immunohistochemistry study of m-DTD (mouse DTD) protein in the adult mouse CNS (Figure 1b). The most intense expression was observed in hippocampal pyramidal cells, cerebellar purkinje cells and most layers of the cerebral cortex, and condensed staining was observed at the nuclear envelope region of neurons. However, a signal was absent from the white matter, suggesting that mammalian DTD protein expression may be highly enriched in neurons, which was verified by immunohistochemistry of MAP-2 (neuron marker) and GFAP (glial cell marker).

Expression pattern of h-DTD and M-DTD (mouse DTD)

Figure 1
Expression pattern of h-DTD and M-DTD (mouse DTD)

(a) Multi-tissue expression pattern of h-DTD. (b) Immunohistochemistry study of m-DTD. Adult mouse brain was immunostained with h-DTD polyclonal antibody, MAP-2 monoclonal antibody or GFAP monoclonal antibody. Slides were pictured of 100x magnification.

Figure 1
Expression pattern of h-DTD and M-DTD (mouse DTD)

(a) Multi-tissue expression pattern of h-DTD. (b) Immunohistochemistry study of m-DTD. Adult mouse brain was immunostained with h-DTD polyclonal antibody, MAP-2 monoclonal antibody or GFAP monoclonal antibody. Slides were pictured of 100x magnification.

h-DTD is enriched in the nuclear envelope region

h-DTD was initially named as HARS2 (histidyl-tRNA synthetase 2) for its similarity to HARS [11], HARS has been reported to co-localize with the NPC marker [16]. To investigate the subcellular localization of h-DTD, HeLa cells were transfected with plasmids coding the full-length and C-terminal deletion of h-DTD. The fusion proteins with C-terminal Myc tag and N-terminal FLAG tag both presented similar peri-nuclear enriched localizations; immunofluorescence signal can also be seen in the cytoplasm (Figure 2). Deletion of the C-terminal domain, which is responsible for DNA binding, showed no significant influence on the subcellular localization of h-DTD. As it has been reported that translocation of h-DTD occurs during cell cycle progress [12], we used the h-DTD polyclonal antibody to visualize the subcellular localization of endogenous h-DTD in HeLa cells during the cell cycle. As shown in Figure 3, the h-DTD signal was seen within the nucleus during the G1- and S-phases, with condensation of the signal in the nuclear envelope region. In the G2-phase, h-DTD displayed peri-nuclear distribution in cells. In the M-phase cells, h-DTD displayed cytoplasmic distribution, similar to the NPC marker. Using an immunoelectron microscopy assay, we confirmed that the nuclear envelope contains h-DTD. As shown in Figure 4, colloidal gold particles labelled both the cytoplasmic periphery and nuclear periphery of the nuclear pore (arrowheads), with some directly on the nuclear pore and some adjacent to the pore. The nuclear pore localization of h-DTD indicates that it may be functioning during tRNA export.

Subcellular localization of overexpressed h-DTD

Figure 2
Subcellular localization of overexpressed h-DTD

Fragments coding for full-length and the C-terminal deletion of h-DTD were subcloned into pcDNA4-myc (C-terminal tag) and pcDNA3-flag (N-terminal tag) vectors and transfected to HeLa cells. Rabbit anti-Myc or anti-FLAG polyclonal antibodies and FITC-labelled secondary antibody were used to visualize tagged proteins. The NPC marker and TRITC-labelled secondary antibody were used to show the NPC. DAPI (4′,6-diamidino-2-phenylindole) was used to stain the nucleus. Further study of locations of full-length h-DTD and deletion mutants are shown in Supplementary Figure S2, at http://www.BiochemJ.org/BJ/417/bj4170085add.htm.

Figure 2
Subcellular localization of overexpressed h-DTD

Fragments coding for full-length and the C-terminal deletion of h-DTD were subcloned into pcDNA4-myc (C-terminal tag) and pcDNA3-flag (N-terminal tag) vectors and transfected to HeLa cells. Rabbit anti-Myc or anti-FLAG polyclonal antibodies and FITC-labelled secondary antibody were used to visualize tagged proteins. The NPC marker and TRITC-labelled secondary antibody were used to show the NPC. DAPI (4′,6-diamidino-2-phenylindole) was used to stain the nucleus. Further study of locations of full-length h-DTD and deletion mutants are shown in Supplementary Figure S2, at http://www.BiochemJ.org/BJ/417/bj4170085add.htm.

Immunofluorescence of endogenous h-DTD in cell cycle progress

Figure 3
Immunofluorescence of endogenous h-DTD in cell cycle progress

HeLa cells were synchronized in four cell cycle phases and immunostained with h-DTD polyclonal antibody and NPC marker. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 3
Immunofluorescence of endogenous h-DTD in cell cycle progress

HeLa cells were synchronized in four cell cycle phases and immunostained with h-DTD polyclonal antibody and NPC marker. DAPI, 4′,6-diamidino-2-phenylindole.

Immunoelectron microscopy study of endogenous h-DTD in HeLa cells

Figure 4
Immunoelectron microscopy study of endogenous h-DTD in HeLa cells

A stretch of the nuclear envelope is shown. Endogenous h-DTD was labelled with h-DTD polyclonal antibody (arrowheads).

Figure 4
Immunoelectron microscopy study of endogenous h-DTD in HeLa cells

A stretch of the nuclear envelope is shown. Endogenous h-DTD was labelled with h-DTD polyclonal antibody (arrowheads).

h-DTD silencing and D-Tyr treatment reduce tRNATyr availability

As DTD deletion and D-Tyr treatment have been reported to affect the tRNATyr availability in E. coli [7], we studied the tRNATyr distribution and level in HeLa cells in response to h-DTD silencing and D-Tyr treatment. As shown in Figure 5(a), a similar silencing efficiency was achieved as previously reported [12]. A 59-base Alexa Fluor®555-tagged DNA probe was used to visualize mature tRNATyr in HeLa cells, and the same digoxin-labelled probe specifically hybridized to one band in Northern blotting analysis of total HeLa RNA (Figure 5b). Using the FISH assay, we observed nuclear accumulation of human mature tRNATyr in response to treatment of D-Tyr. Similar responses were found under the condition of L-amino acid deprivation in both yeast and mammalian cells [17,18]. In h-DTD-silenced cells, the nuclear accumulation of human mature tRNATyr was not as obvious as that seen in non-silenced cells (Figure 5a). Then we detected the total tRNATyr level. Both h-DTD silencing and D-Tyr treatment reduced the level of tRNATyr (Figure 5b). A similar tRNA depletion was observed in Drosophila when exportin 5, which is responsible for tRNA nuclear export, is deleted [19]. The nuclear aminoacylation level was also determined; 70–80% of the nuclear tRNATyr is aminoacylated, as previously found in yeast [14]. Neither h-DTD silencing nor D-Tyr treatment significantly influenced the nuclear aminoacylation levels. Taken together, the tRNA availability for cytoplasmic protein synthesis is influenced by h-DTD silencing and D-amino acid treatment.

D-Tyr treatment and h-DTD silencing influences tRNATyr distribution and level

Figure 5
D-Tyr treatment and h-DTD silencing influences tRNATyr distribution and level

(a) Nuclear accumulation of tRNATyr following D-Tyr treatment. Scale bar, 19.08 μm. (b) Total tRNATyr level in HeLa cells and aminoacylation status analysis of nuclear tRNATyr.

Figure 5
D-Tyr treatment and h-DTD silencing influences tRNATyr distribution and level

(a) Nuclear accumulation of tRNATyr following D-Tyr treatment. Scale bar, 19.08 μm. (b) Total tRNATyr level in HeLa cells and aminoacylation status analysis of nuclear tRNATyr.

h-DTD silencing and D-amino acid treatment inhibits protein synthesis and cell viability

tRNA availability is crucial for protein synthesis and cell viability in E. coli [7]. We used a [3H]leucine incorporation assay to investigate the influence of h-DTD silencing and D-amino acid treatment on protein synthesis of mammalian cells. When treated with D-Tyr, the h-DTD-silenced HeLa cells showed increased inhibition in protein synthesis compared with the non-silenced cells (94.8% to 50.3% compared with 100% to 66.4%) (Figure 6a). The MTT assay showed that D-amino acid (D-Tyr, D-Asp and D-Ser) treatment resulted in decreased cell viability, and the decrease was dose-dependent (Figures 6b–6d). For example, in non-silenced cells, 1.25 mM, 2.5 mM, 5 mM and 7.5 mM D-Tyr inhibited cell viability by 0.3%, 1.6%, 5.5% and 11.6%. Overexpression of h-DTD decreased the inhibition rate, while h-DTD-silenced cells became more sensitive to D-amino acid treatment (Figures 6b–6d). As shown in Figure 6(b), when treated with 7.5 mM D-Tyr, h-DTD-overexpressed cells displayed only 8.7% inhibition rate of cell viability, whereas in empty vector transfected cells the value is 16.6%. h-DTD silencing caused approx. twice the inhibition in non-silenced cells (23.8% compared with 11.6%). These results indicate that h-DTD, similar to its homologues in E. coli and yeast [47], contributes to mammalian cellular resistance to D-amino acids.

h-DTD and D-amino acids treatment influences protein synthesis and cell viability

Figure 6
h-DTD and D-amino acids treatment influences protein synthesis and cell viability

(a) h-DTD silencing increases the inhibition by D-Tyr of protein synthesis. (bd) h-DTD overexpression plasmid (h-DTDv5) compared with control pcDNA3.1v5 empty vector plasmid (Vector)-transfected HeLa cells and h-DTD-silenced against non-silenced HeLa cells were treated with 0–7.5 mM D-Tyr (b), D-Asp (c) or D-Ser (d). Values are the means of three independent experiments±S.D. **P<0.01, *P<0.05.

Figure 6
h-DTD and D-amino acids treatment influences protein synthesis and cell viability

(a) h-DTD silencing increases the inhibition by D-Tyr of protein synthesis. (bd) h-DTD overexpression plasmid (h-DTDv5) compared with control pcDNA3.1v5 empty vector plasmid (Vector)-transfected HeLa cells and h-DTD-silenced against non-silenced HeLa cells were treated with 0–7.5 mM D-Tyr (b), D-Asp (c) or D-Ser (d). Values are the means of three independent experiments±S.D. **P<0.01, *P<0.05.

DISCUSSION

Being different from that in prokaryotes, tRNA synthesis and protein synthesis are separated by the nuclear envelope in eukaryotes. In yeast, a nuclear pool of aminoacyl-tRNA synthetases was found [20] and the nuclear aminoacylation of tRNA was thought to be an important procedure for tRNA maturation [21]. Furthermore, the nuclear tRNA aminoacylation-dependent pathway is thought to be the principal route for tRNA export from the nucleus [14]. Further reports have shown that tRNA shuttles actively between the nucleus and cytosol in yeast and mammalian cells [17,18,21,22]. Two possibilities for this phenomenon were suggested: tRNA cytoplasmic splicing and tRNA quality control [21]. In mammalian cells, splicing of tRNAs is likely to be restricted to the nucleus with only mature tRNAs being exported [21], so a quality control system may exist to repair or filter out dysfunctional tRNAs during shuttling and permit only active tRNAs to enter the cytosol [22]. On the other hand, protein synthesis exhibits strict homochirality, showing preference for L-amino acids. In vitro experiments using RNA minihelix (progenitor of the modern tRNA) as substrate indicated that chiral selection in aminoacylation is probably determined by the pre-existing chirality of the RNA [23,24], so the quality control of tRNA may be very important in maintaining the homochirality in aminoacylation. DTD in all three kingdoms maintains this homochirality via a deacylase activity [49]. Kemp et al. [13] showed that the deacylase activity of h-DTD was by hydrolysing E. coliD-[3H]Asp-tRNA. In the present paper, we found that at least some of the mammalian DTD is localized to the nuclear pore (Figures 2, 3 and 4). On the basis of its activity and position, mammalian DTD may play a role in deacylating D-aminoacyl-tRNA during tRNA exporting or shuttling. The accumulation of tRNA during D-amino acid treatment (Figure 5a) is the same as the response observed during L-amino acid deprivation [17]. In both cases, the tRNA is more difficult to be L-aminoacylated than under normal conditions. Further investigation is necessary to determine whether there is nuclear aminoacylation and that the aminoacylation-dependent pathway is the principal pathway for tRNA export, as it is in yeast and mammalian cells. However, it is clear that nuclear tRNATyr aminoacylation in HeLa cells is similar to that observed in yeast (Figure 5b) [14]. The nuclear aminoacylated tRNA could also be imported from the cytoplasm. The cytoplasmic D-aminoacyl tRNAs could be deacylated by either cytoplasmic or the nuclear envelope h-DTD. tRNA level decrease caused by h-DTD silencing and D-Tyr treatment was also observed in our investigation (Figure 5b). Our results suggest that h-DTD is involved in a novel mechanism keeping homochirality of tRNA aminoacylation and protein synthesis in mammalian cells. We suppose that h-DTD may control the tRNA quality though the aminoacylation-dependent pathway by keeping tRNA with D-amino acid preference from being exported, while it could allow tRNA with L-amino acid preference to be exported (Figure 7). There may be other members involved, for silencing of h-DTD did not cause significant nuclear accumulation of tRNATyr nor effectively abolish the tRNA nuclear accumulation under D-Tyr treatment. The downregulation of tRNATyr in response to h-DTD silencing and D-Tyr treatment may be a protection reaction, keeping D-Tyr from aminoacylation and following protein incorporation. tRNA nuclear accumulation and downregulation together inhibited protein synthesis and this is due to the reduction of tRNA availability for cytoplasmic protein synthesis (Figure 6a). By silencing and overexpressing h-DTD, we found h-DTD plays an important role in the resistance of mammalian cells to D-Tyr, D-Asp and D-Ser (Figure 6b). These results demonstrated that h-DTD still plays its initial role of D-amino acid resistance in mammalian cells as its homologues do in other species.

Overview of h-DTD in tRNA metabolism

Figure 7
Overview of h-DTD in tRNA metabolism

In mammalian cells, tRNAs are transcribed and spliced within the nucleus, and perhaps exported mainly through an aminoacylation-dependent pathway. D-Aminoacylated tRNAs are probably deacylated during export by h-DTD.

Figure 7
Overview of h-DTD in tRNA metabolism

In mammalian cells, tRNAs are transcribed and spliced within the nucleus, and perhaps exported mainly through an aminoacylation-dependent pathway. D-Aminoacylated tRNAs are probably deacylated during export by h-DTD.

The conserved N-terminal tRNA-editing domain of h-DTD is responsible for deacylation, and the C-terminal DNA-binding domain of h-DTD allows it DNA replication-related functions in addition to deacylation [13]. Deletion of the C-terminal did not significantly influence the subcellular localization (Figure 2), probably because the dimerization of the fused protein between endogenous h-DTD or the C-terminal domain is not required for h-DTD's nuclear envelope localization. The overexpressed fusion h-DTD displayed more cytoplasmic distribution than the endogenous h-DTD, probably due to the influence of overexpression. It is reported that h-DTD is involved in the G1- to S-phase progression and plays an important role in DNA replication [12], we found h-DTD within the nucleus and showed condensation in the nuclear envelope region in G1- and S-phase (Figure 3). In the G2-phase, when DNA replication is over, h-DTD displayed a mainly perinuclear localization. When the nuclear envelopes have broken down in M-phase, h-DTD displayed cytoplasmic distribution, similar to the NPC marker. Viewed via electron microscopy, we have found that h-DTD was labelled with colloidal gold particles near the nucleolonema in some nucleoli beside the nuclear pore (Supplementary Figure S3, at http://www.BiochemJ.org/bj/417/bj4170085add.htm), where DNA replication was going to happen. D-Amino acids were also found within the eukaryotic nucleus and assumed to be involved in some biological function [25]. It is also assumed that tRNA may participate in progresses including DNA replication and transcription other than translation in an uncharged form [26], so deacylation of nuclear aminoacylated tRNA by h-DTD may help tRNA to carry out DNA replication function. In a recent report [27], a relationship between tRNA export and cell cycle check point was discovered in yeast. The nuclear accumulation of un-spliced tRNA resulted in cell cycle arrest at the G1-phase via activating the Gcn4 transcription factor and delaying the accumulation of cyclin Cln2. In our investigation, translocation of h-DTD during cell cycle progress was observed (Figure 3). Treatment of HeLa cells with 7.5 mM D-Tyr resulted in increased populations of S- and G2/M-phase cells. h-DTD-silenced cells had a similar effect. h-DTD-silenced cells treated with 7.5 mM D-Tyr resulted in an increase in populations of S- and G2/M-phase cells (Supplementary Figure S4, at http://www.BiochemJ.org/bj/417/bj4170085add.htm). There is also possibly an unknown mechanism linking tRNA editing and traffic between cell cycle progress in mammalian cells, in which h-DTD is probably involved. However, h-DTD highly expressing neurons are at G0-phase and do not divide. Within these cells, h-DTD is concentrated in the nuclear envelope region (Figure 1b), so the deacylase activity may be principal.

We also found that h-DTD is enriched in the CNS (Figure 1a), where high concentrations of free D-amino acids are present with physiological functions. In brain, levels of D-Ser are up to one-third that of L-Ser, and D-Asp levels are 20–30% of L-Asp levels [28]. D-Ser was recognized as an endogenous ligand of the NMDA (N-methyl-D-aspartate) receptor [29], and D-Asp was shown to influence the secretion of several hormones [30]. We assume that cells in an environment of high D-amino acid concentration need to be more resistant to D-amino acids. Although D-amino acid oxidase and D-Asp oxidase in glia cells can detoxify D-amino acids by oxidation [31], the oxidation product H2O2 influences the oxidation state and causes oxidative stress. Nephrotoxicity caused by oxidation cannot be afforded by neurons [32], so the neurons may simply keep D-amino acids from charging tRNAs by neuron-enriched h-DTD, while allowing the excess D-amino acids oxidized in glia cells. This possible mechanism probably makes neurons more resistant to D-amino acids in order to adapt to the high D-amino acid concentration environment in CNS. Although the chances of incorporation of D-amino acids are very little, they cannot be ignored in the CNS. Depositions of D-Asp and D-Ser in β-amyloid in neurons during aging are possibly related to Alzheimer's disease [33,34], and the ratio of D- to L-amino acids in proteins may be used as a marker of aging [33]. Recently, it was reported that an editing-defective tRNA synthetase caused protein misincorporation, misfolding, neuronal loss and finally neurodegeneration [35]. Since the neurons do not divide, there is no dilution of the misfolded protein deposition. Preventing deposition of D-amino acids in proteins in long-living, non-dividing neurons by h-DTD, a tRNA-editing molecule, could be a vital anti-neurodegeneration mechanism.

We thank Professor Tohru Yoshihisa and Professor Dev Mangroo for kindly providing the aminoacylation analysis protocol. We thank Dr Wei Dai of PUMC (Peking Union Medical College) for his expert technical assistance in immunoelectronmicroscopy assay. Our work has been funded by the National Sciences Foundation of China (30421003, 30430200, 30608022 and 90612019), the grant from the National Program for the Key Basic Research Project ('973′ 2004CB518604, 2005CB522507, 2006CB504100, 2007CB946902 and 2007CB946900), grants from the Hi-Tech Research and Development program of China ('863′2006AA02Z137 and 2006AA02A304) and a grant from Program for New Century Excellent Talents in University (grant number NCET-07-0505).

Abbreviations

     
  • CNS

    central nervous system

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • DTD

    D-Tyr-tRNATyr deacylase

  •  
  • FISH

    fluorescence in situ hybridization

  •  
  • GFAP

    glial fibrillary acidic protein

  •  
  • h-DTD

    human DTD

  •  
  • MAP

    microtubule-associated protein

  •  
  • m-DTD

    mouse DTD

  •  
  • MTT

    3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide

  •  
  • NPC

    nuclear pore complex

  •  
  • siRNA

    small interfering RNA

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Supplementary data