Fig 1.
TPR orthologs in yeast contribute to tRNA export.
(A) Binding of Nup60 at tRNA genes. ChIP-qPCR of Nup60-TAP was performed with strains MC177 (wt), MC273 (Δmlp1), MC276 (Δmlp1,2), and MC172 (no TAP) after M phase arrest by CDC20 depletion, when Nup60 was known to have increased association with tRNA genes in the wt strain. Data (mean ± SD, n = 3) were analyzed by Student’s t-test in paired comparison with the same tRNA gene in the wt strain; *P < 0.05. (B) tRNA gene-NPC association. Strains MC199 [tS(CGA)C::256lacop], MC200 [tT(UGU)G1::256lacop], MRG7425 [tS(CGA)C::256lacop Δmlp1,2], and MRG7426 [tT(UGU)G1::256lacop Δmlp1,2] were examined after M-phase arrest. Colocalization was defined as GFP and RFP foci were separated by no more than the width of the GFP spot. Scale bar = 10 μm. N denotes the number of cells analysed. Pairwise χ2-tests, *P < 0.05. (C) Nascent tRNA levels. RT-qPCR was done with strains MC177 (wt) and MC276 (Δmlp1,2) after arrest in M phase to measure the levels of three representative pre-tRNAs and nascent ACT1 mRNA. Values were normalized to mature ACT1 mRNA. (D) Western blot to detect the expression of Gcn4-TAP in asynchronously grown cultures of strains MC341 (wt), MC343 (Δlos1), MC349 (Δnup2), MC350 (Δnup60), MC344 (Δmlp1) and MC345 (Δmlp1,2). Coomassie blue staining was used to show even amount of protein loaded in gels.
Fig 2.
Mlp1/2 are required for tRNA export.
(A) Schematics of unprocessed pre-tRNASer(CGA) with and without synthetic MS2 sites. Color code of nucleotides is shown. The MS2 sites are bound by an MS2-GFP chimera that is expressed in yeast. The intron containing MS2 sites is spliced from the tRNA when it reaches the mitochondrial surface in the cytoplasm. (B) Representative images of cells expressing pre-tRNASer(CGA) with MS2 sites, MS2-GFP, as well as Nab2-mCherry to image the nucleus. Asynchronously grown cultures of strains MRG5788 (wt), MRG5789 (Δlos1), MRG5787 (Δnup60), MRG5816 (Δmlp1), and MRG5817 (Δmlp1,2) were used. Scale bar = 10 μm. (C) Quantification was done by calculating the ratio of nuclear / cytoplasmic GFP signal intensity and shown in the column scatter graph. N denotes the number of cells examined, and *P < 0.05 by Student’s t-test when compared with the wt strain. (D) Nucleic acid staining (top) and Northern analysis (bottom) of RNAs isolated from yeast cells carrying the original wildtype tS(CGA)C [ori, strain MRG7400 (wt)] or the modified tS(CGA)C-2×MS2bs [mod, strains MRG5788 (wt), MRG5789 (Δlos1), MRG5816 (Δmlp1) and MRG5817 (Δmlp1,2)] gene. A single 3’-DIG labeled LNA-modified oligonucleotide probe was used to detect both pre-tRNASer(CGA)-2×MS2bs (pre, 5-minute exposure) and mature tRNASer(CGA) (mature, 20-second exposure). The ratio of pre / mature is indicated below each lane. A single 2-minute exposure of the whole blot is shown in S1B Fig. (E) FISH analysis for pre-tRNAIle(UAU) (red in the merged image) in strains BY4741 (wt), MC343 (Δlos1), MC344 (Δmlp1) and MC345 (Δmlp1,2). DAPI staining (green in the merged image) of DNA shows the nuclei. Scale bar = 10 μm.
Fig 3.
Elevated expression of TPR in lung cancer cell lines and tissues.
(A) Western blot to detect the expression of TPR in lung cancer cell lines. HLF (human lung fibroblast) served as the normal control, while A549 (lung adenocarcinoma cell), H322 (bronchioalveolar carcinoma cell), H460 (large cell lung cancer cell) and H1299 (lung carcinoma cell) were representative lung cancer cells. β-tubulin was used as the loading control. (B) TPR mRNA level in human lung cancer (T, n = 356) and non-tumor (N, n = 161) tissues. Data were extracted from GSE7670, GSE10072, GSE19188 and GSE31210, normalized to the median value of the non-tumor tissues in each dataset, and compiled for analysis. *P < 0.05 by Student’s t-test. (C) Left: Western blot to detect the expression of TPR in paired tumor (T) and adjacent non-tumor (N) tissues from lung cancer patients. β-tubulin served as the loading control. Right: Quantification of TPR expression relative to β-tubulin in patient samples. *P < 0.05 by Student’s t-test for pairwise comparison. (D) The prognostic value of TPR mRNA level in lung cancer patients from KM plotter. Overall survival (OS, left) and progression-free survival (PFS, right) curves were plotted using 201731_s_at, which was selected as the optimal microarray probe to represent TPR by JetSet. The low and high TPR expression patient cohorts were compared by a Kaplan-Meier survival analysis, and the hazard ratio (HR) with 95% confidence intervals and logrank P value were calculated.
Fig 4.
Growth inhibition and nuclear accumulation of tRNAs in lung cancer cells with TPR knockdown.
(A) Western blot of TPR in A549, H322, H460 and H1299 cells transfected with TPR-specific siRNAs (Tsi, Tsi-1 and Tsi-2) or a control siRNA. (B) Cell viability measured by MTS assay. Data (mean ± SD, n = 3) were analyzed by paired Student’s t-test; *P < 0.05. (C) Cell proliferation capacity evaluated by colony formation assay. Representative images were shown on the top panel. Quantification was presented on the bottom panel. Data (mean ± SD, n = 3) were analyzed by paired Student’s t-test; *P < 0.05, **P < 0.01. (D) Nuclear enrichment of tRNAs in TPR knockdown cells relative to control cells. The nrStar Human tRNA PCR Array was used to measure the levels of nuclear-encoded tRNAs and U6 small nuclear RNA included in the array in the nuclear (ctrl_nuc) and whole cell extracts of A549 cells (ctrl_WCE), as well as A549 cells after TPR knockdown (Tsi_nuc and Tsi_WCE, respectively). The values of the tRNAs were normalized to that of U6, which was known to remain in the nucleus after transcription by RNA polymerase III. Nuclear enrichment of each tRNA in Tsi cells relative to control cells was reported as the ratio of (Tsi_nuc/Tsi_WCE): (ctrl_nuc/ctrl_WCE), as shown in the graph. (E) Direct measurement of nuclear tRNA enrichment by RT-qPCR. Nuclear and whole cell RNA extracts were prepared from A549 cells (control) and A549 cells with TPR knockdown (Tsi-1 and Tsi-2). RT-qPCR was performed for each extract to measure the level of tRNAGlu(CTC), tRNATyr(GTA), tRNAHis(GTG), tRNALeu(CAG), tRNAGly(CCC) and tRNAGly(GCC), using U6 as an internal control. Relative nuclear enrichment of each tRNA was determined by its nuclear / total expression normalized to the same ratio of the tRNA within the control. Data (mean ± SD, n = 3) were analyzed by paired Student’s t-test; *P < 0.05, **P < 0.01.
Fig 5.
A role for NXF1 in tRNA nuclear export.
(A) Interaction between NXF1 and tRNAs. RIP assay was performed with α-NXF1 or α-XPO1 on A549 cell lysates. RIP with IgG served as control. RT-qPCR was performed to measure the amount of tRNAGlu(CTC), tRNATyr(GTA), tRNAHis(GTG), tRNALeu(CAG), tRNAGly(CCC) and tRNAGly(GCC) in the precipitates. Data (mean ± SD, n = 3) were analyzed by Student’s t-test in paired comparison; *P < 0.05. (B) Western blot of NXF1 in A549, H322, H460 and H1299 cells transfected with NXF1-specific siRNAs (Nsi-1 and Nsi-2) or a control siRNA. (C) Nuclear enrichment of tRNAs in A549 cells (control) and A549 cells with NXF1 knockdown (Nsi-1 and Nsi-2). Relative nuclear enrichment of each tRNA tested was determined by its nuclear / total expression normalized to the same ratio of the tRNA within the control, shown as mean ± SD (n = 2), and analyzed by Student’s t-test; *P < 0.05. (D) Cell viability measured by MTS assay. Data (mean ± SD, n = 3) were analyzed by paired Student’s t-test; **P < 0.01. (E) Cell proliferation capacity evaluated by colony formation assay.
Fig 6.
Suppression of protein synthesis by TPR knockdown.
(A) Western blot to detect the phosphorylation of eIF2α. Protein extracts were prepared from A549 and H1299 cells without (control) or with TPR knockdown (Tsi, Tsi-1 and Tsi-2). Specific antibodies were used to detect the cellular levels of TPR, β-tubulin, phosphorylated eIF2α (p-eIF2α) and total eIF2α. Numbers indicate the ratios of TPR / β-tubulin (top) and p-eIF2α / eIF2α (bottom). (B) FUNCAT assay followed by flow cytometry analysis of the fluor 488 signal intensity. A549 and H1299 cells without (control) or with TPR knockdown (Tsi-1 and Tsi-2) were incubated with 4 mM azidohomoalanine (AHA) or methionine (met, as negative control) for 2 hours, and tagged with fluor 488-alkyne for detection. At least 10,000 cells were analyzed for each condition. (C) Correlation of cytoplasmic abundance of tRNAAla, tRNAArg and tRNALeu isoacceptors in control (ctrl) and TPR knockdown (Tsi) A549 cells to codon usage of the top 10 up-regulated (Up) and the 10 most down-regulated (Down) genes in lung cancer tissues with high TPR expression. Cytoplasmic abundance of tRNA was calculated as tRNAcyt = tRNAtot - tRNAnuc, using the data from the tRNA PCR array.