EMBO/soda-nel huggingface dataset:
A dual role of human tRNA methyltransferase hTrmt13 in regulating translation and transcription (2021)
Figure 3

Figure 3. hTrmt13 binds tRNA in cytoplasm and directly binds DNA in nucleus.

A, B. Representative images showing endogenous hTrmt13 (A) and HA-tagged hTrmt13 (HA-hTrmt13, B) subcellular localization by confocal microscopy analysis.

C. Representative confocal microscopy images showing the subcellular localization of hTrmt13 in MDA-MB-231 cells cultured with 10% FBS (left) or no FBS (right).

D. The distribution of hTrmt13 analyzed by subcellular fraction assay. β-actin as a marker of cytosol, Lamin A/C as a marker of nucleoplasm, Histone H3 as a marker of chromatin.

E. Confocal microscopy images of MDA-MB-231 cells were co-immunolabeled with antibodies against hTrmt13 and histone H3 (left). The images were captured by GE DeltaVision OMX SR. Co-localization of H3 and hTrmt13 was analyzed by ImageJ (right, n = 20 cells, center line, the median of the data).

In A, B, C and E, DAPI staining was included to visualize the cell nucleus (Blue).

F. Schematic diagram illustrating the principle of nucleic acids-IP performed in nuclear extract and cytoplasm without adding RNase or DNase.

G. hTrmt13 bound nucleic acids from the cytosol and nuclear extract were stained by GoldView. Transcribed HctRNAGly(GCC) was used as a size marker for tRNAs.

H. Genomic DNA was sheared to fragments by Biorupt (left). EMSA analysis of hTrmt13 and DNA fragments (15 ng/μL DNA was used).

I. The binding profiles of hTrmt13, H3K4me3, H3K27Ac, and other transcription factors (MYC, E2F1, TP53, FOSL1, EZH2, FOXM1) from ChIP-seq and ATAC-seq on representative target genes TGFB1, MEF2A, and PKN2. hTrmt13 track were average across 2 biological replicates.

J. Genomic distribution of hTrmt13 reproducible (N=2) binding sites determined by ChIP-seq analysis in MDA-MB-231 cells.

k, hTrmt13 binding de novo motif for reproducible (N=2) binding sites.

L. hTrmt13 reproducible (N=2) binding sites enriched for known motifs for FOSL1, FOSL2, JunB, BATF, and AP-1.

M. The binding affinities of wild type and mutant hTrmt13s for synthesized DNA (0.25 μM) analyzed by EMSA. The concentration of hTrmt13-dZ2 or hTrmt13 used in the system was 1 μM.

N. EMSA of hTrmt13 for synthesized DNA (0.25 μM) carrying consensus motif or mutated motif.
A dual role of human tRNA methyltransferase hTrmt13 in regulating translation and transcription (2021)
Figure 4

Figure 4. hTrmt13 promotes cell migration independent of tRNA modification activity.

A. MTT assay for cell proliferation in MDA-MB-231 cells infected with lentiviruses carrying control shRNA (shSCR) or shRNA against hTrmt13 (sh-1, sh-2). Error bars represent mean ± SD for three independent experiments.

B. Transwell migration assays in MDA-MB-231 cells. Control with siRNA scramble (NC), treated with siRNA against hTrmt13 (si-1, si-2) and stably expressing wild-type hTrmt13 (si-1+WT) or enzymatic inactive hTrmt13 mutant (si-1+E463A). Representative images (left panel) and statistical analysis (middle panel) are shown.

C. Transwell migration assays in HCC38 cells infected with scrambled siRNA (NC) or siRNA against hTrmt13 (si-1, si-2).

D. Western blot of the expression of Vimentin, N-cadherin, and E-cadherin in MDA-MB-231 cells with the indicated treatments. See also Fig EV4D.

E. Volcano plot of differential expressed genes analysis results for RNA-seq data comparing hTrmt13 knockdown MDA-MB-231 cells (N=2) to control cells (N=2), the dots for significant regulated genes (FDR corrected p-value < 0.05) were colored red, the dots and labels of interesting genes with oncogenic implications have been colored in blue or red.

F. Functional categories of downregulated genes in hTrmt13 knockdown MDA-MB-231 cells showing the p-value for the enrichment of biological process GO term.

G. GSEA plot showing an interesting gene set from MSigDB were enriched for gene down-regulated in hTrmt13 knockdown MDA-MB-231 cells (hTrmt13 KD) for POOLA_INVASIVE_BREAST_CANCER_UP and SARRIO_EPITHELIAL_MESENCHYMAL_TRANSITION_UP (extracted from GSE47203).

H. Heatmap of expression level for highlighted genes and core enriched genes in EMT gene set.

Data information: In B and C, for each group, five different fields were chosen and counted using a microscope with 20-fold magnification. Statistical analysis was performed using t-tests. Error bars represent mean ± SD for four (B) or three (C) independent experiments.
A dual role of human tRNA methyltransferase hTrmt13 in regulating translation and transcription (2021)
Figure 6

Figure 6. hTrmt13 interacts with FOSL1, E2F1 and USF1 to regulate transcription.

A. The Venn diagram of overlapping genes targeted by hTrmt13, FOSL1, and E2F1 in MDA-MB-231 cells. Among 1136 (220+32+371+513=1136) hTrmt13 binding sites, ~35% were also bound by FOSL1(32+371=403), ~78% were also bound by E2F1 (513+371=884), while 92% of hTrmt13 and FOSL1 co-binding sites (403) also have E2F1 binding (371).

B. Interaction of FLAG-hTrmt13 with endogenous FOSL1, E2F1, and USF1. Whole-cell lysates from MDA-MB-231 cells stably expressing FLAG-hTrmt13 were prepared and immunoprecipitation was performed with anti-FLAG followed by immunoblotting with antibodies against indicated proteins.

C. Interaction of endogenous hTrmt13 with FOSL1, E2F1, and USF1.Whole-cell lysates from MDA-MB-231 cells were immunoprecipitated with antibodies against hTrmt13 or IgG followed by immunoblotting with the antibodies against the indicated proteins. * and ** are nonspecific bands.

D. A graphical scheme illustrating PLA analysis of protein-protein interaction.

E. PLA analysis showing representative images obtained from imaging confocal microscopy of the interaction of hTrmt13 and FOSL1, hTrmt13 and E2F1, hTrmt13 and USF1. Red dots indicate that the detected protein interacts with hTrmt13, and DAPI staining was included to visualize the cell nucleus (Blue). The anti-hTrmt13 and IgG was used as a negative control.

F. ChIP-qPCR analysis of the selected promoters was performed using antibodies against indicated proteins in control and hTrmt13 knockdown MDA-MB-231 cells. Results are represented as fold change over control IgG. Statistical analysis was performed using t-tests. Error bars indicate mean ± SD for three independent experiments.
A dual role of human tRNA methyltransferase hTrmt13 in regulating translation and transcription (2021)
Figure 7

Figure 7. hTrmt13 directly interacts with USF1 to exclude binding with tRNAs in nucleus

A. In vitro pull-down assay of hTrmt13 with USF1. USF1 with an N-terminal GST tag (GST-USF1) co-purified by hTrmt13 with a C-terminal His tag (hTrmt13-His) using Ni-NTA resin. GST-USF1 and hTrmt13-His were expressed separately in E. coli BL21.

B. The gel filtration chromatography of hTrmt13-His or USF1-GST or their mixture on Superdex 200 Increase.

C. The binding shift assays of hTrmt13, USF1, and their mixture for synthesized DNA from the TGFB1 promoter. In the reaction system, 10 ng/μL DNA was used, and 1 μM hTrmt13 or 1 μM USF1 or a mixture (0.5 μM hTrmt13 + 0.5 μM USF1) was applied.

D. The binding shift assays of hTrmt13 for HctRNAGly(GCC) with the presence of various concentrations of USF1. In the reaction system, 0.3 μM HctRNAGly(GCC) and 0.5 μM hTrmt13 were used, and a varied amount of USF1 (0, 0.5, 1, 2, 3, 4 μM) were used.

E. The binding shift assays of hTrmt13 (0.5 μM), or USF1 (0.5 μM) or the hTrmt13-USF1 complex (0.5 μM) for both tRNA (0.1 μM) and DNA (15 ng/μL) in the same reaction.

F. A proposed model based on the above results showing that standalone hTrmt13 binds either to tRNA or synthesized DNA, while the complex of hTrmt13 and TFs (e.g., USF1) excludes the binding with tRNA but not DNA.
A dual role of human tRNA methyltransferase hTrmt13 in regulating translation and transcription (2021)
Figure 8

Figure 8. hTrmt13 regulates in vivo cancer progression.

A. Volcano plot of differential expressed genes analysis results for RNA-seq data on TCGA comparing hTrmt13 expression low group patients (first quartile) versus high group patients (fourth quartile). Significant differentially expressed genes (FDR corrected p-value < 0.05) were colored by red, interesting genes with oncogenic implications are labelled in blue. Pseudo values 1e-15 were added to FDR correlated p-value for better visualization.

B. Stacked bar plot of patients number diagnosed as various breast cancer subtype in TCGA group by the expression level of CDH1 or MMP1 or hTrmt13 (low as the first quartile, high as the fourth quartile).

C. GSEA plot showing an interesting gene set from MSigDB were enriched for genes expressed higher in hTrmt13 low group for SOTIRIOU_BREAST_CANCER_GRADE_1_VS_3_UP (upper) and enriched for genes expressed higher in hTrmt13 high group for TURASHVILI_BREAST_LOBULAR_CARCINOMA_VS_LOBULAR_NORMAL_UP (down).

D. Lung metastasis assay in mice model. MDA-MB-231-Luc-D3H2LN cells infected with lentivirus carrying control (shSCR) or shRNA against hTrmt13 (sh-1) were injected through the tail vein of 6-week-old female SCID mice (n=8). Lung metastasis was monitored by bioluminescent imaging after three weeks of injection. Representative in vivo bioluminescent images are shown (left); Bioluminescent quantitation of lung metastasis (right). Statistical analysis was performed using t-tests.

E. Immunohistochemical staining of tissue arrays containing 24 breast carcinoma samples paired with adjacent normal mammary tissues for hTrmt13 expression. Representative images are shown (left), and the mean staining intensity was calculate using Image-Pro Plus software (right). Statistical analysis was performed using t-tests.

F. Kaplan-Meier survival analysis of Kaplan-Meier plotter (http://kmplot.com/analysis/) for the relationship between the relapse-free survival (RFS) or overall survival time (OS) of breast cancer patients (BRCA, n=349 (low)/205 (high)), liver cancer patients (LIHC, n=270 (low)/100 (high)), patients with kidney renal clear cell carcinoma (KRIC, n=156 (low)/374 (high)), and patients with kidney renal papillary cell carcinoma (KIRP, n=208 (low)/79 (high)) and their expression of hTrmt13.
Inactivation of the type I interferon pathway reveals long double stranded RNA-mediated RNA interference in mammalian cells (2016)
Figure 2

Figure 2. Sequence-specific gene silencing by long dsRNA in cells deficient in the IFN response.

(A) Mavs+/- and Mavs-/- MEFs stably expressing GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs (top panels) or siRNAs (bottom panels). GFP level was measured in Cy5+ cells by flow cytometry 72 hours (hrs) post-transfection. (B) Mavs+/- and Mavs-/- MEFs stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs. The level of d2GFP in Cy5+ cells was measured at various timepoints post-transfection as indicated. Histograms plots show d2GFP level 48 hrs post-transfection. Bar graphs show 3 timepoints as indicated. (C) WT, Ifnar1+/- or Ifnar1-/- MEFs stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+ cells was monitored by flow cytometry at 48 hrs post-transfection. All histograms plots are representative of 10 (A-B), 4 (C). Each histogram and bar represents a sample size of 10 000 cells. Bar graphs depict the median fluorescence values normalised to those in mock-transfected samples. Mean values and SD of 3 independent experiments are shown (B, C). Statistical analysis was performed using Two-Way ANOVA with Sidak’s multiple comparisons test as post-test for pairwise comparisons. Significant differences with Sidak’s multiple comparisons test are shown (ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).
Inactivation of the type I interferon pathway reveals long double stranded RNA-mediated RNA interference in mammalian cells (2016)
Figure 3

Figure 3. The IFN pathway negatively influences the detection of the sequence-specific gene silencing.

(A) Mavs+/- and Mavs-/- MEFs stably expressing GFP were incubated with either a neutralising antibody against IFNAR (α-IFNAR Ab), an isotype control antibody (IgG1) or no antibody (no Ab). Cells were then were transfected or not (mock) with Cy5-labeled dsRNA as indicated and GFP level monitored in Cy5+ cells 72 hrs post-transfection. (B) Mavs-/- MEFs stably expressing d2GFP were treated with recombinant IFN (200 U/ml) for 24 hrs prior to transfection or not (mock) with Cy5-labeled dsRNA, as indicated. IFN treatment was maintained for 2 days post-transfection and then GFP level monitored in Cy5+ cells. All histograms plots are representative 2 independent experiments. Each histogram and bar represents a sample size of 10 000 cells. Bar graphs depict median fluorescence values normalised to those in mock-transfected samples. Mean values and SD of 3 independent experiments are shown (B). Statistical analysis was performed using Two-Way ANOVA with Sidak’s multiple comparisons test as post-test for pairwise comparisons. Significant differences with Sidak’s multiple comparisons test are shown (ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).
Inactivation of the type I interferon pathway reveals long double stranded RNA-mediated RNA interference in mammalian cells (2016)
Figure 4

Figure 4. Sequence-specific gene silencing by long dsRNA depends on Dicer and Ago2.

(A) Northern blot analysis of Ifnar1-/- MEFs at 24 h.p.t with the indicated dsRNA or transfection reagent alone (mock) using a probe specific for dsRNA-GFP (top) or dsRNA-RL (bottom). Reactions from a dicing assay using the indicated dsRNA incubated in vitro with immunoprecipitated FLAG-human Dicer were loaded in parallel. Two lanes (delimited by dashed lines) were left empty between the Ifnar1-/- MEFs transfected samples and the in vitro dicing assay reactions. A miRNA marker containing 3 synthetic ssRNA oligonucleotides of 17, 21 and 25-nt in length was run in parallel. Endogenous U6 was used as a loading control. Signals corresponding to the dsRNA-derived siRNAs are indicated with an arrow. The membrane was first probed for dsRNA-GFP (top), then probed for the miRNA marker (top left) and subsequently stripped and reprobed for dsRNA-RL (middle) and finally stripped and reprobed for both U6 (bottom) and for the miRNA marker again (middle left). (B) Western blot of Dicer in d2GFP-expressing Mavs-/- MEFs either not transduced (NT) or transduced to express a non-targeting shRNA control (CTL shRNA) or two different Dicer-specific targeting shRNAs (DICER shRNA 1 and 2). P97 served as loading control. (C) Cells described in (B) were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+ cells was monitored by flow cytometry 48 hrs later. Please note that the biphasic pattern of d2GFP level observed in dsRNA-GFP transfected cells is caused by a lower transfection efficiency of dsRNA in these cells giving rise to significant amounts of lowly transfected cells that do not display a decrease level of d2GFP (D) Western blots of Ago2 in parental Mavs-/- MEFs stably expressing d2GFP and three Mavs-/- Ago2-/- clones generated by CRISPR/CAS9-mediated genome engineering. P97 served as loading control. (E) Mavs-/- (parental line) and Mavs-/- Ago2-/- MEFs (clone 5.1) stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+ cells was monitored by flow cytometry 48 hrs later. Histograms plots are representative of 2 independent experiments (C) or of 2 independent CRISPR clones (E). Each histogram and bar represents a sample size of 10 000 cells. Bar graphs depict median fluorescence values normalised to those in mock-transfected samples. Mean values and SD of 2 independent experiments (C) or of 2 independent CRISPR clones (B) are shown. Statistical analysis was performed using Two-Way ANOVA with Sidak’s multiple comparisons test as post-test for pairwise comparisons. Significant differences with Sidak’s multiple comparisons test are shown (ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).
Inactivation of the type I interferon pathway reveals long double stranded RNA-mediated RNA interference in mammalian cells (2016)
Figure 5

Figure 5. The endonucleolytic activity of Ago2 is essential for sequence-specific gene silencing by long dsRNA.

(A) Western blots for Ago2 (top) and HA tag (bottom) of parental Mavs-/- MEFs and two Mavs-/- Ago2-/- clones (clone 5.2 and 5.3; see Fig 4A) stably expressing d2GFP and transduced with an empty vector or a vector encoding HA-tagged wild-type (HA-mAgo2 WT) or catalytic mutant (HA-mAgo2 D597A) versions of mouse Ago2. β-actin served as loading control. The membrane was first analysed for Ago2 and β-actin, stripped and then analysed for HA tag. (B) Parental non-transduced (NT) Mavs-/- MEFs and complemented Mavs-/- Ago2-/- MEFs as described in (A) were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+ cells was monitored by flow cytometry 48 hrs later. All histograms plots are representative of 2 independent experiments and a sample size of 10 000 cells. Bar graphs depict median fluorescence values normalised to those in mock-transfected samples. Mean values and SD of 2 independent CRISPR clones transduced with an empty vector or a vector encoding HA-mAgo2 WT or HA-mAgo2 D597A (B) are shown. Statistical analysis was performed using Two-Way ANOVA with Sidak’s multiple comparisons test as post-test for pairwise comparisons. Significant differences with Sidak’s multiple comparisons test are shown (ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).
Inactivation of the type I interferon pathway reveals long double stranded RNA-mediated RNA interference in mammalian cells (2016)
Figure 6

Figure 6. Long dsRNA vaccinates cells against viruses bearing a homologous sequence.

(A) Ifnar1-/- MEFs were transfected with the indicated Cy5-labeled dsRNA and the next day were infected with SFV-Rluc at the indicated multiplicities of infection (MOI). Renilla luciferase activity was measured 24 hours later. Each bar represents mean + SD of biological duplicates and numbers above each pair of bars depict the fold-reduction in viral activity achieved with dsRNA-RL vs. dsRNA-GFP treatment. (B) As in (a) but SFV-Rluc virus titre was measured by plaque assay and expressed as plaque forming units (PFU) per ml of supernatant at 24 h post-infection (MOI=0.1). (C) As in (a) but with Ifnar1-/- or Mavs-/- MEFs and a single dose of SFV-Rluc (MOI=0.1). (D) As in (a) but with two Mavs-/- Ago2-/- clones (clone 5.2 and 5.3) individually complemented to express wild-type (HA-mAgo2 WT) or a catalytic mutant (HA-mAgo2 D597A) version of mAgo2 or transduced with a control vector (as described in Fig 5). SFV-Rluc was used at an MOI=0.1. Data are representative of 4 (A), 2 (B, C) and 6 (D) independent experiments.
SIRT3‐dependent GOT2 acetylation status affects the malate–aspartate NADH shuttle activity and pancreatic tumor growth (2015)
Figure 2

Glucose and glutamine promote GOT2 acetylation and GOT2–MDH2 association

A, B Glucose and glutamine increase GOT2 K159 acetylation and GOT2–MDH2 association in HEK293T cells. Flag‐GOT2 and Myc‐MDH2 were overexpressed in cells treated with increased concentrations of glucose (A) or glutamine (B) for 6 h. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 and its protein association with Myc‐MDH2 were determined by Western blot analysis.
C, D Glucose and glutamine increase GOT2 K159 acetylation in Panc‐1 cells. Flag‐GOT2 was overexpressed in cells treated with different concentrations of glucose (C) and glutamine (D) for 4 h. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 was determined by Western blot analysis. Relative GOT2 K159 acetylation levels were normalized against Flag protein levels.
E, F Quantification of the percentage of K159‐acetylated endogenous GOT2 in Panc‐1 cells. Recombinant fully K159‐acetylated GOT2 was loaded onto the same gel, together with endogenous GOT2 from Panc‐1 cells treated without or with glucose (12 mM) (E) or glutamine (2 mM) (F) for 4 h. GOT2 protein and K159 acetylation were detected by Western blot. The percentages of K159 acetylation in GOT2 were calculated after normalizing against GOT2 protein levels.
G, H 3K mutant GOT2 displays negligible response in changing protein association with MDH2 after glucose or glutamine treatment. Panc‐1 cells with GOT2 knockdown and re‐expression of wild‐type or 3K mutant GOT2 were treated with glucose (G) or glutamine (H) at the indicated concentrations for 4 h. The protein association between Flag‐tagged wild‐type or 3K mutant GOT2 and endogenous MDH2 was determined by Western blot analysis.
SIRT3‐dependent GOT2 acetylation status affects the malate–aspartate NADH shuttle activity and pancreatic tumor growth (2015)
Figure 3

SIRT3 is the major deacetylase of GOT2

A GOT2 interacts with SIRT3, but not SIRT4 and SIRT5. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells together with the individual HA‐tagged SIRT as indicated. Proteins were purified by IP with Flag beads, following Western blot to detect SIRTs with an HA antibody.
B Endogenous proteins of GOT2 and SIRT3 interact with each other. SIRT3 protein in HEK293T cells was purified by IP with an anti‐SIRT3 antibody, following Western blot to detect GOT2 with an anti‐GOT2 antibody.
C GOT2 deacetylation is dependent on SIRT3 catalytic activity. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells together with HA‐tagged wild‐type SIRT3 and a catalytically inactive mutant, SIRT3 H248Y. GOT2 proteins were purified by Flag beads, following Western blot to detect GOT2 K159 acetylation.
D SIRT3 impairs the protein interaction between GOT2 and MDH2. HEK293T cells were transfected with the plasmids as indicated. GOT2 proteins were purified by Flag beads, and their protein interaction with Myc‐MDH2 was determined by Western blot.
E Knockdown of SIRT3 increases GOT2 K159 acetylation and enhances GOT2–MDH2 association. HEK293T cells with or without transient SIRT3 knockdown were transfected with the plasmids as indicated. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 and its protein association with endogenous MDH2 were determined by Western blot analysis.
F, G Knockdown of SIRT3 diminishes the effect of glucose or glutamine on changing GOT2 K159 acetylation. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells without or with transient SIRT3 knockdown. These cells were treated with different concentrations of glucose (F) and glutamine (G) as indicated. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 was determined by Western blot analysis.
H, I Up‐regulation of Sirt3 leads to decreased Got2 K159 acetylation in mouse tissues during fasting. C57BL/6 mice (male, 20 weeks; n = 2 per group) were fed a normal chow diet or fasted overnight. Upon sacrifice, mouse liver and white adipose tissues were harvested, and the levels of the indicated proteins were determined by Western blot analysis.