EMBO/soda-nel huggingface dataset:
NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation (2016)
Figure 3

Figure 3. NSUN3 modifies the wobble position of mt-tRNAMet.

A. In vitro methylation reactions were performed using recombinant His14-MBP-NSUN3 (NSUN3) or the catalytically inactive mutant His14-MBP-NSUN3-C265A (C265A), 3H-labelled S-adenosyl methionine as a methyl group donor and in vitro transcribed mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. The RNA was then separated on a denaturing polyacrylamide gel, stained with ethidium bromide (EtBr) to indicate inputs and exposed to an X-ray film to analyse methylation (3H-Me).

B. The distribution of Illumina sequence reads along the mt-tRNAMet sequence obtained from CRAC experiments with NSUN3 after UV (light grey) or 5-AzaC crosslinking (dark grey) is given as reads per million mapped reads. The position of the anticodon is indicated by a bar.

C. Cloverleaf scheme of the mt-tRNAMet sequence. Nucleosides that were exchanged in the mutational analysis shown in the following panels are marked with arrows and the nucleotide positions in the tRNA are given.

D. In vitro methylation assays were performed as described in (A) with His14-MBP-NSUN3 and in vitro transcribed wildtype mt-tRNAMet and cytidine mutants of the anticodon stem and loop region indicated in (C). Two exposure times of X-ray films are shown: 16 hours (short), 3 days (long).

E. In vitro methylation assay of in vitro transcribed mt-tRNAMet and chemically synthesized mt-tRNAMet containing an m5C modification at the wobble position. The experiment and analysis were performed as described in (A).
NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation (2016)
Figure 5

Figure 5. ABH1 localises to mitochondria in HEK293 cells and specifically interacts with mt-tRNAMet.

A. The localization of ABH1 was analysed by immunofluorescence in HEK293 cells. ABH1 (green) localisation and mitochondria stained with a Mitotracker (red) are shown separately and in an overlay with DAPI to indicate nuclei. The scale bar represents 5 m.

B. HEK293 cells expressing ABH1-HisPrcFLAG (ABH1) or the HisPrcFLAG-tag alone (FLAG) were UV crosslinked (UV) and protein-RNA complexes were affinity purified. Co-precipitated RNA was isolated and analysis by northern blot using probes against the mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. Inputs (0.1%) are shown on the left and eluates (50%) on the right.
NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation (2016)
Figure 6

Figure 6. ABH1 can oxidise m5C34 in mt-tRNAMet in vitro.

A. In vitro transcribed mt-tRNAMet was methylated at C34 using recombinant NSUN3 and 3H-labelled S-adenosyl methionine as a methyl group donor. Radiolabelled mt-tRNAMet was re-extracted and then subjected to oxidation assays without protein (-), with maltose binding protein (MBP), with the dioxygenase FTO or using wildtype (ABH1) or mutant (R338A, D233A) His14-MBP-ABH1. Besides ABH1 controls lacking -ketoglutarate (KG) or Fe2+ ions, all samples contained -ketoglutarate and Fe2+ ions. After oxidation, RNA was precipitated and tritium released upon oxidation of radiolabelled mt-tRNAMet was quantified in the supernatant and counts per minute (CPM) are shown for experiments performed in triplicate with error bars indicating ± SD (upper panel). Pelleted RNA was separated on a denaturing polyacrylamide gel and exposed to an X-ray film to analyse the tritium retained (3H-Me).

B. Synthetic anticodon stem loop (ASL) was radioactively labelled and subjected to oxidation assays that were performed and analysed as described in (A) using no protein (-), MBP or wildtype His14-MBP-ABH1 (ABH1). Experiments were performed in triplicate with error bars indicating ± SD.

C. Anion exchange HPLC analysis was performed on synthetic m5C containing ASL (20nt) before and after oxidation by ABH1. The small shift in retention time indicates formation of f5C modified RNA. The ABH1 oxidation product was then treated with NaBH4 to generate hm5C-modified RNA. All three samples were analysed by ESI-MS and the molecular weight (m.w.) is indicated on the HPLC trace. Only the f5C-containing RNA was labelled efficiently with 1-ethyl-2,3,3-trimethylindoleninium-5-sulfonate (TMI).

D. The different retention times of m5C, hm5C and f5C-modified ASL RNA were confirmed by co-injection of samples shown in (C). HPLC was performed as in C.
NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation (2016)
Figure 8

Figure 8. Knockdown of NSUN3 or ABH1 leads to a reduction in the modification of cytosine 34 in mt-tRNAMet in vivo.

A. HeLa cells were transfected with two different siRNAs against NSUN3 (siNSUN3_1, siNSUN3_2), ABH1 (siABH1_1, siABH1_2) or with nontarget (siNT) siRNA and the knockdown efficiency was analysed by quantitative PCR. The relative abundance of the NSUN3 or ABH1 mRNA was normalized to GAPDH levels. Data are presented as mean ± SD.

B. Chemically synthesised f5C modified mt-tRNAMet and total RNA from wild-type (WT) cells or those transfected with siRNAs as in (A) were treated with TMI to specifically label f5C residues. Primer extension, using a radiolabelled antisense primer, was performed under limited dNTP conditions. Products were separated on a denaturing polyacrylamide gel alongside a sequencing ladder and RNAs were detected using a phosphorimager.

C. Primer extension reactions were performed on total RNA from cells transfected with siRNAs as described in B. Stops corresponding to position C34 in mt-tRNAMet were quantified in three independent experiments and results are shown graphically as mean ± SD.

D. RNA from wild-type HeLa cells and cells treated with siRNAs against NSUN3 or ABH1 (as in A) was either first reduced with NaBH4 or directly treated with bisulfite. After deamination and desulfonation, mt-tRNAMet RNAs were reverse transcribed, amplified, cloned and sequenced. The proportions of thymine (grey) generated by bisulfite-conversion or non-converted cytosine (black) at position 34 of mt-tRNAMet are shown. Note that for sequences from non-reduced samples, thymine can originate from unmodified or f5C-containing mt-tRNAMet, while in reduced samples it originates from unmodified cytosine.
NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation (2016)
Figure 9

Figure 9. NSUN3 and ABH1 are both required for efficient mitochondrial translation in vivo.

A. HeLa cells were treated with non-target siRNAs (siNT) or those targeting NSUN3 (siNSUN3_1 or siNSUN3_2) or ABH1 (siABH1_1 or siABH1_2) for 72 h before labelling of mitochondrial translation products with [35S]methionine. Protein samples were separated by SDS-PAGE then transferred to a membrane. Labelled proteins were detected using a phosphorimager and the levels of tubulin were determined by western blotting using an antibody against the endogenous protein for normalisation.

B. Mitochondrially-translated proteins that could be clearly detected were quantified in three independent experiments and the results are shown graphically as mean ± SD.

C. Overview of the modification pathway of C34 in mt-tRNAMet. NSUN3 introduces an m5C methylation on C34 using S-adenosyl methionine as methyl group donor and this can be further oxidised by ABH1 in the presence of O2, Fe(II) (Fe2+) and alpha-ketoglutarate (αKG) to produce f5C34.
LncRNA-dependent nuclear stress bodies promote intron retention through SR protein phosphorylation (2019)
Figure 1

Figure 1. HSATIII lncRNAs control intron retention of a specific set of genes

A Outline of the screening for HSATIII-regulated genes during thermal stress recovery. HeLa cells were transfected with a HSATIII ASO (HSATIII KD) or HSATIII sense oligonucleotide (control), exposed to thermal stress. Nuclear polyA(+) RNAs were analyzed by next-generation sequencing (NGS). NGS data have been deposited in the DDBJ Sequence Read Archive (DRA) (accession number: DRA007304).

B HSATIII ASO-mediated depletion of nSBs. Thermal stress-exposed HeLa cells (42°C for 2 h and recovery for 1 h at 37°C) were visualized by HSATIII-FISH and immunofluorescence using an anti-SAFB antibody or anti-HNRNPM antibody. The nuclei were stained with DAPI. Scale bar: 10 μm.

C qRT-PCR-validation of HSATIII knockdown. The graph shows the qRT-PCR level of HSATIII RNAs in control and HSATIII knockdown cells under three conditions: 37°C, 42°C for 2 h, and thermal stress followed by recovery at 37°C for 1 h (Recovery). Expression levels were calculated as ratios to GAPDH mRNA and were normalized to the levels in control cells under thermal stress conditions (42°C for 2 h). Data are shown as the mean±SD (n=3). HSATIII RNAs ratios (%) are indicated.

D MA-plot (log2 fold-change over the average expression level) of all detected introns in HSATIII KD and control cells (n=3). The introns with significant changes in their expression levels (adjusted p-value < 0.01) are shown as green dots. Among these introns, up-regulated (log2 fold-change > 1) and down-regulated (log2 fold-change < -1) introns are shown with magenta and light blue dots, respectively. Genes and introns that were experimentally validated in subsequent experiments are indicated as gene symbol-i (intron) #. Total numbers of introns and genes utilized were 144769 and 32623, respectively.

E The numbers of introns and exons that were affected by HSATIII knockdown (fold change>2).

F, G Examples of RNA-seq read maps of HSATIII target RNAs. Affected introns and exons are indicated by blue (down-regulated upon HSATIII knockdown) and magenta (up-regulated) boxes.

H Cumulative frequency curves of the lengths of adjacent exons of 399 HSATIII-up-regulated internal introns. The lengths of whole annotated internal introns are shown as a reference.
LncRNA-dependent nuclear stress bodies promote intron retention through SR protein phosphorylation (2019)
Figure 3

Figure 3. The HSATIII lncRNA is necessary and sufficient to promote nuclear intron retention

A Splicing isoforms of the CLK1 pre-mRNA. The retained introns are indicated by red lines. An asterisk indicates the position of the premature termination codon in the nonsense-mediated mRNA decay-targeted isoform.

B Time course analysis of the splicing pattern of CLK1 pre-mRNAs in control and HSATIII knockdown (HSATIII KD) cells by semi-quantitative RT-PCR. Arrows indicate the positions of PCR primers. The GAPDH mRNA was used as an internal control.

C Quantification of the data shown in (B). Data are shown as the mean±SD (n=3); *p<0.05 (Sidak's multiple comparison test).

D Thermal stress-induced expression of HSATIII in CHO (His9) cells. The cells were visualized by HSATIII-FISH (green), and the nuclei were stained with DAPI (blue). Scale bar: 10 μm.

E Ectopic HSATIII-induced nSB assembly in CHO (His9) cells. The nSBs (yellow arrowheads) were visualized by HSATIII-FISH and immunofluorescence using an anti-SRSF1 antibody. The nuclei were stained with DAPI. Scale bar: 10 μm.

F Time course analysis of the splicing pattern of Chinese hamster Clk1 pre-mRNAs in control (CHO) and CHO (His9) cells by semi-quantitative RT-PCR. Arrows indicate the positions of PCR primers. The GAPDH mRNA was used as an internal control.

G Quantification of the data shown in (F). Data are shown as the mean±SD (n=3); *p<0.05 (Sidak's multiple comparison test).

H The effect of HSATIII knockdown on pooling of intron-retaining RNAs in control (CHO) and CHO (His9) cells (42°C for 2 h and recovery for 2 h at 37°C). Arrows indicate the positions of PCR primers.
LncRNA-dependent nuclear stress bodies promote intron retention through SR protein phosphorylation (2019)
Figure 4

Figure 4. Identification of HSATIII-interacting proteins by ChIRP.

A Overview of the ChIRP procedure used to identify HSATIII-interacting proteins in thermal stress-exposed cells. HSATIII-interacting proteins were crosslinked and captured with a biotinylated HSATIII ASO. As a negative control, the same procedure was performed using control (37°C) cells and/or a control probe.

B RT-PCR validation of the specific precipitation of HSATIII by ChIRP. The NEAT1 ncRNA and GAPDH mRNA were used as negative controls. Input: 100%.

C Silver staining of the coprecipitated proteins. The predicted mobilities of five known nSB proteins are indicated. Input: 0.1%.

D Western blot analyses of proteins identified by the HSATIII-ChIRP experiment. Known nSB proteins (SAFB, SRSF1, SRSF7, SRSF9, and HNRNPM) were used as positive controls, and ELAVL1, PSPC1 and α-tubulin were used as negative controls. SRSF3, SRSF5, TRA2B and THRAP3 are newly identified nSB components.

E-G Colocalization of novel nSB proteins with HSATIII. Endogenous TRA2B (E), THRAP3 (F), and FLAG-tagged PPHLN1 (G) were stained with anti-TRA2B, anti-THRAP3, and anti-FLAG antibodies, respectively. HSATIII was visualized by RNA-FISH using a dig-labeled HSATIII ASO, and the nuclei were stained with DAPI. Scale bar: 10 μm.
LncRNA-dependent nuclear stress bodies promote intron retention through SR protein phosphorylation (2019)
Figure 5

Figure 5. CLK1 is recruited to nSBs during thermal stress recovery

A Specific interaction of CLK1 with HSATIII during thermal stress recovery. HSATIII-ChIRP at various time points during and after thermal stress exposure. Endogenous CLK1, SRSF7, and SRSF9 were detected using anti-CLK1, anti-SRSF7, and anti-SRSF9 antibodies, respectively. HSATIII was detected by ChIRP-RNA purification, followed by RT-PCR. Input: 0.1% for CLK1, 1% for SRSF7 and SRSF9, and 100% for HSATIII.

B The domain structures of wild-type (WT) CLK1, the catalytically inactive mutant (CLK1 KR), and the N- and C-terminal partial fragments (CLK1∆C and CLK1∆N).

C Specific interaction of HSATIII with CLK1 proteins during stress recovery. HSATIII-ChIRP/western blotting was performed using HeLa cells expressing FLAG-CLK1 WT or the KR mutant. FLAG-tagged CLK1 and SRSF9 were detected by western blotting using an anti-FLAG and an anti-SRSF9 antibody, respectively. HSATIII was detected by ChIRP followed by RT-PCR. Input: 1% for western blotting, 100% for RT-PCR.

D Re-translocation of FLAG-CLK1 to nSBs after thermal stress removal. HeLa cells were transfected with a FLAG-CLK1 expression vector, cultured for 16 h, and then exposed to thermal stress (42°C for 2 h) with or without recovery (37°C for 1 h). FLAG-CLK1 was visualized with an anti-FLAG antibody. Nuclear stress bodies and nuclei were visualized by HSATIII-FISH and DAPI staining, respectively. Scale bar: 10 μm.

E Box plot of colocalization correlation coefficients between HSATIII and FLAG-CLK1 WT. The first and third quartiles are the ends of the box, the median is indicated by the vertical line in the box, and the minimum and maximum are the ends of the whiskers.

(n=20 nuclei for each condition). *p<0.05 (Mann-Whitney U-test).

F Recovery phase-specific interaction of HSATIII with the CLK1 N-terminal region. HSATIII-ChIRP/western blotting was performed on HeLa cells expressing FLAG-CLK1∆C or FLAG-CLK1∆N using the same procedure as in (C). Asterisk: an unidentified partial fragment of FLAG-CLK1∆N.

G, H Dependency of SRSF9 on the interaction of CLK1 (G) and CLK1∆C (H) with HSATIII. HSATIII-ChIRP was performed using control and SRSF9-depleted cells expressing FLAG-CLK1 proteins (42°C for 2 h and recovery for 1 h at 37°C). Input: 1%. Graphs represent coprecipitation ratios of FLAG-CLK1 proteins to HSATIII in the control and SRSF9-depleted cells. Data are shown as the mean±SD (n=3); *p<0.05 (Welch's t-test).
LncRNA-dependent nuclear stress bodies promote intron retention through SR protein phosphorylation (2019)
Figure 7

Figure 7. The role of SRSF9 re-phosphorylation in controlling HSATIII-dependent intron retention

A, B Validation of the effect of SRSF9 on retention/splicing of HSATIII target introns by qRT-PCR. The relative expression levels of intron-retaining (IR) forms (A) and spliced forms (B) of newly transcribed RNAs during the recovery period are shown. See also the scheme in Figure 2C. Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the control. Data are shown as the mean±SD (n=3); *p<0.05 (multiple t-test modified by Holm-Sidak's method).

C, D Time course analysis of the splicing patterns of CLK1 and TAF1D intron-retaining RNAs in control and HSATIII knockdown cells by semi-quantitative RT-PCR. Arrows indicate the positions of primers for RT-PCR. GAPDH mRNA was used as an internal control. The experiment was performed in triplicate. Quantification of the data shown in (C), Data are shown as the mean±SD (n=3); *p<0.05 (Sidak's multiple comparison test). E Scheme for analyzing the effect of CLK1 inhibition on intron retention by newly transcribed RNAs after stress removal. CHX, Cycloheximide; EU, 5-Ethynyl uridine.

F Validation of the requirement of CLK1 catalytic activity for promotion of intron retention by qRT-PCR. Data are shown as the mean±SD (n=3). P values (multiple t-test modified by Holm-Sidak's method) are shown.

G The diagrams show the domain structures of wild-type (WT) and mutant (mut) SRSF9, in which all SR/SP dipeptides were replaced by AR/AP. FLAG-SRSF9-WT and FLAG-SRSF9-mut were extracted from transfected HeLa cells under normal (37°C) and thermal stress (42°C for 2 h) conditions, separated in a Phos-tag gel, and detected by western blot using an anti-FLAG antibody. The arrowhead indicates the thermal stress-induced de-phosphorylated form of FLAG-SRSF9-WT.

H, I The role of phosphorylation of SRSF9 on splicing of the TAF1D (H) and DNAJB9 (I) reporters. Each splicing reporter plasmid was transfected into HeLa cells along with a siRNA (control or SRSF9-specific) and a siRNA-resistant SRSF9-WT or SRSF9-mut expression plasmid (or pEGFP-C1 vector as the empty vehicle (-)) 48 h before the assay. Semi-quantitative RT-PCR analyses revealed the splicing patterns of the reporter RNAs purified from the thermal stress-exposed cells (42°C for 2 h and 37°C for 1 h). The positions of the PCR primers are indicated by arrows. Graphs represent the relative changes in the spliced/unspliced ratios, normalized to the control sample with control siRNA and empty plasmid (-). Data are shown as the mean±SD (n=3); *p<0.05 (Dunnett's multiple comparison test).

J A model for the function of nSBs in splicing control. Thermal stress-induced HSATIII integrates subsets of de-phosphorylated SRSFs and SR-related proteins. After stress removal, CLK1 relocates to nSBs to promote rapid re-phosphorylation of the sequestrated SRSFs, especially SRSF9. Re-phosphorylated SRSF9 suppresses splicing of target introns to accumulate intron-retaining RNAs in the nucleus.

Expanded View Figure legends
STAT3-dependent analysis reveals PDK4 as independent predictor of recurrence in prostate cancer (2020)
Figure 3

Fig 3: Proteomics show TCA/OXPHOS upregulation in low STAT3 human FFPE- PCa

A. STAT3 immunohistochemistry- staining of low STAT3 and high STAT3 PCa samples. Red arrows indicate transformed PCa glands, black arrows indicate pre-transformed normal prostate glands. Scale bar = 100µm. # = sample-IDs.

B. Significantly enriched KEGG pathways in low STAT3 versus high STAT3 groups. Dotted line: adj. p-value = -log10(0.05).

C. Significantly enriched Hallmark gene sets in low STAT3 versus high STAT3 groups. Dotted line: adj. p-value = -log10(0.05). Red = up-regulated, blue = down-regulated.

D. Simplified scheme of the TCA cycle and associated metabolic pathways. TCA cycle = Tricarboxylic acid cycle, PDC = Pyruvate dehydrogenase complex, PDK = Pyruvate dehydrogenase kinase.
STAT3-dependent analysis reveals PDK4 as independent predictor of recurrence in prostate cancer (2020)
Figure 4

Fig 4: Proteomics and metabolomics show enhanced ribosome- and TCA/OXPHOS- activity in PtenStat3pc-/- -mice

A. Differentially expressed proteins in PtenStat3pc-/- vs. Ptenpc-/- proteomic samples. Colors indicate adj.p-value and log2-FC. (Black = Log2-FC ≤ 1 & adj. p-value ≥ 0.05, orange = Log2-FC > 1 & adj. p-value ≥ 0.05, red = Log2-FC > 1 & adj. p-value < 0.05). FC = fold change.

B. STAT3 immunohistochemistry- staining of wild type, Ptenpc-/-, PtenStat3pc-/- mouse prostates. Scale bar = 100µm.

C. Significantly enriched KEGG pathways in PtenStat3pc-/- versus Ptenpc-/- groups. Dotted line: adj. p-value = -log10(0.05).

D. Significantly enriched metabolic KEGG pathways in PtenStat3pc-/- versus wild type (=WT) groups. Dotted line: adj. p-value = -log10(0.05). Red = up-regulated, blue = down-regulated.

E. Metabolite concentrations in nmol/µg of 5 metabolites in WT, Ptenpc-/- and PtenStat3pc-/- prostates. Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents biological replicates. ANOVA tests and Tukey multiple comparisons were applied. Red = PtenStat3pc-/- (n = 3), blue = Ptenpc-/- (n = 5), grey = wild type (WT, n=5), q = adj. p.-value, n.s. = not significant.
STAT3-dependent analysis reveals PDK4 as independent predictor of recurrence in prostate cancer (2020)
Figure 5

Fig 5: SDHB and IDH2 protein levels are associated with Gleason grade

A. SDHB protein expression levels in a TMA (n = 83) detected by immunohistochemistry (IHC). Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents single values in groups. Kruskal-Wallis test and Dunn's all pairs test were applied. Q = adj. p.-value, n.s. = not significant. TMA = Tissue Micro Array.

B. Representative IHC- staining of SDHB in normal prostate glands and Gleason grade (GL) 3 - 5 PCa glands. Scale bar = 100µm.

C. IDH2 protein expression levels in a TMA (n = 83) detected by IHC. Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents single values in groups. Kruskal-Wallis test and Dunn's all pairs test were applied. Q = adj. p.-value, n.s. = not significant.

D. Representative IHC staining of IDH2 in normal prostate glands and GL 3 - 5 PCa glands. Scale bar = 100µm.

E. Nuclear (N) and cytoplasmatic (C) STAT3 protein expression levels in a TMA (n = 83) detected by IHC. Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents single values in groups. Kruskal-Wallis test and Dunn's all pairs test were applied. Q = adj. p.-value, n.s. = not significant.