EMBO/SourceData huggingface dataset:
Figure 3A 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.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.
Figure 4B 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.Colocalization of novel nSB proteins with HSATIII. Endogenous TRA2B (E) were stained with anti-TRA2B HSATIII was visualized by RNA-FISH using a dig-labeled HSATIII ASO, and the nuclei were stained with DAPI. Scale bar: 10 μm.Colocalization of novel nSB proteins with HSATIII. THRAP3 (F), were stained with anti-THRAP3, HSATIII was visualized by RNA-FISH using a dig-labeled HSATIII ASO, and the nuclei were stained with DAPI. Scale bar: 10 μm.Colocalization of novel nSB proteins with HSATIII. FLAG-tagged PPHLN1 (G) were stained with anti-FLAG antibodies HSATIII was visualized by RNA-FISH using a dig-labeled HSATIII ASO, and the nuclei were stained with DAPI. Scale bar: 10 μm.
Figure 5Specific 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.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.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.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.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 Asterisk: an unidentified partial fragment of FLAG-CLK1∆N.Dependency of SRSF9 on the interaction of CLK1 (G) 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.H Dependency of SRSF9 on the interaction of 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.
Figure 6A Localization of SRSF9 with HSATIII during thermal stress and recovery.B Recovery phase-specific colocalization of CLK1 and SRSF9. HeLa cells were transfected with a FLAG-CLK1 expression vector, cultured for 16 h, and exposed to thermal stress (42°C for 2 h) followed by recovery (37°C for 1 h). FLAG-CLK1 and SRSF9 were co-stained using anti-FLAG and anti-SRSF9 antibodies, respectively, and the nuclei were stained with DAPI.C Box plot of colocalization correlation coefficients between SRSF9 and FLAG-CLK1 WT. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum are the ends of the whiskers.F The effect of HSATIII knockdown on time course-dependent changes in SRSF9 phosphorylation. The phosphorylation states were estimated by Phos-tag SDS-PAGE, followed by western blotting using an anti-SRSF9 antibody. SRSF9 detected by western blotting of a normal SDS-PAGE gel is shown in the bottom panel. The arrows and arrowheads indicate the normal phosphorylated forms and thermal stress-induced hypo- and de-phosphorylated forms, respectively.F The effect of HSATIII knockdown on time course-dependent changes in SRSF9 phosphorylation. The phosphorylation states were estimated by Phos-tag SDS-PAGE, followed by western blotting using an anti-SRSF9 antibody. SRSF9 detected by western blotting of a normal SDS-PAGE gel is shown in the bottom panel. The arrows and arrowheads indicate the normal phosphorylated forms and thermal stress-induced hypo- and de-phosphorylated forms, respectively. G Quantification of the data shown in (F). Graphs show the time-course changes in four representative SRSF9 phosphorylated forms in (F). Relative levels of band intensity were calculated as the ratio of each band to α-tubulin and were normalized to those of control at 37°C (bands (a) and (b)) and 42°C (bands (c) and (d)). Since band (d) partially overlapped with the other band (asterisk in (F)) at 37°C and recovery 4h, band (d) at those time points was not measured. H The re-phosphorylation status of SRSF9 within nSBs. Phosphorylated (arrows) and hypo- and de-phosphorylated (arrowheads) SRSF9 in the nSB fractions (lanes 4, 6, and 8) were analyzed by western blotting on Phos-tag gels. KH-CB19 (10 μM) or DMSO as a control was administrated during the recovery period. The lower panel shows a shorter exposure image of the phosphorylated forms. The asterisks indicate bands different from band (a) or (d). I Quantification of the data shown in (H). Relative band intensities were calculated as the ratios of each band to total SRSF9 detected by western blotting of a standard SDS-PAGE, and were normalized to those at 42°C.
Figure 7A, 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. Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the control.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),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).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.The role of phosphorylation of SRSF9 on splicing of the TAF1D (H) 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 (-).I The role of phosphorylation of SRSF9 on splicing of the 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 (-).
Figure 2Chromosome parameters are shown in A-C on the vertical axis, and time after anaphase I onset is shown in min on the horizontal axis. Thin grey lines in A-C represent individual chromosomes; the distantly observed chromosome is highlighted by dashed line; the mean value for each timepoint is indicated by thick black line. The stages between anaphase I and metaphase II are color-coded and labelled on the top of the charts. On the schemes above the charts in (A) and (C), chromosomes are red with green centromeres. Changes in the distance to the centre for all chromosomes in a representative oocyte undergoing transition from anaphase I to metaphase II from Fig 1. The black arrow on the scheme above the chart indicates chromosome-centre distance.Chromosome parameters are shown in A-C on the vertical axis, and time after anaphase I onset is shown in min on the horizontal axis. Thin grey lines in A-C represent individual chromosomes; the distantly observed chromosome is highlighted by dashed line; the mean value for each timepoint is indicated by thick black line. The stages between anaphase I and metaphase II are color-coded and labelled on the top of the charts. Changes in the speed of all chromosomes observed for the oocyte from Fig 1.Chromosome parameters are shown in A-C on the vertical axis, and time after anaphase I onset is shown in min on the horizontal axis. Thin grey lines in A-C represent individual chromosomes; the distantly observed chromosome is highlighted by dashed line; the mean value for each timepoint is indicated by thick black line. The stages between anaphase I and metaphase II are color-coded and labelled on the top of the charts. On the schemes above the charts in (A) and (C), chromosomes are red with green centromeres. (C) Changes in inter-centromere distance (indicated on a scheme by the black arrow), observed for the oocyte from Fig 1.Kinetochore-MT attachments at the prometaphase II stage were visualized using an anti-tubulin antibody (white), an anti-centromeric ACA antibody (green) and DAPI to label chromatin (red). The image represents maximum intensity projection through all z-planes containing MTs. Representative chromosomes displaying syntelic, amphitelic or merotelic/lateral attachments are enclosed in white frames and their enlarged single z-plane images are shown to the right. PB: polar body. Scale bar, 10μm. Chromosomes with syntelic (red), amphitelic (green) and merotelic/lateral (grey) attachments were plotted according to their inter-centromere distance (on the horizontal axis) and distance to spindle equator (vertical axis). Data shown for 156 chromosomes taken from 8 prometaphase II oocytes.
Figure 4Chromosome parameters obtained for a representative oocyte with normal segregation pattern are shown in A-D on the vertical axis, and time in min on the horizontal axis relative to the anaphase II onset. Thin grey lines in A-D represent individual chromosomes; thick black line in C-D shows the mean value for each timepoint. On the schemes near the charts chromosomes are red with green centromeres; spindle equator in A and spindle axis in B-C are indicated by dashed lines. Changes in distance to the equator plane for all chromosomes in the representative oocyte with normal segregation pattern, shown in Fig 3. The analysed distance is indicated by a black arrowed line on the scheme above the chart. Changes in distance to the spindle axis (shown on the scheme by a black arrowed line) for all chromosomes in oocyte from Fig 3. Changes in chromosome orientation for all chromosomes in oocyte from Fig 3. The orientation is characterized by the angle (highlighted black) between the spindle axis and the line connecting the sister centromeres (black solid line on the scheme).Chromosome parameters obtained for a representative oocyte with normal segregation pattern are shown in A-D on the vertical axis, and time in min on the horizontal axis relative to the anaphase II onset. Thin grey lines in A-D represent individual chromosomes; thick black line in C-D shows the mean value for each timepoint. (D) Changes in inter-centromere distance, shown on the scheme by black arrowed line, for all chromosomes in oocyte from Fig 3.Kinetochore-MT attachments in oocytes released from the CSF-dependent metaphase arrest were visualized using an anti-tubulin antibody (white), an anti-centromeric ACA antibody (green) and DAPI to label chromatin (red). The image represents maxiumum intensity projection through all z-planes containing MTs. Single z-planes of representative chromosomes displaying amphitelic or merotelic/lateral attachments are shown to the right. Scale bar, 10μm. Chromosomes with amphitelic (green) and merotelic/lateral (grey) attachments were plotted according to their inter-centromere distance (on the horizontal axis) and distance to spindle equator (vertical axis). The inter-centromere distance in fixed oocytes is 1.2±0.2 μm for amphitelic and 1.0±0.2 μm for merotelic attachments (Mean±SD). Data shown for 95 chromosomes taken from 5 oocytes released from the CSF-dependent metaphase II arrest.
f1(a) RT-PCR analysis of LC3, IL-8, CHOP and GADD34 in U2OS cells subjected to glutamine deprivation (−Q) or exposed to TPG (1 μM). ((b) Immunoblot analysis of ER stress- and autophagy-related proteins in U2OS cells subjected to glutamine deprivation or TPG treatment. Samples are run in triplicate.(c) Cytokine array analysis of conditioned media from U2OS and A549 cells grown in the presence (+Q) or absence (−Q) of glutamine.(d) IL-8 enzyme-linked immunosorbent assay of conditioned media from U2OS and A549 cells grown in the presence (+Q) or absence (−Q) of glutamine. Error bars in all figures represent s.d. of three biological replicates.
f2(a) Autophagosomes were visualized in U2OS cells stably expressing a GFP-LC3 reporter construct. GFP-labelled puncta were examined after 24 h in the presence (+Q) or absence (−Q) of glutamine, with or without co-addition of 100 nM CCI-779 (CCI) and 400 nM BafA1. Scale bar, 10 μm. (b) Graphical summary of experiments performed as described in a. Percentage of cells with >10 puncta per cell from three independent experiments is depicted. Bars represent mean±s.d. from three independent experiments (>50 cells per experiment). The statistical significance (P value) was determined by a two-tailed, paired Student's t-test. *P0.05.(c) Immunoblot analysis of U2OS cells subjected to glutamine deprivation with or without 400 nM BafA1. Cells were pretreated with BafA1 for 1 h before and during exposure to glutamine-deficient medium. Autophagic activity was monitored by detection of p62 and LC3-II proteins.d) U2OS mCherry-GFP-LC3 cells were cultured in the presence (+Q) or absence (−Q) of glutamine for 18 h. Red vesicles denote autolysosomes, whereas yellow vesicles represent autophagosomes. Bars indicate numbers of yellow vesicles (autophagosomes) or red vesicles (autolysosomes) per cell±s.d. (e) Images of U2OS mCherry-GFP-LC3 cells cultured for 18 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm.(f) Phagophore formation in mCherry-ULK1. Scale bar, 10 μm. (g) mCherry-ATG5-expressing U2OS cells after 24 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm.
f3(a) Colocalization of lysosomes (Lysotracker Green) with phagophores (mCherry-ULK1 and mCherry-glutamine puncta) in glutamine-deprived U2OS cells. Scale bar, 25 μm.(b) Colocalization of LAMP1-positive lysosomes with GFP-LC3-labelled autophagosomes after glutamine deprivation in the absence or presence of 400 nM BafA1 in U2OS cells. Scale bar, 15 μm.(c) Immunofluorescence images of U2OS cells stained for p62 expression and LAMP1 following glutamine deprivation and BafA1 treatment. Nuclei were stained with DAPI. Scale bar, 15 μm.(d) Immunofluorescence images of cells stained with LAMP1 (lysosomes) and the Golgi marker, RCAS1, following glutamine deprivation. Nuclei were stained with DAPI. Scale bar, 15 μm.(e) U2OS cells were treated for 24 h with cell culture medium containing the indicated amino acids. Cell lysates were immunoblotted to detect changes in S6K phosphorylation and p62. ((f) Immunofluorescence staining for LAMP1 (green) and mTOR (red) in U2OS cells subjected to glutamine deprivation (24 h), in the presence of 100 nM WYE-125132 (WYE-132). −AA denotes cells deprived of all amino acids. Scale bar, 15 μm. Error bars in all figures represent s.d. of three biological replicates. Ess., essential.
f4(a) Immunoblot of autophagy-related proteins in U2OS cells expressing a control TALEN (U2OS-TAL2-C1) or two independent subcloned TALEN cell lines targeting ATG7 (U2OS-TAL2-A7, U2OS-TAL2-A8). p62 long and short refer to either long or short exposure times during film development.(b) U2OS cells expressing an ATG7-TALEN (TAL2-ATG7) or control TALEN (TAL2-CTL) were subjected to glutamine starvation for 6 days followed by 6 days recovery in glutamine-replete medium. Cells were then stained with SRB and absorbance was read at 540 nM. Graph shows average results from five clonal sub-lines for each condition. The statistical significance (P value) was determined by a two-tailed, paired Student's t-test. P0.05. (c) Representative images of SRB-stained clones in bd) IL-8 enzyme-linked immunosorbent assay (ELISA) of U2OS-TAL2-CTL-C1 (control) and U2OS-TAL2-ATG7-A8 (ATG7 knockout) following 24 h glutamine deprivation.(e) IL-8 U2OS of U2OS parental cells starved of glutamine for the indicated times with or without BafA1. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.
f5(a) immunofluorescence microscopy images of U2OS cells stained for LAMP1 and the Golgi marker RCAS1 after 24 h glutamine deprivation with or without treatment with 1 μM BrefA or 100 nM WYE-125132. Scale bar, 15 μm.(b) Cytokine array analysis of conditioned medium from U2OS cells subjected to glutamine deprivation in the presence of 1 μM BrefA or 100 nM WYE-125132.(c) IL-8 enzyme-linked immunosorbent assay in U2OS cells after 24 h glutamine deprivation with or without CCI-779 or WYE-125132.(d) RT-PCR analysis of IL-8 mRNA expression in U2OS cells after 24 h glutamine deprivation with or without CCI-779 or WYE-125132. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.
f6(a) Immunoblot analysis of autophagy, ER stress- and mTOR-related proteins, and JNK in U2OS cells after 24 h glutamine deprivation with or without CCI-779.(b) RT-PCR analysis of IL-8 mRNA expression in U2OS cells transfected with two different siRNAs targeting JNK (JNK #1 and JNK #2) and subjected to 24 h glutamine deprivation.(c) IL-8 enzyme-linked immunosorbent assay (ELISA) of conditioned media from the experiment described in b.(d) Immunoblot analysis of JNK protein expression in JNK siRNA-treated cells.e) RT-PCR analysis of IL-8 expression in U2OS cells after 24 h glutamine deprivation with or without the JNK inhibitor SP600125 (JNKi).(f) IL-8 ELISA of conditioned medium from the experiment described in e.(g) IL-8 ELISA of U2OS cells transfected with two different siRNAs targeting IRE1 (IRE-A and IRE-B) and subjected to glutamine deprivation for 24 h.(h) RT-PCR analysis of IL-8 mRNA expression from experiment described in g. (i) RT-PCR analysis of CHOP mRNA expression from experiment described in g. (j) RT-PCR analysis of IRE1 mRNA expression from experiment described in g.(k) Immunoblot analysis of experiment described in g showing phosphorylation of JNK. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.
f7(a) IL-8 enzyme-linked immunosorbent assay (ELISA) on U2OS cells starved of glutamine or treated with BPTES (10 μM) for 24 h.(b) RT-PCR analysis of IL-8 and CHOP mRNA expression in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG.(c) Metabolite analysis of U2OS cells starved of glutamine, treated with BPTES or glutamine-starved co-treated with DMαKG.(d) IL-8 ELISA of U2OS cells starved of glutamine or treated with BPTES for 24 h and co-treated with DMαKG.(e) Immunoblot analysis of pS6K and pJNK in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.
Figure 2Representative density plots of Ca2+ flux from conjugated OptoCAR‑T cells over time at 37ºC. Conjugated cells were stimulated with dimerizer addition for 0 (F), 5 (G), 10 (H), 15 (I) or 30 (J) minutes prior to disrupting signaling by illuminating cells.
Figure 3OptoCAR‑T cells were conjugated with ligand-presenting cells and activated as in the upper regime shown in (A). The median of ppERK intensity distribution was plotted with time, after subtraction of background intensity. Bars show mean ± SEM (n=3). An equivalent experiment was performed as in (B) but following the middle regime shown in (A), with sample illumination after 5 minutes for the remainder of the dataset. Indicated half-life was calculated from the time taken for the median ppERK to decrease to 50% of maximum. Bars show mean ± SEM (n=4). An equivalent experiment was performed as in (B) but following the lower regime shown in (A), with sample illumination after 5 minutes for 3 minutes. Bars show mean ± SEM (n=3).