EMBO/SourceData huggingface dataset:
Figure 2 (A) Temperature-dependent poison events in Hnrnpdl (left, see also Figure 1C), Hnrnph3 (middle) and Cirbp (right). For each target, on top a simplified exon-intron structure is given and below Sashimi plots show the distribution of raw sequencing reads. Exon-Exon junction reads are indicated by the numbers connecting the exons. Below, sequence conservation across placental species is indicated. Hnrnpdl exhibits a heat-included exon that leads to a PTC, Hnrnph3 a cold-skipped exon that leads to a frameshift, and in Cirbp, heat leads to inclusion of an alternative transcript end, which leads to formation of a PTC. (B) For each gene from A, the triplicate normalized read counts in DMSO (left y-axis, green) and the percentage of the poison isoform in CHX (right y-axis, orange) are plotted at the two temperatures. Line represents mean PSI.  (C) Correlation of poison isoform inclusion and GE levels for all RBPs with temperature-dependent AS-decay skipped exon events. Shown are the log2 fold change (FC) in GE versus the ∆PSI of the poison isoform between the CHX samples. Top shows the GE change between the DMSO samples and bottom between the CHX samples. R² and P (deviation from zero slope) are indicated. N=24.  (E) Splicing reporters, containing only an intron (GFP ctrl, #1) or additionally temperature-dependent exons with ~1kB surrounding introns. Representative radioactive RT-PCRs (bottom) confirm cold-induced inclusion of Ythdf3 exon 7 (#2) and human Synerg exon 21 (#3) after 24 hours at the indicated temperatures. Representative images are derived from three independent gels. See SourceDataForFigure2E for uncropped versions of these gels.  (F-G) GFP expression was quantified using FACS and is shown relative to GFP ctrl. In (F) transfected cells were incubated at the indicated temperatures for 24 hours (n=3, mean +/- SD). In (G) cells were incubated directly after transfection for 12 hours at 39°C (blue) or 33°C (red) and then shifted for 2, 4, 8 and 12 hours (n=3, mean +/- SD).
Figure 3 (A) SR protein specific temperature-dependent AS-decay. Sashimi plots are shown for heat-induced poison exon inclusion for Srsf2 (left) and cold-induced poison isoform formation for Srsf10 (right). Quantification of GE and AS are presented in the center (as in Figure 2B), lines represent mean.  (B) Poison exon inclusion for Srsf2 (left) and Srsf10 (right) in a 24h temperature-rhythm in human cells. Hek293 cells were pre-entrained with square-wave temperature cycles (12h 34°C/ 12h 38°C) for 48h. For the last 24h, cells were treated with DMSO or CHX every 4h and harvested after 4h and analyzed by splicing sensitive RT-PCR (n=3, mean ± SD). White area: 34°C; Red area: 38°C. In Hek293 cells, inclusion of Srsf10 exon 3 is coupled to polyadenylation making it a weak decay target.  (C) AS-decay of human targets at 34°C (blue) and 38°C (red) was investigated after a square-wave temperature regime (see B). RNAs were harvested after 56 and 68 hours, respectively (n=3, mean ± SD). Statistical significance was determined by 2-way ANOVA and is indicated by asterisks: adjusted p values: not significant (ns), **p<0.01, ***p<0.001, ****p<0.0001. Box plots show median PSI of validation PCRs, whiskers show min to max. Individual data points are shown as circles.  (E) Quantification of Srsf2 AS-decay in hamster, rabbit and chicken cells (n=2, mean ± SD). Statistical significance was determined by 2-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Box plots show median PSI of validation PCRs, whiskers show min to max. Individual data points are shown as circles.
Figure 4 Rhythmic Srsf2 productive mRNA levels (relative to Gapdh) were analyzed by RT-qPCR. For each clone, expression is normalized to time point 52h,  Rhythmic Srsf10 productive mRNA levels (relative to Gapdh) were analyzed by RT-qPCR. For each clone, expression is normalized to time point 52h For Srsf10 two independently generated clones were investigated (∆E3 A and B). Statistical significance was determined by unpaired t-test and is indicated by asterisks: p values: **p<0.01, ****p<0.0001.  Comparing temperature-induced expression of the core clock components Bmal1 in WT and Srsf10 ∆E3 cells (relative to Gapdh, normalized to time point 52h). Statistical significance was determined by unpaired t-test and is indicated by asterisks: p values: *p<0.05, ***p<0.001, ****p<0.0001. n=3, mean ± SEM.  Comparing temperature-induced expression of the core clock components Per1 in WT and Srsf10 ∆E3 cells (relative to Gapdh, normalized to time point 52h). Statistical significance was determined by unpaired t-test and is indicated by asterisks: p values: *p<0.05, ***p<0.001, ****p<0.0001. n=3, mean ± SEM.
Figure 5 (A) Normalized GE values of plant SR proteins in a 48-hour time course. In the first day, plants were kept at 20°C (red) and shifted to 4°C (blue) for the second day. Plants were under a 12-hour dark/light regimen (gray/yellow bars). Warm- and cold-expressed genes are indicated.  (C) Sashimi plots as shown in Figure 2A (without conservation score) for decay-inducing AS events upon cold (At-SCL33, left) or warm temperature (At-SR34, right). Data shown represent, from top to bottom, warm-dark, warm-light, cold-dark and cold-light conditions as indicated on the right of the plots. (D) For validation, A. thaliana were incubated for 3 days either at 20°C or 4°C and RNA was investigated for gene expression (green, left y-axis) and poison isoform formation (orange, right y-axis). Gene expression of At-SCL33 (left) or At-SR34 (right) is shown relative to Ipp2. Lines represent mean, individual data points are shown.  (E) Correlation of poison isoform inclusion and GE levels for temperature-dependent poison skipped exon events. Shown are the log2 fold change (FC) in GE versus the ∆PSI of the poison isoform. Decay targets were identified by using data from CHX-treated plants. R² and P(deviation from zero slope) are indicated. N=80
Figure 6 (B) Correlation of rhythmic Srsf10 expression (orange, left y-axis) and exon 3 inclusion (black, right y-axis) in mouse liver samples from the indicated ZTs (n=4, mean +/- SEM). Quantifications were obtained by RNA-seq analysis of datasets from (Atger et al., 2015).  (C) Representative Western blot of SRSF10 protein levels from mouse liver nuclear extracts (NX) from different ZTs. The different SRSF10 variants are highlighted on the right. SRSF10-fl and SRSF10-2 are the result of differential last exon usage. hnRNP L was used as a loading control. The asterisks could represent hyperphosphorylated SRSF10-fl through higher CLK activity during the day. (D) Quantification of SRSF10-fl + SRSF10-2 relative to ZT0 and hnRNP L (mean of at least 3 mice ± SEM). Student's unpaired t test-derived p values ***p<0.001.
Figure 1(A and B) Root length (averages ± SD, n ≥ 15) (A) and cell death staining of cotyledons and primary root tips (B) of Arabidopsis seedlings that were exposed to 100 nM Pep2 for 7 days, 2 days after germination. BAK1 (D416N) is a kinase-dead variant.(C) Overview of 3-week-old plants.(D) Pep2 induces PEPR1-FLAG association with SERK1-, SERK2-, BKK1-, and BAK1-HA in N. benthamiana. Experiments were repeated at least three times, with the same conclusions. IP and IB denote imunoprecipitation and immunoblotting with the indicated antibodies, respectively. NT indicates non-transformed plants. (*, p < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of WT plants.)
Figure 2(A) Immunoblot analysis for BIK1-HA in Arabidopsis protoplasts exposed to Pep2 or flg22 for 10 min. Arrowheads indicate phosphorylated BIK1-HA.(B) Immunoblot analysis for Pep2-triggered MAPK activation. Ponceau S-stained loading controls are shown (bottom).(C) Root length (averages ± SD, n ≥ 15) of Arabidopsis seedlings that were exposed to 100 nM Pep2. (*, p < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of bak1-3 plants. Two independent experiments were combined for statistical analysis.)
Figure 3(A) Scatter plots for log2 fold changes of gene expression in WT plants versus bak1-3 plants treated with Pep2 for 2 and 10 h. The regression of these scatter plots is indicated by the red lines.(B) Hierarchical analysis of genes exhibiting a >2-fold change in expression level in bak1-3 plants after Pep2 application for 10 h in a whole-genome microarray analysis. Using Genevestigator v3, these genes were separately cross-referenced to public databases to show their expression responses to salicylate (SA), methyl-jasmonate (JA), or ethylene (ET).(C and D) qRT-PCR analysis of defense-related genes in 10-day-old seedlings exposed to 0.5 µM Pep2 for 10 h. Data are averages (±SD) of three biological replicates. (*, p < 0.05 in two-tailed tests compared to the corresponding bak1-4 and bak1-3 values in C and D, respectively. Relative cycle threshold (Ct) values of two independent experiments were combined for statistical analysis.)
Figure 4(A) qRT-PCR analysis of PROPEP genes in 10-day-old seedlings exposed to 0.5 µM Pep2 for 10 h. *, p < 0.05 in two-tailed tests compared to the corresponding WT values. Relative Ct values of two independent experiments with three biological replicates each were combined for statistical analysis.(B) Anti-GFP (upper) and anti-PROPEP3 (middle) immunoblot analysis of PROPEP3-Venus in seedlings submerged in liquid medium and treated with (+) or without (-) 0.5 M Pep2 for the indicated times. The positions of the molecular weight markers are shown on the left. Arrowheads and asterisks indicate PROPEP3-Venus and cross-reactive bands, respectively.(C, D and E) Immunoblot analysis of PEPR1-FLAG in 10-day-old seedlings following Pep1 or flg22 application. (C and D) Plants were exposed to 1 M Pep1 or flg22 for the indicated times. (E) Plants were pre-exposed to MG132 (50 µM), Wortmannin (Wm) (30 µM) and Tyrphostin A23 (TyrA23) (100 µM) for 1 h before being exposed to Pep1 for 10 h. The numbers below the immunoblots represent relative intensities of the PROPEP-Venus (B) and PEPR1-FLAG (C-E) bands normalized to the backgrounds in the Ponceau S-stained loading controls (bottom), with the band/background values in WT plant extracts 24 h (upper) and 0 h (middle) after Pep2 application, respectively, set as 1.0.
Figure 5(A) qRT-PCR analysis of defense-related genes in 10-day-old seedlings exposed to 0.5 µM Pep2 for 10 h. (*, p < 0.05 in two-tailed tests compared to the corresponding WT values. Relative Ct values of two independent experiments with three biological replicates each were combined for statistical analysis.)(B) Root length (averages ± SD, n ≥ 15) of Arabidopsis seedlings exposed to 100 nM Pep2. (*, p < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of WT plants. Two independent experiments were combined for statistical analysis.)(C) Fresh weight (averages ± SD, n ≥ 25) of 2-week-old plants grown on soil. (*, p < 0.05 in two-tailed tests compared to the corresponding values of WT plants. ■, p < 0.05 in two-tailed tests. Two independent experiments were combined for statistical analysis.)(D) Immunoblot analysis for BAK1 in 10-day-old seedlings with or without 0.5 M Pep1 for 24 h. The numbers below the upper panel represent relative intensities of the BAK1 band normalized to the backgrounds in the Ponceau S-stained loading controls, with the value in non-elicited WT plants set as 1.0.
Figure 6(A) qRT-PCR analysis in 4-week-old plant leaves challenged with the indicated P. syringae pv. tomato (Pst) DC3000 strains for the indicated times.(B) Anti-GFP immunoblot analyses in seedlings challenged with Pst DC3000 (DC3000) and Pst DC3000 ∆hrcC (∆hrcC) for the indicated times.. (C) Anti-PROPEP3 immunoblot analysis of PROPEP3-Venus (upper) in plant tissues (expressed in plants) and in the extracellular fraction (released into medium), 24 h and 48 h after the inoculation with (+) or without (-) Pst DC3000. Lower panels indicate Ponceau S-stained loading controls. The numbers below the upper panel represent relative intensities of the PROPEP3-Venus band normalized to the backgrounds in the Ponceau S-stained loading controls. NT denotes non-transgenic WT plants.(D) Trypan blue cell death staining of Arabidopsis seedlings challenged with Pst DC3000 or Pst DC3000 AvrRpm1 (AvrRpm1) for 48 h.(E) Anti-PROPEP3 immunoblot analyses in the seedlings exposed to the indicated Pst DC3000 strains or 1 M Pep1 for 48 h.(F) Anti-GFP immunoblot analysis for the extracellular fraction of the seedlings exposed to Pst DC3000 AvrRpm1 for 48 h. (B, C, E and F) Arrowheads and arrows indicate an apparently full-length and processed form of PROPEP3-Venus, respectively. Asterisks indicate cross-reactive bands. The positions of the molecular weight markers are shown on the left of the immunoblots.
Figure 7(A) Growth of H. arabidopsidis (Hpa) Noco2 in 2-week-old plants. Spores were counted at 6 days post inoculation (dpi). Results are averages ± SE of three biological replicates. (*, p < 0.05 in two-tailed tests compared to the corresponding values of Col plants. ■, p < 0.05 in two-tailed tests between the indicated values. Three independent experiments were combined for statistical analysis.)(B) Growth of syringe-infiltrated Pst DC3000 in rosette leaves of 4-week-old plants pretreated with water (Mock) or 1 M flg22 for 24 h. Bacterial titers were determined at 3 dpi. Results are averages ± SE of three independent replicates. (*, p < 0.05, **, p < 0.01 in two-tailed tests compared to the corresponding values of WT plants. ▲, p < 0.01 in two-tailed tests compared to the differences (± flg22) from the corresponding values of WT plants. Three independent experiments were combined for statistical analysis.)(C) Total SA levels were determined in the rosette leaves of 4-week-old plants with or without 1 µM flg22 application for 24 h. Data are averages (±SD) of material from three independent plants. The experiments were repeated twice, with similar results. (▲, p < 0.01 in two-tailed tests compared to the differences (± flg22) from the corresponding values of WT plants. Two independent experiments were combined for statistical analysis.)(D) qRT-PCR analysis of the PR1 gene in 4-week-old plants exposed to 1 µM flg22. Data are averages (±SD) of three biological replicates. (*, p < 0.05 in two-tailed tests compared to the corresponding WT values. Relative Ct values of four independent experiments were combined for statistical analysis.)(E) Growth of syringe-infiltrated Pst DC3000 in rosette leaves of 4-week-old plants pretreated with water (Mock) or 1 M flg22 for 24 h. Bacterial titers were determined at 3 dpi. Results are averages ± SE of three independent replicates. (**, p < 0.01, ***, p < 1.0e-10 in two-tailed tests compared to the corresponding values of WT plants. ▲, p < 0.01 in two-tailed tests compared to the differences (± flg22) from the corresponding values of WT plants. Three independent experiments were combined for statistical analysis.)
Figure 8(A) Lesion sizes (left) were determined 5 days after C. higginsianum (Ch) inoculation. Invasion rates (right) of Ch on Arabidopsis leaves were determined 3 days after spore inoculation. An average of 3 replicates is shown (±SD) (left) and (±SE) (right). (■, p < 0.05 in two-tailed tests between the indicated samples. *, p < 0.05 in two-tailed tests compared to the corresponding WT values. Two independent experiments were combined for statistical analysis.)B) Immunoblot (left) and qRT-PCR (right) analyses following Ch challenge for the indicated times (dpi). The numbers below the immunoblots represent relative intensities of the indicated bands normalized to the backgrounds, with the values at 0 dpi set as 1.0. Experiments were performed three times with the same conclusions.
Figure 1E. Microscale thermophoresis binding experiment for PlexA1-4-mVenus and Sema1becto (red) or Sema1becto-mutA (blue) or Sema1becto-mutB (green,) or Sema1becto-mutA+B (orange). The Sema1becto wild-type binding results are as reported previously (Rozbesky et al., 2019). All data were collected at the same time, error bars represent s.d. of three technical replicates.
Figure 2A. FRET-FLIM in live COS-7 cells shows cell surface PlexA-Sema1b cis interaction. Sema1b mutants reveal that both head-to-head and side-on orientations of the PlexA-Sema1b complex are involved in the cis interaction. The cis interaction between Sema1a and PlexA is not observed. The boxcharts represent the average lifetime. Box limits indicate the 25th and 75th percentiles, centred lines show the median, squares represent sample means, whiskers extend 1.5-fold the interquartile range from the 25th and 75th percentiles, p-value was calculated by one-way analysis of variance (ANOVA). *P<0.05, ***P<0.001B. Representative negative stain class averages of PlexAecto correlate well with the ring-like conformation of mouse PlxnA1. The ring-like conformation of mouse PlxnA1 is shown in surface representation rainbow colour ramped from blue (N terminus) to red (C terminus). Scale bar, 10 nm.E. In vitro FRET-FLIM measurement of the PlexA-FRET Biosensor shows that the PlexA ectodomain undergoes a conformational change upon treatment with Sema1b resulting in more open conformation. The boxcharts represent the average lifetime. Box limits indicate the 25th and 75th percentiles, centred lines show the median, squares represent sample means, whiskers extend 1.5-fold the interquartile range from the 25th and 75th percentiles, p-value was calculated by one-way analysis of variance (ANOVA). ***P<0.001.F. Relative change of COS-7 cells surface area upon treatment with Sema1a-Fc. Cells expressing PlexAFL-mClover or PlexAFL-mCover and Sema1b-mRuby2 wild-type or mutants were treated with purified Sema1a-Fc at a final concentration of 5.8 µM. Images were acquired every minute for 30 minutes. Cell surface area was calculated using ImageJ before and after stimulation. Data are presented as means ± sem. *P<0.05, ***P<0.001, p-value calculated by one-way analysis of variance (ANOVA).
Figure 3A. Cultured dorsal root ganglion (DRG) neurons from E12.5 Sema6A knockout (KO) embryos were transfected with EGFP, EGFP-tagged wild-type (WT) Sema6A (Sema6A-WT) or different EGFP-tagged Sema6A mutants (Sema6A-mutA, Sema6A-mutB, and Sema6A-mutA+B) at 1 day in vitro (DIV1) and treated with purified Sema6A-Fc at a concentration of 75 nM for 1 hour at 37°C at DIV4. Transfection of EGFP was used as a control (full collapse). DRG neurons from Sema6A KO embryos acquired sensitivity to Sema6A and growth cone collapse was observed in three independent experiments. Transfection of Sema6A-WT, Sema6A-mutA and Sema6A-mutB but not of EGFP or Sema6A-mutantA+B prevented Sema6A-mediated growth cone collapse (Fisher's exact test p<0.0001). Quantification of growth cone collapse was performed using a growth cone morphology matrix (lower part panel A). Data is presented as percentage of morphologically distinct growth cones, divided (represented by the dotted line) in two categories, from uncollapsed (1-4) to fully collapsed (5-8). Totally, 20-40 growth cones were analyzed for each condition per experiment (n=3 experiments). Scale bar 20 µm.B. Representative images of growth cones of mouse DRG neurons transfected with WT or mutant constructs treated with 75 nM Sema6A-Fc and visualized with immunofluorescent staining to detect GFP (green) and phalloidin (red). Average intensity images for all different constructs are shown in grey color. Scale bar 20 µm.
Figure 1.(A) Hep3B hepatocytes were treated with nontargeting control siRNA (siNT) or siRNA targeting Dyn2 (siDyn2), followed by re-expression of either vector alone, or GFP-tagged versions of Dyn2, loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS. LDs were visualized by immunofluorescence using Oil Red O staining. Cell boundaries are outlined and those cells re-expressing GFP, GFP-wtDyn2, or GFP--K44A are denoted with asterisks. Bars, 20 µM. (B) Representative blot showing the efficiency of the Dyn2 knockdown in these cells. (C and D) Quantitation of the average LD number and area (in pixels2) per cell from three independent experiments. The data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01. NS, not significant.
Figure 2.HuH-7 (A) and Hep3B (B) hepatocytes were loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS in the presence of Dyn2 inhibitors or DMSO as indicated. Representative images of inhibitor-treated and control cells (stained with Oil Red O) are shown in A and B together with the quantitation of the average LD area per cell from three independent experiments. Pharmacological inhibitors used were: Dynasore (inhibits Dyn2 GTPase activity), MiTMAB (targets PH domain and interferes with membrane binding), Dynole 34-2 (allosteric GTPase inhibitor), and Dynole 31-2 (negative control for Dynole 34-2). Bars, 20 µM.(C) Representative images from control and Dyn2 knockout MEFs after an overnight lipid loading with 400 µM Dyn2 for 17 h. Knockout of Dyn2 was induced by treatment with 2 µM 4-hydroxy-tamoxifen for 7 d and was confirmed by immunostaining of endogenous Dyn2 (top row) and by immunoblot (D). Bars, 20 µM. (E and F) Average LD number (E) and area (F) per cell from five independent experiments. All data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01.(G) Whole-cell lysates and LD fractions isolated from HuH-7 hepatocytes under resting or starved (2 h HBSS starvation) conditions.(H) Primary hepatocyte expressing Dyn2-GFP, showing an absence of colocalization with the LD surface (stained with Oil Red O). Bar, 20 µM. Inset shows magnification of boxed region (bar, 2 µM).
Figure 3.Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and co-stained with antibodies specific for LAMP1 (red) and LC3 (green). After Dyn2 knockdown, a juxtanuclear aggregation and enlargement of the LAMP1-positive compartment is observed (C and D, arrows). Increased labeling of LC3 is also detectable after knockdown of Dyn2.(E) Western blotting of Hep3B lysates after a 3-d treatment with either the control or Dyn2-targeted siRNA and further treatment with or without 50 µM leupeptin. Densitometry-based analysis of six independent experiments is shown at the bottom of the figure.(F) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore, in the presence or absence of 50 µM leupeptin. Quantitation of LC3-II levels relative to control are shown below the blots. The data are represented as mean ± SE; *, P ≤ 0.05.
Figure 4.Dyn2 knockdown results in the formation of enlarged autophagic structures. (A-D) Transmission electron micrographs (TEMs) of oleate-loaded Hep3B hepatocytes treated with nontargeting control (siNT) or Dyn2 (siDyn2) siRNA for 72 h. Bars: (A and B) 2 µm; (A′ and B′) 1 µm; (C and D) 0.5 µm. Insets in A and B show fluorescent micrographs of LAMP1-stained cells (bars, 10 µM). Control cells (A) contain an abundance of small (<1 µm) electron-dense lysosomes and autolysosomes (arrowheads). Under conditions in which Dyn2 expression is suppressed (B-D), far fewer small lysosomes are observed. Instead, the cells are populated by larger autolysosomes (≥1 µm) with aberrant morphologies and containing putative LDs (*). (E) Quantitative measure of autolysosomal size from cells in which Dyn2 was knocked down as compared with control cells. Values represent the fold-increase in the number of autophagic structures of a given diameter (measured in microns) observed over siNT-treated control cells. Data were obtained from a single experiment with n = 6 cells examined by electron microscopy from each condition.