EMBO/soda-nel huggingface dataset:
Genetic screen of yeast environmental stress response dynamics uncovers distinct regulatory phases (2019)
Figure 2

Figure 2: Partially decoupled regulation of transcriptional dynamic parameters inferred from protein and RNA measurements

Summary of Hsp12-GFP dynamics single mutant screen. 68 mutants were divided into three groups: increased/decreased/did not change the total GFP produced. Each group was independently hierarchically clustered. The values shown are the Z-Scores of each parameter: ton- response onset time; production rate; total open time - length of transcriptional window (toff − ton); Basal GFP - GFP levels measured before exposure to stress; Delta GFP - amount of GFP produced throughout the experiment (Max GFP - Basal GFP).

The dynamic parameters are regulated by partially independent mechanisms. Mutant strains can be roughly divided to three groups: affecting total open time, affecting production rate and affecting both.

Outline of mRNA-Seq experiment. 16 yeast strains were grown to mid-log phase, exposed to 0.4M KCl and sampled in the indicated time points after exposure to stress.

mRNA levels of HSP12 (Y axis) as a function of time (X axis) in six representative strains.

Comparison of RNA levels to protein levels. X axis is the sum over the HSP12 mRNA counts (see D). Y axis is the maximum over the Hsp12-GFP values. The red line indicates the best linear fit.

Comparing the total open time parameter inferred from mRNA data (X axis) to the one inferred from protein data (Y axis).

The same as (F) for the production/transcription rates.

Time-lapse microscopy of Hsp12-GFP in response to stress (0.4M KCl). Examples of raw images (top) and traces of individual cells (bottom). Traces are colored according to their basal GFP level.

Scatter plot showing the production rate estimated from single traces vs. the production rate estimated from the population average. Each point represents a different yeast strain.
Genetic screen of yeast environmental stress response dynamics uncovers distinct regulatory phases (2019)
Figure 3

Figure 3: Double mutant screen for regulators of dynamic parameters

Double mutant library, containing ~1600 strains with Hsp12-GFP reporter, was screened using automated flow-cytometry in six timepoints following exposure to stress (0.4M KCl).

Estimated parameters (median over repeats) of all ~1600 double mutant strains. The color represents the total Hsp12-GFP produced. The same amount of GFP can be produced with multiple parameter combinations. The ellipses illustrate iso-expression areas in the graph.

Three examples of individual strains (marked in B) with nearly the same total expression but dramatically different dynamics.

Illustration of the genetic interaction trends of individual mutants. For each single mutant we plot the median of all the double mutants that contain it (purple dots). The gray dots are all individual double mutant strains (B).

Illustration of the interactions of Δhog1 in the production rate parameter. Shown the production rate of the single mutant (X axis) versus the production rate of the double mutant with Δhog1 (Y axis). The gray line marks the Hog1 single mutant levels. Points close to the gray line are ones where the value of the double mutant is close to the value of Δhog1 and defined as epistasis of Δhog1 (pink dots).

The same as (E) for the total open time parameter.

Venn diagram comparing the number of epistatic pairs detected in various phenotypes.
Genetic screen of yeast environmental stress response dynamics uncovers distinct regulatory phases (2019)
Figure 4

Figure 4: MCK1 is necessary for secondary wave of Msn2/4 activity

Epistatic interactions of Δmck1 in the production rate parameter (See 3E).

Epistatic interactions of Δmck1 in the total open time parameter (See 3E).

Upper: HSP12 mRNA in response to osmotic stress (0.4M KCl). Lower: Estimate of Msn2/4 dependent production of HSP12 mRNA over time (difference between WT and Δmsn2Δmsn4 production profiles, Figure EV4A, Methods).

Δmck1 effect on the response of all stress genes. The integral over mRNA values in WT strain (X axis) vs. the integral in the Δmck1 strain (Y values). The inset shows the integral for the HSP12 gene.

Heatmap showing expression of stress responsive genes in WT and two mutants over eight timepoints (See 2C). Each gene is presented relative to its pre-stress level and normalized by the median over WT values to visualize the dynamic range in all genes. The genes are separated into MSN dependent and independent genes (Figure EV4B). The MSN-dependent genes are sorted based on the onset time to three groups (Early, Average and Late onset).

The behaviour of early and late onset genes. Similar to (C) but showing the average over the genes in each group.
Genetic screen of yeast environmental stress response dynamics uncovers distinct regulatory phases (2019)
Figure 5

Figure 5: Two regulatory phases in Msn2 response

Msn2-GFP localization following stress (0.4M KCl) in selected strains. Top: The percentage of nuclear cells (Y axis) over time (X axis). Bottom: count of the number of nuclear import events in live-cell time-lapse microscopy at the two relevant periods.

Same data as 4C, with the addition of the Δmsn5 strain.

Same data as 5F, with the addition of the Δmsn5 strain.

Top: Schematic model of Msn2 localization in different genetic backgrounds and phases of the response. Bottom: Summary of our observations in terms of nuclear localization, Msn2 activity and the effect on gene expression.

Expanded View Figure Legends:
CD1d-mediated activation of group 3 innate lymphoid cells drives IL-22 production (2016)
Figure 1

Figure 1. CD1d expression on ILCs

(A) Flow-cytometry for CD1d expression (top) and CD1d mean fluorescence intensity (MFI; bottom) on CD45- cells, ILCs (Lin-CD45+CD127+), DCs (CD11c+) and B cells (B220+) of the depicted tissues from WT mice (n=4).

(B) Flow-cytometry profiles showing gating strategy (dot-plots), CD1d expression (histograms) and CD1d MFI (bars) in T-bet+ (empty profile), GATA-3+ (dark grey), RORγt+ (light grey) and T-bet+RORγt+ (dotted line) ILCs in mLN, spleen, PP and SI-LP (n=5). Numbers indicate percentage of cells in the depicted gates.

(C) Quantitative RT-PCR analysis of mRNA encoding CD1d in freshly isolated ILC3s (from mLN of CD1d-deficient mice and mLN and spleen of WT mice), B cells and DCs (n=3). Results are normalized to those of GAPDH; RE=relative expression. **p< 0.01 (two-tailed unpaired t-test).

(D) Flow-cytometry profiles showing NKp46 and RORγt expression in Lin-CD45+ cells; CD1d and CCR6 expression in NCR- ILC3s and CD1d and CD4 expression in NCR-CCR6+ ILC3s from mLN and SI-LP (n=4). Numbers indicate percentage of cells in the depicted gates.

(E) CD1d expression (histograms) and CD1d MFI in NCR+ (empty profile), NCR-CCR6- (dark grey) and NCR-CCR6+ (light grey) ILC3s from mLN and SI-LP (n=4).

Graphs represent mean +/- SEM. Data are from three (A, C-E) or five (B) independent experiments.
CD1d-mediated activation of group 3 innate lymphoid cells drives IL-22 production (2016)
Figure 2

Figure 2. ILC3s internalise and present lipid antigens to iNKT cells

(A) Cells were cultured in the presence or absence of BODIPY-αGalCer at 4oC or 37oC as indicated. Flow cytometry profiles and percentages of BODIPY-αGalCer+ DC or ILC3 from the indicated tissues incubated at 4oC (white bars) or 37oC (grey bars) are shown (n=3).

(B) WT mice were intravenously injected with BODIPY-αGalCer or PBS (control); lipid uptake was analysed by flow-cytometry in the depicted populations from the spleen 16 h after lipid injection (n=3). Numbers indicate percentage of cells in the depicted gates.

(C) IL-2 secretion by DN32.D3 cells co-cultured with ILC3 sort-purified from mLN or spleen as indicated, pulsed (+) or not (-) with αGalCer and with or without antiCD1d-blocking antibody.

(D-F) Sort-purified ILC3s were preincubated with αGalCer (ILC3(αGal)) or PBS (ILC3(cont)) and adoptively transferred into WT recipients. (D) Experimental protocol; (E) Fold change in mRNA expression for the indicated cytokines in iNKT cells sort-purified from spleen, mLN or SI-LP of mice injected with αGalCer-loaded ILC3s. Gene expression was measured by qPCR, normalized to GAPDH and presented as expression relative to iNKT cells sort-purified from mice injected with control ILC3s (n=3-5). (F) Intracellular cytokine staining (left) and percentage of cytokine+ cells (right) in splenic iNKT cells from ILC3-recipient mice (n=3). Numbers indicate percentage of cells in the depicted gates. **p<0.01, two-tailed unpaired t-test.

Graphs represent mean +/- SEM. Data are from more than two independent experiments.
CD1d-mediated activation of group 3 innate lymphoid cells drives IL-22 production (2016)
Figure 3

Figure 3. Engagement of CD1d on ILC3s induces IL-22 production

(A-B) Flow-cytometry plots showing gating strategy (A) and percentage of ILC populations (from Lin-CD45+CD127+ cells, B) in mLN from WT (n=4) and CD1d-deficient (n=3) mice.

(C) Fold change in mRNA expression for the indicated cytokines in ILC3s after antibody-mediated CD1d crosslink (n=3). Gene expression was measured by qPCR and normalized to GAPDH and to the mRNA expression levels in control ILC3s. *p<0.05 two-tailed unpaired t-test.

(D) Fold change in IL22 mRNA expression in ILC3s after antibody-mediated CD1d crosslink and/or IL-23 stimulation (n=4). Gene expression was measured by qPCR and normalized to GAPDH and to the mRNA expression levels in control ILC3s. **p<0.01 two-tailed unpaired t-test.

(E) IL-22 detection by ELISA in the supernatant of ILC3s cultured in the presence of αCD1d and/or IL-23 (n=3). *p<0.05 two-tailed unpaired t-test

(F-G) Intracellular cytokine staining (F) and percentage of IL-22+ ILC3s (G) after stimulation with αCD1d, IL-23 and/or IL-1β. *p<0.05, **p<0.01 two-tailed unpaired t-test (n=4-8)

(H) WT mice were i.v. injected with αGalCer or PBS (control) and activation of splenic ILC3s was assessed by flow-cytometry as CD69 up-regulation at 6 h or 16 h after injection. Grey filled profile= ILC3 from αGalCer-injected mice; Empty profile= ILC3 from PBS-injected mice; Dotted line= CD45- cells. Right graph, percentage of CD69+ ILC3s 6 h after injection of αGalCer (grey) or PBS (white). (n=3) *p<0.05 two-tailed unpaired t-test.

(I-J) Fold change in mRNA expression for the indicated cytokines in ILC3s sort-purified from spleen (I) or SI-LP (J) 6 h after intravenous (I) or oral (J) αGalcer administration. Gene expression was measured by qPCR and normalized to GAPDH and to the expression levels in ILC3s sort-purified from PBS-injected mice (n=3). *p<0.05, **p<0.01 two-tailed unpaired t-test.

Graphs represent mean +/- SEM.
Poly(U)-specific endoribonuclease ENDOU promotes translation of human CHOP mRNA by releasing uORF element-mediated inhibition (2020)
Figure 1

Figure 1. Method of searching for candidate genes identified as potentially involved in translational control mediated by huORFchop transcript through their upregulation in GFP-expressing cells.

A. Schematic diagram illustrating the separation of GFP-expressing (+) and non-GFP-expressing (-) cells from stress-treated zebrafish embryos of transgenic line huORFZ using LMD methods followed by microarrays.

B. Graphical representation of two microarray expression analyses. A total of 298 over-represented transcripts and 104 under-represented transcripts were obtained from both microarrays.

C. Upper panels: top view of EGFP expressed in zebrafish embryos derived from zebrafish transgenic line huORFZ under non-stress and heat-shock treatment. Middle panels: lateral view of endouc expression patterns in embryos under non-stress condition and treated with heat-shock. Lower panels: top view of endouc expression patterns.

D. Quantitative RT-PCR determined the relative expression level of endouc mRNA in non-stress condition and heat-shock-treated embryos. Values representing the non-stress and Heat-shocked conditions were calculated from three independent experiments and presented as mean ± S.D. (n=3). Student's -t test was used to determine significant differences between each group (***: P <0.001).

E. The endouc and ela3l (negative control) mRNAs were separately microinjected into zebrafish embryos from huORFZ at a one-cell stage. Upper panels: observation of GFP signal expressed in huORFZ embryos at 72 hpf. Lower panels: magnification of pictures corresponding to the upper panels.
Poly(U)-specific endoribonuclease ENDOU promotes translation of human CHOP mRNA by releasing uORF element-mediated inhibition (2020)
Figure 3

Figure 3. Identification of Endouc functional domain involved in regulating huORFchop-TI.

A. Schematic representation of zebrafish Endouc protein and its internal deletion mutants.

B. Histograms presenting the relative luc activity obtained from zebrafish embryos microinjected simultaneously with puORFchop-luc, phRG-TK, and each of the expression plasmids as indicated followed by analysis of luc activity at 96 hpf. Embryos microinjected with pCS2 vector during normal condition served as a control group (non-stress; blank column), while the microinjected embryos treated with 40℃ at 72 hpf for one hour were the heat-shocked group (solid column). The relative luc activity of microinjected embryos shown on the right was normalized by the amount of RNA in the injected embryos shown on the left. The relative luc activity is represented by the fold increase in Fluc/Rluc ratio over that obtained from control group. The luc activity mediated by the huORFchop element was measured by a dual-luciferase assay, and its corresponding huORFchop-luc transcript was measured by RT-qPCR.

C. Histograms presented the luc activity obtained from HEK 293T cells co-transfected with puORFchop-luc, phRG-TK, and each one of the plasmids as indicated and treated with either DMSO (control group) or Thapsigargin (TH; stress group) followed by analysis of luc activity. The luc activity of cells transfected with pCS2 vector and kept at normal condition served as a control group. The relative luc activity of transfected cells shown on the right was normalized by the amount of RNA in the transfected cells shown on the left. The relative luc activity is represented by the fold increase of Fluc/Rluc ratio over that obtained from pCS2-transfected control group normalized as 1. The luc activity mediated by the huORFchop transcript was measured by the dual-luciferase assay, and its corresponding huORFchop-luc transcript was measured by RT-qPCR.

D. Zebrafish embryos at one-cell stage were microinjected with plasmids as indicated, followed by extraction of embryonic proteins at the 96-hpf stage. Protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants were detected using Western blot analysis. GAPDH served as an internal control.

E. HEK 293T cells were transfected with indicated plasmids followed by analysis of the protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants using Western blot. The α-tubulin served as an internal control. Protein levels relative to each internal control are presented in each lane.
Poly(U)-specific endoribonuclease ENDOU promotes translation of human CHOP mRNA by releasing uORF element-mediated inhibition (2020)
Figure 4

Figure 4. The positive effect of Endouc on p-eIF2α and CHOP expressions is dependent on its endoribonuclease activity.

A. Schematic representation of zebrafish Endouc protein and its internal deletion mutants.

B. Histograms presented the relative luc activity obtained from zebrafish embryos microinjected simultaneously with puORFchop-luc, phRG-TK, and each one of the expression plasmids as indicated followed by analysis of luc activity at 96 hpf. Embryos microinjected with pCS2 vector during normal condition (non-stress; blank column) served as a control group while the microinjected embryos treated with 40℃ at 72 hpf for 1 hr were the heat-shocked group (solid column). The relative luc activity of microinjected embryos shown on the right was normalized by the amount of RNA in the injected embryos shown on the left. The relative luc activity was represented by the fold increase of Fluc/Rluc ratio over that obtained from control group. The luc activity mediated by the huORFchop transcript was measured by dual-luciferase assay, and its corresponding huORFchop-luc transcript was measured by RT-qPCR.

C. Histograms presented the luc activity obtained from HEK 293T cells co-transfected with puORFchop-luc, phRG-TK, and each one of the plasmids as indicated; these were treated with either DMSO (control group) or thapsigargin (TH; stress group) followed by analysis of luc activity. The luc activity of cells transfected with pCS2 vector and kept at normal condition served as a control group. The relative luc activity of transfected cells shown on the right was normalized by the amount of RNA in the transfected cells shown on the left. The relative luc activity was represented by the fold increase of Fluc/Rluc ratio over that obtained from pCS2-transfected control group normalized as 1. The luc activity mediated by the huORFchop transcript was measured by the dual-luciferase assay, and its corresponding huORFchop-luc transcript was measured by RT-qPCR.

D. Zebrafish embryos at one-cell stage were microinjected with plasmids as indicated followed by extraction of embryonic proteins at the 96-hpf stage. Protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants were detected using Western blot analysis. GAPDH served as an internal control.

E. HEK 293T cells were transfected with indicated plasmids followed by analysis of the protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants using Western blot. Here, α-tubulin served as an internal control. Protein levels relative to each internal control were presented in each lane.
Poly(U)-specific endoribonuclease ENDOU promotes translation of human CHOP mRNA by releasing uORF element-mediated inhibition (2020)
Figure 5

Figure 5. Besides p-eIF2α, Endouc is another key protein that causes an increase in the CHOP protein level.

A. HEK 293T cells were transfected with plasmids as indicated for 24 hr and treated with DMSO or 200 nM ISRIB for 2 hr followed by analysis of the protein level of CHOP, p-eIF2α, total eIF2α, and Endou-Flag using Western blot. The α-tubulin served as an internal control. Protein levels relative to each internal control are presented in each lane.

B. HEK 293T cells were transfected with plasmids as indicated for 24 hr and treated with DMSO, thapsigargin (TH), ISRIB, or TH plus ISRIB for 2 hr followed by analysis of the protein level of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants using Western blot. The α-tubulin served as an internal control. Protein levels relative to each internal control were presented at each lane.

C. MEF wild-type (WT) and its eIF2αS51A mutant (S51A) cells were treated with 1 μg of TH for 4 hr followed by Western blot analysis of p-eIF2α, total eIF2α, and ENDOU. GAPDH served as an internal control. Protein levels relative to each internal control are presented in each lane.

D, E. MEF WT and mutant S51A cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or TH for 4 hr followed by analysis of the protein levels of CHOP, p-eIF2α, total eIF2α, GADD34, and Endouc-Flag using Western blot. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane.

F. HEK 293T cells were transfected with negative-control siRNA and ENDOU-siRNA for 48 hr. After treatment, protein lysates were prepared and subjected to Western blot analysis for ENDOU-1 and CHOP detection using antibodies. The α-tubulin served as an internal control. Protein levels relative to each internal control were presented in each lane.
Poly(U)-specific endoribonuclease ENDOU promotes translation of human CHOP mRNA by releasing uORF element-mediated inhibition (2020)
Figure 6

Figure 6. Endouc induces the phosphorylation of eIF2α through the PKR pathway.

A. MEF wild-type (WT) cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or Thapsigargin (TH) for 4 hr followed by analysis of the protein level of p-PKR, total PKR, and Endouc-Flag. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane.

B. MEF WT cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or TH for 4 hr followed by analysis of the protein levels of p-eIF2α, total eIF2α, CHOP, and Endouc-Flag. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane. The results obtained from the two trials are shown on the left and right.
Poly(U)-specific endoribonuclease ENDOU promotes translation of human CHOP mRNA by releasing uORF element-mediated inhibition (2020)
Figure 7

Figure 7. Endouc can bind and cleave the huORFchop transcript.

A. Northern blot analysis to detect the endogenous huORFchop-tagged gfp (uORF-gfp) mRNA and its cleaved form in zebrafish embryos of transgenic line huORFZ treated with non-stress condition (NS) or heat-shocked (HS) stress. Zebrafish 18S rRNA served as an internal control. The RNA fragments cleaved from huORFchop transcript were detected.

B. Northern blot analysis to detect endogenous CHOP mRNA in HEK 293T cells treated with either DMSO (control group) or thapsigargin (TH). Human GAPDH RNA served as internal control. The RNA fragments cleaved from CHOP mRNA were detected.

C. The predicted RNA sequences of Endouc recognition site on huORFchop transcript from 72 to 91 nts are illustrated. The predicted cleavage sites at uridylates (U) are indicated by arrowheads.

D, E. The cleavage activity of Endouc was studied using materials presented at each lane. EndoucK242A: Endouc mutant; Extract: incubated in cell extracts; and Buf: reaction buffer. RNA probe without adding reaction buffer served as a negative control. The predicted cleavage sites (U) were mutated to generate mutant huORFchop transcript (unlabelled huORF-mt) and labelled by biotin (biotin-huORF-mt). Unlabelled competitor RNA was added in reactions at five times higher than that of the biotinylated probe.

F. Electrophoretic mobility shifted assay of RNA. Biotin-labelled huORFchop transcript was reacted with the increased amount of Endouc. The positions of free RNA (free probe) and RNA-protein complex (shifted band) were indicated on the right. The luc 105-nt RNA and single-stranded huORFchop 105-nt DNA served as negative controls.