EMBO/soda-nel huggingface dataset:
CDK9 and PP2A regulate RNA polymerase II transcription termination and coupled RNA maturation. (2022)
Figure 4

Figure 4. CDK9 phosphorylates several transcription and splicing factors in vivo and CDK9 inhibition promotes the loss of interaction of SF3B1 and CPA factors with pol II.

A. Distribution of the proteins found to be phosphorylated by CDK9 in SILAC phosphoproteomics in HeLa cells treated or not with 100 µM DRB for 30 minutes (fold change > 1.5 in both biological replicates, number of phosphopeptides decreased shown in brackets).

B. Western blot of total SF3B1, SF3B1 T142P, and histone H3 as a loading control, on the chromatin fraction of CDK9as cells treated for 30 minutes with DMSO, NA, tautomycetin (TT), calyculin A (CA), NA+TT, or NA+CA. The histone H3 loading control (CA samples) is the same as the loading control shown in Appendix Figure S3B as the same western blot experiment is shown in two different figures.

C. Co-immunoprecipitation of CPSF2 in the CDK9as cell treated for 30 minutes with DMSO or NA followed by western blot with total pol II, CPSF2, CPSF3, SF3B1, SF3B3 and β-actin (negative control) antibodies.

D. Co-immunoprecipitation of SF3B1 in the CDK9as cell treated for 30 minutes with DMSO, NA, or DRB followed by western blot with total pol II, SF3B1, SF3B2, and GAPDH (negative control) antibodies.
CDK9 and PP2A regulate RNA polymerase II transcription termination and coupled RNA maturation. (2022)
Figure 7

Figure 7. CDK9 and PP2A regulate mRNA cleavage and polyadenylation and alternative poly(A) site usage.

A. Schematic of the nuclear and cytoplasmic qRT-PCR experiments.

B. qRT-PCR of nuclear polyadenylated mRNAs of several TNFα induced or non-induced genes with a 30 minutes DMSO, DRB, CA, or DRB+CA treatment. n=6 biological replicates, mean ± SEM, p-value: * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical test: two-tailed unpaired t test.

C. qRT-PCR of cytoplasmic polyadenylated mRNAs of several TNFα induced or non-induced genes with a 30 minutes DMSO, DRB, CA, or DRB+CA treatment. n=4 biological replicates, mean ± SEM, p-value: * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical test: two-tailed unpaired t test.

D. ChIP-qPCR of Ser2P, CPSF73, or CPSF2 ratioed to total pol II after 30 minutes treatment with DMSO, DRB, CA, or DRB+CA on KPNB1. n=3 biological replicates, mean ± SEM, p-value: * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical test: two-tailed unpaired t test.

E. Co-immunoprecipitation of total pol II from HeLa cells treated for 30 minutes with DMSO, DRB, CA, or DRB+CA followed by western blot with total pol II, Ser2P, SF3B1, CPSF2, Xrn2, and GAPDH antibodies.
TOC1 clock protein phosphorylation controls complex formation with NF-YB/C to repress hypocotyl growth (2021)
Figure 1

Figure 1. Phosphorylation-dependent protein mobility shift is eliminated or strongly reduced in 5X and S175A.

A, Schematic diagrams of full-length (FL), N-terminus (NT) and C-terminus CCT domain of TOC1 used in this manuscript. The 5 phosphorylated residues identified from our mass spectrometry were progressively substituted by alanine, and their positions are displayed on top of TOC1FL.

B, Mutations in S175 and 5X attenuate PRR5's enhancement on TOC1 phosphorylation. Wild type and phosphosite mutants of TOC1NT were transiently expressed with or without PRR5 in N. benthamiana leaves, total proteins were extracted 3 days after infiltration.

C, Phosphorylation of TOC1 is eliminated or largely reduced in 5X and S175A. Total protein extracts (ZT13) from 10-day-old seedlings grown under 12h/12h light/dark cycles were subject to heat (30ºC 20min), pyrophosphatase (PP) and NaF/Na3VO4 (phosphatase inhibitors) treatments. TOC1-GFP at different phosphorylation states were separated by 8% SDS-PAGE (acrylamide:bisacrylamide 150:1) and subsequently detected by α-GFP.
TOC1 clock protein phosphorylation controls complex formation with NF-YB/C to repress hypocotyl growth (2021)
Figure 2

Figure 2. 5X and S175A fail to fully rescue the hypocotyl phenotype displayed in toc1-101.

A-F Shown are hypocotyl lengths of cRL-grown 4-day-old seedlings of toc1 complemented with wild-type TOC1 (A), 5X (B), T135A (C), S175A (D) driven by TOC1 native promoter and wild-type TOC1 (E), 5X (F) driven by 35S promoter at sequential fluent rates. Different letters indicate statistically significant differences between genotypes (p<0.01, one-way ANOVA followed by Tukey-Kramer HSD test), error bars indicate SEM. Data are representative of three biological trials with similar results.
TOC1 clock protein phosphorylation controls complex formation with NF-YB/C to repress hypocotyl growth (2021)
Figure 3

Figure 3. Mutations in 5X and S175A incompletely rescue toc1-101 defects in period length, expression phase and hypocotyl growth.

A and B, Hypocotyl lengths in SD condition (A) and free-running period (B) of Col-0, toc1-101 and two independent native or 35S promoter lines of wild-type TOC1 and each phosphosite mutants. The middle line of the box represents the median, the x in the box represents the mean. The bottom line and the top line of the box represent 1st and 3rd quartile, respectively. The whiskers extend from the ends of the box to the minimum value and maximum value.

C-E, Average CCA1:LUC bioluminescence traces of indicated plant lines.

Data information: For A, seedlings were grown under white light and SD (8h/16h L/D) conditions for 3 days. 100 seedlings from 4 independent trials were averaged. For B-E, 18 to 27 seedlings per line were entrained in 12h/12h light/dark cycles for 7 d and next transferred to constant red light at ZT2 for image acquisition with 2-h intervals for 1 week. White and gray regions indicate subjective light and dark period. Data are representative of at least two independent trials with similar results. Different letters indicate statistically significant differences in period between genotypes (p<0.01, one-way ANOVA followed by Tukey-Kramer HSD test).
TOC1 clock protein phosphorylation controls complex formation with NF-YB/C to repress hypocotyl growth (2021)
Figure 4

Figure 4. Mutations in 5X de-repress the pre-dawn hypocotyl-growth-related genes (PHGs).

A-C, PIL1, AT5G02580 and CDF5 expression pattern in 3-d-old SD-grown seedlings from indicated plant lines at night. Data is representative of 3 biological trials.

D-F, Expression of PIL1, AT5G02580 and CDF5 in 3-d-old SD-grown seedlings at ZT18. 5 biological trials were averaged, error bars indicate SEM. Different letters denote statistically significant differences based on Wilcoxon test (p<0.05).
TOC1 clock protein phosphorylation controls complex formation with NF-YB/C to repress hypocotyl growth (2021)
Figure 5

Figure 5. 5X interacts more poorly with PIF3 and is present to a lesser extent at PIF3 target promoters.

A, Co-IP assay of PIF3-TAP with TOC1-GFP vs. 5X-GFP in N. benthamiana. Protein extracts were immunoprecipitated by IgG resins followed by HRV-3C protease digestion. PIF3 and TOC1 were detected by anti-MYC and anti-GFP, respectively.

B, Quantification of protein interaction in A. Results from 3 independent trials were averaged. Error bars indicate SEM, asterisks indicate significant difference (*p<0.05, student's t test).

C, ChIP-qPCR assay of TOC1 binding to promoter of pre-dawn-phased PIF3 target genes at ZT14 in Col-0, native promoter TOC1 and 5X lines.

D, ChIP-qPCR assay of the chromatin residence of native promoter TOC1-GFP at target promoters in toc1 vs. pif3/4/5 toc1 (pif345t1) mutant backgrounds.

Data information: For C and D, 3-d-old SD-grown seedlings were harvested at ZT14, TOC1-GFP was immunoprecipitated by α-GFP and magnetic protein G beads. Data from 3 independent trials were averaged, error bars indicate SEM. Asterisks indicates significant differences (** p<0.001, *** p<0.0001, Student's t test), n.s=not significant.
TOC1 clock protein phosphorylation controls complex formation with NF-YB/C to repress hypocotyl growth (2021)
Figure 6

Figure 6. TOC1 phospho-mutants show early-phased protein expression and reduced stability.

A, TOC1 protein expression pattern in 3-d-old seedlings in native promoter TOC1/5X/T135A/S175A lines grown in SD. White and black regions indicate light and dark period. Data from 3 independent trials were averaged; error bars indicate SEM. Statistical analyses were performed by Students's t test. Asterisks indicate significant differences between TOC1 wild type and its phosphosite mutants (*, p<0.05).

B, Measurement of TOC1-GFP protein turn-over in TOC1, 5X, T135A, S175A native promoter lines by cycloheximide (CHX) treatment. 10-d-old seedlings grown in 12h/12h light/dark cycles were subject to cycloheximide (CHX) or ethanol (Mock) treatment for indicated time length. Data from 3 independent trials were fitted to non-linear Weibull regression curves.

C, Estimates of TOC1-GFP protein half-life in B. Error bars indicate SEM, statistical analyses were performed with student's t test, different letters indicate significant differences (p<0.05). Data from 3 independent trials were averaged.

Data information: For A and B, TOC1 protein was detected by α-GFP, ADK was used as loading control. TOC1/ADK ratio was calculated by the intensities of TOC1-GFP bands normalized by the intensities of ADK bands using ImageJ.
TOC1 clock protein phosphorylation controls complex formation with NF-YB/C to repress hypocotyl growth (2021)
Figure 7

Figure 7. NF-YB/C forms trimeric complex with TOC1 and stabilize TOC1 at target promoters.

A, Yeast two-hybrid (Y2H) assay of interactions between the Gal4 DNA binding domain BD-NF-YB2/3, BD-NF-YC3/4/9 and Gal4 activation domain AD-TOC1/5X fusions. Yeast growth shown on leucine-, tryptophan-deficient plate indicates successful plasmid transformation, and yeast growth shown on leucine-, tryptophan-, histidine-deficient and 3-AT plate indicates positive protein interactions.

B, Quantification of interactions between TOC1/5X-TAP and NF-YC proteins in presence or absence of NF-YB subunits. Proteins were transiently expressed in N. benthamiana. TOC1/5X were immunoprecipitated with IgG resins followed by HRV-3C protease digestion. TOC1/5X and NF-YB/C were detected by α-MYC and α-HA, respectively. Protein band intensity was measured by ImageJ and interactions were exhibited as ratio of co-immunoprecipitated NF-YC to immunoprecipitated TOC1/5X to NF-YC input. Results from 3 independent trials were averaged. Error bars indicate SEM, different letters denote significant differences (p<0.05, Student's t test).

C, ChIP-qPCR assay of native promoter driven TOC1-GFP and 5X-GFP's residence at target promoters in toc1 vs. nf-yc3/4/9 toc1 (c349t1) mutants. 3-d-old SD-grown seedlings were harvested at ZT14, TOC1 and 5X were immunoprecipitated by α-GFP and magnetic protein G beads. Data from 3 independent trials were averaged. Error bars indicate SEM, asterisks indicate significant differences (* p<0.05, *** p<0.0001, Student's t test), n.s= not significant.
TOC1 clock protein phosphorylation controls complex formation with NF-YB/C to repress hypocotyl growth (2021)
Figure 8

Figure 8. NF-YB/C and HDA15 repress downstream hypocotyl genes together with TOC1.

A, Co-IP assay of HDA15-TAP with TOC1-GFP and 5X-GFP in N. benthamiana. HDA15-TAP was immunoprecipitated with IgG resins and digested by HRV-3C protease. HDA15 and TOC1 were detected by α-MYC and α-GFP, respectively.

B, Quantification of the interaction strength in A. Results from 5 independent trials were averaged. Error bar indicates SEM, asterisk indicates significant difference (*p<0.05, student's t test).

C and D, Hypocotyl lengths and free-running periods of indicated plants lines. Data of ~75 plants from 3 independent experiments were averaged, different letters indicate statistically significant differences (p<0.01, one-way ANOVA followed by Tukey-Kramer HSD test). The middle line of the box represents the median, the x in the box represents the mean. The bottom line and the top line of the box represent 1st and 3rd quartile, respectively. The whiskers extend from the ends of the box to the minimum value and maximum value.

E, Expression of PIL1, AT5G02580 and CDF5 in 3-d-old SD-grown seedlings at ZT18. Data from 3 independent experiments were averaged. Error bars indicate SEM. Different letters denote statistically significant differences based on Wilcoxon test (p<0.05).
TOC1 clock protein phosphorylation controls complex formation with NF-YB/C to repress hypocotyl growth (2021)
Figure 9

Figure 9. TOC1 and PRR5 anchor HDA15 and NF-YC9 to the target promoters.

A and B, ChIP-qPCR assay of HDA15's (A) and NF-YC9's (B) residence at target promoters in presence or absence of TOC1 and PRR5. GFP-HDA15 and GFP-NF-YC9 driven by 35S promoter were transiently expressed in protoplasts isolated from Col-0, toc1 single and prr5 toc1 double mutant grown in SD condition. Results were normalized by % input of PIL1 coding region (PIL1 CDS). Data from at least 3 independent experiments were averaged, error bars indicate SEM. Asterisks indicate significant differences (* p<0.05, ** p<0.001, *** p<0.0001, Student's t test).

C and D, Dual luciferase assay of the repression activity of GFP-TOC1 and GFP-5X on promoter of AT5G02580 (C) and CDF5 (D) in protoplasts of toc1 and hda15toc1 (h15t1) mutants. AT5G02580:LUC, CDF5:LUC reporters and the effectors (empty GFP, GFP-TOC1, and GFP-5X) were co-transfected to toc1 and hda15 toc1 protoplasts with 35S:Renilla-LUC as internal control. Data are means ± SEM (n = 5 biological trials) relative to empty GFP which is set as 1. Different letters indicate statistically significant differences (student's t test, p<0.05).
TOC1 clock protein phosphorylation controls complex formation with NF-YB/C to repress hypocotyl growth (2021)
Figure 10

Figure 10. NF-YB/C subunits and HDA15 physically interact with PRR5.

A, Quantification of interactions between TAP-PRR5 and NF-YC proteins in presence or absence of NF-YB subunits. Proteins were transiently expressed in N. benthamiana. TAP-PRR5 was immunoprecipitated with IgG resins followed by HRV-3C protease digestion. PRR5 and NF-YB/C were detected by α-MYC and α-HA, respectively. Protein band intensity was measured by ImageJ and interactions were exhibited as ratio of co-immunoprecipitated NF-YC to immunoprecipitated PRR5 to NF-YC input. Results from 3 independent trials were averaged. Error bars indicate SEM, different letters denote significant differences (p<0.05, student's t test).

B, Co-IP assay of HDA15-TAP and GFP-PRR5 in N. benthamiana. HDA15-TAP was immunoprecipitated with IgG resins and digested by HRV-3C protease. HDA15 and PRR5 were detected by α-MYC and α-GFP, respectively. Results represent two independent trials.

C, ChIP-qPCR assay of native promoter TOC1-GFP and 5X-GFP binding to the target promoters at ZT14 in toc1 vs. prr5 toc1 (p5t1) mutant. 3-d-old SD-grown seedlings were harvested at ZT14, TOC1 and 5X were immunoprecipitated by α-GFP. Data from 3 independent trials were averaged, error bars indicate SEM. Asterisks indicate significant differences (Student's t test; * p<0.05), n.s= not significant.

D, Expression of PIL1, AT5G02580 and CDF5 in 3-d-old SD-grown seedlings at ZT18. Data from 3 biological trials were averaged. Error bars indicate SEM. Different letters denote statistically significant differences based on Wilcoxon test (p<0.05).
Mutations in pregnancy-associated plasma protein A2 cause short stature due to low IGF-I availability (2016)
Figure 3

Figure 3: In vitro characterization of mutated PAPP-A2

• A Assessment of proteolytic activity present in culture media of human embryonic kidney 293T cells transfected with cDNA encoding PAPP-A2, PAPP-A2(D643fs) (family 1), or PAPP-A2(A1033V) (family 2). Radiolabeled substrates tested include IGFBP-3, IGFBP-4, and IGFBP-5. Cleavage was visualized by autoradiography following SDS-PAGE. Positions of intact IGFBPs (i) and cleavage products (c) are indicated.

• B Western blots following nonreducing or reducing SDS-PAGE of media from transfected cells, as indicated. Polyclonal antibodies raised against the N-terminal portion of PAPP-A2 were used for detection.

•C Comparison of proteolytic activity similar to the experiment of panel A, except that equimolar concentrations of PAPP-A2 and PAPP-A2 (A1033V) were used. The variant from Family 1, carrying a frameshift mutation N-terminal to the proteolytic domain of PAPP-A2, did not show any detectable expression. All gels and blots are representative of three independent experiments.
Mutations in pregnancy-associated plasma protein A2 cause short stature due to low IGF-I availability (2016)
Figure 4

Figure 4: IGF-I Neutral Chromatography and Proposed Mechanism of Action of PAPP-A2 Deficiency

• A Concentrations of endogenous insulin-like growth factor (IGF)-I in the different fractions obtained by neutral chromatography on a Superdex 200 column. Serum samples from the four siblings of family 1 were analyzed and compared to a non-related prepubertal control, as all siblings were prepubertal. The amount of IGF-I detected in the fractions corresponding to the ternary complex (TC) was markedly greater in the homozygous affected subjects (II·1 and II·3) compared to the unaffected siblings (II.2 and II.4) and the prepubertal control. No IGF-I was detected in the fractions corresponding to the binary complex (BC) or free IGF-I in any of the subjects. This is most likely due to the fact that under physiological conditions 80-85% of IGF-I circulates in the TC and after the separation procedure, the amount of IGF-I in the BC or free was below the level of detection of our IGF-I assay. These results clearly demonstrate that in the affected patients the amount of IGF-I circulating in the TC is markedly elevated.

•B Cartoon of hypothesized mechanism underlying growth failure due to lack of PAPP-A2. There is an increase in the formation of ternary complexes, due to decreased proteolysis subsequent to the lack of PAPP-A2 activity. This results in reduced concentrations of free IGF-I (fIGF-I) in serum and most likely at specific target tissues. This also decreases the negative feedback effect of IGF-I on growth hormone (GH) production, contributing to the increased circulating concentrations of GH. Increased serum GH concentrations cause increased IGF-I, IGFBP-3 and ALS concentrations. This increase in IGFBP-3 and ALS would then contribute to further ternary complex formation and an increase in total IGF-I and IGF-II concentrations.
In situ imaging of the bacterial flagellar motor disassembly and assembly processes (2019)
Figure 3

Figure 3: Flagellar sub-complexes in P. aeruginosa

(A-C) Slices through electron cryo-tomograms showing fully-assembled motors without the hook and filament. The dashed-yellow circles indicate the IM sub-complex.

D) Schematic representation of the P. aeruginosa motors lacking the hook and filament shown in (A-C).

E-G) Slices through electron cryo-tomograms showing fully-assembled motors with the hook and lacking the filament.

H) Schematic representation of the motors with the hook shown in (E-G).

I-K) Slices through electron cryo-tomograms of intact P. aeruginosa ∆flgI cells showing the presence of the inner-membrane sub-complex with the rod.

L) Schematic representation of the inner-membrane sub-complex with the rod shown in (I-K).

M-O) Slices through electron cryo-tomogram of lysed P. aeruginosa ∆flgI cells showing the presence of the sub-complex constituting the MS-ring and the rod.

P) Schematic representation of the sub-complex described in (M-O).

Q) A slice through an electron cryo-tomogram of a P. aeruginosa ∆flgG cell highlighting the presence of the inner-membrane sub-complex.

R) Schematic representation of the inner-membrane sub-complex shown in (Q).

Data information: All scale bars 25 nm.
In situ imaging of the bacterial flagellar motor disassembly and assembly processes (2019)
Figure 4

Figure 4: Flagellar sub-complexes in S. oneidensis wild type and mutant cells.

(A-C) Slices through electron cryo-tomograms of wild type cells showing fully-assembled motors without the hook and filament.

D) Schematic representation of the motors lacking the hook and filament shown in (A-C).

E) Sub-tomogram average of the motors of fully-assembled flagella.

F) Slice through an electron cryo-tomogram of a ΔflgH cell showing an IM sub-complex, indicated by the dashed-yellow circle.

G) Schematic representation of the IM sub-complex shown in (F).

H, I) Slices through electron cryo-tomograms of ΔflgH cells showing the IM sub-complex with the rod and the P-ring, indicated by the dashed-yellow circles.

J) Schematic representation of the structures shown in (H and I).

K-M) Slices through electron cryo-tomograms of ΔflaA/B cells highlighting the flagellar motor and the hook (without the filament).

N) Sub-tomogram average of the flagellar motor and the hook structure found in ΔflaA/B cells.

O) Schematic representation of the sub-tomogram average shown in (N).

P-R) Slices through electron cryo-tomograms of ΔflaA/B cells illustrating a disassembly product constituting the PL sub-complex, the rod and the hook.

S) Sub-tomogram average of the disassembly complex shown in (P-R). The dashed-orange arrow indicates the absence of the IM sub-complex in this structure.

T) Schematic representation of the disassembly product shown in (P-S).

U-W) Slices through electron cryo-tomograms of ΔflaA/B cells highlighting PL sub-complexes (dashed-yellow circles).

X) Sub-tomogram average of the PL sub-complexes in ΔflaA/B cells.

Y) Schematic representation of the sub-tomogram average shown in (X).

Data information: Scale bars: (red) 25 nm, (orange and white) 20 nm.
Metabolic remodeling maintains a reducing environment for rapid activation of replication checkpoint (2021)
Figure 1

Figure 1. Replication stress induces the pentose phosphate pathway through Snf1 and Mig1.

(A) The relative fold change of carbon metabolites with or without HU treatment for 60 min. Error bars represent standard deviations (SD) from six independent experiments.

(B) The diagram of carbon metabolism rewiring focused on glycolysis and PPP (pentose phosphate pathway).

(C) The relative ZWF1 mRNA levels were measured by qPCR. Cells were grown into an exponential phase and treated by 200 mM HU for 0, 30, 60 min. mRNA were prepared just as described in Methods and Materials. The relative mRNA levels of ZWF1 to ACTIN1 were determined by qPCR analyses. The value of the untreated WT sample was normalized to 1. Error bars represent standard deviations (SD) from at least three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01< P-values <0.05).

(D, E) The relative levels of endogenous Zwf1 protein with a GFP tag were analyzed by immunoblots in WT, snf1Δ or snf1Δmig1Δ cells (D). Protein extracts were probed with an anti-GFP antibody. Mcm2 was applied as a loading control. Three independent experiments were carried out and the relative fold changes were measured by Image J (E). Error bars represent standard deviations (SD) from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01< P-values <0.05; **0.001< P-values <0.01).