EMBO/soda-nel huggingface dataset:
Regulated splicing of large exons is linked to phase-separation of vertebrate transcription factors (2021)
Figure 3

Figure 3. Interactome analysis for SRSF3 and hnRNP K

Schematic diagram of the identification of protein complexes associated with SRSF3 and hnRNP K. Nuclei were extracted from HEK 293 cells overexpressing FLAG-tagged SRSF3 (3XFLAG-SRSF3-IP) or FLAG-tagged hnRNP K (3XFLAG-HNRNPK-IP), followed by isolation of the high molecular weight (HMW) fraction (Damianov et al., 2016). After mild treatment with MNase to release proteins from chromatin, immunoprecipitation (IP) was performed using anti-FLAG antibody and mass spectrometry analysis was performed. Naïve cells not expressing a FLAG-tagged protein were used for control IP (Cont-IP).

Volcano plot showing fold changes versus P-values of the normalized MASCOT scores (see Materials and Methods) of identified proteins between 3XFLAG-SRSF3-IP and Cont-IP. Each experiment was triplicated. The members of the representative families of splicing factors are highlighted by colors (right upper box).

Volcano plot showing fold changes versus P-values of the normalized MASCOT scores of identified proteins between 3XFLAG-HNRNPK-IP and Cont-IP. Each experiment was triplicated. The members of the representative families of splicing factors are highlighted by colors (right upper box).

Bubble plot of MASCOT scores of representative proteins identified in 3XFLAG-SRSF3-IP (red) and 3XFLAG-HNRNPK-IP (green). P-values by paired t-test are indicated by bubble sizes.
Regulated splicing of large exons is linked to phase-separation of vertebrate transcription factors (2021)
Figure 4

Figure 4. The SRSF3-hnRNP K axis regulates global selection of polyadenylation sites

Scatter plots of percent distal polyA site usage indices (PDUIs) in control cells (siCont), Srsf3-silenced cells (siSrsf3; left panel), and both Srsf3- and Hnrnpk-silenced cells (siSrsf3 + siHnrnpk; right panel). PDUIs of all 3′ UTRs are plotted. The 3′ UTRs shortened and extended by siSrsf3 exceeding the thresholds of |ΔPDUI| > 0.2 and p < 0.01 are indicated in red and blue, respectively.

3′ UTRs that were shortened by Srsf3 silencing were first selected. PDUIs of these 3′ UTRs in C2C12 cells treated with control siRNA (C), Srsf3 siRNA (S), Hnrnpk siRNA (K), or both Srsf3 and Hnrnpk siRNAs (S+K) are plotted. ****p < 0.0001 by Steel-Dwass test. The box plot shows the interquartile range (boxes), the median (central band) and the minimum and maximum except for the outliers at the ends of whiskers (n = 965).

mRNA expression levels of seven CPSF factors in C2C12 cells treated with the indicated siRNA normalized for those treated with the control siRNA. **p < 0.01 by CuffDiff (Trapnell et all., 2010).

Western blots showing protein expression levels of CPSF6 and β-actin (ACTB). Whole cell lysates were harvested 42 h after siRNA transfection.

Distributions of interactions between RNA and CPSF6 (red), hnRNP K (black), or SRSF3 (blue) around the distal polyA sites that are downregulated in Srsf3-silenced cells. The standard error of the average density of tRIP reads is shown as a semi-transparent shade around the average curve.
Regulated splicing of large exons is linked to phase-separation of vertebrate transcription factors (2021)
Figure 6

Figure 6. Srsf3 silencing disrupts transcription assemblies

Representative transcriptional factor genes containing the SRSF3-dependent exons. Edges show protein-protein interaction (PPI) as annotated within the STRING database (Szklarczyk et al., 2019). Solid lines and dotted lines indicate PPIs within a cluster and over a cluster, respectively. Genes are clustered by the Markov cluster algorithm.

SRSF3-responsive alternative 3′ ends of the Med1 gene. (Top panel) Disorder analysis for MED1 using PONDR VL-XT (deep blue) and IUPred2 (light blue). The scores in the ordinate indicate disordered tendencies between 0 and 1 (a score of more than 0.5 indicates disordered). (Middle panel) Protein and gene structures of MED1. The protein structure was segmented into individual exonic regions. The SRSF3-responsive last exon is indicated in pink. (Bottom panel) Sashimi plots of RNA-seqs from Srsf3- and/or Hnrnpk-silenced cells.

Immunofluorescence images of MED1 in C2C12 cells treated with siCont (C), siSrsf3 (S), siHnrnpk (K), or siSrsf3+siHnrnpk (S+K). A white dotted contour outlines the nucleus. Box plots (right) show quantification of nuclear (NUC) and cytoplasmic (CYT) localization of MED1. The average intensity of one nucleus or a cytoplasmic segment is individually plotted.

The intrinsically disordered region (IDR) in MED4. Disorder analysis, protein and gene structures, and Sashimi plots are shown in (B). Splicing of these genes was not affected by the siRNA treatment.

Immunofluorescence images of MED4 in C2C12 cells treated with the indicated siRNAs. Box plots show quantification of nuclear localization of MED4. Each dot represents one nucleus.

Immunofluorescence images of MED1 and MED4 in C2C12 cells The arrows denote representative puncta, including both MED1 and MED4.

The number of MED1 puncta (left) and MED4 puncta (right) per nucleus in cells treated with the indicated siRNA.

MED1 signals on MED4 puncta normalized to nuclear MED1 signals. MED1 signal intensity on each MED4 punctum was individually plotted.

Data information: *p < 0.01, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Steel-Dwass test. In (C, D, G, and H), box plots show the interquartile range (boxes), the median (central band) and the minimum and maximum except for the outliers at the ends of whiskers. More than 50 nuclei in more than five randomly selected visual fields of at least two independent immunostainings were analyzed in each experiment.
Re-equilibration of imbalanced NAD metabolism ameliorates the impact of telomere dysfunction (2020)
Figure 2

Figure 2. CD38 depletion ameliorates dysregulated NAD metabolism in DC cells.

Quantitative RT-PCR and immunoblots of the expression of CD38 and SARM1 in DC and age-matched health control fibroblasts. All values are presented as mean ± SD of three replicates in quantitative RT-PCR. Protein levels are normalized to GAPDH, and mean (± SD) quantification values of 4 controls vs 5 DC samples are shown. Student's t test was performed on DC cells vs controls. Irrelevant intervening lanes have been removed for clarity (full blots are available online as Source Data).

Immunoblots and quantification of indicated proteins in DC fibroblasts treated with DMSO and with 2 μM ATM inhibitor, KU-55933 for 48 hours. Mean (± SD) quantification values of three DC lines with and without ATMi treatment are shown. P values on the basis of Student's t test.

The efficacy of CD38 shRNA (sh-1 and sh-2) was verified by quantitative RT-PCR in DC fibroblasts and by RT-PCR and immunoblots in A549 cells. The relative CD38 mRNA values in the CD38 knockdown cells were normalized to the scrambled shRNA. All values are presented as mean ± SD of three replicates. Student's t test was performed on DC and A549 cells in indicated conditions.

D, E Intracellular NAD levels and immunoblots of the expression of indicated proteins in the scramble and CD38 knockdown DC fibroblasts. The CD38 knockdown DC fibroblasts were treated with the PARP1 inhibitor Olaparib (400nM) and the SIRT1 inhibitor EX 527 (1 μM) for 24 hours. Data are representative four replicates. All values are presented as mean ± SD of four replicates. One-way ANOVA was performed on DC cells in indicated conditions.
Re-equilibration of imbalanced NAD metabolism ameliorates the impact of telomere dysfunction (2020)
Figure 3

Figure 3. G3 Tert-/- mice display defects in the NAD metabolism.

A, B Intracellular NAD levels and immunoblots of the expression of NAD consuming related proteins and their activities in brain tissues from 6-month- old G1 and G3 Tert-/- female mice. The CD38 antibody was validated using the CD38-/- mouse brain tissues.

C Quantification of immunoblots from the indicated proteins in B.

D NADase activity in G1 and G3 Tert-/- mouse brain lysate was determined at various time points during the reaction. NADase activity was not detectable in the extracts treated with the CD38 inhibitor, 78c, at a concentration of 50nM.

Data information: All values are presented as mean ± SD of G1 Tert-/- mouse brain tissues versus G3 Tert-/- mouse brain tissues. n=4 in each group. P values on the basis of Student's t test.
Re-equilibration of imbalanced NAD metabolism ameliorates the impact of telomere dysfunction (2020)
Figure 4

Figure 4. NAD intervention impacts mitochondrial parameters of DC fibroblasts.

A Representative immunoblots of the expression of PGC1-1α in DC cells treated with vehicle or 3 mM NR. Quantification of the indicated proteins is from three immunoblots.

B-D Cellular and mitochondrial ROS in DC and age-matched healthy control cells supplemented with vehicle and NR (B and C) and in the scramble and CD38 knockdown DC cells (D) were measured by flow cytometry from three replicates.

E Representative images captured by transmission electron microscopy of DC1 cells treated with vehicle or NR. White arrows: mitochondria. Yellow arrow: autophagosome-like structures with engulfed mitochondria. Enlarged image within white frame is shown.

F-H Quantifications of percentage of damaged mitochondria per cell (F), mitochondrial length (G), and mitochondrial diameter (H). A minimum of 200 mitochondria counted per group.

Data information: All data are represented as mean ± SD. Student's t test was performed for individual pairs in indicated conditions. Con: control.
Re-equilibration of imbalanced NAD metabolism ameliorates the impact of telomere dysfunction (2020)
Figure 5

Figure 5. Impaired mitophagy process in DC fibroblasts is improved by NR supplementation.

Representative images of mitophagy showing co-localization between the mitophagy dye (red) and the lysosome dye (green) in DC and control fibroblasts treated with vehicle or 3 mM NR. White arrows: mitophagy.

B The mean value of fluorescence intensity/cells in each image was scored. At least 15 images (~ 200 cells) were counted per group. The relative values in each group was normalized to Con1.

C Representative immunoblots of the expression of PINK1 and PARKIN in DC cells treated with vehicle or 3 mM NR. Quantification of immunoblots from the indicated proteins is from three replicates.

Data information: All values are represented as mean ± SD. One-way ANOVA (B) and Student's t test (C) were performed for each individual pair. Con: age-matched healthy controls.
Re-equilibration of imbalanced NAD metabolism ameliorates the impact of telomere dysfunction (2020)
Figure 6

Figure 6. NR supplementation reduces telomeric oxidative DNA lesions and TIF formation in DC cells.

A PCR amplification efficiency at the telomere in the mock- and FPG-treated DC and age-matched healthy control fibroblasts supplemented with vehicle or 3 mM NR. All values are presented as mean ± SD of three replicates. The relative telomere PCR amplification in each sample was normalized to the 36B4 reference gene.

B Telomere restriction fragment analysis in DC and control fibroblasts treated with vehicle or 3 mM NR. Genomic DNA was isolated from the indicated cell lines at 0 d or 14 d, with or without 3 mM NR treatment. At left, DNA molecular mass markers, in kilobase pairs (Kb).

C, D Representative images of γH2AX (green), telomere immuno-FISH (red) in DC cells treated with vehicle or 3 mM NR. White arrows: co-localization of γH2AX foci and telomeric DNA (or TIF). Enlarged views of co-localizing foci are shown at the right panel and in Figure EV1. Percentage of DC and control cells with indicated TIFs per nuclei. ~ 100 cells per condition were scored.

Data information: All values are represented as mean ± SD. P values were calculated using One-way ANOVA (A and D). Con: age-matched healthy controls.
Re-equilibration of imbalanced NAD metabolism ameliorates the impact of telomere dysfunction (2020)
Figure 7

Figure 7. NAD intervention improves the proliferative capacity and suppresses cellular senescence and SASP in DC fibroblasts.

A Cumulative population doubling analysis of the proliferation of representative scramble and CD38 knockdown DC fibroblasts or DC fibroblasts treated with vehicle or 3 mM NR. Each data point is represented as mean ± SD of three replicates.

B, C Representative images of BrdU (red) and DAPI (blue) staining of DC and age-matched healthy control fibroblasts treated with vehicle or NR. Percentage of BrdU -positive cells per condition. Each dot represents the percentage of cells with BrdU staining per image. ~400 cells were counted per condition. All values are presented as mean ± SD.

D, E Representative images of SPiDER-β-gal (green) and DAPI (blue) staining of DC and age-matched healthy control cells treated with vehicle or 3 mM NR. Percentage of SA-β-gal-positive cells per condition. Each dot represents the percentage of cells with SA-β-gal staining per image. ~400 cells were counted per condition. All values are presented as mean ± SD.

F The levels of IL-6, IL-8, and MCP-1 in both supernatant and cell lysate of indicated DC fibroblasts treated with vehicle or 3 mM NR. All values are represented as mean ± SD of three replicates.

G, H Representative immunoblots of p21 and p16 in DC fibroblasts treated with vehicle or 3 mM NR. Quantification of the indicated proteins are presented as mean ± SD from three immunoblots.

Data information: P values were calculated using one-way ANOVA for multiple comparisons (C) and Student's t test between two groups (C, E, F and H). Con: control.
Regulation of protein quality control by UBE4B and LSD1 through p53-mediated transcription. (2015)
Fig 1

Identification and characterization of a robust suppressor that ameliorates the locomotion defects in the <italic>C</italic>. <italic>elegans</italic> model of SOD1-associated ALS.

(A) Workflow of the suppressor screen identifying mutant C. elegans (red) with saliently improved movement. (B) Locomotor behavior, measured by thrashing rates in liquid medium, in the C. elegans strains with neuronal expression of human WT SOD1 or ALS-linked mutant SOD1G85R, in the presence (M1/M1) or absence (+/+) of the suppressor mutation (n = 16). (C) Northern (top panel) and western (middle, bottom panels) blot analyses of total RNA and protein from SOD1G85R strains, with (M1/M1) or without (+/+) suppressor mutations, demonstrating that the levels of SOD1G85R mRNA and total protein are not changed by the suppressor mutation (M1/M1). The western blot lanes are from the same gel and exposure. (D) Sequence analysis of the M1 strain, revealing that independent mutations in two genes, a lysine-specific demethylase, spr-5 (R646Q), and a ubiquitin ligase, ufd-2 (W824X), are required for the full suppressor phenotype. (E) C. elegans SPR-5 and UFD-2 and their mammalian homologs lysine-specific demethylase 1 (LSD1) and ubiquitination factor E4 B (UBE4B) share all major protein domains. These include the Swi3-Rsc8-Moira (SWIRM), amine oxidase-like (AOL), and TOWER domains in LSD1/SPR-5 and the Ufd2 Core and U-box domains in UBE4B/UFD-2. Positions of the missense, nonsense, and deletion mutations in mutant C. elegans are indicated. (F) Locomotor behavior, measured by thrashing rates, of the C. elegans carrying the SOD1G85R transgene on the normal background (WT), with the null mutation of either ufd-2(tm1380) or spr-5(by134), the double mutation ufd-2(tm1380);spr-5(by134), or the M1 suppressor ufd-2(W824X);spr-5(R646Q) (n = 16). (G) Western blotting of the supernatant (S) and pellet (P) protein fractions from the C. elegans carrying the SOD1G85R transgene with the double mutation ufd-2(tm1380);spr-5(by134) compared with controls. While SOD1G85R protein levels are unchanged in the S fraction, those in the P fraction are decreased in the double suppressor mutant. The P fraction represents only about 1.7% of total SOD1G85R in the WT sample; therefore, a higher ratio of the P fraction relative to the S fraction (approximately 2:1) is used for the western analysis. (H) The overexpression (OE) of WT UFD-2 or SPR-5 in the C. elegans nervous system blocks the protection conferred by the double mutation ufd-2(tm1380);spr-5(by134) (n = 16). Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.
Regulation of protein quality control by UBE4B and LSD1 through p53-mediated transcription. (2015)
Fig 2

Suppression of neurodegeneration associated with diverse misfolded proteins in invertebrate models by <italic>ufd-2</italic> and <italic>spr-5</italic> loss-of-function mutations.

(A) Top: schematic drawing depicts pan-neuronal expression of YFP in head and ventral neurons in the context of the C. elegans body plan. Left: micrographs show the SOD1G85R-YFP proteins expressed in WT or the spr-5(by134);ufd-2(tm1380) mutant background. The double-mutant worms show a marked decrease in protein aggregation in neurons. Enlarged sections of head neurons (red framed) and ventral cord neurons (white framed) are shown. Middle: quantification of locomotion in the spr-5(by134);ufd-2(tm1380) and the WT C. elegans with neuronal expression of SOD1G85R-YFP (n = 30). Right: a decrease in the protein levels of SOD1G85R-YFP in the presence of spr-5(by134);ufd-2(tm1380) is shown by western blots of the supernatant (S) and the pellet (P) fractions. (B and C) Analyses of the spr-5(by134);ufd-2(tm1380) and the WT C. elegans with neuronal expression of TDP-43-c25-YFP (n = 30) or PolyQ-YFP (n = 12) as in (A). (D) Neurodegenerative rough-eye phenotype in adults is alleviated by the knockdown of the Drosophila homologs of ufd-2/UBE4B and spr-5/LSD1—CG9934 and Su(Var)3-3, respectively, compared to the control (CTRL). Eye-specific expression of TDP-43M337V, FUSR521C, and RNA interference (RNAi) was driven by GMR-Gal4. (E) Drosophila eye pigment quantitation. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.
Regulation of protein quality control by UBE4B and LSD1 through p53-mediated transcription. (2015)
Fig 3

UBE4B and LSD1 double-knockdown accelerates SOD1<sup>G85R</sup> protein degradation.

(A) Western blots of cell lysates derived from mock (CTRL), single UBE4B or LSD1, or double UBE4B and LSD1 knockdowns. Supernatant (S) and pellet (P) fractions were probed with indicated antibodies. While the LSD1 or UBE4B single-knockdown reduces the SOD1G85R aggregates in both supernatant and pellet fractions, the combined knockdown produces the strongest reduction in the aggregates. (B) Quantification of SOD1G85R protein levels by western blotting (A). n = 3 (supernatant); n = 8 (pellet). (C) Western blots of a representative cycloheximide chase experiment to determine SOD1 protein half-lives in the double UBE4B and LSD1 knockdown cells versus controls. (D) Quantification of SOD1G85R clearance, as analyzed by western blotting in (C). The graph indicates the relative band intensity of SOD1G85R at each chase time point. n = 5; Overall p = 0.02 (paired t test, CTRL versus UBE4B and LSD1 double knockdown). Individual p = 0.03 (3 h), p = 0.003 (6 h), p = 0.06 (9 h), and p = 0.004 (12–21 h). (E) The half-life of SOD1G85R is reduced from 8.5 h to 5 h upon knockdown of UBE4B and LSD1. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.
Regulation of protein quality control by UBE4B and LSD1 through p53-mediated transcription. (2015)
Fig 4

UBE4B and LSD1 knockdown activates transcription mediated by p53.

(A) Venn diagram of upstream activators (z-score ≥ 2) that are differentially activated in single—UBE4B or LSD1—knockdowns and double UBE4B and LSD1 knockdowns, compared with the control. Activation state of an upstream regulator is predicted from differential mRNA levels of its downstream target genes. (B) The volcano scatter plot indicates fold changes in the levels of gene transcripts affected differentially by the UBE4B and LSD1 double knockdown versus control shRNA. Gray spots represent 22,148 annotated transcripts. Red spots are predicted p53-activated targets, and blue spots are predicted p53-inhibited targets. The enrichment of red spots in the up-regulated genes (right upper quadrant, >1.2-fold change, p ≤ 0.04) and blue spots in the down-regulated genes (left upper quadrant) indicates that the p53-mediated transcription is activated by the UBE4B and LSD1 double knockdown. (C) RT-qPCR validation of the expression levels of representative p53 target genes in the UBE4B and LSD1 double-knockdown samples. n = (2 to 6), p ≤ 0.04. (D) The p53 protein level is significantly increased by UBE4B and LSD1 double knockdown. Left: representative western blots showing p53 levels, the knockdown of UBE4B and LSD1, and the actin control in HEK293T cells. Right: quantification of the p53 protein levels normalized against actin (n = 3). (E) The p53-mediated transcriptional activity is measured by a luciferase reporter under the control of a p53-response element promoter, which was transfected into HEK293T cells 72–96 h after the initiation of the UBE4B and LSD1 single or double knockdowns (n = 6). (F) Increased interaction of p53 with its activating partner 53BP1 in response to UBE4B and LSD1 knockdown. Left: representative western blots of 53BP1 co-immunoprecipitation. Right: quantification of the 53BP1 co-immunoprecipitation (n = 2). Data represent means ± SEM. (G) RT-qPCR validation of the expression levels of FOXO3a, FOXO4, and PSMD11 (n = 6). (H) The FOXO3a-mediated transcriptional activity was measured with a luciferase reporter under the control of a FOXO-response element promoter. HEK293T cells were transfected with shRNAs for control, LSD1, UBE4B, or both LSD1 and UBE4B, followed by transfection of the luciferase reporter together with a constitutively active form of FOXO3a (TM) 72–96 h later (n = 6). Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.
Regulation of protein quality control by UBE4B and LSD1 through p53-mediated transcription. (2015)
Fig 5

UBE4B and LSD1 double knockdown activates both proteasomes and autophagy.

(A) Increased protein levels of proteasome subunits upon the UBE4B and LSD1 double knockdown in HEK293T cells. (B) The UBE4B and LSD1 single or double knockdowns increase the proteasomal degradation of a reporter substrate, Suc-LLVY-Luciferin, whose degradation is measured in a luciferase release assay. The double knockdown shows synergistic proteasomal activation when compared with the individual knockdowns (n = 6). (C) The autophagy activity is significantly increased in cells with the UBE4B and LSD1 double knockdown. The in vivo activity of the ATG4B protease, which cleaves LC3 precursors, was quantified via a Gaussia luciferase release assay (see S5D Fig., S5E Fig., and Materials and Methods for details). The HCT116 cells were analyzed 48 h after the initiation of the knockdown. (D) Quantification of LC3-II levels in HEK293T cells with the UBE4B and LSD1 double knockdown or the mock control. The cells were treated with or without 10 mM 3-methyladenine (3-MA) for 48h before lysis. Western blots indicate an increase in LC3 levels (top panels) in the double UBE4B and LSD1 knockdown cells (middle panels), while the actin control is unchanged (bottom panels). The graph shows the quantification of LC3-II levels as normalized to actin levels (n = 3). A one-way ANOVA test with matched multiple comparisons and Tukey correction was used for statistics. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.