EMBO/soda-nel huggingface dataset:
Arginine methylation of the DDX5 helicase RGG/RG motif by PRMT5 regulates resolution of RNA/DNA hybrids (2019)
Figure 5

Figure 5. The RGG/RG motif of DDX5 is a substrate for PRMT5 and is required for R-loop resolution in vivo.

(A) U2OS cells were transfected with a Flag-DDX5 expression vector in the presence of siCTL or siPRMT5. Lysates were immunoprecipitated with IgG or anti-Flag antibodies as indicated. The WCE and the bound proteins were Western blotted with anti-symmetrical dimethylarginine antibody or anti-Flag antibodies.

(B) A schematic of DDX5 helicase domain and RGG/RG motifs is shown.

(C) GST-fusion of DDX5 fragments (F1-F4) and the RK mutant used in this study. RK (*) indicates R to K substitutions of 5 arginines (R478, R482, R484, R486, R502).

(D and E) Coomassie blue (left panel) and in vitro methylation assay (right panel) of indicated GST-DDX5 fragments and the GST-DDX5-RK mutant.

(F) Immunofluorescence analysis with S9.6 and anti-Flag antibodies of U2OS cells transfected with Flag-tagged DDX5, DDX5-RK or DDX5-XD (helicase dead). Nuclear S9.6 signal was counted in both Flag-negative and Flag-positive cells. The Flag-negative cells were considered as untransfected cells. The graphs shown represent the quantification with the S.E.M from 3 independent experiments. Statistical significance was assessed using one-way ANOVA t-test. ****, p < 0.0001.

(G) Immunofluorescence analysis with S9.6 and anti-Flag antibodies of U2OS cells transfected with siCTL or siDDX5-1 and Flag-tagged DDX5 (+WT) or DDX5-RK (+RK) as indicated. The graphs show the average and S.E.M. from three independent experiments. Statistical significance was assessed using one-way ANOVA t-test. ****, p < 0.0001.

(H) HEK293 cells were transfected with siCTL, or siDDX5. The next day, the cells that received siDDX5 were subsequently transfected with empty pcDNA3 vector (-), expression vectors encoding Flag-DDX5 (WT), or Flag-DDX5-RK (RK). Forty eight hours later, all the cells were subjected to DRIP-qPCR analysis. The average with the S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and n.s.: not significant.
Arginine methylation of the DDX5 helicase RGG/RG motif by PRMT5 regulates resolution of RNA/DNA hybrids (2019)
Figure 6

Figure 6. DDX5 physically associates with the 5'-3' exonuclease XRN2, functioning together to repress R-loops.

(A) HEK293 cells were transfected with empty pcDNA3 vector (-) or Flag-DDX5 (+). Whole cell lysates (WCE) and anti-Flag immunoprecipitations (IP) were immunoblotted with anti-Flag and anti-XRN2 antibodies.

(B) Untransfected U2OS cells were lyzed and subjected to immunoprecipitation with control immunoglobulin G (IgG) or anti-XRN2 antibodies. The bound proteins were separated by SDS-PAGE followed by Western blotting with anti-DDX5 or anti-XRN2 antibodies. The migration of DDX5 and XRN2 is depicted with an arrow.

(C) U2OS cells were transfected with empty pcDNA3 vector (pcDNA3) or expression vectors encoding Flag-DDX5 WT (1-614) or truncated Flag-DDX5 proteins (60-614; 1-554; 1-435). WCE of the transfected cells (left panel) were Western blotted with anti-Flag and anti-XRN2 antibodies to confirm equivalent expression. The transfected cells were lysed and anti-Flag immunoprecipitations (right panel) performed for the presence of co-immunoprecipitating endogenous XRN2 by Western blotting with anti-XRN2 antibodies. M denotes molecular mass markers in kDa.

(D) Untransfected and U2OS stably expressing Flag-DDX5 WT or Flag-DDX5-RK were subjected to immunoprecipitation with anti-Flag antibody. The WCE and the anti-Flag immunoprecipitated proteins were Western blotted with anti-monomethylarginine (MeR), anti-Flag and anti-XRN2 antibodies, respectively. M denotes molecular mass markers in kDa.

(E) U2OS cells transfected with siCTL, siDDX5, or siXRN2 were subjected to DRIP-qPCR analysis with anti-IgG, and anti-S9.6 antibodies with or without RNaseH treatment. The average and S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01.
Arginine methylation of the DDX5 helicase RGG/RG motif by PRMT5 regulates resolution of RNA/DNA hybrids (2019)
Figure 7

Figure 7. Depletion of DDX5 or XRN2 causes enrichment of R-loops and RNA Pol II at the transcriptional termination pause site.

HEK293 cells were transfected with siCTL, siDDX5, or siXRN2. The gene location and qPCR amplification region are shown at the top of each panel. The identified R-loop peaks were extracted from R-loop database (R-loop DB) for each gene region. R-loop: DRIP denotes anti-S9.6 DRIP-qPCR and Pol II ChIP denotes RNA pol II ChIP. The bar graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001. B, E, H, S and X denote the location of the BsrGI, EcoRI, HindIII, SspI, and XbaI.
Arginine methylation of the DDX5 helicase RGG/RG motif by PRMT5 regulates resolution of RNA/DNA hybrids (2019)
Figure 8

Figure 8. DDX5 cooperates for XRN2 recruitment and facilitates the release of RNA Pol II from β-actin transcriptional termination pause site.

(A) Schematic representation of the β-actin locus and the location of the genomic PCR primers used for ChIP-qPCR and DRIP-qPCR. Prom (promoter), RB (region B), poly(A) and RD (region D) indicate the regions analyzed by qPCR.

(B) HEK293 cells were transfected with siCTL or siDDX5 and XRN2 ChIP assays were performed. The XRN2 ChIP signal was normalized to the Pol II ChIP signal. WCE were immunoblotted with anti-DDX5 and α-tubulin antibodies, the latter was used as a loading control. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01.

(C) HEK293 cells were transfected with siCTL, siDDX5 or siXRN2. The cells were subjected to Western blotting, RNA Pol II ChIP and DRIP analysis of β-actin gene. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001.

(D) HEK293 cells were transfected with siCTL or siDDX5. The next day, the cells that received siDDX5 were subsequently transfected with empty pcDNA3 vector (-), expression vectors encoding Flag-DDX5 (WT), or Flag-DDX5-RK (RK). The cells were subjected to Western blotting to visualize Flag-DDX WT and RK expression. Pol II ChIP and DRIP analysis of β-actin gene were performed. For the ChIP analysis, the Y-axis shows the signal-to-noise ratio of RNA Pol II IP relative to control IgG IP. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001.

(E) HEK293 cells were transfected with siCTL or siPRMT5. The cells were subjected to Western blotting, RNA Pol II ChIP and DRIP analysis of β-actin gene. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01.

(F) HEK293 cells were transfected with empty plasmid (-), wild type Flag-DDX5 (WT) or its RK mutant (RK) and subjected to western blot and Flag-DDX5 ChIP-qPCR analysis of β-actin gene. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001; n.s. not significant.

9
A multifunctional locus controls motor neuron differentiation through short and long non coding RNAs (2022)
Figure 1

Figure 1. LncMN2 KO affects MN differentiation.

A. Block-and-line model of lncMN2 gene structure. Exons specific for lncMN2-202/204 isoforms are in yellow, lncMN2-203 specific exon is in blue. Shared exons are gray. MiR-325 and miR-384 are represented as red and green blocks, respectively.

B. qRT-PCR expression analysis of lncMN2 isoforms (left panel) and embedded miRNAs (right panel) in mESC and EBs differentiated for 6 days (EB6). Data, that are expressed as mean (error bars represent SD), were normalized over Atp50 mRNA (left) and snoRNA U25 (right) levels and reported as RNA fold over the control (mESC), set as 1. N=3 biological replicates.

C. qRT-PCR expression analysis of lncMN2-202/203/204 isoforms (left panel), miR-325-3p (middle panel) and miR-384-5p (right panel) in WT, KO1 and KO2 EB6. Details as in B.

D. Cytofluorimetric analysis of cells from dissociated EB6. The left diagram reports the distribution of cells (percentage of the peak) according to GFP levels, from WT (gray), KO1 (orange) and KO2 (blue) EB. In the histogram aside, the percentage of GFP+ cells in WT, KO1 and KO2 is reported. Data are expressed as mean and error bars represent SD. N=3 biological replicates.

E. Left: representative immunofluorescence of the MN marker Isl1 (red signals) in cells maintained in vitro for 14 days after EB6 dissociation (DIV14). DAPI counterstaining in blue. Scale bar=100 mm.. Right: box plot shows the distribution of Isl1+ nuclei with respect to the total cell number marked with DAPI staining in WT and in lncMN2 KO mutants (clone 1 and clone 2). Data are expressed as mean and error bars represent SD. The central band represents median and the whiskers represent the distribution that goes from the minimum value to the lower quartile and from the upper quartile to the maximum value. N=3 biological replicates and 10 fields were analyzed for each replicate. Scale bar=100 mm.

Data information: histogram Histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).
A multifunctional locus controls motor neuron differentiation through short and long non coding RNAs (2022)
Figure 3

Figure 3. LncMN2 ∆SP mutation impairs MN production.

A. Block-and-line model of the lncMN2 edited locus. Details as in Figures 1A and 2A. Predicted miRNA binding sites deletion is represented as ∆SP.

B. qRT-PCR expression analysis of lncMN2-203 isoform, lncMN2-202/204, miR-325-3p and miR-384-5p embedded miRNAs in WT, ∆SP1 and ∆SP2 EB6. Data, that are expressed as mean (error bars represent SD), are normalized over Atp5o mRNA levels (for lncRNA isoform quantification) or over snoRNA U25 levels (for miRNA quantification) and are reported as RNA fold over the control (mESC), set as 1. N=3 biological replicates.

C. Cytofluorimetric analysis of cells from dissociated EB6. The right diagram reports the cell distribution (percentage of the peak) according to GFP levels, from WT (gray line), ∆SP1 (purple line) and ∆SP2 (blue line) EB6. In the histogram aside, the percentage of GFP+ cells in WT, ∆SP1 and ∆SP2 is reported. Data are expressed as mean and error bars represent SD. N=3 biological replicates.

D. Left: representative immunofluorescence of the MN marker Isl1 (red signals) in cells maintained in vitro for 14 days after EB6 dissociation (DIV14). DAPI counterstaining in blue. Scale bar=100 mm. Right: box plot shows the distribution of Isl1+ nuclei with respect to the total cell number marked with DAPI staining in WT and in ∆SP mutants (clone 1 and clone 2). The central band represents median and the whiskers represent the distribution that goes from the minimum value to the lower quartile and than from the upper quartile to the maximum value. N=3 biological replicates and 10 fields were analyzed for each replicate.

Data are expressed as mean and error bars represent SD. Scale bar=100 mm. Data information: histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).
The C-degron pathway eliminates mislocalized proteins and products of deubiquitinating enzymes (2020)
Figure 3

Figure 3. The subcellular distribution of a protein dictates its accessibility to the C-degron pathway.

(A) The relative frequency of Gly in the final 50 residues of functional human proteins with different subcellular localizations. The number on each graph denotes the left-tailed p-value of the -1 residue. CT-inside and CT-outside represent membrane proteins with their C-termini facing the cytosol or extracellular space, respectively.

(B) Comparison of stabilities between wild-type and diGly degron-tagged membrane proteins. The C-terminal topology of tested proteins is indicated at top.

(C) GPS reporter cells expressing indicated membrane proteins with a C-terminal diGly degron were treated with or without KLHDC2 overexpression or knockdown and then analyzed.

(D) Stability analysis of C-terminal diGly degron-tagged membrane proteins with or without KLHDC2 overexpression and CB-5083 treatment (to inhibit p97/VCP).

(E) Abundance analysis of GFP-diGly degron fusion proteins with or without N-terminal tagging with the MTSs of PDHA1, COX8A or GLUL, and with (+) or without (-) blockage of C-degron pathways by dominant-negative Cul2 (DNCul2).

(F) Abundance comparison of MTS-GFP-degron fusion proteins in cells treated with shRNAs against various BC-box proteins. The MTS and C-degrons used are labeled at top and left, respectively. The MTS and C-degron sequences are provided in the Materials & Methods.

(G) Confocal images of U2OS cells expressing MTSGLUL-GFP proteins with or without a C-terminal diGly degron. MitoTracker staining shows mitochondria. Scale bar = 20 µm. DAPI staining and merged images are shown in Appendix Fig. S1.

(H) Confocal images of U2OS cells expressing MTSGLUL-GFP-diGly degron fusion proteins co-stained with MitoTracker, and with or without DNCul2 treatment. Scale bar = 20 µm.
The C-degron pathway eliminates mislocalized proteins and products of deubiquitinating enzymes (2020)
Figure 4

Figure 4. C-degron pathways clear mislocalized cellular proteins.

(A) The extreme C-terminal sequence of indicated secretory and mitochondrial proteins. The diGly, R__G and R_G_ motifs are labeled red.

(B) Stability analysis of indicated proteins in cells treated with shRNAs against various BC-box proteins. Since GFP was tagged at the protein N-terminus and signal peptide and MTSs need to be exposed to be functional, all tested proteins were mislocalized.

(C, D) Protein stability analyses of wild-type (WT) and mutant proteins lacking their extreme terminal Gly (∆G) or having it masked by adding Leu (+L) in cells with or without BC-box protein knockdown (C) or overexpression (D).

(E) Western blot analysis of Flag-tagged proteins in cells with or without KLHDC3 or APPBP2 expression. The N-terminal Flag abolished protein targeting. GAPDH serves as a loading control.

(F) Fractionation and Western blot analysis of wild-type and Flag-tagged PDP2 (top), and wild-type and MTS-deleted (∆MTS) MRPL28 (bottom). Flag-PDP2 and ∆MTS MRPL28 were mislocalized. W, C, and M denote whole cell lysates, cytosol fraction and mitochondrial fraction, respectively. Tubulin and VDAC serve as fractionation quality controls.

(G) Protein abundance analysis of HA-tagged wild-type or mutant secretory proteins with their signal peptide deleted (∆SP) in cells with or without KLHDC3 or APPBP2 expression. The HA epitope was inserted immediately after the signal peptide (SP), which had no impact on protein secretion.
The C-degron pathway eliminates mislocalized proteins and products of deubiquitinating enzymes (2020)
Figure 5

Figure 5. MIC19 is regulated by both N-degron and C-degron pathways.

(A) Schematic diagram of the MIC19 protein. MIC19 contains an N-terminal myristoylation motif followed by a DUF37 domain (domain of unknown function) and a CHCH (coiled-coil helix-coiled-coil helix) domain, and is terminated by a diGly motif. Myristic acid was co-translationally attached to the N-terminal Gly2 residue by N-myristoyltransferases (NMTs) after removal of the initiating Met by methionine aminopeptidases. The N-terminal sequence of MIC19 is indicated at bottom with the myristoylation site, Gly2, and residues crucial for myristoylation labeled turquoise or underlined, respectively.

(B) Live-cell images of U2OS cells stably expressing GFP or GFP N-terminally tagged with the wild-type or mutant N-terminal motif of MIC19 (aa. 1-24). Scale bar = 20 µm.

(C) Fractionation and Western blot analysis of endogenous MIC19 in U2OS cells with or without DNKLHDC2, DNCul2 or IMP-1088 treatments. IMP-1088 inhibits NMTs. C and M denote cytosol and mitochondrial fractions, respectively. Tubulin and VDAC serve as fractionation quality controls. The relative abundance of cytosolic MIC19/tubulin was normalized to that of lane 1 and is indicated under each lane in red. Another replicate of this experiment is shown in Fig. EV3B.

(D) Abundance comparison of GFP N- or C-terminal tagged with the wild-type or mutant N-terminal (aa. 1-24) or C-terminal (a.a. 218-227) motif of MIC19 in cells with indicated treatments. Data from sh#1 is shown here.

(E) Live-cell images of U2OS cells expressing indicated GFP fusion proteins with or without DNCul2 and IMP-1088 treatments. The complete set of images containing degron mutant controls is shown in Appendix Fig. S3. Scale bar = 20 µm.

(F) Representative confocal immunofluorescence images of HA-tagged MIC19 variants with MitoTracker staining in U2OS cells. The fluorescence intensities along the dashed lines are shown as line profile graphs on the right. Scale bar = 20 µm.
The C-degron pathway eliminates mislocalized proteins and products of deubiquitinating enzymes (2020)
Figure 6

Figure 6. Mitochondrial localization of MIC19 is ensured by dual-end inspections.

(A) Representative confocal immunofluorescence images of HA-tagged MIC19 variants with MitoTracker staining in U2OS cells. All MIC19 variants examined carried the S6W,T7P mutation. The fluorescence intensities along the dashed lines are shown as line profile graphs on the right . Scale bar = 20 µm.

(B) Fractionation analysis of wild-type HA-MIC19 in U2OS cells treated with IMP-1088 and with or without inhibition of ZYG11B or KLHDC2. The normalized relative abundance of cytosolic HA-MIC19/tubulin is indicated under each lane in red.

(C) MitoTracker staining of IMP-1088-treated U2OS cells with or without inhibition of ZYG11B or KLHDC2. The complete set of images containing MIC19 localizations is shown in Fig. EV4C. Scale bar = 20 µm.

(D) Proposed model. CRL2-operated "double-end" inspections safeguard the mitochondrial localization of MIC19.
The C-degron pathway eliminates mislocalized proteins and products of deubiquitinating enzymes (2020)
Figure 7

Figure 7. The diGly/C-degron pathway eliminates cleavage products of deubiquitinating enzymes.

(A) The cleavage sequence of indicated proteins.

(B) Western blot analysis of HEK293T cells expressing GFP-tagged wild-type (WT) or an uncleavable mutant (diGly to GA) full-length (FL) version or the N-terminal domain (NTD) of indicated proteins. Full-length and NTD proteins are marked by * or # on the blot, respectively.

(C) Cycloheximide (CHX)-chase analysis of GFP-tagged FL and NTD versions of indicated proteins.

(D) GPS analysis of wild-type and mutant forms of FL or the NTD of indicated proteins with or without dominant-negative KLHDC2 (DNKLHDC2) treatment. A C-terminal Leu was added (+L) to NTDs to cap the diGly degron.

(E) GST pull-down assay using cells expressing GST or GST-tagged wild-type or mutant KLHDC2 and HA-tagged SDE2, FAU or TUG.
The C-degron pathway eliminates mislocalized proteins and products of deubiquitinating enzymes (2020)
Figure 8

Figure 8. Coronaviral diGly-ending polypeptides resist C-degron pathway-mediated proteolysis.

(A) Schematic depiction of the proteolytic process of coronaviral replicase protein 1a and 1ab by proteases PLpro/NSP3 (labeled red) and 3CLpro (labeled blue). PLpro/NSP3-mediated cleavage generates NSP1, NSP2 and PLpro/NSP3 proteins ending in the diGly motif. The cleavage sequences of indicated viruses are shown.

(B) Stability analysis of FL SARS-CoV NSP1 and NSP2 proteins with or without DNKLHDC2 treatment. We were unable to analyze NSP3 due to its large size (214 kDa).

(C) Relative stability analysis of GAPDH C-terminally tagged with the last 12 residues of indicated proteins with or without DNKLHDC2 treatment by GPS assays. The -3, -4 or -5 residue of SARS-CoV NSP proteins was mutated to Ala (A) as indicated. The experiment was done in triplicate using three independently prepared viruses to express DNKLHDC2. Data are presented as the mean value of GFP/RFP +/- standard deviation from triplicate experiments.

(D) Global protein stability profiling of GPS reporter cells expressing random peptide libraries ending with a diGly motif or the last six residues of SARS-CoV NSP1 protein with or without DNKLHDC2 treatment.
The cellular modifier MOAG-4/SERF drives amyloid formation through charge complementation (2021)
Figure 5

Figure 5. SERF2 drives amyloid formation via charge interactions.

(A,B) Net charge distribution (black) of αSyn (A) and Aß (B), plotted together with the binding distribution of wild-type SERF2 (blue) and the SERF2 charge mutant (red). Blue boxes represent the SERF2 interacting regions.

(C) ThT-monitored amyloid kinetics of 50uM alpha-synuclein in the presence of either wild-type SERF2 or mutant SERF2 in a 1:1 ratio. The average of three biological replicates is represented and error bars indicate mean ± SD. Normalized aggregation data in Appendix Figure S3A.

(D) ThT-monitored amyloid kinetics of 1uM amyloid-beta in the presence of either wild-type SERF2 or mutant SERF2 in a 1:4 ratio. The average of four technical replicates is represented and error bars indicate mean ± SD. Normalized aggregation data in Appendix Figure S3B.

(E) Net charge distribution of hIAPP (black), plotted together with the binding distribution of wild-type SERF2 (blue) and the SERF2 charge mutant (red).

(F) ThT-monitored amyloid kinetics of 2uM human islet amyloid polypeptide in the presence of either wild-type SERF2 or mutant SERF2 in a 1:4 ratio. The average of five technical replicates is represented and error bars indicate mean ± SD. Normalized aggregation data in Appendix Figure S3C.

(G) Model and charge distribution graph of HA-Q74 and muHA-Q74 constructs.

(H) Mutations and charge distribution in the SERF2 control mutant and SERF2 charge mutant proteins.

(I-K) Filter Trap with 5-fold serial dilution and quantification of crude protein extract from SERF2 CRISPR deletion mutant cells transiently transfected with HA-polyQ74 and either wild-type SERF2, empty vector or SERF2 charge mutant (I), wild-type SERF2, empty vector or SERF2 control mutant (J) or transfected with muHA-Q74 and either wild-type SERF2, empty vector or SERF2 charge mutant. Western blots for HA-Q74, muHA-Q74, SERF2 and Tubulin expression were included as controls (see Appendix Figure S3K-M for quantification). For all Filter Trap assays, the depicted blots are from one representative experiment of four biological replicates. The average of four biological replicates with each three or four technical replicates is represented in the graphs. Data are represented as mean ± SD, significance was calculated using a one-way ANOVA, followed by a post-hoc Bonferroni multiple comparisons test. (I) p<0.0001, (J)=0.0003, (K) p=0.9130 **p < 0.01; ***p < 0.001.