thomas cooper  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: U2AF2 controls alternative splicing in speckle-proximal regions in an RS domain-dependent manner

doi: 10.1093/nar/gkag143

Figure Lengend Snippet: Effects of RS domain mutants of U2AF2 on splicing of a minigene reporter. (A) Schematic representation of the RG6 splicing reporter minigene used for the analysis of alternative splicing. Exon inclusion induces the formation of the GFP-fused product, whereas exon skipping leads to the production of RFP-fused protein. Both protein products are localized in the nucleus due to an NLS (black bar). (B) Removal of the RS domain of U2AF2 affects exon inclusion. Normal and CRISPR-modified U2AF2-dRS HEK293 cells (cell clones G2 and 33; Fig. and ) were grown in a 96-well plate and transfected with the minigene-encoding plasmid, fixed, and analyzed by HCS fluorescence microscopy. Left panel: Representative immunofluorescence images of cells expressing RG6 splicing reporter. Scale bar: 50 µm. Right panel: A plot representing the ratio of nuclear GFP to nuclear RFP in the indicated HEK293 cells expressing RG6 minigene. Calculated ratios are averaged for all cells in a single well of a 96-well plate; data from three wells per condition are shown. N = 73 000 cells. *** P <0.001, two-sample t -test. (C) Exon inclusion of RG6 minigene depends on the length and arginine residues in RS dipeptides of the RS domain of U2AF2. CRISPR-modified HEK293 cells expressing U2AF2-dRS (clone 33) were cotransfected with the RG6 minigene-encoding plasmid and one of the indicated Myc-tagged U2AF2 mutants. Cells were fixed and analyzed by HCS fluorescence microscopy. The plot shows the ratio of nuclear GFP to nuclear RFP in cells cotransfected with RG6 minigene and indicated U2AF2 mutants. Calculated ratios are averaged for all cells in a single well of a 96-well plate. Data from four wells per condition are shown. N = 330 000 cells. ns, nonsignificant, ** P <0.01, **** P <0.0001, one-way ANOVA versus U2AF2 WT. (D) Representative immunofluorescence images of cells co-expressing RG6 splicing reporter and Myc-tagged U2AF2 with deletions in RS domain and R-to-A (AS) or R-to-K (KS) substitutions in RS dipeptides. Scale bar: 50 µm. ( E ) Mutants analysis supports an RS domain–phosphorylation-dependent role of U2AF2 in exon inclusion. CRISPR-modified HEK293 cells expressing U2AF2-dRS (clone 33) were cotransfected with the RG6 minigene-encoding plasmid and one of the indicated Myc-tagged U2AF2 mutants. Cells were fixed and analyzed by HCS fluorescence microscopy. A plot represents the ratio of nuclear GFP to nuclear RFP in cells cotransfected with RG6 minigene and indicated U2AF2 mutants. Calculated ratios are averaged for all cells in a single well of a 96-well plate; data from three wells per condition are shown. N = 220 000 cells. ns, nonsignificant, ** P <0.01, *** P <0.001, **** P <0.0001 one-way ANOVA versus U2AF2 WT. ( F ) Representative immunofluorescence images of cells cotransfected with plasmids for expression of the RG6 splicing reporter and Myc-tagged U2AF2 with S-to-A (RA) or S-to-D (RD) substitution in RS dipeptides and R452D substitution in the UHM domain. Scale bar: 50 µm.

Article Snippet: RG6 splicing reporter construct was a gift from Thomas Cooper [ ] (Addgene plasmid #80167). pEGFPC1-based expression plasmids for GFP-fused U2AF2 mutants (RS-domain deletions and R-to-A, R-to-K, S-to-A, or S-to-D mutations) were prepared using restriction-free cloning and a template construct GFP-U2AF2-WT that was obtained by cloning the corresponding human cDNA sequence into the pEGFPC1 vector.

Techniques: Alternative Splicing, CRISPR, Modification, Clone Assay, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Immunofluorescence, Expressing, Phospho-proteomics

Journal: bioRxiv

Article Title: The proteasome maturation factor POMP moonlights as a stress-induced transcriptional regulator

doi: 10.1101/2025.04.25.650603

Figure Lengend Snippet: (A) Scheme of siRNA-based approach to isolate POMP’s proteasome independent actions on the transcriptome by creating two conditions where proteasome activity is reduced to the same extent: one in which POMP is absent (POMP-) and one in which POMP is present (POMP+). (B) Analysis showing that proteasome activity in the different siRNA conditions (see ) was reduced to the same extent across conditions. For every gene, results obtained with two different siRNAs were pooled together for analysis. ns=p>0.05, one-way ANOVA and post-hoc Tukey’s multiple comparison test, n=8 (PSMB1/7), 6 (POMP), mean±SD. FC=fold change. (C) Differential expression analysis of the HEK293 cell transcriptome under conditions where the proteasome was inhibited and POMP was either present (POMP+) or absent (POMP-). Y-axis depicts the differential expression of transcripts; x-axis depicts the average expression level. Transcripts that were significantly upregulated in POMP+ and POMP- are labelled in gold and blue, respectively. (D) Gene Ontology (GO) molecular function (MF) analysis of transcripts upregulated in POMP-condition. The x-axis depicts the statistical significance of the enrichment (-log 10 FDR) and the colour depicts fold enrichment. FDR<0.05, Fisher’s exact test. (E) Gene Ontology (GO) molecular function (MF) analysis of transcripts upregulated in POMP+ condition. The x-axis depicts the statistical significance of the enrichment (-log 10 FDR) and the colour depicts fold enrichment. FDR<0.05, Fisher’s exact test. (F) Representative images of HEK293 cells transfected with either a construct encoding a nucleolar-targeting signal (NoTS) fused to the fluorescent protein Neon (control) or to POMP-Neon and then probed for the CXCL10 or Myc mRNA, using in situ hybridization (black signal). Dashed lines outline the perimeter of transfected cells for comparison to neighboring, untransfected cells. Scale bars = 5 µm. (G) Analysis of the experiments like that shown in F. The expression of NoTS-POMP-Neon significantly reduced both CXCL10 and c- Myc mRNA. ****p≤0.0001, unpaired two-tailed t-tests, n=100, mean±SD, FC=fold change. (H) Representative images of HEK293 cells transfected with either a construct encoding a nucleolar-targeting signal (NoTS) fused to the fluorescent protein Neon (control) or to POMP-Neon cultured for 72 hr prior to 1 hr incubation with 5-EU to label nascent RNA (black signal). The green signal indicates Neon fluorescence. Scale bars= 5 µm. (I) Analysis of the nucleolar nascent RNA experiments like that shown in H. The expression of NoTS-POMP-Neon significantly enhances the nascent RNA signal in the nucleolus compared to NoTS-Neon. ****p<0.0001, unpaired two-tailed t-tests, n=92 (NoTS-Neon), 74 (NoTS-POMP-Neon) cells from 3 experiments. Violin plots show median, interquartile range, minimum and maximum values, FC=fold change. (J) Analysis of the splicing reporter experiments in which expression of NoTS-POMP resulted in significant altered splicing (exon retention; higher GFP:RFP ratio) of the TDP-43-sensitive (IBB) but not the general (RG6) splicing reporter. ****p≤0.0001, Mann-Whitney U tests, n = 2852 (NoTS-BFP, RG6), 3843 (NoTS-POMP-BFP, RG6), 3480 (NoTS-BFP, IBB), 4025 (NoTS-POMP-BFP, IBB) cells from 3 experiments. (K) Western blot analysis for the indicated proteins under control (untreated and scramble siRNA) or PSMB1/7 or POMP siRNA knockdown conditions. Letters refer to different siRNA constructs, numbers refer to biological replicates. Arrowheads point to the cleaved products of Caspase 7 or PARP, * points to non-specific bands in the vicinity of the target band. (L) Analysis of the experiment shown in K. For all of the proteins or protein forms indicated on the x-axis, POMP siRNA, but not PSMB1/7 siRNA, led to a significant increase in the protein. ****p≤0.0001, one-way ANOVA with post-hoc Dunnett’s test, n= 6, 2 different siRNA constructs with 3 biological replicates, mean±SD. FC=fold change. (M) Analysis of cell death experiments using propidium iodide (PI). POMP siRNA led to a significant increase in the number of PI+-cells whereas PSMB1/7 siRNA had no effect. ****p<0.0001, one-way ANOVA with post-hoc Dunnett’s test, n=113 (Scrm), 114 (PSMB1/4, POMP), mean±SD. FC=fold change.

Article Snippet: The constructs HA-POMP (86765), pHBS1389 IBB-GFP-mCherry3E (118803) and RG6 (80167) were purchased from Addgene.

Techniques: Activity Assay, Comparison, Expressing, Transfection, Construct, Control, In Situ Hybridization, Two Tailed Test, Cell Culture, Incubation, Fluorescence, MANN-WHITNEY, Western Blot, Knockdown

Journal: bioRxiv

Article Title: The proteasome maturation factor POMP moonlights as a stress-induced transcriptional regulator

doi: 10.1101/2025.04.25.650603

Figure Lengend Snippet: (A) Density plots comparing transcriptomic features of differentially regulated genes (blue, “List”) to background gene sets (red, “Background”) in PSMB KD (POMP present, left) and POMP KD (POMP absent, right) conditions. Features include coding sequence length, transcript length, genome span, UTR lengths, and GC content. (B) Linear discriminant analysis (LDA) based on sequence and structural features shows partial separation between gene sets regulated upon PSMB KD (magenta) and POMP KD (green). (C) Feature importance plot from a random forest classifier trained to distinguish between POMP-dependent and POMP-independent gene sets. Top contributing features include CDS GC content, minimum free energy (mfe), exon/intron lengths, and sequence skew metrics. (D) Feature enrichment analysis histograms showing distributions of exon counts (top panels) and transcript isoforms per coding gene (bottom panels) in PSMB KD (left, pink) and POMP KD (right, green) compared to genome-wide expectations (light blue). Chi-squared p-values indicate deviation from expected distributions. (E) Juxtaposed transcript usage over gene expression plots for POMP KD (left) and PSMB KD (right). The x-axis reports differential gene expression and the y-axis differential transcript usage. Genes showing differential gene expression (DGE), differential transcript usage (DTU), or both (DTE) are color-coded; selected genes that show differential transcript usage regulation and are involved in DNA-repair and stress response are labeled. (F) Venn diagram depicting the overlap between genes that, by DRIM-Seq analysis, show differential transcript usage in POMP KD and PSMB KD conditions. (G) Gene ontology (GO) enrichment analysis of biological process (BP) terms for genes that show differential transcript usage uniquely in the POMP-dependent condition (PSMB KD only,1055 genes). Top terms are related to DNA damage response and cellular stress response. (H) Schematic diagrams of splicing reporters used to monitor exon retention vs. exon skipping. Upper one is the RG6 general splicing reporter, which incorporates an artificial version of the chicken cardiac troponin (cTNT) exon 5 and is not selective for specific splicing factor. Lower one is the IBB reporter, which contains exon 9 from the CFTR gene and is selective for TDP-43-mediated splicing events. The two reporters work in the way that exon retention/skipping events will lead to different ratios of GFP:RFP signal. (I) DRIMseq analysis of differential isoform usage for PARP3 in HEK293 cells transfected with siRNAs against PSMB1, PSMB7, POMP and scrambled control (Scrm) for 72 hr. n=3 (Scrm), 6 (PSMB1, PSMB7 and POMP). Boxplots show the median (line), interquartile range (box), and Min-Max whiskers. (J) Western blot analysis of PARP3 isoform usage in HEK293 cells transfected with siRNAs against PSMB1, PSMB7, POMP and scrambled control for 72 hr. Knock-down of proteasome subunits, but not POMP, leads to an increase in the levels of one of the lower molecular weight isoforms of PARP3 (indicated as iso_1 and _2). Total protein stain is reported and was used as loading control. The dashed lines mark where the gel was spliced. (K) Quantifications of the relative abundance of PARP3 FL and its two isoforms in experiments like the one shown in I. PSMB1 and PSMB7 knock-down data were merged into a common PSMB term. The data show that in response to proteasome subunit knock-down expression of PARP3 FL decreases slightly in favour of its alternative isoforms, in particular iso_2. By contrast, POMP knock-down leads to a reduction in the levels of the two isoforms compared to scrambled control. n=2 (Scrm, POMP) and 4 (PSMB) biological replicates.

Article Snippet: The constructs HA-POMP (86765), pHBS1389 IBB-GFP-mCherry3E (118803) and RG6 (80167) were purchased from Addgene.

Techniques: Sequencing, Genome Wide, Gene Expression, Labeling, Transfection, Control, Western Blot, Knockdown, Molecular Weight, Staining, Expressing