miniturbo egfp  (Addgene inc)

Journal: bioRxiv

Article Title: RNA-coupled CRISPR Screens Reveal ZNF207 as a Regulator of LMNA Aberrant Splicing in Progeria

doi: 10.1101/2025.04.25.648738

Figure Lengend Snippet: (A) Heatmap showing ΔPSI values of mutant LMNA CRASP-Seq screen hits in HAP1, RPE1, and HepG2 cells. (B) Schematic representation of the ZNF207 protein structure, highlighting the C2H2 zinc finger (ZnF) domain, the charged helical structure, low-complexity regions, and the Gle2-binding-sequence (GLEBS) motif. (C) RT-PCR analysis of aberrant LMNA splicing in RNA from HGPS patient-derived immortalized fibroblasts treated with three independent siRNAs targeting ZNF207. Quantifications of PSI values from three independent experiments are shown below the gel. Data are presented as mean ± standard deviation (SD). ****p-value < 0.0001; Dunnett’s multiple comparisons test following one-way ANOVA. (D) RT-PCR analysis of LMNA splicing in HGPS patient fibroblasts treated with ZNF207-targeting siRNA and/or ectopically expressing a ZNF207 ORF resistant to siRNA for nine days. Quantification of PSI values from three independent experiments are shown below the gel. Data are presented as mean ± SD. ****p-value < 0.0001; Dunnett’s multiple comparisons test following one-way ANOVA. (E) Western blot analysis of lamins in HGPS patient-derived immortalized fibroblasts transduced with either a siRNA-resistant 3×FLAG C-terminal-tagged ZNF207 ORF or an empty vector. Cells were treated with either non-targeting control siRNA or siRNA targeting endogenous ZNF207 (siZNF207) for 16 days. Blots were probed with antibodies against lamins, ZNF207, and GAPDH (loading control). Relative progerin expression was quantified and normalized to β-actin, with values further normalized to the corresponding siControl-treated sample. Quantifications from three independent experiments are presented on the right. Data are presented as mean ± SD. *p-value < 0.05, ****p-value < 0.0001; Uncorrected Fisher’s LSD following one-way ANOVA.

Article Snippet: After 48 hours, doxycycline was added to the media at a final concentration of 0.0005 µg/mL for miniTurbo-Empty and miniTurbo-EGFP (Addgene #209059), and 0.5 µg/mL for ZNF207-miniTurbo (Addgene #231938).

Techniques: Mutagenesis, Binding Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Standard Deviation, Expressing, Western Blot, Transduction, Plasmid Preparation, Control

Journal: bioRxiv

Article Title: RNA-coupled CRISPR Screens Reveal ZNF207 as a Regulator of LMNA Aberrant Splicing in Progeria

doi: 10.1101/2025.04.25.648738

Figure Lengend Snippet: (A) Heatmap showing ΔPSI values for wild-type LMNA CRASP-Seq screens for the hits identified in the mutant LMNA screens across HAP1, RPE1, and HepG2 cell lines. (B) Gene Ontology (GO) enrichment analysis of biological processes among LMNA CRASP-Seq screen hits with adjusted p-value < 0.01. (C) Real-time quantitative RT-PCR measuring ZNF207 transcript depletion in HEK293 cells transfected with three independent siRNAs. Quantification of transcript levels from three independent experiments are displayed. Data are presented as mean ± SD. ****p-value < 0.0001; Dunnett’s multiple comparisons test following one-way ANOVA. (D) RT-PCR analysis of aberrant LMNA splicing in RNA from HEK293T cells transduced with the mutant LMNA minigene reporter and treated with three independent siRNAs targeting ZNF207. Quantification of PSI values from three independent experiments are shown below the gel. Data are presented as mean ± SD. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001; Dunnett’s multiple comparisons test following one-way ANOVA. (E) Western blot analysis of ZNF207 in HEK293T cells expressing the LMNA minigene reporter, transiently transfected with either a siRNA-resistant 3×HA C-terminal-tagged ZNF207 ORF or an empty vector. Cells were treated with either non-targeting siRNA control or siRNA targeting endogenous ZNF207 (siZNF207). Blots were probed with antibodies against ZNF207, HA tag, and GAPDH (loading control). (F) RT-PCR analysis of LMNA splicing in HEK293 cells expressing the LMNA minigene reporter and treated with ZNF207-targeting siRNA and/or ectopically expressing an siRNA-resistant ZNF207 ORF. Quantification of PSI values from three independent experiments are shown below the gel. Data are presented as mean ± SD. **p-value < 0.01, ****p-value < 0.0001; Dunnett’s multiple comparisons test following one-way ANOVA. (G) Western blot analysis of ZNF207 in HGPS patient-derived immortalized fibroblasts stably transduced with either a siRNA-resistant 3×FLAG C-terminal-tagged ZNF207 ORF or an empty vector. Cells were treated with either non-targeting siRNA control or siRNA targeting endogenous ZNF207 (siZNF207). Blots were probed with antibodies against ZNF207, FLAG, and GAPDH (loading control). (H) Single cell high throughput immunofluorescence quantification of LAP2α, H3K27me3, H4K16ac and LB1 expression levels in immortalized human fibroblasts treated with siZNF207 or siCTL for 15 days. At least 600 cells were analyzed in each experiment and represented as the distribution of average mean fluorescence intensity. The values from three independent experiments ± SD are plotted (see Methods). Statistical differences were analyzed by one-way ANOVA followed by the Dunnett’s test using the WT siCTL cell line as the negative control for WT siZNF207 and the HGPS siCTL as the negative control for HGPS siZNF207.

Article Snippet: After 48 hours, doxycycline was added to the media at a final concentration of 0.0005 µg/mL for miniTurbo-Empty and miniTurbo-EGFP (Addgene #209059), and 0.5 µg/mL for ZNF207-miniTurbo (Addgene #231938).

Techniques: Mutagenesis, Quantitative RT-PCR, Transfection, Reverse Transcription Polymerase Chain Reaction, Transduction, Western Blot, Expressing, Plasmid Preparation, Control, Derivative Assay, Stable Transfection, High Throughput Screening Assay, Immunofluorescence, Fluorescence, Negative Control

Journal: bioRxiv

Article Title: RNA-coupled CRISPR Screens Reveal ZNF207 as a Regulator of LMNA Aberrant Splicing in Progeria

doi: 10.1101/2025.04.25.648738

Figure Lengend Snippet: (A) Distribution of alternative splicing event types (CE: cassette exons, Alt3: alternative 3′ splice sites, Alt5: alternative 5′ splice sites, IR: retained introns) detected by RNA-seq following ZNF207 knockdown for 48 hours with three independent siRNAs in HEK293T cells. Events with significant splicing changes (|ΔPSI| ≥ 10; probability ≥ 0.95) upon siZNF207 knockdown are included. (B) Western blot analysis of ZNF207 in HEK293 Flp-In cells expressing doxycycline-inducible, siRNA-resistant 3×FLAG C-terminal tagged ZNF207 ORF. Cells were treated with control siRNA or siRNA targeting endogenous ZNF207 (siZNF207) for 48 hours. Blots were probed with antibodies against ZNF207, FLAG, and GAPDH (loading control). (C) RNA-seq analysis of splicing changes (ΔPSI) upon ZNF207 knockdown in HEK293 Flp-In cells. Comparisons include knockdown of ZNF207 relative to non-targeting siRNA treatment without induction (siZNF207) and ZNF207 ORF rescue expression in siRNA-treated cells compared to expression of empty vector (siZNF207 + rescue). Events with significant splicing changes (|ΔPSI| ≥ 10; probability ≥ 0.95) upon siZNF207 knockdown and rescue by ZNF207 ORF expression are highlighted. (D) RT-PCR analysis of ACLY (left) and SRRM1 (right) alternative splicing in HEK293 Flp-In cells treated with ZNF207-targeting siRNA and/or expressing siRNA-resistant C-terminally 3xFLAG-tagged ZNF207 ORF. Quantification of PSI values from three independent experiments are shown below the gel. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ****p < 0.0001; Dunnett’s multiple comparisons test following one-way ANOVA. (E) Gene expression analysis (Z score normalized) from RNA-seq profiling of HEK293 Flp-In cells treated with ZNF207-targeting siRNA and/or rescued with siRNA-resistant ZNF207 ORF expression. Only genes with significant expression changes (adjusted p < 0.05; |log2FC| ≥ 0.35) upon siZNF207 and rescued by ZNF207 expression are shown. (F) RT-PCR analysis of LUC7L, LUC7L2, SNRNP70, and RBM3 alternative splicing in HEK293 Flp-In cells treated with ZNF207-targeting siRNA and/or expressing siRNA-resistant ZNF207 ORF. Experiments were conducted with and without the NMD inhibitor SMG1i (0.5 µM, 6 hrs). Quantification of PSI values from three independent experiments are shown below the gel. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Šídák’s multiple comparisons test following one-way ANOVA.

Article Snippet: After 48 hours, doxycycline was added to the media at a final concentration of 0.0005 µg/mL for miniTurbo-Empty and miniTurbo-EGFP (Addgene #209059), and 0.5 µg/mL for ZNF207-miniTurbo (Addgene #231938).

Techniques: Alternative Splicing, RNA Sequencing, Knockdown, Western Blot, Expressing, Control, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Gene Expression

Journal: bioRxiv

Article Title: RNA-coupled CRISPR Screens Reveal ZNF207 as a Regulator of LMNA Aberrant Splicing in Progeria

doi: 10.1101/2025.04.25.648738

Figure Lengend Snippet: (A) Venn diagram illustrating the overlap of regulated splicing events (|ΔPSI| ≥ 10 and probability ≥ 0.95) across the three independent siRNA sequences targeting ZNF207. ****p-value < 0.0001; Fisher’s exact test. (B) Correlation of PSI changes (ΔPSI) in HEK293T cells transfected with three independent siRNA sequences targeting ZNF207. Significant events for each siRNA treatment are highlighted using distinct colors. “r” denotes the Pearson correlation coefficient and “ρ” represents the Spearman’s rank correlation coefficient. (C) Western blot analysis of ZNF207 in HEK293 Flp-In cells expressing doxycycline-inducible, siRNA-resistant 3×FLAG N-terminal tagged ZNF207 ORF. Cells were treated with non-targeting siRNA control or siRNA targeting endogenous ZNF207 (siZNF207). Blots were probed with antibodies against ZNF207, and GAPDH (loading control). (D) RT-PCR analysis of alternative splicing for ACLY (left) and SRRM1 (right) in HEK293 Flp-In cells treated with ZNF207-targeting siRNA and/or expressing siRNA-resistant N-terminally tagged ZNF207 ORF. Quantification of PSI values from three independent experiments are shown below the gel. Data are presented as mean ± SD. ****p < 0.0001; Dunnett’s multiple comparisons test following one-way ANOVA. (E) Enrichment of KEGG biological pathways among genes showing differential expression after ZNF207 depletion, which are rescued by ZNF207 reintroduction (as shown in ). Only terms with adjusted p-value < 0.05 are shown. (F) Real-time quantitative RT-PCR analysis of transcript levels for LUC7L , LUC7L2 , RBM3 , and SNRNP70 in HEK293 Flp-In cells expressing a doxycycline-inducible, siRNA-resistant 3×FLAG C-terminal tagged ZNF207 ORF. Cells were treated with either non-targeting siRNA controlor siRNA targeting endogenous ZNF207 (siZNF207) and further treated with either 0.5 µM SMG1 NMD inhibitor (SMG1i) or DMSO for 6 hours prior to RNA extraction. Quantification of transcript levels from three independent experiments are displayed. Data are presented as mean ± SD. ****p-value < 0.0001, ***p-value < 0.001, **p-value < 0.01, *p-value < 0.05; Šídák’s multiple comparisons testing following one-way ANOVA. (G) Genome browser tracks highlighting alternative splicing events in LUC7L , SNRNP70 , and RBM3 , genes whose expression is regulated by ZNF207. Tracks represent data from two independent RNA-Seq replicates under three conditions: cells treated with control siRNAs (siControl), siRNAs targeting ZNF207 (siZNF207), and siZNF207-treated cells rescued by induced ZNF207 expression. Normalized reads per million (RPM) are displayed on the left of each track. The regulated splicing events are highlighted.

Article Snippet: After 48 hours, doxycycline was added to the media at a final concentration of 0.0005 µg/mL for miniTurbo-Empty and miniTurbo-EGFP (Addgene #209059), and 0.5 µg/mL for ZNF207-miniTurbo (Addgene #231938).

Techniques: Transfection, Western Blot, Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Alternative Splicing, Quantitative RT-PCR, RNA Extraction, RNA Sequencing

Journal: bioRxiv

Article Title: RNA-coupled CRISPR Screens Reveal ZNF207 as a Regulator of LMNA Aberrant Splicing in Progeria

doi: 10.1101/2025.04.25.648738

Figure Lengend Snippet: (A) Overview of the TurboID experimental pipeline. HEK293 Flp-In cells expressing C-terminal miniTurbo-tagged ZNF207 were induced for 24 hours and incubated with biotin for 2 hours. Proximal biotinylated proteins were purified and analyzed via mass spectrometry. (B) Proximal interaction network of ZNF207 identified by TurboID. Only significant interactors consistently detected across all three replicates are shown (Table S7). (C) Gene Ontology (GO) enrichment analysis of biological processes among ZNF207 TurboID proximal interactors with FDR < 0.05. (D) Western blot analysis of total cell lysates (input) treated with benzonase, and FLAG immunoprecipitates (IP: FLAG-M2) from HEK293 Flp-In cells expressing 3×FLAG-ZNF207. Blots were probed with antibodies against FLAG, SNRPA, SNRNP70, RBM25, SF3B1, SNRNP200, PRPF6, SNRPF, and GAPDH (negative control). (E) Bar plot showing the distribution of ZNF207 eCLIP peaks across RNA biotypes. Only biotypes with at least 10 ZNF207 peaks are included. eCLIP reads were normalized to input, and peaks were called using the DEWSeq pipeline (see Methods for details). (F) Stacked bar chart showing the percentage of intronic and exonic ZNF207 binding peaks. (G) Average eCLIP signal profiles of C-terminal FLAG-tagged ZNF207 for ZNF207-regulated cassette exons (n = 715) and unchanged alternative exons (n = 3,745). Profiles include the negative control GFP and N-terminal FLAG-tagged ZNF207 (devoid of splicing activity) for the same exon subsets.

Article Snippet: After 48 hours, doxycycline was added to the media at a final concentration of 0.0005 µg/mL for miniTurbo-Empty and miniTurbo-EGFP (Addgene #209059), and 0.5 µg/mL for ZNF207-miniTurbo (Addgene #231938).

Techniques: Expressing, Incubation, Purification, Mass Spectrometry, Western Blot, Negative Control, Binding Assay, Activity Assay

Journal: bioRxiv

Article Title: RNA-coupled CRISPR Screens Reveal ZNF207 as a Regulator of LMNA Aberrant Splicing in Progeria

doi: 10.1101/2025.04.25.648738

Figure Lengend Snippet: (A) Western blot analysis of HEK293 Flp-In cells expressing 3×FLAG-miniTurboID-tagged constructs, including an empty vector, EGFP, or ZNF207 (C-terminal tagged with 3×FLAG-miniTurboID). The blot was probed with streptavidin (for biotinylation detection), ZNF207, FLAG, and GAPDH antibodies. (B) CORUM complex enrichment analysis of ZNF207 proximal interactors identified via TurboID experiments. All enriched terms have adjusted p-value < 0.0001. (C) Average eCLIP signal profiles for C-terminal FLAG-tagged ZNF207 over exons positively (top; n = 639) or negatively (bottom; n = 76)) regulated by ZNF207 and over unchanged alternative exons (n = 3,745). Signal profiles are compared to the negative control GFP and N-terminal FLAG-tagged ZNF207 (lacking splicing activity) for the same exon subsets.

Article Snippet: After 48 hours, doxycycline was added to the media at a final concentration of 0.0005 µg/mL for miniTurbo-Empty and miniTurbo-EGFP (Addgene #209059), and 0.5 µg/mL for ZNF207-miniTurbo (Addgene #231938).

Techniques: Western Blot, Expressing, Construct, Plasmid Preparation, Negative Control, Activity Assay

Journal: bioRxiv

Article Title: RNA-coupled CRISPR Screens Reveal ZNF207 as a Regulator of LMNA Aberrant Splicing in Progeria

doi: 10.1101/2025.04.25.648738

Figure Lengend Snippet: (A) Schematic of the CRASP-Seq base editor tiling approach. A lentiviral vector enables constitutive expression of sgRNAs while a doxycycline-inducible TRE promoter drives the expression of a mutant LMNA minigene reporter. Splicing-derived mRNA products are analyzed via Illumina paired-end high-throughput sequencing. (B) ΔPSI scores from gene-level analysis of all guides in the base editor library, including those targeting splice sites, across the 39 profiled genes. Intergenic and non-targeting controls are also included. The genes are arranged in ascending order of their ΔPSI scores, from left to right. (C) ΔPSI scores for base editor sgRNAs targeting ZNF207 coding sequence, excluding splice-site-overlapping guides. Average ΔPSI scores for individual sgRNAs within 10-amino-acid windows are plotted, with the scores of individual sgRNAs depicted as black dots. The ZNF207 protein is illustrated below, showing ZnF domains (blue), GLEBS motif (orange), and low-complexity regions (purple). (D) Schematic representation of ZNF207 constructs: full-length wild-type ZNF207 (top), full-length K42E point mutant (middle), and the ΔZnF truncation mutant lacking the zinc finger domain (bottom). (E) RT-PCR analysis of ACLY (left) and SRRM1 (right) alternative splicing in HEK293 Flp-In cells treated with ZNF207-targeting siRNA and/or expressing siRNA-resistant wild-type or truncation mutant ZNF207 ORFs. Quantification of ΔPSI values from three independent experiments are shown below the gel. Data are presented as mean ± SD. Statistical comparisons were performed relative to the siZNF207 sample without ZNF207 ORF expression. ****p < 0.0001; Dunnett’s multiple comparisons test following one-way ANOVA. (F) Western blot analysis of total cell lysates (input) treated with benzonase, and FLAG immunoprecipitates (IP: FLAG-M2) from HEK293 Flp-In cells expressing 3×FLAG-tagged ZNF207 variants. Blots were probed with antibodies specific for FLAG, SNRPA, SNRNP70, RBM25, SF3B1, and GAPDH (negative control). (G) Quantification of co-immunoprecipitated spliceosome components, normalized to FLAG-ZNF207 pulldown efficiency and wild-type (WT) ZNF207 levels. Data are presented as mean ± SD. Statistical comparisons were performed relative to WT-ZNF207. ***p < 0.001, **p < 0.01, *p < 0.05; One-way ANOVA with Dunnett’s multiple comparisons correction.

Article Snippet: After 48 hours, doxycycline was added to the media at a final concentration of 0.0005 µg/mL for miniTurbo-Empty and miniTurbo-EGFP (Addgene #209059), and 0.5 µg/mL for ZNF207-miniTurbo (Addgene #231938).

Techniques: Plasmid Preparation, Expressing, Mutagenesis, Derivative Assay, Next-Generation Sequencing, Sequencing, Construct, Reverse Transcription Polymerase Chain Reaction, Alternative Splicing, Western Blot, Negative Control, Immunoprecipitation

Journal: bioRxiv

Article Title: RNA-coupled CRISPR Screens Reveal ZNF207 as a Regulator of LMNA Aberrant Splicing in Progeria

doi: 10.1101/2025.04.25.648738

Figure Lengend Snippet: (A) AlphaFold prediction of the full-length ZNF207 protein. Key structural regions are highlighted: the C2H2 zinc finger domain in blue, the helical structure in brown, the GLEBS motif in orange, and the C-terminal regions, predicted to be disordered and RNA-binding, in pink. Corresponding features are similarly annotated in the schematic below. (B) HydRA analysis predicting RNA-binding regions within ZNF207. Occlusion maps illustrate regions with differential RNA-binding activity: purple regions represent Δscore < 0, while blue regions represent Δscore > 0. Statistically significant regions (p < 0.05 or p < 0.001) are highlighted in light blue and dark blue, respectively, on the bottom track. The coordinates of protein domains and annotated regions are displayed at the top. (C) Schematic representation of ZNF207 truncation mutants used in the figure. (D) Western blot analysis of ZNF207 truncation mutants transiently transfected into HEK293T cells. Cells were treated with control siRNA (siControl) or siRNA targeting endogenous ZNF207 (siZNF207). Blots were probed with antibodies against ZNF207, and FLAG. (E) RT-PCR analysis of ACLY (top) and SRRM1 (bottom) alternative splicing in HEK293 cells treated with ZNF207-targeting siRNA and transiently transfected with siRNA-resistant, C-terminally 3×FLAG-tagged ZNF207 truncation mutants. Quantification of PSI values are shown below the gel. (F) Western blot analysis of siRNA-resistant 3×FLAG C-terminal tagged ZNF207 truncation mutants expressed in HEK293 Flp-In cells under doxycycline induction. Cells were treated with non-targeting siRNA control or siRNA targeting endogenous ZNF207 (siZNF207). Western blots were probed with antibodies against ZNF207, FLAG (to detect tagged constructs), and GAPDH (loading control). (G) RT-PCR analysis of ACLY (left) and SRRM1 (right) alternative splicing in HEK293 Flp-In cells treated with ZNF207-targeting siRNA and/or expressing siRNA-resistant wild-type or truncation mutant ZNF207 ORFs. Quantification of ΔPSI values from three independent experiments are shown below the gel. Data are presented as mean ± SD. Statistical comparisons were performed relative to the siZNF207 sample without ZNF207 ORF expression. ****p < 0.0001; Dunnett’s multiple comparisons test following one-way ANOVA. (H) Western blot analysis of ZNF207 mutants at residues H41, K42, and K43 (wild-type: HKK), transiently expressed in HEK293T cells. Cells were transfected with either control siRNA (siControl) or siRNA targeting endogenous ZNF207 (siZNF207). Blots were probed with antibodies against ZNF207, FLAG, and GAPDH (loading control). (I) RT-PCR analysis of ACLY (left) and SRRM1 (right) alternative splicing in HEK293 cells treated with ZNF207-targeting siRNA and transiently transfected with siRNA-resistant, C-terminally 3×FLAG-tagged ZNF207 mutants. Quantification of ΔPSI values from three independent experiments are shown below the gel. Data are presented as mean ± SD. Statistical comparisons were performed relative to the siZNF207 sample without ZNF207 ORF expression. ****p < 0.0001; Dunnett’s multiple comparisons test following one-way ANOVA.

Article Snippet: After 48 hours, doxycycline was added to the media at a final concentration of 0.0005 µg/mL for miniTurbo-Empty and miniTurbo-EGFP (Addgene #209059), and 0.5 µg/mL for ZNF207-miniTurbo (Addgene #231938).

Techniques: RNA Binding Assay, Activity Assay, Western Blot, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Alternative Splicing, Construct, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: RNA-coupled CRISPR Screens Reveal ZNF207 as a Regulator of LMNA Aberrant Splicing in Progeria

doi: 10.1101/2025.04.25.648738

Figure Lengend Snippet: (A) Schematic representation of ZNF207 variants. (B) RT-PCR analysis of ACLY (top) and SRRM1 (bottom) alternative splicing in HEK293 cells treated with ZNF207-targeting siRNA and transiently transfected with siRNA-resistant, C-terminally 3×FLAG-tagged ZNF207 mutants. Quantification of ΔPSI values from three independent experiments are shown below the gel. Data are presented as mean ± SD. Statistical comparisons were performed relative to the siZNF207 sample without ZNF207 ORF expression. ****p < 0.0001; Dunnett’s multiple comparisons test following one-way ANOVA. (C) Western blot analysis of siRNA-resistant 3×FLAG C-terminal tagged ZNF207 mutants expressed in HEK293 Flp-In cells under doxycycline induction. Cells were treated with non-targeting siRNA control or siRNA targeting endogenous ZNF207 (siZNF207). Western blots were probed with antibodies against ZNF207, FLAG (to detect tagged constructs), and GAPDH (loading control). (D) RT-PCR analysis of ACLY (top) and SRRM1 (bottom) alternative splicing in HEK293 Flp-In cells treated with ZNF207-targeting siRNA and/or expressing siRNA-resistant wild-type or mutant ZNF207 ORFs. Quantification of ΔPSI values from three independent experiments is shown below the gels. Data are presented as mean ± SD. Statistical comparisons were performed relative to the siZNF207 sample without ZNF207 ORF expression. ****p < 0.0001; Dunnett’s multiple comparisons test following one-way ANOVA. (E-F) Biotin-based RNA labeling of UV-crosslinked RNA bound to immunoprecipitated ZNF207 truncation mutants. Pull-down efficiency of each construct was assessed by FLAG Western blot. Quantification of relative RNA signal from three independent experiments is shown below the gel. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test. (G) Representative western blot analysis of total cell lysates (input) treated with benzonase, and FLAG immunoprecipitates (IP: FLAG-M2) from HEK293 Flp-In cells expressing 3×FLAG-tagged ZNF207 truncation mutants shown in S8C. Blots were probed with antibodies specific for FLAG, SNRPA, SNRNP70, RBM25, SF3B1, and GAPDH (negative control). (H) Quantification of co-immunoprecipitated spliceosome components, normalized to FLAG-ZNF207 pulldown efficiency and wild-type (WT) ZNF207 levels. Data are presented as mean ± SD. Statistical comparisons were performed relative to WT-ZNF207. ***p < 0.001, **p < 0.01, *p < 0.05; Šídák’s multiple comparisons test following two-way ANOVA.

Article Snippet: After 48 hours, doxycycline was added to the media at a final concentration of 0.0005 µg/mL for miniTurbo-Empty and miniTurbo-EGFP (Addgene #209059), and 0.5 µg/mL for ZNF207-miniTurbo (Addgene #231938).

Techniques: Reverse Transcription Polymerase Chain Reaction, Alternative Splicing, Transfection, Expressing, Western Blot, Control, Construct, Mutagenesis, Labeling, Immunoprecipitation, Negative Control

Journal: bioRxiv

Article Title: RNA-coupled CRISPR Screens Reveal ZNF207 as a Regulator of LMNA Aberrant Splicing in Progeria

doi: 10.1101/2025.04.25.648738

Figure Lengend Snippet: Illustration of the proposed mechanism by which ZNF207 regulates alternative splicing. ZNF207 interacts with U1 snRNP and other core spliceosome components, binding directly to target pre-mRNAs. These interactions influence splice site selection, promoting the activation of specific exons.

Article Snippet: After 48 hours, doxycycline was added to the media at a final concentration of 0.0005 µg/mL for miniTurbo-Empty and miniTurbo-EGFP (Addgene #209059), and 0.5 µg/mL for ZNF207-miniTurbo (Addgene #231938).

Techniques: Alternative Splicing, Binding Assay, Selection, Activation Assay

Journal: bioRxiv

Article Title: RNA-coupled CRISPR Screens Reveal ZNF207 as a Regulator of LMNA Aberrant Splicing in Progeria

doi: 10.1101/2025.04.25.648738

Figure Lengend Snippet: (A) Western blot analysis of BUB3 and ZNF207 in HEK293T cells treated with non-targeting siRNA control (siCTL), four independent siRNAs targeting BUB3 (siBUB3), or a pool of these siRNAs. Blots were probed with antibodies against BUB3, ZNF207, and GAPDH (loading control). (B) Real-time quantitative RT-PCR analysis of BUB3 and ZNF207 transcript levels in HEK293T cells treated with either control siRNA (siCTL) or siRNAs targeting BUB3 (siBUB3), as in panel B. Quantification of transcript levels from three independent experiments are displayed. Data are presented as mean ± SD. ****p-value < 0.0001; one-way ANOVA with Dunnett’s multiple comparisons test. (C) RT-PCR analysis of LMNA minigene reporter (left), endogenous ACLY (middle), and endogenous SRRM1 (right) alternative splicing in HEK293T cells treated with BUB3-targeting siRNAs, as in panel B. Quantification of PSI values from three independent experiments are shown below the gel. Data are presented as mean ± SD. *p-value < 0.05; one-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: After 48 hours, doxycycline was added to the media at a final concentration of 0.0005 µg/mL for miniTurbo-Empty and miniTurbo-EGFP (Addgene #209059), and 0.5 µg/mL for ZNF207-miniTurbo (Addgene #231938).

Techniques: Western Blot, Control, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Alternative Splicing