Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) RIP schematic. (B) SPEN-Repeat A association across genotypes by input-normalized RIP-qPCR. SM33 WT vs. Δ Spen ESCs confirm specificity of SPEN antibody. Srsf2 mRNA, control. Dots, triplicate qPCR values from two independent experiments. (C) Median silencing vs. SPEN-Repeat A association across genotypes. r , p : data fit to linear regression. (D) Specificity vs. signal for SPEN RIP-seq peaks in SM33 ESCs. (E) Motifs from top 1500 non- Xist , non- Spen SPEN RIP-seq peaks. (F) Overview of equilibrium RBNQ. SBP, streptavidin-binding peptide. (G) RBNQ with SPEN RRM2-4 and Repeat A. Dots, values from two independent experiments. Error bars, standard deviation of qPCR triplicates. CI, confidence interval. (H) RBNQ across Repeat A RNAs using fixed SPEN RRM2-4 concentration. Dots, qPCR triplicates from ≥2 independent experiments. For all panels vs. WT (two-sided t-test): (*), p < 0.05; (**), p < 0.01. See .

Article Snippet: Male E14 mouse embryonic stem cells (mESCs, kind gift of D. Ciavatta), male B6/CAST F1-hybrid Rosa26 -RMCE mESCs, and male SM33 mESCs (with dox-inducible promoter at the endogenous Xist locus; kind gift of K. Plath; ), were grown on gelatin-coated plastic dishes in a humidified Thermo Fisher Forma Series II water-jacketed incubator at 37 °C and under 5% CO 2 .

Techniques: Control, Binding Assay, Standard Deviation, Concentration Assay

Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) Genotyping PCR for independent clonal lines of SM33 Δ Spen cells generated by CRISPR-Cas9. PCR1 was performed using primers flanking either side of the deletion; PCR2 and PCR3 were performed using primers flanking the upstream and downstream gRNA target sites. (B) RT-qPCR measuring Spen mRNA in SM33 WT and Δ Spen cells. Values are relative to SM33 WT. Dots represent average qPCR-replicate values for independent cell lines. (C) Top 15 most significant sequence motifs enriched in the 1500 most abundant non- Xist , non- Spen SPEN-specific peaks from SPEN RIP-seq, determined by STREME. (D) Promega Diamond-stained urea-PAGE gels of in-vitro-transcribed Repeat A RNAs used for equilibrium RBNQ experiments. (E) Coomassie blue-stained SDS-PAGE gel of two independent preps of SBP-tagged SPEN RRM2-4 used for replicates of equilibrium RBNQ experiments.

Article Snippet: Male E14 mouse embryonic stem cells (mESCs, kind gift of D. Ciavatta), male B6/CAST F1-hybrid Rosa26 -RMCE mESCs, and male SM33 mESCs (with dox-inducible promoter at the endogenous Xist locus; kind gift of K. Plath; ), were grown on gelatin-coated plastic dishes in a humidified Thermo Fisher Forma Series II water-jacketed incubator at 37 °C and under 5% CO 2 .

Techniques: Generated, CRISPR, Quantitative RT-PCR, Sequencing, Staining, In Vitro, SDS Page

Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) SRSF1-Repeat A association in SM33 or RMCE- Xist ESCs by input-normalized RIP-qPCR. Srsf2 mRNA, control. Dots, qPCR triplicates from two independent experiments. (B) SPEN- vs. SRSF1-Repeat A association (RIP-qPCR). r , p : data fit to linear regression. (C) RBNQ with SRSF1 RRM1-2 and Repeat A. Dots, values from two independent experiments. Error bars, standard deviation of qPCR triplicates. (D) RBNQ across Repeat A RNAs using fixed SRSF1 RRM1-2 concentration. Dots, qPCR triplicates values, ≥2 independent experiments. (E) FKBP1A F36V -SRSF1 degradation by dTAG V -1 in cells derived from RMCE- Xist WT. (F) SPEN-Repeat A association in WT or FKBP1A F36V - Srsf1 cells by input-normalized RIP-qPCR. Dots, qPCR triplicates from independent clonal lines. (G) Xist abundance (RNA-seq) in FKBP1A F36V - Srsf1 cells, with/without dox and with/without dTAG V -1. Dots, independent lines, relative to dox(+), dTAG V -1(-) condition. (H) Xist silencing activity as in , for FKBP1A F36V - Srsf1 cells treated as in (G). (I) MCP-MS2 tethering to assess SPEN recruitment to Xist ΔRepA-XB-6x(U-spacer-3xMS2). (J) SPEN-6x(U-spacer-3xMS2) association across MCP genotypes by input-normalized RIP-qPCR. Dots, qPCR triplicates from ≥2 independent experiments. (K) Xist abundance (RNA-seq) across genotypes. Dots, independent experiments. (L) Xist silencing activity as in across MCP genotypes. “n.s.,” not significantly different. P -values, two-sided t-tests relative to WT or EGFP unless specified by black bars: (*), p < 0.05; (**), p < 0.01. See .

Article Snippet: Male E14 mouse embryonic stem cells (mESCs, kind gift of D. Ciavatta), male B6/CAST F1-hybrid Rosa26 -RMCE mESCs, and male SM33 mESCs (with dox-inducible promoter at the endogenous Xist locus; kind gift of K. Plath; ), were grown on gelatin-coated plastic dishes in a humidified Thermo Fisher Forma Series II water-jacketed incubator at 37 °C and under 5% CO 2 .

Techniques: Control, Standard Deviation, Concentration Assay, Derivative Assay, RNA Sequencing, Activity Assay