Journal: Cellular and Molecular Life Sciences

Article Title: PRMT5-mediated homologous recombination repair is essential to maintain genomic integrity of neural progenitor cells

doi: 10.1007/s00018-024-05154-x

Figure Lengend Snippet: RNA-sequencing analysis shows an abnormal transcriptomic signature in the cKO-Emx1 neocortex. A Total RNA from E12.5 WT and cKO-Emx1 cortices were used for RNA sequencing. B Venn diagrams comparing genes differentially expressed in WT (pink) and cKO-Emx1 (cyan) cortices at E12.5. C Volcano plots illustrate differentially expressed genes between WT and cKO-Emx1 cortical samples. The number of significantly changed genes ( p -adj < 0.05, ≥ 0.2-fold change in either direction, red dots are upregulated genes, and blue dots are downregulated genes compared to WT) is indicated. The violin plot shows the absolute change for the genes changing by ≥ 0.2-fold (WT: N = 3, cKO-Emx1: N = 3). D GO process enrichment analysis of DEGs between WT and cKO-Emx1 samples. E “DNA double-strand break response” gene expression signature was tested for enrichment by GSEA in WT and cKO-Emx1 cortices. F Quantification of RT-qPCR results in WT and cKO-Emx1 cortices at E12.5 (WT: N = 3, cKO-Emx1: N = 3, data shown as the mean ± SEM). Genes related to “DSBs repair” and “cortical development” were downregulated, and genes related to the “p53 pathway” and “apoptosis” were upregulated in cKO-Emx1 cortices. Relative mRNA levels were normalized to Gapdh . The relative mRNA level in WT mice was set to 1. Two-tailed unpaired t test. G Western blotting and quantification results showed that the expression levels of HR repair-associated proteins were decreased and the expression levels of NHEJ repair-associated proteins were increased in cKO-Emx1 cortical samples (E12.5, WT: N = 3, cKO-Emx1: N = 3, data shown as the mean ± SEM). Expression levels were normalized to GAPDH. The ratio in WT mice was set to 100%. Two-tailed unpaired t test. For quantification, at least 3 brains for each genotype were analyzed. Mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: AAV_Actb HR donor_U6_sgRNA_EF1a_GFP_polyA (RRID: addgene_97309) and AAV_Actb NHEJ donor_U6_sgRNA_EF1a_GFP_polyA (RRID: addgene_97310) were used to provide the HR donor and NHEJ donor for DSBs repair, respectively.

Techniques: RNA Sequencing, Gene Expression, Quantitative RT-PCR, Two Tailed Test, Western Blot, Expressing

Journal: Cellular and Molecular Life Sciences

Article Title: PRMT5-mediated homologous recombination repair is essential to maintain genomic integrity of neural progenitor cells

doi: 10.1007/s00018-024-05154-x

Figure Lengend Snippet: Prmt5 deletion from NSCs disrupts homologous recombination DNA repair. A Schematic illustration of in vitro DSBs repair assay using DR-GFP/EJ5-GFP detection system. DR-GFP contains a SceGFP gene and an iGFP gene. SceGFP is a modified GFP gene and contains an I- Sce I site and an in-frame termination codon. iGFP is a 5′ and 3′-truncated GFP gene. I- Sce I nucleases specifically recognized the I- Sce I site on DR-GFP and generate a DSB. When HR repair occurred, the DSB will be repaired from the iGFP gene on the same chromatid or sister chromatid, resulting in a functional GFP gene expression. EJ5-GFP contains a promoter that is separated from a GFP gene by a puro gene which is flanked by two I- Sce I sites. When I- Sce I nucleases exist, the puro gene will be cut off and GFP gene expressed by NHEJ repair. B Representative fluorescence staining images of DMSO (Mock) or PRMT5 inhibitor (PRM5i) treated NSCs after transfection for 72 h. Scale bar, 40 μm. C Quantification of the percentage of GFP + cells among total transfected HA + cells from ( B ). Two-tailed unpaired t test. D Representative fluorescence staining images of DMSO (Mock) or PRMT5 inhibitor (PRM5i) treated NSCs after transfection for 72 h. Scale bar, 20 μm. E Quantification of the percentage of mCherry + GFP + cells among all the GFP + cells from ( D ). F Schematic of DNA double-strand break repair. G Representative images of γH2AX (red), 53BP1 (green, upper), BRCA1 (green, medial) and RAD51 (green, lower) immunostaining results during the DNA repair process. WT and cKO-Emx1-derived NSCs were treated with 50 μm etoposide for 2 h and then incubated with fresh medium for the times indicated. Representative images are shown. Scale bar, 5 μm. H–K Quantification of the percentage of cells with > 5 γH2AX foci ( H ), cells with > 5 53BP1 foci ( I ), cells with > 5 BRCA1 foci ( J ), cells with > 5 RAD51 ( K ) foci from ( G ). One-way ANOVA with Tukey’s post hoc test. Mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: AAV_Actb HR donor_U6_sgRNA_EF1a_GFP_polyA (RRID: addgene_97309) and AAV_Actb NHEJ donor_U6_sgRNA_EF1a_GFP_polyA (RRID: addgene_97310) were used to provide the HR donor and NHEJ donor for DSBs repair, respectively.

Techniques: Homologous Recombination, In Vitro, Modification, Functional Assay, Gene Expression, Fluorescence, Staining, Transfection, Two Tailed Test, Immunostaining, Derivative Assay, Incubation