Journal: Nature Communications

Article Title: Modulation of Nudt21 levels reveals dose-dependent roles of alternative polyadenylation in tissue regeneration

doi: 10.1038/s41467-026-68630-x

Figure Lengend Snippet: a Generation of Nudt21 conditional KO esophageal basal stem cells with dox-inducible HA-Nudt21 transgene. b Top: FACS-based EdU incorporation assay. Bottom: Western blot analysis for CFIm complex members Nudt21, Cpsf6, Cpsf7, and transgenic HA-Nudt21 (HA-tag), (n = 4 biol. replicates). c Immunofluorescence (top) and proteomic (bottom) analyses for differentiation markers (Krt13, Ppl), stem cell markers (Trp63, Sox2) and CFIm complex members (Nudt21, Cpsf6 and Cpsf7), in cultures exposed to calcium for 6 days (n = 3 biol. replicates). d, e Changes to APA in Nudt21 KO and Hypo cells relative to control, as measured by PAS-seq analysis. Plots display the log2-fold ratios of proximal PAS (“P”) to distal PAS (“D”) usage for each comparison. APA events determined by a significant false discovery rate (FDR) < 0.05 and changes in distal PAS usage > 14.5%. Cells were harvested for analysis after 4 days of ETOH, 4OHT or 4OHT+Dox treatment. f Euler plot showing common and distinct changes to PAS usage. Commonly shortened 3’UTRs (944 mRNAs) were used for subsequent analysis (g-j). g mRNAs undergoing 3’UTR shortening and containing putative miRNA target sites. Only binding sites for expressed miRNAs were considered. h Model: 3’UTR shortening eliminates miRNA binding sites, leading to increased protein levels and a stem cell differentiation block. i Relative mRNA and protein expression levels of Nudt21 targets that follow the model in (h), with the number of cognate miRNAs shown on the right. Analysis was performed after 4 days of ETOH (Control) or 4OHT (KO) or 4OHT+Dox (Hypo) treatment. Cut-offs: log2FC ≥ 1.3, and FDR ≤ 0.05. j PAS-seq gene-tracks for Jmjd1c and Sec24a , including examples of miRNA binding sites. k Effect of Jmjd1c and Sec24a suppression on differentiation. l Quantification of Trp63 signal from images generated in (k) (n = 3 biol. replicates). Statistical analysis: Box plots show median (center), 25th–75th percentiles (box), and whiskers to 1.5×IQR; outliers plotted individually; point inside boxplot signifies geometric mean. Error bars represent standard deviation of the mean for biological replicates. Statistical significance was determined using a non-parametric unpaired, two-tailed, Wilcoxon signed-rank test (l).

Article Snippet: Primary antibodies used were: Nudt21 (Santa Cruz, sc-81109, 1:100), β-Actin (Santa Cruz, sc-47778, 1:1000), Cpsf7 (Santa Cruz, sc-393880), Cpsf6 (Bethyl, 50-156-0173), Krt13 (abcam, ab92551), P63 (abcam, ab53039), Seh1l (abcam, ab218531), Nup107 (Invitrogen, PA5-20562), Nup133 (abcam, ab155990), Nup155 (Proteintech, 66359-1-1 g), Nup96 (Bethyl, A301-784A), Nup35 (abcam, ab169162), Gapdh (Fisher Scientific, NC0330823), Phospho-ATR (Ser428, Cell Signaling, 2853), ATR (C-1, sc-515173, Santa Cruz).

Techniques: Western Blot, Transgenic Assay, Immunofluorescence, Control, Comparison, Binding Assay, Cell Differentiation, Blocking Assay, Expressing, Generated, Standard Deviation, Two Tailed Test

Journal: mBio

Article Title: HIV-1 infection regulates gene expression by altering alternative polyadenylation correlated with CPSF6 and CPSF5 redistribution

doi: 10.1128/mbio.02865-25

Figure Lengend Snippet: Loss of CPSF6 expression globally shortens the 3’UTR of cellular mRNAs. ( A ) A549 WT, NT#H1, CPSF6-KO#B4, CPSF6-KO#B7, and CPSF6-KO#C8 cells were lysed, and proteins were analyzed by Nu-PAGE, followed by western blot using anti-CPSF6, anti-CPSF5, anti-CPSF7, and anti-GAPDH antibodies. Experiments were repeated at least three times, and a representative image is shown. Graphs show the average densitometry quantification of three replicates with standard deviation. Significance was determined using ANOVA multiple comparisons tests; *** P < 0.001; ns, not significant. ( B ) Total RNA from CPSF6-KO and WT A549 cells was prepared, and polyadenylated transcripts were identified by PAC-seq, followed by APA analysis using PolyAMiner. ( C ) A549 WT, NT#H1, CPSF6-KO#B4, CPSF6-KO#B7, and CPSF6-KO#C8 cells were infected with increasing amounts of HIV-1-GFP for 24 or 48 h. Infection was assessed as the percentage of GFP-positive cells by flow cytometry. ( D ) WT and CPSF6-KO#B7 A549 cells were infected with HIV-1-GFP at an MOI of 2 for 48 h. Cells were fixed, permeabilized, and stained using the following antibodies: (i) anti-CPSF5 (red) and anti-CPSF6 (green); (ii) anti-SC-35 (red) and anti-CPSF6 (green); (iii) anti-LEDGF/p75 (red) and anti-CPSF6 (green); and (iv) anti-LEDGF/p75 (red) and anti-SC35 (green). Nuclei were stained with DAPI (blue). Scale bar, 10 µm. ( E ) Percentage of A549 cells containing CPSF6, CPSF5, or CPSF7 in nuclear speckles (condensates) upon HIV-1 infection (average of three independent experiments with standard deviation). Cells containing CPSF6, CPSF5, or CPSF7 in nuclear speckles were determined by visual examination of 200 cells. Significance was determined using unpaired t -test; *** P < 0.001; ns, not significant. APA, alternative polyadenylation; CPSF5, cleavage and polyadenylation specificity factor subunit 5; CPSF6, cleavage and polyadenylation specificity factor subunit 6; CPSF7, cleavage and polyadenylation specificity factor subunit 7; DAPI, 4’,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; KO, knockout; LEDGF/p75, lens epithelium-derived growth factor; NT, non-targeting; PAC-Seq, Poly(A)-Click-Sequencing; UTR, untranslated region; WT, wild-type; hpi, hours post-infection.

Article Snippet: We used rabbit polyclonal antibodies against the following proteins: CPSF6 (Cat# ab99347, Abcam), CPSF7 (Cat#A301-359A, Bethyl Laboratories, Inc), LEDGF/p75 (Cat# A300-847A, Bethyl Laboratories, Inc), SLFN5 (Cat#PA5-53638, Invitrogen), and MxA (Cat#PA5-22101, Invitrogen) and VMA21 (Cat#21921-1-AP, Proteintech).

Techniques: Expressing, Western Blot, Standard Deviation, Infection, Flow Cytometry, Staining, Knock-Out, Derivative Assay, Sequencing