Fig 1: SKIV2L2 binds to and modulates the half-life of replication dependent histone mRNAs. (A) Western blot to detect immunoprecipitated SKIV2L2 in N2A cells. Western blots to detect SKIV2L2 were performed on the following samples from N2A cells. (Lane 1) Whole-cell extract incubated with anti-goat IgG. (Lane 2) Immunoprecipitate with anti-goat IgG. (Lane 3) Whole-cell extract incubated with anti-SKIV2L2. (Lane 4) Immunoprecipitate with SKIV2L2. The top band indicates the position of SKIV2L2, and the lower band represents the heavy chain from the antibody used for immunoprecipitation. (B) Western blot to detect immunoprecipitated SKIV2L2 in P19 cells. Western blots to detect SKIV2L2 were performed in P19 cells as stated above. (Lane 1) Whole-cell extract incubated with anti-goat IgG. (Lane 2) Immunoprecipitate with anti-goat IgG. (Lane 3) Whole-cell extract incubated with anti-SKIV2L2. (Lane 4) Immunoprecipitate with SKIV2L2. (C) qRT-PCR of histone RNAs bound to immunoprecipitated SKIV2L2 in N2A cells. Proteins were extracted from N2A cells irradiated with UV light. SKIV2L2 was immunoprecipitated with its cross-linked RNAs using anti-SKIV2L2, with anti-goat used as a negative control. RNA was isolated from both anti-SKIV2L2 and anti-goat immunoprecipitates. Using qRT-PCR, RNA abundance levels of histone mRNAs immunoprecipitated in either sample were calculated using ?Cq values, and normalized to the amplification of Skiv2l2 mRNA, which was unbound in both samples (error bars represent ±SD for n = 3). Immunoprecipitated retro-Phgdh represents an RNA precipitated equally by anti-goat and anti-SKIV2L2. (D) qRT-PCR of histone RNAs bound to immunoprecipitation SKIV2L2 in P19 cells. Performed as stated in C on immunoprecipitate from P19 cells. (E) Northern blot demonstrating H4 mRNA turnover following Dactinomycin treatment. Following transfection with control or Skiv2l2 siRNA, RNA was extracted at 0, 1, 2, 3, and 4 h following application of Dactinomycin. Northern blotting detected H4 mRNA, which was quantified and normalized to ribosomal RNA levels to calculate half-life.
Fig 2: Zcchc8-null mice have TR insufficiency. (A–C) Immunoblot for ZCCHC8, SKIV2L2, and RBM7, respectively, on lysates from mouse ear fibroblasts. (D,E) Northern blot for mouse TR and quantification. For E, mean reflects mice Zcchc8+/+ (n = 4, 2M/2F), Zcchc8+/− (n = 4, 2M/2F), Zcchc8−/− (n = 3M), mTR+/− (n = 2, sex unknown) and mTR−/− (n = 2, sex unknown). (F) TR 3′ extended levels (>20 bp) relative to Hprt as measured by qRT-PCR. Mouse numbers and M/F designations as in E. Data are expressed as mean ± SEM. (*) P < 0.05; (**) P < 0.01 (Student's t-test, two-sided).
Fig 3: ZCCHC8 loss of function is sufficient to cause low TR levels. (A) Immunoblot of ZCCHC8 in lymphoblastoid cell lines (LCLs) from healthy controls (C1 and C2) and unaffected relatives and mutation carriers labeled with pedigree identifiers from Figure 1A. Quantification from one blot and result replicated twice from independently harvested protein lysates. (B) Immunoblot of ZCCHC8, SKIV2L2, and RBM7 levels in proband's primary skin fibroblasts. (C) Immunoblot of transfected Myc-tagged (293FT cells) and endogenous ZCCHC8. (D) Mean ZCCHC8 mRNA levels ± SEM from LCLs in unaffected family members (n = 4) and ZCCHC8 p.P186L mutation carriers (n = 3). (E) Chromatogram showing that ZCCHC8 p.P186L mutation is expressed in LCL mRNA from proband (also verified in two other mutation carriers). (F) Immunoblot showing efficiency of shRNA knockdown of Luciferase (Luc), ZCCHC8, and NAF1 in HeLa cells. (G) Total TR levels measured by qRT-PCR (mean ± SEM from three independent knockdowns and RNA isolations). (H) Northern blot of TR after stable knockdown of ZCCHC8 and NAF1 (replicated twice with independent RNA isolations). (**) P < 0.01; (***) P < 0.001 (Student's t-test, two-sided).
Fig 4: Skiv2l2 knockdown results in delayed proliferation attributed to slowed G2/M phase progression. (A) MTT time point assay in N2A cells. Absorbance of formazan (570 nm) at 24, 28, and 32 h following treatment with control siRNA, ATRA, or Skiv2l2 siRNA (n = 4 for each time point, [*] represents P-value <0.05). (B) MTT assay in P19 cells. Absorbance of formazan was measured after 48 h of treatment with control siRNA, ATRA, or Skiv2l2 siRNA (error bars represent ±SD for n = 9). (C) Propidium iodide viability staining of N2A cells. Fluorescing cells (dead or necrotic) detected by the FL-2 filter are circled, and the Accuri C6 software was used to quantify the percentage of control, ATRA treated, and Skiv2l2 knockdown cells staining with propidium iodide. (D) Cell-cycle analysis with propidium iodide staining of fixed N2A cells. Cells treated with control siRNA, Skiv2l2 siRNA, or ATRA were excited at 488 nm and sorted using FL-2 on the Accuri C6 based on propidium iodide fluorescence. The sorted cells corresponded to G1, S, and G2/M phase as denoted. (E) Quantification of cell-cycle profile. Based on the cell-cycle landscapes generated in D, the Accuri C6 software was used to calculate the percentage of cells in G1, S, and G2/M phase, where n = 3 sets of 50,000 cells and statistically significant differences (P-value <0.05) are denoted with an asterisk (*).
Fig 5: Replication-dependent histone mRNAs accumulate in Skiv2l2 knockdown cells. (A) Transcripts up-regulated in P19 cells following Skiv2l2 knockdown as measured by RNA-seq. Transcripts up-regulated in Skiv2l2 knockdown cells were categorized based on coding and noncoding annotations (q-value <0.05, >1.5-fold change). (B) Transcripts down-regulated in P19 cells following Skiv2l2 knockdown as measured by RNA-seq. Transcripts down-regulated in Skiv2l2 knockdown cells categorized based on coding and noncoding annotations (q-value <0.05, >1.5-fold change). (C) qRT-PCR of histone mRNAs in N2A cells. Histone mRNA levels were calculated using ?Cq values and normalized to ß-actin mRNA (error bars represent ±SD for n = 3). (D) qRT-PCR of histone mRNAs in P19 cells. Histone mRNA levels calculated as in C (error bars represent ±SD for n = 3).
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