Journal: bioRxiv

Article Title: ZCCHC4 Promotes Translation of Replication-dependent Histone mRNAs by Recruiting Cytoplasmic eIF3 complex

doi: 10.1101/2025.06.21.660898

Figure Lengend Snippet: a, Venn diagram showed the overlap of ZCCHC4 PAR-CLIP-seq target genes between two biological replicates. b, Distribution plots (up) and sequence motif enrichment analysis (down) of ZCCHC4-bound histone mRNAs. ZCCHC4-binding motifs were identified by HOMER findMotifsGenome.pl from the PAR-CLIP-seq peaks of two biological replicates. The motif length was restricted to 4-8 nucleotides. The P -value was calculated from the random background sequences with a ZOOPS score. c, RIP/qPCR analyses showed the binding of ZCCHC4 and potential targets in wild-type HeLa cells. d, Volcano plot depicting down-regulated (blue) and up-regulated (red) genes identified by Ribo-seq in ZCCHC4 KO cells compared with WT cells. e, Integrated Genome Viewer (IGV) showing the mapped reads of the binding peaks identified by ribosome-bound fragments identified by Ribo-seq of H2BC6 after ZCCHC4 knockout. The histograms represent the sum of mapped reads along the genome. The mapped reads of Ribo-seq were normalized by the sequencing depth. f, Volcano plot depicting down-regulated (blue) and up-regulated (red) genes identified by TMT in ZCCHC4 KO cells compared with WT cells. g, Cumulative distribution of translation efficiency with histone mRNA (red) and non-histone (blue) mRNA by TMT (up) and Ribo-seq (down). h, Volcano plot depicting replication-dependent (blue, RD-Histone) and replication-independent (red, RI-Histone) histone identified by Ribo-seq (left) and TMT (right) in ZCCHC4 KO cells compared with WT cells. i, Western blot analysis to validate the expression of RD-histone (H2A and H2B) and RI-histone (H2AZ) in ZCCHC4 knockout and control cells. β-actin was used as internal control. j, The broken line chart showed the relative distribution of the mRNA level of H2B, H2AC and H4A in the different fractions isolated by the sucrose gradient. The same volume (50 µL) of the fractions was loaded to perform RT-qPCR. k, RT-qPCR analysis of polysome-or monosome (80S)-bound histone mRNA in WT (left) ZCCHC4 knockout (right) HeLa cells. Note: c , k Two-tailed unpaired Student’s t tests. Data were presented as mean ± SD (n=3). * P <0.05, * * P <0.01, ** * P <0.001, **** P <0.0001. ns: not significant.

Article Snippet: The corresponding antibodies included anti-ZCCHC4 antibody with 1:1000 dilution (Abcam, ab209901), anti-RPS6 antibody with 1:1000 dilution (Proteintech, 66886-1-Ig), anti-RPL14 antibody with 1:1000 dilution (Proteintech, 14991-1-AP), anti-CDKN1A antibody with 1:1000 dilution (Proteintech, 10355-1-AP), anti-puromycin antibody with 1:1000 dilution (ABclonal, A21205), anti-H2B antibody with 1:2000 dilution (PTM-1007), anti-H2A antibody with 1:2000 dilution (ABclonal, A3692), anti-H2AZ antibody with 1:2000 dilution (Proteintech, 16441-1-AP), anti-HistoneH1.2 antibody with 1:1000 dilution (Proteintech, 19649-1-AP), anti HistoneH3 antibody with 1:2000 dilution (ABclonal, A22348), anti-α-Tubulin antibody with 1:3000 dilution (Proteintech, 80762-1-RR), anti-EIF3H antibody with 1:1000 dilution (Proteintech, 11310-1-AP), anti-EIF3A antibody with 1:1000 dilution (Proteintech, 26178-1-AP), anti-PDCD4 antibody with 1:1000 dilution (Proteintech, 12587-1-AP), anti-CyclinB1 antibody with 1:1000 dilution (ABclonal, A19037), anti-GAPDH antibody with 1:5000 dilution (Proteintech, 10494-1-AP), anti-β-actin antibody with 1:3000 dilution (SAB Signalway Antibody, 52901), and anti-Flag antibody with 1:2000 dilution (Sigma, F1804).

Techniques: Sequencing, Binding Assay, Knock-Out, Western Blot, Expressing, Control, Isolation, Quantitative RT-PCR, Two Tailed Test