Fig 2: PCIF1 is located at the promoter regions of its target genes. Chromatin immunoprecipitation (ChIP) analyses of RAB23 (A–C) and CNOT6 (D–F) genes. (A,D) Schematic representations of the indicated gene structures; the transcription start sites are indicated by arrows, the exons are shown as black boxes, and the polyadenylation signals are indicated by arrowheads. The positions of the PCR amplicons for ChIP are indicated by the numbers below each diagram. The position of the center of each amplicon relative to the transcription start site (+1) is indicated on the right. The lower panel shows the results of the ChIP analyses of the indicated regions of the RAB23 (B,C) and CNOT6 (E,F) genes in HeLa cells using antibodies against PCIF1 (B,E) and Pol II (C,F). The y-axis represents the ChIP signal relative to promoter (number 2 position). Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between position 1 and position 2 (Student’s t-test, *** p < 0.001).

Fig 3: CNOT7v2 is a CCR4–NOT subunit. (A) Interaction of CNOT7v2 with CCR4–NOT components. Vector expressing V5-CNOT7v2 (or control plasmid) was transfected into MCF7 cells. Lysates of transfectants were immunoprecipitated (IP) with either normal mouse IgG or anti-V5 antibody. Total lysates (input) and eluted fractions were analyzed on a 4–12% SDS-PAGE gel, followed by immunoblotting with the indicated antibodies. (B) Interaction of CNOT7 isoforms with endogenous CNOT1. Vectors expressing V5-CNOT7v1 or v2 (or control plasmid) were transfected into MCF7 cells. Extracts were immunoprecipitated with anti-CNOT1 antibody. Total lysates (input) and eluted fractions were analyzed with the indicated antibodies. (C) Interaction between CNOT7v2 and CNOT6 was analyzed by GST pull-down experiments. In vitro translated biotin-labelled CNOT6 was incubated with an equivalent amount of GST, GST-CNOT7v1 or GST-CNOT7v2 bound to glutathione-Sepharose beads. The input (10%) and eluted proteins were analyzed by SDS-PAGE, electroblotted on a membrane and detected by streptavidin-mediated chemiluminescence. Biotin-labelled luciferase was tested in parallel as a negative control. GST fusion proteins used in this assay were shown by Coomassie blue staining (Supplementary Figure S6A). (D) A mammalian two-hybrid assay was used to analyze the interaction of CNOT7 variants with BTG proteins. Coding sequences of BTG1 and 2 were inserted in frame with the GAL4 binding domain, and CNOT7v1/v2 coding sequences with the VP16 transactivation domain. The pG4-TK-Luc reporter plasmid contains 6 GAL4 binding elements, upstream of the thymidine kinase minimal promoter region fused to the luciferase reporter gene. The interaction between the GAL4 and VP16 fusion proteins results in an increase in luciferase expression. Luciferase activities were measured in MCF7 cells 24 h post transfection and are expressed as the fold increase over the luciferase activity of the reporter vector alone.

Fig 4: PCIF1 suppression resulted in a significant decrease in m6A levels of both RAB23 and CNOT6 mRNAs. MeRIP-qPCR analysis was performed using HeLa cells treated with control siRNA (siNC) or PCIF1-targeted siRNA (siPCIF1: siPCIF1 #3 was used for PCIF1 suppression). RT-qPCR analysis of RNAs purified from the immunoprecipitates of HeLa cell extracts using the anti-m6A antibody, using the specific primer set detecting RAB23 (A), CNOT6 (B), and ACTB (C) mRNAs. The y-axis represents the fold change relative to the RNA levels in the immunoprecipitate by anti-m6A antibody from HeLa cells treated with control siRNA (siNC). Normal rabbit IgG (IgG) was used as a negative control. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the indicated pairs (Student’s t-test, n.s. p > 0.05, *** p < 0.001).

Fig 5: PCIF1 does not regulate expression of its target genes at the transcriptional level. (A,E) Schematic illustrations of the RAB23 (A) and CNOT6 (E) genes. The transcription start sites are indicated by arrows, the exons are shown as black boxes, and the polyadenylation signals are indicated by arrowheads. The positions of the PCR primer set for RT-qPCR amplification of the precursor and mature mRNAs are indicated by arrows. (B–D,F–H) RT-qPCR analysis of total RNAs isolated from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #3) using the indicated primer sets. (I,J) ChIP analyses of the RAB23 (I) and CNOT6 (J) gene promoters (position 2 in Figure 4) using antibodies against Pol II in HeLa cells treated with control siRNA (siNC) and two distinct PCIF1 targeted siRNAs (siPCIF1 #1 and #3). Normal rabbit IgG was used as the negative control. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the control siRNA treatment and the indicated PCIF1 targeted siRNA (Student’s t-test, n.s. p > 0.05, *** p < 0.001).