Fig 2: siRNA-based high-throughput screen identifies CNOT6 as a potential MMR regulator. (A) Schematic depiction of the screening procedure combining custom made siRNA libraries of 160 human nucleases, MNNG treatment. The screening endpoint was cell survival. (B) The top five candidates from two independent experiments are shown. (C) SiRNA-mediated knockdown efficiency was quantified for the indicated genes by real-time quantitative PCR. GAPDH was the reference gene (n = 3). (D) Representative immunoblots of MSH2, CNOT6 and Actin in extracts of U2OS cells treated with siRNA targeting CNOT6 or MSH2. (E) The viability of siRNA- transfected cells treated with MNNG at the indicated dose is shown. Data are shown as mean ± SEM, n = 3. Statistical significance (** p < 0.01, *** p < 0.001) was determined using an unpaired two-tailed Student’s t test. (F) Cell viability in the presence of the indicated dose of MNNG is shown; relative survival is shown for cells overexpressing CNOT6 or control cells. Data are shown as mean ± SD, n = 3, ** p < 0.01, using unpaired two-tailed Student’s t test. (G) Representative immunoblots of CNOT6 and Actin in extracts of U2OS cells overexpressing CNOT6 or control cells.

Fig 3: Proposed model for the regulation of MMR by CNOT6. (A) The stability of MMR transcripts under normal condition. (B) Depletion of CNOT6 leads to enhanced MMR, follow by increased DSBs and apoptosis. To explain the increased DSBs and apoptosis in CNOT6-depleted cells after MNNG treatment, there are several hypotheses. (a) The depletion of CNOT6 leads to unbalanced increase of MMR proteins, due to the different extension of mRNA half-life. (b) Increased MMR proteins may lead to more proteins that bind to the mispairs, which could impede the repair progress. (c) MMR can suppress recombination, which is important in DSB repair. (d) Processing by multiple repair pathways at the same site could slow down or interfere the repair. All these could activate a G2 checkpoint and subsequent cell cycle arrest.

Fig 4: Depletion of CNOT6 increases apoptosis after exposure to MNNG, stimulates MMR and reduces mutation frequency in MMR-proficient cells. (A) Representative scatter plots of PI vs. Annexin V staining of U2OS cells transfected with siRNA targeting LUC, MSH2 or CNOT6 and treated with or without 0.5 µM MNNG as described in Materials and Methods. (B) Quantification of flow cytometry data. Data are shown as mean ± SEM, n = 3; “ns” signifies “not significant”, * p < 0.5, ** p < 0.01, using unpaired two-tailed Student’s t test. (C) MMR activity in extracts from MSH2-depleted or CNOT6-depleted or control siLUC-treated U2OS cells. MMR was quantified using in vitro MMR assay (see Materials and Methods). Data are shown as mean ± SEM, n = 3; Statistical significance (*** p < 0.001) was determined using unpaired two-tailed Student’s t test. (D,E) HPRT assay was used to estimate mutation frequency in CNOT6-depleted (D) HCT116 + Chr3 (MMR-proficient) and (E) HCT116 (MMR-deficient) cells. Data are shown as mean ± SEM, n = 3; statistical significance (* p < 0.5) was determined using unpaired two-tailed Student’s t test.

Fig 5: CNOT6 deficiency stabilizes MMR mRNA transcripts. (A) U2OS cells transiently overexpressing Flag-tagged-CNOT6 were collected and lysed, and cell extracts used for anti-Flag immunoprecipitation. Representative immunoblots for the indicated proteins are shown. (B) Representative immunoblots for the indicated MMR factors using extracts from CNOT6-depleted cells. (C) Real-time quantitative PCR was performed to quantify expression of the indicated MMR genes in CNOT6-depleted cells. Data are shown as mean ± SEM, n = 3; statistical significance (* p < 0.5, ** p < 0.01, *** p < 0.001) was determined using unpaired two-tailed Student’s t test. (D) The half-life of MMR gene transcripts in control and CNOT6-depleted cells with the treatment of transcriptional inhibitor Actinomycin D is shown. n = 3. (E) Data from panel D are compared to demonstrate the effect of CNOT6-depletion on the half-life of MMR gene transcripts.