Fig 2: 5'tsRNA-Gly-GCC targets 3'UTR of Sirt6 mRNA. (A) An illustration of the putative site of 5'tsRNA-Gly-GCC binding within the 3'UTR of Sirt6 predicted by bioinformatics software RNAhybrid, with green used to highlight those nucleotides mutated for a mutant version of this sequence. (B) Luciferase activity assay revealed that 5'tsRNA-Gly-GCC mimic transfection into Hepa1-6 cells resulted in a 40% reduction in luciferase activity for constructs containing a WT but not a mutated version of this 3'UTR of Sirt6 binding site (n = 7). (C) Luciferase activity assay revealed that 5'tsRNA-Gly-GCC inhibitor transfection into Hepa1-6 cells significantly increased WT Sirt6 3'UTR activity, while 5'tsRNA-Gly-GCC inhibitor also had no significant effect on MUT Sirt6 3'UTR (n = 7). ****p < 0.0001.

Fig 3: APC/C leads to SIRT6 decrease. (a) HeLa cells were synchronized to the G2 phase and samples were collected with the procedure shown in Supplementary Fig. S4. G2 samples were collected without release from RO-3306 and other cells were released. The time when most cells entered mitosis was designated as 0 h when mitotic cells were physically detached, and detached cells were cultured separately and sampled at 0.5 h intervals from 0 to 2.5 h. Changes in the indicated proteins were detected by immunoblotting. Actin was used as a loading control. (b) 293 T and HeLa cells were treated with 10 µM proTAME or DMSO for 12 h. SIRT6 levels were examined by immunoblotting. Actin was used as a loading control. (c) 293 T and HeLa cells were transfected with non-specific control (NC) or APC1 knockdown siRNAs. SIRT6 levels were examined by immunoblotting. Actin was used as a loading control.

Fig 4: Overexpression of SIRT6 leads to up-regulation of various APC/C substrates and defective mitosis. (a) 293 T and HeLa cells stably expressing empty vehicle (EV), SIRT6-FLAG (SIRT6), SIRT6-FLAG, and CDH1-HA simultaneously (ST6 + CDH1) were treated with thymidine block and subjected to western blotting. Levels of TPX2, cyclin B1, AURKA, AURKB, securin, FLAG, and CDH1 were detected using the corresponding antibodies. Actin was used as a loading control. Short exposures of 293 T CDH1 are shown in the original image section of the supplementary file. (b) Proportions of cells with three centrosomes or more were counted in control and SIRT6 overexpressing 293 T and HeLa cells (n = 100). (c) Proportions of cells with two or more nuclears were counted in control and SIRT6 overexpressing 293 T and HeLa cells (n = 100). (d) Mechanistic illustration of the current study.

Fig 5: SIRT6 overexpression leads to abnormal mitosis. (a) Growth curves of control and SIRT6 overexpressing 293 T and HeLa cells. (b) Validation of SIRT6-FLAG by anti-FLAG blotting. Phosphorylation of histone H3 at Ser10 and cyclin B1 were also detected. HSP70 was used as a loading control. (c) Cell cycle distribution of control and SIRT6 overexpression in 293 T and HeLa cells. (d) Box-and-whisker plot showing the duration from NEB to completion of cytokinesis in 293 T stably expressing H2B.1-mCherry, which were transfected with empty vehicle (EV, n = 62) or SIRT6 overexpressing (ST6, n = 62) lentivirus. (e) Box-and-whisker plot showing the duration from NEB to chromosome alignment completion (P-PM), chromosome alignment maintenance (Meta), and anaphase onset to cytokinesis completion (Ana-telo) of cells described in d. (f) MCF10A cells were infected with SIRT6 overexpressing (n = 58) or empty vehicle (n = 63) lentivirus. Chromosomes of metaphase spreads were counted within 50 generations.