Intracellular distribution of human SIRT7 and mapping of the nuclear/nucleolar localization signal

Sirtuins belong to a class of NAD-dependent deacetylases, and include seven distinct isoforms, of which SIRT7 is the least studied member. In the present study, the subcellular expression of SIRT7 in primary fibroblasts undergoing senescence was evaluated by immunocytochemistry and immunoblot assay. Expression of nucleolar SIRT7 in young fibroblast was very prominent, decreased in pre-senescent cells, and became undetectable in the senescent cells. Interestingly, we observed previously unreported staining for cytoplasmic SIRT7 in fibroblasts, and report the existence of a steady-state level of SIRT7 in cytoplasm. Selective localization of the high-molecular-mass (47.5 kDa) SIRT7 in the cytoplasmic fraction and the low-molecular-mass (45 kDa) SIRT7 in the nuclear fraction was observed in the immunoblot analysis of various cell types. The specificity of the N-terminal antibodies for detection of cytoplasmic SIRT7 was confirmed by RNAi and peptide competition assays. The two forms of SIRT7 showed reciprocal expression following serum starvation, nocodazole and okadaic acid treatments, and also during senescence. Using a combination of deletion constructs and site-directed mutagenesis, we defined the role of two distinct SIRT7 sequences in the N-terminal region (amino acids 61-76, LQGRSRRREGLKRRQE) and the C-terminal region (amino acids 392-400, KRTKRKKVT) for nuclear and nucleolar import, respectively. In conclusion, we report for the first time the existence of a cytoplasmic pool of SIRT7 in addition to its well-known nucleolar form, identify distinct localization signals for its nuclear/nucleolar targeting, and describe an association between loss of nucleolar SIRT7 and replicative senescence.