Abstract
SNPs (single nucleotide polymorphisms) on a chromosome 16 locus encompassing FTO, as well as IRX3, 5, 6, FTM and FTL are robustly associated with human obesity. FTO catalyses the Fe(II)- and 2OG-dependent demethylation of RNA and is an AA (amino acid) sensor that couples AA levels to mTORC1 (mammalian target of rapamycin complex 1) signalling, thereby playing a key role in regulating growth and translation. However, the cellular compartment in which FTO primarily resides to perform its biochemical role is unclear. Here, we undertake live cell imaging of GFP (green fluorescent protein)-FTO, and demonstrate that FTO resides in both the nucleus and cytoplasm. We show using ‘FLIP’ (fluorescence loss in photobleaching) that a mobile FTO fraction shuttles between both compartments. We performed a proteomic study and identified XPO2 (Exportin 2), one of a family of proteins that mediates the shuttling of proteins between the nucleus and the cytoplasm, as a binding partner of FTO. Finally, using deletion studies, we show that the N-terminus of FTO is required for its ability to shuttle between the nucleus and cytoplasm. In conclusion, FTO is present in both the nucleus and cytoplasm, with a mobile fraction that shuttles between both cellular compartments, possibly by interaction with XPO2.
Highlights
GWAS (Genome-wide association studies) have indicated that SNPs on a chromosome 16 locus encompassing FTO, as well as IRX3, 5, 6, FTM and FTL are robustly associated with human obesity, with carriers of the risk alleles reported to have increased appetite [1,2,3]
We have previously reported that FTO is an amino acid (AA) sensor that couples AA levels to mTORC1 signalling, thereby playing a key role in regulating growth and translation [12]
FTO resides in both the nucleus and cytoplasm To mitigate against the possibility of cell fixation disrupting the dynamic physiological balance of FTO’s cellular compartmentalization, we performed live cell imaging of COS7 cells transiently expressing green fluorescent protein (GFP)-tagged FTO (GFP-FTO)
Summary
GWAS (Genome-wide association studies) have indicated that SNPs (single nucleotide polymorphisms) on a chromosome 16 locus encompassing FTO, as well as IRX3, 5, 6, FTM and FTL are robustly associated with human obesity, with carriers of the risk alleles reported to have increased appetite [1,2,3]. Transgenic manipulation of Fto in mice supports the notion that it regulates body size, with overexpression resulting in obesity [6], whereas Fto null mice [7] and humans homozygous for a loss-of-function allele [8] display post-natal growth retardation and have high early mortality. We have previously reported that FTO is an AA (amino acid) sensor that couples AA levels to mTORC1 (mammalian target of rapamycin complex 1) signalling, thereby playing a key role in regulating growth and translation [12]. Cells lacking FTO display decreased activation of the mTORC1 pathway, decreased rates of mRNA translation and increased autophagy all of which are likely to contribute to the phenotype of stunted growth seen in humans and mice homozygous for loss-of-function mutations in FTO [12]
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