FTO levels affect RNA modification and the transcriptome

Abstract

A block of single-nucleotide polymorphisms within intron 1 of the FTO (fat mass and obesity associated) gene is associated with variation in body weight. Previous works suggest that increased expression of FTO, which encodes a 2-oxoglutarate-dependent nucleic acid demethylase, leads to increased body weight, although the underlying mechanism has remained unclear. To elucidate the function of FTO, we examined the consequences of altered FTO levels in cultured cells and murine brain. Here we show that a knockdown of FTO in HEK293 cells affects the transcripts levels of genes involved in the response to starvation, whereas overexpression of FTO affects the transcript levels of genes related to RNA processing and metabolism. Subcellular localization of FTO further strengthens the latter notion. Using immunocytochemistry and confocal laser scanning microscopy, we detected FTO in nuclear speckles and--to a lesser and varying extent--in the nucleoplasm and nucleoli of HEK293, HeLa and MCF-7 cells. Moreover, RNA modification analyses revealed that loss of Fto affects the 3-methyluridine/uridine and pseudouridine/uridine ratios in total brain RNA. We conclude that altered levels of FTO have multiple and diverse consequences on RNA modifications and the transcriptome.

Figure 1

Overexpression of FTO in Flp-In 293 T-Rex cells. (a) Real-time PCR analyses of FTO1_C1 and FTO2_D4 clones. Means±SD of three independent analyses using duplicates are given. ni, not induced, Flp-In, unmodified Flp-In 293 T-Rex cells. (b) Western blot analysis demonstrated overexpression of FTO protein in both clones. (c) Immunostaining using an antibody against FTO showed that overexpressed FTO is present in the nucleus. In addition, dot-like enrichment of FTO on evenly distributed nucleoplasmic background is visible.

Figure 2

Knockdown of FTO in Flp-In 293 T-Rex cells. (a) qPCR showed decreased FTO mRNA levels in both FTO-specific siRNA transfections. Three independent experiments were performed (mean values with SD are depicted). (b) Reduced protein levels were revealed by western blot.

Figure 3

Intranuclear localization of FTO in Hela (a) and MCF-7 cells (b). RNA-FISH for the non-coding long RNA MALAT1, which is a component of nuclear speckles, and immunostaining for FTO protein showed that FTO is enriched at nuclear speckles. Note that FTO is also present in nucleoli. DNA was counterstained with DAPI.

Figure 4

Effect of loss of FTO on normalized 3-methyluridine/uridine and pseudouridine/uridine ratios in total brain RNA. Each RNA sample was measured at least three times. The mean ratios of modified/unmodified U in wild-type animals were set to 1 and used to normalize the ratios in Fto ^−/− mice. Mean±SD are given.