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-oxoglutaratedependent 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. Keywords: FTO; RNA modifications; nuclear speckles; transcriptome INTRODUCTION Genome-wide association studies have revealed a strong association between a block of single-nucleotide polymorphisms (SNPs) in intron 1 of the fat mass and obesity-associated (FTO) gene, body mass index and other obesity-related traits in children and adults of different populations. 1-3 Stratigopoulos et al 4 have suggested that one of the SNPs (rs8050136) affects binding of the transcriptional regulator CUX1. By studying allelic expression levels in heterozygous individuals, we have found that the risk allele of the FTO gene makes more transcripts and have proposed that increased expression of the FTO gene leads to increased body weight. 5 This hypothesis is supported by the clinical findings in rare patients and in mouse models with an FTO/Fto mutation. Homozygous loss-of-function of FTO was reported to cause severe growth retardation and multiple malformations, 6 whereas a duplication of FTO was found to be associated with morbid obesity. 7 Fto-knockout mice 8 and mice with a missense mutation in exon 6 9 showed leanness, postnatal growth retardation and a higher metabolic rate. Mice with one or two additional copies of Fto had a gene-dosage-dependent increase in body weight. 10 FTO is a member of non-heme Fe(II)-and a-ketoglutaratedependent oxygenase superfamily and is found in vertebrates and green algea, but not in invertebrate animals, fungi and green plants. 11 By in vitro studies, FTO was shown to function as a demethylase with a strong preference for 3meU and 3meT in single-stranded RNA and DNA, respectively. 12,13 Han et al 14 have provided structural evidence for understanding the substrate specificity of FTO and have suggested

N6-Adenosine Methylation in MiRNAs

“…In addition to let-7b, we also analyzed let-7e, although it was reduced by only 21% (S2 Table), because in a previous study [18] we had observed reduced transcript levels of LIN28B , which is a negative regulator of this particular family of miRNAs [27]. We confirmed reduced levels of let-7e, miR-7–5p and miR-22–3p miRNAs (Fig.…”

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA Met to expand codon recognition in mitochondrial translation

“…While the Ten‐Eleven Translocation (TET) protein family of dioxygenases primarily mediates oxidation of m 5 C in nuclear DNA and has also been implicated in histone modification, most of the members of the AlkB‐like Fe(II)/alpha‐ketoglutarate‐dependent dioxygenases (ALKBH) have been shown to act on RNA (reviewed in Shen et al , 2014; Fedeles et al , 2015; Li et al , 2015; Ougland et al , 2015). These include FTO (ALKBH9) that is implicated together with ALKBH5 in the oxidative removal of several modifications including 6‐methyladenosine (m 6 A) from RNA and ALKBH8 that is involved in the generation of 5‐methoxycarbonylmethyluridine (mcm 5 U) in cytoplasmic tRNAs (Fu et al , 2010a,b; Songe‐Møller et al , 2010; Jia et al , 2011; Thalhammer et al , 2011; Berulava et al , 2013; Zheng et al , 2013). So far, only ALKBH7, which was suggested to act on protein substrates during necrosis (Fu et al , 2013; Solberg et al , 2013; Wang et al , 2014), and ALKBH1/ABH1 have been reported to localise to mitochondria; however, the cellular localisation of ABH1 has been a matter of debate (Pan et al , 2008; Westbye et al , 2008; Ougland et al , 2012).…”

What a difference a hydroxyl makes: mutant IDH, (R)-2-hydroxyglutarate, and cancer

“…Other potential 2OG-dependent targets of (R)-2HG include FIH1 (factor inhibiting hypoxia-inducible factor 1), an asparaginyl hydroxylase that regulates the transcriptional activity of HIF (Mahon et al 2001); the ABH family of DNA demethylases that are involved in DNA damage repair (Lee et al 2005); and the RNA demethylase FTO (fat mass and obesity-associated), which is believed to be important for the regulation of cellular metabolism (Jia et al 2008;Berulava et al 2013). …”