BioProject

Purpose: FTO is an N6-methyladenosine (m6A) RNA demethylase. The goals of this study are to to identify functional mRNA targets of FTO m6A demethylase activity in VHL deficient ccRCC cells. We performed transcriptome wide m6A sequencing and RNA sequencing analysis of two independent FTO deficient and wild type ccRCC cell lines. Methods: We performed transcriptome wide m6A sequencing and RNA sequencing analysis of two independent FTO deficient (shFTO#2 and shFTO#5) and wild type ccRCC cell lines. Results: Analysis using exomePeak identified a total of 20,168 m6A peaks that are different between the shFTO cells and the control cells. A total of 2,680 and 4,049 m6A peaks showed a significant increase and decrease (p < 0.05), respectively, in abundance (normalized to input), in shFTO cells relative to Ctrl_cells, and they were thus termed hyper- and hypo-methylated m6A peaks, respectively. Through integrative analysis of the RNA-seq data, we identified 459 hypermethylated m6A peaks the RNA transcripts of which were significantly (p < 0.05) downregulated (255; Hyper-down) or upregulated (204; Hyper-up) in shFTO cells relative to Ctrl cells (Figure XB). Conclusions: Our study represents the first detailed analysis of FTO driving transcriptomes, with biologic replicates, generated by RNA-seq technology and m6A sequencing. Our results confirmed the functional role of FTO in mRNA demethylase and identify the targets of FTO. We conclude that FTO plays important role in mRNA destiny determination by m6A demethylase activity. Overall design: m6A-specific antibodies are used to immunoprecipitate RNA. RNA is reverse-transcribed to cDNA and deep sequenced. In paralal mRNA of UMRC2 cell transfected with shSCM, shFTO#1 and shFTO#2 were generated by deep sequencing.