RNA-Seq data of ALKBH5 and FTO double knockout HEK293T human cells

Abstract

N6-methyladenosine (m6A) is the most abundant, highly dynamic mRNA modification that regulates mRNA splicing, stability, and translation. The m6A epigenetic mark is erased by RNA demethylases ALKBH5 (AlkB Homolog 5) and FTO (Fat mass and obesity-associated protein). The ALKBH5 and FTO RNA demethylases recognize m6A in similar nucleotide contexts. Therefore, these proteins can partially substitute for each other. To assess the impact of total m6A demethylation failure we performed high-throughput sequencing of cytoplasmic RNA from ALKBH5 and FTO double knockout and wild type HEK293T cells. The RNA-Seq libraries were sequenced on Illumina NextSeq 500, trimmed, and mapped to the human genome. The consequent read counting and differential expression analysis in the R environment detected 5871 differentially expressed and 166 alternatively spliced genes comparing double knockout against wild type HEK293T cells. Raw data are deposited in NCBI Gene Expression Omnibus (GEO) repository under GEO accession GSE198050.

Keywords: ALKBH5; Demethylase; Epitranscriptomics; FTO; RNA modifications; Transcriptome; m6A demethylation; m6A methylation.

Fig. 1

Evaluation of ALKBH5 and FTO knockout. (A) Schematic representation of guide RNA (gRNA) locations. Two gRNAs targeting the third exon of FTO (for nickase CRISPR/Cas9) and two gRNAs targeting the first and third exons of ALKBH5 (for CRISPR/Cas9) are shown. The coordinates are according to the hg38 genome assembly. (B) Western blot of wild type and ΔALKBH5ΔFTO HEK293T cells using anti-ALKBH5, anti-FTO, and anti-ACTB as a loading control.

Fig. 2

The data are suitable for analysis of differential gene expression under FTO and ALKBH5 knockout. (A) Principal component analysis for three replicates of HEK293T (WT) and three double knockout (KO) samples, X and Y axes correspond to the first two principal components. (B) Barplot illustrating FTO and ALKBH5 expression in counts per million (CPM). (C) Volcano plot of differential expression analysis. The red line indicates 5% FDR, gray lines indicate -1 and 1 log₂(Fold Change).

Fig. 3

The data are suitable for analysis of differential exon usage under FTO and ALKBH5 knockout. (A) Volcano plot of differential splicing analysis. The red line indicates 5% FDR, several illustrative examples of alternative splicing are highlighted. (B-F) Genomic views of RNA-Seq profiles for the selected genes with alternative splicing events.