N6-Adenosine Methylation in MiRNAs

<div><p>Methylation of N6-adenosine (m6A) has been observed in many different classes of RNA, but its prevalence in microRNAs (miRNAs) has not yet been studied. Here we show that a knockdown of the m6A demethylase FTO affects the steady-state levels of several miRNAs. Moreover, RNA immunoprecipitation with an anti-m6A-antibody followed by RNA-seq revealed that a significant fraction of miRNAs contains m6A. By motif searches we have discovered consensus sequences discriminating between methylated and unmethylated miRNAs. The epigenetic modification of an epigenetic modifier as described here adds a new layer to the complexity of the posttranscriptional regulation of gene expression.</p></div

miRNAs immunoprecipitated by the anti-m6A antibody.

<p>Seventeen miRNAs with >100 fold enrichment after immunoprecipitation in m6A RNA samples compared to IgG RNA samples are listed. Means and standard deviations of fold enrichments in three independent experiments are given. The complete list of immunoprecipated miRNAs can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118438#pone.0118438.s003" target="_blank">S3 Table</a>.</p><p>miRNAs immunoprecipitated by the anti-m6A antibody.</p

<i>K</i>nockdown of <i>FTO</i> does not significantly change mRNA levels of genes involved in miRNA biogenesis.

<p>The steady-state mRNA levels of <i>DICER</i>, <i>DROSHA</i>, <i>DGCR8</i> and <i>ADAR</i> were analyzed by qRT-PCR in cells treated with scrambled (scr) and <i>FTO-</i>specific siRNAs, respectively. <i>GAPDH</i> was used as a reference gene. The observed changes were not significant. Merged values of mean ± SD from triplicates per assay for the three independent cell lines FTO1C1, FTO2D4 and FTO3C3 are depicted. <i>FTO</i> kd, <i>FTO</i>-specific siRNA treated cells, scr siRNA, scrambled siRNA treated cells.</p

Effect of <i>FTO</i> knockdown on the steady state levels of methylated miRNAs.

<p>X-axis, log2 fold-changes (log2fc) of enrichment after imuunopreciptation with an anti-m6A antibody; y-axis, log2 fold-changes of steady state miRNA levels after <i>FTO</i> knockdown. The values of all 239 methylated miRNAs are shown. The red dotted line is the regression line.</p

Deregulation of miRNAs in <i>FTO</i> knockdown cells.

<p>Mature miRNAs showing increased (A) and decreased (B) steady state levels in <i>FTO</i> knockdown cells. Normalized RNA-seq read numbers of individual miRNAs in <i>FTO</i> knockdown and scrambled siRNA treated cells were compared. Mean ± SD of three independent experiments are depicted. For verification and further studies, qRT-PCR analyses of selected mature miRNAs (C) and primary miRNA transcripts (D) were performed. We did not use other small RNAs as a reference gene for measuring mature miRNAs levels (as suggested by Life Technologies), since depletion of <i>FTO</i> might have an impact on their levels. Therefore, luciferase RNA was used to generate a standard curve and added to the qRT-PCR assays. <i>GAPDH</i> was used as a reference gene for measuring primary miRNAs transcript levels. Mean ± SD from quadruplicates per assay for three independent cell lines (FTO1C1, FTO2D4 and FTO3C3) are depicted. kd, <i>FTO</i> specific siRNA treated cells, scr, scrambled siRNA treated cells.</p