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. 2017 Sep;23(9):1444-1455.
doi: 10.1261/rna.061044.117. Epub 2017 Jun 13.

N6-methyladenosine is required for the hypoxic stabilization of specific mRNAs

Nate J Fry  1 , Brittany A Law  2 , Olga R Ilkayeva  3 , Christopher L Holley  2 , Kyle D Mansfield  1
Affiliations

N6-methyladenosine is required for the hypoxic stabilization of specific mRNAs

Nate J Fry et al. RNA. 2017 Sep.

Abstract

Post-transcriptional regulation of mRNA during oxygen deprivation, or hypoxia, can affect the survivability of cells. Hypoxia has been shown to increase stability of a subset of ischemia-related mRNAs, including VEGF. RNA binding proteins and miRNAs have been identified as important for post-transcriptional regulation of individual mRNAs, but corresponding mechanisms that regulate global stability are not well understood. Recently, mRNA modification by N6-methyladenosine (m6A) has been shown to be involved in post-transcriptional regulation processes including mRNA stability and promotion of translation, but the role of m6A in the hypoxia response is unknown. In this study, we investigate the effect of hypoxia on RNA modifications including m6A. Our results show hypoxia increases m6A content of poly(A)+ messenger RNA (mRNA), but not in total or ribosomal RNA in HEK293T cells. Using m6A mRNA immunoprecipitation, we identify specific hypoxia-modified mRNAs, including glucose transporter 1 (Glut1) and c-Myc, which show increased m6A levels under hypoxic conditions. Many of these mRNAs also exhibit increased stability, which was blocked by knockdown of m6A-specific methyltransferases METTL3/14. However, the increase in mRNA stability did not correlate with a change in translational efficiency or the steady-state amount of their proteins. Knockdown of METTL3/14 did reveal that m6A is involved in recovery of translational efficiency after hypoxic stress. Therefore, our results suggest that an increase in m6A mRNA during hypoxic exposure leads to post-transcriptional stabilization of specific mRNAs and contributes to the recovery of translational efficiency after hypoxic stress.

Keywords: N6-methyladenosine; hypoxia; mRNA stabilization; methyltransferase; post-transcriptional regulation.

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Figures

FIGURE 1.
FIGURE 1.
Hypoxia increases m6A in poly(A)+ RNA but not in total or ribosomal RNA. RNA isolated from HEK-293T cells grown in normoxic (Normox) or hypoxic (Hypox) conditions for 24 h. (A) LC-MS/MS of mRNA from HEK-293T cells. Values represent the amount of m6A divided by total adenosine (N of 3). (*) P ≤ 0.05 by paired Student's t-test. Error bars represent standard error of the mean (SEM). (B) LC-MS/MS of total RNA from HEK-293T cells. Values represent the amount of m6A divided by total adenosine (N of 2). (C) LC-MS/MS of 18 and 28S rRNA from HEK-293T cells. Values represent the amount of m6A divided by total adenosine (N of 3). (D) MeRIP of 100 ng of mRNA from HEK-293T cells grown in normoxic or hypoxic conditions for 24 h quantified by qRT-PCR. Fold enrichments calculated from immunoprecipitated mRNA levels normalized to input and bead-only negative control IP and expressed as a ratio of hypoxia/normoxia. (*) P ≤ 0.05 by paired Student's t-test. Error bars represent SEM of five experiments.
FIGURE 2.
FIGURE 2.
Individual mRNAs are stabilized under hypoxia in an m6A-dependent manner. Total RNA from HEK-293T cells was harvested after 72 h transfection with METTL3/14 siRNA (M3/14 siRNA) or negative control siRNA (Neg siRNA) and 24 h of normoxic or hypoxic conditions and half-life determined via 4sU. (#) denotes P ≤ 0.05 by paired Student's t-test between negative siRNA samples in normoxic and hypoxic conditions while (*) denotes P ≤ 0.05 by paired Student's t-test between negative and M3/14 knockdown siRNAs in the hypoxic condition. Error bars represent SEM of five experiments.
FIGURE 3.
FIGURE 3.
Translation rates and protein output are not affected by loss of m6A. HEK-293T cells harvested after 72 h transfection with METTL3/14 siRNA (M3/14 KD) or negative control siRNA (neg) and 24 h of normoxic (normox) or hypoxic (hypox) conditions. (A) Polysome profiling of extracts separated by differential centrifugation through sucrose gradients. qRT-PCR analysis of the fractions shows percentage of individual mRNA in each fraction. Error bars represent SEM of three experiments. Fraction containing 80S peak is marked. (B) Western blots of 50 µg of protein lysates of normoxic negative control siRNA (NNeg), normoxic METTL3/14 knockdown (NKD), hypoxic negative control (HNeg), hypoxic METTL3/14 knockdown (HKD) (representative of three experiments).
FIGURE 4.
FIGURE 4.
METTL3/14 Knockdown decreased the ability of messages to recover from hypoxic stress after 4 h reoxygenation. (A) HEK-293T cells harvested after 72 h transfection with METTL3/14 siRNA (M3/14 KD) or negative control siRNA (neg) and 24 h of hypoxic conditions and either 30 min, 1, or 4 h of room level reoxygenation recovery (reoxy). Polysome profiling of extracts separated by differential centrifugation through sucrose gradients. qRT-PCR analysis of the fractions shows percentage of individual mRNA in each fraction. Error bars represent SEM of three experiments in the 1 and 4 h reoxygenation experiments and SEM of two experiments in the 30 min reoxygenation experiment. Fraction containing the 80S peak is marked. Paired Student's t-test indicates significant (P ≤ 0.05) decrease (*) or increase (#) compared to negative control siRNA. (B) Western blots of 50 µg of protein lysates of normoxic negative control siRNA (NNeg), normoxic METTL3/14 Knockdown (NKD), hypoxic negative control (HNeg), hypoxic METTL3/14 Knockdown (HKD), and 1, 2, 4, or 8 h reoxygenation after hypoxia (representative of three experiments).
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