ALKBH10B Is an RNA N6-Methyladenosine Demethylase Affecting Arabidopsis Floral Transition

Abstract

N⁶-methyladenosine (m⁶A) is the most abundant, internal, posttranscriptional modification in mRNA among all higher eukaryotes. In mammals, this modification is reversible and plays broad roles in the regulation of mRNA metabolism and processing. Despite its importance, previous studies on the role and mechanism of m⁶A methylation in Arabidopsis thaliana have been limited. Here, we report that ALKBH10B is a demethylase that oxidatively reverses m⁶A methylation in mRNA in vitro and in vivo. Depletion of ALKBH10B in the alkbh10b mutant delays flowering and represses vegetative growth. Complementation with wild-type ALKBH10B, but not a catalytically inactive mutant (ALKBH10B H366A/E368A), rescues these effects in alkbh10b-1 mutant plants, suggesting the observed phenotypes are controlled by the catalytic action of ALKBH10B We show that ALKBH10B-mediated mRNA demethylation affects the stability of target transcripts, thereby influencing floral transition. We identified 1190 m⁶A hypermethylated transcripts in the alkbh10b-1 mutant involved in plant development. The discovery and characterization of the archetypical RNA demethylase in Arabidopsis sheds light on the occurrence and functional role(s) of reversible mRNA methylation in plants and defines the role of m⁶A RNA modification in Arabidopsis floral transition.

Figure 1.

ALKBH10B Transcripts Are Abundant in Most Arabidopsis Organs. Relative gene expression levels were determined using RT-qPCR with TUBULIN4 as a reference gene, prior to normalization to ALKBH10B expression levels in seedlings. The amplification efficiencies of the primers we used are approximately equal. Data are represented as means ± se. n = 3×2 (n = biological replicates × technical replicates).

Figure 2.

ALKBH10B Demethylates m⁶A in Vitro. (A) Proposed reaction mechanism of oxidative demethylation of m⁶A to adenosine byALKBH10B. (B) LC-MS/MS chromatograms of digested substrates revealing that an equal amount of recombinant ALKBH10B protein demethylates 50% of m⁶A in 42-mer ssRNA in 1 h. (C) Demethylation activity of an equal amount of recombinant ALKBH10B or the inactive ALKBH10B H366A/E368A using m⁶A-containing RNA as a substrate. Data are presented as means ± se. n = 3×2 (n = biological replicates × technical replicates). (D) Comparative analysis of Arabidopsis ALKBH10B- and human ALKBH5-mediated demethylation (%) versus time (min) using structured and nonstructured 42-mer m⁶A-modified RNA. Data are presented as means ± sd. n = 3×2 (n = biological replicates × technical replicates). (E) Representative LC-MS/MS chromatograms of m¹A and m⁶A nucleoside standards. The LC-MS/MS chromatograms of m¹A and m⁶A are indicated in red, while the A/U/C/G nucleosides are represented in black. (F) ALKBH10B-mediated demethylation (%) of m⁶A- or m¹A-modified 42-mer ssRNA versus time. Data are presented as means ± sd. n = 3×2 (n = biological replicates × technical replicates). All reactions were performed at pH 6.8 and 20°C for 1 h.

Figure 3.

m⁶A-Modified mRNA Is the Major Physiological Substrate of ALKBH10B. (A) Quantification of the m⁶A/A ratio of mRNA in the indicated plant lines by LC-MS/MS. (B) to (E) Quantification of the RNA modification ratios (m⁶A/A, m¹A/A, m⁵C/C. and m¹G/G) in tRNA (B), 28S rRNA (C), 18S rRNA (D), and mRNA (E) in wild-type and alkbh10b-1 mutant seedlings. Data are presented as means ± se. n = 3×3 (n = biological replicates × technical replicates). *P < 0.05 and **P < 0.01.

Figure 4.

Floral Transition Is Disrupted in alkbh10b Mutants. (A) Late flowering phenotype of alkbh10b-1. (B) Leaf number (rosette and main stem) of wild type (Col-0), alkbh10b-1, ALKBH10B:ALKBH10B/alkbh10b-1, ALKBH10B:ALKBH10Bm/alkbh10b-1, and 35S:ALKBH10B at flowering time. (C) Successive leaves per plant at flowering time for the indicated lines. Data are presented as means ± se. n = 15. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 5.

Upregulation of FT, SPL3, and SPL9 Is Correlated with ALKBH10B-Mediated m⁶A Demethylation. (A) Diurnal time course of FT expression in seedlings. (B) m⁶A levels of FT, SPL3, and SPL9 in wild-type and alkbh10b-1 plants. (C) Relative mRNA levels of SPL3 and SPL9 in indicated seedlings. (D) Immunoblot analysis using anti-myc antibody shows the expression of 6×myc-ALKBH10B in ALKBH10B:6×myc-ALKBH10B/alkbh10b-1 plants (input) and in the immunoprecipitated fraction (Anti-myc-IP), but not in wild-type Col-0 and IgG-IP fraction. (E) RIP-qPCR assays in ALKBH10B:6×myc-ALKBH10B/alkbh10b-1 plants show that 6×myc-ALKBH10B directly binds FT, SPL3, and SPL9 transcripts in vivo. (F) m⁶A levels of m⁶A peaks in FT, SPL3, and SPL9 in the wild type and alkbh10b-1 showing m⁶A positions demethylated by ALKBH10B. Data are presented as means ± se. n = 3×2 (n = biological replicates × technical replicates). *P < 0.05 and **P < 0.01 for paired samples; Col-0 versus alkbh10b-1 in dark color and ALKBH10B:ALKBH10B/alkbh10b-1 versus ALKBH10B:ALKBH10Bm/alkbh10b-1 in orange.

Figure 6.

m⁶A Demethylation by ALKBH10B Increases mRNA Stability of FT, SPL3, and SPL9. (A) and (B) Measurement of FT mRNA precursors. (A) RT-PCR analysis confirming the termination time point of FT transcription. (B) Diurnal time course of FT pre-mRNA. (C) Degradation of FT transcripts at night. FT mRNA levels in wild-type, alkbh10b-1, ALKBH10B:ALKBH10B/alkbh10b-1, and ALKBH10B:ALKBH10Bm/alkbh10b-1 plants were normalized to ZT16, respectively. (D) FT, SPL3, and SPL9 mRNA lifetimes in the wild type and alkbh10b-1. The transcription inhibition assays were repeated three times. (E) RNA gel blot analysis showing the abundance of mature miRNAs (miR156 and miR172) in wild-type and alkbh10b-1 plants. One representative result is shown. 5S rRNA was used as the loading control. (F) Proposed model of floral transition mediated by ALKBH10B-catalyzed demethylation. Data are presented as means ± sd. n = 3×3 (n = biological replicates × technical replicates).

Figure 7.

Global m⁶A Methylation Is Altered in alkbh10b-1 Plants. (A) Overlapping m⁶A peaks identified in the wild type and alkbh10b-1 (R1-R2). (B) Heat map of IP enrichment values of hypermethylated m⁶A peaks identified in alkbh10b-1 and wild-type seedlings. (C) Volcano plot of hypermethylated m⁶A peaks identified in alkbh10b-1 and the wild type across two biological replicates. (D) and (E) Metegenomic profiles of read distributions (D) and m⁶A peak distributions (E) along transcript segments in peak groups. (F) Fractions and relative enrichment of m⁶A peaks in each nonoverlapping transcript segment within peak groups. (G) RRACH consensus motifs in m⁶A-containing peak regions. (H) RRACH motif-containing peaks in different groups. *P < 1e-10 and **P < 1e-20. P values were determined by χ² test.