Differential m6A, m6Am, and m1A Demethylation Mediated by FTO in the Cell Nucleus and Cytoplasm

Abstract

FTO, the first RNA demethylase discovered, mediates the demethylation of internal N⁶-methyladenosine (m⁶A) and N⁶, 2-O-dimethyladenosine (m⁶A_m) at the +1 position from the 5' cap in mRNA. Here we demonstrate that the cellular distribution of FTO is distinct among different cell lines, affecting the access of FTO to different RNA substrates. We find that FTO binds multiple RNA species, including mRNA, snRNA, and tRNA, and can demethylate internal m⁶A and cap m⁶A_m in mRNA, internal m⁶A in U6 RNA, internal and cap m⁶A_m in snRNAs, and N¹-methyladenosine (m¹A) in tRNA. FTO-mediated demethylation has a greater effect on the transcript levels of mRNAs possessing internal m⁶A than the ones with cap m⁶A_m in the tested cells. We also show that FTO can directly repress translation by catalyzing m¹A tRNA demethylation. Collectively, FTO-mediated RNA demethylation occurs to m⁶A and m⁶A_m in mRNA and snRNA as well as m¹A in tRNA.

Keywords: FTO; cap m(6)A(m); cytoplasmic demethylation; m(6)A; nuclear m(6)A demethylation; snRNA demethylation; tRNA m(1)A demethylation; translation regulation.

Figure 1.. FTO Demethylates both m⁶A and m⁶A_m inside Cells.

See also Figure S1. (A) A sketch that FTO mediates demethylation of m⁶A and cap m⁶A_m in polyadenylated RNA. (B) Quantification of the m⁶A/A and cap m⁶A_m/A ratio in polyadenylated RNA by LC-MS/MS. In comparison to controls, significant increases in the m⁶A/A ratio were consistently observed among HeLa, HEK293T, and 3T3-L1 cells upon transient knockdown of FTO (blue bars). Increases of the m⁶A_m/A ratio were consistently observed among HeLa, HEK293T, and 3T3-L1 cells upon transient knockdown of FTO (green bars). P values were determined using Student’s unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.s. means not significant. Error bars, mean ± s.d. for n = 4 experiments in (B).

Figure 2.. Subcellular Distribution of FTO Differs among Cell Lines and the Spatial Localization of FTO Influences its Demethylation of Target Substrates.

See also Figure S2. (A) Subcellular localization of FTO (green) among HeLa, Mel624, HepG2, HEK293T, and 3T3-L1 cell lines. The nucleus was stained with DAPI (blue). Scale bar = 10 μm. Representative images are selected from three independent experiments. (B) Quantification of the m⁶A/A and cap m⁶A_m/A ratios in polyadenylated RNA from HEK293T cells by LC-MS/MS. Transient knockdown of FTO led to a significant increase of nuclear polyadenylated RNA m⁶A/A ratio but not the cytoplasmic polyadenylated RNA m⁶A/A ratio (blue bars). Transient knockdown of FTO led to a significant increase of cytoplasmic polyadenylated RNA m⁶A_m/A ratio but no noticeable change of the nuclear polyadenylated RNA m⁶A_m/A ratio (green bars). (C) Quantification of the m⁶A/A and cap m⁶A_m/A ratios in polyadenylated RNA from NB4 cells (left) and MONOMAC-6 cells (right) by LC-MS/MS. Stable knockdown of FTO led to both increased m⁶A/A and cap m⁶A_m/A ratios in total polyadenylated RNA; P values were determined using Student’s unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.s. means not significant. Error bars, mean ± s.d. for n = 6 experiments in (B); for n = 4 experiments in (C)

Figure 3.. FTO-mediated Demethylation of m⁶A but not m⁶A_m in Polyadenylated RNA More Affects Transcript Expression Level Changes.

See also Figure S3 and Table S1. (A) mRNA transcripts are classified into four groups: transcripts containing only m⁶A_m, containing only internal m⁶A, containing both m⁶A and m⁶A_m (overlap), and transcripts that contain neither m⁶A nor m⁶A_m (labeled as the rest). The global expression level of m⁶A_m-alone transcripts did not significantly change upon FTO knockdown while the global transcript level of other three groups increased significantly. (B) The cumulative fraction of gene expression shows that compared to the transcripts containing only m⁶A, containing both m⁶A and m⁶A_m, and containing no m⁶A or m⁶A_m, the transcripts containing only m⁶A_m but not m⁶A showed significantly less transcript level change with FTO knockdown compared to the control (in A, P values were determined using the Wilcoxon test; in B, P values were determined using Mann-Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.s. means not significant. Error bars, mean ± s.d. for n = 3987, m⁶A only; n = 2016, m⁶A only; n = 1307, m⁶ m A and m⁶A_m; n = 5598, the rest). (C) The cumulative fraction of gene expression shows that FTO CLIP targets showed significantly increased expression level upon transient knockdown of FTO but not non-targets for transcripts containing only m⁶A compared to the rest genes. (In C, P values were determined using Mann-Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.s. means not significant. Error bars, mean ± s.d. for n = 1895, m⁶A genes overlapped with FTO targets; n = 2092, non-targets; n = 8896, the rest).

Figure 4.. FTO-mediated Internal m⁶A, Internal and Cap m⁶A_m Demethylation in snRNAs.

See also Figure S4. (A) Bar graph summarizing the reads distribution of the FTO target RNA species in the 3T3-L1 cells; donut graph showing the percentage of the FTO-bound mRNA in all FTO CLIP-seq targets in the 3T3-L1 cell line. (B) An increased m⁶A/A ration in the U6 RNA was observed by LC-MS/MS in Fto^−/− MEF cells compared to the control wild-type MEF cells. (C) Quantification of the m⁶A_m/A ratio of the U1/5.8s RNA and U2 RNA by LC-MS/MS after decapping, showing increased m⁶A_m/A ratios in the Fto^−/− MEF cells compared to the wild-type control. (D) Quantification of the m⁶A_m/A ratio in U1/5.8s RNA and U2 RNA by LC-MS/MS without decapping, showing increased internal m⁶A_m/A ratios in Fto^−/− MEF cells compared to the wild-type control. P values were determined using Student’s unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.s. means not significant. Error bars, mean ± s.d. for n = 6 experiments in (B) to (D).

Figure 5.. FTO-mediated tRNA m¹A Demethylation in vitro, inside Cells, and in Brain Tissues.

See also Figure S5, Table S2, and Table S3. (A) Quantification of the m¹A/G ratio in total tRNA purified from HeLa, HEK293T, and 3T3-L1 cells by LC-MS/MS. The transient knockdown led to an increase of at least ~15% of the m¹A/G ratio in total tRNA compared to control cells. (B) A consistent decrease (~20%) of the total tRNA m¹A/G ratio was observed upon overexpression of ΔNLS-FTO compared to the control. (C) Transient knockdown of FTO led to a significant increase of both nuclear and cytoplasmic total tRNA m¹A/G ratio in HEK293T cells. (D) A significant increase of m¹A in tRNA but not m⁶A or m⁶A_m in the polyadenylated RNA was observed in the Fto^−/− MEF cells compared to the wild-type MEF cells. (E) Knockout of Fto led to significant increases of both nuclear and cytoplasmic tRNA m¹A levels in tRNA in MEF cells. (F) A significant increase of the total tRNA m¹A but not polyadenylated RNA m⁶A or m⁶A_m could be observed in Fto^−/− mouse brain tissues compared to wild-type mouse brains. (G) Demethylation of m¹A in tRNA isolated from HEK293T cells by recombinant FTO. EDTA chelates cofactor iron and inactivates FTO. The ratio of m¹A/G was shown. P values were determined using Student’s unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.s. means not significant. Error bars, mean ± s.d. for n = 4 experiments in (A) to (E); for n = 5 experiments in (F); for n = 3 experiments in (G).

Figure 6.. FTO Mediates Specific tRNA m¹A Demethylation and Suppresses Translation.

See also Figure S6. (A) Pie chart showing the top FTO-bound tRNAs identified by CLIP-seq with FLAG-tagged FTO in 3T3-L1 cells. (B) Quantification of the m¹A/G ratio in tRNA targets bound to FTO by LC-MS/MS. In comparison to the control, m¹A/G ratios of tRNA^Glu(CUC), tRNA^His(GUG), tRNA^Gly(GCC), tRNA^Gly(ACC), tRNA^Asp(GUC), tRNA^Lys(CUU), tRNA^Gln(CUG), and tRNA^Leu(CAA) noticeably increased upon transient knockdown of FTO in 3T3-L1 cell. (C) Quantification of total protein synthesis in MEF cells by flow cytometry. HPG incorporation into wild-type MEF cell was recorded in gray and the Fto^−/− MEF cells in red. (D) Supplementation of total tRNA purified from Fto^−/− MEF cell leads to increased translation efficiency in vitro compared to using the total tRNA from wild-type MEF cells. (E) Cartoon illustration of the reporter assays was shown: firefly luciferase (F-luc) was used as the reporter and Renilla luciferase (R-luc) on the same plasmid was used as the internal transfection control. 6 × GUG(Glu)-coding sequences (recognized by tRNAGlu(CAC)) and 6 × CAC(His)-coding sequences (recognized by tRNAHis(GUG)) were inserted after the PLK promoter region of F-luc as the positive reporter, respectively. The effects of tRNA^Glu(CUC) and tRNA^His(GUG) were revealed by the reporter assay. The reporter assay showed significant increases of protein synthesis in the Fto^−/− MEF cell compared to the wild-type MEF cell when the control reporter and test reporters (with inserted Glu(CUC)-coding sequence or His(GUG)-coding sequence) are transiently expressed in the wild-type and Fto^−/− MEF cell, respectively. P value was determined using Student’s unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.s. means not significant. Error bars, mean ± s.d. for n = 3 experiments in (B) and (C); n = 8 experiments in (D) and (E).

Figure 7.. A Model for FTO-mediated RNA Demethylation.

Nuclear m⁶A is the main substrate of FTO in the cell nucleus. FTO also mediates m¹A tRNA demethylation as well as m⁶A (U6) and m⁶A_m (U2 in particular but also U1) snRNA demethylation in the nucleus. FTO can localize to the cytoplasm and mediate mRNA m⁶A and cap m⁶A_m demethylation as well as tRNA m¹A demethylation in the cytoplasm.