A cleaved METTL3 potentiates the METTL3–WTAP interaction and breast cancer progression
- Department of Thyroid and Breast Surgery, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, China
- Department of Thyroid and Breast Surgery, Zhongnan Hospital of Wuhan University, China
- Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, China
- State Key Laboratory for Cellular Stress Biology, Innovation Center for Cell Signaling Network School of Life Sciences, Xiamen University, China
Figures
Figure 1 with 1 supplement
Identification of two novel C-terminal short forms of METTL3.
(a) Immunoblot of lysates from paired breast cancer patient non-tumor (N) and tumor (T) tissues. (b) Immunoblot of T47D cells infected with lentivirus encoding METTL3 shRNA or control shRNA by …
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Figure 1—source data 1
Unedited western blot images for Figure 1.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig1-data1-v1.pdf
Figure 1—figure supplement 1
The short forms bands exist in lung cancer cells and other cell lines.
Immunoblot of lysates from lung cancer cells (a) or renal cancer cells (b) or HCT116 and MEF cells (c).
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Figure 1—figure supplement 1—source data 1
Unedited western blot images for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig1-figsupp1-data1-v1.pdf
Figure 2 with 2 supplements
METTL3a (residues 239–580) and METTL3b (residues 197–580) are post-translational products through highly conserved residues.
Sequence alignments of the conserved residues on (234–244) (a) and (192–201) (c) of METTL3. (b, d) Immunoblot of 293T cells transfected with empty vector (Vec) or METTL3-WT (wild-type) or the …
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Figure 2—source data 1
Unedited western blot images for Figure 2.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig2-data1-v1.pdf
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Figure 2—source data 2
Table related to Figure 2i.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig2-data2-v1.zip
Figure 2—figure supplement 1
Distribution of METTL3-WT and its mutants in breast cancer cells.
Immunofluorescence analysis of T47D cells stably expressing METTL3-WT or mutants under METTL3 knockout. Scale bars, 10 μm.
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Figure 2—figure supplement 1—source data 1
Images related to Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig2-figsupp1-data1-v1.zip
Figure 2—figure supplement 2
The short form METTL3c is dispensable for METTL3-mediated functions.
(a) Purified recombinant METTL3-WT-His, METTL3-Δ198, METTL3-Δ238, or METTL3-(Δ198+Δ238)-His protein were incubated with or without T47D cell lysates at 37°C for 1 hr followed by immunoblot with …
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Figure 2—figure supplement 2—source data 1
Unedited western blot images for Figure 2—figure supplement 2a–d, f–h, and j–l.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig2-figsupp2-data1-v1.pdf
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Figure 2—figure supplement 2—source data 2
Table related to Figure 2—figure supplement 2i.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig2-figsupp2-data2-v1.zip
Figure 3
METTL3a contributes to cell proliferation and breast cancer progression.
Immunoblot (a) and cell proliferation (b) of T47D cells infected with retrovirus encoding sgMETTL3 resistant METTL3 variants or control vector (Vec) followed by another infection with sgMETTL3 or …
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Figure 3—source data 1
Unedited western blot images for Figure 3a, c, e, and g and picture related to Figure 3j.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig3-data1-v1.pdf
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Figure 3—source data 2
Tables related to Figure 3b, d, f, h, i, and k.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig3-data2-v1.zip
Figure 4
METTL3a is required for METTL3–WTAP interaction.
Co-immunoprecipitation analyses of T47D cells (a) and 293T cells (b) transfected with Vector (Vec), METTL3-WT or its mutants. (c) Immunoblot analysis of 293T cells infected with or without …
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Figure 4—source data 1
Unedited western blot images for Figure 4.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig4-data1-v1.pdf
Figure 5 with 1 supplement
METTL3a mediates the METTL3–METTL3 interaction, a prerequisite step for WTAP recruitment in methyltransferase complex (MTC).
(a) Immunoblot of whole cell extracts (WCE) and immunoprecipitations (IP) of 293T transfected with METTL3 containing different tag. (b) Immunoblot analysis showing the binding between purified …
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Figure 5—source data 1
Unedited western blot images for Figure 5a–c, e–l, and n.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig5-data1-v1.pdf
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Figure 5—source data 2
Tables related to Figure 5m and o.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig5-data2-v1.zip
Figure 5—figure supplement 1
The METTL3–METTL3 interaction depends on its C-terminal region.
(a–d) Immunoblot of whole cell extracts (WCE) and immunoprecipitations (IP) of 293T cells infected with lentivirus encoding METLL3 shRNA followed by transfection with the mutants of METTL3 as …
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Figure 5—figure supplement 1—source data 1
Unedited western blot images for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig5-figsupp1-data1-v1.pdf
Figure 6 with 1 supplement
METTL3a is essential for the global METTL3-mediated N6-methyladenosine (m6A) deposition and mTOR activation.
(a) Cumulative distribution function of log2 peak intensity of m6A-modified sites in T47D cells infected with retrovirus encoding sgMETTL3 resistant METTL3-WT, METTL3-Δ198, METTL3-Δ238, or control …
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Figure 6—source data 1
Unedited western blot images for Figure 6e, h, and i.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig6-data1-v1.pdf
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Figure 6—source data 2
Tables related to Figure 6c, d, g, j.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig6-data2-v1.zip
Figure 6—figure supplement 1
METTL3a is essential for the global METTL3-mediated N6-methyladenosine (m6A) deposition and mTOR activation.
Consensus motifs (a), and the distribution (b) of m6A-modified sites in T47D cells infected with retrovirus encoding sgMETTL3 resistant METTL3-WT, METTL3-Δ198, METTL3-Δ238, or control vector (Vec) …
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Figure 6—figure supplement 1—source data 1
Unedited western blot images for Figure 6—figure supplement 1d.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig6-figsupp1-data1-v1.pdf
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Figure 6—figure supplement 1—source data 2
Table related Figure 6—figure supplement 1c.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig6-figsupp1-data2-v1.zip
Figure 7 with 1 supplement
Proteasome mediates METTL3 cleavage in an mTOR-dependent manner.
(a) Immunoblot of T47D cells treated with indicated inhibitors. Immunoblot of T47D (b) and MDA-MB-231 (c) cells treated with MG132 for 8 hr. (d) Immunoblot of T47D cells infected with METTL3-Flag …
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Figure 7—source data 1
Unedited western blot images for Figure 7.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig7-data1-v1.pdf
Figure 7—figure supplement 1
Proteasome mediates METTL3 cleavage in an mTOR-dependent manner.
(a) Information on protease inhibitors utilized in Figure 7a. Immunoblot analyses of T47D cells (b) or MDA-MB-231(c) cells infected with shControl, shRaptor, or shRictor.
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Figure 7—figure supplement 1—source data 1
Unedited western blot images for Figure 7—figure supplement 1b and c.
- https://cdn.elifesciences.org/articles/87283/elife-87283-fig7-figsupp1-data1-v1.pdf
