Figures and data
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 antibodies recognizing N-terminal or C-terminal of METTL3 as indicated.
C Immunoblot of T47D cells infected with retrovirus encoding shMETTL3 resistant pMSCV-METTL3 without tag or control vector followed by METTL3 shRNA or control shRNA infection.
D Immunoblot of T47D cells infected with retrovirus encoding pMSCV-METTL3 with C-terminal Flag tag.
E Purified recombinant METTL3-His protein was incubated with or without T47D cell lysates at 37 °C for 1 hour followed by immunoblot with anti-His antibody.
F Ponceau S staining and the N-terminal sequence of the short forms of METTL3 determined by Edman sequencing are shown.
FL indicates the full-length of METTL3. The short forms are labeled as a and b.
METTL3a (residues 239-580) and METTL3b (residues 197-580) are post-translational products through highly conserved residues
A and C Sequence alignments of the conserved residues on (234-244) (A) and (192-201) (C) of METTL3.
B and D Immunoblot of 293T cells transfected with empty vector (Vec) or METTL3-WT (wild type) or the indicated mutants.
E-F Immunoblot analysis of 293T cells with CRISPR knock-in (KI) mediated deletion of Q238 (E) or L198 (F).
G Purified recombinant METTL3-WT-His or METTL3-(Δ198+Δ238)-His protein were incubated with or without T47D cell lysates at 37 °C for 1 h followed by immunoblot with anti-His antibody.
H-J The in vitro protein methylation activity was tested using purified METTL3-WT-Flag and its mutant proteins in combination with co-purified Flag-METTL14 and RNA-probe. The methylation activity was measured by using dot blot (H) or the Promega bioluminescence assay (I), and the immunoblot of those purified proteins were shown in (J). Error bars represent mean ± SD, unpaired t-test. *** P< 0.001, ns denotes no significance.
FL indicates the full-length of METTL3. The short forms are labeled as a and b. se and le indicated short exposure and long exposure, respectively.
METTL3a contributes to cell proliferation and breast cancer progression
A-B 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 sgControl (sgCtrl).
C-D Immunoblot (C) and cell proliferation (D) of MDA-MB-231 cells infected with retrovirus encoding sgMETTL3 resistant METTL3 variants or control vector (Vec) followed by another infection with sgMETTL3 or sgControl (sgCtrl).
E-F Immunoblot (E) and cell proliferation (F) of 293T cells with WT or CRISPR knock-in mediated deletion of Q238.
G-H Immunoblot analysis (G) cell proliferation (H) of Δ238 knock-in 293T cells rescued with METTL3a or METTL3b.
I-K Mouse xenograft experiments were performed with MDA-MB-231 cells infected with retrovirus encoding sgMETTL3 resistant METTL3-WT, METTL3-Δ198 or METTL3-Δ238 followed by another infection with sgMETTL3 (n=8 mice per group). Tumor growth curve (I), tumor (J) and tumor weight
(K) were recorded. Error bars represent mean ± SEM, unpaired t-test. *** P< 0.001, ns denotes no significance. FL indicates the full-length of METTL3. The short forms are labeled as a and b.
METTL3a is required for METTL3-WTAP interaction
A-B 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 lentivirus encoding METLL3 shRNA.
D Immunoblot of whole cell extracts (WCE) and immunoprecipitations (IP) of 293T cells infected with lentivirus encoding METLL3 shRNA followed by transfection with METTL3-WT-Flag, METTL3-Δ238-Flag combining with or without METTL3a (no flag tag), or control vector as indicated.
METTL3a mediates the METTL3-METTL3 interaction, a prerequisite step for WTAP recruitment in MTC complex
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 METTL3-Flag and GST-METTL3.
C Immunoblot of whole cell extracts (WCE) and immunoprecipitations (IP) of 293T transfected with METTL3 containing different tag with or without RNase or DNase.
D A schematic representation of METTL3 and its truncations used for immunoprecipitation.
E Immunoblot of whole cell extracts (WCE) and immunoprecipitations (IP) of 293T cells infected with lentivirus encoding METLL3 shRNA followed by transfection with full-length (FL) or truncation of METTL3.
F Immunoblot of whole cell extracts (WCE) and immunoprecipitations (IP) of 293T cells infected with lentivirus encoding METLL3 shRNA followed by transfection with METTL3-WT-flag, METTL3-Δ238-Flag combining with or without METTL3a (no flag tag) or control vector as indicated.
G-H Immunoblot of whole cell extracts (WCE) and immunoprecipitations (IP) of 293T cells infected with or without lentivirus encoding METLL3 shRNA (G) or METTL14 shRNA (H) followed by transfection with METTL3 or METTL14 as indicated.
I Immunoblot of whole cell extracts (WCE) and immunoprecipitations (IP) of 293T cells infected with lentivirus encoding METTL3 shRNA followed by transfection with WTAP containing different tag.
J-K Immunoblot of whole cell extracts (WCE) and immunoprecipitations (IP) of 293T cells infected with lentivirus encoding WTAP shRNA followed by transfection with METTL3 or METTL14 as indicated.
L-M m6A dot blot (L) and quantification (M) of 293T cells infected with lentivirus encoding METLL3 shRNA followed by transfection with METTL3-WT, METTL3 variants, or control vector as indicated. Methylene blue (MB) is used as a loading control.
N-O m6A dot blot (N) and quantification (O) of 293T cells with WT or CRISPR knock-in mediated deletion of Q238 with or without transfection of METTL3a.
FL indicates the full-length of METTL3. The short forms are labeled as a and b. no tag represents exogenous METTL3a containing no flag tag.
METTL3a is essential for the global METTL3-mediated 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 vector (Vec) followed by another infection with sgMETTL3 or sgControl (sgCtrl).
B Overlaps of m6A peaks among METTL3-WT, METTL3-Δ198 and METTL3-Δ238 in METTL3 KO T47D cells.
C GSEA with GO terms of differentially m6A methylated peaks in METTL3-WT and METTL3-Δ238 for molecular functions.
D Heat map of m6A peaks related to mTOR pathway in T47D cells infected with retrovirus encoding sgMETTL3 resistant METTL3-WT, METTL3-Δ198, METTL3-Δ238 or control vector (Vec) followed by another infection with sgMETTL3 or sgControl (sgCtrl).
E Immunoblot analysis of T47D cells infected with sgControl or sgMETTL3.
F Integrative genomic viewer (IGV) plots of m6A peaks at TMEM127 mRNA in T47D cells as described in (A).
G m6A-qPCR showing the m6A enriched TMEM127 transcripts from T47D cells.
H Immunoblot of cell lysates from T47D cells as described in (A).
I-J Immunoblot (I) and cell proliferation (J) of T47D cells infected with shMETTL3 or shControl followed by transfection with siTMEM127 or siControl.
Error bars represent mean ± SEM, unpaired t-test. *** P < 0.001, ns denotes no significance.
Proteasome mediates METTL3 cleavage in an mTOR-dependent manner
A Immunoblot of T47D cells treated with indicated inhibitors.
B-C Immunoblot of T47D (B) and MDA-MB-231 (C) cells treated with MG132 for 8 hours.
D Immunoblot of T47D cells infected with METTL3-Flag followed by treatment with MG132 (10 μM) for 8 h.
E Immunoblot of purified recombinant METTL3-His protein incubated with or without T47D cell lysates wherein with or without MG132 treatment (20 μM) at 37 °C for 1 h.
F List of the proteasome components identified by LC-MS/MS analysis of the tandem affinity purification with METTL3 antibody in 293T cells.
G Immunoblot of whole cell extracts (WCE) and immunoprecipitations (IP) of T47D cells infected with METTL3-Flag or control vector.
H-J Immunoblot of MDA-MB-231 cells infected with shControl, shPSMC3 (H), shPSMC5 (I), shPSMD10 (J).
K Immunoblot of the purified HA-METTL3-Flag WT or indicated mutant from 293T cells using HA-magnetic beads followed by incubation with 26S at 37 °C for 1 h.
L-M Immunoblot of MDA-MB-231 cells infected with shmTOR or shControl.
N Immunoblot of T47D cells or MDA-MB-231 cells with time course treatment of rapamycin (100 nM) as indicated.
O Immunoblot of Tsc2 WT (Tsc2+/+) or Tsc2 KO (Tsc2-/-) MEF cells treated with or without MG132 (10 μM) for 8 h.
P A model for METTL3a-mediated MTC complex assembly.
FL indicates the full-length of METTL3. The short forms are labeled as a and b. se and le indicated short exposure and long exposure, respectively.
The short forms bands exist in lung cancer cells and other cell lines
A-C Immunoblot of lysates from lung cancer cells (A) or renal cancer cells (B) or HCT116 and MEF cells (C).
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.
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 h followed by immunoblot with anti-His antibody.
B Immunoblot of T47D cells infected with pHAGE-METTL3-Flag.
C-D, F-G Immunoblot of 293T cells transfected with pHAGE-METTL3-WT or its indicated mutants. E Sequence alignment of conserved amino acids on (150-160) of METTL3.
H-J Immunoblot analysis (H), cell proliferation (I) and soft agar growth (J) of T47D cells infected with retrovirus encoding Vector (Vec), sgMETTL3 resistant METTL3-WT or its indicated mutants followed by another infection with sgMETTL3 or sgControl (sgCtrl).
K-L Immunoblot analysis (K) and m6A dot blot (L) in 293T cells infected with shMETTL3 or shControl (shCtrl) followed by transfection with indicated plasmids. Methylene blue (MB) was used as a loading control. Error bars represent mean ± SEM, unpaired t-test. ns denotes not significant. FL is the full-length of METTL3. The short forms are labeled a, b and c.
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 indicated.
METTL3a is essential for the global METTL3-mediated m6A deposition and mTOR activation
A-B 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) followed by another infection with sgMETTL3 or sgControl (sgCtrl).
C RIP-qPCR showing the m6A enriched TMEM127 transcripts from MD-MB-231 cells infected with retrovirus encoding sgMETTL3 resistant METTL3-WT, METTL3-Δ198, METTL3-Δ238 or control vector (Vec) followed by another infection with sgMETTL3 or sgControl (sgCtrl).
D Immunoblot of cell lysates from T47D cells as described in (C). Error bars represent mean ± SEM, unpaired t-test. *** P< 0.001, ns denotes no significance.
Proteasome mediates METTL3 cleavage in an mTOR-dependent manner
A Information on protease inhibitors utilized in Figure 7A. B-C Immunoblot analyses of T47D cells (B) or MDA-MB-231(C) cells infected with shControl, shRaptor or shRictor.
