Figure 7. 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 followed by treatment with MG132 (10 μM) for 8 hr. (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 hr. (f) List of the proteasome components identified by liquid chromatography–tandem mass spectrometry (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. Immunoblot of MDA-MB-231 cells infected with shControl, shPSMC3 (h), shPSMC5 (i), and 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 hr. (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 hr. (p) A model for METTL3a-mediated methyltransferase complex (MTC) 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.

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.