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. 2023 Aug 17;12:RP87283. doi: 10.7554/eLife.87283

Figure 5. 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 METTL3-Flag and GST-METTL3. (c) Immunoblot of WCE and 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 IP. (e) Immunoblot of WCE and IP of 293T cells infected with lentivirus encoding METLL3 shRNA followed by transfection with full-length (FL) or truncation of METTL3. (f) Immunoblot of WCE and 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. Immunoblot of WCE and 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 WCE and IP of 293T cells infected with lentivirus encoding METTL3 shRNA followed by transfection with WTAP containing different tag. (j, k) Immunoblot of WCE and IP of 293T cells infected with lentivirus encoding WTAP shRNA followed by transfection with METTL3 or METTL14 as indicated. N6-methyladenosine (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. 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.

Figure 5—source data 1. Unedited western blot images for Figure 5a–c, e–l, and n.
Figure 5—source data 2. Tables related to Figure 5m and o.

Figure 5.

Figure 5—figure supplement 1. The METTL3–METTL3 interaction depends on its C-terminal region.

Figure 5—figure supplement 1.

(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.
Figure 5—figure supplement 1—source data 1. Unedited western blot images for Figure 5—figure supplement 1.