Figure 3.

METTL3 binds to the coding region of PGC-1α mRNA and decreases its stability. A, RNA immunoprecipitation of various mRNAs with an anti-METTL3 antibody (n = 3). B, prediction sites of PGC-1α mRNA binding to METTL3 as produced from SRAMP. C, schematic of PGC-1α mRNA segments. D, RNA pull-down assays using THP-1 cell lysates and in vitro-transcribed RNA segments. Antisense sequence of METTL3 served as the negative control [N], as well as a 5-μg aliquot input [Inp.] and GAPDH (n = 2). E, relative luciferase activities with in vitro-transcribed RNA segments in control and METTL3 knockdown cells (n = 3). F, PGC-1α mRNA collected from a m⁶A RNA immunoprecipitation assay with control and METTL3 knockdown THP-1 cells using an IgG antibody or an anti-m⁶A antibody (n = 3). G, degradation of PGC-1α mRNA and 12S rRNA in control and METTL3 knockdown cells, which was used to determine half-life (n = 3). H, immunoblot detection of various proteins in control, METTL3 knockdown, PGC-1α knockdown, and METTL3/PGC-1α knockdown cells, with GAPDH as a loading control (n = 3). Data are represented as mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.