MAT2A hp1, but not hp2–6, requires the presence of the detained intron to regulate mRNA levels. (A) Full-length gene diagram of MAT2A, approximately to scale. Hp1–6 are denoted with red boxes, DI marks the regulated detained intron. (B) Diagrams of the eight β-MAT reporters used throughout this study (not to scale; Fl, flag-tag). The reporters are driven by a CMV immediate-early promoter and have a bovine growth hormone poly(A) signal. Asterisks show mutant hairpin sequences in the inset. (C) Representative northern blot data using RNA from HEK293 cells transfected with the indicated reporters and conditioned in media ±Met for 4 h. The mobilities of the detained intron transcript and mRNA are denoted with DI and m, respectively. (D) Quantification of the β-globin mRNA first normalized to GAPDH then expressed relative to the β-MAT-WT −Met signal, which was set to one for each replicate. (E) Quantification of the Met responsiveness of each reporter. The same data used in panel D were compared in −Met to +Met conditions for each construct. (F) Quantification of protein expression from each of the reporters after overnight incubation with the MAT2A inhibitor AGI-24512. The β-globin signal was first normalized to a cotransfected GFP control, and the β-MAT-WT values were set to one in each replicate (n = 3). The color scheme is the same as panel D above. (G) Representative western blots with anti-Flag antibodies to detect β-globin and anti-GFP which served as a cotransfected loading control. The same exposure is shown for both β-MAT and β-MAT-ΔDI blots. These experiments were performed using quantitative near-infrared fluorescence western blotting (LI-COR Odyssey). For panels D–F data are represented as mean ± standard deviation (SD). For panel E, the data were analyzed by a two-tailed, unpaired Student's t-test comparing each value to the matched wild-type control, β-MAT-WT (left) or β-MATΔDI-WT (right). Significance is annotated as (*) P < 0.05, (**) P < 0.01, or (***) P < 0.001.
