Hp2–6 control stability of the spliced mRNA. (A) Representative northern blots using RNA from cells transfected with the indicated β-MAT reporters after ActD treatment for the indicated times. Cells were conditioned in the presence (top) or absence (bottom) of Met for 4 h then kept in the same media for an additional 1, 2, or 4 h after ActD. GAPDH half-life is significantly greater than 4 h, so it was used as a loading control. (B) Quantification of decay experiments. β-globin signal was first normalized to GAPDH, and samples were quantified relative to the t = 0 sample which was set to 100. Each data point is mean value ± SD (n = 3). (C) Representative northern blots of a decay assay with RNA from HCT116 cells or 116-ΔDI. The latter are HCT116-derived cells that lack the MAT2A detained intron at the endogenous locus. Cells were conditioned in the presence (top) or absence of Met (bottom) for 4 h then kept in the same media for an additional 0, 1, 2, or 4 h with ActD. Methylene blue staining of the membrane serves as a loading control (bottom). The bands with decreased mobility over time in ActD are nuclear RNAs undergoing nuclear hyperadenylation as previously reported (Bresson et al. 2015). (D) Quantification of the decay experiments as in panel C (n = 3). For panels B and D, the data were analyzed by a two-tailed, unpaired Student's t-test comparing each time point ±Met. Significance is annotated as (*) P < 0.05, (**) P < 0.01, or (***) P < 0.001.
