Fig. 3: PRMT1 mRNA is a de novo target of LC3B-mediated mRNA decay (LMD).
From: LC3B is an RNA-binding protein to trigger rapid mRNA degradation during autophagy
a Read densities of the LC3B CLIP-seq (LC3B CLIP1 and LC3B CLIP2) and mRNA sequencing (input mRNA1 and input mRNA2) for PRMT1 mRNA in two biological replicates. The map for PRMT1 is shown at the bottom. b Schematic representation of RLuc reporter mRNAs harboring the full-length PRMT1 3′UTR of either WT or U/A substitution. The relative positions of the LC3-binding site (AAUAAA) and its variant (AAAAAA) are indicated by arrows. c In vivo CLIP of endogenous LC3B. HEK293T cells expressing the RLuc reporter mRNA and FLuc mRNA (which served as a negative control) were either treated or not treated with Rapa + CQ. The cells were subjected to in vivo CLIP using α-LC3B antibody or nonspecific rabbit IgG (rIgG). The amounts of coimmunoprecipitated reporter mRNAs were normalized to those of the FLuc mRNAs. Then, the normalized levels obtained in IPs using rIgG in the untreated cells were arbitrarily set to 1.0. n = 3; Data are presented as mean values ± SD; p values were analyzed using two-tailed and equal-variance Student’s t test; *p < 0.05; **p < 0.01 (The exact p values are provided in Source Data file). d Effect of LC3B downregulation on the abundance of the RLuc-P3′ reporter mRNAs. HEK293T cells either undepleted or depleted of LC3B were transiently transfected with plasmids expressing RLuc and FLuc reporter mRNAs. The cells were either treated or not treated with Rapa + CQ for 12 h before cell harvest. The amounts of the RLuc mRNAs were normalized to those of FLuc mRNAs. Then, the normalized levels in the untreated cells were arbitrarily set to 100%; n = 3; Data are presented as mean values ± SD; p values were analyzed using two-tailed and equal-variance Student’s t test; **p < 0.01 (The exact p values are provided in Source Data file). e Half-life measurement of the RLuc reporter mRNA, RLuc-P3′-WT. As performed in d except that the cells were harvested at the indicated time points. The y axis represents the level of mRNA remaining (percentage) on the logarithmic scale (log2); n = 4; Data are presented as mean values ± SD. f, g Half-life of endogenous LMD substrates after treatment with Rapa + CQ or LC3B downregulation. Two endogenous LMD substrates, PRMT1 mRNA (f) and MARS1 mRNA (g) were analyzed. n = 3; Source data are provided as a Source Data file.
