Fig. 6: Effects of VIR-mediated m6A modification on the stability and translation of photoprotection-related gene mRNAs.
From: N6-methyladenosine RNA modification regulates photosynthesis during photodamage in plants
a Analysis of protein abundance in Col-0 and vir-1 mutants. Protein samples from Col-0 and vir-1 seedlings were separated by 12% SDS-urea-PAGE and probed with antisera against specific proteins. CBB was used to estimate loading. Similar results were obtained from three independent biological replicates. b Proteins immunodetected from (a) were quantified with Phoretix 1D Software (Phoretix International, UK). Values (means ± SE; n = 3 independent biological replicates) are given relative to protein levels of Col-0 before HL treatment. *P < 0.05; **P < 0.01, by two-sided Student’s t test. c, Relative mRNA levels of HHL1 and MPH1 in Col-0 and vir-1 seedlings. UBQ10 was used as an internal control. Values are means ± SE (n = 3 biological replicates). *P < 0.05; **P < 0.01, by two-sided Student’s t test. d mRNA lifetimes of HHL1 and MPH1 in Col-0 and vir-1 seedlings. Seven-day-old Col-0 and vir-1 seedlings treated with actinomycin D for 0, 2, or 6 h were used for transcription inhibition assays. 18S ribosomal RNA was used as the internal reference. Values are means ± SE (n = 3 biological replicates). *P < 0.05; **P < 0.01, by two-sided Student’s t test. e Predicted cleavage site within m6A peaks in HHL1 and MPH1 transcripts and primer design. f Ratio of relative mRNA amounts at cleavage sites and control sites in Col-0 and vir-1. Values are means ± SE (n = 3 biological replicates). *P < 0.05; **P < 0.01, by two-sided Student’s t test. g Normalized distribution of Ribo-seq reads in Col-0 and vir-1 along HHL1. h Schematic representation of the positions of m6A motifs within HHL1. i Analysis of HHL1-FLAG accumulation in hhl1 protoplasts transfected with equal amounts of plasmids overexpressing wild-type or mutant HHL1. A641,661,670-G mutant, mutant harboring the transition mutations A641, A661, A670-G; A670-G mutant, mutant with A670-G transition mutation; A661-G mutant, mutant with A661-G transition mutation; A641-G mutant, mutant with A641-G transition mutation. j Proteins immunodetected from (i) were quantified with Phoretix 1D Software (Phoretix International, UK). Values (means ± SE; n = 3 independent biological replicates) are given relative to protein levels of wild-type HHL1. *P < 0.05; **P < 0.01, by two-sided Student’s t test. k, Analysis of HHL1 mRNA lifetimes in hhl1 protoplasts transfected with equal amounts of wild-type or mutant HHL1 plasmids. The overnight cultured protoplasts were treated with actinomycin D for 0, 2.5, or 5 h for transcription inhibition assays. 18S ribosomal RNA was used as the internal reference. Values are means ± SE (n = 3 biological replicates). *P < 0.05; **P < 0.01, by two-sided Student’s t test.
