Extended Data Fig. 8: MTC promotes miRNA production.
From: SERRATE drives phase separation behaviours to regulate m6A modification and miRNA biogenesis
a, Linear regression analysis did not detect significant correction between expression and methylation profiles of methylated pri-miRNAs identified by exomePeak2 in se-2. b, Statistical analysis showed that enrichment efficiencies of m6A (+) vs m6A (–) spike-ins were inversely correlated with the endogenous m6A levels in m6A-IP-qPCR. Enrichment efficiency of methylated spikes was divided by the one of unmethylated spikes in individual samples and then normalized to that of Col-0 in which the value was arbitrarily set as 1. P values for relative enrichment of standards at se-2 and mta are 0.040 and 0.0001, respectively. c – f, Bar graphs showed that reduction of miRNA (c, d), accumulation of miRNA target transcripts (e) and pri-miRNAs (f) in indicated plants. In (c), our sRNA-seq results and public sRNA-seq24 were exhibited in parallel. In (d), small RNA RT-qPCR analysis of indicated miRNAs. In (e), RNA-seq analysis of miRNA target genes. In (f), RT-qPCR analysis of pri-miRNAs. U6 and UBQ10 served as internal controls for normalization of miRNAs in (d) and pri-miRNAs in (f), respectively. For (c), p values for relative expression of miR156, miR158, miR159, miR164, miR166, miR167, miR171, miR319, and miR161.1, at the sRNA-seq of mta; pABI3::MTA vs Col-0 are 0.15, 0.12, 0.0004, 0.0002, 0.014, 0.0023, 0.0008, 0.0035, and 0.37, at the sRNA-seq mta-a vs Col-0 are 0.16, 0.081, 0.038, 0.10, 0.019, 0.057, 0.0013, < 0.0001, and 0.43, respectively. For (d), p values for relative expression of miR158, miR164, miR166, miR167, miR171, miR319 are all < 0.0001, for miR156 and miR161.1, at mta vs Col-0 are 0.014 and 0.86, at mtb vs Col-0 are 0.76 and 0.76, at fip37 vs Col-0 are 0.87 and 0.87, at se-2 vs Col-0 are both < 0.0001, respectively. For (e), p values for relative expression of genes at mta vs Col-0 are 0.0010, < 0.0001, 0.052, < 0.0001, 0.10, 0.015, 0.060, < 0.0001, 0.0003, 0.034, 0.053, 0.0011, and 0.019, at se-2 vs Col-0 are < 0.0001, < 0.0001, < 0.0001, 0.0003, 0.0037, 0.015, 0.018, 0.018, 0.027, 0.043, 0.061, 0.066, and 0.090, respectively. For (f), p values for relative expression of pri-miRNAs at mta vs Col-0 are 0.0017, 0.0015, < 0.0001, < 0.0001, 0.0047, 0.035, 0.015, and 0.0059, at se-2 vs Col-0 are 0.0012, < 0.0001, < 0.0001, < 0.0001, 0.0015, 0.0054, < 0.0001, and < 0.0001, respectively. g, h, MTA does not impact the transcription of MIR167a locus. Both histochemical staining analysis (g) and RT-qPCR of GUS activity (h) showed comparable transcriptional levels of MIR167a in mta vs Col-0. Two-week-old seedlings were analyzed. Scale bar, 0.5 cm. For (h), P value is 0.95. i, RT-PCR analysis showed that the patterns of pri-miRNA alternative splicing are comparable in Col-0 and mta. j, RNA-seq analysis showed that the transcript levels of microprocessor components are not decreased in mta vs Col-0. P values for relative expression of miRNA pathway genes at mta vs Col-0 are 0.018, 0.98, 0.69, 0.019, 0.71, 0.082, 0.99, 0.88, 0.69, and 0.12, at se-2 vs Col-0 are 0.0038, 0.99, 0.74, < 0.0001, 1.00, 0.0001, 0.11, 0.84, 0.68, and 0.24, respectively. The experiments were replicated three times and representative results are shown (i, g). Data are mean ± s.d. of three independent experiments (b – f, h, and j). P values, one-way (b) and two-way (c – e, and j) ANOVA with Dunnett’s multiple comparison test, and unpaired two-sided t-test (f, h).
