Fig. 2: Plant pri-miRNAs are processed co-transcriptionally.

a, Detection of nascent pri-miRNA processing intermediates associated with RNAPII as detected by 5’-RACE. The fraction of clones with the expected 5’-end (among all the sequenced products) is indicated next to the predicted pri-miRNAs secondary structures. The miRNA-5p and miRNA-3p positions are noted in red and green, respectively. The values displayed were obtained from two independent experiments. b,c,d,g, Metagene analysis of nascent BTL pri-miRNAs processing fragments associated to RNAPII. b–d, Metagene analysis of nascent BTL pri-miRNAs processing intermediated associated to RNAPII as determined by scoring the 5’-end (b and c) or 3’-end (d and g) nt of plaNET-seq reads. Pri-miRNAs were scaled from the beginning of miRNA-5p to the end of miRNA-3p (b and d) or using only the mature miRNAs sequences (c and g). Cyan and orange arrows indicate the processing fragments detected also marked in the schemes illustrated next to b and d. e, Levels of the 5’-arm processing by-product of different pri-miRNAs associated with the chromatin in Col-0 and abh1-285 mutants as measured by RT–qPCR. Each processing by-product associated with the chromatin in the RIP experiment was normalized by the values in the input sample and relative to the normalized unprocessed pri-miRNAs in the same IP fraction. Values of n = 3 biologically independent samples are expressed relative to the Col-0 plants. Data are presented as mean values ± s.d. P values were calculated with two-tailed unpaired t-test with Welch’s correction. f, Co-transcriptional pri-miRNA processing score as measured by RT–qPCR in H3 or IgG IP RNA-samples from Col-0 and hyl1-2. Data are presented as mean values ± s.d. P values were calculated with two-tailed unpaired t-test with Welch’s correction and are noted for each comparison. RT–qPCR using primer pairs A, B and C in the IP fraction were normalized to U6 transcript and to the input levels using the same primers. Co-transcriptional processing was measured as a reduction in the hairpin amplicon (primer B) relative to the 5′ region (amplicon A). Non-detected RNA levels are displayed as ND; n = 3 biologically independent samples.