Fig. 3: Co-transcriptional processing of BTL pri-miRNAs involves a second nucleoplasmic processing step.

a,b,g, Metagene analysis of processing fragments on nascent LTB (a), LTBs (b) and BTLs (g) pri-miRNAs as determined by scoring the 3′-end nt of plaNET-seq reads. c,d, Metagene analysis of processing fragments on nascent LTB (c) and LTBs (d) as shown in a and b but excluding selected miRNA loci. Pri-miRNAs were scaled from the beginning of miRNA-5p, or from the first DCL1 cleavage site for g, to the end of miRNA-3p. Arrows indicate the processing fragments detected for each processing type as illustrated in the schematic representation of LTB and LTBs and BTLs pri-miRNAs. A peak corresponding to an exon donor site of AT1G12290 is noted with a black arrow in g and Extended Data Fig. 3f. e, Metagene analysis of plaNET-seq LTB and LTBs scaled using the miRNA-3p sequence (top). Orange arrow matches the arrow in b. Metagene of entire plaNET-seq reads coverage over LTB and LTBs MIRNA loci, at the miRNA-3p encoding regions, showing the accumulation of 21-nt long reads corresponding to the miRNA-3p mature sequences (bottom). f, Mean plaNET-seq signal at the MIR156E and MIR159B loci (red bars). Coverage of plaNET-seq reads over the same loci (grey). The sequence corresponding to the mature miRNA-3p is noted on top of each panel highlighted in yellow. h, Relative abundance of BTL and LTB pri-miRNA hairpin region in the nucleoplasm compared to the H3 RIP fraction as measured by RT–qPCR in Col-0 samples. Data are presented as mean values ± s.d.; n = 3 biologically independent samples. P values were calculated with two-tailed unpaired t-test with Welch’s correction and are noted for each comparison. Processed hairpins measurements in the nucleoplasm and H3 IP fraction were normalized to the input levels for each samples. Quantifications were then expressed as a nucleoplasm/IP ratio of the values corrected by relative amount of unprocessed pri-miRNAs quantified with primers flanking the DCL1 cleavage site. i, Schematic summary of the co-transcriptional processing mechanisms of Arabidopsis pri-miRNAs. Coloured asterisks in each panel denote the plaNET-seq scored nts for each processing type and match the arrows of the same colours displayed in the previous panels and in Fig. 2.