Paralleled degradome-seq and DMS-MaPseq largely revise the miRNA biogenesis atlas in Arabidopsis

Due to exceeding heterogeneity of structures, initial processing sites of primary substrates of microRNAs (pri-miRNAs) and underlying determining elements are unknown. Here, we pinpointed their first cleavage sites of pri-miRNAs and determined that only 147 of 326 annotated miRNAs are bona fide ones. We revised or deciphered the processing patterns for 95 of 147 pri-miRNAs. Approximately 95% of detected pri-miRNAs exhibited RNA secondary structures (RSS) in vivo distinct from in silico predicted, enabling better interpretation of underlying processing sites and patterns. Whereas pri-miRNAs undergo first cleavages, predominantly at unpaired regions in their duplexes, via the canonical 15~17-nt molecular ruler, they tended to harbor additional internal loops/bulges being 9~11-nt away from the initial processing sites and fitting into a binding pocket of DCL1. Finally, DCL1 partners could synergistically and independently impact processing patterns and in vivo RSS of pri-miRNAs. Together, our work shed light into precise processing mechanisms of plant pri-miRNAs.