RNA Secondary Structural Determinants of miRNA Precursor Processing in Arabidopsis

MicroRNAs (miRNAs) are excised from hairpin structures within primary miRNAs (pri-miRNAs). Most animal pri-miRNAs are processed by two cleavages, the first at a loop-distal site similar to 11 nucleotides (nt) from the end of the hairpin and the second similar to 22 nt beyond the first [1-3]. To identify RNA structural determinants of miRNA processing in plants, we analyzed the functional consequences of changing the secondary structure of the lower (loop-distal), middle (miRNA:miRNA(star)), and upper (loop-proximal) stems of the hairpin in two different pri-miRNAs. Closing bulges immediately below the loop-distal cleavage sites increased the accumulation of accurately cleaved precursor miRNAs but decreased the abundance of the mature miRNAs. A pri-miRNA variant with an unpaired lower stem was not processed, and variants with a perfectly paired middle or upper stem were processed normally. Bioinformatic analysis of pri-miRNA structures, together with physical mapping of initial cleavage sites and in vitro processing of pri-miRNA, reveals that the first, loop-distal cleavage is often at a distance of similar to 15 nt from an unpaired region. Hence, a common determinant of the rate and location of the initial pri-miRNA cleavage is an imperfectly base-paired duplex of similar to 15 nt between the miRNA:miRNA(star) duplex and either a less structured region of the lower stem or its end.