The high-throughput shRNA cleavage assays on 46,000 variants

DICER-LIKE1 (DCL1), a plant-specific RNase III enzyme, is fundamental to post-transcriptional gene regulation mediated by microRNAs (miRNAs). DCL1 processes precursor miRNAs into mature miRNAs, typically 20–22 nucleotides in length. Despite its crucial role, the RNA elements that guide DCL1's cleavage site selection have remained largely uncharacterized. In this study, we employed a high-throughput sequencing approach to analyze Arabidopsis thaliana DCL1 cleavage patterns on over 46,000 short-hairpin RNA (shRNA) sequences previously studied with human DICER. Our analyses revealed that DCL1 cleavage preferences are governed by specific secondary RNA structures and sequence motifs, among which a particular RNA element, designated the GHR motif, emerged as pivotal. This motif significantly influences cleavage site selection independently of the dsRBD and helicase domains of DCL1, operating primarily through the RNase IIIDa domain. Notably, the GHR motif is evolutionarily conserved across plant species and is essential for the precise cleavage of various plant pre-miRNAs. Our findings also suggest a novel role for the GHR motif in the biogenesis of non-canonical 22-nt miRNAs, expanding its functional impact. These insights deepen our understanding of the molecular mechanisms underlying DCL1's specificity and highlight its integral role in miRNA maturation and gene regulatory networks in plants. Overall design: 46,000 shRNA variants were processed by plant DCL1 in vitro. The original substrates and cleaved products were cloned and sequenced.