Transcriptome-wide analysis of Arabidopsis DICER-LIKE1 RNA substrates

In plants, DICER-LIKE1 (DCL1) orchestrates microRNA (miRNA) biogenesis by cleaving imperfect stem-loop precursors within primary transcripts (pri-miRNAs). However, the full spectrum of DCL1 RNA substrates remains unexplored. Here, we report transcriptome-wide RNA immunoprecipitation and deep-sequencing (RIP-Seq) analyses of the Arabidopsis catalytically inactive DCL1 (DCL1ci), designed to bind but not cleave its targets. In inflorescences, DCL1ci-RIP retrieved nearly all evolutionarily conserved MIRNA loci and uncovered many hitherto unknown young MIRNA loci. Extensive interactions with both pre-miRNA stem-loops and flanking single-stranded regions were detected, suggesting that DCL1 scans pri-miRNAs prior to stem-loop cleavage. Quantitative binding profiles resolved the specific contribution of paralogous MIRNA family members in inflorescences, enabling tissue-level discrimination of pri-miRNA engagement. The analysis also identified hundreds of DCL1ci-interacting non-MIRNA loci, including protein-coding genes, transposons, and intergenic regions, with many lacking canonical stem-loop structures. We show that DCL1 promotes 24-nt small RNA biogenesis mostly from helitron-derived transcripts via a pathway genetically distinct from RNA-directed DNA methylation. Moreover, we identify a conserved stem-loop in the DCL1 5′-UTR suggesting autoregulatory feedback control. Collectively, our study establishes DCLci-RIP as a robust noninvasive approach for profiling DCL substrates, broadens DCL1’s functional landscape, and provides a foundation for dissecting dynamic DCL–RNA interactions across developmental and stress contexts.