Extended Data Fig. 1: High-throughput cleavage assays for DCL1.
From: Key RNA elements influencing DCL1 cleavage in plant microRNA biogenesis
(a) The biogenesis of animal and plant miRNAs. The blue arrowheads indicate the cleavage sites of enzymes on pri-miRNAs or pre-miRNAs. (b) The two directions of DCL1 cleavage on pri-miRNAs. The blue and red arrowheads indicate the first and second cleavages, respectively. (c) Schematic diagram of DCL1. The numbers indicate the amino acid positions. DExD helicase domain; DUF283, an unknown function domain; PAZ (Piwi/Argonaut/Zwille); RIIIDa and RIIIDb, two RNase III domains; and dsRBDs, double-stranded RNA-binding domains. (d) The purity of DCL1 was assessed by SDS-PAGE. The DCL1 purification experiments were repeated two times with similar results. (e) Diagrammatic structures of pri-miR166f and pri-miR166c. The blue arrowheads indicate DCL1 cleavage sites. (f) In vitro DCL1 cleavage assays were performed for the two pri-miRNAs shown in (e). The assays were repeated two times with similar results. (g) Diagrammatic structures of shRNAs with or without a 32-nt barcode. The blue arrowheads indicate the DCL1 cleavage sites. (h) In vitro DCL1 cleavage assays were performed for the two shRNAs shown in (g). The blue arrowheads indicate the DCL1 cleavage sites. The alternative cleavages of DCL1 are marked by asterisks. The assays were repeated two times with similar results. (i) A schematic cloning scheme was used for randomized shRNA substrates and DCL1-cleaved products. RA3 and RA5 represent sequencing adapters. CirRTP refers to a reverse transcription primer that incorporates both RA3 and RA5 adapter sequences. (j, m) Structural diagrams of shRNA and pre-miRNAs without or with a bulge at different positions. (k, n) In vitro DCL1 cleavage assays for shRNAs in (j), pre-miR166c variants in (m). Grey, blue and red arrowheads indicate DC20, D21, and D22, respectively. The alternative cleavages of DCL1 are marked by asterisks. (l, o) A quantitative analysis of cleavage accuracy at different sites, based on three repeated assays from (k) and (n). Bars indicate the precision of cleavage at each position, data values are shown as dots and error bars represent mean ± SD.
