Lariat RNAs competitively inhibit DCL1/HYL1 binding to pri-miRNAs.

(A) Lariat RNAs associate with DCL1 using a RIP assay performed as in Fig 2B. Immunoprecipitated RNAs were analyzed by qRT-PCR with divergent primers to detect the indicated lariat RNAs. UBQ5 was used as the loading and negative control. Error bars show SE calculated from three biological replicates. (B) Lariat RNAs associate with HYL1 using a RIP assay performed as in Fig 2C. qRT-PCR was performed according to (A). hyl1-2 was used as the negative control. UBQ5 was used as the loading and negative control. Error bars show SE calculated from three biological replicates. (C) R-EMSA to determine HYL1 binding to pri-miR167b in the presence of circular RNAs from Col-0 or dbr1-2 plants. Recombinant MBP-HYL1-D1D2 (MBP-HYL1) was incubated with a 5’_biotin_labeled pri-miR167b probe after addition of different amounts of circular RNAs isolated from Col-0 or dbr1-2 inflorescences, respectively. The arrow indicates the HYL1-pri-miR167b complex. (D) Hybridization intensities were quantified and normalized to the controls (lane 2 in C), and are shown in the line graph. Bars represent the average normalized intensity of three biological replicates.