Figure 1.
In Vitro Cleavage Assays with Random Hairpin RNA
(A) Four Dicer-like proteins from Paramecium tetraurelia are shown schematically.
(B) SDS-PAGE analysis of immunopurified Dcl3 and Dcl4 proteins and purified recombinant Dcl2 and Dcl5 from insect cells. 500 ng of purified proteins and 20 μL beads were loaded on 8% SDS-PAGE gels and stained with either Coomassie or silver. Dcl3-3xFlagHA and Dcl4-3xFlagHA have a size of 88 kDa, Dcl2 has a size of 74 kDa, and Dcl5 has a size of 97 kDa.
(C) A schematic of DNA oligo used to generate a hairpin RNA with random nucleotides for in vitro cleavage assays.
(D) 15% polyacrylamide-urea gel with small RNAs 5′-labeled with 32P. M is the small RNA ladder. No enzyme controls are marked as (−). “Dcl3 beads only” is a no template RNA control for Dcl3.
(E) Size distribution graphs of Dcl2, Dcl3, and Dcl5 cleavage products based on sRNA sequencing.
See also Figure S1.