Fig. 4.
In vitro processing of scaRNAs by Drosha/DGCR8. (A) Schematic of scaRNA 2, 9 and 17 substrates used for in vitro processing assays with immunoprecipitated Drosha/DGCR8. Substrates were obtained by in vitro transcription of linearized DNA encoding the indicated scaRNA followed by RNA gel purification. (B) In vitro transcribed scaRNA9 (left), scaRNA2 (middle) and scaRNA17 (right) substrates were used in a processing assay with immunopurified (FLAG) Drosha/DGCR8. RNA isolated from the processing reactions was subjected to Northern blotting with probes that detect the various full-length scaRNAs and fragments. Substrate was incubated with FLAG beads from Drosha/FLAG-DGCR8 transfected lysate (Trans), FLAG beads from un-transfected cell lysate (UT), or water (H2O). Note that two different Trans processing reactions are shown for each substrate (lanes 4 and 5 for scaRNA9 and lanes 3 and 4 for scaRNA2 and scaRNA17). A positive control of ectopically expressed scaRNA9 RNA is shown in the left panel, lane 6 (+sca9). Ectopically expressed scaRNA2 and scaRNA17 are shown in lane 5 of the middle (+sca2) and right (+sca17) panels, respectively.