The effect of simultaneous and individual MRB8170/4160 RNAi silencing on minicircle-encoded gRNAs. (A) RNA from the double MRB8170/4160 knockdown grown in the presence (+) and absence (−) of the RNAi-induction agent tetracycline was capped with [α-32P]GTP by the recombinant guanylyltransferase enzyme. The homogeneous population of small gRNA molecules in the samples was resolved as a ladder of bands on a denaturing 7 M urea/8%-acrylamide gel (bottom panel). A cytosolic RNA that is simultaneously labeled by this reaction is shown (top panel) as a loading control. (B) Individual gRNAs directing editing of RPS12, MURF2, co3, and A6 mRNAs (using the abbreviations as defined in Fig. 9) were detected by Northern blot. A probe against a cytoplasmic tRNACys (GCA) was used as a loading control. The “+” and “−” above the lanes indicate the state of tetracycline induction.
