Northern validation of the incorporation of mirtron-derived miRNAs into Ago2 complexes. The structures of small RNA precursors of various biogenesis classes are shown at far left and far right. (A) Mirtron expression constructs were transfected into HeLa cells along with myc-tagged Ago2 vector. Endogenous miR-19 was detected in input samples and Ago2 immunoprecipitate, but not IgG immunoprecipitate. We detected mature mirtron-derived small RNAs from each of the three biogenesis classes in input samples. These were strongly enriched in Ago2 immunoprecipitates but absent from control IgG immunoprecipitates, demonstrating their specific residence in effector complexes. (White bar) Designates that the mirtron lanes were not adjacent to the ladder lanes, although they were taken from the same blots in all cases. The data for miR-1226 and miR-19 were generated by sequential stripping and reprobing of the same blot as an internal control; the other blots also showed mature miR-19 (data not shown). (B) Mirtron expression constructs were transfected into Dicer-KO MEFs along with a Mir144/451 expression construct and myc-tagged Ago2 vector. While the Dicer-independent species miR-451 was successfully processed in Dicer-KO cells, none of the three mirtrons generated mature small RNAs in either total RNA or Ago2 complexes. The data for miR-1226 and miR-451 were generated by sequential stripping and reprobing of the same blot as an internal control; the other blots also showed mature miR-451 (data not shown).
