Biogenesis and function of a Drosha/Dicer-independent miRNA generated by the tRNA pathway. (A) Design of tRNA–miRNA fusion constructs in which either a Dicer-dependent (dd) or Ago2-dependent (ad) pre-mir-451 hairpins reside downstream from the RNase Z cleavage site of a tRNAlys construct; mature miR-451 is represented by the red box. (B) Functionality of mir-451 expression constructs on perfect (si) and bulged (mi) miR-451 luciferase sensors. Compared with control mir-1-2 construct, the endogenous mir-144/451 and both “dd” and “ad” versions of tRNA-mir-451 fusions could repress these sensors. As expected, repression of the perfect sensor was more efficient with all constructs. (C) Functionality of miRNA expression constructs in Dicer−/− MEFs. Only Dicer-independent hsa-mir-144/451 and tRNA-451-ad constructs were functional in this setting. (D) Drosha-independent processing of tRNA-mir-ad. Drosha-fl/fl Cre-ER mouse ES cells were mock-treated (−) or treated (+) with 4-OH-Tamoxifen (tam) for 72 h, followed by cotransfection of tRNA-mir-ad, mir-142, and EBER1. Only miR-451 derived from tRNA-mir-ad, but not miR-142-3p, accumulated in Drosha knockout cells. (E) Dicer-independent processing of tRNA-mir-ad. Northern blot analysis of Hela cells and Dicer−/− MEFs transfected with tRNA-mir-dd or tRNA-mir-ad constructs; mir-375 was cotransfected as a control. (F) tRNA-mir-ad matures exclusively within Ago2. Northern blot showing coimmunoprecipitation of tRNA-mir-dd or tRNA-mir-ad with control (IgG), anti-human Ago1 (hAgo1), or anti-human Ago2 (hAgo2).
