Skip to main content
. 2012 Dec;18(12):2166–2173. doi: 10.1261/rna.036194.112

FIGURE 2.

FIGURE 2.

Biogenesis and function of a Drosha/Dicer-independent miRNA generated by the Integrator pathway. (A) A minigene in which the normally Dicer-dependent (dd) pre-mir-HSUR4 hairpin (U1-mir-dd) (see also Supplemental Fig. 2) has been shortened to Ago2-dependent (ad) length (U1-mir-ad); mature miR-HSUR4-5p is indicated by the red box. (B) Functionality of U1-mir-dd and U1-mir-ad against a luciferase sensor bearing bulged miR-451 sites, assayed in 293T cells. Averages of three independent experiments with SD are shown. (C) Accumulation of mature miR-HSUR4-5p following transfection of U1-mir-dd and U1-mir-ad into 293T cells; EBER1 was cotransfected and detected as a control. (D–F) Integrator-dependent, microprocessor-independent processing of U1-mir constructs. 293T cells were transfected with control siRNA (siControl) or siRNA against Integrator11 (siInt11) for 48 h, followed by retransfection with siRNA and a mixture of expression plasmids for EBER1, mir-142, and either U1-mir-dd or U1-mir-ad. Expression of mature miR-HSUR4-5p from both U1-mir-dd and U1-mir-ad was lost following knockdown of Int11, while miR-142-3p and EBER1 were unaffected. (E) The same experimental setup as in D, except using siRNA against DGCR8. Mature miRNAs were effectively produced from both U1-mir constructs, but not from canonical mir-142. (F) 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 U1-mir-ad, mir-142, and EBER1. miR-HSUR4-5p but not miR-142-3p accumulated in Drosha knockout cells. (G) Dicer-independent processing of U1-mir-ad. Dicer knockout MEFs were transfected with U1-mir-dd and U1-mir-ad; biogenesis of the former was arrested at the pre-miRNA stage while the latter continued to generate mature miRNAs in the absence of Dicer. (H) Slicer-dependent processing of U1-mir-ad. Northern blot showing coimmunoprecipitation of miR-HSUR4-5p or endogenous miR-16 from 293T cells cotransfected with either U1-mir-dd or U1-mir-ad, and either FLAG-Ago1, FLAG-Ago2, or FLAG-Ago2D669A. (I) Input (5%); (P) Pellet (100%). Dicer-dependent mature miRNAs associate with all of these tagged Ago proteins, but Ago2-dependent mature miRNAs are present only in wt Ago2 immunoprecipitates.