An in vivo Dicer reconstitution assays for the functional analysis of dmDcr1–Loqs interactions. (A) Schematic overview of full length Loqs-PB. Point mutations are indicated as red lines. (B) HA-dmDcr1 (lanes 4–11), wt Loqs (lane 5) as well as the indicated Loqs mutants (lanes 6–11) were cotransfected into Dicer-deficient MEFs. Additionally, HA-GFP and HA-hDcr (lanes 2,3) were transfected as controls. Endogenous let-7a from Dcr+/+ MEFs (lane 1) was loaded as a marker and processing in the deficient cells was analyzed by Northern blotting (upper panel). HA-dmDcr1 (middle panel) or HA-Loqs (lower panel) expression was analyzed by Western blotting using anti-HA antibody. Loading of the Northern blot samples was controlled by EtBr staining and loading of Western blots via an actin blot. (C) Comparison of the Loqs-PB and Loqs-PA isoforms in the rescue assay. (D) Influence of the dimerization mutants L426R, K460E, and KI460/61EA on miRNA processing.
