Fig 6. miRNA and splicing machineries are physically and functionally distinct.

(A) S2 cells were treated with control or SmD1 dsRNAs and analyzed for expression of various canonical miRNA pathway components by immunoblotting. Tubulin protein served as controls. Note identical copies of samples were subject to immunoblotting using various antibodies. (B) S2 cells were treated with various dsRNAs (top). Levels of miR-2b, esi-2.1 and the control 2S rRNA were measured by Northern blot and quantified (bottom). (C,D) Splicing activity was assessed in various knockdown cells (top) by examining splicing pattern of the CG13887 pre-mRNA by RT-PCR. The RB splice variant retains an intron (a straight line between exons shown as open boxes), whereas the RC/RD variants lack the intron. Ratio of intron retention/excision was quantified and shown in D (n = 3). (E) Lysates from cells expressing various Flag-tagged Sm proteins or the control protein Ran together with T7-tagged Pasha were subject to immunoprecipitation using anti-Flag antibody. Both input samples and isolated complexes were probed with anti-T7 (top panel), anti-SmD1 (middle) and anti-Flag antibodies (bottom). (F) Lysates from cells expressing various Flag-tagged Sm proteins or the control protein were subject to immunoprecipitation using anti-Flag antibody. Total RNA was extracted from various immunopurified Flag-tagged protein complexes and subject to RT-qPCR to measure levels of various pri-miRNAs and the U4 snRNA. (G) Total RNA was extracted from immunopurified TAP-Pasha or control samples (TAP) and subject to RT-qPCR to measure levels of various snRNAs. Fold change relative to levels of the control rp49 mRNA are shown (n = 3; n.s., non-significant). (H) A schematic depicting the proposed roles of SmD1 in the initiation (miRNA biogenesis) and effector (miRISC assembly and/or mRNA cleavage) steps of the miRNA pathway.