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. 2012 Jul 1;26(13):1459–1472. doi: 10.1101/gad.189001.112

Figure 3.

Figure 3.

QKI associates with and stabilizes miR-20a. We investigated whether QKI-5, QKI-6, and QKI-7 isoforms each associate with and stabilize miR-20a and focused on QKI-6 for in-depth analysis. QKI-6 is the isoform previously shown to have evolutionarily conserved function in Caenorhabditis elegans (Schumacher et al. 2005). (A) In vitro UV cross-linking was performed using normal human astrocytes treated with or without doxorubicin, and QKI-5, QKI-6, or QKI-7 was immunoprecipitated by isoform-specific antibodies targeting each isoform. QKI-5, QKI-6, QKI-7 each associated with miR-20a in doxorubicin-treated samples but not with pre-miR-20a or pri-miR-20a. Ten percent of the Input cross-linked samples and 50% of the immunoprecipitates were analyzed by Western blotting for QKI (WB) to ensure similar immunoprecipitation of each QKI isoform. Each QKI isoform migrated in SDS-PAGE gel at their estimated sizes: QKI-5, 45 kDa; QKI-6, 38 kDa; QKI-7, 40 kDa. (*) P < 0.05. (B) In vivo UV cross-linking using V5 antibody to immunoprecipitate V5-QKI-6 or V5-QKI-6 mutant from transiently transfected 293T HEK as described in the Materials and Methods. Wild-type (WT) V5-QKI-6 associated with miR-20a but not pre-miR-20a or pri-miR-20a, while V5-QKI-6 mutant did not associate with any miRNA. (**) P < 0.01. Ten percent of the Input cross-linked samples and 50% of the immunoprecipitates were analyzed by Western blotting with QKI antibody to ensure similar expression and immunoprecipitation of the in vivo cross-linked V5-QKI-6 and V5-QKI-6 mutant proteins but not by the control Flag (Ctrl) antibody (the proteins' migration is affected due to the cross-linking incomplete nucleic acid removal). (C) In vitro UV cross-linking was performed using purified V5-QKI-6 and V5-QKI-6 mutant proteins with radio-labeled miRNAs, followed by immunoprecipitation with V5 agarose or control Flag agarose as described in the Materials and Methods. The immunoprecipitates were separated by SDS-PAGE, Western blotted, and analyzed by PhosphorImaging. Wild-type (WT) V5-QKI-6 purified protein associated with miR-20a but not with miR-18a, miR-20a*, or pre-miR-20a; V5-QKI-6 mutant purified proteins did not associate with any miRNA. To ensure similar immunoprecipitation of V5-QKI-6 and V5-QKI-6 mutant purified proteins and not with control Flag (Ctrl) antibody, these Western blots were reprobed with QKI antibody. (D) 293T HEK cells were transfected with vector, QKI-5, QKI-6, and QKI-7 all together (QKI) or mutant QKI-5, QKI-6, and QKI-7 all together (QKI-mut) for 24 h and then treated with 20 μg/mL α-amanitin. Cells were lysed every 3 h for up to 12 h. QKI-transfected cells showed miR-20a stabilization relative to vector-transfected cells and QKI-mut-transfected cells. No difference in stabilization was observed for pre-miR-20a, pri-miR-20a, miR-18a, and miR-20a* mRNA. (*) P < 0.05 compared with vector control. QKI expressions were displayed by Western blot.

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