α-Actinin TvACTN3 of Trichomonas vaginalis Is an RNA-Binding Protein That Could Participate in Its Posttranscriptional Iron Regulatory Mechanism
Figure 8
Interaction of TvACTN3r with trichomonad IRE RNA. (a) Representative α-TvACTN3r antibody supershift assays. A radiolabeled IRE-tvcp4 RNA probe (lane 1) was incubated with cytoplasmic proteins from T. vaginalis grown under iron-depleted conditions without (lane 2) or with different quantities of α-TvACTN3r antibody (lanes 3–5) or an unrelated antibody (α-TvTIM2r; lane 6). Arrowheads indicate the RNA-protein complexes. A representative result of three independent experiments with similar results is shown. (b) Northwestern blot assay. The recombinant proteins TvACTN3r; DIr, DIIr, and DIIIr and the controls hIRP-1r; and BSA were separated by SDS-PAGE on 10% polyacrylamide gels (panel I) and transferred onto NC membranes, which were incubated with a radiolabeled IRE-tvcp4 RNA probe (panel II). The controls included a radiolabeled nonrelated RNA (panel III) and a radiolabeled deletion mutant that disrupts IRE-tvcp4 RNA (panel IV). (c) UV cross-linking assays of the recombinant proteins TvACTN3r, DIr, DIIr, and DIIIr and radiolabeled IRE-tvcp4 RNA probes resolved by SDS-PAGE on 10% polyacrylamide gels (panel II). As controls, TvACTN3r and its domains were incubated with radiolabeled nonrelated RNA (panel III) and a radiolabeled deletion mutant that disrupted IRE-tvcp4 RNA (panel IV). In addition, a control from a representative gel of the different recombinant proteins in cross-linking mix was CBB-stained (panel I); kDa, molecular weight standards in kilodaltons. A representative result of three independent experiments with similar results is shown. (d) Identification of putative RNA-protein interaction motifs in TvACTN3 by in silico analysis. The complete protein and the different domains of TvACTN3 are illustrated with brackets, and the possible motifs for RNA-protein interactions are indicated. The boxes show the location of the motif described on top of the figure and in Table 3.
