Figure 1.

Identification of the IRES-binding site in Gemin5. (A) Schematic of HIS-tagged polypeptides used in RNA-binding assays. Numbers indicate amino acid residues referred to the full-length protein. Grey ovals depict WD motifs located within the N-terminal region of the protein. A triangle within the C-terminal region of the protein depicts the position of the epitope recognized by Gemin5 antibody. (B) UV-crosslinking (UV-XL) assay conducted with increasing amounts of purified HIS-tagged Gemin5 polypeptides depicted at the top and radiolabelled domain 5, fractionated in SDS-PAGE and visualized by autoradiography. In each case, the mobility of the protein detected by WB using anti-Gemin5 or Coomassie Blue staining of the purified protein is shown on the right. Mobility of Mw markers is indicated at the left. (C) Gemin5-RNA binding assay. Autoradiograph of denaturing 6% acrylamide gels, 7 M Urea loaded with RNAs isolated from Ni-agarose beads coupled to the indicated proteins (0.07 pmol). D5 and RNAc are used for radiolabelled domain 5 and total cytoplasmic RNA, respectively.