FIGURE 4.

RioK1 and pICln compete for binding to PRMT5. A, GST-tagged RioK1 (lane 2) and pICln (lane 3) or GST alone (lane 4), covalently cross-linked to GSH-Sepharose were incubated with HeLa extract in a pulldown assay. After extensive washing, the precipitated proteins were eluted from the matrix by sample buffer, resolved by SDS-PAGE, and visualized via Coomassie staining. Intervening lanes have been spliced out. Lane 1 shows protein standard (M). B, in a competition assay, the PRMT5 complex was immunoprecipitated with anti-WD45/MEP50 antibodies from HeLa cell extract. The purified complex was incubated with increasing amounts (0.1 to 10 μg) of GST-tagged RioK1 (aa 1–242, left panel) or pICln (right panel). The complex composition was afterward analyzed by Western blotting using the indicated antibodies. C, HeLa cell extract was incubated with GST (20 μg, upper panel), GST-RioK1 (20 μg, middle panel), or GST-pICln (<20 μg, lower panel) and was afterward fractionated by size exclusion chromatography. The collected fractions were analyzed by Western blotting with the indicated antibodies. D, GST-tagged pICln (lanes 2–4) or GST (lanes 5–7) immobilized on glutathione-Sepharose were incubated with in vitro translated, [³⁵S]methionine-labeled, full-length PRMT5 or truncations thereof in an interaction assay. After extensive washing, the precipitated proteins were eluted from the matrix in loading buffer, resolved by SDS-PAGE, Coomassie stained, and visualized by autoradiography. For comparison, 10% of in vitro translated proteins were loaded (lanes 8–10). E, schematic overview of the interacting domains of PRMT5, RioK1, and pICln. Interactions are indicated by arrows. Interacting domains are colored black. fl, full length; nt, N-terminal; ct, C-terminal; TCL, total cell lysate.