FIGURE 3.
RioK1 directly interacts with PRMT5 via its very N-terminal region. A, in an interaction assay, in vitro translated, radioactively [35S]methionine-labeled RioK1 was incubated with GST-tagged PRMT5 (lane 2), WD45/MEP50 (lane 3), pICln (lane 4), and GST (lane 5) bound to GSH-Sepharose. After extensive washing, the precipitated proteins were eluted with sample buffer, resolved by SDS-PAGE, and visualized by Coomassie staining. The in vitro translated protein was visualized by autoradiography of the dried gel. For comparison, 10% of in vitro translated RioK1 was loaded (lane 6). B, GST-tagged truncations of RioK1 comprising aa 1–242 (lanes 2–4), aa 227–568 (lanes 5–7), aa 1–120 (lanes 8–10), or GST (lanes 11–13) bound to GSH-Sepharose were incubated with in vitro translated, [35S]methionine-labeled full-length (fl), N-terminal (nt; aa 1–291), or C-terminal (ct; aa 295–637) versions of PRMT5 in an interaction assay. For comparison, 10% of in vitro translated proteins were loaded (lanes 14–16). The interaction assay was analyzed as described in A. C, GST-PRMT5 (lanes 2–6) and GST (lanes 7–11) bound to GSH-Sepharose were incubated with an in vitro translated, [35S]methionine-labeled version of RioK, including full-length RioK1, aa 1–242, aa 227–568, aa 1–120, and aa 121–242. For comparison, 10% of in vitro translated RioK1 was loaded (lanes 12–16). D, GST-tagged pICln (lanes 2 and 3), RioK1 (lanes 4–6), GST alone (lanes 7 and 8), RioK2 (lanes 9 and 10), or RioK3 (lanes 11 and 12), covalently cross-linked to GSH-Sepharose were incubated with or without HeLa extract in a pulldown assay. As a control, non-cross-linked protein was analyzed by SDS-PAGE. After extensive washing, the precipitated proteins were eluted from the matrix by sample buffer, resolved by SDS-PAGE, visualized via Coomassie staining, and analyzed by mass spectrometry. Note, that lanes containing non-cross-linked, recombinant proteins exhibit degradation products of these proteins.