Figure 5.

Rio1 associates at a very late step of pre-40S biogenesis via its conserved C-terminal region. Rio1 associates with a very late pre-40S particle lacking most late SSU ribosome biogenesis factors. Wild-type yeast cells (BY4741-Euroscarf) transformed with a plasmid carrying the indicated bait protein fused either with N-terminal ProtA-TEV-Flag (Nob1 and Rio1 constructs) or C-terminal Flag-TEV-ProtA (Rio2) under the control of the ADH1 promoter were grown in SDC-Leu up to OD₆₀₀ = 1.5. Bait proteins were purified by standard tandem affinity purification (see the Materials and Methods section). Co-immunoprecipitated proteins from Flag eluates were precipitated by addition of TCA and resolved on a 4–12% Nupage gel (Invitrogen) and analyzed either by coomassie staining and mass-spectrometry or by western blotting using the indicated antibody. RNA was extracted by hot-phenol extraction and resolved on a 1.2% agarose Formaldehyde/MOPS gel, and transferred to a positively charged Nylon membrane. For northern blot analysis membrane was hybridized with the indicated probes (see the Materials and Methods section) and exposed to a Phosphorimager screen. Signals were quantified with ImageJ.