Figure 5. Gemin5 binds directly to the m⁷G cap structure.

(A) FLAG-tagged gemin5 was immunoprecipitated and released from the resin with FLAG peptide. Eluted protein was subsequently applied to cap-sepharose in the presence of free m⁷GpppG or GpppG competitors. Precipitation of FLAG-gemin5 was monitored by western blot and silver stain analysis. (B) A representation of the m⁷G cap structure is shown with position of radiolabeled α-phosphate indicated by an asterisk. A short RNA transcript was synthesized and then modified with an m⁷G cap using [α-³²P]-GTP and guanylyltransferase (see experimental procedures for details). Purified capped RNA was incubated in the presence or absence 0.1 mM free cap analog with 1) 293T cytoplasmic lysate, 2) eluate from α-FLAG IP of lysate from pcDNA3-transfected cells (mock IP), or 3) FLAG-tagged gemin5 or gemin5(Δ25) immunopurified as in (A). Binding reactions were irradiated with UV light, incubated with RNase cocktail, and then analyzed by SDS-PAGE followed by autoradiography as indicated.