FIGURE 3:

Y14 binds directly to the mRNA cap structure. (A) For UV cross-linking, 5 μg of recombinant GST or GST-Y14 was incubated with ³²P-labeled capped PIP85a∆i RNA (m⁷G*pppG-; lanes 3 and 4) or ³²P-labeled free cap (m⁷G*pppGp; lanes 5 and 6). After UV radiation and RNase digestion, proteins were resolved by SDS–PAGE and detected by autogradiography (lanes 3–6) and Coomassie blue staining (lanes 1 and 2). (B) UV cross-linking of GST or GST-Y14 (full-length, ∆C, or ∆N) proteins was performed as in A using ³²P-labeled m⁷G*pppG-RNA (lanes 5–8) or noncapped RNA (*pG-; lanes 9–12) as substrate. Lanes 1–4, Coomassie blue staining of the recombinant Y14 proteins used. (C) UV cross-linking of GST, GST-Y14, or GST-Y14∆C was performed using the m⁷G*pppG-capped RNA as substrate in the absence (–) or presence (+) of m⁷GpppG. (D) UV cross-linking was performed with the m⁷G*pppG-capped RNA and 5 μg of His-tagged Y14 (lane 1) or His-Magoh alone (lane 5) or 5 μg of His-Y14 together with 1.25, 2.5, or 5 μg of His-Magoh (lanes 2–4, respectively). (E) FLAG-tagged Y14 and CBP80 were each transiently expressed in HEK293 cells and immunopurified from the lysates using anti-FLAG Agarose. Purified proteins (lanes 1–3) were then subjected to affinity chromatography using m⁷GTP-Sepharose in the absence (lanes 4–6) or presence (lanes 7–9) of m⁷GpppG. Chromatography was performed using GTP-agarose (lanes 10–12). FLAG-tagged protein input (lanes 1–3), and proteins bound to the m⁷GTP (lanes 4–9) or GTP (lanes 10–12) were analyzed by immunoblotting with anti-FLAG.