FIG. 2.

Recruitment of Aly/REF onto RNA by UAP56. (A) The same ³²P-labeled RNA mixture as in Fig. 1A, except that CTE was omitted but intronless β-globin mRNA and U5ΔSm RNA were supplemented, was incubated with (+) or without (−) recombinant Aly/REF (10 nM) and recombinant Flag-tagged UAP56 (10 nM), either wild type (+) or GET mutant (GET, K95E), in either the absence (−) or presence (+) of 2 mM ADP, ATP, or ATP-γS at 30°C for 15 min. After the incubation, immunoprecipitation was performed with anti-Aly/REF antibody (anti-REF, 11G5; ImmuQuest) or anti-Flag antibody (M2; Sigma) and the coprecipitated RNA was analyzed as described for Fig. 1. (B) The same experiment as in panel A, lanes 1, 2, 4, and 5, except that a higher concentration (50 nM) of Aly/REF was employed. (C) Recombinant Flag-UAP56 (1 μg) and 3,000 Ci/mmol [α-³²P]ATP (1 μl; Amersham) were incubated in the absence or presence of increasing concentrations of unlabeled ATP, ADP, ATP-γS, AMP-PNP, and AMP-PCP as competitors and irradiated with UV light as described in Materials and Methods. The cross-linked protein was analyzed by SDS-PAGE and autoradiography. (D) A UV cross-linking experiment similar to that shown in panel C was performed with wild-type UAP56 (UAP-WT), UAP56 LAT mutant (UAP-LAT, S228L), or UAP56 GET mutant (UAP-GET, K95E), without nucleotide competitors, in the absence (−) or presence (+) of yeast RNA (1 mg/ml; Sigma).