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2]. Beads were preblocked with glycogen (0.1 mg/ml), bovine serum albumin (1 mg/ml), and tRNA (0.1 mg/ml) in WB-50 buffer for 20 min at 4°C, then washed four times with 1 ml of WB-50 and once with WB-250 (WB-50 brought to 250 mM NaCl). After resuspension in an equal volume of WB-250, 50-mg aliquots of beads were gently rocked for 1 hour at 4°C with 25 μl of a standard splicing reaction containing 60% v/v HeLa nuclear extract but no substrate or, in later experiments, with either 250 μl of each glycerol gradient fraction. Beads were washed four times with 0.5 ml WB-250 buffer, and bound RNA was purified by digestion with proteinase K, phenol extraction, and ethanol precipitation. Affinity selection on a monomeric avidin column was performed according to the manufacturer (Pierce) with WB-500 as the wash buffer. The three RNA oligonucleotides used in Fig. 4 were 5′-CAGGUAAGUAdT-3′ (5′ SS), 5′-CAUACUUAUUCCdU-3′ (BP), and 5′-CCCUUUUUUCCACAGCUdC-3′ (Py-3′ SS). The sequence of α-Py is 5′-GGAAAAAAGGG-3′.
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Both labeled and unlabeled Py-3′ SS oligo support 5′ SS selection, although labeled oligo is present at 20-fold lower concentration than unlabeled oligo.
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We thank J. A, Steitz for many useful suggestions, oligonucleotides, and Y12 antibody; members of her laboratory for suggestions; A. D. Bailey and T. A. Pavelitz of our laboratory for discussions and critical reading of the manuscript; and M. Moore for the gift of p220 antibody. G.A. was supported by a Human Frontiers Science Program Organization Long-Term Fellowship. This work was funded in part by NIH award GM31073 to A.M.W.
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