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. 1999 May;19(5):3561–3570. doi: 10.1128/mcb.19.5.3561

FIG. 5.

FIG. 5

Formation of a stable complex containing the SLBP and the U7 snRNP on the pre-mRNA substrate. (A) Sequences of the substrates used for assaying U7 snRNP binding. The sequence of the parental H2a pre-mRNA comprising the stem-loop structure, the cleavage site represented by an arrow, and the U7 binding site are shown at the top. The nucleotides at the 5′ and 3′ ends of the RNA encoded in the pGEM3 vector are not included. The nucleotide substitutions introduced into the H2a pre-mRNA in order to generate the other pre-mRNA substrates are shown below the H2a sequence. The unchanged sequences are represented by solid lines. The RS mutant of the H2a-614 pre-mRNA contains reversed sequence of the stem-loop structure, as shown in Fig. 1B. (B) The H2a/4G (lane 1) and HDE− (lane 2) substrates shown in panel A were incubated in a nuclear extract under standard processing conditions. The RNA was analyzed as described in the legend to Fig. 2. (C) The H2a-614 substrate (lanes 2 to 6) and the indicated mutant substrates (lanes 7 to 10) were briefly incubated in nuclear extract to allow the formation of processing complexes. The complexes were immunoprecipitated with anti-SLBP antibody, RNA prepared, resolved by electrophoresis on 8.5% polyacrylamide gel containing 7 M urea, and assayed for the presence of U7 snRNA by Northern blotting. Immunoprecipitation was carried out in the presence of the H2a-614 pre-mRNA (lanes 2 to 6) or mutant pre-mRNAs, as indicated (lanes 7 to 10). Lane 1, no substrate added; lane 3, 10 μg of antigenic peptide was added to the reaction mixture prior to addition of the antibody; lanes 4 and 5, immunoprecipitation in the presence of 0.5 μg of a nonspecific 2′-O-methyl oligoribonucleotide or 2′-O-methyl oligoribonucleotide complementary to the 5′ end of U7 snRNA, respectively. Lane 6, immunoprecipitation in the presence of a 100-fold excess of the 30-nucleotide RNA containing the stem-loop sequence. Control, 0.1 ng of a synthetic 77-nucleotide RNA containing the complete sequence of the U7 snRNA added to each sample as an internal standard for RNA recovery and hybridization efficiency.