FIG. 4.

Alpha-globin G triplets psoralen cross-link to nucleotides U10 and U22 of U1 snRNA. (A) Sequence of U1 snRNA. The sequences targeted by the U1 5′ and loop 2 oligonucleotides are shaded. The Sm protein binding region is indicated by a stippled box. The positions of mapped cross-links (U10 and U22) are indicated. (B) Psoralen cross-linking reactions using the wild-type G1-G2 RNA were carried out in nuclear extracts treated with 2′-O-methyl RNA oligonucleotides complementary to U3 RNA or U1 RNA nucleotides 1 to 11. The cross-linking products were analyzed on a 5% polyacrylamide–8.3 M urea denaturing gel and visualized by autoradiography. The labeled band corresponds to the cross-linked species in Fig. 1. (C) Analysis of U1 snRNA in nuclear extract after incubation with RNase H (lane 1) or RNase H plus a DNA oligonucleotide complementary to U1 nucleotides 1 to 11 (lane 2). RNAs were separated on a 10% polyacrylamide–8.3 M urea denaturing gel and visualized by ethidium bromide staining. The positions of U2 snRNA, 5.8S RNA, and cleaved and uncleaved U1 are indicated. (D) Primer extension analysis of U1 snRNA in nuclear extract after incubation with RNase H (lane 1) or RNase H plus a DNA oligonucleotide complementary to U1 nucleotides 1 to 11 (lane 2). The U1 loop 2 oligonucleotide was used to prime RT, and the products were analyzed on a 6% polyacrylamide–8.3 M urea sequencing gel and visualized by autoradiography. The positions of uncleaved and cleaved U1 molecules are indicated. Nucleotide numbers were assigned by comparison to an adjacent U1 sequencing reaction (not shown). (E) Analysis of the G1-G2–U1 cross(X)-linked species by DNA oligonucleotide-directed RNase H cleavage using the U1 5′ oligonucleotide (lane 2), loop 2 oligonucleotide (lane 3), or both (lane 4). Treatment with the 5′ oligonucleotide was done before cross-linking, and treatment with the loop 2 oligonucleotide was done after cross-linking and RNA purification. RNAs were analyzed on 5% polyacrylamide–8.3 M urea denaturing gels and visualized by autoradiography. (F) Primer extension analysis of cross-linked RNAs. Cross-linked RNAs were purified and analyzed by primer extension using the U1 loop 2 oligonucleotide. The positions of two transcription stops (U10 and U22) and the 5′ end of U1 RNA are indicated. In lanes 1 and 2, 5 or 10 μg of nuclear RNA was analyzed by primer extension. The U10 and U22 stops were not observed when RNAs were analyzed following psoralen cross-linking in the absence of substrate RNA and therefore are not intramolecular cross-links (data not shown).