FIGURE 2.

Internal modifications in the first 24 nucleotides of U2 snRNAs are required for splicing. (A) The sequence composition of the chemically synthesized oligonucleotides corresponding to the first 24 nucleotides of U2 snRNA. The following notation is used: ΔΨm, replacement of all pseudouridines (Ψ) by uridines (U) and deletion of all 2′O-methylations (2′O-Me); (Ψm) retention of all Ψ and 2′O-Me; (ΔΨ) replacement of all Ψs by U; (ΔΨn) replacement of Ψ at position n with U; (Δm) deletion of all 2′O-Me; (Δmn) deletion of 2′O-Me at position n. The oligonucleotides were ligated to in vitro-transcribed U2 starting at G25. For comparison, sequences of purified HeLa U2 and U2 transcript are shown. (B) Internal modifications within the first 24 nucleotides of U2 snRNA are required for efficient complementation of splicing. Splicing complementation of U2 snRNAs differing in the number and type of internal modifications was assayed in the two-step reconstitution system by monitoring splicing of ³²P-labeled pre-mRNA (lane 1). Untreated (lane 2), mock-depleted (lane 3), and U2-depleted (lane 4) nuclear extract are shown as controls. (Lanes 5–18) Complementation of U2-depleted nuclear extract with U2 snRNPs reconstituted with the U2 snRNA indicated above each lane. RNA was analyzed by denaturing PAGE and visualized by autoradiography.