Structural and hydrolytic features of DiGIR2. (A) Secondary structure model of DiGIR2. Structural segments important in this study (P1 and P9.2) are boxed, and specificities of the peptide nucleic acids PNA1283, PNA1487, and PNA1488 are indicated. (B) Incubation of 3′ SS hydrolyzed precursor RNA generates the full-length circular intron RNA. Individual RNA species were purified from gels and incubated under splicing conditions without GTP for various times (0, 30, and 60 min, indicated by triangles above lanes). RNAs were subsequently separated on an 8 M urea/4% polyacrylamide gel in 0.4× TBE buffer. The RNAs are the 5′-exon-intron RNA (5′E-Int), the intron RNA (Int), and the full-length circular intron RNA (FLC). The circularization junction in FLC was amplified by RT-PCR, sequenced, and verified to correspond to full-length intron circularization (data not shown).
