Figure 8

In vivo template jump to the small nuclear RNA, snU12. (A) 5' R2 junction products from PCR amplification in Drosophila ambigua separated on a native, 8% polyacrylamide gel. Lane M, DNA length markers. (B) Diagrams of sequenced PCR products: 28S sequences (gray boxes); R2 sequences (blue boxes); snU12 sequences, yellow boxes. Long PCR products (12 clones) had a 48 bp deletion of upstream 28S sequences, 156 bp with sequence identity to the 5' end of snU12, and a 6 bp repeat at the snU12/R2 junction (arrowheads). Short products (eight clones) had typical 5' junctions that differed by zero to two non-templated nucleotides. (C) In vitro cotranscription/cleavage assay of RNA containing R2 sequences with the snU12 extension indicated self-cleavage only immediately upstream of the R2 sequences (lane 1, open circles). RNA constructs (see (D)) designed to promote self-cleavage upstream of the snU12 sequences did not self-cleave (lanes 2 and 3, solid circles). (D) Secondary structures of R2 with U12 extension (1) and two modified constructs. The substitution of two C’s in the P1 stem (2) and deletion of the 5' end of R2 (3) are highlighted in gray. Structure number corresponds to lane number in (C). Nomenclature and highlighted nucleotides are as described in Figure 4.