Figure 3

RNA processing activity by AspGIR2. (A) Schematic organization of Asp.S516 intron and corresponding splicing RNA construct (Asp.S516Δ742 RNA) and lariat capping RNA construct (AspLC-191.28 RNA). SSU; small subunit ribosomal RNA exons. (B) Time-course gel analysis of Asp.S516 self-splicing products. In-vitro transcribed ³²P-labelled RNA from Asp.S516Δ742 was incubated at self-splicing with and without exoG. RNA species generated are indicated to the right. RNA#1, circular intron; RNA#2, precursor transcript; RNA#3, 5′SS or 3′SS processed precursor transcript; RNA#4, excised linear intron; RNA#5, IPS processed 5′ precursor; RNA#6, IPS processed 5′ intron; RNA#7, IPS processed 3′ precursor; RNA#8, IPS processed 3′ intron; RNA#9, ligated exon; RNA#10, free 5′ or 3′ exons. Exons, AspLC and AspGIR2 are indicated as dark grey, black and light grey boxes, respectively. (C) ExoG-labelling of Asp.S516Δ742 RNA compared with uniformly labelled time-course experiment of the same RNA. RNA#4 (excised linear intron) is the main labelled RNA species. (D) Ligation junction sequence of ligated exons generated in self-splicing reaction with exoG. Sequences were determined from RT-PCR generated products of ligated exons (RNA#9, Figure 3B) using primer combinations C716/C736 in amplification and C736 in sequencing reactions. DNA sequence reads from the reactions (right) and is complementary to that of the RNA sequences presented. Arrow marks intron insertion site or ligated exon site. (E) Full-length circle-junction sequences of Asp.S516 generated in self-splicing reaction without exoG. Sequences were determined from RT-PCR generated products of spliced Asp.S516Δ742 RNA using primer combination C734/C735 in amplification and C734 in sequencing reactions. The signal one nucleotide above the branch point is due to a capping-independent primer extension stop. Note that the sequencing ladder is generated from the opposite strand. (F) Dominating circle junction generated with exoG in the self-splicing reaction. The sequence corresponds to truncated circles lacking the first two nucleotides of the intron.