Competition between added exogenous guanosine (GTP) and terminal guanosine (ωG) in DiGIR2 intron splicing and 3′ hydrolysis. The primary transcripts (Pre) were incubated under splicing conditions at varying GTP concentrations (0, 2, 20, 200, and 2000 μM) in time course experiments (0, 2, 5, 15, 30, and 60 min). RNAs were subsequently separated on an 8 M urea/5% polyacrylamide gel. The positions in the gels of the relevant RNA species are indicated. Note that the RNA species slightly smaller than RNA 3 present at early time points and high GTP concentrations corresponds to an Intron-3′-exon (Int-3′E) RNA. The circular species (RNA1) was eluted from the gels and purified. The circularization junction was amplified by RT-PCR and sequenced and found to correspond to a full-length intron circle (data not shown).
