Self-splicing of a mitochondrial group I intron from the cytochromeb gene of the ascomycetePodospora anserina

Summary

We have shown that the second intron of thePodospora mitochondrial gene coding for cytochromeb (Cytb 12) splices autocatalytically, using in vitro transcripts generated from the T7 promoter. The reaction takes place at 37° C in the presence of 50 mM TRIS-HCl pH 7.5, 60 mM MgC1₂ and 1 mM GTP but shows a low efficiency even at high KCl concentrations of up to 1.2 M. Under these conditions, intron bI2 follows the conventional pathway of group I splicing, and all characteristic products, with regard to both transesterification and hydrolysis, could be identified. Moreover, the intron is capable of undergoing cyclization, thereby releasing the noncoded G and one additional nucleotide (U) from the 5′ end. The 5′ cleavage site is preceded by the same two nucleotides, indicating a base-pairing at the same site of the internal guide sequence (IGS) for both splicing and cyclization (“one-binding-site model”). In addition, products resulting from site-specific hydrolysis 138 nucleotides downstream of the 5′ splice site were detected. Unusually, the shortened intron is also able to form a circular RNA and an alternative sequence that aligns the cyclization site to the catalytic core of the intron must be assumed.