Models for incorporation of non-encoded nucleotides in intron RNA circles. Non-encoded nucleotides (gray box) may be incorporated during splicing using variations of the hydrolytic (A) (Fig. 1B) or circularization pathway (B) (Fig. 1C). (A) Non-encoded nucleotides may be added by an unknown mechanism to the 3′ end of linear introns released by the hydrolytic splicing pathway before circularization. The external nucleophilic attack model (B) consists of the nucleophilic attack of the 3′-OH residue of a block of non-encoded RNA nucleotides (gray box) at the intron 5′ splice site, ligating it to the intron 5′ end while concurrently displacing exon 1 (step 1). The 3′-OH residue of exon 1 would then attack the 3′ splice site releasing ligated exons and a linear intron harboring non-encoded nucleotides at its 5′ end (step 2). The third transesterification reaction would be initiated by the 2′-OH of the last nucleotide at the 3′ end of the intron (step 3). The position of this final nucleophilic attack would thus dictate how many non-encoded nucleotides are incorporated at the junction of intron circles.
