Scheme 1. (A) Usually accepted mechanism for the ligation by Rnl2, and (B) the mechanism proposed here for the connection of two ssRNA strands by Rnl2. In (A), both the nick-sealing (upper part) and the connection of two ssRNAs (lower part) involve the corresponding enzyme–substrate complexes, which are significantly different from each other. In the mechanism proposed in (B), however, the connection of two ssRNA strands also proceeds via “nick-like intermediate” (upper), rather than direct connection of two strands by the enzyme (lower part). The “nick-like intermediate” allows mismatches in both sides. Note that the present figure is never restricted to the intramolecular cyclization to rings and rather also applicable to the ligation of two ssRNA strands to longer ssRNA. “C-RNA” and “L-RNA” refer to circular RNA and linear RNA, respectively.