Fig. 3. RNA ligase activity of C12orf29.

RNA/DNA oligonucleotides are schematically depicted in blue/green, respectively. The ³²P-labelled 5′-ends are depicted in red. The radioactive oligonucleotides were resolved by denaturing PAGE and analysed by phosphorimaging. a (Top) Schematic display of two nucleic acid substrates used. (Bottom) PAGE analysis of reaction products after incubation with C12orf29 for various time points as indicated. Ligation was observed for 5′-phosphorylated RNA (representative images of n = 3). b Reaction of C12orf29 with indicated RNA constructs using various nucleotides (representative images of n = 3). Besides ATP, C12orf29 is able to efficiently catalyse ligation by processing GTP, dATP, Ap₃A, and Ap₄A. Nuc., nucleotide. c (Left) C12orf29 promoted ligation reaction at various concentrations of ATP and GTP (representative images of n = 3). (Right) Michaelis-Menten fits to initial rates providing K_M, k_cat and k_cat/K_M for ATP and GTP. Plotted data represent the mean value ± SD for three biological replicates. d (Left) Proposed ligation scheme starting from 2′,3′-cyclic phosphorylated RNA by the sequential action of ANGEL2-ΔN and C12orf29^WT. (Right) PAGE analysis of the depicted reaction (representative images of n = 3). All oligonucleotide sequences are provided in Supplementary Table 1. Source data are provided as a Source Data file.