Circularization of pre-miRNAs by T4 RNA ligase 1. (A) Diagram to illustrate microRNA biogenesis. Each pre-miRNA contains a monophosphate at its 5′-end (5′-P) and a hydroxyl at its 3′-end (3′-OH). (B) Circularization of the pre-miRNA by T4 RNA ligase 1. Each pre-miRNA (1 pmol) was incubated with 16 units of T4 RNA ligase 1 at 25°C for 2 h in ligation buffer (NEB, M0204L) alone or supplemented with 5% or 20% polyethylene glycol 8000 (PEG 8000). The ligation reaction mixtures were then analyzed via 12% urea-PAGE. The ligated products and original substrates are indicated by red and blue arrowheads, respectively. (C) The ligation efficiency of the pre-miRNAs was calculated from three repeated experiments as the percentage of the original substrate that was converted into ligated product. Two-tailed two-sample assuming unequal variances t-tests for statistical analysis were used to calculate the P-value. (*) P-value <0.05. (D) The diagram shows ligated or unligated pre-miRNAs being treated with RNase R. The pre-miRNAs were ligated by T4 RNA ligase 1 as described in B. The ligated RNAs were purified by isopropanol and were subsequently treated with five units of RNase R at 37°C for 1 h. (E,F) The T4 RNA ligase 1-circularized pre-miRNAs were resistant to RNase R. One pmol of each pre-miRNA was used in these RNase R incubation experiments. Primer sequences used for RNA preparation are shown in Supplemental Table S1.
