In vitro mRNA transcription

To express Regnase-1 and -3, pUC57-mini plasmids harboring the WT or mutated coding sequence downstream of the T7 promoter were first transcribed into mRNA using HiScribe T7 ARCA mRNA Kit (New England BioLabs) according to the manufacturer’s protocol. Briefly, the plasmids were linearized by digestion with SpeI (New England BioLabs), followed by purification using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel). Linearized DNA templates (1 μg) were then assembled together with ARCA/NTP mix, T7 RNA polymerase mix (both included in the kit), and pseudo-UTP (Jena Bioscience) for the IVT reaction. mRNA synthesis was completed by removing the DNA template through DNase I treatment, followed by poly(A) tailing by adding poly(A) polymerase and buffer (all included in the kit) to the reaction mix. All reactions were each done at 37°C for 30 min. Resulting mRNA products were purified using Monarch RNA Cleanup Kit (New England BioLabs), visualized on 1% TBE gel, and quantified using NanoDrop 2000. BMMCs were transfected with Regnase-1 or Regnase-3 (WT or RNase-inactive mutant D141N or D252N) IVT mRNA using 10 μl Neon Transfection System Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. IVT mRNA from the pUC57-mini plasmid expressing ZsGreen alone was used as a control. Briefly, cells were washed with PBS and resuspended in 10 μl of buffer R. IVT mRNA (3 pmol for BMMCs and 0.25 pmol for HMC-1.1 and HMC-1.2 cells) and 10U RNase inhibitor (Promega) were then added to the cell suspension. Cell electroporation was performed with one pulse at 1,600 V and 30 ms of width for BMMCs, and one pulse at 1,700 V and 20 ms of width for HMC-1.1 and HMC-1.2 cells. Transfected cells were kept in antibiotic-free medium, and downstream experiments were performed within 24 h.