Fig. 5: TUT7 binds and uridylates Zc3h12a.

a, b RAW 264.7 cells were treated with 100 ng/ml LPS for 2 h and cell lysates were incubated with control IgG or protein A-agarose beads-conjugated anti-TUT7 antibody at 4 °C for 4 h. TUT7-interacting RNAs were extracted and analyzed by RT-PCR (a) and RT-qPCR (b). c Synthetic RNAs containing Il6 or Zc3h12a wild-type stem-loop or mutant variants were incubated with immunopurified Flag-tagged wild-type TUT7 at 37 °C for 30 min. d Cy3-labeled RNA containing Zc3h12a stem-loop was incubated with immunopurified TUT7 in the presence or absence of 1× or 5× fold excess of unlabeled RNA containing Zc3h12a wild-type or the indicated stem-loop mutant at 37 °C for 30 min. All RNA samples were resolved on a 10% native PAGE and visualized by SYBR Green II staining (c) and Cy3 fluorescent signal (d). e Schematic diagram of different constructs used for in vitro transcription. Flowchart on the right shows the procedure for in vitro uridylation. RNA transcripts were prepared by in vitro transcription. Transcripts were incubated with Flag-tagged wild-type (WT) or enzyme-dead (DADA) TUT7 mutant prepared by immunoprecipitation of HEK293T cells transfected with Flag-tagged TUT7- or DADA mutant-expressing plasmids in the presence of α-³²p-UTP at 37 °C for 1 h. f–h Immunoprecipitation of flag-tagged wild-type and DADA mutant TUT7 was confirmed by immunoblotting (the upper panel of f). RNA samples were separated on a 1% formaldehyde-agarose gel and visualized by SYBR Green II staining and autoradiography (the bottom panel of f, g, h). Data presented are representative of three independent experiments. Source data are provided as a Source Data file. Asterisk (*) represents the nonspecific binding.