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. 2018 Apr 30;8:6748. doi: 10.1038/s41598-018-25137-w

Figure 6.

Figure 6

TARG1 is an RNA binding macrodomain. (a) TARG1 wildtype, -G43E, -I44E and -G123E were expressed as N-terminal His6-tag fusion proteins in bacteria and purified by immobilized metal affinity chromatography (IMAC). ALY, fused to an N-terminal glutathione S-transferase (GST)-tag, was expressed in bacteria and purified by glutathione affinity chromatography. Aliquots of the eluates, together with a bovine serum albumin (BSA) standard, were separated on an SDS-gel and stained with Coomassie blue. (b) Electrophoretic mobility shift assay (EMSA) using RNA Pentaprobe oligonucleotides41. Purified 32P-labeled Pentaprobe 9 (32P-PP9) was incubated with the indicated amounts of purified GST, GST-ALY, His-TARG1 wildtype (WT), -G123E or -G43E (2.5–20 pmol corresponding to 83.3–666 nM, respectively). Free RNA and RNA-protein complexes were separated on native 7% poly-acrylamide gels. Mobility shifts were analyzed by auto-radiography. (c) EMSA performed as described in panel b with 32P-PP9 and His-TARG1 (5 pmol, 0.16 µM), His-TARG1 in complex with a polyclonal antibody raised against TARG1 (Eurogentec) or with antibody alone. (d) EMSA of 32P-PP7 that was incubated with constant amounts of His-TARG1 (5 pmol, 0.16 µM) and increasing amounts of cellular RNA (5–150 pmol, corresponding to 0.16–5 µM, calculated per nucleotide). (e) Thermal shift assay of 2 µM His-TARG1 WT or -G123E together with increasing amounts of cellular RNA (5–100 µM, calculated per nucleotide). Melting temperatures were determined according to25 and are presented as ΔTM to H2O control (ctrl; mean ± SD of 3 experiments). (f) Thermal shift assay of 2 µM His-TARG1 WT or -G123E in the presence of constant amounts of cellular RNA (100 µM, calculated per nucleotide) together with increasing amounts of ADPr. Melting temperatures are expressed as ΔTM to H2O control (ctrl; mean ± SD of 3 experiments). (g) EMSA of 32P-PP7 that was incubated with constant amounts of His-TARG1 WT or -G123E (5 pmol, 0.16 µM) and increasing amounts of purified PAR (0.5–10 pmol, calculated per ADPr unit, corresponding to 0.016–0.33 µM) or ADPr (50–200 pmol, corresponding to 1.66–6.66 µM).