Figure 7

Effects of tristetraprolin (TTP)-related tandem CCCH zinc finger (TZF) proteins to bind AU-rich element (ARE)-containing probes and to promote their deadenylation. HEK-293 cells were maintained, and transient transfection of 1.2 × 10⁶ cells with expression plasmid constructs in calcium phosphate precipitates was performed, as described [22]. To each plate of HEK-293 cells was added 0.2 μg of the TZF protein expression constructs CMV.hTTP.tag (hTTP), a human TTP (hTTP) zinc finger mutant (C124R), CMV.cMG1.tag (cMG1), CMV.mTis11D.tag (mTis11D), 0.1 μg of human poly(A) exonuclease (hPARN) expression plasmid CMV.hPARN.flag (hPARN), or plasmid DNA alone (BS+). The zinc finger protein expression constructs were transfected either with vector alone or together with CMV.hPARN.flag; vector DNA (BS+) was added to each transfection to make the total amount of co-transfected DNA 5 μg per plate. Cytosolic extracts were prepared and used in deadenylation assays as described [23]. (a) Extracts (10 μg of protein per sample) were incubated with probes ARE or ARE-A50 at 37°C for 60 min in the presence (+) or absence (-) of 20 mM EDTA, as indicated. The samples were processed as described previously [23]. The arrow indicates the migration position of the ARE probe (lanes 1–6) and the deadenylated product of probe ARE-A50 (lanes 9, 11, 12, 14, 16 and 17). (b) The extracts used in lanes 7–13 of (a) were incubated with the ARE-A50 probe and used in a gel-shift assay. Lane 7 (P') was loaded with probe alone (digested with RNase T1). The migration positions of the zinc finger protein-RNA complexes are indicated by the bracket to the right of the gel, and the position of the free probe (FP) is also indicated. The bands present in the gel in lane 1 represent endogenous HEK-293 cell proteins shifting the probe; note that this pattern is identical in lane 3, representing a zinc finger mutant of TTP, and in lane 6, representing hPARN alone.