GTP binding is required for TIF-IA regulation of rRNA synthesis. (A) GTP binding motif in TIF-IA at amino acids 333 to 340 (top) and GTP binding by endogenous TIF-IA in primary T cells (bottom). T cells were isolated from PBMCs or were cultured in medium with anti-CD3/CD28. Lysates were incubated with GTP-agarose beads and the blots were probed with anti–TIF-IA antibody. (B) Effect of excess GTP on TIF-IA binding to GTP-agarose beads. Lysates from Jurkat or primary T cells were incubated with GTP-agarose beads in the presence or absence of GTP (10 mM). Blots were probed with anti–TIF-IA antibody. (C) Effect of a T340N mutation of TIF-IA on GTP binding. Jurkat T cells were transfected with Myc–TIF-IA or Myc–T340N–TIF-IA, and cultured for 24 hours. The lysates were incubated with GTP-agarose beads and blots were probed with anti-Myc antibody. (D) Effect of the T340N TIF-IA mutation on rRNA synthesis and Pol I binding to rDNA. Jurkat T cells were transfected with Myc–TIF-IA or Myc–T340N–TIF-IA. Lysate was incubated with anti-Myc antibody and a western blot of the precipitate probed with anti-Pol I antibody (left), densitometry measurements of Pol I binding are shown in supplemental Figure 3D, ChIP assay with Pol I antibody and western blot (middle), and 5′ETS pre-rRNA levels and RNA labeling (right). (E) Effect of TIF-IA or T340N TIF-IA overexpression on MPA-induced inhibition of rRNA synthesis and cell proliferation. Jurkat T cells were transfected with Myc–TIF-IA or Myc–T340N–TIF-IA and treated with MPA for 3 hours (left) or 24 hours (right). 5′ETS pre-rRNA and ChIP assay with Pol I antibody (left) and MTS assay, Annexin V staining, and western blot (right).