Fig. 1. Amino acid sequence of human TIF-IA (H. s.) aligned with Rrn3 from S. cerevisiae (S. c.). Identical residues are shaded black and conserved residues grey. The DDBJ/EMBL/GenBank database accession No. for TIF-IA is AJ272050.

Fig. 2. Nucleolar localization of TIF-IA. NIH 3T3 cells were transfected with CMV-FLAG-hTIF-IA, fixed in methanol for 1 min at –20°C, washed once with –20°C acetone and several times with phosphate-buffered saline. TIF-IA was visualized by indirect immunofluorescence using M2 antibodies (Sigma, 1:200) and fluorescein isothiocyanate-conjugated goat anti-mouse IgGs (Dianova, 1:300). UBF was stained with anti-UBF serum (1:500) and Texas red-conjugated goat anti-human IgGs (Dianova, 1:200).

Fig. 3. TIF-IA augments transcription of a Pol I reporter gene. HeLa and NIH 3T3 cells were transfected with 10 µg of pHr-CBH or pMr1930-BH and increasing amounts of pCMV-FLAG-hTIF-IA. Transcripts from the reporter plasmid were visualized on northern blots. To normalize for variations in RNA loading, the filter was hybridized with a riboprobe against cytochrome c oxidase.

Fig. 4. TIF-IA activates transcription in vitro. (A) Complementation of transcriptional activity in extracts from cycloheximide-treated cells. Transcription assays contained nuclear extract (40 µg protein) from mouse cells that were cultured either in the absence of cycloheximide (NE) or for 2 h in the presence of 0.1 mg/ml of cycloheximide (NE_CHX). Reactions were supplemented with 0.5 (lanes 2 and 5) and 1 µl (lanes 3 and 6) of cellular TIF-IA (PL-650 fraction) or 2.5 (lanes 8 and 11) and 5 ng (lanes 9 and 12) of recombinant TIF-IA. (B) Transcription in a reconstituted system. The assays contained 20 ng of template DNA, 4 µl of Pol I (H-400 fraction), 3 µl of TIF-IB (CM-400 fraction) and 2 ng of recombinant UBF. The reactions were complemented with 1 µl of partially purified cellular TIF-IA (PL-650 fraction, lane 1) or increasing amounts (0.5, 1.5 and 3 ng) of recombinant TIF-IA (lanes 3–5).

Fig. 5. TIF-IA interacts with Pol I. (A) Pull-down assay. Immobilized TIF-IA or control beads were incubated with Pol I (MonoS-250) and bead-bound Pol I was detected on immunoblots with antibodies against RPA116. (B) Co-immunoprecipitation of Pol I and TIF-IA. Pol I was incubated with ³⁵S-labeled TIF-IA and immunoprecipitated with either pre-immune (lane 2) or anti-RPA53 serum (lane 3).

Fig. 6. Antibodies against the recombinant protein recognize cellular TIF-IA. (A) Western blot. Fractions from a MonoS column were assayed on immunoblots for the distribution of Pol I and TIF-IA with antibodies against RPA116 and TIF-IA. One nanogram of recombinant TIF-IA is shown in the first lane (+). (B) Transcriptional activity. Individual fractions were assayed for TIF-IA activity by testing their capability to restore transcription of extracts from cycloheximide-treated cells. As a positive control, the extract was complemented with 3 ng of TIF-IA (+).