TIF-IA, the factor mediating growth-dependent control of ribosomal RNA synthesis, is the mammalian homolog of yeast Rrn3p

Abstract

Cells carefully modulate the rate of rRNA transcription in order to prevent an overinvestment in ribosome synthesis under less favorable nutritional conditions. In mammals, growth-dependent regulation of RNA polymerase I (Pol I) transcription is mediated by TIF-IA, an essential initiation factor that is active in extracts from growing but not starved or cycloheximide-treated mammalian cells. Here we report the molecular cloning and functional characterization of recombinant TIF-IA, which turns out to be the mammalian homolog of the yeast factor Rrn3p. We demonstrate that TIF-IA interacts with Pol I in the absence of template DNA, augments Pol I transcription in vivo and rescues transcription in extracts from growth-arrested cells in vitro.

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 (+).