Nucleolar Arf Tumor Suppressor Inhibits Ribosomal RNA Processing Masataka Sugimoto, Mei-Ling Kuo, Martine F Roussel, Charles J Sherr  Molecular Cell  Volume 11, Issue 2, Pages 415-424 (February 2003) DOI: 10.1016/S1097-2765(03)00057-1

Figure 1 Arf Induction and Cell Cycle Arrest in MT-Arf Cells (A) MT-Arf and NIH-3T3 cells were treated with zinc sulfate for the indicated times or exposed to 5-FU, and lysates were immunoblotted with antibodies to the proteins indicated at the left of the panel. (B) Two hours prior to lysis for protein and RNA analysis, parallel zinc-induced cell cultures were either pulsed with BrdU or metabolically labeled with [3H]-uridine. BrdU incorporation into DNA was measured by immunofluorescence (light gray bars), and the number of untreated cells in S phase (0 hr) was normalized to 100%. RNA was extracted and quantified by absorbance measurement, and radioactivity in aliquots was determined by scintillation counting (dark gray bars). Incorporation (cpm/μg RNA) into untreated cells (0 hr) was normalized to 100% (see Table 1). Molecular Cell 2003 11, 415-424DOI: (10.1016/S1097-2765(03)00057-1)

Figure 2 RNA Processing in MT-Arf Cells (A) Schematic illustrating steps in rRNA processing adapted from Eichler and Craig (1994) with cleavage steps indicated by vertical arrows. Sedimentation coefficients (S) of intermediates and mature rRNA products (filled bars) are shown, as are the locations of external (ETS) and internal (ITS) transcribed spacer sequences in the 47S precursor. (B) Extracted [3H]-uridine-labeled RNAs from cells treated with zinc for the indicated times were separated on a gel, transferred to membrane, and subjected to fluororadiography. Minor radiolabeled bands migrating between the 47S and 32S species (best visualized in lanes 4–7) represent less stable 41S and 36S intermediates. (C) Following zinc treatment (24 hr), cells were labeled for 30 min with [methyl-3H]-methionine and chased for the indicated times (min). Two thousand 3H counts per minute were loaded per lane. Brackets (MT-Arf, right) point to improperly processed intermediates. (D) Northern blot analysis of total RNA extracted from untreated (−) or zinc-treated (24 hr) (+) NIH-3T3 and MT-Arf cells. Equivalent amounts of RNA were loaded in each lane. The radiolabeled probe corresponded to tandem 5.8S-ITS2 sequences. Molecular Cell 2003 11, 415-424DOI: (10.1016/S1097-2765(03)00057-1)

Figure 3 Cell Cycle Arrest by Low Serum or Aphidicolin Does Not Inhibit rRNA Processing (A) NIH-3T3 or MT-Arf cells growing in medium containing 10% FCS (lanes 1–6) or arrested for 24 hr in medium containing 0.1% FCS (lanes 7–10) were treated with zinc for 24 hr or with 5-FU as indicated, and [3H]-uridine-labeled rRNA was analyzed (upper bracket) as in Figure 2B. Lysates from parallel cultures were separated on gels, transferred to membrane, and blotted with antibodies to p19Arf, cyclin D1, and Cdk4, as indicated at the left (lower bracket). (B) Quiescent NIH-3T3 or MT-Arf cells stimulated with serum to reenter the cell cycle synchronously were left untreated (lanes 1–6) or were treated with aphidicolin to rearrest them at the G1/S boundary (lanes 7–12). Cells were then exposed to zinc or 5-FU and incorporation of [3H]-uridine into rRNA was determined as above. (C) Nuclear run-on assays were used to measure transcription of 47S rRNA. The relative ratios of hybridization signals (47S rRNA/β-actin) for each sample provide an index of transcription rate in response to the indicated treatments (Table 1). Arf-specific exon-1β sequences were used to confirm Arf induction. Molecular Cell 2003 11, 415-424DOI: (10.1016/S1097-2765(03)00057-1)

Figure 4 Interference with rRNA Processing Is Not Mimicked by and Does Not Depend on p53, and Requires Arf Residues 2–14 (A) MEFs lacking both Arf and p53 were transduced with a control retroviral vector or with those encoding either p19Arf or p53. Lysates prepared 4 days later were assayed by immunoblotting for the indicated proteins. In the absence of p53, p19Arf was unable to induce the p53-responsive gene product, p21Cip1. By contrast, p53 induction of p21Cip1 does not depend on Arf. (B) Cells harvested as in (A) were metabolically labeled with [3H]-uridine, and radiolabeled rRNA species were separated on gels, transferred to membrane, and detected by fluororadiography. (C) NIH-3T3 cells treated with 5-FU or infected for 24 hr with the indicated retroviral vectors were studied as in (B). Arf mutants lacking amino acids 26–37 (d26-37) did not localize to nucleoli, whereas Arf (d2-14) did. (D) TKO MEFs infected with a retrovirus encoding wild-type (WT) Arf undergo arrest and remain viable, whereas those infected with the Arf (d2-14) mutant continue to proliferate. (E) At 3 days postinfection, cells were pulsed with BrdU and assayed by immunofluorescence for its incorporation into DNA. The number of BrdU-positive cells in S phase following infection with the control retroviral vector was normalized to 100%. (F) Arf-infected TKO MEFs were labeled 3 days postinfection with with [3H]-uridine, and radiolabeled rRNA species were separated on gels, transferred to membrane, and detected by fluororadiography. Molecular Cell 2003 11, 415-424DOI: (10.1016/S1097-2765(03)00057-1)

Figure 5 Arf Binds to 5.8S rRNA (A) Proliferating NIH-3T3 and MT-Arf cells were either left untreated (−) or were induced for 24 hr (+). Whole-cell lysates (10% of the total) were immunoblotted with antibodies to fibrillarin or p19Arf (lanes 1–4). The remaining lysates were divided in two and precipitated with antibodies to fibrillarin (lanes 5–8) or to p19Arf (lanes 9–12), and the immunoprecipitates were separated on denaturing gels, transferred to membrane, and blotted with the same and heterologous antibodies. The Ig symbol in lanes 9–12 indicates the position of immunoglobulin light chains detected with antibody to rabbit Ig, whereas the positions of fibrillarin (Fib.) and p19Arf are indicated at the right. (B) RNA coprecipitating with antibody precipitates prepared from cells as in (A) was 3′ end-labeled using T4 RNA ligase and [32P]pCp. Products were separated on polyacrylamide gels containing urea and detected by autoradiography of the dried slab gel. The major bands coprecipitating with anti-fibrillarin correspond to U3 snoRNA, 5.8S, and 5S rRNA, respectively (Tyc and Steitz, 1989). Only 5.8S rRNA coprecipitated with p19Arf. (C) TKO MEFs lacking endogeneous Arf, Mdm2, and p53 genes cells were infected with a control retroviral vector (Ctl) or vectors encoding either wild-type p19Arf (WT) or the p19Arf (d2-14) mutant. Cell lysates were prepared 3 days after infection, and 15% of the lysates (lanes 1–3) were taken for immunoblotting (top panel) and RNA analyses (bottom panel). The remaining lysates were divided in two and precipitated with antibodies directed to the p19Arf C terminus (anti-Arf) or with control rabbit serum (NRS Ctl). RNA extracted from immune precipitates was 3′ end-labeled and analyzed as in (B). Molecular Cell 2003 11, 415-424DOI: (10.1016/S1097-2765(03)00057-1)