A Cell Cycle–Dependent Internal Ribosome Entry Site Stéphane Pyronnet, Lucien Pradayrol, Nahum Sonenberg  Molecular Cell  Volume 5, Issue 4, Pages 607-616 (April 2000) DOI: 10.1016/S1097-2765(00)80240-3

Figure 1 Expression of ODC during the Cell Cycle (A) Flow cytometry analysis of HeLa cells following release from the sequential thymidine and aphidicolin block (see Experimental Procedures). Arrows indicate G1 (1c) and G2/M (2c) DNA content. X axis, fluorescence; y axis, cell number. (B) Protein synthesis during the cell cycle. [35S]methionine incorporated into TCA-precipitable material was measured and expressed as a percentage of the level at time 0. (C) ODC activity (top), biosynthesis (middle), and mRNA (bottom). (D) Results shown in (C) were quantified as described in Experimental Procedures and are representative of three independent experiments. (E) Pattern of 4E-BP1 phosphorylation in cells treated (rapa) or untreated (cont) with rapamycin (20 ng/ml). The slowest migrating forms represent hyperphosphorylated 4E-BP1 (β and γ), while the fastest form (α) is hypophosphorylated. (F) ODC activity in cells treated (rapa) or untreated (cont) with rapamycin. Results are representative of three different experiments. Molecular Cell 2000 5, 607-616DOI: (10.1016/S1097-2765(00)80240-3)

Figure 2 ODC Induction Is Prevented by Rapamycin at G1/S but Not at G2/M (A) Flow cytometry analysis of HeLa cells following release from the nocodazole block (see Experimental Procedures). Arrows and axes are as in Figure 1. (B) Pattern of 4E-BP1 phosphorylation in cells treated (rapa) or untreated (cont) with rapamycin. (C) ODC activity in extracts prepared from cells treated (rapa) or untreated (cont) with rapamycin. (D) Results shown in (C) were quantified (see Experimental Procedures), and values are representative of three separate experiments. (E) Biosynthesis of ODC (top) and actin (middle) at different phases of the cell cycle. Actin total amount was measured in a separate gel (bottom). G1/S and G2 cell extracts were prepared from cells scraped following a block with thymidine and etoposide, respectively. M cell extracts were prepared from cells arrested with nocodazole and collected by gentle pipetting. (F) Results shown in (E) were quantified (see Experimental Procedures) and expressed as a ratio relative to the value obtained for G1/S cells, which was set at 1. Values are representative of two separate experiments. Molecular Cell 2000 5, 607-616DOI: (10.1016/S1097-2765(00)80240-3)

Figure 3 Sequence Similarities among ODC and Picornavirus 5′UTRs (A) (Top) Alignment of the pyrimidine-rich sequence of picornavirus 5′UTRs (Nicholson et al. 1991). PV, poliovirus; EMCV, encephalomyocarditis virus; HRV, human rhinovirus; CV, coxsackie virus; HAV, hepatitis A virus; FMDV, foot and mouth disease virus. (Bottom) Sequences flanking the initiator AUG of mammalian ODC mRNAs. The first column shows the nucleotides encompassing the pyrimidine stretch (UUUC) (box A); the second column shows the nucleotides flanking the initiator AUG (box B). Nucleotides that match the UUUC sequence in box A are in bold letters. The number of nucleotides between the 3′ border of the A box and the AUG is in parentheses. (B) ODC 5′UTR monocistronic (top) and dicistronic (bottom) constructs. The thin line represents the ODC 5′UTR; the thick line represents the ODC 5′UTR placed in the antisense orientation. Gaps represent deletions. The open and closed boxes represent the CAT and LUC coding sequence, respectively, and the hatched boxes represent ODC coding sequence. For each construct, the length of the 5′UTR is indicated. One stretch of pyrimidines (Py1) or both stretches (Py2) were mutated into AAAC. Molecular Cell 2000 5, 607-616DOI: (10.1016/S1097-2765(00)80240-3)

Figure 4 The ODC 5′UTR Contains an IRES (A) Mono- and dicistronic capped mRNAs were translated in control or 2Apro-treated RRL. Results are expressed as percentage of the value obtained in the absence of 2Apro, as described in Experimental Procedures. (B) Capped methylated or unmethylated CAT mRNAs were generated by in vitro transcription and translated in RRL. Results were quantified as described in Experimental Procedures and are expressed as percentage of the value obtained for capped mRNA. (C) Dicistrinic capped mRNAs were tested as described in (A). ND, not determined. (D) Dicistronic constructs were transfected in HeLa cells, and CAT or LUC activities were assayed as described in Experimental Procedures. Results are expressed as a ratio relative to the activity measured for the CAT-LUC construct containing no 5′UTR, which was set at 1. (E) Northern blot analysis of mRNAs from cells transfected with dicistronic constructs. Molecular Cell 2000 5, 607-616DOI: (10.1016/S1097-2765(00)80240-3)

Figure 5 ODC mRNA 5′UTR that Affect Cap- and IRES-Dependent Translation (A) Monocistronic capped CAT mRNAs containing various fragments of the ODC 5′UTR were translated in control or 2Apro-treated RRL, as described in Experimental Procedures. Results are expressed as a percentage of translation measured in control RRL. (B) Monocistronic constructs tested in (A) were transfected into HeLa cells, and CAT activity was assayed as described in Experimental Procedures. (C) Monocistronic capped CAT mRNAs containing point mutants of the ODC 5′UTR were translated as in (A). Results are expressed as a percentage of the value measured in the absence of 2Apro. Molecular Cell 2000 5, 607-616DOI: (10.1016/S1097-2765(00)80240-3)

Figure 6 The ODC mRNA IRES Functions in G2/M HeLa cells were transfected with monocistronic constructs carrying different fragments of the ODC 5′UTR upstream of CAT. Cells were synchronized at the G1/S boundary as in Figure 1 12 hr after transfection. CAT synthesis was measured at various times after release from the sequential thymidine and aphidicolin block, as described in Experimental Procedures. CAT synthesis was quantified using a PhosphorImager and is expressed as a ratio relative to the value obtained at time 0, which was set at 1. Molecular Cell 2000 5, 607-616DOI: (10.1016/S1097-2765(00)80240-3)

Figure 7 The c-myc mRNA IRES Functions in G2/M (A) c-myc 5′UTR constructs. The Myc-CAT fused ORF is under the control of the c-myc IRES (P2 leader). A hairpin structure (HP, ΔG = ∼−40 kCal/mol) was inserted upstream of the first cistron to generate HP-CAT-myc-CAT (Nanbru et al. 1997). (B) c-myc IRES activity in synchronized cells. Cells were transfected and synchronized as in Figure 6. M cells were collected by shake off 8 hr after release from the G1/S block. To obtain G1 cells, a fraction of M cells was replated and grown for an additional period of 10 hr. G1 and M cells were then pulse labeled for 20 min with [35S]methionine. Cell cycle distribution was monitored by FACS analysis (left). CAT and Myc-CAT synthesis was measured in G1 and M cells (right) as described in Figure 6. Values obtained using a PhosphorImager are shown. Molecular Cell 2000 5, 607-616DOI: (10.1016/S1097-2765(00)80240-3)