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J. Biol. Chem., Vol. 279, Issue 14, 13721-13728, April 2, 2004

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Preferential Translation of Internal Ribosome Entry Site-containing mRNAs during the Mitotic Cycle in Mammalian Cells^*

Xiaoli Qin and Peter Sarnow

From the Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305

Received for publication, November 25, 2003 , and in revised form, January 21, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
A cell synchronization protocol was established in which global and individual mRNA translational efficiencies could be examined. While global translational efficiency was reduced in mitotic cells, 3% of mRNAs remained predominantly associated with large polysomes during mitosis, as determined by cDNA microarray analyses. The 5'-non-coding regions of six mRNAs were shown to contain internal ribosome entry sites (IRES). However, not all known mRNAs that contain IRES elements were actively translated during mitosis, arguing that specific IRES sequences are differentially regulated during mitosis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cells of higher organisms can regulate gene expression at the translational level in response to a wide variety of stimuli and conditions. While much is known about the control of translational initiation and elongation in response to nutritional deprivation (1, 2) and environmental stress (2), the role of translational control in mammalian cell cycle progression is not well understood. It has long been known that specific proteins are needed at specific times during the cell cycle to ensure cell cycle progression. For the most part, the expression of these cell cycle-specific proteins is thought to be regulated at the transcriptional or posttranslational level (3).

In cultured mammalian cells arrested at G₂ and M phases, the rate of total protein synthesis was markedly decreased to about 25% of the rate in non-arrested, cycling cells (4). This reduction was shown to be, at least partly, due to inhibition of the initiation step of polypeptide synthesis (4–6). Subsequently, several eukaryotic initiation factors that regulate the assembly of 40 S ribosomes at the 5' ends of capped mRNAs were observed to change their phosphorylation status (7–9). Therefore, it seemed likely that inhibition of translation initiation was caused by diminishing ribosome recruitment to capped mRNAs during mitosis. Viral mRNAs whose translation is initiated by an internal ribosome entry site (IRES)¹ mechanism (10), such as polioviral and hepatitis C viral mRNAs (4, 11–14) or mRNAs which lack significant structures in their 5'-non-coding regions (5'-NCRs), such as the mRNAs containing the late leader of adenovirus (15), were found to be selectively translated during mitosis due to their lessened requirement for eIF4F. More recently, additional IRES-containing mRNAs, those encoding ornithine decarboxylase and kinase p58^PITSLRE, have been reported to be selectively translated during G₂/M of the cell cycle in cultured cells (11, 12). These findings raise the question whether all IRES-containing genes are preferentially translated during mitosis and whether any of these encoded products play roles in cell cycle progression. We have begun to address these questions by genomic analysis of cellular mRNAs that are associated with mitotic polysomes and are, thus, predicted to be translated during the overall translation repression in mitosis. We determined that many, but not all, IRES elements are present in mRNAs which are selectively translated during mitosis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Synchronization and Transfection Protocols—HeLa cells were maintained in Dulbecco modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (Invitrogen). To synchronize cells at the G₁/S boundary, cells were treated at 50% confluence with 2 mM thymidine (Sigma) or 5 µg/ml aphidicolin (Sigma). After 16 h, cells were washed and fed with fresh medium for 6 h before the addition of either 0.6 µg/ml colcemid (Invitrogen) or nocodazole (Sigma; Ref. 16). Synchronization was monitored by flow cytometry analysis after DNA staining with propidium iodide. To measure protein synthesis rates in non-synchronized and mitotic arrested cells, cells were incubated in DMEM with 100 µCi/ml [³⁵S]methionine/cysteine for 10 min. Extracts were prepared and processed as described previously (17).

DNA plasmids were transfected into 50–60% confluent cells with the Lipofectin reagent (Invitrogen). After a 24-h incubation, luciferase assays were performed or RNA samples for Northern analyses were prepared.

Indirect Immunofluorescence Analysis—Cells were grown on 12-mm coverslips, fixed for 5 min in 100% methanol at -20 °C, and permeabilized in phosphate-buffered saline with 0.2% Triton X-100. Following fixation and permeabilization, cells were washed with PBS and incubated in PBS containing 3% bovine serum albumin, 0.1% Triton X-100, and 5% calf serum for 40 min at 25 °C. Coverslips were then incubated with diluted rat antibodies against -tubulin (a gift from P. Jackson, Stanford University) in IF buffer (PBS with 3% bovine serum albumin and 0.1% Triton X-100) for 1 h at 25 °C. After washing, coverslips were inverted into the IF buffer with Texas Red-conjugated donkey anti-rat antibody for 30 min at 25 °C. Cover slips were washed twice with PBS, and once with PBS containing 0.2 µg/ml Hoechst 33258, drained, and mounted in vectashield mounting medium (Vector Laboratories) onto glass slides. Cells were viewed using an Olympus BX-60 fluorescent microscope. Pictures were acquired using a Hamamatsu Orca digital camera and Image Pro plus software with 40x or 60x objectives.

5' RACE and Dicistronic Plasmid Constructions—The 5' NCRs of selected mRNA species were converted to cDNA copies using the First-Choice RLM-RACE Kit (Ambion). The DNA fragments were amplified by the Advantage-GC PCR kit (Clontech) and directly cloned into PCR2.1-topo vector (Invitrogen). The nucleotide sequences of all inserted fragments were obtained. The 5' NCRs were amplified by the PCR with 5' primer containing EcoRI sequences and 3' primers containing NcoI sequences. These fragments were inserted into dicistronic SV40/T7 plasmid, c-53, between EcoRI and NcoI sites, or a T7 promoter-containing dicistronic vector, c-84, as described previously (18).

Northern Blot Analyses—Total RNA was isolated using the TRIzol Reagent (Invitrogen) and analyzed as described previously (17). Radio-labeled DNA hybridization probes were generated using the RadPrime Kit (Invitrogen).

Polysomal Analysis—Polysomal mRNA was prepared as described previously (17). Briefly, cells were incubated with 0.1 mg/ml cycloheximide for 3 min at 37 °C before being harvested. Non-synchronized control cells were washed with PBS, harvested directly on the plate in lysis buffer (15 mM Tris-Hcl, pH 7.4, 15 mM MgCl₂, 0.3 M NaCl, 1% Triton X-100, 0.1 mg/ml cycloheximide, 1 mg/ml heparin), and transfered to Eppendorf tubes. The mitotic cells were shaken off from plates and pelleted at 1000 rpm for 3 min. Mitotic cells were washed in PBS and lysed in lysis buffer for 10 min on ice. The nuclei and debris were removed by centrifugation at 12,000 x g for 10 min, and the supernatants were loaded onto 10–50% sucrose gradients composed of same extraction buffer lacking Triton X-100. The gradients were sedimented at 35,000 rpm for 160 min in a SW41 rotor at 4 °C. Fractions of equal volumes were collected from the top using an ISCO fraction collector system. RNAs were collected in 4 M guanidine HCl, precipitated after addtion of ethanol, and resuspended in 1 mM Tris-HCl, pH 8.

Western Blot and Immunoprecipitation Analyses—Proteins were separated by SDS-PAGE and transferred to Immobilon-P membrane (Millipore). Poly(ADP-ribose) polymerase (PARP) was detected with antibody number SC7150 (Santa Cruz Biotechnology) as described previously (19). To examine newly synthesized proteins, cells were grown in DMEM lacking methionine and cysteine (Invitrogen) with 10% fetal bovine serum for 1 h and metabolically labeled with 100 µCi/ml [³⁵S]methionine-cysteine in DMEM (lacking methionine-cysteine) with 10% fetal bovine serum for 30 min. Labeled cells were lysed in a buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 0.5% Nonidet P-40, and protease inhibitor mixture. Equal amount of protein extracts, determined with the Bio-Rad protein reagent, were incubated with mouse anti-nucleophosmin (Zymed Laboratories Inc.) and anti-vimentin (Santa Cruz Biotechnology) antibodies and agarose-protein A/G plus beads according to the procedure recommended by Santa Cruz Biotechnology. The agarose beads-attached proteins were recovered by boiling in Laemmli sample buffer and separated on SDS gels. The gels were dried and enhanced with Amplify solution (Amersham Biosciences) before exposed to a phosphoimager screen. Quantification was performed using a STORM phosphoimager and ImageQuant software (Amersham Biosciences).

Preparation of Fluorescently Labeled cDNA for Microarray Hybridization and Data Analysis—Equal volume amounts of RNAs from pooled polysomal fractions were used to generate fluorescently labeled DNA probes as described (www.cmgm.stanford.edu/pbrown). The human cDNA microarrays contained a set of 43,000 human cDNA clones, manufactured at Stanford University as described (www.cmgm.stanford.edu/pbrown). The microarrays were scanned on a Axon laser scanner and analyzed using ScanAlyze (available at www.rana.lbl.gov/). The computed data were entered and further analyzed in Stanford Microarray Data Base (www.genome-www5.stanford.edu/MicroArray/SMD/). Only genes for which the fluorescence signal in each channel exceeded certain cutoffs (including regression correlation, signal-noise ratio, signal intensity, and the tolerance of missing data among multiple measurements) were retrieved from the Stanford Microarray Data Base for the deterimation of the microarray distribution score S(x) (see Fig. 3) and significant analysis of microarray.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Representative histogram displaying the polysomal distribution of mRNA species (x) in mitotic cells compared with that in non-synchronized cells. The microarray score index S(x) was calculated as follows. Non-synchronized: Ii(x) = LOG2 x (pol(x)int/nonpol(x)int); mitosis: Mj(x) = LOG2 x (pol(x)mit/nonpol(x)mit) (where i indicates an individual array measurement using RNA extracted from non-synchronized cells (i = 1, 2, 3), j indicates an individual array measurement using RNA extracted from mitosis cells (j = 1, 2, 3), and x indicates an individual species with a specific IMAGE ID on each array: ; is the standard deviation. The arrows point to the positions of the mean S(x) and 2.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reduction of Protein Synthesis in G₂/M-arrested HeLa Cells—To investigate translational control during mitosis, HeLa cells were synchronized in mitosis by a sequential block with thymidine and colcemid or with thymidine and nocodazole. Mitotic cells were shaken off from the plates, and the efficiency of synchronization was monitored by flow cytometry after propidium iodide staining. About 80–90% of cells treated with thymidine-colcemid or thymidine-nocadozole displayed a double content of DNA, confirming their arrest (Fig. 1A). To identify the specific mitotic phase in which cells were arrested, the microtubule structure of thymidine/colcemid-arrested cells and non-synchronized cells were visualized by immunofluorescence staining using antibodies directed against -tubulin (Fig. 1B). Loss of functional spindle structures and condensed chromosomes in treated cells revealed that these mitotic cells were arrested at prophase or premetaphase of mitosis.

View larger version (51K): [in this window] [in a new window]   FIG. 1. Translation reduction during mitosis in HeLa cells. A, flow cytometry analysis of HeLa cells that were arrested by the sequential treatments of thymidine and colcemid or thymidine and nocodazole. x axis, Cy5PE propidium iodide; y axis, cell numbers. B, non-synchronized cells and thymidine/colcemid-arrested cells. Left, x100 image of non-synchronized and thymidine/colcemid-arrested HeLa cells. Right, non-synchronized cells and thymidine/colcemid-arrested cells stained with antibodies against -tubulin and Hoechst; x100 fluorescence image. C, protein synthesis reduction in mitotic arrested cells. [35S]Methionine/cysteine incorporation into non-synchronized and mitotic cells, arrested with either thymidine/colcemid (T/C) or thymidine/nocodazole (T/N), was determined by trichloroacidic acid precipitation. The percentage of the radiolabel incorporation compared with of non-synchronized cells is shown. D, Western blot analysis of PARP protein in non-synchronized and mitotic-arrested cells. Cells were arrested at mitosis by treatment with aphidicolin/colcemid (A/C), thymidine/colcemid (T/C), or thymidine/nocodazole (T/N) in the presence or absence of apoptosis inhibitor benzyloxycarbonyl-VAD-fluoromethylketone. Non-synchronized HeLa cells were used as control. The apoptosis inhibitor benzyloxycarbonyl-VAD-fluoromethylketone was added to the culture 30 min before harvest at a concentration of 50 µM. The arrow refers to the intact PARP proteins.

It has been recently reported that many synchronization protocols can induce apoptosis (20, 21). To examine whether the synchronization protocols used here triggered cells to enter apoptosis, caspase-induced cleavage of PARP was measured. Fig. 1D shows that little cleavage of PARP occurred in either thymidine/colcemid- or thymidine/nocodazole-treated cells. In contrast, notable amount of PARP was cleaved in another synchronization protocol, i.e. treatment of cells with aphidicolin and colcemid. These findings suggest that the global reduction of protein biosynthesis that was observed in thymidine/colcemide- and thymidine-nocodazole-treated cells was not a result of ongoing apoptosis.

Reduced Association of Cellular mRNAs with Mitotic Polysomes—To examine the translation status of both total and individual mRNAs, cytoplasmic extracts from interphasic- and mitotic-arrested cells were fractionated in sucrose gradients (17). Fig. 2 shows that, in non-synchronized cells, most ribosomes were associated with mRNAs, sedimenting with polysomal fractions 8–10. In contrast, most ribosomes in mitotic extracts had dissociated from mRNAs, sedimenting as free 40 and 60 S subunits (Fig. 2). This finding argues for mitotic inhibition at the initiation step of translation.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Association of mRNAs with polysomes in non-synchronized and mitotically arrested HeLa cells. Lysates were fractionated by sucrose gradient centrifugation. The fractions from the top to the bottom of the sucrose gradient are displayed from left to right. The absorbance profile at 260 nm is shown, and the arrow denotes the migration of 40 and 60 S ribosomal subunits. The distributions of -actin and transforming growth factor- mRNAs in each fraction, obtained after Northern analysis, are shown. The Northern analyses were performed three times.

Identification of Mammalian mRNAs Species That Are Preferentially Associated with Mitotic Polysomes—To identify individual mRNA species which are selectively translated during mitosis, the polysomal distribution of mRNAs was examined using human cDNA microarray analyses (22, 23). Specifically, the amount of individual cDNAs obtained from RNA in fractions 4–7 was compared with that obtained from RNA in polysomal fractions 9 and 10 (Fig. 2). Statistical analyses indicated (Fig. 3) that the majority of mRNAs shifted toward the lower molecular weight polysomal fractions during mitosis, arguing that the mitotic sedimentation profiles of -actin and transforming growth factor- mRNAs (Fig. 2) reflected an overall diminished association with ribosomes of mRNAs. However, at least 3% of the mRNA species (49 genes out of 1494 genes) continued to be associated with similar or more numbers of ribosomes in mitotic extracts, determined both by statistical analysis (Fig. 3) and Northern blot analysis (Fig. 4). Selected positive candidate genes are listed in Table I. Approximately one-third of these genes encode nuclear proteins, many of which are predicted to play roles in RNA metabolism (Table I).

View larger version (28K): [in this window] [in a new window]   FIG. 4. Polysomal distribution of selective mRNAs in non-synchronized and mitotic arrested cells. A, polysomal distributions of -actin, GRP58, NAP1L1, NPM1, La, and ODC mRNAs were displayed after Northern hybridization as described in the legend to Fig. 2. Note that mRNAs were visualized after reprobing of the blot shown in Fig. 2. Thus, data showing distribution of -actin are identical in Figs. 2 and 4. B, quantification of mRNA distributions of -actin, ODC, NAP1L1, and GRP58. The data points represent the percent intensity of each fraction relative to the total combined intensity of all fractions for each gradient. The Northern analyses were performed three times.

To determine whether mRNAs associated with polysomes during mitosis were translated, we measured the relative translational efficiencies of nucleophosmin, vimentin, and La mRNAs in non-synchronized and mitotis-arrested cells. Translation efficiency was defined as the amount of newly synthesized protein compared with total translational efficiency during mitosis. The amounts of total nucleophosmin, vimentin, and La mRNAs were very similar in non-synchronized and mitotic extracts. While the translational efficiencies of nucleophosmin and La mRNAs were somewhat enhanced during mitosis, the mitotic translational efficiency of vimentin mRNA was more pronounced (Fig. 5, A and B) compared with the bulk of cellular mRNAs. These findings argue that the polysomal association of these mRNAs reflected translation by ribosomes.

View larger version (23K): [in this window] [in a new window]   FIG. 5. Measurement of relative translational efficiency of mRNAs encoding vimentin, nucleophosmin, and the La autoantigen. A, total mRNAs were visualized by Northern analysis using gene-specific hybridization probes. Newly synthesized nucleophosmin, vimentin, and La proteins were visualized after labeling with [35S]methionine/cysteine for 30 min followed by immunoprecipitation. Phosphoimages of the gels are shown. B, relative translational efficiencies of nucleophosmin, vimentin, and La mRNAs. Incorporation of radioactive amino acids was quantified by phosphoimaging and normalized to the incorporation of radioactive amino acids into total protein in mitotic extracts (which 30% compared with non-synchronized extracts; see Fig. 1C). Each bar represents the average of three independent measurements.

View larger version (25K): [in this window] [in a new window]   FIG. 6. Measurements of IRES activity within the 5'-NCRs of selected mitotically translated mRNAs. A, schematic representation of dicistronic constructs. Construct contain a SV40 promoter for in vivo expression studies and a T7 promoter for generation of transcripts in vitro (23). The Renilla luciferase gene serves as a first cistron and the firefly luciferase gene as a second cistron. B, IRES activity in the 5'-NCRs of selective mRNAs. HeLa cells were transiently transfected with the indicated plasmids and Renilla (Rluc) and firefly luciferase (Fluc) activities were measured. Fluc/Rluc ratios are displayed in reference to the construct with 5'-NCR of -actin, whose Fluc/Rluc ratio was set to 1. Error bars indicate measurements from three independent experiments. Note, that the NPM1 cDNA comprises the predicted 97-bp 5'-NCR plus the first 9 codons, which contain 4 in-frame ATG codons. C, integrity of dicistronic mRNAs in transfected HeLa cells. Total RNA was isolated from transfected cells and analyzed by Northern blot hybridization using hybridization probes complementary to firefly luciferase sequences. The migrations of RNA markers are indicated. D, translation of selected dicistronic mRNAs in HeLa S10 lysates. Dicistronic RNA was in vitro transcribed with T7 RNA polymerase. A dicistronic construct lacking a 5'-NCR insert was used as a negative control. 20 ng/ml of the dicistronic RNAs were added to the lysate and incubated at 30 °C for 1 h followed by dual luciferase assays (29). The ratios of Fluc/Rluc and error bars were determined by three independent measurements.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

However, not all of the known cellular IRES-containing messages were associated with mitotic polysomes. For example, whereas IRES-contanining La (18) and Cyr61 (23) mRNAs were preferentially detected in polysomal fractions during mitosis, IRES-containing c-Myc (27, 28) (not shown) and ODC (12) mRNAs were not. These findings seem to be, at first glance, at odds with observations reported by Pyronnet and Sonenberg (8) who used a sequential thymidine and aphidiolin block to synchronize HeLa cells at S phase, following cell cycle progression through the G₂/M phase after release of the drugs. Synthesis of ODC protein peaked 8 h after release from S phase, but dropped dramatically thereafter (12). It is possible that the up-regulation of ODC mRNA and protein occurred in G₂, before cells entered mitosis. Because we focused in the present study on the translation activity of mRNAs in cells arrested in mitosis, up-regulation of ODC in G₂ would have not been scored in our assays. Nevertheless, it is clear from all studies (8, 11) that IRES element can be regulated in a cell cycle-specific manner and that IRESs are preferentially used by the mitotic translation apparatus. The task will now be to identify the different regulatory elements in mitotically active IRES elements and the proteins and cellular structures that modulate their activity. The outcome of these lines of investigation will determine roles for the internal initiation mechanism in cell cycle progression and point to posttranscriptional circuits that regulate cell growth.

	FOOTNOTES

 
^* This work was supported by Grant GM55979 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Molecular & Cell Biology, University of California, Berkeley, CA 94720.

To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Stanford University School of Medicine, Fairchild Science Bldg., Stanford, CA 94305. Tel.: 650-498-7076; Fax: 650-498-7147; E-mail: psarnow{at}stanford.edu.

¹ The abbreviations used are: IRES, internal ribosome entry site; NCR, non-coding region; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; RACE, rapid amplification of cDNA ends; PARP, poly(ADP-ribose) polymerase; ODC, ornithine decarboxylase.

	ACKNOWLEDGMENTS

 
We are grateful to Karla Kirkegaard for critical reading of the manuscript.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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