Identification and Characterization of a Novel Cell Cycle–Regulated Internal Ribosome Entry Site

Molecular Cell, Vol. 5, 597–605, April, 2000, Copyright 2000 by Cell Press

Identification and Characterization of a Novel Cell Cycle–Regulated Internal Ribosome Entry Site Sigrid Cornelis,* Yanik Bruynooghe, Geertrui Denecker, Sofie Van Huffel, Sandrine Tinton, and Rudi Beyaert Department of Molecular Biology Flanders Interuniversity Institute for Biotechnology and University of Gent K. L. Ledeganckstraat 35 B-9000 Gent Belgium

Summary PITSLRE protein kinases are related to the large family of cyclin-dependent kinases. They have been proposed to act as tumor suppressor genes and have been shown to play a role in cell cycle progression. We report that two PITSLRE protein kinase isoforms, namely p110PITSLRE and p58PITSLRE, are translated from a single transcript by initiation at alternative in-frame AUG codons. p110PITSLRE is produced by classical capdependent translation, whereas p58PITSLRE results from internal initiation of translation controlled by an internal ribosome entry site (IRES) with unique properties. The IRES element is localized to the mRNA coding region, and its activity is cell cycle regulated, which permits translation of p58PITSLRE in G2/M. Introduction PITSLRE protein kinases are encoded by the duplicated genes cell division cycle 2–like 1 (Cdc2L1) and Cdc2L2, which span approximately 140 kb on human chromosome 1p36.3 (Gururajan et al., 1998). These genes result in the expression of almost identical protein kinases of 110 kDa, which contain at their C-terminal end the open reading frame of a smaller isoform of 58 kDa, p58PITSLRE. While p110PITSLRE isoforms are ubiquitously expressed in asynchronous cell populations, overexpression of p58PITSLRE in CHO fibroblasts leads to a late mitotic delay due to an apparent failure of cytokinesis (Bunnell et al., 1990) and to a reduced rate of cell growth (Lahti et al., 1995). Conversely, diminished p58PITSLRE mRNA levels in CHO fibroblasts are associated with enhanced cell growth (Meyerson et al., 1992). The RNA-binding protein RNPS1 specifically interacts with p110PITSLRE isoforms, suggesting that the latter regulate some aspect of RNA splicing/transcription during the cell cycle (Loyer et al., 1998). Interestingly, the chromosome region 1p36.3 is often deleted in neuroblastomas (Lahti et al., 1994), childhood endodermal sinus tumors (Perlman et al., 1996), a subset of malignant melanoma (Nelson et al., 1999), and in non-Hodgkin lymphoma (Dave et al., 1999). Since deletion of this chromosome region occurs late in oncogenesis and is correlated with aggressive tumor * To whom correspondence should be addressed (e-mail: sigrid. [email protected]).

growth, a potential role for PITSLRE protein kinases as tumor suppressor genes has been suggested (Eipers et al., 1991). Hence, understanding the mechanisms controlling the expression of PITSLRE protein kinase isoforms might contribute to the development of new cancer therapies. We report that PITSLRE mRNA produces two different PITSLRE proteins, namely p110PITSLRE and p58PITSLRE. Translation of p58PITSLRE is initiated at an internal in-frame AUG and is mediated by an internal ribosome entry site (IRES) present in the coding region of PITSLRE mRNA. Moreover, we found that this posttranscriptional mechanism is used to express p58PITSLRE specifically in the G2/M phase of the cell cycle, indicating that the activity of the IRES element is controlled by cell cycle–specific factors. These observations reveal a novel mechanism of translational regulation during cell cycle progression. Results p58PITSLRE Is Specifically Expressed in G2/M of the Cell Cycle To determine whether specific PITSLRE isoforms are expressed in different phases of the cell cycle, an antibody raised against a C-terminal peptide was used to analyze lysates of cells in specific cell cycle phases. The antibody recognizes all isoforms because the latter share a conserved kinase domain–containing C-terminal end (Xiang et al., 1994). An interleukin-3 (IL-3)-dependent pre-B-cell line, Ba/F3, was synchronized in G1 by IL-3 depletion for 14 hr. Subsequent stimulation with IL-3 drives the cells simultaneously through further phases of the cell cycle. Cell cycle progression was followed by FACS analysis in which the DNA content was determined using propidium iodide staining (data not shown). Western blot analysis revealed that the p110PITSLRE isoform was expressed in the different phases of the cell cycle, whereas expression of the smaller p58PITSLRE isoform was at least 20-fold increased in the G2/M phase (Figure 1A). Cyclin B1 expression was used as a marker for G2/M in these studies (Figure 1B). We further investigated the cell cycle dependence of p58PITSLRE expression by Northern blot analysis using a fragment corresponding to the 3⬘ end of the coding region of mouse p110PITSLRE (Malek and Desiderio, 1994) as a probe. Based on the high conservation in this region between the different human isoforms, we assumed this probe would recognize all murine PITSLRE transcripts. This analysis indicated a single transcript of approximately 3.2 kb in all phases of the cell cycle (Figure 1C), which is in agreement with previously published observations in mouse tissues (Malek and Desiderio, 1994). No additional G2/M-specific transcript accounting for expression of the 58 kDa product was detectable in Ba/F3 cells, suggesting that G2/M-specific expression of p58PITSLRE was regulated at a posttranscriptional level. To investigate the origin of p58PITSLRE in G2/M, an expression plasmid for the p110PITSLRE isoform fused to an E tag at its C-terminal end was stably transfected in

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Figure 1. Expression of PITSLRE Protein Kinases during Cell Cycle Progression in Ba/F3 Cells (A and B) Western blot analysis with anti-PITSLRE antibodies (A) or with anti-cyclin B1 antibodies (B) of Ba/F3 cells. Cells were first synchronized in G1 by IL-3 depletion and released from this G1 block by subsequent stimulation with IL-3 for the times indicated. The corresponding cell cycle phases are indicated. The upper band in (B) is a-specific and serves as an internal control for equal loading. (C) Northern blot analysis of total RNA preparations (30 ␮g) of Ba/ F3 cells. The RNA samples were prepared at the same time points as the samples used for Western blot analysis. A PstI restriction fragment (1072 bp) of the mouse p110PITSLRE cDNA was used as a probe. Numbers to the left indicate the length of RNA markers (kb).

the Ba/F3 cell line (Ba/F3-p110PITSLRE). The expression pattern of the exogenous PITSLRE protein kinase during cell cycle progression was analyzed via immunoblotting and detection with anti–E tag antibodies (Figure 2A). Corresponding to the endogenous PITSLRE kinase expression pattern (Figure 1A), also in the transfected cells an E-tagged p58PITSLRE product was mainly expressed in G2/M (Figure 2A). These results further show that alternative splicing as a possible source for p58PITSLRE expression is very unlikely. As an alternative for cell synchronization in G2/M, we studied the effect of nocodazole on the expression of p58PITSLRE in both the Ba/F3 and Ba/F3-p110PITSLRE cells. Nocodazole is an inhibitor of microtubule polymerization that blocks mitotic spindle formation, resulting in the accumulation of cells in mitosis. The expression of endogenous and exogenous p58PITSLRE increased considerably after incubation of the cells with 0.8 ␮M nocodazole for 6 hr and 14 hr, respectively (Figures 2B and 2C). This correlated with an increased number of M phase– specific cells as demonstrated by Western blotting with anti-cyclin B1 antibodies and by FACS analysis (Figures 2D and 2E). Removal of nocodazole allows the cells to leave mitosis and to finish the cell cycle. This resulted in a decrease in M phase–specific cells by approximately 30% after 12 hr (Figure 2E) and was also reflected in a lower expression level of p58PITSLRE (Figures 2B and 2C).

Figure 2. Cell Cycle–Dependent Expression of Transfected p58PITSLRE (A) Western blot analysis with anti–E tag antibodies of synchronized Ba/F3-p110PITSLRE cells prepared at different time points after IL-3 stimulation. Corresponding cell cycle phases are indicated (upper panel). The percentage of cells that are in a specific phase of the cell cycle was determined by FACS analysis of the DNA content after staining with propidium iodide (lower panel) (diamond, G1; square, S; triangle, G2/M). Results are shown for a representative cell clone. (B–D) Exponentially growing Ba/F3 (B) and Ba/F3-p110PITSLRE cells (C) were either untreated, treated with nocodazole (NOC.) (0.8 ␮M) for respectively 6 hr and 14 hr, or treated for 14 hr with nocodazole followed by 12 hr incubation without nocodazole. Endogenous (B) and exogenous (C) PITSLRE protein kinase expression, as well as cyclin B1 (D) expression, were analyzed by Western blot detection with anti-PITSLRE protein kinase, anti–E tag, and anti cyclin-B1 antibodies, respectively. (E) The percentage of cells that are in a specific phase of the cell cycle was determined by FACS analysis.

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Table 1. Survey of AUG Codons and Their Flanking Nucleotides in p110PITSLRE cDNA AUG Number

AUG Position

AUG Context (CC[A/G]CCAUGG)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

112 (p110) 119 152 227 283a 328a 350 382a 416 440 519 544a 578 581 646a 757a 874a 1126a (p58)

CUCAAAUGG GGGUGAUGA UUUAGAUGA UUCUGAUGA ACUGCAUGG ACUCUAUGG AGAAGAUGA AGCAAAUGU AAAAGAUGA AAAGCAUGC CGGGAAUGG GGGAAAUGG GGGGAAUGA GAAUGAUGG GCAAGAUGC GAACGAUGA AGAAAAUGG AAGAAAUGA

Matches with the consensus sequence are shown in italics. a In-frame AUG.

cDNA a frameshift by deletion of two guanosine nucleotides at positions 926 and 927 upstream of the internal AUG (fs-p110PITSLRE). This mutation led to a short open reading frame of 867 nucleotides (Figure 3C). Western blot analysis of stable Ba/F3 transfectants of fsp110PITSLRE revealed that the p58PITSLRE was still produced in G2/M in the absence of p110PITSLRE expression (Figure 3C). These observations clearly demonstrate that p58PITSLRE originates from internal initiation of translation on PITSLRE mRNA and not from proteolytic cleavage of p110PITSLRE. Figure 3. p58PITSLRE Is Expressed in G2/M by Internal Initiation of Translation on the Full-Length PITSLRE mRNA Western blot analysis with anti–E tag antibodies (A, B, and C) or anti-cyclin B1 antibodies (D) of Ba/F3-p110PITSLRE cells (A), Ba/F3mut-p110PITSLRE cells (B and D), or Ba/F3-fs-p110PITSLRE cells (C) prepared at different time points during cell cycle progression. Cells were synchronized in G1 by IL-3 depletion and released from this G1 block by subsequent stimulation with IL-3 for the times indicated. “c” indicates the control experiment with Ba/F3-p110PITSLRE in G2/M.

p58PITSLRE Is Produced from the p110PITSLRE mRNA by a Mechanism of Internal Initiation The C-terminal end of p110PITSLRE contains the open reading frame of p58PITSLRE (Xiang et al., 1994). We hypothesized that p58PITSLRE is synthesized by internal initiation of translation at an in-frame AUG codon in the full-length PITSLRE transcript (position 1126). Therefore, a mutagenesis experiment was performed in which the AUG codon has been replaced with a GCG codon. The mutant cDNA was stably transfected into the Ba/F3 cell line (Ba/F3-mut-p110PITSLRE), and p58PITSLRE expression was analyzed by Western blotting. This mutation completely knocked out p58PITSLRE expression in G2/M, whereas mutp110PITSLRE expression remained unchanged (Figure 3B). To rule out the possibility that we mutated a potential proteolytical cleavage site in p110PITSLRE capable of generating a 58 kDa product, we introduced in the p110PITSLRE

Internal Initiation of Translation on p110PITSLRE mRNA Is Mediated by an IRES Element Present in the Coding Region A possible mechanism accounting for the synthesis of p58PITSLRE is leaky ribosome scanning (Kozak, 1989, 1991). According to this model, the small subunit of the ribosome first recognizes the 5⬘ terminal cap structure of a mRNA and then scans the mRNA sequence in a 5⬘ to 3⬘ direction for potential AUG initiation codons. Often, but not always, the first AUG is used. Whether this AUG is selected or ignored depends largely on the sequence context surrounding it. An optimal nine nucleotide consensus sequence (5⬘-CC[A/G]CCAUGG-3⬘) has been proposed on the basis of extensive mutagenesis experiments (Kozak, 1986). The presence of a purine at position ⫺3 is most important for efficient AUG usage. In the absence of a ⫺3 purine, the presence of a guanosine at position ⫹4 is essential. Ribosomal subunits that fail to initiate at the first AUG can continue their search for an AUG in a more favorable sequence context. Inspection of the PITSLRE mRNA sequence reveals a moderate match with the consensus sequence for the nucleotides flanking the first AUG and the AUG of p58PITSLRE (Table 1). If the scanning mechanism would be used for translational initiation of p58PITSLRE, the ribosomal complex that binds at the 5⬘ end should scan 1126 nucleotides and

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bypass 17 AUG codons to initiate protein synthesis at AUG 18. Moreover, several of the upstream AUG codons are in a more favorable context to initiate protein synthesis than the AUG 18 (Table 1). Because these requirements are not compatible with the leaky scanning model, we considered the presence of an IRES structure in a region upstream of the initiation codon for p58PITSLRE expression. Cellular IRES elements described so far are all localized to the 5⬘UTR of the mRNA. In the case of p58PITSLRE, however, the potential IRES has to be present in the coding region of the PITSLRE mRNA, because the p110PITSLRE gene that was transfected in the Ba/F3 cells contained only the coding region of this gene. To demonstrate the presence of an IRES in the PITSLRE mRNA sequence, we made use of a dicistronic expression vector Di-1, in which the SV40 early promoter drives transcription of a capped dicistronic transcript. Both cistrons, respectively luciferase (LUC, first cistron) and ␤-galactosidase (LACZ, second cistron), are separated by an intercistronic spacer (ICS). The latter corresponds in the Di-1 vector to a fragment of 1005 nucleotides from the p110PITSLRE cDNA starting from position 121 and ending up at the internal initiation codon ATG(p58) at position 1126 (Figure 4A). Di-1 was transiently transfected into the 293T cell line; translation of luciferase and ␤-galactosidase was monitored by enzymatic activity assays (Figure 4B), as well as by Western blotting (Figure 4C). As expected, Di-1 produced luciferase by the conventional cap-dependent scanning mechanism. However, ␤-galactosidase will be translated only if the preceding sequence (ICS) contains an IRES structure. The same cell lysate was also positive for ␤-galactosidase (Figure 4B), suggesting that the cloned PITSLRE protein kinase–specific fragment contains a functional IRES element. Western blot analysis showed that both translation products were of the correct size, excluding the presence of fusion proteins in 293T cells (Figure 4C). To exclude the possibility that the potential IRES element in the ICS promotes the transfer of initiation-competent ribosomes from the termination codon of the upstream cistron to the initiation codon of the downstream cistron, we inserted a hairpin just upstream of the initiation codon of the luciferase gene (HPDi-1). This modification only negatively affected the translation of luciferase, whereas ␤-galactosidase expression remained unaffected. If enhanced ribosomal readthrough would be responsible for the ICS-mediated stimulation of ␤-galactosidase expression, then this activity should have been reduced by an equivalent amount (Figure 4D). To exclude that ␤-galactosidase was expressed from a monocistronic mRNA that might have been generated if the IRES element had sites for cleavage by a specific ribonuclease or if the IRES element would have a cryptic promoter element, we performed a Northern blot analysis of 293T cells transfected with the dicistronic reporter Di-1. The same mRNA was detected both by luciferaseand ␤-galactosidase-specific probes, indicating that both cistrons were translated from an intact dicistronic mRNA (Figure 4E). To rule out the possibility that the expression of the second protein, ␤-galactosidase, results from translation of a mRNA cleavage product that may not be detected on the Northern blot, we performed a control experiment to correlate LACZ mRNA levels on the Northern blot with ␤-galactosidase activities in the

cell extract (Figure 4F). Therefore, we transfected 293T cells with a mixture of monocistronic expression plasmids (pSV-Sport-LUC and pSV-Sport-LACZ) or with the dicistronic construct Di-1. Half of the cell suspension was used to prepare total RNA, the other part was used to measure luciferase and ␤-galactosidase activity. Transfection efficiencies were comparable as monitored by luciferase activity (monocistronic LUC/LACZ transfection: 31,614,592 cpm ⫾ 3,600,427 in a 40 ␮g protein extract; dicistronic Di-1 transfection: 33,186,412 cpm in a 40 ␮g protein extract). To correlate LACZ mRNA levels with ␤-galactosidase activity, we made a serial dilution (1/3) starting from 40 ␮g down to 0.165 ␮g of a protein extract from monocistronic LACZ transfectants and measured ␤-galactosidase activities. For the activity resulting from the dicistronic Di-1 transfectants, a 40 ␮g protein extract was used. In parallel, we prepared a serial dilution (1/3) of total RNA starting from 4 ␮g down to 0.0165 ␮g and measured LACZ mRNA expression. It is important to note that the amount of total RNA loaded on the Northern blot corresponds to the amount of protein used in the activity tests. On the same gel, 4 ␮g of Di-1 total RNA was loaded. In this experiment, the detection limit of ␤-galactosidase activity corresponds well to the detection limit of LACZ mRNA on the Northern (overnight exposure) (Figure 4F). Therefore, if the LacZ protein expression by Di-1 was derived from a cleaved mRNA species, we should be able to see it on the Northern, or, as we cannot detect it, it should be translated much more efficiently than this monocistronic LACZ mRNA, which is very unlikely. To further delineate the functional IRES element in the region upstream of the internal initiation codon on PITSLRE mRNA, a series of dicistronic plasmids containing deletions of the p110PITSLRE fragment cloned in Di-1 was generated (Figure 4A). The ability of the truncated sequences to promote internal ribosomal entry on the dicistronic mRNA and to promote translation of ␤-galactosidase was compared to Di-1, the relative ␤-galactosidase activity of which was set as “1.” Luciferase activity was used as an internal control to correct for variation of the transfection efficiencies. The dicistronic constructs Di-2 and Di-3 contain in their ICS the 5⬘ end of the p110PITSLRE region cloned in Di-1. Both vectors did not score positive in the ␤-galactosidase activity test (Figure 4B) nor in a Western blot analysis (Figure 4C), indicating that big deletions at the 3⬘ end completely abrogated internal initiation. This suggests that the IRES element is situated immediately upstream of the internal initiaton codon in the PITSLRE mRNA. This observation further excludes readthrough of the ribosomes from the luciferase to the ␤-galactosidase cistron. In contrast to Di-2 and Di-3, Di-4 and Di-5 contain in their ICS fragments that were able to promote internal ribosome entry and translation of ␤-galactosidase (Figure 4B). These fragments correspond to the 3⬘ end of the p110PITSLRE region cloned in Di-1. Although internal ribosome entry on Di-4 and Di-5 was reduced by approximately 20% and 50%, respectively, as compared to Di-1, the IRESmediated translation was still 40-fold and 25-fold higher than that of a dicistronic reporter containing no IRES (Di-2, Di-3). The integrity of the dicistronic mRNAs was

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Figure 4. Identification and Mapping of an IRES Element in the Coding Region of PITSLRE mRNA (A) Schematic representation of a dicistronic mRNA and of different p110PITSLRE-specific sequence elements that were cloned as an ICS between the coding regions for luciferase and ␤-galactosidase. Nucleotide numbers indicate the positions based on the p110PITSLRE cDNA. (B and C) The dicistronic plasmids (Di-1-Di-5) depicted in (A) were transiently transfected in 293T cells, and expression of luciferase and ␤-galactosidase was analyzed by measurement of their enzymatic activity (B) and by Western blot analysis (C). In the latter case, 293T cells transfected with pSV-Sport-LUC (LUC) or pSV-Sport-LACZ (LACZ) served as positive control. The upper and the lower panels show detection with anti-luciferase and anti-␤-galactosidase antibodies, respectively. IRES-mediated internal initiation activity was expressed as the ratio between ␤-galactosidase and luciferase activities (REL LACZ) (bars are representative of four independent transfections) (B). (D) Comparison of luciferase and ␤-galactosidase expression in 293T cells transiently transfected with Di-1 or HPDi-1, which carries an additional hairpin downstream from the SV40 early promoter. (E) Northern blot analysis of dicistronic mRNA expression in 293T cells transfected with the dicistronic constructs indicated on top of each lane (5 ␮g total RNA/lane), as revealed with LUC- (left) and LACZ- (right) specific probes. Detection of LUC mRNA and LACZ mRNA induced by overexpression of pSV-Sport-LUC and pSV-Sport-LACZ served as a positive control. (F) Correlation between LACZ mRNA levels and ␤-galactosidase activity. (Top) Northern blot analysis of a serial dilution (1/3) of total RNA prepared from monocistronic LACZ transfectants starting from 4 ␮g down to 0.0165 ␮g. On the same gel, 4 ␮g total RNA from dicistronic Di-1 transfectants was loaded. (Bottom) ␤-galactosidase activities of a serial dilution (1/3) starting from 40 ␮g down to 0.165 ␮g protein extract from monocistronic LACZ transfectants. For the activity resulting from the dicistronic Di-1 transfectants, a 40 ␮g protein extract was used. It is important to note that the amount of total RNA loaded on the Northern blot corresponds to the amount of protein used in the activity tests.

verified by Northern blot analysis, which showed anticipated reductions in length of the dicistronic mRNAs produced (Figure 4E). The Activity of the PITSLRE IRES Is Upregulated in G2/M To determine whether the cellular environment in G2/M is more supportive for internal initiation of translation mediated by the PITSLRE IRES element, we stably transfected the dicistronic expression vector Di-1 into Ba/F3

cells (Ba/F3-Di-1). Several luciferase-producing clones were obtained, all of which clearly expressed ␤-galactosidase (Figure 5A). To analyze the IRES activity on Di-1 mRNA during cell cycle progression, we synchronized several clones in G1 or G2/M by depletion and restimulation with IL-3, respectively. In each case, ␤-galactosidase activity was measured and corrected for the amount of dicistronic mRNA as deduced from the luciferase activity. In all clones analyzed, the relative ␤-galactosidase activity is four to six times enhanced in G2/M

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Figure 5. Upregulation of Internal Ribosome Entry on Dicistronic mRNA Di-1 in G2/M-Specific Ba/F3 Cells (A) Ba/F3 cells were stably transfected with the dicistronic expression plasmid Di-1, and expression of luciferase (left) and ␤-galactosidase (right) was analyzed by Western blotting in two representative nonsynchronized clones. (B) Four different Ba/F3-Di-1 clones were synchronized in G1 (open bars) and G2/M (closed bars) by IL-3 depletion (14 hr) and restimulation (24 hr) with IL-3, respectively. Specific activity of ␤-galactosidase and luciferase was analyzed and expressed as the ratio between ␤-galactosidase and luciferase activities. Data are the mean ⫾ SD of triplicates.

versus G1, demonstrating that G2/M functionally activates the PITSLRE IRES element in Ba/F3 cells (Figure 5B). The observation that ␤-galactosidase activity was also detected in G1, though we never observed p58PITSLRE in G1, is most likely due to a higher stability of ␤-galactosidase as compared to p58PITSLRE. Discussion In this report, we describe the regulated expression of PITSLRE protein kinase isoforms, p58PITSLRE and p110PITSLRE, during cell cycle progression. p110PITSLRE can be detected in all phases of the cell cycle, whereas p58PITSLRE is mainly expressed predominantly in G2/M. We found that the cell cycle–dependent expression of p58PITSLRE is mediated by an IRES element present in the coding region of PITSLRE mRNA. This IRES-mediated initiation of translation is activated specifically in mitosis, when conventional cap-dependent translation is inhibited. Several experiments support the use of an IRES to establish cell cycle–dependent expression of p58PITSLRE. The possibility that the latter is produced by proteolytical cleavage of p110PITSLRE is excluded by the observation that a frameshift mutant of PITSLRE mRNA can still produce p58PITSLRE in G2/M, whereas expression of p110PITSLRE is completely inhibited. Alternative splicing is also very unlikely because only one PITSLRE-specific mRNA could be detected during cell cycle progression. The observation that p58PITSLRE is expressed from a transfected p110PITSLRE cDNA in Ba/F3 transfectants also argues against an alternative spliced p58PITSLRE. Decreased levels of p58PITSLRE in G1 and S are also not due to instability of p58PITSLRE during G1 and S, since expression of p58PITSLRE open reading frame linked to a non-IRES leader did not reveal increased protein turnover in G1 and S (data not shown). The presence of an IRES in the PITSLRE coding region was shown by means of a dicistronic vector approach,

in which the complete p110PITSLRE coding sequence immediately upstream of the p58PITSLRE internal initiation codon was able to direct G2/M-specific translation of a downstream cistron encoding ␤-galactosidase. Deletion analysis showed that a small region of 219 nucleotides immediately upstream of p58PITSLRE still contained IRES activity. However, it should be noted that the IRES activity mediated by this short region was reduced as compared to the internal initiation of translation provided by the complete sequence. This partial loss of IRES activity may reflect removal of protein-binding sites or structural elements that contribute positively to internal initiation of translation. The PITSLRE IRES element contains a purine-rich tract (93% A/G) of 90 nucleotides, which is situated 60 nucleotides upstream of the AUG(p58). A similar polypurine motif has also been found in a tobamoviral IRES (Ivanov et al., 1997). The functional significance of this motif is still unclear. Possibly, it plays a functional role in analogy with the oligopyrimidine motif that has been described for the picornavirus IRES (Pilipenko et al., 1992; Jackson et al., 1994). An exceptional feature of the PITSLRE IRES is its cell cycle–dependent control, leading to G2/M-specific expression of p58PITSLRE. The underlying mechanism for this phenomenon is still unclear. We were unable to demonstrate activation of the PITSLRE IRES element in in vitro translation assays in the presence of rabbit reticulocyte lysate, regardless of using capped or uncapped p110PITSLRE transcripts (data not shown). Nevertheless, p110PITSLRE was efficiently translated from the capped transcript. This observation suggests that transacting factors might be missing or limiting in the lysate of “nonproliferating” reticulocytes. Such a role for transacting factors has already been demonstrated for viral IRESs (Meerovitch et al., 1989, 1993; Borovjagin et al., 1994; Borman et al., 1997). Alternatively, binding of inhibitory molecules to the IRES structure during G1 cannot be excluded. What might be the biological significance of IRESmediated translation of p58PITSLRE? Only a few other cellular mRNAs have been shown to possess an IRES structure. These include immunoglobulin heavy chain binding protein (Macejak and Sarnow, 1991), Drosophila antennapedia (Oh et al., 1992), fibroblast growth factor 2 (Prats et al., 1992; Vagner et al., 1995, 1996; Arnaud et al., 1999), platelet-derived growth factor B (Bernstein et al., 1997), insulin-like growth factor II (Teerinck et al., 1995), the translation initiation factor eIF4G (Gan and Rhoads, 1996), c-myc (Nanbru et al., 1997; Stoneley et al., 1998), vascular endothelial growth factor (Akiri et al., 1998; Huez et al., 1998; Miller et al., 1998; Stein et al., 1998), and X-linked inhibitor of apoptosis (Holcik et al., 1999). It should be mentioned that the IRES structure of these cellular mRNAs is always located to the 5⬘ untranslated region, whereas PITSLRE IRES is the first IRES element found in the coding region of a gene. Strikingly, most of the cellular mRNAs mentioned above code for proteins involved in growth control. PITSLRE kinases are related to the master mitotic protein kinase p34cdc2. Based on this relationship and the observed deletions of the PITSLRE locus on chromosome region 1p36.3 in many cancers, it is likely that PITSLRE kinases play an important role in growth control or mitosis. It is well known that during mitosis cap-dependent translation in mammalian

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cells is extremely inefficient due to the presence of underphosphorylated and therefore nonfunctional eIF4F (Bonneau and Sonenberg, 1987; Huang and Schneider, 1991). The presence of an IRES structure to ensure translation of p58PITSLRE protein kinase in G2/M suggests an important role for p58PITSLRE in mitosis or in cell cycle progression. In this context, it should be noted that growth inhibitory effects of constitutive overexpression of p58PITSLRE in CHO cells have already been described (Bunnell et al., 1990; Lahti et al., 1995). Similarly, our preliminary results show that abrogation of the IRESmediated G2/M-specific expression of p58PITSLRE by constitutive overexpression of p58PITSLRE in Ba/F3 cells tempers cell proliferation, whereas G2/M-specific expression of p58PITSLRE induced by p110PITSLRE overexpression did not (data not shown). So far, mechanisms involved in the control of protein expression during cell cycle progression have been reported to occur at the transcriptional level or at the level of protein stability. Here, we propose translational regulation mediated by G2/M-specific activation of a cellular IRES as a novel mechanism. More specifically, the latter leads to G2/M-specific expression of a member of the cdc2-related protein kinase family, in casu p58PITSLRE. Since IRES-mediated translation does not require capping of the mRNA, we expect that similar IRESs will be discovered in transcripts encoding proteins that need to be expressed under conditions where cap-independent translation is inefficient, such as mitosis, apoptosis, or other stress conditions. Finally, since the PITSLRE IRES structure is remarkably efficient and specifically active in G2/M, it might be a useful tool for the development of novel polycistronic vectors or gene therapy approaches that are targeted to dividing cells. Experimental Procedures Plasmid Constructions p110PITSLRE cDNA (␣2-2 isoform) was obtained by reverse transcription with Superscript reverse transcriptase (Life Technologies, Paisly, UK) and polymerase chain reaction (PCR) amplification with high fidelity DNA polymerase (Roche Molecular Biochemicals, Basel, Switzerland) of polyA⫹ mRNA from human HL-60 cells. The primers 5⬘-TGACCGGAATTCATGGGTGATGAAAAGGACTCTTGG-3⬘ and 5⬘-TGACCGGAATTCTGACCTTCAGAACTTGAGGCTGAAGCC-3⬘ used for this amplification resulted in a cDNA fragment of 2480 bp, which was digested with EcoRI and cloned into the pMA58 plasmid (a gift from Dr. Patrick Stanssens). The resulting plasmid (pMAp110PITSLRE) was used to perform site-directed mutagenesis by a chloramphenicol selection procedure using a Transformer SiteDirected Mutagenesis Kit of Clontech Laboratories (Palo Alto, CA). Briefly, this method involves the simultaneous annealing of two oligonucleotide primers to one strand of the denatured doublestranded pMA-p110PITSLRE plasmid. One primer introduces the desired mutation; the second primer (5⬘-CCGTAATATCCAGCTGA ACGGTCTGG-3⬘) induces a gain-of-function mutation in the gene encoding chloramphenicol resistance for the purpose of selection. To fuse an E tag at the 3⬘ end of p110PITSLRE cDNA, we introduced an in-frame NotI restriction site at the stop codon by using the mutation primer 5⬘-AGCCTCAAGTTCGCGGCCGCAGAGTGGACC-3⬘. The p110PITSLRE cDNA was inserted as an EcoRI-NotI fragment in the pSV–Sport–E tag expression plasmid. The latter was obtained by insertion of the E tag as a NotI-XbaI fragment in the pSV-Sport expression plasmid (Life Technologies,). Mutation of the internal initiation codon was obtained by using the mutation primer: 5⬘-GAGGAAGAAGCGAGTGAAGAT-3⬘. The frameshift mutation was induced by using mutation primer: 5⬘-GACAGCG AGAAAGACCAGCTCG-3⬘.

The dicistronic pSV-Sport expression vectors (Di-1-Di-5) with different p110PITSLRE fragments in the ICS were made by first cloning the PITSLRE-specific PCR fragments (XbaI-NcoI digested) and the lacZ gene as a NcoI-SalI fragment from pIRES-lacZ (gift from Dr. D. Huylebroeck) into the pUC19 plasmid by a three-point ligation. In a subsequent three-point ligation, the complete PITSLRE-LacZ insert was cloned as an XbaI-SalI fragment together with the firefly luciferase gene as a KpnI-XbaI fragment from the pGL3-basic vector (Promega, Madison, WI) in the KpnI-SalI opened pSV-Sport expression plasmid. The 5⬘ end and 3⬘ end primers used for amplification of the different PCR fragments were as follows: Di-1 sense, 5⬘-CTAGTCTA GAAAAGTGAAAACTTTAGATGAAATTC-3⬘, antisense, 5⬘-TTCTTCA TCTTCACCCATGGCTTCCTCACTTAC-3⬘; Di-2 sense, (idem Di-1), antisense, 5⬘-TGCATGCCATGGTCCTCTCTCATCGTTCGGTGATG-3⬘; Di-3 sense, (idem Di-1), antisense, 5⬘-TGCATGCCATGGATGTCG TTTCCGACGTTCGTGC-3⬘; Di-4 sense, 5⬘-CTAGTCTAGACATC ACCGAACGATGAGAGAGG-3⬘, antisense, (idem Di-1); Di-5 sense, 5⬘-CTAGTCTAGAGACATCAGCGACAGCGAGAGGAAGACCAGC-3⬘, antisense, (idem Di-1). A stable hairpin (⌬G ⫽ ⫺40 kcal/mol) was created by cloning of a double-stranded oligonucleotide between the BglII and HindIII sites of pGL3-basic (5⬘-GATCTTTACCAACAGTACCGGAATGCCA AGAAA-3⬘; 5⬘-AAATGGTTGTCATGGCCTTACGGTTCTTTTCGA-3⬘). pSV-Sport-LUC was created by cloning luciferase (from pGL-3 basic) as a KpnI-XbaI fragment in the pSV-Sport expression vector. pSV-Sport-LACZ was created by cloning ␤-galactosidase in pBluescript as a HindIII-SalI fragment from which ␤-galactosidase was recloned as an EcoRI-SalI fragment into the pSV-Sport expression plasmid. Cells and DNA Transfection The IL-3-dependent mouse pre–B cell line Ba/F3 (Palacios and Steinmetz, 1985) was maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) heat-inactivated fetal calf serum and 10% (v/v) conditioned medium from the WEHI-3B cells as a source of mouse IL-3. Ba/F3 cells were stably transfected by electroporation. Before transfection, cells were collected and resuspended at 1 ⫻ 107 cells per milliliter of medium. An expression plasmid (20 ␮g) carrying the gene of interest and 5 ␮g pBSpac⌬p carrying a puromycine resistance gene (De la Luna et al., 1988) were added to a 0.8 ml cell suspension. Electroporation was performed using the Easy Ject apparatus (Eurogentec, Seraing, Belgium) at 1500 ␮F and 300 V. Subsequently, the cells were resuspended in growth medium, and selection was initiated 48 hr after transfection in medium containing 1 ␮g/ml puromycin (Sigma Chemical Co., St. Louis, MO). After 1 week, surviving cells were subcloned by limiting dilution. Positive transfectants were selected on the basis of expression as revealed by immunoblotting or reporter gene expression measured by enzymatic assays (see below). Human embryonic kidney 293T cells (a gift from Dr. M. Hall) were transiently transfected by the calcium phosphate precipitation method (O’Makoney and Adams, 1994). Cells were incubated for at least 4 hr with the transfection solution followed by adding fresh Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) heat-inactivated fetal calf serum. Cells were collected by centrifugation at 48 hr posttransfection and were further analyzed as described below. Cell Cycle Synchronization Ba/F3 cells were IL-3 depleted for 14 hr or treated with 0.8 ␮M nocodazole (Sigma Chemical Co.) to arrest cells in G1 or G2/M, respectively. DNA content was measured by freezing cells in the presence of propidium iodide and subsequent FACS analysis. Western Blot Analysis Cells were lysed in a buffer containing 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 1% NP40, 1 mM Pefablock, 200 U/ml aprotinin, 10 mM EDTA, and 10 ␮g/ml leupeptin. Protein concentration was quantified in the cell lysates by the Bradford assay. Proteins (50 ␮g) were subjected to SDS-PAGE and transferred by electroblotting onto a nitrocellulose membrane. E tag–fused proteins were immunodetected with mouse monoclonal anti–E tag antibodies

Molecular Cell 604

(1/1000 dilution) (Pharmacia Biotech, Uppsala, Sweden). ␤-galactosidase and firefly luciferase were immunodetected with mouse monoclonal anti-␤-galactosidase (1/1000 dilution) (Roche Molecular Biochemicals, Basel, Switzerland) and rabbit polyclonal anti-luciferase antibodies (1/2000 dilution) (Promega), respectively. PITSLRE protein kinases and cyclin B1 were immunodetected by the rabbit polyclonal anti-PITSLRE antibodies (1/1000) (Santa Cruz Biotechnology, Santa Cruz, CA) and the rabbit polyclonal anti-cyclin B1 antibodies (dilution 1/1000) (Santa Cruz Biotechnology), respectively. Immunoreactivity was revealed with an enhanced chemiluminescence kit (Amersham Life Science, Amersham, UK). Reporter Gene Assays Cells were lysed in 25 mM Tris phosphate (pH 8), 2 mM DTT, 2 mM CDTA, 10% glycerol, and 1% Triton X-100. Firefly luciferase activity was assayed in a total volume of 30 ␮l. The reactions were initiated by addition of 15 ␮l luciferase assay/substrate buffer (40 mM Tricine, 2 mM [MgCO3]4Mg[OH]2. H2O, 5 mM MgSO4, 66 mM DTT, 0.2 mM EDTA, 0.5 mM CoA, 1 mM ATP, 1 mM D-luciferin) to 15 ␮l cell lysate. Light emission was measured by a Topcount scintillation counter (Packard Instrument Co., Meriden, CT). ␤-galactosidase activity was measured in a total volume of 200 ␮l. Cell lysate (20 ␮l) was added to 160 ␮l substrate buffer (60 mM Na2HPO4, 10 mM KCl, 1 mM ␤-mercaptoethanol), and the reaction was initiated by adding 20 ␮l of 50 mM chlorophenolred-␤-D galactopyranoside. The colorimetric signal was measured at 595 nm using spectrophotometry. Cellular RNA Purification and Northern Blotting Total cellular RNA was isolated with the RNeasy kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. RNA was denatured in formaldehyde and separated on a 1.2% formaldehyde– agarose gel. RNAs was transferred onto a nylon membrane (Amersham Life Science) by the capillary blot procedure. The filters were UV cross-linked using a UV Stratalinker apparatus (Stratagene, La Jolla, CA) and were hybridized with the indicated cDNAs that were labeled with ␣-[32P]ATP by randomly primed DNA synthesis. The hybridization probes were luciferase (a 1700 bp cDNA NcoI-XbaI restriction fragment), ␤-galactosidase (an 800 bp cDNA NcoI-ClaI restriction fragment), and mouse p110PITSLRE (a 1072 bp cDNA PstI restriction fragment). Acknowledgments We are indebted to Dr. Ivan Shatsky for his critical and helpful comments on the manuscript. We thank Dr. W. Fiers for helpful discussions. The technical assistance of A. Meews and W. Burm is gratefully acknowledged. This work was supported by the FWO (Fonds voor Wetenschappelijk Onderzoek), the Sportvereniging tegen Kanker, and the IUAP (Interuniversitaire Attractiepolen). S. V. H. and G. D. are predoctoral fellows and S. C. and R. B. are postdoctoral research associates with the FWO, respectively.

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Received July 19, 1999; revised February 17, 2000.

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