The Chicken C-Jun 5' Untranslated Region Directs Translation by Internal Initiation

Oncogene (2000) 19, 2836 ± 2845 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc The chicken c-Jun 5' untranslated region directs translation by internal initiation Anil Sehgal1,2, Joe Briggs1, Janet Rinehart-Kim1, Johnny Basso1,3 and Timothy J Bos*,1 1Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, PO Box 1980, Norfolk, Virginia, VA 23501, USA The 5' untranslated region (UTR) of the chicken c-jun Because of its role in controlling gene expression, message is exceptionally GC rich and has the potential much eort has been expended studying the regulation to form a complex and extremely stable secondary and function of the c-Jun protein. Like other nuclear structure. Because stable RNA secondary structures can oncogenes, c-jun is tightly regulated at a number of serve as obstacles to scanning ribosomes, their presence dierent levels. Transcriptional regulation is well suggests inecient translation or initiation through documented in response to a wide variety of growth alternate mechanisms. We have examined the role of factors (Lamph et al., 1988; Ryder and Nathans, 1988; the c-jun 5' UTR with respect to its ability to in¯uence Vogt and Bos, 1990). Post-translational regulation of translation both in vitro and in vivo. We ®nd, using Jun activity occurs through speci®c phosphorylation rabbit reticulocyte lysates, that the presence of the c-jun and dephosphorylation events which regulate DNA 5' UTR severely inhibits translation of both homologous binding as well as transcriptional regulatory activity and heterologous genes in vitro. Furthermore, transla- (Adler et al., 1992; Baker et al., 1992; Boyle et al., tional inhibition correlates with the degree of secondary 1991; Franklin et al., 1992; Pulverer et al., 1991; structure exhibited by the 5' UTR. Thus, in the rabbit Smeal et al., 1991, 1992). Activity can also be reticulocyte lysate system, the c-jun 5' UTR likely regulated by a variety of dierent protein-protein impedes ribosome scanning resulting in inecient interactions with non-leucine zipper transcription translation. In contrast to our results in vitro, the c-jun factors (Bengal et al., 1992; Boise et al., 1993; SchuÈ le 5' UTR does not inhibit translation in a variety of et al., 1990; Stein et al., 1993a,b; Touray et al., 1991; dierent cell lines suggesting that it may direct an Yang-Yen et al., 1990). alternate mechanism of translational initiation in vivo. The c-jun message is produced from an intronless To distinguish among the alternate mechanisms, we gene (Hattori et al., 1988) and has an interesting generated a series of bicistronic expression plasmids. structure which suggests additional levels of control. In Our results demonstrate that the downstream cistron, in addition to the protein coding region, the c-jun message the bicistronic gene, is expressed to a much higher level contains long 5' and 3' untranslated regions (UTRs). when directly preceded by the c-jun 5' UTR. In addition, The 3' UTR contains several AUUUA motifs, adding inhibition of ribosome scanning on the bicistronic an additional layer of complexity to c-jun regulation at message, through insertion of a synthetic stable hairpin, the level of mRNA stability (Aharon and Schneider, inhibits translation of the ®rst cistron but does not inhibit 1993; Curatola et al., 1995; Savant-Bhonsale and translation of the cistron downstream of the c-jun 5' Cleveland, 1992). The 5' UTR of c-jun is extremely UTR. These results are consistent with a model by which long and GC rich, ranging in size from 301 nucleotides the c-jun message is translated through cap independent in chicken to 974 bases in human (see Table 1). internal initiation. Oncogene (2000) 19, 2836 ± 2845. Typically, messenger RNAs with long GC rich 5' UTRs are translated ineciently by cap dependent Keywords: c-Jun; translation; internal initiation; 5' scanning ribosomes. Translation of these messages UTR often requires alternative mechanisms of translational initiation. Most eukaryotic messenger RNAs contain 5' UTRs Introduction less than 100 bases long and are translated by a mechanism referred to as ribosome scanning (reviewed v-Jun was originally discovered as the oncogenic in Gray and Wickens, 1998; Hershey, 1991; Kozak, component of Avian Sarcoma Virus 17 (Maki et al., 1991a). RNA secondary structures with standard free 1987). The cellular homologue, c-Jun, is a sequence energy values (DG) up to about 730 kcal/mole can speci®c DNA binding protein that binds to promoters easily be negotiated by the scanning 43S ribosome in responsive genes to activate transcription (Angel et subunit, allowing progression until the AUG start al., 1988; Bohmann et al., 1987; Vogt and Bos, 1990). codon in the proper context is found (Kozak, 1989, 1991b). When more stable structures are encountered, the 43S subunit stalls and can not proceed. RNAs that *Correspondence: TJ Bos contain these stable structures will likely not be Current addresses: 2Department of Neurological Surgery, University translated eciently by the ribosome scanning mechan- of California at San Francisco, 1855 Folsom Street, MCB 230, San ism (Kozak, 1989). Interestingly, the 5' UTRs of many Francisco, CA 94103; 3University of Ottawa, Department of Biology, growth factor and proto-oncogene messages are long Gendron Bldg., 550 Cumberland Street, Ottawa, Ontario, Canada, K1N 6N5 and have the potential to form stable secondary Received 7 January 2000; revised 23 March 2000; accepted structures (Kozak, 1991a). A growing number of these 28 March 2000 proteins have been shown to initiate translation Internal initiation of c-Jun translation A Sehgal et al 2837 Table 1 Comparison of 5' untranslated sequences of Jun family presence of motifs which have the potential to play mRNAs across species roles in RNA structural stability as well as RNA- Length Free energy protein interactions. Several other potential structures Species (bases) % GC Accession # kcal/mole within this optimal free energy range are depicted in c-Jun Figure 1c. All of these potential secondary structures Chicken 301 81 M57467 7181.4 are extremely stable with optimal free energy values Quail 354 73 X15547 7173.6 well beyond that shown to inhibit ribosome scanning. Rat 917 63 X17215 7402.1 Secondary structures with free energy values as low as Mouse 614 63 X12740 7255.8 Human 974 65 J04111 7444.6 750 kcal/mole have been shown to inhibit cap dependent translation (Kozak, 1989, 1991b). Transla- Jun B tion of messages with stable secondary structures Rat 289 70 X54686 7138.2 generally occurs through alternative mechanisms such Mouse 287 70 J03236 7127.6 Human 287 74 M29039 7135.1 as internal initiation and ribosome jumping (Gray and Wickens, 1998). From this structural analysis, we Jun D predicted that the full length c-jun message would fall Mouse 121 83 J04509 774.1 into this category. To test this hypothesis, we have Human 174 86 X56681 7103.2 analysed the mechanism of translation of the chicken c- Chicken 165 84 X60063 791.2 jun message both in vitro and in vivo. Effect of the c-jun 5' UTR on translation from rabbit through alternate mechanisms. For example, c-Myc reticulocyte lysates in vitro (Nanbru et al., 1997; Stoneley et al., 1998), bFGF (Vagner et al., 1995), PDGF-2 (Sella et al., 1999), We utilized an in vitro transcription and translation GRP78/BIP (Macejak and Sarnow, 1991; Sarnow, system to assay the eect that the 5' UTR has on 1989), eIF4G (Gan and Rhoads, 1996), Apaf1 translation of c-jun.c-jun cDNA with (pG5'CJ3) and (Coldwell et al., 2000) and VEGF (Akiri et al., 1998; without (pGCJ3) the 5' untranslated region were Stein et al., 1998) are each capable of bypassing the cloned into pGEM4 such that they were under the impediment of RNA secondary structure in the 5' UTR control of the SP-6 promoter. Because 3' untranslated by translation initiation through cap independent sequences of c-jun have been implicated in mRNA internal ribosome entry. This capability extends stability, we prepared templates for transcription such another level of complexity to the regulation of the that transcripts would run o shortly after the stop expression of these proteins allowing their expression codon (Figure 2a). RNA levels were assayed by even under conditions where cap dependent ribosome synthesis in the presence of [32P]UTP or [32P]CTP scanning is inhibited. followed by separation on 6% urea-acrylamide gels We have examined the 301 nucleotides of the 5' and quantitated (Figure 2b, bottom panel) by UTR of the chicken c-jun message and found it to phosphorimage analysis. Equivalent values of RNA contain 81% G or C residues. Algorithmic analysis were used in subsequent translation reactions. Transla- predicts that this sequence will form a stable and tion of in vitro transcribed RNA was carried out in complex secondary structure with an optimal standard rabbit reticulocyte lysates in the presence of free energy of 7181.4 kcal/mole. The predicted [35S]methionine. Labeled proteins were then separated stability of this structure far exceeds that which can by SDS ± PAGE and quantitated by phosphorimage be melted by a scanning 43S ribosome subunit, analysis. The results are presented in Figure 2b suggesting that translation will either be extremely (compare lanes 1 and 6). Clearly, the presence of the inecient or proceed through an alternative mechan- 5' UTR has a dramatic inhibitory eect on translation ism of initiation. We demonstrate here that c-jun is of c-Jun in rabbit reticulocyte lysates. part of a growing list of important cellular genes whose To examine this eect in more detail, we generated a translation is directed through cap independent internal series of deletion mutations within the c-jun 5' UTR ribosome entry. designed to reduce the potential structural complexity and stability.

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