Homeotic Gene Antennapedia Mrna Contains 5'-Noncoding Sequences That Confer Translational Initiation by Internal Ribosome Binding

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Homeotic gene Antennapedia mRNA contains 5'-noncoding sequences that confer translational initiation by internal ribosome binding

Soo-Kyung OH, 1 Matthew P. Scott, 2 and Peter Sarnow 1'3 ~Department of Biochemistry, Biophysics and Genetics, 3Department of Microbiology and Immunology, University of Colorado Health Sciences Center, Denver, Colorado 80262 USA; 2Departments of Developmental Biology and Genetics, Stanford University School of Medicine, Stanford, California 94305-5427 USA

The Antennapedia (Antp) homeotic gene of Drosophila melanogaster has two promoters, P1 and P2. The resulting Antp mRNAs contain 1512-nucleotide (P1) and 1727-nucleotide (P2) 5'-noncoding regions, composed of exons A, B, D, and E (PI) or exons C, D, and E (P2), respectively. Multiple AUG codons are present in exons A, B, and C. We have found that 252-nucleotide exon D, common to mRNAs from both transcription units and devoid of AUG codons, can mediate initiation of translation by internal ribosome binding in cultured cells. Many mRNAs in Drosophila contain long 5'-noncoding regions with apparently unused AUG codons, suggesting that internal ribosome binding may be a common mechanism of translational initiation, and possibly its regulation, in Drosoptu'la. [Key Words: Internal ribosome binding; homeotic gene; Antennapedia mRNA; dicistronic mRNA; RNA transfection] Received May 18, 1992; revised version accepted July 7, 1992.

The homeotic genes required for segments in Drosophila similarly, exon C contains 15 upstream AUG codons. All melanogaster to have different developmental fates are of these upstream AUG codons are followed by very expressed in some segment primordia but not in others. short open reading frames that could potentially encode Each segmented part of the embryo expresses a different peptides ranging from 6 to 44 amino acids. Noncoding homeotic gene, or combination of them (for review, see exon D and the 5' half of exon E, common to mRNAs Duncan 1987; Mahaffey and Kaufman 1988). Transcrip- initiated at both P1 and P2, are devoid of AUG codons tion of the homeotic genes is tightly regulated by the (Laughon et al. 1986; Stroeher et al. 1986). It is remark- activities of a large number of regulators, including able that the large number of total upstream AUG many of the segmentation genes (Scott and Carroll 1987; codons in these 5'-noncoding regions does not prevent Ingham 1988; Irish et al. 1989). Transcription of a ho- translation of the main open reading frame. According to meotic gene in a location where the gene is normally the scanning model for translational initiation (Kozak silent can lead to transformations of segments (Frischer 1989, 1991), translational initiation of Antp mRNA et al. 1986; Schneuwly et al. 1987a, b). Thus, transcrip- should be very inefficient. tional regulation is clearly involved in the spatially reg- If the scanning mechanism were used for translational ulated activities of homeotic genes. The sequence orga- initiation of Antp mRNA, derived from the P2 promoter, nization of certain homeotic mRNAs suggests that there for example, the ribosomal 43S ternary complex would may be an additional level of regulation. bind at the 5' end of the mRNA and would scan 1730 Transcription of the homeotic Antennapedia (Antp) nucleotides of the 5' NCR, bypassing 15 AUG codons, to gene is initiated at two promoters (P1 and P2), producing initiate protein synthesis at the sixteenth AUG codon. transcripts that differ in their 5'-noncoding regions (5' Several of the 15 upstream AUG codons are in a favor- NCR) (Laughon et al. 1986; Schneuwly et al. 1986; Stroe- able context to initiate protein synthesis in Drosophila her et al. 1986). Figure 1 illustrates that Pl-initiated tran- cells (Table 3, below; Cavener 1987). In addition, RNA scripts contain a 1512-nucleotide 5' NCR derived from structures in this long leader may render the scanning of sequences in non-protein-coding exons A, B, D, and E, the ribosomal subunits inefficient. whereas P2-initiated transcripts contain a 1727-nucle- There are precedents for translational initiation in otide 5' NCR derived from exons C, D, and E. The non- mRNAs with multiple AUG codons in their 5' NCRs, by coding exons A and B contain 8 AUG codons preceding both ribosomal reinitiation and internal ribosome bind- the translation initiator AUG codon located in exon E; ing mechanisms. For example, yeast GCN4 mRNA con-

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OH et al.

ORF and Sonenberg (1988). Briefly, polymerase II in cells PI: transfected with these plasmids should initiate transcription at the SV40 promoter, producing a capped dicistronic transcript that contains 3'-terminal polyadenosine. The first cistron, encoding chloramphenicol P2: I acetyhransferase (CAT), lies downstream of a short, capped 5' NCR and should be translated by the conven- tional cap-dependent scanning mechanism (Kozak 1989). However, the second cistron, encoding luciferase (LUC), t I should be translated only if the preceding intercistronic ' kO spacer (ICS) insert contains an IRES, a sequence that can Figure 1. Codingand noncoding exons of Antp mRNAs. Struc- mediate internal ribosome binding. ture of Antp transcripts derived from the P 1 and P2 promoters. We engineered different parts of the 5' NCR of Antp P2 (Open rectangles) Noncoding exons; (solid rectangles) coding mRNA, as well as control sequences, into the ICS region exons. Exons are numbered according to Laughon et al. (1986). of pSVACAT/ICS/LUC (Fig. 3) and transfected the individual plasmids into Drosophila Schneider line 2 (SL2) cells. The translation products of the first (CAT) and the tains 4 AUG codons upstream of the initiator AUG second (LUC) cistrons were monitored during the tran- codon, and this property is responsible for the regulation sient expression of the transfected plasmids. As ex- of GCN4 translation initiation in response to amino acid pected, all dicistronic mRNAs directed the synthesis of availability (for review, see Hinnebusch 1988). Transla- similar amounts of active CAT protein in transfected tional initiation by internal ribosome binding was first cells (Table 1). Therefore, the different amounts of CAT observed in two picornaviral mRNAs. Pelletier and protein that accumulated in individual transfection ex- Sonenberg (1988) and Jang et al. (1989) found that the 5' periments can be used as an internal control for the dif- NCRs of poliovirus and encephalomyocarditis virus ferent transfection efficiencies of the plasmids and for (EMCV), respectively, could mediate translational initi- the amount of translation-competent mRNA. If these ation by internal ribosome binding. Because internal ri- mRNAs are intact, relative CAT activity can serve as a bosome binding to viral RNAs was found to function control for RNA stability as well. both in uninfected and infected cells, it was concluded The accumulation of LUC activity in the same lysates that the mammalian cells possess all of the factors re- was then determined (Table 1). Dicistronic mRNAs con- quired for this mechanism. Recently, the 5' NCR of a taining the entire 5' NCR of Antp P2 (AP2, exons C, D, cellular mRNA was found to direct translation by inter- and E) in the ICS directed the translation of the LUC nal ribosome binding in human cells (Macejak and Sar- protein at a 240-fold level higher than dicistronic now 1991). mRNAs containing control sequences in the ICS (exons We tested whether ribosomes could use internal se- D-E inverted, I~; Table 1). Monocistronic mRNAs con- quences within the 5' NCR of Antp P2 mRNA to initiate taining the 5' NCR of Antp upstream of LUC produced translation in dicistronic transcripts. We found an appar- the same amount of active LUC (not shown), indicating ent internal ribosome entry site (IRES) sequence element that translation of both monocistronic and dicistronic within exon D that can direct translation initiation at mRNAs was initiated by the same major route. When the most proximal downstream AUG codon in cultured the activity of translation products from dicistronic Drosophila cells. That at least 20% of Drosophila mRNAs bearing only exons D and E in the ICS was mon- mRNAs contain long leaders with multiple upstream itored, LUC activity was found to be 30% the level re- AUG codons suggests that internal ribosome binding sulting from mRNAs containing exons C, D, and E in the may be an important mechanism in the expression of ICS (Table 1). Thus, RNA sequences in exons D and E are regulatory genes in the organism. sufficient to direct translation of the second cistron in dicistronic mRNAs in SL2 cells.

Results Direct transfection of dicistronic mRNAs The 5' NCR of Antp P2 mRNA mediates translation into cultured cells: exon D of Antp is sufficient of the second cistron in dicistronic mRNAs for translation of the second cistron in cultured Drosophila cells To substantiate these data further and to exclude the The 5' NCR of Antp mRNAs revealed a sequence orga- possibihy that cryptic promoter sequences in the 5' nization similar to that of the 5' NCR of poliovirus; both NCR of the Antp cDNA produced functionally monocis- 5' NCRs are unusually long [1730 (P2) and 743, repec- tronic LUC mRNA, we transfected dicistronic mRNAs, tively] and contain multiple upstream AUG codons [15 synthesized in vitro, directly into SL2 cells. Capped di- (P2) and 8, respectively]. To test whether sequences in cistronic mRNAs containing 3'-terminal polyadenosine the 5' NCR of Antp enable translation to be initiated by tails were synthesized in vitro by use of T7 RNA poly- internal ribosome binding, we constructed plasmid vec- merase and transfected directly into SL2 cells. All tors (Fig. 2) similar to the ones first described by Pelletier mRNAs tested were functionally associated with ribo-

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Internal ribosome binding of Antp mRNA

Amp r

/\ Figure 2. Structure of DNA expression vec- tors used to produce dicistronic mRNA mole- ICS cules. Structure of a generic vector to express dicistronic mRNAs in cultured cells. The Transfection of plasmid into cells plasmid, pSVACAT/ICS/LUC, contains promoter and enhancer elements from SV40, fol- Transcription lowed by the coding region for CAT, an ICS, and a second cistron encoding LUC. [ivs and I Transport of RIgA into cytoplasm poly{A)] Cassettes containing sequences medi- ating splicing and polyadenylation, respectively, of the primary transcript. The predicted structure of the primary transcript is shown. (Cap) The m7GpppN structure (N can be any nucleotide) found at the 5' end of polymerase Cap L CAT I 'cs I LUC J----A n II transcripts.

somes and were translated to produce CAT, the product ciencies of RNA uptake into the cells or in different sta- of the first cistron (Table 2). The amount of CAT activity bilities of the RNAs in the cells. In any case, determina- in cell lysates varied slightly between different RNA spe- tion of LUC activity in the same lysates revealed much cies tested. Most likely, differences in the sequences and greater differences. RNAs containing exons C, D, and E structures of the RNA species resulted in different effi- (AP21, exons D and E (DE), or exon D alone (D) in the ICS

Intercistronic Structure of Intercistronic Spacer Spacer

CAT Exon C D E LUC 1321 nl 252 nt 157 nt Ant P2 ,i V V ,1r3o

Exons D-E Figure 3. Structures of the ICS regions in dicistronic mRNAs. Dicistronic mRNAs containing different parts of the Antp P2 5' NCR are shown. The 3' and 5' ends of the Exons D-E CAT- and LUC-coding regions, respectively, are indicated. Antp P2 contains the 11730 #1282 entire 5' NCR of Antp P2 (exons C, D, and AUG 111766 E); exons ~-7-E contain exons D and E inverted; exons ~-E + AUG contain an ~ ~ Exons D-~E + AUG AUG triplet in the same translational reading frame as the AUG codon used to trans- late LUC. (L) AUG codons. Upstream AUG in frame

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Table 1. Translation of dicistronic mRNAs after was intact. Furthermore, no protection was observed transfection of DNA plasmids into cultured D. melanogaster when mRNAs from nontransfected cells were used in SL2 cells the protection assay (Fig. 4B, lane 3), excluding the pos- Chloramphenicol sibility that protection from fortuitously cross-hybridiz- conversion/106 LUC light ing cellular mRNA was monitored. Similarly, transfec- Plasmid cells (%) units/106 cells tion of pSVnCAT/exons DE/LUC into mammalian COS cells resulted in the production of intact dicistronic AP2 a 10 - 2 220,000 +_ 20,000 mRNAs (Fig. 4C, lane 3) which could be translated to D~ b 12 -+ 2 76,500 +_ 6,500 I~ a 9 +-- 2 900 +- 300 produce LUC (not shown). D-~ + AUG a 12---3 190-+ 10 Furthermore, we transfected gel-purified, dicistronic 5' CAT/exon DE/LUC 3' mRNAs directly into SL2 cells structural features of plasmids are shown in Fig. 3. Experiments and analyzed the integrity of the dicistronic RNAs at 19 were repeated three (a) or six (b) times. hr after transfection. Figure 4C (lane 5) shows that a 635- nucleotide fragment could be protected from ribonuclease digestion, indicating that dicistronic mRNAs con- region, mediated high levels of translation of the second taining intact intercistronic spacer regions were present LUC cistron (Table 2). Also, uncapped AP2 RNAs medi- at that time after transfection. ated high levels of translation of LUC, but not of CAT, suggesting that the second cistron can be translated independently of the first cistron. However, exon E (E), Translation of the second cistron in dicistronic exon C (C), inverted exons D and E (I~), or DE se- mRNAs is due to internal ribosome binding quences containing an upstream AUG codon (see Fig. 3) To determine whether the second cistron in the dicis- did not direct translation of LUC when present in the tronic mRNA is translated by ribosomal readthrough or ICS region. The entire 5' NCR of Antp P2 (exons C, D, by internal ribosome binding, we constructed a dicis- and E) again mediated translation of the second LUC tronic vector containing exon DE sequences in the ICS cistron most efficiently, but exon D alone was clearly but also bearing an inverted repeat DNA element up- sufficient to mediate translation of LUC. Thus, these stream of the CAT-coding region. After transfection of results exclude the possibilty that cryptic promoter ele- this DNA into cultured cells, the transcript produced ments present in the first cistron or in the ICS region in should contain a stable RNA hairpin in the 5' NCR of the DNA transfection experiments could have produced CAT. Because stable RNA hairpins in 5' NCRs are functionally monocistronic mRNAs. known to inhibit translation, the CAT cistron of this There are three possible mechanisms by which the mRNA should be poorly translated, as we have shown 252-nucleotide RNA sequence (exon D) of the 5' NCR of previously (Macejak and Sarnow 1991). Antp P2, when present as an ICS, could stimulate trans- We reasoned that if exon DE sequences mediate translation of the second cistron. First, a nuclease-sensitive lation of LUC by internal ribosome binding, an RNA site in the ICS could allow the production of functionally hairpin structure upstream of CAT, to inhibit the trans- monocistronic mRNAs. Second, the 5' NCR could pro- lation of CAT, should not affect the translation of LUC. vide RNA structures or sequences that mediate In contrast, if translation of LUC in dicistronic con- readthrough of ribosomes from the first (CAT) to the structs is accomplished by ribosomal readthrough, sec- second LUC cistron. Alternatively, the Antp sequences could provide an IRES element for the LUC cistron (Pel- letier and Sonenberg 1988; Jang et al. 1989). Table 2. Translation of dicistronic mRNAs after direct transfection of RNA molecules into cultured D. melanogaster Intact dicistronic 5' CAT/exon DE/LUC 3' mRNA SL2 cells is present in transfected SL2 cells Chloramphenicol We analyzed the integrity of dicistronic mRNAs ex- conversion/106 LUC light pressed from pSVnCAT/exon DE/LUC-transfected SL2 RNA cells 1%1 units/10 6 cells cells. Briefly, SL2 cells were transfected with pSVACAT/ AP2 68 -+ 2 16,000 ± 2,000 exons DE/LUC containing an inverted repeat upstream D~ 67 -+ 3 7,000 ± 1,000 of CAT, which is predicted to result in the formation of D 49 +- 5 3,500 --- 500 an RNA hairpin structure in the 5' NCR of CAT, as E 49 - 2 240 ± 200 discussed in the next section. At 48 hr after transfection, C 30-+2 300+- 100 polyadenylate-containing RNA was prepared and was ex- 1~ 60 + 3 240 ± 40 amined by a ribonuclease protection assay (Zinn et al. D-)E + AUG 66 -+ 3 170 -+ 60 1983). A 711-nucleotide radiolabeled RNA probe (Fig. Uncapped AP2 9 13,600 4A) was hybridized to oligo(dT)-selected mRNA ob- All dicistronic mRNAs contained a mTGpppG cap structure, tained from transfected lysates, and an -635-nucleotide- except uncapped AP2. Structural features of dicistronic mRNAs long RNA was protected from nuclease degradation {Fig. are shown in Fig. 3. Experiments were repeated three times, 4B, lane 4), demonstrating that the dicistronic mRNA except the transfection with uncapped AP2 {1 x }.

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Internal ribosome binding of Antp mRNA

A Figure 4. Ribonuclease protection analysis of dicistronic N X 1140 450 454 CAT/exon DE/LUC mRNA containing an RNA hairpin up- CAT ' LUC stream of the CAT-coding region. (A) Diagram of dicistronic

3,/ 635 5' CAT/exon DE/LUC mRNA hybridized to antisense RNA probe. Uniformly 32p-labeled RNA probe (771 nucleotides), PROBE[ 711 ntl complementary to 635 nucleotides of the dicistronic mRNA and corresponding to the plasmid DNA between the NcoI site (N) in CAT and the XbaI site (X) in LUC, was synthesized in ! 2 3 4 ! 2 3 4 5 6 ? vitro. The ICS region containing exons D and E is 450 nucleotides in length. (B) Ribonuclease protection analysis of dicis- B C tronic mRNAs obtained from SL2 extracts, transfected with pSVA hairpin CAT/exon DE/LUC plasmids. Polyadenylate-containing mRNAs were isolated from transfected SL2 cell extracts and treated as indicated in Materials and methods. The reaction products were analyzed in denaturing polyacrylamide gels. A 711 -- composite autoradiograph of the gel is shown. Lanes 1-3 were 711 - 635 - exposed for 14 hr; lane 4, for 4 days. Note that the gel displaying 635 - 600- lanes 1-3 was left in developing solution longer than the gel 600- 521 - showing lane 4. (Lane I) RNA probe alone; (lane 2} RNA probe after ribonuclease treatment; (lane 3) RNA probe hybridized to total RNA isolated from mock-transfected SL2 cells; (lane 4) RNA probe hybridized to poly(A)-containing RNA isolated from cell extracts, transfected with pSVnCAT/exon DE/LUC containing an RNA hairpin upstream of the CAT-coding region. The migration of in vitro-synthesized, labeled RNA molecules of known length (in nucleotides) is shown at left. (C) Ribonu- clease protection analysis of dicistronic mRNAs obtained from COS or SL2 cell extracts. COS cells were transfected with pSVA hairpin CAT/exon DE/LUC plasmids; alternatively, in vitro- synthesized, gel-purified dicistronic mRNAs were transfected into SL2 cells. Cell extracts were prepared and RNAs were analyzed by ribonuclease protection assays, as described for B. A composite autoradiograph (lanes 1-4 were exposed for 14 hr; lanes 5-7 for 30 hrl of the gel is shown. (Lane 1} RNA probe 150 - 150 - alone; (lane 2) RNA probe after nuclease treatment; (lane 3) RNA probe hybridized to mRNA isolated from transfected COS extracts; (lane 4) no sample; (lane 5) RNA probe hybridized to RNA isolated from transfected SL2 extracts; (lanes 6,7) in vitro- synthesized, labeled RNA molecules of known length. The sizes (nucleotidesl of the marker RNAs are indicated at left. The RNA species in lane 5, migrating below the 600-nucleotide marker RNA, resulted from ribonuclease cleavage in an AU-rich region in the CAT gene (Macejak and Samow 1991). This RNA species can also be seen in lane 3 after longer exposure of this part of the gel.

ond cistron translation should be inhibited by the RNA in mammalian cells independent of an intact eIF-4F, hairpin at the 5' end of the first cistron. Thus, we tested then intact dicistronic 5' CAT/exon DE/LUC 3' mRNA whether dicistronic 5' (RNA hairpin) CAT/exon DE/ should be associated with polysomes in poliovirus-in- LUC 3' mRNAs are associated with polysomes under fected mammalian cells. conditions where no ribosome binding would occur at COS cells were transfected with pSVACAT/exons DE/ the 5' ends of the mRNAs. Such a condition can be mim- LUC containing an RNA hairpin upstream of CAT. At icked by infection of cells with poliovirus. Infection of 48 hr after transfection, cells were either mock infected mammalian cells with poliovirus is known to result in or infected with poliovirus. Four hours later, some of the the dramatic inhibition of host cell protein synthesis (for cells from both treatments were pulse labeled with review, see Sonenberg 1987). This inhibition is the result ['~SS]methionine, and the labeled proteins were analyzed of the proteolytic cleavage of the p220 component of the in SDS-polyacrylamide gels to confirm the efficiency of cap-binding protein complex eIF-4F (Etchison et al. the viral infection. Translation of cellular mRNAs was 1982). As a consequence, it is thought that the 43S ter- drastically inhibited in infected cells, and only known nary complex cannot be recruited to the 5' end of capped viral polypeptides IRueckert and Wimmer 1984) were cellular mRNAs, and cellular mRNAs are not associated synthesized during the labeling period, indicating that with polysomes in poliovirus-infected cells. However, cap-independent translation of mRNAs could be selec- poliovirus mRNA, translated by internal ribosome bind- tively monitored at that time (not shown). Polysomal ing, is found to be associated with polysomes when eIF- fractions were prepared from the unlabeled remainder of 4F is not intact. If exon DE-containing mRNAs, like the cells. The optical density profile of extracts prepared those containing the poliovirus IRES, can be translated from mock-infected and poliovirus-infected lysates after

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OH et al. chromatography on Bio-Gel A15m resin {Calzone et al. both CAT and LUC RNA probes hybridized to a similar- 1982) was used to analyze polysomes {Fig. 5A). Polyade- size transcript, ~3700 nucleotides in length, demon- nylate-containing mRNA was isolated from polysomal strating that the polysomal RNA fraction was intact. In fraction A and nonpolysomal fraction B (Calzone et al. mock-infected lysates, most of the dicistronic mRNA 1982), separated on a formaldehyde-containing agarose was associated with polysomes (Fig 5B, fraction A, lanes gel, and transferred to nitrocellulose. Two identical ni- M). In lysates from poliovirus-infected cells, most of the trocellulose blots were prepared and incubated individ- dicistronic mRNA was again present in the polysomal ually with radioactive RNA probes complementary to fractions. Association of intact dicistronic mRNA con- CAT and LUC, respectively. As can be seen in Figure 5B, taining both CAT and LUC sequences with polysomes in

1 2 3 4 5 6 7 8 C A

711- 635- A B -600 -521 495-

E 7,

~3 ;J

-150

I " I ' I " I 0 10 20 30 40 50 Fract ions

Figure 5. Association of dicistronic mRNAs with polysomes in mock-infected and B poliovirus-infected COS cells. (A) Optical density profiles of fractions obtained from M I M I A 8 A B A B A B separation of cellular lysates by Bio-Gel Al5m chromatography (Calzone et al. 1982). I I P I J I I I Both polysomal {fraction A] and nonpolysomal fractions (fraction B), from mock-infected {El) and poliovims-infected (I~) lysates are indicated. {B) Northern blot analysis of mRNAs isolated from fractions A and B after Bio-Gel A15m chromatography. Polyade- nylate-containing RNA was separated on an agarose gel and transferred to nitrocellulose paper. Two blots were prepared and hybridized with uniformly aZP-labeled ant{sense [. :i :i - CAT and ant{sense LUC probes. The positions of unlabeled 18S [1900 nucleotides) and il . 28S (4800 nucleotides) rRNA, present in the original agarose gel, are indicated. (C) Ri- -28S bonuclease protection analysis. Polysomal RNA was analyzed in denaturing polyacrylamide gels. An autoradiograph of such a gel is shown. (Lane I ) RNA probe; (lane 21 RNA -18S probe after ribonuclease treatment; (lane 31 RNA probe hybridized to mRNA from polysomal fraction A (B; see I); {lane 4), RNA probe hybridized to RNA from pSVA exon , ii DE/LUC-transfected lysates, containing monocistronic DE-LUC RNA; {lane 5) RNA probe hybridized to total RNA isolated from mock-transfected COS cells. Lanes 6-8 display the position of in vitro-synthesized, labeled RNA molecules of known length. Sizes of RNAs are indicated in nucleotides. CAT LUC

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Internal ribosome binding of An~ mRNA poliovirus-infected cells is a strong indication that the ond cistron. Instead, sequences in exon D confer to second LUC cistron was translated independently of the RNAs the ability to be translated by internal ribosome first CAT cistron. binding. To provide further evidence that the polysome-associated dicistronic mRNA was intact, the integrity of the The 5'-terminal sequences of noncoding exon D sequences between the translational termination codon are highly conserved between Drosophila species in CAT and the translation initiation codon in LUC was examined by a ribonuclease protection assay, by use of Noncoding exon D is sufficient to mediate internal ribo- the RNA probe shown in Figure 4A. An -635-nucle- some binding. The sequence is present in transcripts ini- otide-long RNA was protected from nuclease degrada- tiated at both the P1 and P2 promoters, indicating that tion by hybridization to mRNA from fraction A (Fig. 5C, all Antp mRNAs could use an internal ribosome-binding lane 3), demonstrating that the polysome-associated di- mechanism for translational initiation. The first 55 nu- cistronic mRNA was intact. Furthermore, no protection cleotides of exon D (252 nucleotidesl are highly con- was observed when mRNAs from nontransfected cells served among D. melanogaster, D. virilis, and D. subob- were used in the protection assay (lane 5). Thus, func- scura (Hooper et al. 1992). In contrast, the sequences tionally dicistronic mRNA was associated with poly- flanking this element are highly variable. The common somes under conditions in which the bulk of cellular ancestor of D. melanogaster and D. virilis is estimated to mRNA was not translated. Although COS cells are not a have existed 60 million years ago (Beverley and Wilson normal cell line for Antp gene expression, it is clear from 1982), and the ancestor of D. melanogaster and D. sub- these experiments that exon DE sequences can direct obscura, 20--50 million years ago. The sequences may translation of the second cistron, independent of the have been conserved because they serve important func- translation of the first cistron, in dicistronic RNAs. tions in regulating transcription or translation. Prelimi- nary experiments have shown that the first 55 nucleotides of exon D can mediate internal ribosome binding Discussion when placed into the ICS region of dicistronic mRNAs Sequences in exon D of the Antp gene of D. melano- (S.-K. OH and P. Sarnow, unpubl.), indicating that this gaster were shown to mediate initiation of translation by 55-nucleotide sequence is one of the smallest IRES ele- providing an IRES in cultured SL2 cells. Translation of ments reported so far. LUC from dicistronic mRNAs, in which the LUC-coding region was the second cistron, was demonstrated by en- Many Drosophila mRNAs contain complex 5' NCRs zymatic assays following DNA transfection (Table 1) and RNA transfection {Table 2). Several lines of evidence in- We would like to determine whether translational initi- dicate that the 5' NCR of Antp P2 mRNA mediates in- ation by internal ribosome binding may be commonly ternal ribosome binding, as opposed to promoting used in Drosophila mRNAs. Up to 20% (38 of 192; readthrough of ribosomes from the CAT to the LUC cis- Cavener and Ray 1991; D.R. Cavener, pers. comm.) of tron in cultured ceils. First, dicistronic mRNAs contain- identified Drosophila cDNAs predict long 5' NCRs with ing the 157-nucleotide-long exon E as an ICS did not multiple upstream AUG codons. In contrast, only mediate translation of the second cistron (Table 2}. Be- 5-10% of vertebrate mRNAs have upstream AUG cause exon E is smaller than combined exons DE and is codons (Kozak 1989, 1991). Interestingly, vertebrate devoid of AUG triplets, one would predict enhanced mRNAs with unusual 5' NCRs belong predominately to rather than blocked translation of the second cistron by genes involved in growth control (Kozak 1991), indicat- a termination/reinitiation event. Second, uncapped di- ing that certain mRNAs, whose translation products are cistronic mRNAs supported translation of the second, involved in modulating growth and development, may but not the first, cistron, demonstrating the independent be subject to translational control. Most Drosophila translation of the two cistrons. A termination-reinitia- genes have been cloned on the basis of mutant pheno- tion mechanism would predict a coupled translation of types, suggesting that many are regulators. Regulatory both cistrons. Furthermore, that uncapped dicistronic genes may be subject to a more precise regulation than, mRNAs failed to be translated to produce CAT suggests for example, housekeeping genes. that the translation of LUC was unlikely to be mediated Several features of the nucleotide sequences of key reg- by a scanning mechanism operating on broken dicis- ulatory Drosophila mRNAs with long 5' NCRs are listed tronic mRNAs within the cell. A third and most impor- in Table 3. The transcription initiation sites for the tant line of evidence for internal ribosome binding is that mRNAs shown have been determined, excluding the intact dicistronic 5' CAT/exon DE/LUC 3' mRNAs con- possibility that the cDNA sequence was incomplete. taining an RNA hairpin upstream of CAT were associ- Most of these 5' NCRs contain hundreds of nucleotides, ated with polysomes in poliovirus-infected cells, sub- in contrast to the 40- to 80-nucleotide-long leader of stantiating the fact that the second LUC cistron could be most Drosophila mRNAs; other exceptions are the 110- translated independently of the first CAT cistron. Thus, to 250-nucleotide 5' NCRs of heat shock mRNAs {Ingo- the data are not consistent with a model in which se- lia and Craig 1981). Furthermore, most of the noncoding quences present in exons D (but not in E) facilitated ef- regions summarized in Table 3, contain multiple up- ficient ribosomal readthrough from the first to the sec- stream AUG codons, many of them located within se-

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Table 3. Structural feature of 5'-leader sequences of Drosophila mRNAs Length Upstream Consensus Stop Embryonic mRNA (nucleotides) AUGs AUGs a codons References Abdominal-B {pill89) 2800 31 13 108 DeLorenzi et al. (1988) Abdominal-B (pH200) 494 1 0 22 DeLorenzi et al. (1988) Antennapedia P1 1512 8 2 78 Laughon et al. (1986) Stroeher et al. {1986) Antennapedia P2 1727 15 6 72 Laughon et al. (1986) Stroeher et al. (1986) bicoid 171 2 1 5 Berleth et al. (1988) caudal (maternal) 275 3 2 11 Mlodzik et al. {19871 caudal {zygotic) 461 4 2 21 Mlodzik et al. (1987) Deformed 490 4 0 23 Regulski et al. (1987) E74 A 1891 17 4 95 Burtis et al. (1990) E74 B 794 6 4 34 Burtis et al. (1990) fushi tarazu 120 1 0 3 Laughon and Scott (1984) hunchback 511 1 0 13 Tautz et al. (1987) {3.2 kb transcript) Krfippel 185 2 2 9 Rosenberg et al. {1986) labial 239 0 0 11 Diederich et al. (1989) Mlodzik et al. (1988) nanos 262 1 0 11 Wang and Lehmann ( 1991 ) Notch 799 7 1 21 Kidd et al. (1986) Sex combs reduced 626 5 1 23 Lemotte et al. (1989) sevenless 824 10 4 22 Bowtell et al. (1988) terminus 155 0 0 6 Baldarelli et al. (1988) Ultrabithorax 965 2 0 47 Komfeld et al. (1989) The 5' ends of the mRNAs have been determined experimentally. aDrosophila consensus sequence for translation initiation is C/AAAA/c AUGN (Cavener 1987).

quences that are predicted to be optimal for translational ing nuclei. Translation by the cap-dependent scanning start sites in Drosophila (Cavener 1987). Again, this model is known to be inhibited during mitosis in mam- raises the question of how these mRNAs can be trans- malian cells as a result of the underphosphorylation of lated efficiently according to the scanning model of the cap-binding protein eIF-4E (Bonneau and Sonenberg translation initiation (Kozak 1989, 1991), if they are in- 1987; Huang and Schneider 1991). If this is also the case deed translated efficiently. Recently, we have found that in Drosophila cells, it is not clear how early embryonic the 5' NCR of Uhrabithorax (Kornfeld et al. 1989) can Drosophila mRNAs, such as bicoid or nanos, can be also confer internal ribosome binding to a heterologous translated efficiently in the syncytial embryo. A cap-in- mRNA in cultured cells (S. Hoover, A. Rudie, and P. dependent translational mechanism could, in principle, Sarnow, unpubl.). That the 5' NCRs of two homeotic be used by mRNAs that need to be translated during mRNAs can initiate translation by internal ribosome mitosis or at times when the cap-binding protein com- binding raises the possibility that this mechanism may plex is not functional (Bonneau and Sonenberg 1987; be commonly used in Drosophila. Huang and Schneider 1991). One mechanism of cap-independent translation is translation by internal ribosome binding. Translational initiation by internal ribosome Is there any evidence for post-transcriptional control binding in Drosophila of Antp expression? It has been observed that Antp Transcriptional regulation of the zygotic genome at the mRNA and protein do not always appear at the same cellular blastoderm stage has been studied extensively. time during embryonic development (Carroll 1986; Ber- In contrast, only a few examples of translational regula- mingham 1989), implying at least some post-transcription of early embryonic mRNAs have been reported tional regulation. Such translational regulation could be (Macdonald and Struhl 1986; Mlodzik and Gehring under the control of factors that may be under temporal 1987), with the exception of translational control in control themselves. To test whether internal ribosome heat-shocked Drosophila embryos (Lindquist and Craig binding occurs in the fly, we are in the process of con- 1988; Maroto and Sierra 1988). structing transgenic flies expressing dicistronic mRNAs It is not known how many maternal and early zygotic containing exons D and E as an ICS. Monitoring the pro- mRNAs are translated before and during the cellular tein production of the first and second cistron, respec- blastoderm stage. At these times, the embryo is still a tively, will reveal whether this mechanism is used in the single-cell syncytium that contains many rapidly divid- organism.

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Internal ribosome binding of Antp mRNA

Materials and methods leting the SalI-AfllItsso fragment from pSVACAT/APJLUC: Following digestion with SalI and AflII, the single-stranded ends Cell culture and transfections of the large vector fragment were removed with mung bean D. melanogaster SL2 cells {Schneider 1972) were grown in nuclease and the plasmid was recircularized. Plasmid pSVA- Schneider's Drosophila medium supplemented with 17% fetal CAT/exon C/LUC was obtained by deleting a ClaI-NcoI frag- bovine serum. SL2 cells were passaged every 2-3 days and main- ment {see above) from plasmid pSVACAT/AP2/LUC. In addi- tained at a density of 1 x 10 6 tO 8 X 10 6 cells/ml at 25°C. COS-1 tion, plasmids were constructed that contained the promoter for cells (obtained from Robert Schneider, New York University, T7 RNA polymerase upstream of the CAT cistron. First, a 45-bp New York) were grown in Dulbecco's modified Eagle medium fragment containing 30 adjacent adenosine residues was excised (DMEM) supplemented with 10% bovine serum. Wild-type Ma- from pSP64 [poly(A)] (Promega) and ligated into plasmid honey type-1 poliovirus was isolated from HeLa cells trans- pGEM-4 (Promega} at unique Sinai and EcoRI sites to create fected with a pSV2 poliovirus plasmid as described (Simoes and pGem-4 CAn) (constructed by L. Najita, UCHSC, Denver). To Sarnow 1991). obtain T7-LUC (An)(constructed by S. Hambidge, UCHSC, Cells were transfected with 5 lag of plasmid DNA per plate Denver), the 296-bp XbaI-NheI fragment from pGem-4 {An) was with calcium phosphate, as described (Ausubel et al. 1989), ex- ligated into the XbaI-NheI sites located downstream of the cept that the "glycerol shock" was omitted for SL2 cells. Cyto- LUC-coding region in pGEM-LUC (Macejak et al. 1990). Fi- plasmic cell extracts were prepared 48 hr after transfection, and nally, plasmid T7 CAT/ICS/LUC was obtained by replacing the CAT (Ausubel et al. 19891 and LUC (De Wet et al. 1987) activity HindIII-BstEII fragment from TT-LUC CAn) with HindIII-BstEII were measured in the same extracts. The amount of acetylated fragments from various pSVACAT/ICS/LUC plasmids. In vitro- chloramphenicol was measured by computing densitometry synthesized RNAs were purified in low-melting agarose gels in (Molecular Dynamics, Sunnyvale, CA) of X-ray films. CAT ac- some experiments to exclude the possibility that broken RNAs tivity is displayed as the percent of [14C]chloramphenicol con- were transfected into cells. Transfection into SL2 cells of puri- version to monoacetylated chloramphenicol. fied and nonpurified RNAs yielded similar amounts of LUC, Transfection of SL2 cells with RNA was initiated by mixing excluding the possibility of promoter-like sequences in exon D. 10 tzg of in vitro-synthesized, m7GpppG-capped RNA with a lipofectin solution (Betheda Research Laboratories). The mix- ture was then added to cells as described (Simoes and Sarnow Isolation and analysis of RNA 1991). Extracts were prepared 19 hr after transfection, and CAT Polysome-associated RNA was isolated from COS cells that and LUC activities were measured. were transfected with plasmid pSVACAT/exon DE/LUC containing an RNA hairpin upstream of the CAT-coding region. Cells were mock infected or infected with poliovirus (m.o.i. of Construction of plasmids 100) at 48 hr after transfection, as described {Simoes and Sarnow 1991). Four hours later, cycloheximide was added to the culture The construction of plasmid pSVACAT/ICS/LUC has been de- medium (100 lag/ml) for 15 min to inhibit ribosome movement, scribed {Macejak et al. 1990). Plasmid pSVACAT/AP2/LUC con- lysates were prepared, and polysomes were separated from the tains a cDNA of the entire 1730-bp 5'-noncoding region (see Fig. nonpolysomal fraction by chromatography on Bio-Gel Al5m as 1) of Antp P2 mRNA inserted between the CAT- and LUC- described {Calzone et al. 1982). Polyadenylate-containing coding region. Briefly, the 5'-noncoding region of Antp P2, con- mRNAs from each fraction were isolated by oligo(dT) affinity taining SalI and NcoI restriction sites at the 5' and 3' termini, chromatography and analyzed by Northern blot or by a ribonu- respectively, was obtained by polymerase chain reaction (Ausu- clease protection assay (Zinn et al. 1983). bel et al. 1989). The YE10 cDNA of Antp (Laughon et al. 1986) was used as template for the amplification reaction. The Antp P2 cDNA was then ligated by the SalI and NcoI sites at the termini of the amplified fragment into the ICS region of pSV A- Acknowledgments CAT/ICS/LUC (Fig. 2A). An ATG codon provided by sequences P.S. thanks John Bermingham for a stimulating introduction to derived from the NcoI restriction site was used to initiate trans- the "Drosophila world." We are grateful to Karla Kirkegaard for lation of LUC. Plasmid pSVA CAT/exon DE/LUC contains a continued discussions throughout this work and for comments 448-bp ClaI-NcoI fragment spanning exons D and E (Laughon et on the manuscript. We thank Joan Hooper and Manuel Alonso al. 19861 in the ICS region, pSVACAT/exon ED/LUC, contain- for communicating the Antp sequence comparison. P.S. ac- ing exons DE in inverted orientation in the ICS, was con- knowledges the receipt of a Faculty Research Award (FRA 413) structed by first repairing the ends of the 448-bp ClaI-NcoI from the American Cancer Society. M.P.S. was an investigator fragment with Klenow enzyme. The blunt-ended fragment was of the Howard Hughes Medical Institute during the early part of then ligated to pSVACAT/ICS/LUC, which had been linearized this work. This work was supported in part by a grant from the with SalI, and the ends were repaired with Klenow polymerase. Lucille P. Markey Charitable Trust, The Council for Tobacco Insertion of the ClaI-NcoI fragment in one orientation pro- Rescarch (U.S.A.) (P.S.), and grants AI 25105, AG 07347 (P.S.), duced pSVACAT/exon ED/LUC. Insertion of the fragment in and 18163 (M.P.S.) from the National Institutes of Health. the other orientation produced pSVACAT/exon DE + AUG/ The publication costs of this article were defrayed in part by LUC, containing an ATG codon 36 bp upstream of the initiation payment of page charges. This article must therefore be hereby codon used to express LUC. A derivative of pSV^CAT/exon marked "advertisement" in accordance with 18 USC section DE/LUC was constructed that contained an RNA hairpin in the 1734 solely to indicate this fact. 5'-noncoding region of CAT (Macejak and Sarnow 1991). Plas- mid pSVACAT/exon D/LUC was obtained by deleting a 180-bp AflIIlsso-NcoI fragment (exon E) from pSVACAT/exon DE/ References LUG: Following digestion with AflII and NcoI, the ends of the large vector fragment were repaired with Klenow and recircu- Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seid- larized. Plasmid pSVACAT/exon E/LUC was constructed by de- man, J.A. Smith, and K. Struhl. 1989. Current protocols in

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Homeotic gene Antennapedia mRNA contains 5'-noncoding sequences that confer translational initiation by internal ribosome binding.

S K Oh, M P Scott and P Sarnow

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