Proc. Natl. Acad. Sci. USAVol. 86, pp. 3997-4001, June 1989Biochemistry

An element downstream of the cap site is required for transcriptionof the gene encoding mouse ribosomal protein L32

(promoter/transcription factor/internal element/vector compensation)

RODRIGO MOURA-NETO*, KALIN P. DUDOVt, AND ROBERT P. PERRYInstitute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111

Contributed by Robert P. Perry, February 27, 1989

ABSTRACT To identify the elements that regulate tran-scription of the mouse gene encoding ribosomal protein L32(rpL32), we transfected monkey kidney (COS or CV-1) cellswith mutants bearing progressive 5' deletions or an internaldeletion in exon I and measured their transient expression byS1 nuclease protection analysis. When the mutant genes weretested in the vector 1rSVHSplac, which contains a short seg-ment of the on region of simian virus 40, maximum expressionwas observed with as little as 36 base pairs of 5' flankingsequence, and the mutant bearing the exon I deletion wasexpressed very efficiently. However, when the genes weretested in a simple prokaryotic (pUC) vector, the expression wasincreased 3- to 4-fold by sequences between -36 and - 159, andthe exon I segment was absolutely required for expression. Gelmobility-shift and methylation interference analyses revealedthat a nuclear factor specifically binds to a GGCTGCCATCsequence within this exon I segment. These results, takentogether with other recent findings, indicate that the elementsinvolved in transcriptional regulation of the rpL32 gene aredistributed over a 200-base-pair region that spans the cap site.The contributions of some of these elements are apparentlymasked in the presence of simian virus 40 on-region elements.

The gene encoding the mouse ribosomal protein L32 (rpL32),like those encoding other mouse ribosomal proteins, has apromoter region with several distinctive features (1). Theseinclude the lack of a canonical TATA box, a transcriptionalstart (cap) site that is embedded in a pure pyrimidine tract,and sequence blocks of high C+G content including a re-markably high frequency of unmethylated CpG doublets. Asa housekeeping gene encoding a relatively abundant protein,it needs to be efficiently expressed in all types of tissue. Theprinciples that govern the transcription of this type of genehave yet to be clearly defined.

In an earlier study designed to identify the transcriptionalregulatory elements of the rpL32 gene, we constructed a setof 5' deletion mutants and examined their transient expres-sion in transfected primate cells (1). When these constructswere inserted into the expression vector 7rSVHSplac, whichcontains a short enhancerless segment of the simian virus 40(SV40) ori region (2), maximum levels of expression wereobserved with as little as 36 base pairs (bp) of rpL32 5'flanking sequence. Although this brief stretch of sequencewas also found to be sufficient for the expression of an rpL32gene inserted into a pUC vector, we did not accuratelycompare the level of activity with that of counterpartscontaining additional rpL32 upstream sequence. Moreover,in this initial study, we did not investigate the importance ofinternal sequences for rpL32 expression.

In the present series of experiments, the relative expres-sion of the various 5' deletion mutants and of a gene that has

an internal deletion in the first exon has been quantitativelyevaluated in both vector systems. With constructs insertedinto the pUC vector, we observe a significant increase in thelevel of expression when rpL32 sequences upstream of -36are present. Furthermore, expression is absolutely depen-dent on a segment of the first exon that contains a nuclearfactor binding site. Interestingly, the contributions of theseupstream and exonic elements are masked in the rSVHSvector, apparently due to a compensatory effect by SV40ori-region sequences. These findings, together with otherrecent studies (3, 4), indicate that transcription of the rpL32gene is regulated by a complex array of elements distributedon both sides of the cap site.

MATERIALS AND METHODSPlasmid Construction. The 5' deletion mutants of rpL32

inserted into the BamHI site of the frSVHSplac vector(irSA5' series) were constructed by BAL-31 nuclease resec-tion as described (1). These mutants, designated frS(-109),irS(-79), frS(-68), 7rS(-36), IrS(-13), and frS(+ 11) accord-ing to the 5' limit of retained rpL32 sequence, extendedthrough the remainder of the rpL32 gene to a position 300 bpbeyond the poly(A) site (Fig. 1A). For the internal deletionmutant AE1, a BAL-31-resected gene beginning at position+46 was joined by means of a BamHI linker to an rpL32fragment that extends from the Nru I site at -456 to the HaeII site at + 11. The 6-bp linker which replaces the 34-bp exonI sequence from position +12 to +45 restores one correctnucleotide at + 12 so that the actual deleted sequence is + 13to +45. In irSAE,, this construct was inserted between thePvu II and Xba I sites of frSVHSplac. All inserts wereoriented so that the 5' end of the rpL32 gene is adjacent to thesupF segment of the ISVHS vector (Fig. 1B).For transfer of A5' mutants to the pUC vector (pA5' series),

the inserts were excised from the irSA5' constructs byBamHI digestion, purified by agarose gel electrophoresis,and inserted into BamHI-digested pUC18. To construct thepAE1 mutant, an EcoRI-BamHI fragment containing thesequence -456 to + 11 and a BamHI fragment containing thesequence +46 to +3568 were excised from irSAE1 andsequentially inserted into the corresponding sites of pUC18.The p(-159) construct was described previously (1). Forp(-316), a fragment encompassing the -316 to +77 sequencewas excised from a subclone ofRPL32-3A (5) and substitutedfor the -159 to +77 sequence of p(-159). The recombinantpS16 contains a 2.5-kbp BamHI-Sac I fragment encompass-ing the entire rpS16 gene (6) and including 400 bp and 160 bpof 5' and 3' flanking sequence, respectively.

Abbreviations: rpL32 and rpS16, ribosomal proteins L32 and S16;SV40, simian virus 40.*Present address: Institute of Biology, Federal University of Rio DeJaneiro, Rio De Janeiro, RJ 21914, Brazil.tPresent address: Institute of Molecular Biology, Bulgarian Acad-emy of Science, Sofia 11113, Bulgaria.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. Diagram of the rpL32 gene and the constructs used in the transfection experiments. (A) The gene, drawn to scale with exons asfilled boxes, introns and flanking regions as thin lines, and the site of transcriptional initiation (cap site) indicated by a bent arrow. The 5' endis enlarged with the locations of the 5' deletions and AEI deletion [A] marked by vertical arrows. (B) The expression vectors 1TSVHSplac andpUC18, showing the sites of insertion of the rpL32 mutant genes with the 5' -. 3' orientation indicated by a horizontal arrow. MCS, multiplecloning site; B, BamHI; H, Hindll; N, Nco I; P, Pvu II; R, EcoRI; X, Xba I. (C) The 243-bp probe used for the S1 nuclease protectionexperiments includes 5' flanking sequence (thin line), exon I sequence (hatched bar), and a portion ofexon II sequence (open bar). The fragmentsprotected by properly initiated and spliced transcripts of genes with an intact exon I (83 nucleotides) and the +13 to +45 AE1 mutant (39nucleotides) are diagramed below. A, Acc I; Sa, Sau3A.

Cell Culture and DNA Transfection. Monkey kidney cells(COS-7 and CV-1), obtained from the American Type CultureCollection and maintained in Dulbecco's modified Eagle'smedium (GIBCO) containing 10% fetal bovine serum, weretransfected by the DEAE-dextran (Pharmacia) procedurefollowed by treatment with chloroquine diphosphate (Sigma)(7, 8). Each plate, containing about 106 adherent cells and 5ml of medium, was transfected with 5 gg each of plasmidscontaining rpL32 and rpS16 genes.RNA Preparation and Analysis. For transient-expression

assays, the cells from three plates were harvested 36-40 hr

after transfection, and total cell RNA, total cytoplasmicRNA, or poly(A)+ cytoplasmic RNA was isolated (9).For S1 nuclease protection analysis, 30-100 Ag of RNA

was hybridized at 460C for 3-6 hr with 2-4 ng of double-stranded, 5'-end-labeled probe in 50 jul of 80% (vol/vol)formamide/0.4 M NaCl/10 mM Pipes, pH 6.5/1 mM EDTA.Prior to hybridization the reactants were incubated for 10 minat 80°C in the absence of salt. The restriction fragments usedas S1 probes were dephosphorylated with calf intestinephosphatase (Boehringer Mannheim) and labeled with ['y-32P]ATP and T4 polynucleotide kinase (Pharmacia) to spe-

t k\ I L;-

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cific activities of 105 cpm/pmol. The 243-bp rpL32 Si probe(Fig. iC) was derived from A123, an rpL32 pseudogenederivative (4) kindly provided by S. Chung. The rpS16 S1probe was a 244-bp Ava II fragment containing 5' flankingsequence and 68 bp ofexon I sequence (6). Total cytoplasmicRNA from untransfected COS cells, or wheat germ tRNA,was used to adjust the total amount of RNA in each sampleto 100 pug. Probe excess was verified by calibration withgraded amounts ofRNA from exponentially growing mouseplasmacytoma cells. A standard sample of mouse RNA wasincluded with each transfection series. After hybridization,samples were diluted with 0.45 ml of S1 buffer (3 mM zincacetate/30 mM sodium acetate, pH 4.5/250 mM NaCl) anddigested with 75 units of S1 nuclease (Pharmacia) for 30 minat 300C. Protected DNA fragments were then analyzed in 8%polyacrylamide gels containing 7 M urea.For Northern blot analysis, 1.0-,tg samples of poly(A)+

RNA were electrophoresed in 1.2% agarose/2 M formalde-hyde gels and then transferred to Nytran filters as describedby the manufacturer (Schleicher & Schuell). Blots werehybridized with nick-translated probes in 50% formamide/0.75 M NaCl/0.75 M sodium citrate, pH 7.0/50 mM sodiumphosphate, pH 6.5/0.1% sodium dodecyl sulfate containingheparin at 500,g/ml and denatured, sonicated salmon spermDNA at 100 ,g/ml. The rpL32 probe was the insert of A123,which contains the entire sequence of rpL32 mRNA. TheirSVHS probe was the complete vector.Gel Retardation and Methylation Interference Analysis.

These analyses were carried out as described (3, 10) with a-36 to +72 fragment from the rpL32 gene and a nuclearextract from S194 mouse plasmacytoma cells.

RESULTSIn our earlier studies, the relative expression of a set of 5'deletion mutants inserted into the irSVHSplac expressionvector (Fig. 1) was assayed by Northern blot analysis ofpoly(A)+ RNA from transfected COS cells. To establishwhether the RNA transcripts produced by these mutants areinitiated at the authentic rpL32 cap site, we carried out asimilar set of experiments using an S1 nuclease protectionassay, as well as a Northern blot analysis, to monitorexpression. The 243-bp SI nuclease probe, which was de-rived from a fusion product of the rpL32 gene and anunmutated rpL32 processed pseudogene (4), consists of 5'flanking sequence, the first exon, and a portion of the secondexon (Fig. iC). It yields an 83-nucleotide fragment whenprotected by a properly spliced transcript that is initiated atthe authentic cap site. In this series of transfection experi-ments we also examined the expression of an internal dele-tion mutant, AE1, which contains ample 5' flanking sequence(456 nucleotides) but lacks about three-fourths of the firstexon (Fig. LA). A properly initiated and spliced transcriptfrom this mutant would protect a 39-nucleotide fragment inthe S1 nuclease assay (see Fig. iC).

In agreement with our previous results, mutant constructscontaining 109, 79, 68, and 36 bp of 5' flanking sequenceproduced equal amounts of properly initiated and splicedRNA, whereas deletions that extend to -13 or +11 com-pletely abolished normal expression. This was demonstratedby the S1 nuclease protection assay (Fig. 2A, lanes 1-7) aswell as a Northern blot analysis (Fig. 2B, lanes 1-5). The -13and +11 constructs produced aberrant transcripts (Fig. 2B,lanes 4 and 5) that were initiated in the expression vector, asindicated by their strong hybridization to a vector probe (Fig.2B, lanes 10 and 11). A small fraction of these transcriptsprotected the S1 probe to the limits of sequence homology(Fig. 2A, lanes 6 and 7); however, most transcripts appearedto be processed so as to exclude this region, possibly by

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FIG. 2. Expression of the rpL32 mutants in the 1rSVHS vectorsystem. (A) S1 nuclease protection assay. Lane M, size markers(Msp I-digested pBR322); lanes 1 and 10, 10 ;kg of mouse cell RNA(i); other lanes, 30 ,ug of RNA from transfected (lanes 2-8) oruntransfected (lane 9) COS cells. Lanes 1-4 and 5-10 are fromdifferent experiments. n, Nucleotides. (B) Northern blot analysis.Cytoplasmic poly(A)+ RNA (1 ,ug) from transfected COS cells wasfractionated by gel electrophoresis, transferred to a nylon sheet, andsequentially hybridized with probes that specifically recognize rpL32sequences (Left) or irSVHS sequences (Right).

splicing from a cryptic site in the vector sequences to rpL32exons III or IV.The internal deletion mutant, AEI, is efficiently expressed

in this vector system. This is evidenced both in the S1nuclease assay, by a substantial yield of the predicted 39-nucleotide resistant fragment (Fig. 2A, lane 8), and in theNorthern blot analysis, by an abundant component that isslightly smaller than normal L32 mRNA (Fig. 2B, lane 6).Given the appropriate size of this RNA component and itslack of vector sequences (Fig. 2B, lane 12), we infer that it isderived from a transcript that is initiated at or near theauthentic rpL32 cap site.To evaluate the possible influence of vector sequences on

the foregoing results, we transferred the set ofrpL32 mutantsto the pUC vector system and reexamined their transientexpression in transfected COS cells. As a control for possiblevariations in transfection efficiency or RNA yield, we mon-itored the expression of an intact rpS16 gene, which wascotransfected with each of the rpL32 mutants. Properlyinitiated transcripts from the rpS16 control gene protect a68-nucleotide segment of the S1 probe used in this assay. Theresults of these experiments were strikingly different fromthose obtained with the irSVHS vector system in two re-spects. First, the progressive deletion of sequences between-159 and -36 resulted in a decrease in activity by a factor of3-4 (Fig. 3A, lanes 3-6, 11, and 12; Table 1). Second, the AE1mutant was completely inactive (Fig. 3A, lane 9). In otherrespects, the results were similar in both vector systems. The-13 and +11 mutants were not expressed (Fig. 3A, lanes 7and 8) and the deletion of sequences upstream of -159 hadno detectable effect on expression (Fig. 3A, lanes 3, 4, 10, and11; Table 1).

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FIG. 3. (A) Expression of the rpL32 mutants in the pUC vectorsystem. S1 nuclease protection assays of RNA produced by rpL32mutants (Upper) and intact rpS16 genes (Lower) cotransfected intoCOS cells. Lane 1, untransfected COS cells; lane 2, 10 ,ug of mousecell RNA (m); lanes 3-12, 100 jug of cytoplasmic RNA from cellstransfected with mutants inserted into the pUC18 vector. Lanes 1-9 and 10-12 are from different experiments. The horizontal lines arereference marks for densitometer scanning. (B) Expression of se-lected rpL32 mutant constructs in CV-1 cells. RNA (150 ,ug) fromuntransfected CV-1 cells (lane 1) or from cells transfected with theindicated mutant constructs (lanes 2-5) was analyzed. Comparablesignals from the cotransfected rpS16 genes within each experimentalseries indicate equivalent transfection efficiencies and RNA yields.n, Nucleotides.

The foregoing results indicate that the contributions ofsequences upstream of -36 and in exon I are masked in theIrSVHS vector system. Since the irSVHS vector replicatesin COS cells, whereas the pUC vector does not, it is ofinterest to know whether this masking effect requires vectorreplication. To this end we compared the expression of theirS(-79), frS(-36), frS(A&EI), and p(-79) constructs in CV-1cells, the COS cell parental line, which does not produceSV40 large tumor (T) antigen and therefore is nonpermissivefor irSVHS replication. Although the vector influence inCV-1 cells was less than in COS cells, it was neverthelesssubstantial (Fig. 3B, Table 2). The expression of the irS(-36)and irS(AE,) constructs in CV-1 cells was 73% and 44% ofmaximum, respectively, compared to 28% and 0% for thecorresponding pUC constructs in COS cells (Table 1). Thus,

Table 1. Relative expression in COS cells of rpL32 mutant genesinserted into the 7rSVHS and pUC vectors

Expression, %Mutant ITSVHS pUC-316 100-159 100 100-109 100-79 100 82-68 100 42-36 100 28-13 0 0+11 0 0AE1 82 0

Autoradiographs from S1 nuclease protection assays such as thoseshown in Figs. 2 and 3 were scanned with a densitometer. Theintensity of the 83-nucleotide protected fragment (or 39-nucleotidefragment for the AEl mutant) is expressed as a percentage of themaximum value for each series. The results of independent trans-fection experiments for any particular mutant agreed to within 10oof the representative values given here. The sensitivity of the assayswas such that any expression >2% of maximum would have beendetected readily.

Table 2. Expression of rpL32 constructs in replicating andnonreplicating vector-host systems

Expression relative to wrS(-79), %Cell line 'S(-36) irS(AEI) p(-79)CV-1 73 44 61COS 100 82 7.7

Data from S1 nuclease protection assays were analyzed as in Table1. The large difference in the p(-79)/rS(-79) expression ratio inCVr1 vs. COS cells confirms the differential replicative behavior ofthe 1rS vector in the two cell lines.

7TSVHS vector sequences can compensate for the loss ofrpL32 sequences in the absence of plasmid replication. Theapparent magnification of this effect in COS cells suggeststhat the vector contribution might be enhanced by replicationor by the interaction of T antigen with SV40 ori-regionsequences.The fact that vector elements known to be involved in

transcriptional regulation can compensate for the lack of theexon I segment suggests that this segment has a transcrip-tional role in rpL32 expression (see Discussion). On the basisof this conjecture, we investigated whether the exon I seg-ment contains a binding site for a nuclear factor. Gel retar-dation analysis revealed that there is indeed a factor-bindingsite in this region (Fig. 4A; also see ref. 3). Furthermore, thespecificity of this binding was verified by appropriate com-petition experiments with homologous and heterologousDNA fragments (3). To localize the binding site more pre-

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FIG. 4. Identification of the nuclear factor-binding site in exonI of rpL32. (A) Gel retardation analysis of a -36 to +72 DNAfragment incubated without (-) or with (+) nuclear extract. Thebands corresponding to bound and free fragment are designated Band F, respectively. (B) Methylation interference analysis. Lanes Fand B, sense and antisense DNA strands of the bound and freefragments shown in A were cleaved at guanine residues. An (A+G)-cleaved strand is included as a marker. The bands corresponding tothe guanine contact points are indicated at right. (C) Factor-bindingsite with the guanine contact residues encircled.

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cisely, we carried out a methylation interference analysis.This analysis identified a total of 7 guanine-residue contactpoints on the sense and antisense DNA strands (Fig. 4B) andlocalized the binding site to the sequence 5'-GGCTGCCATC-3' (Fig. 4C). The presence of this binding site in exon Isupports the idea that elements downstream of the cap siteare critical for rpL32 transcription.

DISCUSSION

The foregoing experiments indicate that efficient transcrip-tion of the rpL32 gene requires sequences both upstream anddownstream of the cap site. The upstream sequences com-prise elements in the -1 to -36 region, which are needed forbasel-level activity and for accurate recognition of the tran-scriptional start site, as well as elements in the -36 to -159region, which stimulate transcription 3- to 4-fold over thebasal level. The relatively sharp decline in activity uponremoval of the -68 to -79 sequence suggests that an impor-tant regulatory element may be located at this position. The-79 deletion might penetrate the 5' boundary of this elementand thus account for the slightly reduced activity of the -79mutant.The most striking finding of these experiments was the

requirement for sequences in the first exon. The first exon ofrpL32 consists of 46 bp of noncoding sequence (5). The AE1mutant lacks 33 bp of this sequence, from positions +13 to+45. When inserted into the rISVHS vector, this mutantproduces transcripts that appear to be initiated at the authen-tic rpL32 cap site and that undergo normal processing.However, when inserted into a pUC vector, this mutant istotally inactive. That the effect of the AE, deletion is vector-dependent argues strongly that this exon I sequence isessential for rpL32 transcription. If the deletion acted post-transcriptionally, its effect could not be masked by a vectorsequence that is not part of the transcript. A recent study (4)evaluating the importance ofintrons for rpL32 expression hasrevealed that sequences in the first intron between positions+47 and +73 are also necessary for efficient transcription.Thus, there are at least two internal elements in the rpL32promoter.The IrSVHS vector is able to compensate for the lack of

upstream and exon I sequences irrespective of whether it isin a replicating or nonreplicating state. The vector elementsresponsible for this compensatory effect are presumably part

of the SV40 ori sequence because the other portions of thevector, being entirely ofprokaryotic origin, should contributeno more than the pUC vector. Most likely, the effect involvesan interaction between proteins bound to the ori region andthose bound to other elements of the rpL32 promoter.The experiments reported here, together with other current

studies (3, 4), have localized the sequences involved intranscriptional regulation of the rpL32 gene to a region ofabout 200 bp roughly centered on the cap site. Within thisregion there are multiple elements that serve as binding sitesfor nuclear protein factors (3). One such binding site islocated in the exon I segment that is encompassed by the AE,deletion. We have now identified this binding site as thesequence GGCTGCCATC, which is located at positions +28to +37. Conceivably, the interaction of a protein bound tothis site with proteins bound to other upstream and intronsites could facilitate the assembly of the transcription com-plex. The elements within the -36 to +46 region mayconstitute a core with a basal activity that can be augmentedby the participation of more peripheral elements. This designmay be particularly well suited to genes that need to beefficiently expressed in different types of cells with diverseassortments of transcriptional factors.

This research was supported by grants from the National ScienceFoundation (DCB-84-13609), the National Institutes of Health(AI17330-07, CA06927, RR05539), and an appropriation from theCommonwealth of Pennsylvania. R.M.-N. acknowledges a fellow-ship from the Conselho Nacional de Desenvolvimento Cientifico eTecnologico (CNPq), Brazil.

1. Dudov, K. P. & Perry, R. P. (1986) Proc. Natl. Acad. Sci. USA83, 8545-8549.

2. Treisman, R., Green, M. R. & Maniatis, T. (1983) Proc. Natl.Acad. Sci. USA 80, 7428-7432.

3. Atchison, M. L., Meyuhas, 0. & Perry, R. P. (1989) Mol. Cell.Biol., in press.

4. Chung, S. & Perry, R. P. (1989) Mol. Cell. Biol., in press.5. Dudov, K. & Perry, R. P. (1984) Cell 37, 457-468.6. Wagner, M. & Perry, R. P. (1985) Mol. Cell. Biol. 5, 3560-3576.7. Sompayrac, L. M. & Danna, K. J. (1981) Proc. Natl. Acad.

Sci. USA 78, 7575-7578.8. Bienz, M. & Pelham, H. R. B. (1986) Cell 45, 753-760.9. Schibler, U., Marcu, K. B. & Perry, R. P. (1978) Cell 15, 1495-

1509.10. Siebenlist, U. & Gilbert, W. (1980) Proc. Natl. Acad. Sci. USA

77, 122-126.

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