Vol. 11, No. 12

Sequences Regulating Temporal Poly(A) Site Switching in theAdenovirus Major Late Transcription Unit

JAMES D. DEZAZZO,l ERIK FALCK-PEDERSEN,2 AND MICHAEL J. IMPERIALE1*Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor,

Michigan 48109-0620,1 W. R. Hearst Department of Microbiology, Cornell UniversityMedical College, New York, New York 100282

Received 30 July 1991/Accepted 12 September 1991

Temporal regulation of poly(A) site choice occurs in an adenovirus recombinant encoding a miniature ver-

sion of the major late transcription unit with two poly(A) sites, Li and L3. Using deletion mutagenesis, we havelooked directly for cis-acting elements regulating poly(A) site choice in this recombinant. From this work, we

draw two main conclusions. First, elements other than the AAUAAA and downstream sequences of the Lipoly(A) site are required for temporal regulation of poly(A) site choice during infection. Second, these regionsfunction in two distinct modes during infection. The two regions enhance selection of the Li poly(A) site in anadditive manner during an early infection, but deletion of either element abolishes the switch in poly(A) sitechoice during a late infection. This work documents the first example of a regulatory element downstream ofa core poly(A) region.

Complex transcription units produce multiple mRNAsfrom a single promoter via alternative use of splicing andpolyadenylation signals in the primary transcript (for areview, see reference 35). Alternative RNA processing tendsto be limited, however, in that most signals are used consti-tutively, while only a few are used selectively. Conse-quently, different mRNAs made from the same complexlocus usually have extensive regions of sequence identityand only limited regions of sequence heterogeneity. Notsurprisingly, many complex units, such as the troponin-Tlocus, encode multiple protein isoforms which are function-ally related but structurally distinct (6). In contrast, othercomplex units, such as the calcitonin/calcitonin gene-relatedpeptide locus, exploit differential RNA processing to pro-duce functionally distinct proteins (2).An important theme emerging from studies of many com-

plex units is that alternative processing signals can beregulated both temporally and spatially, so that differentmRNAs are produced only in specific tissues or duringspecific stages of development or phases of viral infection.Such tissue-specific and temporal regulation results in func-tional modulation of many cellular and viral products,including contractile proteins, hormones, antibodies, ionchannels, and transcription factors (35). Regulated RNAprocessing can also lead to tissue-specific expression ofpremature translation-stop signals, providing a posttran-scriptional mechanism for selectively turning off proteinexpression. By this mechanism, sex-specific splicing in theDrosophila tra and sxl loci controls somatic sexual differen-tiation (for a review, see reference 3).

Tissue-specific and temporal regulation of RNA process-ing presumably reflects corresponding changes in levels ortypes of trans-acting factors. Such factors have been directlyimplicated in sex-specific splicing of Drosophila tra pre-mRNA (21, 36), cell-specific splicing of simian virus 40 earlypre-mRNAs (11), and late infection-specific activation of aherpes simplex virus poly(A) site (25). To understand howtrans-acting factors in these and other systems regulate

* Corresponding author.

alternative processing, it will be important to generate adetailed map of the involved cis-acting sequences. Giventhat sites of regulated processing and sites of unregulatedprocessing share strongly conserved consensus sequences,such as the AAUAAA and GU-rich elements found atpoly(A) sites, it is plausible that regulated processing mayinvolve additional, nonconserved elements. Indeed, regu-lated splicing can require cis-acting control sequences dis-tinct from the splice acceptor, splice donor, polypyrimidinetract, and branch point sequence (4, 15, 29, 37).

In contrast to regulated splice site selection, controlelements required for a switch in poly(A) site use in complextranscription units have not yet been fully documented. Anattractive place to look for such signals, however, is theadenovirus major late transcription unit (MLTU), whichencodes five sets of 3' coterminal mRNAs, Li through L5.Through regulated choice of these poly(A) sites duringinfection, the virus modulates the levels of both structuralcomponents and additional proteins involved in virion mat-uration (1, 30, 31, 34). This regulation can be convenientlystudied in an adenovirus recombinant encoding a miniatureversion of the MLTU (mini-MLTU) with just the Li and L3poly(A) sites (10). In this recombinant, the Li poly(A) site isselected about eight times as frequently as the L3 site earlyin infection, but with almost equal frequency late in infec-tion. Two basic requirements for this processing switch havebeen established: physical linkage of the processing signalsand replication of the viral template (10).Given that cis-acting mechanisms are involved in regulat-

ing poly(A) site use in the MLTU, it seems plausible that theMLTU may encode cis-acting regulatory signals in additionto core processing elements. Analysis of the Li poly(A) siteon plasmids encoding simple and complex transcription unitsreveals a bipartite genetic structure. In simple transcriptionunits, a core region is necessary and sufficient for efficient 3'end processing; this core spans the AAUAAA signal andcleavage site and extends into a U-rich region 33 nucleotides(nt) past the site (14). In complex transcription units encod-ing two poly(A) sites, efficient processing at the Li siterequires additional sequences upstream of AAUAAA, re-ferred to as the selector element (8). However, a regulatory

5977

MOLECULAR AND CELLULAR BIOLOGY, Dec. 1991, p. 5977-59840270-7306/91/125977-08$02.00/0Copyright © 1991, American Society for Microbiology

5978 DEZAZZO ET AL.

role for the selector element, or other sequences outside thecore, has not been tested on the viral chromosome. In thisstudy, we have looked directly in the adenovirus recombi-nant for elements regulating the processing switch duringinfection. We find that the consensus processing signals atthe Li site are flanked by two regulatory regions whichcontrol poly(A) site selection in a complex transcription unit.

MATERIALS AND METHODS

Reagents. Enzymes and molecular linkers were purchasedfrom New England Biolabs, Inc., and Bethesda ResearchLaboratories, Inc. Radiochemicals were obtained from Am-ersham Corp.

Transfections, infections, and RNA isolation. Maintenanceand transfection ofhuman 293 cells and isolation of poly(A)+RNA were as previously described (8). Cells were infectedwith recombinant adenoviruses either at a multiplicity of 50PFU per cell for 6 h in the presence of 5 ,ug of cytosinearabinoside per ml (early) or at a multiplicity of 20 PFU percell for 22 to 26 h (late).

Plasmids and bacteria. All plasmids were grown, analyzed,and quantitated as previously described (8).

Mutagenesis strategy and plasmid constructions. The par-ent construct dlpMLP6-L13 (10) is designated pML1+170-L3 in this study. The name of each plasmid indicates thepresence or absence of sequence elements as follows: (i) thenumber following the plus sign indicates the number ofnucleotides downstream of the Li poly(A) site; (ii) A63indicates a deletion of the upstream selector element (nt -50to -113 with respect to the Li site); and (iii) KL3 indicatesthe presence of additional L3 sequences upstream of the L3poly(A) site, to control for possible spacing effects (seebelow).To engineer changes in the parent plasmid in sequences

flanking the poly(A) sites, an EcoRI-EcoRV fragment span-ning the Li and L3 regions was first isolated frompML1+170-L3 and cloned into the EcoRI and SmaI sites ofpGEM2. Serving as a shuttle vector, this construct,pGEML1 (not shown), allowed independent manipulation ofthe sequences flanking the Li poly(A) site via three uniquerestriction sites: PstI [316 bp 5' to the poly(A) site], NcoI [1bp 5' to the poly(A) site], and KpnI [170 bp 3' to the poly(A)site]. In a stepwise manner, each of the following constructswas derived from pGEML1.

(i) Upstream deletion mutants. The PstI-NcoI fragment ofpGEML1 was replaced with the corresponding PstI-NcoIfragment, containing a deleted upstream selector element,from pEGCA63 (8).

(ui) Downstream deletion mutants. The NcoI-KpnI frag-ment in pGEMLi was replaced with the NcoI-BamHI frag-ment from pE2L1P+33 or pE2L1P+52 (14). The BamHI sitein these plasmids defines the endpoint of the Li downstreamsequences. This cloning step required the addition of a KpnIlinker (5'-CGGTACCG-3') to the BamHI site and, for the+33 mutant, the additional insertion of a i9-bp spacersegment, derived from pGEM2, into the BamHI site. Toconstruct the +33/+59 internal deletion, a BamHI-KpnIinsert spanning the Li downstream sequences between +59and +170 was cloned into the BamHI and KpnI sites of the+33 construct. This insert was obtained through Bal 31mutagenesis of the Li downstream sequences followed byaddition of a BamHI linker to the terminus. The exactendpoint was determined by the chain termination sequenc-ing method, using the Sequenase kit (United States Bio-chemical).

(iii) L3 spacer insertion mutants. The sequences 5' to theL3 poly(A) site were extended by 417 bp in the KL3constructs by inserting into the KpnI site an adenovirus type5 BglII-KpnI fragment [from -204 to -621 relative to the L3poly(A) site] derived from pE2 (20). This cloning steprequired addition of a KpnI linker to the filled-in BglII site.

Following these manipulations on the shuttle plasmid,each modified L1-L3 fragment was excised by digestion withEcoRI and KpnI and cloned into the corresponding sites ofpML1+170-L3, generating the collection of mini-MLTUsshown in Fig. 1. This excision step required partial KpnIdigestion for plasmids containing the L3 spacer.

Virus constructions. Each recombinant virus was gener-ated by the overlap recombination method (7) and is namedafter its plasmid parent. Briefly, viral fragments beginningwith the left end of the genome were made by digesting eachmini-MLTU plasmid with EcoRI (indicated by an E in Fig.1). Viral fragments extending to the right end of the genomewere obtained by digesting full-length sub360 viral DNA (22)with ClaI and XbaI. These fragments are cotransfected intocells and give rise to viable viruses, containing origins ofreplication located at either end of the genome, only ifrecombination takes place between the overlapping ElBregions on both fragments. The EiB region is downstream ofthe mini-MLTU on each plasmid (Fig. 1). Each recombinantstrain was subsequently plaque purified and propagated in293 cells.HIV-L3 constructs. Viruses vEHX-L3 and vEHXAU3p-L3

are identical to the L1-L3 viruses except that the Li frag-ment has been replaced by a human immunodeficiency type1 (HIV-1) fragment containing a wild-type or mutant HIVpoly(A) site, respectively (9).

Quantitative S1 nuclease mapping. Poly(A)+ RNA fromtransfection or infection of each mini-MLTU was assayedwith a DNA probe made from the corresponding plasmid.Each probe was specifically 3' end labeled at the XbaI siteupstream of the Li poly(A) site. Hybridizations and Sidigestions were performed as previously described (8) ex-cept that the temperature for hybridization was optimized at60°C for L1-L3 RNAs and 52°C for HIV-L3 RNAs. Process-ing values in Fig. 2 and 3 were obtained by densitometricscanning of multiple autoradiograms derived from at leasttwo independent experiments for transfections and at leastthree independent experiments for infections. Care wastaken to ensure that the intensities of the bands to bescanned were within the linear range of the film. Typicalexposure times for autoradiograms shown in Fig. 2 and 3were 48 to 96 h for early infections and 3 to 6 h for lateinfections and transfections. In Fig. 4, 40 times less latepoly(A)+ RNA than early poly(A)+ RNA was assayed.

RESULTS

Experimental strategy. To identify sequences regulatingLi mRNA 3' end formation in a viral mini-MLTU encodingthe Li and L3 poly(A) sites, we used a two-step strategy.First, we constructed a set of modified mini-MLTU plasmidswith various deletions in the sequences flanking the Li coreregion. This core, consisting of the AAUAAA signal and 33nt of sequence beyond the poly(A) site, has been previouslyidentified as the minimal region necessary for accurate andefficient 3' end processing in simple transcription units (14).Second, we used this set of plasmids to make a correspond-ing set of recombinant adenoviruses harboring the mini-MLTUs. With this approach, we could compare the 3' endprocessing patterns of mini-MLTU transcripts produced

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FIG. 1. Mini-MLTUs encoding the Li and L3 poly(A) sites. The vector dlpMLP6 was adapted for both transient expression of themini-MLTUs and their subsequent recombination into the adenovirus genome (10). It contains the leftmost 5,580 bp of the adenovirus type5 sub360 genome (solid bars), including the adenovirus type 2 major late transcription control region (MLTCR) in place of the ElA promoter,and lacking the E1B promoter and ElA poly(A) site (open box). Mini-MLTU constructs were made by inserting the appropriate XbaIfragment into the XbaI site of dlpMLP6. Details of plasmid constructions are described in Materials and Methods. Probe and protectedproduct sizes (in nucleotides) for each Si analysis are shown at the right. Deleted Li sequences (..), linker sequences (solid boxes in lines1 and 5), cleavage sites (arrows), AAUAAA signal (filled triangle), and L3 sequences (hatched box) are shown. Restriction sites: E, EcoRI;RV, EcoRV; X, XbaI. SV40, simian virus 40.

from plasmid transfections and from early and late viralinfections.

Therefore, modifications were made in the wild-type mini-MLTU, or pML1+170-L3 (dlpMLP6-L13 in reference 10).The resulting constructs, shown in Fig. 1, can be divided intotwo groups, containing either the original L3 poly(A) sitefragment (lines 1 to 4, 8, and 9) or a longer L3 fragmentwhich includes an additional 417 bp of upstream sequence(lines 5 to 7 and line 10). Possible effects of differentialspacing on poly(A) site selection could therefore be studied.Within each group, there are mini-MLTUs with deletionsupstream (A63) and/or downstream (+52 or +33) of the Licore region.

Processing function of Li flanking sequences in transfectedmini-MLTUs. Each mini-MLTU plasmid was cotransfectedinto human 293 cells, which efficiently drive transcriptionfrom the major late promoter, along with the control plasmidpLlSV, which contains the simian virus 40 early poly(A) sitein place of the Li and L3 poly(A) sites (Fig. 1). Total cellularpoly(A)+ RNA was isolated 48 h later and analyzed with aprobe spanning the two sites and specifically end labeled atthe XbaI site upstream of the Li poly(A) site (Fig. 1). Allprobes were also homologous to sequences upstream of thesimian virus 40 sequence in control RNA, allowing simulta-neous resolution of control and experimental RNAs fromeach transfection. The efficiency of 3' end processing fromeach mini-MLTU was quantitated by determining the rela-tive amounts of control and experimental products, a strat-egy used successfully before in the study of Li and HIV-1poly(A) site selection (8, 9). Figure 1 lists the probe size andpredicted protected band sizes for each Si analysis.The Si analysis of RNA from the mini-MLTU constructs

is shown in Fig. 2. Li sequences preceding AAUAAAenhance selection of the Li poly(A) site in the mini-MLTUs.When these upstream sequences are removed by a 63-bpdeletion in pML1+170A63-L3 and pML1+170A63-KL3, therelative use of the Li site decreases from 7.6 to 1.0 and from20.1 to 2.9, respectively (compare constructs 3 and 9, or 7and 10, in Fig. 2B). Therefore, in these mini-MLTUs, theselector element functions independent of the distance be-tween the Li and L3 poly(A) sites.The results in Figure 2 also implicate Li sequences

downstream of the core region in poly(A) site selection. Withtheir absence in pMLl+ 33-L3, the relative use of the Li sitedecreases from 7.6 to 1.1 (compare constructs 1 and 3).Although it suggests that Li downstream sequences specif-ically enhance Li poly(A) site selection, this finding couldalso indicate that the 140-nt decrease in spacing between theLi and L3 poly(A) sites in pML1+33-L3 causes the reducedlevel of processing at Li. To distinguish between these twopossibilities, a longer L3 fragment was substituted for the L3poly(A) site in pMLi + 33-L3 to give pML1+ 33-KL3, makingthe distance between the Li and L3 sites even greater thanthat in the original wild-type plasmid while maintaining thedeletion of the Li downstream sequences. Despite spacingrestoration, Li poly(A) site function in pML1+33-KL3 isnot returned to the wild-type level (compare constructs 3 and5). Therefore, spacing alone cannot account for the resultseen with pML1 +33-KL3; specific Li sequences down-stream of the core region are also enhancing Li poly(A) siteselection. This downstream region was further analyzed bydeleting a small internal segment (nt +33 to +59), whichincludes a splice acceptor for the adenovirus L2 gene. Thisdeletion has a negative impact on Li poly(A) site selection,

xixixi

VOL. 11, 1991

5980 DEZAZZO ET AL.

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lowering its relative use greater than twofold (compareconstructs 3 and 4).Taken together, these results demonstrate that sequences

flanking the Li core region are required for predominant Lisite use on transfected plasmids. Indeed, disruption of bothof these elements in pML1+52A&63-L3 (construct 8) has thegreatest effect of all upon Li poly(A) site selection, loweringrelative use of the site to 0.21 (Fig. 2B). These Li flankingsequences influence the distribution of processing, not thetotal amount of processing: the total amount of RNA proc-essed at the Li and L3 sites varies less than twofoldcompared with the control. Having established that the Liflanking regions influenced processing choices in transfectedmini-MLTUs, we could now ask the more important anddirect question: do these regions play a regulatory role onthe viral chromosome?Requirement of Li flanking sequences for regulated use of

the Li poly(A) site during infection. To study the potentialregulatory role of the Li flanking regions during infection,we inserted each mini-MLTU described above into the Elregion of the adenovirus chromosome by overlap recombi-nation (7). Because the El region is located over 12 kb awayfrom the endogenous Li poly(A) site, each recombinantvirus encodes both a miniature and an intact version of theMLTU. The mini-MLTU RNAs can be distinguished fromtheir natural counterparts, however, by XbaI linker se-quences present at the 5' border of the Li sequences in eachmini-MLTU. Si probes including these linker sequences,used in the previous analysis of plasmid RNAs, were there-fore used again to mediate exclusive recognition of mini-

MLTU RNAs during infection. The resulting Si analysis ofpoly(A)+ RNA from early and late infections is shown inFig. 3. A comparison of the processing phenotypes fromplasmid transfections and early viral infections reveals onlyslight differences (compare solid bars in Fig. 2B and 3B),supporting our previous finding that predominant selectionof the Li poly(A) site is not dependent upon viral infection(8, 10).The efficiency of Li poly(A) site switching in each viral

mini-MLTU can be defined as the ratio of Li to L3 poly(A)site use early to that ratio late. By this criterion, switching ofthe Li poly(A) site is equally efficient in the two wild-typeconstructs, vML1+170-L3 and vML1+170-KL3, with theprocessing ratios shifting from 7.3 to 1.2 and from 18.3 to3.2, respectively. Temporal regulation of the Li poly(A) siteis therefore independent of both its absolute level of cleav-age and its distance from the L3 poly(A) site. UsingvMLl +170-L3 and vML1+170-KL3 as reference points, weevaluated the potential regulatory role of the two regionsflanking the Li poly(A) site. Deleting the Li selector ele-ment from either virus disrupts poly(A) site regulation,causing the early processing phenotype to persist late (Fig.3, constructs 9 and 10). Similarly, removing the Li se-quences downstream of the core poly(A) region from eithervirus can also prevent poly(A) site switching (e.g., con-structs 5 and 6). The switching efficiency is reduced to lessthan 2 upon removal of a 26-nt sequence within the down-stream region in vML1 +33/+59-L3.

Together, the transfection and infection analyses demon-strate that sequences which influence preferential Li

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FIG. 3. Poly(A) site selection in viral mini-MLTUs in an early or late infection. (A) Si analysis. Probes were hybridized to 2 ,ug of poly(A)+RNA for early infections (part E) and 0.5 F.g of poly(A)+ RNA for late infections (part L) and analyzed by S1 nuclease protection as describedin Materials and Methods. Si products representing poly(A)+ transcripts from viral mini-MLTUs cleaved at the Li poly(A) site (filled circles)or L3 poly(A) site (arrowheads) are shown. Probe DNAs, seen in part E above L3 products, can also be detected in part L upon longerexposure. Markers (M) are shown in nucleotides at the left. (B) Quantitation of results. The L1/L3 ratio for each virus early and late ininfection is shown. The switch is indicated by the relative differences in the sizes of each pair of bars. Values represent the average of at leastthree experiments.

poly(A) site selection on mini-MLTU plasmids also regulatethe site temporally on the viral chromosome. Moreover, twolines of evidence indicate that L3 sequences have no signif-icant regulatory role: first, a general increase of L3 process-ing in the viral constructs during infection does not occur;second, the processing switch in the mini-MLTUs is abol-ished by replacement of the Li poly(A) site with the HIV-1site, as shown in Fig. 4. Here, the ratio of HIV to L3 is thesame early and late, using either a wild-type or mutant HIVsite. Regulated poly(A) site use in the mini-MLTUs istherefore mediated by at least two distinct control sequenceslying outside of the Li core poly(A) region.

Stability of mini-MLTU transcripts. Several lines of evi-dence argue against a primary role for differential RNAstability in temporal regulation of mini-MLTU RNAs. First,the early to late processing switch in vML1+170-L3 is seenwith nascent pulse-labeled RNAs (10). Second, Li se-quences upstream ofAAUAAA do not influence stability ortransport of Li messages from plasmids transfected into 293cells (8). To directly support this contention, we performedan RNA half-life analysis of the KL3 set of mini-MLTUs,comparing the stability of Li and L3 transcripts from trans-fected plasmids and the late viral chromosome. A repre-sentative Si analysis is shown in Fig. 5, and the quantitationis presented in Table 1. The ratio of Li to L3 poly(A)+ RNAmade from each virus remains essentially constant at eachtime point following inhibition of transcription. One cannotargue, therefore, that relative to Li, the L3 messages are

being selectively stabilized during a late infection. Whenexpressed from transfected plasmids, the L3 transcript has ahalf-life equivalent to that of Li in pML1+170-KL3 andslightly less than that of Li in pML1+33-KL3 andpML1+170A63-KL3. This result is reproducible and sug-gests that the L1/L3 processing ratios for the latter twoconstructs are artificially high, given that the decrease in L3is partly due to lower stability. Adjustment of these valueswould therefore further strengthen the proposed direct rolefor these Li sequences in 3' end processing. Thus, differen-tial stability cannot account for the differences in ratiosobtained with these constructs.

DISCUSSION

In this work, we have shown that elements which flank thecore Li poly(A) signals control its predominant use early ininfection. Moreover, these regulatory regions function intwo different modes during infection. In an early infection(or transfection), the two regions enhance selection of the Lipoly(A) site in an additive manner: deletion of either regionimpairs the early processing bias for the Li site approxi-mately 5- to 7-fold, whereas deletion of both elements causesa 15- to 30-fold drop in relative use of the Li site. Bycontrast, in a late infection, disruption of either region alonecan completely prevent an efficient processing switch. Thesesequences do not influence the stability of the processedtranscripts (Fig. 5), nor do they affect nucleo-cytoplasmic

VOL . 1 l, 1991

5982 DEZAZZO ET AL.

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FIG. 4. Poly(A) site selection in viruses containing HIV-L3transcription units. The constructs used are shown at the bottom.HIV and L3 sequences are depicted, as are the poly(A) sites(arrows) and the predicted sizes of the protected bands in the Sianalysis shown at the top. The deletion of U3 sequences invEHXAU3p-L3 is indicated by a break in the bar. E, early; L, late;closed circle, HIV band; arrow, L3 band; M, markers. The bandabove the L3 band in each lane is undigested probe. Two micro-grams of poly(A)+ RNA was assayed in the early samples, and 1/40that amount was assayed in the late samples.

transport of the RNA (data not shown). Moreover, thesesequences do not play a role in premature termination or

pausing, as previous studies have shown that those two

processes do not occur at or near the Li poly(A) site (10, 30).In addition, the ratio of Li to L3 would not be affected bychanges in nuclear stability of the primary transcript causedby these mutations; rather, the total amount of processedRNA would change. Although we did not measure thenuclear stability of the processed RNAs, poly(A)+ RNAfrom the MLTU is transported to the cytoplasm very rapidly(30), lessening the probability that nuclear turnover is play-ing a role. Finally, our experiments using HIV-L3 constructsshow that relative use of the L3 poly(A) site does notincrease late in infection. Therefore, the Li flanking se-

quences appear to be directly affecting the level of RNAprocessing at the Li poly(A) site. Further support for thisconclusion should come from either in vitro studies, in whichprocessing can be examined independently of these othercellular processes, or the ability to transfer regulation to a

second poly(A) site. To date, attempts to accomplish thelatter have not borne fruit, indicating either that the Li sitemust function as an integral unit or that additional elements,not defined in this study, may play a role in regulation.

Since the processing phenotype of each mini-MLTU on anearly viral template is equivalent to that observed on atransfected plasmid (compare Fig. 2 and 3), cellular factorsalone are probably sufficient to control Li processing duringan early viral infection. By contrast, the late processingphenotype of each mini-MLTU is seen only on a late viraltemplate; plasmids transfected or viruses superinfected intolate infected cells give rise to relative mRNA levels whichreflect the early processing phenotype (7a, 10). This resultindicates that trans-acting factors alone cannot account forthe difference in poly(A) site choice. There is a change whichalters the state of the viral chromosome and acts in cis. Thiscould entail either or both of two possibilities: a change inthe physical nature of the viral chromosome, resulting in achange in the dynamics of transcription and how the nascentRNA is recognized by the processing machinery, or a changein subnuclear localization of the chromosome, resulting in adifferent environment of trans-acting factors. Indeed, elec-tron microscopy and sedimentation analyses have revealedstructural and topological changes in the adenovirus tem-plate during infection (45). Furthermore, during infection,there is stable attachment of the adenovirus chromosome tothe nuclear matrix (5, 33, 46), which could serve to compart-mentalize the DNA. Replication of adenovirus takes place inmultiple foci, or replication factories (28), which are local-ized to the nuclear periphery (26), and during infection thenucleous is broken down and nuclear factors, includingprocessing factors, are redistributed (39).Not knowing exactly what accounts for the cis effect noted

above, one can only speculate as to the role of specifictrans-acting factors in the processing switch. Accurate andefficient 3' end processing requires stable and specific for-mation of a large, multicomponent complex at the poly(A)site (for reviews, see references 23 and 42). Complex assem-bly is dependent upon specific contacts between the cis-acting sequences and the protein factors and between thefactors themselves (12, 13, 16, 27, 38, 40, 43, 44). Wepropose that in an early adenovirus infection, the Li regu-latory sequences do not modulate the final stability of the 3'end processing complex but rather modulate the rate of itsassembly. This model is based on two observations. First, ifthe Li regulatory sequences did influence complex stability,then their deletion should reduce the level of 3' end proc-essing in simple transcription units (40). Such a reductionhas not been observed in transfection assays; instead, the Liregulatory regions regulate processing choices only in com-plex units encoding more than one poly(A) site (8). Second,preliminary direct assays of complex stability at the Lipoly(A) site in vitro reveal no differences between a wild-type site and one missing the selector element (lla). Ourlaboratories are currently pursuing these questions.

Since dissection of the Li processing switch has beenperformed in mini-MLTUs, we cannot exclude an accessoryrole for other sequences which are present in the intactMLTU, such as splicing signals. Indeed, splice acceptorsignals can activate poly(A) site use in vivo (17) and in vitro(32). Binding of splicing factors might therefore influence 3'end processing in the MLTU, even though splicing in theMLTU occurs after polyadenylation (30). And, since thepoly(A) sites are much further apart in the MLTU than in themini-MLTUs, a mechanism must be operative which com-pensates for the temporal advantage that the Li poly(A) sitewould be expected to have due to its promoter proximalposition and the observation that 3' processing can occursoon after the poly(A) site is transcribed (30).Sequences upstream of the AAUAAA signal are required

MOL. CELL. BIOL.

POLY(A) SITE REGULATORY SIGNALS 5983

TRANSFECTIONpMLI +33 -KL3 pML1it+177053-KL3 pML1+170-KL3

N W0 816 24 0 8 16 24 0 816 24

LATE INFECTIONML1+33-KL3 ML1+170A63-KLS ML1+170-KL3

M 0 8 16 24 T W16 24 0 8 16 24

0 ee -A _ A A___~~~~o

FIG. 5. Stability of mini-MLTU transcripts from plasmid transfections or late viral infections. Actinomycin D was added 24 h aftertransfection or 22 h after infection to a final concentration of 10 1Lg/ml. Poly(A)+ RNA was isolated at the indicated time points and analyzedby S1 nuclease protection as described in Materials and Methods. Markers and abbreviations are the same as for Fig. 2.

for efficient 3' end formation at several animal and plantvirus poly(A) sites (for a review, see reference 19) as well asat certain yeast poly(A) sites (18, 24). In addition, upstreamsequences are needed at several Xenopus and mouse poly(A)sites for regulation of poly(A) tail length during development(for a review, see reference 41). The Li regulatory regionsdefined in the present study are distinguished from theseother signals in that they include sequences downstream ofthe core processing elements and they regulate poly(A) sitechoice in a complex transcription unit. Furthermore, thisregulation is intertwined with the intricacies of the adenovi-ral life cycle. Clearly, the adenovirus system provides a

TABLE 1. L1/L3 Ratios in mini-MLTUs on transfected plasmidsor during a late viral infection following addition of actinomycin D

Time (h) after L1/L3 ratioaMini-MLTU actinomycin D Late

addition Transfection infection

ML1+33-KL3 0 1.5 0.878 1.7 0.9116 2.0 1.124 2.4 0.88

ML1+170A63-KL3 0 2.5 2.48 3.3 2.816 4.5 3.024 6.2 2.8

ML1+170-KL3 0 22.6 2.38 17.8 2.416 18.3 2.624 21.8 2.4

a Data were obtained by densitometric scanning of bands corresponding toLi and L3 poly(A)+ products.

unique opportunity to study the interplay of multiple factorswhich control gene expression.

ACKNOWLEDGMENTS

We thank Jay Kilpatrick for critical comments on the manuscript,the members of the Imperiale laboratory for stimulating discussionsthroughout this project, and Mike Savageau for help with graphics.J.D. thanks Gretchen Edwalds-Gilbert for valuable assistance dur-ing early phases of this project and Joe Tantravahi for helpful adviceon constructing recombinant viruses.

This work was supported by PHS grants GM34902 (M.J.I.) andGM41967 (E.F.-P.). M.J.I. also acknowledges the support of Fac-ulty Research Award FRA-338 from the American Cancer Society.

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