Splice site selection in polyomavirus late pre-mRNA processing.

0022-538X/94/$04.00+0

Copyright

C) 1994,American

Society

forMicrobiology

Splice

Site

Selection

in

Polyomavirus Late Pre-mRNA Processing

DAVID B. BATT,LISAM. RAPP, ANDGORDON G.CARMICHAEL*

Departmentof Microbiology, UniversityofConnecticutHealth Center, Farmington, Connecticut 06030 Received 23 September 1993/Accepted2 December 1993

Polyomavirus

latepre-mRNAs contain one5' splice site and two message body 3' splice sites,which arenot used atequal frequencies. As a result of alternative splicing, the total latemRNApopulationconsistsof about 5% mVP2(no message bodysplice chosen), about 15% mVP3 (promoter-proximal3' splice site chosen), and about 801%mVP1 (promoter-distal3' splice site chosen).Todetermine whetherit issplicesite strength that determines the ratio ofspliced products, constructs containing duplicated or rearranged 3' splice sites were created. In construct VP1,1, 160 bpsurrounding the VP33' splice site was substituted with the corresponding region of the VP13' splice site. This construct resulted in the duplication of the VP1 3' splicing signal. VP3,3 (two identical VP3 3' splice sites) and VP1,3 (VP1 and VP3 3' splice sites reversed) were similarly created. Each construct maintained wild-type spacingbetween the3' splice sites.

Analysis

of RNAsfrom transfections showed that in each construct,the3' splice site closestto thepolyadenylation site was usedpreferentially. Analysisof anumber of additionalconstructs indicatedthat there are no strongcis-acting positiveornegative regulatorsof

polyomavirus

latesplicing; rather, splicing choices appear to be determined

largely

byrelativeposition of splice sites.

Alternative

splicing of pre-mRNAs is commonly used to regulate cellular and viral gene expression and to generate diversity. Although many of the molecular details of constitu-tive

splicing

have been described

(20),

lessis known about the regulation of alternative splice site selection at the molecular level.

There are a number of ways in which splicing signals can influence alternative splice site choice (41). The strength of 5' splice sites has been shown to influence alternative splicing; 5' splice sites that are nearer to consensus bind

Ul

ribonucleo-protein more efficiently and are used more frequently than nonconsensus sites (9, 16, 19, 24, 28, 45). For example, in Drosophila melanogaster, the muscle myosin heavy-chain premessage is alternately spliced to generate some mRNAs containing exon 18 and some

lacking

it.Animproved 5' splice

site

attheexon18-intron 18 border increased the

efficiency

of

intron

18 removal in an invitro system containing exon 18, intron 18, and exon 19

(24).

In

simian

virus 40 (SV40), alternative

recognition

of two 5'

splice

sites generates large andsmallT-antigenmRNAs.The relativestrengthsof the two

splice

siteswereshowntoinfluence the relative levels oflarge T and small T messages

(16, 45).

In a

synthetic

system

containing

fragments of the human

P-globin

gene, the inclu-sion of an

optional

33-nucleotide

(nt)

internal exon was influenced by the sequence of the 5' splice site, with exon inclusionincreasing sixfold when the sitewas consensus

(9).

Thestrength of 3' splice sites can also influence alternative

splicing.

In the D.

melanogaster

muscle

myosin heavy-chain

system mentioned above, inclusion of exon 18 is also influ-enced

by

the

strength

of the 3'

splice site,

with consensus sequences

leading

togreaterinclusion

(24).

InRoussarcoma

virus,

balanced

splicing

generating

both

unspliced

and

spliced

mRNAsis achieved bytwo weak 3' splice sites, which allows much of thepre-mRNAtoremainunspliced, with the remain-der

being

alternatively spliced

(34).

Inthis system,however, 3'

splice

sites andtheir

surroundings

mayalso influence

splicing

by

a different mechanism. When the 3' splice site for the

*Correspondingauthor. Mailingaddress:Department of

Microbi-ology, University of Connecticut Health Center, Farmington, CT 06030. Phone:(203)679-2259. Fax: (203)679-1239.

promoter-proximalenvgeneisdeleted, the relativeamountof src mRNA

increases;

however, when the 3'

splice

site of the

promoter-distal

srcgeneisdeleted, the relativeamountof env mRNAdoesnotincrease

(3).

The

polypyrimidine

stretch and branchpoint sequencescan influence both

splice

site strength and alternative

splicing.

In Roussarcoma

virus,

a

24-bp

insert

creating

a newbranch

point

increased the amount ofenv mRNAand altered the

ratio

of spliced versus

unspliced

message

(15).

In SV40, multiple branchsitesare

important

in

regulating

theratio of

large

T to small T mRNAs, and mutations in these sites can alter the ratio of mRNAs

by

100-fold

(14,

37).

Branch

point recognition

alsoplaysarole in the alternative choice oftwoclosely spaced 3'

splice

sites in

polyomavirus early pre-mRNA processing

(18).

Intherat

,-tropomyosin

system,the

polypyrimidine

tract andasequence upstreamof the 3'

splice

siteare

important

in

determining

the inclusion or exclusion of the

muscle-specific

exon

(exon 7)

(23).

In asystem

using splicing signals

from the human

,-globin

gene,both in vitro and in

vivo,

theinclusion of a short internal exonwas influenced

by

the sequence of the branch

point

and the

polypyrimidine stretch,

with consensus

signals

increasing

the inclusion ofa 33-ntexon

by

10-foldor more

(9). Finally,

in yeast

cells,

theuseof alternative 3'

splice

sitescanbe influenced

by

the

length

ofaU-richtract

preceding

the 3'

splice

site

(38).

Several

cis-acting

elements other than those

directly

in-volved in

splicing

play a role in

splice

site choice.

Negative

regulators

of

splicing

(NRS)

are

cis-acting

elements found in Rous sarcoma virus which affect the ratio of

spliced

versus

unspliced

mRNA.These sequences work in thesense orienta-tion

only

when

placed

near a5'

splice

site

(35).

Inadenovirus type

2,

a

cryptic

5'

splice

site in

early region

3

is

suppressed by

a 120-nt segment located upstream of the

splice

site

(8).

Removal of this segment resulted ina

competition

betweenthe natural and the

cryptic site,

with the ratio determined

by

the relative

strengths

of the two sites

(8).

Secondary

structurealso has been

reported

to

play

arole in alternative

splicing; however,

its roles in vitroandin vivo may be different

(42, 43).

In aninvitro system,constructs contain-ing palindromic sequences which could sequester

splice

sites revealed that

secondary

structure caninhibit

splicing,

presum-ably by

masking

the

cis-acting signals

(43).

When these con-1797

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1798 BATT ET AL.

late leader

1

i1o

_,oM

480p 46C0 4400 4209 400 3320v3609 3401 300S 2809

mlivr- 4____

mVP3

-

16%

mVPI

-

80%

FIG. 1. Polyomavirus late pre-mRNA processing. Polyomaviruslatepre-mRNAmoleculescanbealternatively splicedtoform threeproducts, mVPI, mVP2,and mVP3. mVP2accountsforabout 5% of total latemessages,mVP3accountsfor about15%, andmVP1accountsforabout80%.

Thetoplinerepresentstheviral lateregionand shows thepositionof the late 5'noncodinglate leaderexonwhich isattachedtoeachlatemessage.

The thicknessof the linesreflects therelative proportions ofthe latemessages.

structswere analyzed in vivo, the secondarystructureseemed incapable of influencing splicing unless the stem structure

exceeded50 bp in length (43). Secondarystructureappearsto

play a role in the selection of splice sites in human growth

hormone (11), chicken 3-tropomyosin (10, 30), adenovirus EIA (5), and immunoglobulin heavy-chain (44) pre-mRNA splicing. In thesecases,the secondarystructure maysequester important cis-acting signals such asbranch points or3' splice

sites.

Splice site spacingcanalso affectsplicesite choice.InSV40, the ratio between large T and small T mRNAs results partly from the short small T intron, which isnotremoved efficiently (16). In a 3-globin gene derivative, the relative use of two

closely spaced competing 5' splice sites could be altered by changing the spacing between them. In this system,theuseof

theproximal splice site declined sharply when itwasseparated

bymore than 40bp from the distal site (7).

In addition to cis-acting signals, trans-acting factors

influ-ence alternative splice site choice. The effect of trans-acting

factors has been studied best in D. melanogaster, in which factors that bind near splice sites have been shown to either

suppress orenhance theiruse(33).Twocellularproteins which

can influence the choice oftwo 5' splice sites in SV40 early pre-mRNA processing have been described. At higher

concen-trations, the essential splicing factor ASF (alternative splicing factor)/SF2 (17, 26) allowed increased use of the promoter-distal smallT5'splice site relativetouseof theproximal large

Tsite (17), while another factor, DSF (distal splicing factor), promoted the useof the large T 5' splice siteoverthe small T

site (21).

Polyomavirus late pre-mRNAs are alternatively spliced to

formmessagesfor thestructural proteins VP1, VP2, and VP3.

Differences in the frequency of useoftwo alternative late 3' splice sites leads to ratios of about 5% mVP2 (no message

body splice chosen), 15% mVP3 (proximal 3' splice site chosen), and 80% mVPI (distal 3' splice site chosen) (Fig. 1). We have investigated the elements that influence the relative

useofeach 3' splice site. Toassesswhethersplice site strength determinesthe ratio ofspliced products,constructscontaining duplicatedorrearranged splice siteswerecreated. To examine

the possibility of positive or negative regulators, an insertion

and several deletion constructs were made. Finally, to deter-mine whether spacing is important, deletions which brought the two 3' splice sites closer to each other orwhich brought

both sites closer to the polyadenylation signalwere created.

Resultsindicate that there are no strongcis-acting regulators ofpolyomavirus late splicing; rather, splicing choicesappearto

be determined largely by the relative positions of splice sites within thetranscription unit.

MATERIALS AND METHODS

Restriction enzymes, T4 DNA ligase, DNA polymerase I

large fragment, T4 DNA polymerase, and T4polynucleotide kinase were from New England Biolabs and were used as

suggested by the manufacturer. RNase T2 was prepared as

describedbefore(31). [_x-32P]UTPwasfrom Amersham. Poly-omavirus strain 59RA was used and has been described

elsewhere(12, 13). The numberingsystemused here has been described elsewhere (4). All recombinant plasmids used here

were propagated in Escherichia coli JM83.

Cell culture and transfections. Mouse NIH 3T3 cellswere

obtained from the American Type Culture Collection. The techniques for their propagation and transfection have been described in detail elsewhere (4). Before transfection, all recombinant plasmids were cut with EcoRI to release intact viral genomes and dilute ligated with T4 DNA ligase as

described previously(1).

Constructs used. The startingconstructfor allwork, pPy*, consisted of the wild-type polyomavirus strain 59RAgenome

insertedatits unique EcoRI site intoamodifiedpUC18vector

that lacked all sitesin the multisite cloning regionexceptthe EcoRI site. Site-directed mutagenesiswasthenusedtocreate

a Sall site 80 bp upstream of the VP3 3' splice site (clone pPy*C3) andto createaBstEII site80bpupstreamof theVP1 3' splice site (clone pPy*C5). PCR was used to amplify

sequencesspanning the VP1 3' splice site (80 bpupstreamand

80bp downstream of the 3' splice site) while also generating SallandBamHIrestriction sitesattheends(Fig. 2). PCRwas

similarly usedtoamplifysequencesspanning the VP3 3' splice site (80 bp upstream and 80 bp downstream of the 3' splice site)whilealsogenerating BstEII and EcoRV restriction sites

at the ends (Fig. 2). Clone VP1,1 was created by digesting

pPy*C3 and the VP1 3' splice site PCR product with Sall and BamHI and ligating the digested PCR product with the large fragment of pPy*C3 (Fig. 2). Clone VP3,3 was created by

digesting pPy*C5 and the VP3 3'splice site PCR product with BstEII and EcoRV andligating the digested PCR product with the large fragment of pPy*C5 (Fig. 2). VP1,3was created by removing the BamHI-BsaBIfragment from VP1,1 and replac-ing it with the samefragment fromVP3,3.

Constructp5'ss/Sacwasmadeby insertingacopyof the late leader 5' splice site into theSacl sitelocated atposition 4367 inthe late region ofpolyoma strain 59RA. The fragment used

wasdescribed elsewhere(32) and contained 6 bpupstreamand 15 bp downstream of the late leader 5' splice site. Construct pC5BBdel is a deletion of the BamHI-to-BstEII fragment of

clone pPy*C5 (Fig. 2). Construct pRVHdel was created by deletionofanEcoRV-HincII fragment, leavingan81-bp VP1

exon. Construct pHdel carries a deletion of the HinclI

frag-*rnlDO

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I---mmmp.

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Wild

type

PCR usedtoremovesplice sites,withlinker ends

VP1 ,1

VP1ssfragmentinserted betweenSallandBamHl sites

VP3,3

VP3 ssfragmentinserted between Bst Elland EcoRVsite

VP33'ss VPI 3'ss

BatEll90X

'%

EcoRV Sall _ BamHl

5005>~~~~~ ~

~~~~~~~~~~~~~~~~~~~---VP13'ss VPI3'ss

500 4009

VP33'ss

s

5N

5000

VP1

,3

BamHl-EcoRV

fragment of VP1 3'ss VP3

3'ss

[image:3.612.94.515.79.389.2]

VP3,3inserted into VP1,1

FIG. 2. Splice site duplication and rearrangement constructs. Constructs

VPl,l,

VP3,3, andVPI,3 were made as described in Materials and Methods. Briefly, PCR was used to amplify theVP1 and VP3 splice sites (80base pairs upstream and 80 base pairs downstream of the splice junctions). PCR primersweredesigned such that the amplified fragments contained convenient restriction enzyme cutting sites at their edges. Site-directedoligonucleotide mutagenesiswasthen usedtointroduce restriction sitesneareachsplice site(circled). Theintroduced restriction site alongwithanaturally occurringoneallowed thesplice sitestobe removed andreplaced with PCR-generated fragments. This allowed the creation ofVP1,1 (twoVPI 3'splice sites[3'ss])andVP3,3 (two VP3 3'splice sites).InbothVP1,1andVP3,3, the spacing between the 3' splice sites was closetothespacingfound in the wild type. ConstructVP1,3wascreatedbyexchangingthe BamHI-BsaBIfragment ofVPl,lfor the BamnHI-BsaBI fragmentofVP3,3.

mentofthe lateregion. ConstructpPY-200Acarries a deletion ofapproximately200bpbetween the 5'splice siteofthe leader exon and the VP 3 3' splice site and was created by oligonu-cleotidemutagenesis.

Harvestingofcytoplasmic RNA.Cellswere harvested 44 to 48 hafter initiationof transfection. Eachplatewasrinsedwith Ix PBS- (phosphate-buffered saline

[PBS]

without

Mg2

and

Ca2+),

and RNA was harvested by the protocol described elsewhere (25). Briefly, cells were scraped into PBS and pelleted bycentrifugation at 1,000 x gfor 3 min. The pellet wasresuspendedinPBSandcentrifugedagain at1,000 xgfor 3 min.The cellpelletwasresuspended in 3 mlof NonidetP-40

lysis

bufferandleftonice for10 min.Nucleiwere removed by centrifugationat

1,000

xgfor 3 min. To the supernatant (the cytoplasmic RNA fraction) was added 1.42 g of guanidine thiocyanate. RNA wasseparatedfrom DNAbycentrifugation througha5.7 M cesium chloridecushionfor 20 h at

110,000

x g(6).The pelletedRNAs were drained well and resuspended

in 300

pl

ofRNase-free water.

Riboprobe preparation and RNase T2 protection assays. All riboprobeswere made fromclones by usinginvitro transcrip-tion withT3 or T7 RNApolymeraseand

[32P]UTP,

creatingan internal labeled probe. For constructs

VP1,1, VP1,3,

and VP3,3, thecorresponding HindIIItoEcoRI fragmentsspanning the latesplice sites weresubcloned into pBS+. The probe for

p5'ss/Sacwascreatedbycloning the EcoNI fragment into the SmaIsite ofpBS+. Theprobeclone forpRVHdelwasmadeby cloning thePstI-ApaI

fragment

surroundingthe altered region into the same sites in pBlueScript SK. Internally labeled riboprobeswereannealedto25 to 50 ,ug ofcytoplasmicRNAat 57°C for 15 to 20 h. Theresulting hybridsweredigestedwith 15

[LI

of RNase

TF/T,

(31)at37°C for2h. Afterphenolextraction and ethanolprecipitation, the samplesweredissolved in dena-turing dye, and protected bandswere resolved on 6% polyac-rylamide-urea sequencing gels. Band intensitieswere quanti-fied with a Betascope Blot Analyzer, and after

adjusting

for uridine content,relative percentageswere

assigned.

RESULTS

Theprocessingofpolyomaviruslatepre-mRNAsis shown in Fig. 1. The mRNA for VP1 is formed when the 57-base noncoding late leader exon isjoined to the 3'

splice

site at position4158. The mRNA for VP3 is formed when the leader isjoined tothe 3' splicesiteat position4733. The mRNA for VP2isformedwhen the leaderexon isnot

spliced

toeither 3' splice site. To measure the

proportion

of mRNAs for

VP1,

VP2, andVP3, we constructed

radioactive,

internally

labeled riboprobes which span the two message

body

3'

splice

sites. RNaseprotectionassayswere

performed

on

cytoplasmic

RNA

VP33'ss

400 N

46,

e

Aco

A

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1800 BATT ET AL.

A

501 489

404 353

242

190

B

DNA

-.o- nomessagebody splice

.m- upstream body splicesiteused

-downstream bodysplicesite used

2 3 4

upstream3'as downstream3' a

5' 5' 5'T

- undigested cRNA probe

nomessagebodysplice

upstreambodysplik siteused

downstreambody spike site used

FIG. 3. Relativesplice site use in thewild type and inconstructs VP1,1, VP3,3, and VP1,3. Viral constructs were liberated fromplasmid backbones,recircularized,and transfected into NIH 3T3 cellsasdescribed in Materials and Methods. (A)CytoplasmicRNA washarvestedat48 h andsubjectedtoRNaseprotectionassaysusingriboprobesdiagrammedinpanelB. Arrowsindicate thepositions expected for protectedbands that representalternativesplicing products. WT,wild type.Markersare32P-labeledMspI fragmentsofpUC18;sizesareindicated in nucleotides.

harvested from cellstransfected with wild-type polyomavirus genomes.Results from RNase

protection

assaysindicated that the relative

wild-type proportions

ofmVP1, mVP3, andmVP2 in

polyomavirus

late mRNA were80, 16,and4%, respectively

(Fig.

3A, lane 2; Table

1).

Todetermine whether the relative strength ofthevarious 3'

splice

sites dictatesthe ratio ofspliced productsin polyomavi-rus latepre-mRNA, constructs containing duplicated or rear-ranged splice sites were created (Fig. 2). First, site-directed mutagenesiswasused tointroduce unique restriction endonu-cleasesites upstream of each of the 3' splice sites. Use of these newsites inconjunction with naturally occurring unique down-streamrestriction sites allowed the release of each splicesite.

Second,

PCRwasusedto

amplify fragments spanning

eachof the wild-type 3' splice sites. The oligonucleotides used to

amplify

thesplice sites were designed to bind 80 bp on either

side

of the

splice

sites and to create convenient restriction

endonuclease sites on the ends of the

amplified fragments.

Finally,

theconstructscontainingnew

unique

restriction sites were digested with the appropriate enzymesto liberate frag-ments containing either the VP1 or the VP3 splice site, and these fragmentswere replaced with the

PCR-generated

ones. Thisallowed the spacing between the 3'

splice

sites in these constructs to be similar to that of the wild type. Construct VP1,1 contains two VP1 3'

splice

sites, construct

VP3,3

containstwoVP3 3'

splice

sites, andconstructVP1,3 contains both

splice

sites but reversed from their wild-type

positions.

Constructs VP1,1 and VP3,3 allowed us to investigate the relative use oftwoidentical 3'splicesites, and construct VP1,3, when compared with the wild type, allowed us to investigate the role of

splice

site

strength.

Results of RNase protection assays are shown in Fig. 3A, and

quantitation

is showninTable1.Values forrelative molar amountsof the variousprotected species have been

adjusted

to

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TABLE 1. Relative splicesitechoice induplication and

rearrangement constructs

Bandintensity(%yc)'

Splicesiteused Wild

type

VPl?l

VPI,3 VP3,3

None (unspliced) 4 1 3 2

Proximal 3' 16 36 44 15

Distal 3' 80 63 53 83

"The intensities of the bands (shown in Fig. 3A) were quantitated with a

Betagen Betascope blot analyzer and thenadjusted foruridine content (bands representinternally labeled fragments) to arrive at the values presented.

take into account the number of uridines present in the internally labeled protected fragments. For construct VPl,1 (Fig. 3A,lane 3), the proximal sitewasused36% of the time and the distal site was used 63% of the time, with the remaining 1% unspliced.Forconstruct VP3,3(Fig. 3A, lane 5), theproximal sitewasused 15% ofthe time and the distal site was used 83% of the time, with the remaining 2% unspliced. For construct VP1,3 (Fig. 3A, lane 4), the proximal site was used 44%ofthe time and the distal sitewas used53% ofthe time, with the remaining 3% unspliced. In repeated RNase protectionassays,percentages veryclosetothe datapresented wereobserved (datanotshown).RNaseprotectionassayswere also carried outwith shorterprobes (400 bp)which spanned onlythe distal splice site. Resultsobtainedwith these probes were consistent with those obtained with the longer probes (datanotshown). The conclusion from theseexperimentswas that splice site strength alone could not account for message ratios observed. This raised the possibility that splice site choice in this system is influenced bycis-acting sequences in the late transcriptional unit which lie outside the regions manipulated above.

The preceding results could be explained if there were an NRS which acted to suppress splicing to the proximal splice site, orif there were a positive cis regulatorofsplicing acting

A

on the distal splice site. Alternatively, 3' splice site choice could be determined by relative position. To determine whether there is a negative or positive regulator element in polyomavirus, several constructs were made. Negative cis regulation of theproximal 3' splice sitewastested byinserting a small DNA fragment containing 21 bp spanning the late leader 5' splicesite (as well as plasmid linker sequences) at a Saclsite 430 ntdownstream of the VP3 3' splice site (Fig.4A). If the VP3 3' splice site is negatively regulated, then this new exon should be skipped frequently, producing a 579-nt pro-tected band; however, if the splice site is not negatively regulated, the exon should not beskipped, producing a 43 1-nt protected band. An RNase protectionassay (Fig.4B) showed prominent protected bands of 431 and 254 nt but no band of 579 nt. The absence of a 579-nt protected band, along with carefulquantitation of thebands observed (almost equimolar levelsof the431- and254-nt species), revealed that theVP3 3' splice site is almost neverskippedinthis mutant.Further,this construct, as well as all others studiedhere, produced wild-type levels of late messages (data not shown). Results obtained from this experiment were consistent with the absence of an NRS for the proximal (VP3) 3' splice site. In a similar experiment reported previously (32), we showed that if the same5'splicesite was inserted within theVP1bodyexon, then the VP1 3' splice site was not skipped during pre-mRNA processing. Although this experiment indicatedthat the VP3 3' splicesite is notnegativelyregulated,itremainedpossiblethat theinsertion of the 5' splicesite in that constructstrengthened the VP3 3' splice site and/or overrode an NRS. Therefore, deletionsonboth sides of the VP3 3' splicesite were created. Construct pPY-200A (Fig. 5A) creates a 200-bp deletion upstream of the VP3 3' splice site, and construct pC5BBdel deletes 500bpdownstream of the VP3 3'splicesite(Fig. 5A). Construct pC5BBdel is discussed in greater detail in the following paragraph. Neither of these constructs significantly altered thesplicingratios (Fig.5B and C), arguing againstan NRS for the VP3 3' splicesite.

B

late VP3 VP1 leader 3 aa Sea 3ass

5'ss

0 CO,

Go

. 0.

Q

Q0

VP3splice only allsplice sites used VP1splice only

-skipleader and

VP3splicesites

mredictedbonds

riboprobe-1096 nt

946nt

41 nt 254n VP3spliceonly

useallsplice sites 254nt VP1spliceonly

579nt 2U nt skip VP3 3splice site only

<- probe-1096nt <- 948nt

-o.- 431nt

.-- 254nt

* S

2

FIG. 4. Analysis ofsplicing inconstructp5'ss/Sac.(A) The late region isdiagrammed, alongwith thepositionof the 5'splicesite(5'ss)inserted

inconstructp5'ss/Sac.An EcoNIfragmentspanningtheVP1 and VP33'splice sites(3'ss)wasusedtogeneratea1,096-ntspecificriboprobe for

RNase protectionassays. Sizes of bandsexpected for varioussplicingevents areshown.(B)Theriboprobeillustrated inpanelAwasused inan

RNaseprotectionassayofcytoplasmicRNAharvested 48 h aftertransfection ofNIH3T3cells withp5'ss/Sac.The absence ofa579-ntfragment indicates that the VP3 3' splicesite isnotskipped in thisconstruct.Almost all messagesappeartohave used allavailable splicesites.

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1802 BATT ET AL.

263

le Lr

5000

lateleader I

mVP33' as rr pPY-200 pCSBBd.l

4000 3000

A~~~~

nVPI3'5ss

pRVHd.l

at~~~~~~~"

I

aI

a.

VP2

-VP3 *.VP2+VP3

_

203nl .0;..

1 2 3 4 5

D

306n

203nr

190t-l

cc

VP1 -PI

[image:6.612.125.498.75.418.2]

ri-1 2 3 4 5

FIG. 5. Analysis of deletionconstructs.(A)Schematicdiagramof thepolyomaviruslateregion (topline)andsequencesdeleted in constructs

pPY-200A, pC5BBdel, pRVHdel,andpHdel.The VP1 and VP3 3'splicesites(3'ss)areindicated,andtheBstEIIsitepresentinconstructPy*C5

isshown inboldface. (B) RNaseprotection assaysofcytoplasmicRNAs fromcellstransfectedwith the wild typeandpPY-200A. Assayswere

performedasdescribed in Materials andMethods, usingthe sameprobeasinFig. 3A, lane 2. Markersare the same as inFig. 3.(C) RNase

protectionassaysofcytoplasmicRNAs from cells transfected with thewildtype(WT),pC5BBdel,andpHdel. Assayswereperformedasdescribed

in Materialsand Methods. Markersarethesame asinFig.3.(D)RNaseprotectionassaysofcytoplasmicRNAs from cells transfected with the

wildtype (WT), pHdel,andpRVHdel. Markersarethe same asin Fig.3.

To determine whether the distal (VP1) 3' splice site is positively cis regulated,several additionalconstructsweremade (Fig. 5A).Figure 5C shows theresults ofanRNaseprotection

assay using two of these constructs and a VP1 3' splice

site-specific probe. Analysis of cytoplasmic RNA from wild-type-transfected cells (Fig. 5C, lane 1) revealed 76% VP1 splicing, with 24% mVP2 plus mVP3. Construct pC5BBdel lackssequencesfromBamHItoBstEIIinconstructpPy*C5.In thisconstruct,theVP1 andVP3 3'splicesitesareseparatedby 136 nt. Analysis of cytoplasmic RNAs from cells transfected with thisconstruct revealed67%VP1splicing,with 33% mVP2 plus mVP3 (Fig. 5C, lane 3). Construct pHdel lacks a504-bp

HincII fragment from the late region (Fig. 5A). Analysis of cytoplasmic RNAs harvested from cells transfected with this

construct(Fig.5C,lane4)revealedthesameratios(76%mVP1 and24% mVP2plus mVP3)asfoundfor thewildtype(lane 1). Figure 5D shows the analysis of splicing ratios in constructs

pRVHdelandpHdel. Construct pRVHdel lacks 1,147 bp of the VP1 bodyexon(Fig. 5A). For thisconstruct,aprotected band

of 81 ntrepresents splicing to the VP1 3' splice site, while a

protected band of 196nt represents unspliced VP2-plus-VP3-spliced molecules. Figure 5D, lane 4, shows that construct

pRVHdel uses the two 3' splice sites almost equally (52%

mVP1and 48% mVP2plus mVP3).Incontrast,pHdelshowed

asplicingratio similar tothatobserved for the wild type(Fig. SD,lanes 2 and3). Theconclusion from these experiments is

thatonlymodesteffectsonVP13'splicesite choiceresultfrom

deletionsinthe polyomavirus lateregion,with theonly signif-icantchangeinrelativesplicinglevelsbeingobserved whenthe

terminal VP1bodyexon wastruncatedtoonly82nt. That these

dataarguefor the absenceofapotentpositive regulatorofVPl

splicingwillbe addressed further below. DISCUSSION

Alternative3'splicesiteselectionduringRNAprocessingof

polyomavirus late pre-mRNA molecules generates multiple mRNAs.The promoter-distal VP1 3' splicesite is chosenthe

majority of the time. Here we have examined how this

pre-ferential choice is made. Is the VP1 3'splicesitestrongerthan

the VP3 3' splice site, thus allowing it to compete more

effectively forthe splicing machinery, or are there cis-acting

sequencesinthe lateregionthatinfluence 3'splicesite choice?

Asimple splice site strength model would predict that when

twoidentical 3' splice sitesare present,such asfor constructs

VP1,1 andVP3,3, theywould be used equally; likewise,when

A

B

J. VIROL.

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the sitesarereversedintheir

order,

then the ratioof

splice

site

useshould alsobe reversed.Thiswas notobserved.

Instead,

in this system,the distal 3'

splice

sitewas

always preferred

over the

proximal

site. That

splice

site

strength probably plays

a minor role can best seen

by comparing

the relative

splicing

frequencies

ofconstructs

VP1,3

and VP3,3

(Fig.

3 andTable

1).

VP1,3

usesthe

proximal

site40% ofthe timeand the distal site 60% ofthe

time,

while

VP3,3

usesthe

proximal

site 15% ofthe time and the distalsite 80% of the time.The

ability

of the VP1 3'

splice

sitetobe usedmore

frequently

thantheVP3 3'

splice

site at the same

position (proximal position)

argues that the VP1 3'

splice

site is somewhatstrongerthan the VP3 3'

splice

site.

However,

it should be

pointed

out that in absolute terms,both 3'

splice

sitesare

likely

tobeweak. This conclusion is basedon the observation that

despite

the

pres-ence oftwo 3'

splice sites,

about 5% of the mRNA remains

unspliced

(mVP2). Further,

in numerous attempts, we have been unabletoobserve

polyomavirus

late pre-mRNA

splicing

in vitro

(32a).

Asa

splice

site

strength

modelcouldnot

explain

the results

observed,

the

question

remained as to

why

the distal site is

preferred.

The ratios observed could result from either cis

downregulation

of

splicing

tothe

proximal

site

(such

as

by

an NRS

element)

or cis

upregulation

of the distal site

(by

a

positive element). Analysis

of a number of additional

con-structs revealed that a strong

positive

or strong

negative

cis

regulator

of either

splice

site is

unlikely.

Insertion ofa5'

splice

site downstream of theVP3 3'

splice

sitebut upstream ofthe VPI 3'

splice

site

(construct p5'ss/Sac; Fig. 4A)

revealed no apparent

downregulation

oftheVP3 3'

splice

site. In

fact,

this 3'

splice

sitewas nowused

virtually

100% of the time

(Fig. 4B),

indicating

that it is an efficient 3'

splice

site in the proper

context. ApotentNRS elementwouldmost

likely

have ledto

frequent

skipping

of the VP3 3'

splice

site. Deletions both upstream and downstream of the VP3 3'

splice

site

(Fig. 5)

confirmed the absence of an NRS. A series of deletion

constructs was then used to argue

against

positive

cis

regula-tion ofthe distal 3'

splice

site. As seenfromthe aggregate of results

presented

in

Fig.

5,

deletion of

essentially

allsequences between the VP3 3'

splice

siteand the late

poly(A)

sitedonot alter the fact that the distal site is chosen

preferentially.

Reducing

the distancebetween the VP3andVPI 3'

splice

sites

(construct pC5BBdel)

resultedina modest

change

in relative

splicing

ratios. Construct

pHdel (500-bp

deletion in the VPI

exon)

gave ratios identical to

wild-type ratios,

while a

large

deletion which truncated the VP1

body

exon to 81 nt

(con-struct

pRVHdel)

resulted in

nearly equal

use of the two

alternative 3'

splice

sites. For this latterconstruct,we donot know whether thesomewhataltered

splicing

ratio is duetothe presenceofaweak

positive regulator (which

has been deleted inthis

construct)

or tothe fact thatnowthe terminalVPI exon is too short

(82 nt)

to be

efficiently

processed

or

accurately

detected in our RNase

protection

assays.

Attempts

to

study

constructswith

intermediate-length

VP1 exons,or exons

con-taining heterologous inserts,

failed due to the activation of

cryptic splice sites,

making analysis

of

splicing

ratios

impossible

to

interpret.

Consistent with the lack ofa

positive

or

negative

regulator

of

polyomavirus

late

splicing,

wehave alsoobserved that a 6-nt deletion of the VPI 3'

splice

site leads to an increase in the amounts of mVP2 and

mVP3,

and a 6-nt deletion of the VP3 3'

splice

site leads to an increase in the

amountsof mVP1 and mVP2

(data

not

shown).

The data

presented

above,

while

allowing

us to rule out

strong

positive

or

negative

cis

regulators,

do not allow us to rule out the existence of several weak

regulators placed

in different areas and which work

additively

or

synergistically.

In

the work describedhere, we also did not rule out thepossibility of splice site regulation by two small regions at the extreme ends of the late viral transcription unit. A 45-bp region immediately downstream of the late leader5' splice site was notremoved, nor was the 50 bp immediately preceding the late polyadenylation signal.

The results discussed above show that the relative position-ing of splice sites in the transcript appears to be the most importantdeterminant ofsplice site choice in the polyomavirus late system. Even when two identical 3' splice sites exist in the same transcript, the distal site is preferred. This isconsistent with theinvivofindingsof Kuhne et al. (27),who found that constructs which contain two rabbit ,3-globin 3' splice sites always used the downstream site. However, it contradicts results of Lang and Spritz (29), who concluded that the upstream 3' splice site, in human

y-globin

constructs contain-ing duplicated 3' splice sites,wasalwayschosenin vivo.Finally, Reed and Maniatis (39) concluded from in vitro studies of human 3-globinconstructscontaining duplicated 3' splicesites thatthe upstream 3' splicesite waschosen ifthe second exon was205 nt inlength,and the downstream site was chosen if the secondexon wasshorter than 55 nt. However, these research-ersalso found that exonic sequences couldinfluence which site waschosen.

One working model, which is consistent with the results reported here, is that the length of the late terminal exon influences theprocessingof the latepre-mRNA.Robberson et al. (40) postulated thatexonsrather thanintronsaretheinitial units of definition in pre-mRNA splicing. In their model, 3' splice sites are not normally used unless the downstream borderof theexon(eithera5' splice site for internalexons or apolyadenylation site for terminal exons) is also recognized. More recentevidencehasbeen presentedthat, in fact, eukary-otic exonsarescanned in a 5'-to-3' directionduring splicesite

selection,

and that exonskippingorsplice site skipping might result when exons are excessively long (36). Interestingly, in p5'ss/Sac,the exon createdbythe insertion ofthe 5'splice site is 431 nt long. While this is somewhat longer than the approximately300-nt length limit reported for most (but not

all)

internalexons(36), it is nevertheless efficiently includedin maturemRNA. Inpolyomavirus late transcripts, theVP1 body exon is greater than 1 kb in length, already longer than the great majority of vertebrate 3' exons (22). This, coupled with the fact that both the VP1 3' splice site and the viral late polyadenylation signal may beweak, wouldlead to inefficient splicing of mVPI molecules, which is observed. However, otherresults shown heresuggestthatamodelrelying solelyon the

spacing

of 3' splice site and the poly(A) signal cannot simply account for the results observed. Relative position, rather than spacing, appears to be the major determinant of splice site choice in the polyomavirussystem.

Although relative splice site position is of primary impor-tance to

splice

sitechoiceinthis system,splicing ratiosarealso subtly influenced byavariety ofotherfactors,such asrelative

splice

site strengths and the spacing between splice sites. We are

currently investigating

the role of each oftheseparameters in polyomavirus late pre-mRNA processing.

ACKNOWLEDGMENTS

This work was supported by grant CA45382 from the National CancerInstitute.

We thankother members of thelaboratoryforhelpfulcomments on

the manuscript and N. Barrett for technical assistance in the early phaseof this work.

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http://jvi.asm.org/

1804 BATT ET AL.

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