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0022-538X/85/050383-09$02.00/0

Copyright C) 1985, American Society for Microbiology

Organization of Early Region lB of Human Adenovirus Type 2:

Identification of Four

Differentially Spliced mRNAs

ANDERS VIRTANEN* AND ULFPETTERSSON

Departmentof Medical Genetics, Biomedical Center, S-751 23 Uppsala, Sweden

Received24October 1984/Accepted 3 January 1985

ThemRNAsfromearlyregion1B of adenovirustype2 have been studied by Northern blot,Si nuclease, and

cDNAanalysis. Two novel mRNAs, designated14Sand14.5S, have been observedinadditiontothepreviously identified 9S, 13S, and 22SmRNAs. They are 1.26and 1.31 kilobases long anddiffer from the13S and 22S

mRNAs in being composed ofthree exons instead oftwo. Their twoterminal exons are thesame as those

present inthe 13S mRNA, whereas themiddleexon is unique toeach of the twonovel mRNA species. The structuresof the14S and 14.5SmRNAs allow theprediction of their coding capacities: bothmRNAspecies, like the 22Sand 13S mRNAs, containanuninterrupted translational readingframeencodinga 21,000-molecular-weight (21K) polypeptide. The 14S mRNA can, in addition, encode a 16.5K polypeptide which shares

N-terminal andC-terminalsequenceswiththe55Kpolypeptide, knowntobeencoded by the 22SmRNA. The

14.5S mRNA species encodes a hypothetical 9.2K polypeptide which has the same N terminus asthe 55K polypeptidebutauniqueC terminus. The two mRNAs differ in their kinetics ofappearance;the14.5SmRNA ispreferentially expressedlate afterinfectionin contrast tothe 14S mRNA,which is present inapproximately equalamountsearlyand late afterinfection. Taken togetherwithpreviouslypublished informationtheresults

suggestthatearly region 1B ofadenovirustype 2encodes fiveproteinsin addition tovirionpolypeptide IX. Thesehavepredicted molecularweights of 55,000, 21,000, 16,500, 9,200, and 8,100.

The DNAtumorviruses are important tools to elucidate the mechanisms ofmammalian cell transformation. Avast

amountof knowledgehas accumulated inrecentyearsabout the transforming region of human adenoviruses (ad). It is nowwellestablishedthat thetransformingregion is located

within the left-most 11% of the adenovirusgenome (18, 22, 45),and thetransforminggenesfromad2,ad5, ad7,andadl2 have beensequenced (11, 15, 20, 25, 41, 46).The

transform-ing region includesseveralgenesandcanbesubdivided into

twoseparatetranscription units, early regions1A (ElA)and

1B (E1B) (51). The mRNAs which are transcribed from

regionsElA andE1B have been studiedbyseveral methods

including electron microscopic heteroduplex analysis (14, 26), S1 nuclease analysis (10), and cDNA analysis (35, 36, 47). Sofar, sixunique mRNAs have beenassignedtothe El

region. Region ElA directsthesynthesis of 13S, 12S, and 9S

mRNAs which are all overlapping. 22S and 13S mRNA

species are transcribed from region E1B. In addition, an unspliced 9S mRNA encoding virion polypeptide IX is transcribed from a separate promoter in region E1B at intermediate and latetimes after infection (3, 37, 38).

In the present study we have reinvestigated the mRNAs fromregionE1B ofad2. Thereasonforthisundertakingwas

that the previously detected EiB mRNAs of ad2 do not account for all polypeptides which have been assigned to regionE1B (17, 23). The results makeit possibletoidentify two hitherto uncharacterized E1B mRNAs each of which appearsto encode aunique polypeptide.

MATERIALS ANDMETHODS

Preparation of RNA from ad2-infected cells. RNA was

extractedasdescribedbyBrawermannet al. (12) from

ad2-infected HeLa cells(39).The RNAwasextractedat8 and 18

hpostinfection. RNAwasalso preparedfrominfected cells which had beenmaintainedinthepresenceof cycloheximide

* Correspondingauthor.

(25 ,ug/ml)between 2 and 10 h after infection. Polyadenylate [poly(A)]-containing RNAwas isolated fromtotal cytoplas-mic RNA by affinity chromatography on oligodeoxythy-midylate-cellulose.

Preparation of plasmidDNA. Plasmid DNAwasextracted from E. coli cells by usingthe potassium-acetate

precipita-tion procedure describedbyTanaka and Weisblum (42). Northern blot analysis.

Formaldehyde-formamide-dena-tured poly(A)-containing RNA was fractionated in a 1.4% agarose gel containing2.2 M formaldehyde (28). The RNA

wassubsequentlyblottedontoanitrocellulosefilter,and the

filter was hybridized at 42°C for 16 h with nick-translated 32P-labeled fragments (21, 43). The hybridization solution contained 50% formamide, 50 mM HEPES (N-2-hydroxyl-piperazine-N'-2-ethanesulfonic acid[pH 7.4]), 1x Denhardt solution (0.02% each ofFicoll400 [Pharmacia Fine

Chemi-cals], bovine serum albumin, and polyvinylpyrollidone-360 [Sigma Chemical Co.]), 250 ,ug ofyeast RNA per ml, 3x SSC(1x SSC consists of 0.15 M sodium chloride and 0.015 M sodium citrate [pH 7.0]), 100 ,ug ofsingle stranded calf

thymus DNA per ml, and 0.1% sodium dodecyl sulfate. Afterhybridization,the filterwaswashed threetimes for 40 min in 0.1x SSC-0.1% sodium dodecyl sulfate at 50°C

beforeautoradiography.

S1 nuclease analysis. The protocolof Berk and Sharp (9) was followed for S1 analysis, using 5'- or 3'-end-labeled fragmentsasprobes (Fig. 1). Nucleotidesequencingladders wereseparated on thesamegelas the Si-resistant material (49).

Primer extension. A synthetic oligodeoxynucleotide (5'-dATGGGTTTCTTCGC-3') (7) was used to prime the syn-thesis of single-stranded cDNA by avian myeloblastosis

viruspolymerase.TheprocedureusedforpreparativecDNA

synthesis was amodification ofpreviouslypublished

meth-ods(1, 19, 31).Theprimer (90 pmol)was5' endlabeled with

32p (32)andsubsequently annealedto 100,ugof RNAat4°C

in200,ulofhybridizationbuffer(5MTris-hydrochloride [pH

383

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(2)

XO 0n (0 c (D X') 0

a) LI C) - _ CM t

uz ,- 0 e~NN\ X

\\\, \ \N \ \

lIIl t I

9S (0.;54)§,

12S (0.8))

I

~~D2'

,

13 S (1.02)'

D3 Al

CD 0

lq

I

'I13S(1.02)

J 14.5S(1.31) q 14S (1.26)

1 22S(2.28)

J 9 S (0.48)

2 3 4 5 6 7 8 9 10 11 m.u.

EIA

Sacl

268 322

tNcol2/Smal

.4.-.--- * 335bp. cDNA

EIB

Ncol / Hinc

11 1

400 164l

/\.4 Ncol/Pst(22ScDNA)

* Bgl II*/Xbal

- Bglll*/Hhal cDNA Sacl*

PstI *

FIG. 1. Aschematic drawingillustrating the mRNAs which are transcribedfrom the r-strand of region El.The three differentreading frames are indicated with open, filled, and hatched boxes. Donor (D) and acceptor (A) sites for splicing are also shown. Numbers at the top of thefigure refertopositions in the sequence ofGingeras et al. (20).Thenumbersinparenthesis indicate the length in kb of each individual mRNA.Atthe bottom of thefigure the probes used for Northernblotanalysis(Fig. 2) and

Si

nuclease analysis (Fig.3and 4) are shown. The strategy for cDNA synthesis (Fig. 5) witha specific oligonucleotideor purified single-stranded fragments as the primers is also shown. Asterisks indicate32plabeling at the 5' end of the probes and theprimers. The5'-and3'-labeledends are locatedatthefollowingpositions:

NcoI,2204 and3664;BgII, 3226; Sacl, 1771;PstI, 1834.Thefragments which were usedasprobes in the Northern blotanalysis are located betweenthefollowing positions: 268, 1780through 2048; 322, 2246 through 2569; 400,2943through 3343; 164, 3343 through 3507.

7.9],

0.25 mM

EDTA). After

20

min,

200 ,u

of

2x reverse

transcriptase buffer (0.2

M

Tris-hydrochloride [pH 8.3],

20 mM

MgCl2,

0.28 M

KCI,

1 mg

of dATP

per

ml,

1 mg

of dGTP

per

ml,

1 mg

of

dCTP

per

ml,

1 mg

of dTTP

per

ml)

was

added. Themixture was

divided

into

50-plI

aliquots, and

30 U

of

AMV

polymerase (obtained from

J.

Beard)

wereadded

toeach

aliquot.

The cDNA

synthesis

was

performed

at

42°C

for

2h. To stop

the

reaction,

the

aliquots

were

pooled,

and 16

pAl of

0.5 MEDTA and 100

pul

of 0.3 M NaOH were

added.

After alkaline hydrolysis

(1 h at

65°C),

the pH was

neutral-ized

with 100pA

of

1 M

Tris-hydrochloride

(pH 7.9) and 100

pA

of

0.3 M

HCI. After

phenol

extraction, the

cDNA was

purified by spun-column chromatography on Sepharose CL-6B

(Pharmacia

Fine

Chemicals).

The

percolate

was

precipi-tated with

ethanol,

and the cDNA was

dissolved

in

sample

buffer

(80% formamide,

10 mM NaOH, 1 mM EDTA) and

separated

on a

6% polyacrylamide gel containing

7M urea as

described

by Maxam and

Gilbert

(32). The sameprocedure wasused when 100 ,ugof sucrose

gradient

size-fractionated

RNAwas

analyzed.

However, RNA from each

fraction

was

hybridized

with 10 pmol of

5'-end-labeled primer

in 25 pAof

hybridization

buffer. After

hybridization,

25pAof 2xreverse

transcriptase buffer

and 30 U of avian

myeloblastosis

virus

polymerase

was added. The

resulting

cDNAs

from

each

fraction

were analyzed by polyacrylamide gel

electropho-resis. A slightly modified procedure was used when

re-striction fragments were used as primers: 5'-end-labeled

single-stranded DNA fragments (4 pmol) were purified on

polyacrylamide gels containing7 M urea(32). After purifi-cation,theprimerwasmixed with50 to100pug ofRNA and ethanolprecipitated.Theprecipitatewassolved in 30ptlof a

buffercontaining 83mMTrishydrochloride(pH 7.5) and167 mM NaCl. Hybridization was performed in sealed glass capillariesfor 2 h at65°C. After

hybridization,

the material

was ethanol

precipitated,

and reverse

transcription

was

performed asdescribed above.

Sequence analysis. The method of Maxam and Gilbert (32)

wasused for sequence analysis. RESULTS

Studies of the E1B mRNAs by Northern blot analysis. Previous studies have identified two major early mRNA

species

in

region

E1B,

a22S

(2.28-kb),

anda 13S(1.02-kb) mRNA

(10,

14, 26,

36).

To

search

for additional mRNA

species

from this

region

of the ad2 genome, we performed

an

analysis by

the Northern blot

technique. Cytoplasmic

poly(A)-containing

RNA was fractionated

by

electropho-resis in a

denaturing

agarose

gel

and

subsequently

blotted

onto a nitrocellulose filter. Two different

preparations

of

RNA were

investigated: early RNA, prepared

10 h

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mRNAs OF ADENOVIRUS TYPE 2

Sac

CH L

400

CH L

922 S S

13S

f

"A

9S _

322

CH L

.

268 164 NcoI/Hincil

CH L CH L CH L

_

.3

t

O

m

Io _6

FIG. 2. Northern blot analysis ofE1BmRNAs. Two different RNA preparations were investigated. CH, RNA preparedfrom infected cells which were maintained in cycloheximide; L, RNA prepared 18 h postinfection. The positions of the probes (Sacl, 400, 322, 268, 164,

NcoI-HincII)areshown inFig.1.Sizes of the RNAs are given in Svalues. The mRNAsin the 14Sregion of lateRNApreparationsareonly revealed afterlong exposure.

fection from cells treated with

cyclohexiimide (25

,ug/ml),

and late

RNA, prepared

18 h

postinfection.

The E1B RNAs were

visualized

by

hybridization

with a set of

32P-labeled

restriction

enzyme

fragments (Fig. 1).

A

Sacl

fragment

located

between coordinates

4.9 and 10.1, thus

covering

almost the

whole

E1B

region, hybridized

tothree mRNAs

in

theearly RNA

preparation.

The late mRNA

preparation,

in contrast,

contained four

species complementary

tothe DNA

probe, corresponding

in

size

to

22S,

14S,

13S,

and 9S

(Fig. 2,

lane

Sacl,

and

data

not

shown). The

22S, 14S,

and

13S

species

werepresentin

both

RNA

preparations, whereas

the

9S

mRNAwas

exclusively

detected

in the late RNA

prepa-ration. The 22S and

13S mRNA

species

arethemajor

early

E1B mRNA

species, which

have

been

characterized in detail

before (36), whereas

the

9S

mRNA encodes the

virion

polypeptide

IX

(3, 38). The 14S

species

represents a

novel

E1B mRNA

which

has not

been characterized.

To mapthe 14S RNA more

precisely,

we used

five different

DNA

fragments which

had been

generated from

region

E1B

by

RsaI and

NcoI-HincII cleavage (Fig.

1)as

probes for

hybrid-ization.

The

fragments, designated

400, 322, 164,

268,

and

NcoI-HincII,

hybridized

to

the 22S

RNA, whereas

fragments

268 and

NcoI-HincII, which

arelocatednearthe 5'end(268)or nearthe 3' end

(NcoI-HincII) of

region

E1B,

were the

only

ones

which

hybridized

tothe13S mRNA

(Fig. 2).

The novel 14S mRNA was revealed

by

three

fragments, 268,

164, and

NcoI-HincII.

Taken

together, the results suggested

thatthe

14S

mRNA

like

the 13S mRNAs

contained

sequences

from

the 5' and the 3' end

of

region

E1B

(Fig. 2,

lanes 268 and

NcoIlHincII)

but also a characteristic set of sequences located around coordinate 9.5

(Fig.

2, lane 164). Itsstructure

resembles that

of

the 13S mRNA, but it includes additional sequences which are located inside the

large

intron of the 13S mRNA. We show below thatthe 14SmRNAconsists

of

twodistinct

species.

Spliced

structures of the novel mRNAs. The results ob-tained

by

Northern blot

analysis

suggested that there

might

bean additional shortexon in the 14S mRNA ascompared

with

the structure

of

the 13S mRNA.

Judging from

the structure

of

the E1B mRNAs we have

observed

in

adl2-infected cells (48),

we

also

had reason to believe that one

intron

removed

from

the

14S

mRNA was the same as that removed

from

the22S mRNA. To

study

the structure

of

the 14S mRNA, we

used

a

cDNA clone

representing the 22S

mRNA

(clone

272) (36)asthe

probe for

Si nuclease analysis;

DNA

from clone

272was 5'end labeled

after

cleavage

with

NcoI,

and the 5' part

of

the cDNA was

isolated after

PstI

cleavage

(Fig.

1). Three RNA

preparations

were

investi-gated:

late

RNA,

RNA

synthesized in

the presence

of

cycloheximide (25

,ug/ml),

and RNA

prepared

8 h

postin-fection

in the

absence of

any

drug.

The

results

(Fig.

3)

confirmed

our

assumption that

a mRNA

species

existed

which lacked the

intervening

sequence that

is

missing in

clone

272 and revealed two novel

splice

acceptor

sites,

located

310 and

370 nucleotides

tothe

left of

the NcoI

site

at

coordinate

10.0. Both

splice

acceptor

sites

were

detectable

in all three RNA

preparations which

were

analyzed,

al-though

the onemore

distant from

the NcoI

site

was much less

frequent

in RNA

samples which

were isolated

early

after

infection

in the absence

of

cycloheximide (Fig. 3).

To

locate

the two

novel

splice

acceptor sites at the

nucleotide level,

we

carried

out

S1

nuclease

analysis

witha

BgIII-XbaI fragment prepared from

the

ad2

genome as the

probe.

The

fragment

was5'end labeledatthe

BglII site (Fig.

1). The

Si-resistant

material

was

separated

on a

gel

in

parallel

with

sequencing ladders starting

at

the

same

BgIII

cleavage site

(Fig.

4).

The

results showed that the

two

splice

acceptor siteswere

located

at

nucleotides

3212

and

3770 in the ad2 sequence (20).

Sequence

combinations which are

characteristic for

splice

acceptor

sites, including

the AG

dinucleotide

(13, 33), were found in the sequence at these

positions.

To

identify

the

splice

donor

site(s)

towhich the novel E1B acceptor sites are

connected,

we

performed S1

nuclease

analysis

with

3'-end-labeled

probes (Fig.

5). The

analysis

identifiedonlyone

unique splice

donor

site, being

thesame asused

for

splicing of

the 13S mRNAs.

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[image:3.612.149.474.67.280.2]
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310-

_I-"' -298

All major E1B transcripts have identical 5' termini. The DNA sequence of region ElB reveals two long open

trans-lational reading frames (ORFs) encoding 21K (21K-ORF) and 55K and(55K-ORF) polypeptides (20). Both ORFs can M be used in their

entirety

in the 22S

mRNA,

whereas

only

the shorter ORF is uninterrupted in the 14.5S, 14S, and 13S mRNAs. Consequently, the ElB mRNAs are structurally polycistronic, and it has been experimentally verified that the 22S mRNA also isfunctionally polycistronic, encoding both a 21K and a 55K polypeptide (11, 17, 30). This is a

surprising finding,

sinceeucaryotic mRNAs arebelieved to

be

functionally monocistronic

(27). Since it cannot be com

-516 pletely

ruled

out

from previous

results that a

small

splice is located near the 5' end, deleting the first AUG in the 21K-ORF from certain mRNAs, we have analyzed the 5'-end structureof the EBB mRNAs in greater detail. Two different methodswere

used,

Si nuclease

analysis

and DNA sequence

analysis

of cDNA copies. For the Si analysis, an

NcoI-SmaI fragment, 5' end labeled at the NcoI cleavage

_~ -396

A-,_

T T

G T C

3 212-C T C

G GA CT

....

..

19

a

7i..-.

ft

..

I

M

9 -154

[image:4.612.60.298.67.436.2]

4

FIG. 3. S1nuclease analysis of E1B mRNAs with fragments272 Ncol/PstI and Ncol-SmaI fragments from cDNA clone 272as the

probes. The following RNA preparations were investigated: lane

CH, RNA prepared from cells maintained in the presence of cycloheximide; lane 8HPI, RNA prepared 8 h postinfection; lane 18HPI, RNA prepared 18 h postinfection; lane-, no viral RNA

added. Lane M, DNA fragments used as size markers. Sizes are

given in nucleotides.

The structures of the two novel mRNAs were moreover confirmedbycDNAanalysis.ABglII-HhaI fragment,5' end labeled at the BgIII site, was used as the primer for this analysis (Fig. 1). Poly(A)-containing RNAs from three

dif-ferent RNA preparations were used as described above.

After the reverse transcription was completed, halfofthe cDNApreparationwasdigested byrestriction endonuclease

HaeIII.HaeIIIdigestionrevealed threemajor products 167, 143, and 85nucleotides long (Fig. 6). Thepredicted sizes of

HaeIIII-digested cDNAs from E1B mRNAs should be 167

nucleotides for the 22S mRNA and 143 and 85 nucleotides, respectively,for thetwonovelE1B mRNAs if thedonorsite atnucleotide2249 is linkedtothetwospliceacceptorsitesat nucleotides 3212 and 3270. Thus, it can be concluded that

thetwonewly identifiedacceptorsiteswereconnected with thesamedonorsite thatwas used tosplice the 13SmRNA. The predicted structures of the two mRNA species are

shown in Fig. 1. Their estimated lengths are 1.26 kb (14S)

and 1.31 kb (14.5S).

of 0 -75

G

A A

C

T

c 3270-G

G

G

[image:4.612.316.554.274.650.2]

0L

FIG. 4. Determinationof thespliceacceptorsites in the 14S and

14.5S mRNAsatthenucleotide levelwith theBgIII-XbaI fragment

as the probeforS1 nuclease analysis (Fig. 1). Sequencingladders

were run in parallel. The nucleotide sequences around the splice

acceptorsitesareshown.CH,RNApreparedfrom cells whichwere

maintainedincycloheximide;-, noviral RNAadded; M,marker DNAfragments.Sizesaregivenin nucleotides.

2

-7

2

t1' C ) 1/F )

II

-L -L D

NCOM I/s) ~vli

C_.

C-GO

1~-r

I-Ico

500

-

430-

410-IIIIIIpl

370-

_l

_.

-344

tfL

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(5)

site

at

coordinate

6.1, was

used

as the probe (Fig. 1). The results revealed a major 500-nucleotide-long fragment (Fig. 3)

with its

3'

end precisely

at the map

position

of the E1B cap

site

(6, 24). A minor (less than

0.1%)

approximately 410-nu-cleotide-long fragment was also observed. However, we were unable to analyze this fragmentfurther due to its low

abundance.

For the cDNA analysis, a synthetic

14-nucleotide-long

oligomer

was

used

as a

specific primer.

The

primer

was complementary to nucleotides 2021 through 2034 of the ad2 genome, a

region located

a

few nucleotides downstream

from

the

first

AUG

in

the 55K-ORF

of region E1B (Fig.

1). Individual fractions from size-fractionated

poly(A)-containing

RNA were used as the template for the avian myeloblastosis virus polymerase. Two different RNA

prep-arations

were

investigated:

RNAprepared 10 h

postinfection

from

cells treated with cycloheximide (25

,ug/ml)

and late

t_ a:

I ---I I

CH

8HPI

18HPI

167

-143

-.

Sk I

t.

85

--

516

too

-

e__

420--a

- 396

FIG. 6. cDNA analysis with a 5'-end-labeled BglII-HhaI frag-ment(Fig. 1)astheprimer. The differentRNAs(CH, 8Hpi, 18Hpi)

usedastemplatesaredefined in thelegendtoFig.3. LaneHaeIII, cDNAdigested by restriction endonucleaseHaeIII;laneTot,cDNA before digestion with HaeIII. The fragment which results from HaeIIIdigestion of thecDNAcorrespondingtothe14SmRNAis85 nucleotides long; it is locatedbetweenthe5'-end-labeledBgIII site atnucleotide3326and theHaeIIIsiteatnucleotide 2220. The cDNA of the 14.5S mRNA gives rise to a 143-nucleotide-long fragment

after HaeIIIdigestion.Thefragmentis located between nucleotides 3226(BglII)and2220(HaeIII).The cDNAof the 22SmRNAgives

rise to a 167-nucleotides-long fragment between nucleotides 3326 (BglII)and3159(HaeIII) after HaeIIIdigestion.

-298

FIG. 5. Identification of the splice donor siteat position 2249.

The donorsite is used for maturation of the 13S, 14S,and 14.5S

mRNAs. Fragments 3' end labeled at the PstI and Sacl cleavage

sites(Fig. 1)wereusedastheprobes for Si nuclease analysis. The

same RNA preparation as described in the legend to Fig. 3 was

analyzed.

RNA

prepared

18 h

postinfection.

The

primer

waslabeled at its 5' end, which made it possible to directly sequence the

extension

products

after

purification

ina6%

polyacrylamide

gel.

The extension

yielded

several bands upon

autoradiog-raphy, the

most

prominent of which

were 75 and

335

nucleotides

long

(Fig. 7).

The20 most

prominent

bands

were

excisedfrom the gel, and their nucleotide sequences were

determined

by

the method

of

Maxam and Gilbert

(32). By

comparing

the cDNA sequences with the DNA sequence

of

region E1B from ad2,

it was

possible

to conclude that the

335-nucleotide-long

extension

product

ended

precisely

atthe

E1B

cap site (6, 24). This extension

product

was

predomi-nantwhen RNAfrom both the 13-to-14S and the 22S

region

of

the

gradient

wasusedasthe

template, suggesting

thatit is common toall size

classes of

E1B mRNA. The

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EARLY

28S 18S 4S

Abe ** _od_ _wMO _m-mdo _*so__00 fto

I *

LATE

28S 185 4S M

_ _0.- - _ft'%do__t_*- 0 - 1

6-5

16

* - 396 * -344 e-293

;-221

'9- 54

s-~ ~t

----

-'-

I

[image:6.612.120.498.65.484.2]

1

-75

FIG. 7. cDNAanalysiswitha 14-oligodeoxynucleotide-long synthetic fragmentasthe primer. Sucrosegradient size-fractionated RNA wasusedas atemplate. Two RNApreparationswereinvestigated:RNApreparedfrom infected cells whichweremaintainedin thepresence

ofcycloheximide; and lateRNAprepared18 hpostinfection.Thepositionsof28S, 18S,and 4S RNAsareindicatedatthetop. LaneM,Size markers. Sizes aregiveninnucleotides.

tide-long extension product, in contrast, was not of viral

origin since its sequence could not be found within the ad2 genome; it probably represents a nonviral cDNA copy caused by cross-hybridization between the primer and a

cellular RNA. The bands migrating between the 335- and

75-nucleotide-long bands were all found to be the result of premature termination, whereas extension products longer

than 335 nucleotides were found to originate from early region 4, because the primercouldcross-hybridize withone or moreof the E4 mRNAs.

From the combined results, we concluded that at least 99.5% of all early E1B mRNAs have a common 5' end,

locatedatnucleotide 1699. This conclusionisalso valid for

mRNAs sedimentingat 13S, 14S, 14.5S, and 22S.

The 14S and 14.5S mRNAs are differentially regulated.

Several studies havedemonstrated apreferential

accumula-tionof the 13S mRNA fromregion ElB late after infection

(14, 40, 50). We have also observed the same preferential

accumulation using twodifferent methods. In the Northern

blot analysis withfragment 268 as aprobe,the 13SmRNA

was revealed as the predominant ElB mRNA late after

infection. The preferential accumulation of the 13S mRNA late during infection was also confirmed by the cDNA analysis (Fig. 7). The Si analysiswith cDNA clone 272 as

the probe revealed large amounts of the 22S mRNA in the

earlyRNApreparationsinvestigated, whereas it diminished lateduring infection(Fig. 3).

Thetwonovel mRNAspecieswerefoundtobe presentin differentamountsin the RNApreparations investigated. The

results of the Northern blotanalysisshowed that the mRNAs

wereexpressedatthehighestlevel in infected cellsthatwere

maintained in thepresenceofcycloheximide (Fig. 2).The Si

analysis of thesame RNApreparation showed that the 14S and 14.5S mRNAswerepresent inalmostequalamountsin the cycloheximide-treated RNA (Fig. 3). However, RNA

prepared 8 h postinfection showed a drastically different picture. In this case the 14.5 RNA was about 20-fold less abundantthan theEBB 14Sspecies(Fig. 3).InthelateRNA

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preparation, the 14S and 14.5S RNAs were present in

approximately equal

amounts, albeit at a reduced level compared with the cycloheximde-treated RNA (Fig. 3). Table 1

summarizes

the relative

abundance

of the

14S

and 14.5S

E1B

mRNAs in the

different

RNA preparations

inves-tigated.

DISCUSSION

Wehave

investigated the mRNAs encoded by early region

1B of

adenovirus

type 2.

Previous studies

have shown that the

E1B

mRNAs are

transcribed

from

acommon promoter

located

at

coordinate

4.7 (6, 24,

51).

The synthesis of the 9S

mRNA

of E1B, in

contrast,

is initiated

at a separate pro-moter at

coordinate

10.0 (3, 51). All

E1B

mRNAs appear to use

the

same

polyadenylation site

at

coordinate

11.3 (3, 10, 14, 26). In

the

present study we have identified two novel

differentially spliced

mRNAs. Their structures have been

determined by

Northern blot,

Si nuclease,

and cDNA

analysis. The

two mRNAs havethe same

5'

and 3' exons as are present

in the 13S

mRNA.

They differ from

the

13S

mRNA

by the

presence

of

an extra exon

which

in the two mRNAshas

slightly different

5'

borders. One intron which is

common to

both

mRNAsis

the

sameone

that

is

spliced

out

in

the

formation of

the

E1B 22S

mRNA

species.

mRNAs

formed

by

elimination of

two

introns from

region

E1B are

not

unprecedented. Chow

et al.

(14;

T.

Broker,

personal

communication) have by electron

microscopic heteroduplex

analysis observed mRNAs

which,

as

do

the two mRNAs

described in the

present

study, have three

exons. We

have,

moreover, in

a

previous communication described E1B

mRNAs

from

adl2-infected

cells

which

have an exon

organ-ization similar

to that

of

the novel mRNAs

from

ad2

(48).

However,

one

of

the

adl2 mRNAs

still differs

from the

ad2 counterparts

in

using

a

unique

acceptor

splice site in the

3'

noncoding region.

It has

been shown

before

that the E1B mRNAs are

subjected

to a

posttranscriptional

control

(14, 40, 50).

The

13S

mRNA

accumulates in large

quantities late after

infec-tion,

whereas the

22S

mRNA

is

preferentially expressed

early

after

infection.

To

investigate the relative abundances

of the

two novel

mRNAs,

we

have

analyzed

mRNAs

pre-pared

at

different times after infection from untreated

and

cycloheximide-treated cells. The 14S and 14.5S mRNAs

were present in almost equal amounts

in

the mRNA from

cycloheximide-treated cells

and also

in

late mRNA

prepara-tions, although in reduced

amounts

(Fig.

3 and Table 1).

However,

at

8

h

postinfection in the absence of drugs,

the

14.5S

mRNA was

approximately

20 times lessabundant than

the 1.26-kb

mRNA.

Since cycloheximide is known

to inhibit

TABLE 1. Relative abundancesoftheE1B 14SmRNAs"

Relative abundance(%) mRNAspecies" RNAfrom

cycloheximide-treated Early RNA" LateRNA"

cells'

1.31 40.5 5.6 57.9

1.26 59.5 94.4 42.1

"The relative amounts of the different 14S E1B mRNA species were

determined by densitometry of autoradiograms resulting from Si nuclease

analysis.

mRNAsarelistedbytheirlengthsin kb.

RNA prepared from ad-infected cells which were maintained in the presenceof cycloheximide(25,g/ml).

dRNApreparedfrom cells 8 hpostinfectionin the absence ofdrugs.

e RNAprepared 18 hpostinfection.

proteinsynthesis, these observations indicate that aprotein is involved in the posttranscriptional regulation of the novel mRNAs (16), either at the level of splicing or mRNA turnover. Regulation at the level of splicing has been ob-served for other ad mRNAs. Akusjarvi and Persson (2) as well as Nevins and Winkler (34) have reported that the mRNAsbelonging to the Li cotermination family in the late transcription unit are subjected to regulation through differ-entialsplicing. Drastic changes in the mRNA turnover have also been observed for the ElB 13S mRNA. The half life of

this

mRNAincreases

approximately

10times during the late phase of the infection (50). Babich and Nevins (5) have previously shown that the regulation of the 13S and 22S mRNAs is controlled by the E2A 72K DNA-bindingprotein. The established structures of the two novel mRNAs enables us to predict their coding capacity. Both mRNAs contain an uninterrupted 21K-ORF. Hence, they have the capacity to encode the 21K polypeptide like the 13S and 22S mRNAs. Parts

of

the

55K-ORF

arealso present in the two

mRNAs. In the 14S mRNA, an internal segment of the

55K-ORF is

removed by the first intron. The mRNA

en-codes a hypothetical 16.5K protein product which is

com-pletely

overlapping

with the 55K ElB

polypeptide,

the

difference being

a

340-amino-acid-long

internal segment

which is

deleted

from

the product

of the 14S

mRNA. The polypeptides which are expressed from region

ElB

have

been studied

by

several

investigators using

immunoprecipi-tation. Green

et al. (23) have

identified

a 20K

polypeptide

which shares tryptic peptides with the 55K

polypeptide.

Furthermore they have shownby

immunoprecipitation

with

antibodies

against synthetic

peptides originating

from the

N-terminal

part

of

the 55K

polypeptide

that the20K

polypep-tidestartswith thesame sequenceasdoes the 55K

polypep-tide (29). Thus it

seems

likely that the

20K

polypeptide

identified

in their

study

is the

polypeptide encoded by

the 14S mRNA, although the

predicted size

does not

fit

per-fectly.

Esche et al. (17) have

by in vitro translation

of

size-fractionated

mRNAs

identified

an

18K

polypeptide

that is encoded

by

a 1.2-kb mRNA. This mRNA is

probably

identical

to

the

14S mRNA

described

in

the

present

study.

Andersonetal.(4) have

recently

assigned

this 18K

polypep-tide to

the

55K-ORF by partial amino acid

sequencing.

This 18K

polypeptide

has thesameN

terminus

and

C terminus

as

the 55K

polypeptide

but lacks sequences

from

the internal part

of

the 55K

polypeptide

chain.

Thus, its

structure

fits

perfectly

with the

product of

the

14S

mRNA.Andersonetal. (4) werein fact able to predict from the protein sequence that the

splice

present in the 14S mRNA

exists.

The 14.5S

mRNAhasa

different

coding capacity; beyond

the

first

intron

it will be translated

in

a

different reading

frame, and translation

will

terminate

only fifteen codons

after the

splice

acceptorsite. Its

predicted coding capacity

is

9,200.

A

sitnilar

polypeptide

canbe translated

from

the 13S mRNA,

having

a

predicted molecular weight of 8,100.

None

of

these

polypeptides

have been identified

by

in

vitro

trans-lation

or

immunoprecipitation.

Doubts can

therefore

be raised astowhether the 55K

reading

frame is

functional in

the13S and14.5S mRNAs. It

would, however,

be

surprising

if itwasnot

used,

since the results discussed above

demon-stratethat parts

of

the

55K-ORF

mustbe translated

from

the 14S mRNA. A

polypeptide which

might

be translated

from

either of those two mRNAs was

recently

discovered

by

Anderson et al. (4). Theyidentified a

16K

EiB

polypeptide

which has the same N terminus asthe 55K

polypeptide

but

a

unique

C terminus. The structure

of

this

polypeptide

fits with the coding potential of either the 13S or the 14.5S

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http://jvi.asm.org/

(8)

Structure of reading frame Predicted coding capacity

Present in mRNA (S)

--77U-21K 55K 16.5K

9.2K 8.1K

13S, 14S,14.5S,22S 22S

14S 14.5S 13S =//////- 14.3K

(poly-peptide IX)

5 6 7 8 6 10 11 m.u.

FIG. 8. Sizes and structures of the polypeptides encoded by region E1BmRNAs.Structures of thereading frames (as defined inFig. 1) andtheir locations in the ad2 genome, are shown to theleft.Thepredicted coding capacities of each reading frame are shownaswellasthe putative mRNAs which express the different reading frames. The polypeptide IX is translated from a unique unspliced mRNA. The three different reading frames are indicated with open, filled, and hatched boxes.

mRNAs. There

is,

however, a

considerable

difference be-tween

the predicted

and

estimated

molecular

weights of

these

polypeptides. Figures

1

and

8

summarize

the struc-tures of the E1B

polypeptides

and their predicted coding

capacities.

An

interesting conclusion of this

study

is

related to the way the ad use splicing as a mechanism to generate protein

diversity by creating different

exon

combinations.

In many small DNA

viruses like

4XX174

(8),

simian

virus 40, and

polyomavirus

(see reference 44 for a

review),

regions are

found

in

which

two

translational reading frames

are used.

Overlapping reading frames

appear to

be

uncommon in the adenovirus genome

(Roberts

et

al.,

manuscript

in

prepara-tion). Instead, it

seems that

the virus through

splicing

generates several combinations of a given set of exons

presumably

to create

related but

different proteins and

hereby expand its genetic

contents.

This

has been observed

before for region

ElA (35, 47). In

this

case three

different

mRNAs encode three

polypeptides which

all have a com-mon N

terminus

but

which differ either internally

or at the

C

terminus.

In

region

E1B,

the

situation

is more

complex.

Two

ORFs

are present in

the

DNA sequence

(20) besides the

ORF for

polypeptide

IX

(3).

The mRNAs

which hitherto

have

been

found

in

region

E1B can

in

theory encode six

different

polypeptides, four of which

are related. It willbe

interesting

in the

future

to

study the

function of the

struc-turally related proteins which

are

generated by differential

splicing.

ACKNOWLEDGMENTS

Wethank Jeanette Backmanand Marianne Gustafson for compe-tent secretarial work and Neil Balgobin for the synthesis of the synthetic oligomer. We aregratefultoIngaLofstrom for technical assistance and toGoranAkusjarviandGoran Magnusson for valu-ablecommentsduringthepreparation ofthemanuscript.

The study was supported by grants from the Swedish Medical Research Council and the Swedish National Board forTechnical Development.

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50. Wilson,M. C., andJ. E.Darnell. 1981. Control of messenger RNAconcentrationby differentialcytoplasmic half-life. Adeno-virusmessengerRNAs from transcriptionunits 1A and 1B. J. Mol. Biol. 148:231-251.

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VOL. 54,1985

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Figure

FIG.1.framesofmRNA.betweenAsterisksNcoI,strategy the A schematic drawing illustrating the mRNAs which are transcribed from the r-strand of region El
FIG. 2.NcoI-HincII)revealedwhich Northern blot analysis of E1B mRNAs. Two different RNA preparations were investigated
FIG. 3.Ncol/PstICH,cycloheximide;givenprobes.added.18HPI, S1 nuclease analysis of E1B mRNAs with fragments 272 and Ncol-SmaI fragments from cDNA clone 272 as the The following RNA preparations were investigated: lane RNA prepared fromcells maintainedin the
FIG. 6.HaeIIIatbeforeofcDNAmentusedafternucleotides3226rise(BglII) nucleotide the cDNA analysis with a 5'-end-labeled BglII-HhaI frag- (Fig
+3

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