Multiple methylated cap sequences in adenovirus type 2 early mRNA.

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JOURNAL OF VIROLOGY, Nov. 1976, P.425-435

Copyright0 1976 American SocietyforMicrobiology Printed inVol.20,U.S.A.No.2

Multiple

Methylated Cap Sequences

in

Adenovirus

Type

2 Early mRNA

SHU-ICHI HASHIMOTO AND MAURICE GREEN*

Institutefor Molecular Virology, Saint Louis University School ofMedicine, St. Louis, Missouri 63110 Received for publication 26 April 1976

The methylated constituents of earlyadenovirus2mRNA werestudied. RNA was isolated from polyribosomes of cells double labeled with

[methyl-3H]methionineand32pO4 from 2 to 7 h postinfectioninthe presence of

cyclohexi-mide.Cycloheximideensuresthatmethylation and processingareperformed by

preexisting host cell enzymes. RNA was fractionated into polyadenylic

[poly(A)I+ and poly(A)- molecules using poly(U)-Sepharose, and undegraded

virus-specific RNA wasisolatedbyhybridizationtoviral DNA in50% formam-ide at3700.Viral mRNA wasdigestedwithRNase T2andchromatographedon

DEAE-Sephadex in7 M urea. Two3H-labeled RNase T2-resistant

oligonucleo-tidefractionswithcharges between -5 and -6wereobtained,consistentwith twoclasses of5'terminalmethyl "cap"structures,

m7G(5')ppp(5')NmpNp

(cap 1)

andm7G(5')ppp(5')NmNmpNp (cap 2) (Nmisaribose2'-O-methylation).The

puta-tive cap 1 contains m7G, Am, Gm, Um, and m6Am. Cap 2 contains all the methylated constituents of cap 1 plus Cm. The molar ratios ofm7G to 2'-O-methylnucleosides is about 1.0 for cap 1 and 0.5 for cap2, consistent with the

proposedcapstructures. Mostsignificant,compositional analysis indicatesfour

different cap 1 structures and at least three different cap 2 structures. Thus

there is a minimum of seven early viral mRNA species with different cap structures, unless each type of mRNAcanhavemorethanone5' terminus.In addition tomethylated caps,earlymRNA containsinternal basemethylations,

exclusively asm6A, asshown byanalyses of the mononucleotide (-2 charge)

fraction. m6A was presentinthe ratio of1mol ofm6Apper450nucleotides.Thus viral mRNA molecules contain two to three internal m6A residues permethyl cap, since there is on the average 1 cap per 1,250 nucleotides.

Thetranscription of DNA tumor virus genes

in infected and transformed cells provides a

model for analyzing mRNA biogenesis and

posttranscriptional regulation in eukaryotic

cells. The human adenoviruses, in particular

the well studied adenovirus2 (Ad2), are

espe-ciallyuseful for such studies. Ad2productively

infectscultured human cells (KBorHeLa)and expresses its genomein twostages,"early" (be-fore viral DNA synthesis at 6 to 7 h postinfec-tion) and "late" (14, 32). Early Ad2 mRNA is transcribed in the cell nucleus and processed in the absence of protein synthesis, so host cell enzymes must perform these functions (al-thoughthepossibilityof a virion methylaseor

RNA polymerase has not been excluded). Therefore, by studying early Ad2 mRNA syn-thesis we can learn about the cellular mecha-nisms that regulate both viral and cell gene expression.

Inthisconnection, the possible role of meth-ylationismostintriguing. It has recently been

demonstrated that mRNA from animal cells

(26) and viruses that replicate in eukaryotic cells (10) ismethylated. With RNA viruses that

multiply in the cytoplasm, methylation is

ex-clusively at the 5' terminus in two classes of

"capped" oligonucleotides, m7G(5')ppp(5')Nm

andm7G(5')ppp(5')NmpNm (Nm is a ribose

2'-O-methylation), respectively, termed cap 1 and

cap 2. The basepenultimate tom7G is Am (or

m6Am) in vesicular stomatitis virus mRNA (in vitroand in vivo) (1, 22, 29), and cytoplasmic

polyhedrosis virus mRNA (in vitro) (13),

whereas it isGm in reovirus mRNA (in vitro) (11). ThemRNA generated invitroby cores of vacciniavirus, acytoplasmic virus with a dou-ble-stranded DNA genome, contains two caps withpenultimate Am and Gm (34). All of these

invitrosynthesized viral mRNA's but vesicular stomatitis virus contain only cap 1 structures. The methylation of mRNA synthesized dur-ing late stages of infection by twoDNA tumor viruses thatreplicate in the cell nucleus, sim-ian virus 40 (SV40) (3, 18) and Ad2 (9, 21, 36), have been studied. Presumptive evidence for

425

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426 HASHIMOTO AND GREEN

SV40 mRNAcap1structureswithpenultimate

Am and Gmwasreported (18). LateAd2mRNA

hasbothcap1andcap2 structures (21,36). The cap 1 fraction was found to consist of 80%

m7Gpppm6Am and20%m7GpppAm (21);the cap 2 fraction wasnot fully characterized and

ap-pears tobe morecomplex. Unlike cytoplasmic

viruses,Ad2 (21) andSV40 (18)mRNA, aswell

ascellRNA(33),containinternal methylations,

exclusively m6A. In contrast to the restricted composition ofviralcaps,cellmRNAcontainsa

wide varietyofmethylcaps,ofbothtype 1and type 2: 27 different caps were isolated from

mRNA ofmousemyelomacells (6), and

multi-plecaps werefoundinmRNA ofHeLacells(12,

33) andmouseLcells (27).

Severalfunctions of the 5' terminal

methyla-tion of cell and viral mRNA have been

sug-gested, including: (i) ribosome binding for the

translation of mRNA (4, 23), (ii) recognition signals for processingof nuclear precursorsto mRNA (30), and(iii)protection against

exonu-cleases. Conceivably, different cap sequences

affect translation control, or reflect different

recognition signals for putative cleavage en-zymes or initiatingsequences forRNA polym-erase. Unfortunately, the large number of

mRNAspecies ineukaryotic cellslimits

experi-mentalapproachestofunctional analysisofcap structures.

The study ofAd2early mRNA methylation

offersanopportunitytoinvestigate thepossible

functions of different caps. Unlike late viral

mRNA methylation which may involve the

functioningof viralgeneproducts,early mRNA

methylation probably involves only host cell

enzymes. The genes encoding early mRNA have been mapped (28, 31), individual mRNA

sequencesfromeachgenehavebeenidentified (5, 7, 8; W. Buttner, Z. Veres-Molnar, and M. Green, J. Mol. Biol., in press), and

proce-dures have beendevelopedtopreparatively

iso-late discrete viral mRNA species (Buttner et

al., J. Mol. Biol., in press; 5a). Early Ad2 mRNAhas beentranslatedinvitro into at least sixpolypeptideswhosegeneshavebeen located (19). From six (Chin and Maizel, in press;

14a) to as many as 11 (W. S. M. Wold, M.

Green, and W. Buttner, in press) early

pro-teins (notnecessarilyviralcoded)have been

de-tected in vivo. Thus it is possible to correlate

the cap structure of each early mRNA with

their position on the viral genome and the

synthesis ofspecific viral proteins. Such

infor-mation can help elucidate possible functional

roles forindividualcaps. Wehaveanalyzedthe methylated components of early Ad2 mRNA, and find that it is methylated internally as

m6A,as areothermRNAmoleculestranscribed

in thenucleus. But unlike previously reported viral mRNA species, Ad2 early mRNA has a variety of different methyl caps, both type 1

and type 2. Compositional analysis indicates four type 1 caps andatleastthreetype2 caps. Both types of caps contain m6fAm. These data

are consistent with the possibility that each Ad2 mRNA species has a different cap struc-ture.

MATERIALS AND METHODS

Cell culture and virus infection. Exponentially

growing KB cells in suspension were cultured in

Eagle minimumessential medium (MEM) contain-ing 5%horseserum. Cells wereconcentratedto 3 x

106 to 5 x 106cells/ml by briefcentrifugation and infected in MEM with 100 PFU/cell of Ad2 (plaque 4, strain 38-2, free of adenovirus-associated viruses).

After 1 h ofadsorption, cells were dilutedwith 10

volumes of MEM (with horse serum) containing 25 ,tgofcycloheximideper ml. At2hpostinfectioncells were concentrated 10-fold, resuspended in phos-phate-free MEM containing one-tenth the concen-trationofmethionine and 25 j,g ofcycloheximideper ml, and labeled from 2 to 7 hpostinfection with 1 mCi Of 32P04 per ml and 200 ,uCi of [methyl-3H]methionineperml (13Ci/mmol, Schwarz/Mann). Cell fractionation and isolation of Ad2-specific RNA. RNA was isolated from thepolyribosomesof infected cells aspreviously described (5). Polyriboso-mal RNA was fractionated on polyuridylic acid

[poly(U)]-Sepharosecolumns intopolyadenylic acid

[poly(A)]+ andpoly(A)- RNA fractions (20) and

an-nealed in 50% formamide at 37°C with Ad2 DNA immobilized on membrane filters (5). In some exper-iments, hybridization-purified RNA was further

purified by rehybridization to Ad2 DNA. Poly(A)+

[3H]RNA used incontrol experiments was isolated

from polyribosomes ofuninfected KB cells labeled for15 hinthe presence of 20 ,uCi of[3H]uridine per ml (39 Ci/mmol, New England Nuclear). In addi-tional control experiments, [methyl-3H]-labeled

poly(A)+ RNA was isolated from polyribosomes of uninfected KB cells labeled for 12 h in the presence of 50 ,uCi of [methyl-3H]methionine (13 Ci/mmol, New England Nuclear). Ribosomal RNA used as markers for gelelectrophoresis wasisolated as de-scribed by Hashimoto andMuramatsu (15).

Polyacrylamide gel electrophoresis of viral mRNA.Hybridization-purifiedviralmRNA was an-alyzed by electrophoresis on gels (0.6 by 20 cm) containing 2.5%polyacrylamide, 0.5%agarose, and 0.2% sodium-N-lauroylsarcosinate (ICN Pharma-ceuticals) (5).Electrophoresis wasperformed for6 h

withconstantvoltage (150 V) at 5C with

recircula-tion of buffer. Ribosomal [3H]RNA markers were

coelectrophoresed with Ad2mRNA. Gels were frac-tionated into 2-mmportions with a Gilson gel frac-tionator (Gilson Medical Electronic Co.) and countedinAquasol (New England Nuclear) usinga

scintillationcounter.

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CAP SEQUENCES IN Ad2 EARLY mRNA 427 RNaseT2digestion andDEAE-Sephadex column

chromatography. 32P_ and [methyl-3H]-labeled vi-rus-specific RNA togetherwith 0.5 to 1 mgof carrier

yeast tRNA weredigestedwith 2unitsofRNase T2 (Sankyo Co., Ltd)irn0.5 mlof 0.05Msodium acetate (pH4.5)at37°C for15 h (15).One unitofRNase T2

was added, and incubation was continued for an additional 5 h. The digest was treated with 0.1 N HCl at 0°C for 5 h to cleave cyclic phosphodiester linkages. After neutralization, the solutionwas di-luted with5ml of 7 Murea-0.03 M Tris-hydrochlo-ride (pH 7.6) andloadedontoaDEAE-Sephadex

A-25column (0.4by30 cm).Fiveoptical density units

at 260 nm

(OD2N)

ofa pancreatic RNase digest of

yeast tRNA was added in the application buffer. Elutionwasperformed withalinear gradient of0to 0.4MNaCl(70mlof each)inapplicationbuffer. In

several experiments, elution with 200 ml of eluant

(100ml each) was utilized (16). TheOD2,0 ofeach fraction (1.4ml) was measured, and 100to 200Ml

wasanalyzedfor32p and3H counts perminute.

Compositional analysis of methylated cap struc-turesisolated from Ad2 mRNA. Cap1 (-5charge) and cap2(-6charge) fractions resolved by

DEAE-Sephadexchromatography were pooled, desalted by elution from DEAE-Sephadex (0.3 by2.5cm)with2 Mtriethylamine bicarbonate, dried, anddissolved

in 20 Mul of 50 mM Tris-hydrochloride (pH 8.5) -5

mMMgCl2 containing nucleoside markers (Am, Um, Cm, and m7G, approximately 1OD2. unitofeach). The mixturewasincubatedat37°C for4 hwith5 Mg each of venom phosphodiesterase andEscherichia

coli alkalinephosphatase (Worthington Corp.). The mixture wasreincubated at37°C for 1h after the addition ofanadditional5 Mgofeachenzyme.

Nu-cleosides were separated by ascending paper chro-matographyonWhatman no. 3MM paper strips (1.5 by46 cm) using 1-butanol-0.8 Mboric acid-concen-trated ammonia (2,000:270:8, vol/vol/vol) (2). The chromatogramwasdevelopedat roomtemperature

until the solvent had ascended 30 to 35 cm. The

paper wasdried, cut into 0.5-cmstrips, and eluted with 0.5 mlof0.01 NHClat37°Cfor 15 h.The four markerpeaks which were detected and identified by absorbance at 260 nm and UV spectrum at pH 2migrated inthe order Am, Um, Cm, m7G. The absorbanceat260 nmshowedthatelutionof

nucleo-sideswasquantitative. Gmwasidentifiedfrom

par-allelchromatograms of phosphodiesterase, alkaline phosphatase digests of methyl-3H-labeled RNase

T2stable dinucleotides, isolated fromKBcells ribo-somalRNA(16). Samples of each

eluan%t

(100 to 200 Ml)weredissolved in5mlofBiofluor(NewEngland

Nuclear) andcounted in ascintillation spectropho-tometer. In some experiments (after samples for

counting were taken), fractions ofm7Gand m6Am peaks werepooled and analyzedon Dowex-50 col-umnsasdescribed below.

Dowex-1 andDowex-50chromatography.

Analy-sisofmononucleotideand capfractionsand

identi-fication ofm7G, m6A, and m6Am. The

mononucleo-tidefractions(-2charge)obtained by

DEAE-Sepha-dex column chromatography were pooled, diluted fivefoldwithwater, and loaded on a Dowex-1 column

(0.3 by 20 cm). The column wasequilibrated with

0.004NHClanddeveloped withaconcavegradient

formedwitha40-mlmixingchamber(0.004 NHCl) and a 20-ml reservoir(0.1MNaCl and0.01NHCl) (15). Constituentseluted in theorderCp, Ap,m6Ap,

inorganic phosphate, Up, and Gp. One-milliliter fractions werecollected and suitablesampleswere

counted.m6Ap-containingfractionswerepooled, re-chromatographed on Dowex-1, depurinated, and chromatographed onDowex-50 asdescribed below. Further identification of m7Gandm6Am fromcaps

and m6Ap fromthe -2fraction was performedby

Dowex-50 column chromatography using a micro modificationoftheprocedure of IwanamiandBrown (17). The nucleosideornucleotide fractionwas

depu-rinated by treatment with 1 N HCl for 60 min at

100°C. The sample was diluted to 0.02 NHCl, ap-pliedonto aDowex-50 column(0.2by20cm), washed

with 0.02 NHCl, and eluted with0.5NHCl. After 20 fractions (0.6ml each) were collected, elutionwas

performed by 0.7 N HCl. The elution sequence of

purineswasGua, m7Gua, m'Ade, Ade, m6Ade. RESULTS

Isolation from polyribosomes of poly(A) terminated early Ad2 mRNA double labeled

with[methyl-3H]methionineand32PO4.To

ob-tain early Ad2 mRNA for analysis of methyl-atedconstituents, cellswerelabeled withhigh concentrations of P2PO4 (1mCi/ml) and

[methyl-3H]methionine (200

ACi/ml)

from 2to7h

post-infection in thepresenceof cycloheximide. Cy-cloheximide was usedbecause it increases the yieldof early viral mRNA(5, 25);it alsoblocks late viral mRNA synthesis, and prevents the synthesis of earlyviral-codedproteins, thus as-suring that host mechanisms were utilized to

process (methylate) early viral RNA tran-scripts (unless there is a virion methylase). From2 to 3 mgofpolyribosomal RNA with 32P specific activity of about 2 x 105 cpm/,ug was

isolated from1liter of infected KB cells (3 x 105

to 5 x 105 cells/ml). Polyribosomal RNA was fractionated into poly(A)+ and poly(A)-

mate-rial using poly(U)-Sepharose; analysis offive preparations showed that about 40% of

[32P]RNA waspoly(A)+ and 60%waspoly(A)-.

Viral mRNA was selected byhybridization to

Ad2DNA in 50%formamideat37°C.In a

repre-sentativeanalysis (Table 1), 15.6% ofpoly(A)+ RNA and 2.0%ofpoly(A)- RNAformedhybrids. In control experiments, generally less than

0.1%ofpoly(A)+RNAisolated fromuninfected

cells hybridized to Ad2 DNA (e.g., Table 1). This indicates that contamination of early poly(A)+ viral mRNA with cell mRNA after

hybridizationshould be less than 1%.

The integrity ofhybridization-purified viral

RNA wasroutinelycheckedby polyacrylamide gel electrophoresis. The typical gel pattern shown inFig. 1Aissimilar to thatreported pre-VOL. 20, 1976

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428 HASHIMOTO AND GREEN J uo~

TABLE 1. Sekectionofpoly(A)+andpoly(A) Ad2 mRNAby hybridization toviral DNA

RNAhybridized

LabeledRNA Input (cpm) -__________

cpm

poly(A)+ (32P, [methyl_3H])a Ad2-infectedKBcells 9,644,000(32p) 1,515,000(32P) 15.6

poly(A)- (uP, [methy1_3H])a Ad2-infectedKBcells 73,670,000(32P) 1,529,000(32p) 2.0

poly(A)+ ([IHluridine)buninfected KBcells 5,516,000(3H) 3,000 (3H) 0.05

poly(A)+ ([methyl_3H])c uninfectedKB cells 43,000 (3H) 50 (3H) 0.11

a PolyribosomalRNA (2to3mg)isolated fromKBcells labeledwith [methyl_3Hlmethionine and

32P04

from 2 to 7 h postinfection with Ad2 was fractionated into poly(A)+ and poly(A)- RNA on a

poly(U)-Sepharosecolumn.One-tenth ofpoly(A)+RNAand one-half ofpoly(A)-RNAwereannealedtoAd2 DNA(50

.tig)

immobilizedonnitrocellulosefiltersin 1ml ofhybridizationbufferat370Cfor15h.

bPolyribosomal RNA (2 to 3 mg) was isolated fromuninfected KB cells labeled with [3H]uridine and

fractionatedintopoly(A)+ andpoly(A)- RNA. One tenth of thepoly(A)+RNAwas annealed with50 /igof Ad2DNA asdescribedabove.

PolyribosomalRNA(2to3mg) wasisolatedfromuninfected KBcells labeledwith [methyl-3 Hlmethio-nine and fractionatedintopoly(A)+andpoly(A)- RNA. One-tenth of thepoly(A)+RNAwasannealedwith 50 .tLgofAd2DNAasdescribed above.

20 13S,andtwo

11S);

Buittner

etal. (J.Mol. Biol.,

A in press) have preparatively isolated and

15

2Rs

18s 5s

mapped

five major early mRNA species (24S,

I

~~~~~~~20.5S,

two 20S, andoneortwo

14S).

10

Only

about 20% of total viral RNA was

poly(A)-

(Table 1).

Rechromatography

on

5

~~~~~~~~~poly(U)-Sepharose

showed that about 40% of

5V

hybridization-purified

poly(A)-

viral RNA

4

~~~~~~~~~~~~eluted

inthe

poly(A)+

fraction; therefore, 10 to

~~0 I

~~~~~20%

of

poly(A)+

viralRNAwasnotretainedon

B the first

poly(U)-Sepharose

column. The

gel

i~80

8 8 s

pattern

of

poly(A)-

early

viral

miRNA

included

28s18s 5s

~~~the

major

20-21S peak

(Fig.

iB)

anddecreased

60 levels of smaller viral mRNA

species.

The

40-

methylated

components of

poly(A)-

mRNA

were

analyzed

in

parallel

with

poly(A)+

viral

20

-MRNA,

as describedbelow.

Resolution of methylated components

re-0 leased from Ad2mRNAbyRNase T2.To

char-0 20 40 60 80 acterize the

methylated

nucleotides of

early

Slice number Ad2 mRNA, RNase T2

digests

were

prepared

FIG. 1.

Polyacrylamide

gel electrophoresis of and fractionated on

DEAE-Sephadex.

RNase

poly(A)+(A) and poly(AP-(B) Ad2-specific early T2

hydrolyzes

phosphodiesterbonds

through

a mRNA. Early polyribosomal RNA labeled with 2',3'-cyclicintermediateso thatphosphodiester 32P04and [methyl-3H]methionine was fr-actionated bonds in caps (pyrophosphate) and those

link-intopoly(A)+andpoly(A)-RNAbychromatography ingthe 3' hydroxylof

internal

2'-O-methylated

on poly(U)-Sepharose, and each fraction was an- nucleotides are stable. Chromatography on nealed to membrane filters containing Ad2 DNA. DEAE-Sephadex in 7 M urea at neutral pH Viral RNA waselutedfr-om thehybridand

electro-reovslinuetdsonhebisfcag.

phoresed on polyacrylamide agarose gels together

Fgresolvsholignuletides

onAtehaexbss

chroarge.

with KB cell ribosomal

[PH]RNA

marker. Fgr hw h EESpae hoao

grams of T2

digests

prepared froM 32P04 PlUS

viously (8, 20, 25). Several small peaks were [

methyl-_3H]methionine

-labeled poly(A)

-se-reproducibly

found between the major 20-215 lected Ad2

nmRNA,

once (Fig. 2A) and twice

peak and 13-148 RNA, and a broad shoulder (Fig. 2B)

hybridization

purified.

Over 98% of

wasfoundbetween 215 and28S. This

complex

the32Pradioactivity

applied

tothecolumn was

pattern includes at least five to seven viral recovered, and no further radioactivity was

mRNA molecules. Craiget al. (7, 8) have de- eluted with 1 M NaCi after completion of the tected atleastsevenearlymRNAspecies byan gradient. The mononucleotide

peak

(-2

analytical

mapping

procedure (209,

19S, three charge)contained 99.6% of the total32P-labeled J. VIROL.

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CAP SEQUENCES IN Ad2 EARLY mRNA 429

0.1

c

0

(.0

C\M

.-O

w

0 z

m

a:

0 C/)

m

0.1

-U

I I 0

K

0

0.2 -2

E

0

0.1 --Ia

3

2

CM I

x

6 4

2

20

40

60

80 TUBE

NUMBER

FIG. 2. Chromatography of RNase T2digest of 32p-,[methyl-3H]labeled Ad2 early mRNAon a

DEAE-Sephadexcolumnin7Murea atpH 7.6. Each enzymedigestwasco-chromatographedwith5OD260 unitsof

pancreaticRNase digest of yeast tRNA toprovideoligonucleotide markers as described in Materials and

Methods.3H cpm due to 32p spillover counts (about2%o)inmononucleotides fraction were notsubtracted.(A) Poly(A)+RNA (once hybridized); (B) poly(A)+RNA(twicehybridized); (C) poly(A)-RNA (oncehybridized).

material eluted from digests ofAd2 poly(A)+ mRNA, the dinucleotide (-3 charge), and

tri-nucleotide (-4 charge) peaks contained no

counts,andoligonucleotide peakswith

approxi-mate charges -5 and -6, respectively,

con-tained about 0.20%and0.25%ofthe 32p radioac-tivity (Fig. 2Aand 2B). From furtheranalysis

ofthe -5and-6chargedfractions(seebelow),

andfrom the positions of elution from the col-umn, wetentativelyconclude that the -5and

-6 fractions, respectively, represent5' termi-nal structures of the general type m7GpppNmpNp (cap 1) andm7GpppNmpNmpNp (cap 2), derived from Ad2 early mRNA. We

calculate 1 cap per 1,250nucleotides, basedon

the ratio ofthe radioactivityin32P-labeled caps

(-5 plus -6 fractions) to that of total Ad2 [32P]mRNA.

Analysis of Ad2 poly(A)- early mRNA (Fig.

2C) showed a slightly higher content of

frac-tions with aproximate charges of -5 (0.25%) and -6 (0.38%),probably because the

poly(A)-fraction containedsomeviral mRNAlacking 3'

sequences, perhaps duetodegradation. About

0.1%of the32Pradioactivity was presentinthe dinucleotide fraction and coeluted with a peak

of 3H radioactivity. This peak was probably

derived from contaminating rRNA; e.g., 3% contaminationwith rRNAwould produce 0.1% RNase-resistantdinucleotides (16).

Methylated constituents of cap1and cap2

fractions in Ad2 mRNA. The 3H-labeled

nu-A

~~-2

A

-4 -5 -6 -7-8

B

40 60_2

80

100

0

60 --2

-3 -4 -5 -6-7-8

- ~

VOL. 20, 1976

0.24-0.2

-4

I

20 40 80

11

II

I

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cleoside componentsofcap 1 andcap 2isolated from hybridization-purified Ad2 mRNA were

analyzed. Each cap fraction wasdigested with bacterial alkaline phosphatase and venom phosphodiesterase, and the resulting 3H-meth-ylatednucleosides were resolved by paper chro-matography.Figures3A-Dshow the chromato-grams of poly(A)+ cap 1, poly(A)+ cap 2,

poly(A)- cap 1, and poly(A)- cap 2,

respec-tively. Peaks 1, 4, 5, 6, and 7 are m7G, Gm, Cm, Um,and Am, respectively, as shown by co-chro-matography with marker nucleosides. Peak 1 waseluted from paper chromatograms and also identified as m7G by depurination and chroma-tography on Dowex-50. The elution position wasidentical to m7Gua prepared by depurina-tionof authentic m7G (Fig. 4A). Usually some

m7Gua in isolated caps was partiallyconverted

to the ring-opened form (m7Gua*, 2-amino-4-

hydroxyl-5-(N-methyl)formamide-6-ribosylam-ino-pyrimidine). This occurs during isolation

andanalysis ofcapsunderslightlyalkalinepH

conditions (pH 8 to 9). m7Gua* was identified

fromthe elution profile ofm7Gua* produced by

alkalinetreatment(1 NNH4OH,37°C,6h)and depurination ofpm7G. m7Gua*waselutedfrom Dowex-50 infraction2to4 after application of 0.5 N HCI. m7Gua*wasobtainedby depurina-tion of alkaline treated mRNA, whereas only m7Gua was obtained from untreated mRNA.

Peak 8 was expected to be a 2'--methylated adenosine derivative, becauseonly 2'-O-meth-ylated nucleosides (or 2'-deoxynucleosides)

would move with this solvent system and N6-methyl-2-deoxyadenosine moves the furthest among2'--blocked nucleosides (2). To charac-terize peak 8, the chromatogram spot was

eluted, depurinated, and chromatographed on

Dowex-50 (Fig. 4b). Equimolar amounts of 3H-labeledm6Ade and 3H-labeled noncharged

ma-terial, 2'-O-methylribosewerefound, asshown by co-chromatography with authentic m6fAde. Therefore, peak 8 is N6, 2'-O-dimethyladeno-sine. Peaks2 and3 were presentinsmall and

variable amounts and were absent from some

preparations;chromatographyonDowex-50

in-dicated thatpeaks2and3 arem6Ade and 2'-O-methylribose, presumably degradation

prod-ucts of N6, 2'-O-dimethyladenosine.

Table 2 gives the radioactivity and molar percent ofeachmethylatednucleosideincap1 andcap 2fractions fromtwodifferent prepara-tions of poly(A)+ and one preparation of poly(A)- Ad2 mRNA. Themolar ratio of m7Gto

2'-O-methylnucleosides was approximately 1.0

for the cap 1 fractions and 0.5 for the cap 2 fractions. Analysesof threeadditional prepara-tionsofearlyAd2poly(A)+mRNA (notshown)

10

10 I

0

E a. to

5

10

5

200

)2±34

X<,₅ ₆

0 10 20 30

DISTANCE FROM ORIGIN

(CM)

FIG. 3. Paper chromatography of methyl-3H-la-beled cap1and cap2fractions of early Ad2 mRNA

after digestion with venom phosphodiesterase and alkaline phosphatase. Enzyme digestion and paper chromatography wereperformed as describedin Ma-terials and Methods. The shadowed circles indicate nucleoside markers, the numbers correspond to those

inTable 2, and thearrowsindicatethe solvent front. The paper was cutinto 0.5-cm pieces, and nucleo-sides were elutedin 0.01 NHCl and counted in a

scintillator.(A) Cap1 of poly(A)+Ad2 mRNA; (B) cap2 ofpoly(A)+Ad2mRNA; (C) cap1 ofpoly(A)-Ad2 mRNA; (D) cap2ofpoly(A)-Ad2 mRNA.

gave similarratios with some variation inthe molar percent ofindividual 3H-methylated

nu-cleosidesbut nodifferencesinthespecific meth-ylated components detected. This variation is

expected ifthe relative amounts of different viral mRNA molecules fluctuated indifferent preparations andifeachniRNAhasadifferent cap. Although trace amounts of Cm (less than 3% oftotal 3Hcpm) were found insome cap 1 J. VIROL.

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CAP SEQUENCES IN Ad2 EARLY mRNA

0

E C 0 cqJ w 0

z

D

03

C,) co en

4

0

x

E a. U

I')

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TUBE NUMBER

FIG. 4. Identificationofm7Gand m6AmbyDowex-50columnchromatography. Peaks1and 8 inFig.3were eluted with 0.01 N HCI, dried,and dissolved in 0.1 (A)or(B)0.2 mlof1NHCl containing1 to 3 OD260

unitseachofpG,pm7G,and m6A markers. Thesamplewasdepurinated, diluted to 0.02 NHCl,andloaded

onaDowex-50column. The column was washed with 1 mlof0.02NHClandelutedwith 0.5NHCIand 0.7N HCI.Fractions (0.6ml) werecollected, OD2.was measured, and eachfraction was dissolved in 10 mlof Biofluorand counted. The position ofeach base wasestablished byitsUV spectrum.(A)Analysisofm7G; (B) analysis ofm6Am.

preparations (Fig. 2), the amount was

varia-ble, and Cm was absent from several

prepara-tions.Cm in cap1mayrepresentminor

contam-ination with cell mRNA ormorelikely

cross-contamination onDEAE-Sephadexwith cap2.

Onthe basis of these nucleosideanalyses, four

cap 1 and atleast three cap 2sequences exist:

m7G(5')ppp(5')Nmp

withNm asAm,

Um,

Gm,or

m6Am; and

m7G(5')ppp(5')NmpNmp

with Nm as

pairs of Am, Um, Cm,Gm, orm6Am.

To ruleoutpossiblecontaminationwithcell

mRNA,poly(A)+ and poly(A)- viralmRNA

se-lected by hybridizationtoAd2 DNA was

puri-fied by a second hybridization to viral DNA.

RNase T2-stable cap 1 and cap 2 fractions wereisolated andanalyzed. Noqualitative

dif-ferences were found in the3H-methylated

nu-cleoside components of once and twice hybrid-ized viral mRNA.

Analysesofthemononucleotidefraction. It

is not possible to determine directly whether 3H-labeled nucleotides are presentinthe

mon-onucleotide fraction (-2 charge) eluted from DEAE-Sephadex (Fig. 2) due to the large 32P counts which can mask small amounts of 3H counts.To detect internal basemethylationsin Ad2 early mRNA, the mononucleotidefraction from DEAE-Sephadex (Fig. 2) was chromato-graphedon aDowex-1 column (15).

The four 3P-labeled ribonucleotides eluted in the orderCp, Ap, Up, and Gp andpossessedno detectable 3H cpm; thus, the incorporation of methyl-3H into purinerings isnegligible (Fig. 5A). A distinct 3H peak was identified that eluted afterAp and beforeinorganicphosphate. The peak was furtherpurified by re-chromatog-raphy on Dowex-1 (Fig. 5B), depurinated, and

chromatographed on Dowex-50 (Fig. 5C). The

resuilting base was identified as m6Ade by com-parison with standards including m'Ade and m6Ade. The molar ratio ofm6Ap to total nucleo-tides in viral mRNA was 1 in 450, in both poly(A)+ andpoly(A)- RNA, asmeasured from the32Pcontent ofm6Ap elution from Dowex-1.

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TABLE 2. Composition of methylated oligonucleotides in Ad 2 early mRNA Cap 1 and Cap 2

poly(A)+

poly(A)-Cap Peaka no. Nucleoside Expt 1 Expt 2

cpm Molar%b cpm Molar % cpm Molar %

1 1 m7G 917 50.5 239 49.7 631 49.6

2 + 3 (m6Ade +2'-O-methylribose)c 0 0 25 5.2 66 5.2

4 Gm 180 9.9 50 10.4 163 12.8

5 Cm 63 3.5 0 0 0 0

6 um 254 14.0 58 12.1 94 7.4

7 Am 153 8.4 43 8.9 62 4.9

8 m6Am 497 13.7 132 13.7 514 20.2

2 1 m7G 396 27.2 404 29.6 916 32.0

2 + 3 (m6Ade + 2'-O-methylribose)C 111 7.6 172 12.6 229 8.0

4 Gm 107 7.4 100 7.3 276 9.6

5 Cm 222 15.3 121 8.9 302 10.6

6 um 283 19.5 315 23.1 500 17.5

7 Am 142 9.8 89 6.5 153 5.4

8 m6Am 388 13.3 323 11.8 971 17.0

aPeak numbers correspond to those in Fig. 3.

bMoles of each methylated nucleoside/100 mol of methylated nucleosides present in the cap. Two moles of

methyl residue of peak 8 were calculated as1mol of m6Am. Putative degradation products (peak 2 and 3) were notadded tom6Am values.

cTentative analysis (see text). Molar percent of these peaks was calculated with one mole of methyl

residue corresponding to one mole of the constituent.

DISCUSSION

Poly(A)-terminated polyribosomal. early mRNA was double labeled with

[methyl-3H]-methionine and32po4 in the presenceof

cyclo-heximide (to enhance the yield of early mRNA) andAd2-specificmRNApurified by hybridiza-tion to and eluhybridiza-tion fromAd2 DNA immobilized

onnitrocellulosefilters. The RNAwasdigested

with RNase T2 and chromatographed on

DEAE-Sephadex in 7M urea atpH 7.6. A

ra-dioactive fraction ofcharge -2 (mononucleo-tide)wasobserved, and twoadditional fractions

of charges -5 (cap 1) to -6 (cap 2). These

fractions were further characterized by paper

and column chromatography. m6Ap was the only methylated constituent in the

mononu-cleotide fraction. m7G andthe2'--methyl

de-rivatives ofA, G, U, andm6Awerefound in the cap 1fraction. Thecap2 fraction contained all

the constituents of the cap 1plus 2'--methyl C. The molar ratiosof m7G to the 2'-O-methyl-ated derivativeswere 1.0 and 0.5 for the cap 1

andcap2fractions, respectively. Sincethe cap

1 and cap 2 fractions apparently represent 5'

termini, these data indicate that there are at

leastseven different 5' termini, and therefore atleast sevenearly viral genes (unless a

spe-cific mRNA molecule can contain more than

onetypeof 5'terminus). Baseduponthe

analy-ses of 5' termini of vesicular stomatitis virus and reovirus mRNA's, we conclude that the.

general structures of these termini are:

m7G(5')ppp(5')Nmp(cap 1) and

m7G(5')ppp(5')-NmpNmp(cap 2). Four cap 1 structures exist with Nm = Gm, Um, Am, and m6Am. Three is a

minimumand25 is a maximum number for cap 2 structures (25 different pairs of five methyl-atednucleosides canexist). The existence of at

leastsevenmethyl caps is interesting in view of

the early Ad2mRNA mapping studies of Craig et al. (7, 8) and Buttner et al. (J. Mol. Biol., in press) which suggest a minimum of five to

eight early mRNAspecies. Inaddition,we have

recently obtained evidence of 11 Ad2-induced

early proteins, eight which were not detected in appreciable quantity in uninfected cells

(Woldet al., inpress). Thus, more than seven

earlygenes mayexist.

The significance of multiplecap structurein early Ad2 mRNA is unknown. Compositional

analysisindicates that differentcap structures

are notminor components.Analysis of lateAd2 mRNAby Moss and Koczot (21) gave a much

simpler pattern of two cap 1 structures

(m7Gpppm6Am and m7GpppAm); thecap 2 frac-tion was not fully characterized. The marked

differencesbetweenearly and late Ad2 mRNA

methylation patterns may suggest viral

in-ducedmodification. Two prominentfeatures of late Ad2 gene expression are the switch from

early to late viral mRNA synthesis and the

block in host cell protein synthesis. Although

most, if not all, late Ad2 RNA sequences are

J.

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20

0 N

0

x

E CM. U

a.

NV _C,

0

x

E

a.

C.

XI

9E

o 10 30" 60 80 100"120 140

c 0.5NHCI Gua 0.7NHCI C

° 0.8 -

m6Ade

4

w 0~I

w

~~~~~~~m

IAde

C-)

Z 04 2

4Zo.0

10 20 30 40 50

TUBE NUMBER

FIG. 5. Analysis of m6Ap in the mononucleotide fraction. The mononucleotide fraction of Fig. 2A was pooled, diluted fivefold with water, and loaded on aDowex-1 column(A). The column was equilibrated with 0.004 NHCland developed starting with fraction 20 with a concave gradient formed with 40 ml of 0.004 N

HClin themixing chamber and 20 mlofO0. MNaCIand 0.01 NHCIin thereservoir. One-milliliter fractions were collected,OD26 wasmeasured, and 0.1 ml ofeach fraction was counted. Each peak was identified by its UV spectrum at pH2 and 12 (0D260is not shown). Fractions 29 to 33 were pooled, neutralized, diluted fivefoldwith watercontaining 1OD26 uniteach of Cp, Ap, Up, andOpasmarkers, and loaded on aDowex-1

column(B). The column was equilibratedwith 0.004 NHCland elutionwas performed with 0.008 NHCl

after Ap was eluted. Fractions(1 ml) were collected and 0.3 ml of each was counted. Fractions 77 to 82 were pooled, dried, and dissolved in0.1 ml of 1 NHClcontaining 1 to 2 01)260 units ofpG, m'A andm6A as markers. The sample was depurinated and analyzed on a Dowex-50 column as shown in Fig. 4.

transcribed during early stages of infection,

they donotmature tomRNA andareretained

(ordegraded) inthe cell nucleus (31, 35). Late

after infection, the sameviral RNA sequences are processed to mRNA and transported to

polyribosomes. Conceivably this switch is a

consequenceofanalteredmethylationpattern.

Forexample, specificmethylated basesmaybe recognition sites for enzymes that transport mRNA out of the nucleus and onto polyri-bosomes, orforcleavage byputativeprocessing enzymes. Methylation may also "restrict"

de-struction of late viral mRNA precursor

mole-cules by nucleases. In addition, methylation

couldparticipate in the block of host cell pro-teinsynthesisthat occurslate after Ad2 infec-tion. Perhaps cell mRNA is not capped late

after infectionor capsare removedby specific pyrophosphatases, such as recently described

by Nuss et al. (24). Such enzymes may attack

cell caps which aresimilartosomeearlyviral mRNAcaps;late mRNA caps maybe different. Viral-induced enzymes that affect or

mod-ify methylation are aninterestingpossibility. VOL. 20, 1976

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The m7Gportion ofthe 5' terminal caphas been reported necessary for translation of mRNA (4, 23), but the biological functions of the penultimate 2'-0-methylation and

inter-nalm6A are not known. Since the methyl caps of each or most early mRNA's are different, perhapseach mRNA is metabolizeddifferently accordingtoits capstructure. Aless interesting explanation of multiple caps is that the type of 2'--methylated constituentsinthe cap is

irrel-evanttothe biological function of the cap. That is,the terminalm7G is presumably added post-transcriptionally, andthe penultimate methyl-ated base may result from nonspecific 2'-O-methylation after the capping reaction. Whether other internal base sequences located close to the 5' terminus serve as signals for capping, cleavage, or methylating enzymes is

unknown. Finally, we pointout that we have notstudiedthe effectofcell culture conditions

on the methylation patterns of early Ad2

mRNA, and it is possible that different results would be obtained underdifferent RNA label-ing conditions. However, preliminary analyses ofAd2 mRNA prepared without use of cyclo-heximide have revealed no substantial

differ-ences in methylation patterns from those

de-scribed here.

ACKNOWLEDGMENTS

Wethank W.Buttnerfor helpful discussion on prepara-tionof Ad2 early mRNA, S. Lowery for technical assistance, and W.S. M. Wold for critical evaluation of the manuscript. This investigation was supported by Public Health Serv-ice grant AI-01725 from the National Institute ofAllergy and Infectious Diseases and by contract NO1 CP43359 within the Virus Cancer Program of the National Cancer Program of the National Cancer Institute. S. H. was partly supported by Naito Research grant for travel ex-penses. M. G. is a Research Career Awardee

(5K6-AI-4739) of the National Institute ofAllergy and Infectious Diseases.

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