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Transcription pattern of in vivo-labeled late simian virus 40 RNA: evidence that 16S and 19S mRNA's are derived from distinct precursor RNA populations.

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Pattern of In Vivo-Labeled Late Simian Virus

40

RNA: Evidence that

16S and 19S mRNA's Are Derived

from Distinct Precursor RNA

Populations

JOHN P. FORD,* JOSEPHCOZZITORTO,JR., ANDMING-TA HSU TheRockefellerUniversity, New York, New York10021

The biosynthesis of the two major simian virus 40 mRNA molecules (19S mRNA and 16S mRNA) madeatlate times in the infectivecycle was reinvesti-gated. Byusingamodified Si nucleasetechnique, wewere abletodifferentiate between pulse-labeled RNA precursorand the spliced mRNA. During a 5-min

pulse-labeling with [3H]uridine in vivo, only precursor RNA molecules were

detected. Experimental results with polyadenylic acid-selected 5-min pulse-la-beled RNAareconsistent with the notion thatsimian virus40late RNAcanbe polyadenylatedbefore finalsplicing. Finally, 19S mRNAwassplicedmuchmore

rapidly andappearedmorequickly in thecytoplasmthan 16S mRNA. Neverthe-less, approximately one-half theprecursor molecules were destined to become 16S mRNA. Thus, foratleast thesetwoviralmRNA's derived fromacommon

transcriptionunit,therateofsplicingand therate of nuclear exitare notmajor determinants of relative mRNA abundance.

Lateduring simian virus40(SV40) lytic infec-tion of monkey kidney cells, two major viral mRNA size classescoding for viral capsid

pro-teins appearin the cytoplasm (1, 15, 19). The

twomajormRNA's,16S and 19SmRNA,share thesame sequences attheir 5' and 3' ends,but differ in theinternalsequenceas aconsequence

of differential RNA splicing (9, 10, 16). In the predominantspeciesof 19SmRNA,an

untrans-lated leader of49 nucleotides from coordinate

0.72 to 0.73issplicedto amaincodingsequence

from coordinate 0.78 to 0.17 (Fig. 1) (9). The majorspeciesof16S mRNA containsaleader of

203 nucleotides with the same 5' end as the major 19S mRNA and issplicedtoRNA

tran-scribed from coordinate 0.94 to 0.17 (16). The

structuresof thesetwomajormRNAmolecules

suggest that they could be produced from a commonunsplicedprecursorRNAby different splicingevents.Previousstudies

by

Weinberget

al. (19) and Aloni et al. (1) with ashort pulse

with[3H]uridineindicated that the 19SmRNA is produced at a faster rate than 16S mRNA.

However, it is notclearwhether the 19S RNA observed by previous workers during a short

pulse with

[3H]uridine

represents the spliced

mature 19S mRNA or alargerpolyadenylated

precursor. These two RNA molecules differby only 180 nucleotides out of a total of -2,550

nucleotides including polyadenylic acid

[poly(A)] atthe3'end. Suchasmall difference

in sizeiscertainly beyondtheresolution power of sucrosegradientandgelelectrophoresis

tech-niquesused in thepreviousstudies. Inthe

pres-entcommunicationwereinvestigatethe kinetics

offormation oftwo spliced latemRNA's by a newtechnique whichcandifferentiate

pulse-la-beledunsplicedRNAfromthe spliced products.

S1 nuclease techniques developed by Berk

and Sharp (2) have been used successfully to

analyze thestructure of unlabeledspliced RNA

molecules. We adopted andmodified this

tech-nique for studying pulse-labeled SV40late RNA

molecules. The modifiedversion employs

unla-beled DNA probe complementaryto the labeled

RNAtobe studied. Excess probeis hybridized tothe labeled RNA, andthe resulting

hybridi-zation mixtureisthen digestedextensively with

both S1 nuclease and pancreatic RNase to re-movethe unhybridizedRNA and DNA. To

en-hancethe detection ofsmalldifferences in sizes

between precursor and spliced mRNA

mole-cules,weusedshortL(late)-strandDNA probes

complementarytothe region ofRNA near the

splice junction.

Specifically, we chose the SV40 DNA frag-ment 0.67-0.83 obtained by double digestion with BglI and HaeII endonucleases. The L strand of thisfragment, complementary to the

late RNA transcript, was obtained by strand

separation inagarose gelas describedby Perl-manand Huberman(14).

Asshown inFig. 2, when this probe was hy-bridized in excess to labeled SV40lateRNA and thendigested withS1andpancreaticnucleases, the sizes of the resultant nuclease resistant RNA-DNA hybrids reflected the sequences present at the 5' end of late RNA molecules

972

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3

IN

FIG. 1. Schematic representation of SV40 genome with the regions coding for late primary transcript and mRNA noted. The genomeof 5,227 nucleotides is by convention divided into 1 unit length beginning at the EcoRIcutsite. The coarse interrupted line rep-resents the uncertainty about the location of the 3' terminusfor the late primary RNA transcript which liesfarther than 1,000 nucleotidesfrom the mRNA 3' terminus. The fine interrupted line represents the variability in 19S leader structure (see reference 5 for moredetail) which has been observed for a minority of 168 RNA as well (12).

studied. The nucleasetreatment didnotcleave the RNAopposite theDNA gapleft after diges-tion of the spliced out region (data not

pre-sented).ExaminationofSV40DNALindmRNA

sequences(6, 9, 10,11,17) shows that themajor late mRNA species, 19S and 16S mRNA, will form RNA-DNAhybrids of280and200 nucleo-tides with thisprobe, respectively, whereas an

unspliced late RNA with thesame5'endasthe majorlate mRNA will formahybrid500 nucleo-tideslong. The size difference between the

RNA-DNA hybrids of the major 19S mRNA and unsplicedprecursor was now 500 versus 280

nu-cleotides instead of 2,550 versus 2,370 for the total RNAlengths.The different sizes of RNA-DNAhybridscanbeeasily separated by electro-phoresis in an agarosegel,and the presenceof labeledspliced 19S mRNAoritsunspliced pre-cursor canbeeasily distinguished. When SV40 bulk poly(A)-containing late mRNA's labeled with[3H]uridinewereanalyzed bythismethod,

twoRNA-DNAspeciesofexpected lengthswere

indeedobserved (Fig. 3B).Controlexperiments

with labeled mRNA from uninfected cells showed nonuclease-resistantlabelinthegel.If this RNA is hybridized withadifferent probe, 1.0/0.0 -- 0.67, one measures the distance

be-tweenthe3'endof late mRNA's and coordinate 1.0/0.0(EcoRIcleavage site).Aspredictedfrom the work of other investigators (20), a hybrid lengthof 875 nucleotides was observed(Fig. 3A).

To examine the biosynthesis of SV40 late RNAs, nuclear and cytoplasmic RNAs were

pulse-labeled for5minin vivo with[3H]uridine

at 48 h postinfection. By using the 0.67-0.83 probe, one detected a single hybrid of500

nu-cleotides in length (Fig. 4A). As mentioned above, this was the expected value for an

un-spliced late RNAwith the same, 5' end as the

major late mRNA. To exclude the possibility

that the hybrid detected was the result ofthe hybridizationwithasmallcompletedRNA

tran-scribed from the region 0.67-0.83, we used

an-other

probe,

0.67 -- 1.0/0.0. With this

probe

a

hybrid of 1,450 nucleotides was observed,

whereas nosignificantradioactivity migratingat

the position of 500 nucleotides in agarose gel could beseen(Fig.5). Thisresult indicated that the SV40-specific late RNA labeled for 5 min

0.67 0.83 DNA

restriction fragment

Strand separation

0.67 0.83

RNA Hybri4ization

Late RNA template

--t%%r mRNA

| Nuclease

digestion

-- RNA-DNA

[image:2.510.59.251.56.232.2]

Hybrid

FIG. 2. Schematicrepresentation ofthetechnique used inthis work. SV40 DNA restrictionfragments complementary to the5' end of late RNAwere hy-bridizedin excess with labeled RNAextractedfrom SV40infected CVJ cells. Theunhybridized portions ofDNAand RNAweredigestedwithSl nucleaseas wellaspancreatic RNase. TheresultingRNA-DNA hybrids were then separated by electrophoresis in agarosegel.

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o

0

x

E

uI

C5

x

E

a.

Nucleotides

B

10 20 30 40 50 60

Fractions

FIG. 3. Gel fractionation of nuclease-resistant hy-bridofpoly(A)-terminatedSV40-infectedcytoplasmic RNA andSV40 late template strand of (A) 0/1.0-0.67 and(B)0.67-0.83.Cells were labeled with[3H]uridine

(100Ci/ml)for5h. The cells werefractionatedinto nuclei andcytoplasm by blending the cells in a Vortex mixer in 0.03M Tris (pH8.5)-0.1 M NaCl-0.01 M CaCl2-25pgof Spermidineperml whichwasmade 0.6% inNonidet P-40 (8). The cytoplasmic fractions werephenol extracted and RNA ethanol precipitated as described previously (4). The RNA waspassed over polyuridylic acid-Sepharose as described by Wilson etal. (18). The single-strand DNA probe was prepared as described by Perlman and Huberman (11). The RNA -DNAhybridizationreactionwas per-formed in 200 liters of 0.2 M NaCl-0.2% sodium dodecylsulfate-0.01MTris(pH7.4)-0.01MEDTA at65°C. Toremovethesodium dodecyl sulfate, the solutions weremade 0.3MKCI and centrifuged at 15,000 rpmfor1mininanEppendorfcentrifuge. The supernatant was then diluted 10times with 0.3M NaCl-0.03 M sodium citrate anddigestedwith2.5g ofpancreatic RNase per mlat30°Cfor30min. The mixture wasphenol extracted andethanol precipi-tated with 100 g of yeast RNA as carrier. After centrifugation at 15,000 rpmfor 15 min the hybrid wasresuspendedin 250ulof0.01M Tris (pH

7.4)-0.01MNaCl. Thesamplewasdiluted10timeswith 0.05Msodiumacetate(pH 5.0)-0.3 MNaCl-0.001M

ZnSO4 andwasdigested with 500 U ofSI per ml at 37°Cfor30 min. The sample was diluted with an equal volume of0.01 M Tris (pH 7.4)-0.2% sodium dodecylsulfate-0.01 M EDTAandphenol extracted. After ethanolprecipitation, the samples were ana-lyzedby electrophoresis through 2% agarose gels in 80 mM Tris-90 mM boric acid (pH 8.3)-2.5 mM EDTA. Thegels were sliced into 2 mM disks and countedaftertreatmentwith Protosol(New England Nuclear).

O 4

x

E

u 2

N8

E 4

CU) cli O

x

E 2

Nucleotides

A 850 500

- B

C

_

30 40 50 60 70

Fractions

FIG. 4. Pulse and chase ofSV40 late RNA. (A) Fractionationofnuclease-resistanthybrid ofnuclear RNA labeledfor5minwith1mCiof[3H]uridinein vivo andSV40latetemplatestrandof0.67-0.83. Nu-clei wereprepared as in Fig. 3. The nuclei were

digestedin0.01MTris(pH 7.4)-0.5MNaCl-0.05 M MgCl2-0.033MCaCl2with100pg ofDNase I per ml for1minat37°C. The mixturewasthenadjustedto 0.05M sodiumacetate(pH 5.1)-0.01M EDTA-0.2% sodium dodecyl sulfate andphenol extracted and ethanol-precipitatedasdescribed elsewhere(4). Re-maining DNA was removed by resuspending the ethanolprecipitated pelletinwateranddilutingwith equalvolumeof4MLiCl.After8hat4°C,the RNA waspelletedbycentrifugation forIh at10,000 rpm. RNApreparedfrom5x107 cellswashybridizedwith single strandofSV40 restrictionfragment0.67-0.83 corresponding to 30pg ofform I SV40 DNA. (B) Countprofile for total cytoplasmic RNA from 108 cells labeledfor5min in vivo with[3H]uridineand analyzedasinA.(C)Parallelsampletothat shown in A andB butonewhich waschasedafter labeling period.Aftera5-min exposureto)mCiof[3H]uridine

perml, the20mMglucosamine,5mMuridine,and5 mMcytidine,and thecellswereincubatedan addi-tional30minbefore harvesting(9). Totalcytoplasmic RNAfrom5 x 107 cellswashybridizedasforA and B. For all thesesamples thematerialwasdigested

with nucleasesasinFig.3and thenfractionatedon

a1.4%agarosegel.

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NOTES 975

the hybrid observed is due to contamination frompoly(A)- fraction. In addition the poly(A)-fraction froma5-min in vivolabeling showedno

peak ofnuclease-resistant hybridization

corre-spondingto thatof eithermaturemRNA.These results therefore suggest thatpoly(A) addition

can precede RNA splicing. This conclusion is consistent with that arrivedatby Laietal.(12)

analyzing bulk unlabeled RNA with the classical Berk-Sharp method.

We then analyzed the fate of newly synthe-sized SV40 RNA.When RNAwaspulse-labeled for 5 min in vivo with

[3H]uridine

and then chased for 30 min in the presence of 20 mM glucosamine,5mMcytidine, and5mMuridine, spliced RNA corresponding to the mature

mnRNAwasdetected (Fig.40). Since during the chaseperiod thespecific activityofSV40 nuclear RNA decreased while that in the cytoplasm increased (Table 1), these results strongly sug-gestthatthespliced late mRNA is derived from theunspliced nuclearprecursorobserved during

10 20 30 40 50 60

Fractions

FIG. 5. Gel fractionation of nuclease-resistant pulse-labeledRNA hybridizedtotheSV40late

tem-plate strand 0.67-1.0/0. The total nuclear RNA from 2x107 cellswaslabeled invivofor 5min withImCi

of[3H]uridineperml. TheRNA, afterLiCl precipi-tation,wasthenhybridized with DNA corresponding

to 10pgof SV40 formIDNA.After digestion of the sample with nuclease, the resistant material was

fractionatedon a2.0%agarosegel. The dashed line representsanequivalent RNA sample which isnot hybridizedtoDNA but issubjectedtodigestion with nucleasebefore fractionationon a2.0%agarosegel.

has its 5' end located atcoordinate 0.72 asthe

major late RNA. This RNAwassynthesizedas aspecies thatwasatleast 1,450nucleotides long.

The data in Fig. 4 suggest that duringa 5-min

pulse with [3H]uridine in vivo, the newly

syn-thesized SV40 late RNA hasnot yetundergone final splicing. This RNA would correspond to

the unspliced RNA detected by using Berk-Sharp alkaline gel analysis in examining bulk SV40 RNA(12). However, thepresenttechnique

can not resolve truly unspliced RNAs from RNAs with less than 10% of the nucleotides removedby splicing.

When the poly(A)-containing fraction of the 5-minpulse-labeled RNAwasselectedby

poly-uridinylic acid-Sepharose chromatography and hybridizedtothe0.67-0.83DNA probe,asingle

500 nucleotidehybrid again wasdetected (Fig.

6). Since the polyuridylic acid column was ex-tensively washed with 10% formamide, a

proce-dure which has been shown to quantitatively

remove poly(A)- RNA (16), it is unlikely that

cm ° 2

x

E

0

cm

x

E

0.

30

500 Nucleotides

A

200 280

B

0

40 50

Fractions

60 70

FIG. 6. Gel fractionation of nuclease-resistant pulse-labeled poly(A)+ andpoly(A)- nuclear RNA. RNA waspreparedasinFig. 4. Polyuridylic acid-Sepharose selection wasperformed after Wilson et al. (16). The column was washed with 10 column volumesof10%formamide-0.01 M Tris(pH 7.4)-0.01 MEDTA-0.2% sodiumdodecyl sulfate beforeelution. RNA washybridizedtoSV40 late template strand 0.67-0.83. (A) Poly(A) nuclear RNA from 5 x 106 cells labeledfor5minin vivo with['H/uridineand hybridizedwith DNAcorrespondingto10 pLgof SV40 formIDNA.(B) Poly(A)+nuclear RNAfrom5x107

cellshybridizedwith DNAcorrespondingto10jgof SV40form I DNA. Bothsamples weresubjectedto nuclease digestion and then fractionation on 1.4%

agarosegels. VOL. 35,1980

bor

9

8

7

x,

E

a

6

5

4

3

2

500Nucleotides

I

,/ I

It

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the5-minpulse (Fig. 4A).

Becauseof the previousreports (1,4) which suggested that 19S mRNA is produced more

rapidly than 16S mRNA but -which involved techniques thatprobably couldnotdistinguish between precursor 19S RNA and mature 19S mRNA, we were encouraged to examine the kinetics of19S and 16S mRNA appearance in thecytoplasm. We chosenot touseglucosamine

in these additional studies since glucosamine

may alter the kinetics of mRNA synthesis or turnover.Theinitialfindingfrom the work

sum-marized in Table 2 is that thepercursor-sized SV40 RNA species in the cytoplasm is not

poly(A) terminated. As work bySawicki et al. has shown de novo polyadenylation in HeLa cells occurs in the nucleus (17). For the

cyto-plasmicprecursor-sizedmoleculetogiveriseto mature 19S mRNA and 16S mRNA, it would have to undergo cytoplasmic polyadenylation. Such a biosynthetic step seems quite unlikely althoughnotimpossible.Morelikelyis the

pos-sibility that thecytoplasmicprecursorsize rep-resentsnuclearleakage.SV40-infectedcells may

havequiteleaky nuclear membranes.By dounc-ingSV40-infectednuclei,it has beenpossibleto recover intactSV40 virions in the cytoplasmic compartment (5, 8). Smallerstructuressuchas

TABLE 1. Pulse and chaseofSV40-specific RNAa Nuclear

Cyto-Method RNA plasmic

(p RNA

(cpm) (cpm)

5-minpulse 8,500 200

5-minpulseand 30-min chase 5,900 1,500 with 20 mMglucosamine

a[3H]uridine-labeledRNAcorresponding to 3x

106

cellswashybridizedwith1,ugofSV40 DNA filters at 60°C for 48 hasin(4).

ribonucleoprotein particles mightemergein the

cytoplasm even more easily. Although we

be-lieve nuclear leakageto occur, we were unable

to eliminate the cytoplasmic precursor RNA

molecule despiteavariety ofnucleus-cytoplasm

fractionation procedures (data notpresented). Thus, we cannot exclude the possibility of a

biologically significant role in the cytoplasm for

theprecursorRNA.

The finalinterpretation of the kinetics of16S

and 19S mRNA cytoplasmic accumulation is dependentonthree facts..

(i) Table 2 shows that after 30 min in the

nucleusthere isnopreferential accumulationof

labeled16SmRNAoverlabeled19SmRNA. If

one compares the molar ratio after 30 min of

labeling for 19S RNA and 16S mRNA in the

nucleustothat for bothspecies in thecytoplasm,

one can see that there is relatively more 19S

than 16S mRNA in the nucleus as compared

with the ratio for both RNA species in the cytoplasm. The lack of 16S mRNA in the

nu-cleusduring shortperiods of incorporation with radiochemical label has been observed before (11; J. Aloni, personal communication).

(ii) Workers using two very different ap-proaches (1, 4) have published data which has been used to determine the half-life of 19S

mRNAtobe 2to3 hand that of16SmRNAto be5to6 h.

(iii) 19S mRNA detected in hybridto 0.67-0.83 DNA aftershortlabeling times (280 nucleo-tides) correspondstothemajority of 19S mRNA inbulk mRNA and bothmusthavealeader -30

to50 nucleotidesinlengthtoaccommodatethe main coding sequence from 0.78-0.83 (250

nu-cleotides). Therefore, therecanbenoconversion

of majority 19S mRNA to 16S mRNA, which requires aleader of 200 nucleotides encoded in

thegenomeat0.72-0.76.

TABLE 2. Kineticsof 19S and 16S mRNA appearance in the cytoplasm Duration

Time Glucosa- ofglucosa- cpm in280 cpmm

of la- mine mine,uri- Poly(A) cpminpre- nucleotide 200-nu- Molar

ra-RNA beling pretreat- dine, cyti- selected cursor

19S

RNA cleotide tio,19S/

(min) ment dinechase leader 16S RNA 16S

(min) leader

Nuclear 5 + - 10,100 <50 <50

30 - - 4,300 1,280 400 2.4

Cytoplasmic 5 + - 1,630 <10 <10

7 - + <10 75 <10 >5.4

10 - - 300 230 50 3.3

30 - - 1,400 1,600 550 2.0

45 - + <10 780 430 1.3

5 + 30 - 1,100 870 500 1.2

RNAwasisolated andhybridizedasinFig.4.Counts perminuterepresent totalradioactivity, after subtractingbackground detectedinappropriategel fraction. All samples represent pulse-labeledRNA from 5 x107cellsexcept for 45-minPoly(A) , whichrepresents RNA from2 x107cells.

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VOL. 35,1980

With these three facts as background, the kinetic data for the cytoplasm from Table 2

show that the molar ratio of 19S/16S mRNA

changesfrom>5.4 to 1.2during30min of chase or atimeequaltofrom 'ksthto'/I2th of the

half-life of the mRNA with the shorter half-half-life (19S mRNA).

Thepastwork(1,4)which suggestedthat 19S

mRNA is produced at a faster rate than 16S

mRNA is bothconfirmed and extended. There was no preferential retention of 16SmRNA in

the nucleus, and only '/12th to 1'kth of the less stable mRNA was degraded daring 30 min of

chase. Thus, theremight beamechanismwhich prevents the precursor molecules from being

converted in thesameratioto 19SmRNA and

16S mRNAat 7 minofpulse-labeling and also

after30minofchase. Suchamechanismwhich

"protects"16SmRNAprecursormolecules from being made into 19S mRNA is evidence for the existence of distinct precursor pools for 19S mRNAand for 16SmRNA.

The data presented in Fig.3and 4 of Chin et

al. (4)areconsistent with the model ofseparate precursorpools for 19S and 16S mRNA. They

showthat in the interval between15and60 min

ofglucosamine chase (13) after a 5-min pulse with [3H]uridine, the cytoplasmic 16S SV40 RNA peak increases from 500 to 1,200 cpm. If

thenuclearprecursorpool is randomly

produc-ing both 19S and 16S mRNA, there shouldbe a

muchlarger decrease in radioactivitytoaccount

for the 19S mRNA which would also bemade

between 15 and 60 min of glucosamine chase. However, the nuclear 19S RNA only declines from 1,300 to 600 cpmduring this timeinterval,

and thecytoplasmic 19S mRNA increases only

from900 to1,100 cpmbetween15and 60minof

chase. Consistent with other evidence that 19S mRNAand 16S mRNAare notrapidlyturning

over(2, 14),there isnosubstantial loss of

radio-activity between the nucleus and cytoplasm at 15min ofchase and the nucleus and cytoplasm after60minof chase. This result indicatesthat

there is little short-lived 19S or 16S mRNA.

These facts are again in agreement with the

notion that 19S mRNAis derived more rapidly

from a pool of precursor molecules than 16S

mRNA, which isproduced more slowly froma separate setofprecursor molecules.

Ifdifferentprecursor RNAs did not exist for

19SmRNA and 16SmRNA, one would expect

the relative abundance oflabeled 19S mRNA and 16S mRNA in the nucleus and cytoplasm

initially to reflect the relative rate ofsplicing

and thenchange accordingto therelative differ-ence in mRNA half-lives between the two mRNAspecies. 19SmRNA, which is more than 5.4times morerapidly spliced and 40 to 50% as

NOTES 977

stable as 168 mRNA, would then become the

major late SV40 mRNA. Since this does not

happen,one canspeculatethat distinct precur-sormRNApoolsserve toeliminate the rateof

splicing, atleast for SV40 lateRNA, as a

deter-miningstepin mRNAabundance.Such a

mech-anism allows for the observed preferential

ac-cumulation of 16S mRNA over 19S RNA in steady-stateRNA(1,20).

Inconclusion, how doesanyapparentlysingle

sizeclass ofprecursormolecules(unspliced19S) include atleast two different precursor pools?

Onemorereasonable possibilityissuggested by the work ofCanaani et al. which showed that 19Sand 16S mRNAaremethylated differently (3). If the methylation pattern is set on the

precursormolecules, then methylation would be

amechanism for establishment ofcompartments

for precursor molecules which lead independ-entlyto16Sand19S mRNA. Another possibility isacritical difference in the protein make-up of theprecursorheterogeneous nuclear riboprotein particle which would direct the RNA molecule

to become either 19S mRNA or 16S mRNA. Finally, the present work can not distinguish

precursormolecules whichhave begun the proc-essofsplicing. Therefore, the pool of 16SmRNA precursor molecules may accumulate because the splicing process is slower for 16S mRNA thanfor19SmRNA.

Inany case, this is the firstreporttosuggest

thatmRNA'sgenerated bythesame transcrip-tion unit are derived from distinct

populations

ofprecursor molecules. Thus, for 16S and 19S mRNA's,therateofsplicingisnot animportant determinant of relative mRNA abundance.

Weappreciatethehelpandencouragementof J. E.Darnell, Jr. throughout this project. We thank A. English and L. Cousseau foraid inpreparingthemanuscript.

This workwassupported byPublicHealth Servicegrants CA 16006-06 and CA 19073-03 from the National Cancer Instituteand theAmericanCancer Society grantVC295H. J.F.helda Public HealthServicefellowship (GM05836-02) fromtheNationalInstitute of General Medical Sciences.

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14.Perlman, D., and J. A. Huberman. 1977. Preparation oflarge quantities of separated strands from simian virus 40 DNA restriction fragments by low temperature low salt agarose gelelectrophoresis. Anal. Biochem. 83: 666-677.

15.Prives,C.L.,H.Aviv,E. Gilboa,M.Revel, and E. Winocour. 1974. The cell-free translation ofSV40 mes-senger RNA. ColdSpring Harbor Symp. Quant. Biol. 35:309-316.

16.Reddy,V.B.,P. K.Ghosh,P.Lebowitz,and S. M. Weissman. 1978.Gaps and duplicated sequences in the leaders of SV40 16S RNA. Nucleic Acid Res. 5:4195-4213.

17.Reddy,V.B.,B.Thimmappaya,R.Dhar, K. N. Sub-ramanian,B.S. Zain, J. Pan, M. L. Celma, P. K. Ghosh,and S. Weissman. 1978. The genome of simian virus 40.Science200:494-502.

18.Sawicki,S.G.,W.Jelinek,and J. E. Darnell. 1977. 3' Terminal addition to HeLa cells nuclear and cytoplas-mic poly(A). J. Mol. Biol. 113:219-235.

19.Weinberg, R., Z. Ben-Ishai, and J. Newbold. 1974. Simian virus 40 transcription in productively infected andtransformed cells. J. Virol. 13:1263-1273. 20.Weinberg,R.A.,S. 0.Warnaar,and E.Winocour.

1972.Isolation andcharacterization of simian virus 40 ribonucleic acid. J. Virol. 10:193-201.

21. Wilson,M.C.,S.G.Sawicki,P. A.White,and J. E. Darnell. 1978. A correlation between the rate of poly(A) shorteningandhalf-life of messenger RNA in adenovirus transformedcells. J. Mol. Biol. 126:23-26.

J. VIROL.

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Figure

FIG. 1.resentsmoreofterminustheterminus.liesvariabilitywithandby 168 convention Schematic representation of SV40 genome the regions coding for late primary transcript mRNA noted
FIG.FractionspreparedZnSO4Afterformeddodecyl37°CdodecylsolutionsEDTA.80RNAnuclei0.6%centrifugationwas0.010.05equalatlyzedoversupernatanttatedNaCl-0.03ofmixturecountedmixerwereasand15,000brid(100(11).CaCl2-25Wilson 65°C
FIG. 5.platepulse-labeled2fractionatedoftosampletation,representshybridizednuclease x 10 [3H]uridine Gelfractionationofnuclease-resistant RNA hybridized to the SV40 late tem- strand 0.67-1.0/0
TABLE 1. Pulse and chase ofSV40-specific RNAa

References

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