0022-538X/82/100226-19$02.00/0
CopyrightC1982,AmericanSocietyfor Microbiology
Adenovirus
Type
2
Fiber
mRNA
Synthesis:
No
Evidence
for a
Cytoplasmic
Processing Pathway
CHARLES B. LAWRENCE* AND W. JAY RAMSEY
Departmentof Cell Biology, Baylor College of Medicine, TexasMedicalCenter, Houston, Texas 77030
Received14May1982/Accepted 24 June 1982
Adenovirustype 2fibermRNAexists in several forms in the cytoplasm which
differ in the presence orabsence ofextra 5'-leader segments (L. T. Chow and
T. R. Broker, Cell 15:497-510, 1978). We have investigated the possibility that
formspossessingextraleadersegmentsserve as precursors to the mature form in
the cytoplasm. Pulse-labeled fibermRNA became considerably shorter (150 to
250 bases) duringachase; however, most ofthe pulse-labeled species failed to
hybridizetoDNAfragments knowntoencodeextra leadersegments. Moreover,
the entire decrease in size appeared to be due to extensive shortening of the
polyadenylic acid
tail. Mature-sized fiber mRNA was synthesized normally incells infected with the nondefective adenovirustype2-simian virus40hybrid virus
Ad2+ND5, in which the region encoding the extra leader segments is deleted.
These resultsindicate that the additional 5'-leadersegments presentin wild-type
adenovirustype2fiber mRNAare notrequired for the productionofmature fiber
mRNAand thatspecies thatpossessthem arenot cytoplasmic precursorsto the
matureform.
The late
phase
of adenovirus type 2(Ad2)
infection of
HeLa cells isaconvenientsysteminwhich to study theregulation and biochemistry
of mRNA
biogenesis.
Most of the viral RNAsynthesized in this
period
is transcribed from asingle
largetranscription
unitapproximately
30kilobases in length (17), which has a capsiteat
16.4 mapunits (m.u.) (39) andatermination site at
approximately
98 m.u. (14) on the Ad2 genome. Theprimary
transcript
of thislarge
transcription
unit isunique
amongeucaryotic
mRNAprecursors in that it is
processed by
anumberof different
pathways
toproduce
atleast14
different
mRNAspecies (8,
11,27, 31).
ThesemRNAscanbe
grouped
into five families whosemembers
haveoverlapping
sequences and share common 3' termini (15, 31, 40). All of themRNAs
specified by
thistranscription
unit arespliced
moleculescomposed
of a 5'-terminaluntranslated leader sequence and a main
body
which specifies a particular
protein
(5, 10, 16,22). Thesameleader sequence ispresentonall
of these mRNAs and is
composed
of threespliced segments coded in the virus genome at
16.4 m.u. (41 bases), 19.6 m.u. (72 bases), and
26.6m.u. (90bases) (2, 3, 37, 38).
The detailed
pathways by
which theprimary
transcript
of thistranscription
unit isprocessed
are not known. Early events in the
processing
pathway
are(i) cleavage
andpolyadenylation
of theprimary transcript
at one of fivepossible
sites which
correspond
to the 3' termini of thefivemRNA families(32) and (ii) methylationof
internal adenosine residues in sequences
des-tinedtobe conserved in themature mRNA (7).
Further processinginvolves splicing together of
the three segments of the 5' leader and the
splicing
of
these leadersegments tothebody ofthemature mRNA. Apparently several of these
splicing
eventsdonotnecessarilyoccurinsinglestepsbutthroughaseries of intermediate
struc-turesin which portions ofinterveningsequences
areremoved. Severalintermediate species have
beendetected in nuclear RNAfrom
Ad2-infect-ed cells (6). Intermediates in the
processing
offiber mRNA have also been demonstrated in
nuclear RNA which are likely torepresent the
partial
removal of theintervening
sequences between the 5'-leader segments and the mainbody of fiber mRNA (30). These various
proc-essing steps involved in maturation of the
pri-mary transcript are thought to occur in the
nucleus.
Several studies havedemonstrated that some
ofthe Ad2 late mRNAs presentin the
cytoplasm
contain leader segments in addition to those
normally present in the 5' leader. Certain
Ad2,
Ad3, and Ad7 late mRNAs can have an extra
segmentlocated between the usual second and
third leadersegments(9,
20, 21).
ThemRNAforpolypeptide
IV(fiber)
can have additional seg-mentsbetweenthe 5' leaderand themainbody.
The most
frequently
observed is81 baseslong
andis codedat78.5 m.u.
(8, 38).
This segment226
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has no AUG codon, and its presence has no effect on the ability of the mRNA to be translat-ed in vitro (12). Chow and Broker (8) and
Kilpatrick et al. (20) have suggested that
mole-cules that contain extra leader segments may be authentic precursors to mature mRNA species,
andbecause such molecules have been isolated
with polyribosomes it is possible that the subse-quent processing of these precursors occurs in the cytoplasm. However, no kinetic evidence supporting this concept has been reported.
We haveinvestigated the possibility that fiber
mRNAheterogeneity in the cytoplasm could be
due to theexistence of such a precursor-product
relationship between the multiple cytoplasmic species containing extra leader segments and the predominant form of fiber mRNA detected in the cytoplasm of Ad2-infected HeLa cells. We
pre-sentkinetic and structural data which render this
possibility highly unlikely for the metabolism of fiber mRNA.
MATERIALS AND METHODS
Cellsandvirus. HeLa S3 cells were grown in
sus-pension culture and infected with Ad2 or the Ad2-simian virus 40 (SV40) hybrid virus Ad2+ND5 as
previously described (24). Allexperiments were initi-ated with cells infected for 24 h.
Radiolabeling of RNA. Infected cells were labeled with
32Pi
(NewEngland NuclearCorp.)aspreviously described(24). RNA was isolated from cells labeled for 2 or 3 h as indicated(pulse)orfrom cellslabeled for 3h inphosphate-deficient mediumfollowedby incuba-tion in normal phosphate-containing medium for anadditional8 h(chase).
[3H]uridine pulselabels and glucosamine-unlabeled-uridine chaseexperimentswereperformed bya modi-fication of the protocol of Levis and Penman (25). Glucosamine(Sigma) (pH7.4) was added to 100 mlof infectedcells at aconcentration of20 mM for 30 min. Cellswerecollectedby low-speedcentrifugation,
sus-pendedin 100mlof fresh mediumcontaining50to100
,uCiof[5,6-3H]uridine(Amersham)perml, and
incu-batedfor60minat37°C. The chase wasinitiatedby the additionofglucosamineto20 mM and uridine and cytidine (Sigma) to 5 mM. To reduce glucosamine toxicity,cells were collected after 30 min and suspend-ed in an equal volume of fresh mediumcontaining 5 mM each of uridine and cytidine. There was no significant increase in radioactivity in polyadenylic acid [poly(A)]-containing RNAafter initiation of the chase.
Isolation of RNA. Poly(A)-containing cytoplasmic RNAwas isolated aspreviously described (24).
To make total cellularpoly(A)-containingRNA,4x 106 cells were harvested, washed once in ice-cold saline, and suspended at room temperature in 2.0 ml of 7 M urea-2% sodium dodecyl sulfate (SDS)-0.35 M NaCl-2 mM EDTA-10 mMTris (pH 7.8). DNA was shearedby repeated passage througha21-gauge nee-dle. The suspension was then extracted with phenol and chloroform as previously described (24), and nucleic acids were precipitated from the aqueous phase by the addition of 2.5 volumes of ethanol
(-20°C). Precipitated nucleic acids were dissolved in 5 ml of water, salt concentrations wereadjustedto 10 mM sodium acetate (pH 5.2)-0.5 M LiCl-1 mM EDTA-0.1% SDS, and poly(A)-containing RNA was isolated by batchwise selection with 0.1 g of oligo-deoxythymidylic acid-cellulose (Collaborative Re-search). The cellulose was washed extensively with the above buffer, and boundRNA was eluted with 1.5 ml of water and precipitated by the addition of 0.2 ml of 2 M sodium acetate (pH 5.2) and 5 ml of ethanol.
Hybridization of RNA to immobilized DNA.Plasmids possessing various Sma andEcoRIfragments of Ad2 DNA were constructed in this laboratory by insertion into the Pst site of pBR322. Plasmids possessing Bal, HindIII,Pst, or Bam fragments of Ad2 DNA were the generousgiftof Sue Berget. TheplasmidpJJ1, contain-ing the entire SV40 genome, was the gift ofJohn Wilson.
PurifiedplasmidDNA wascoupledto diazobenzyl-oxymethyl-paper by the method ofStark and Williams (34). Each coupling reaction contained 100 ,ug of sheared, denaturedplasmidDNAand a 1.0-cm-diame-ter papercircle.
Hybridizations wereperformedin50oformamide, 0.4 M NaCl, 20 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)] (pH 6.4), 5 mM EDTA, 0.2% SDS,and 2 mgof yeast tRNA per ml at42°Cfor 6 to 16 h. RNA to be hybridized was dissolvedin the above buffer in a total volume of 10 .Ll and applied to a
prewashed DNA-papercirclewhich had beenblotted to remove excess buffer and placedin a siliconized glass vial. After hybridization, DNA-paper circles were washed four timesfor 5 mineachwith 3 mlof0.2 M NaCI-10mMPIPES (pH6.4)-5mMEDTA-0.1% SDSat60°C.RNA waselutedby heatingthefilterto
90°C in0.45mlof1mMEDTA(pH 7.0)and precipitat-edbytheadditionof 20p.gofyeasttRNA,50p.l of0.2 Msodiumacetate(pH5.2),and 1 mlof ethanol.
Gel electrophoresis of RNA. All labeled RNAs
ana-lyzed were denatured before electrophoresis with glyoxal by theprocedure of McMaster and Carmichael (28).
3H-and32P-labeledpoly(A)-containingRNAs were resolved onhorizontal4-mm-thick1.2%agarose (Sig-matypeV)gelsinLoeningbuffer E (26). Electropho-resiswasfor 16 h at 2 to 3V/cm. 32P-leadersegments wereresolved on15%acrylamide gels by discontinu-ousgelelectrophoresisasdescribed by Laemmli (23).
32P-labeled
RNAspecies werevisualized as previ-ously described (24). [3H]RNA was visualized by preparing agarose gels for fluorography as follows. The agarose gel was immersed in two changes of methanolfor2 heach at37°C,then in5%(wt/vol) 2,5-diphenyloxazoledissolved in methanol for 4 h at37°C, andthen in water for 16 h at 37°C. The gel was then dried onto Whatman3MMpaper.Removal ofpoly(A) tracts. Poly(A) was removed
from 32P-labeledmRNAsby digesting the RNA with
RNase H in thepresence ofpolydeoxythymidylic acid, using a modification of the procedure of Harris (18). Each 10-,ul digestion contained approximately 5,000 cpm of
[32P]RNA,
10,ug of unlabeled tRNA, 1p.g
of polydeoxythymidylic acid (Collaborative Research), and 0.6 U of Escherichia coliRNase H (Enzo Bio-chem) in 0.05 M Tris (pH 7.5)-0.01 M magnesium acetate-0.001 M EDTA-0.01 M dithiothreitol-10% glycerol. The samples were digested for 30 min at VOL.44, 1982on November 10, 2019 by guest
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FIG. 1. Size analysis of Ad2 mRNAsatshort and
long times after their appearance in the cytoplasm. Poly(A)-containing total cytoplasmicRNAwas
isolat-ed from HeLa cells infected for24 h with Ad2 and
pulse-labeled for 2 h with 32p(lanesP)orpulse-labeled
for 2 handthen chased for 8 h(lanes C).Total poly(A)-containing [32P]RNA and [32P]RNA prepurified by hybridization to various cloned Ad2 restriction frag-mentsweredenatured with glyoxalandanalyzedona
1.2% agarose gel. Lanes ongel (left to right):
14C-labeled HeLa cell rRNAs; total poly(A)-containing RNA; RNA selected by HindIII-D DNA;RNA select-edbyPst-E DNA; RNAselectedbyEcoRI-DDNA; RNA selected by EcoRI-E DNA; [14C]rRNA.
Num-bersindicatem.u. of DNAfragment.
30°C,and RNAwasprecipitated bytheaddition of0.4
mlof 0.2 M sodiumacetate(pH5.2)-0.001 M
EDTA-0.1% SDS and 1.0 ml of ethanol. Precipitated RNA was collected by centrifugation and denatured with glyoxalasdescribedabove.
RESULTS
Size changes in Ad2 late mRNAs in the cyto-plasm. Cytoplasmic processing of an mRNA
species would be indicated by adecrease in its size after its transport from the nucleus. We therefore compared the size of cytoplasmic Ad2 late mRNAs isolated after their initial appear-ance in the cytoplasm and several hours later. Cytoplasmic RNAwas isolatedfrom
detergent-lysed HeLacellsinfectedwith Ad2 and labeled
with 32P,. Under these conditions unprocessed
high-molecular-weight RNAremained
associat-edwith nuclei, andonly mRNA-size molecules
were found in the cytoplasmic extract. RNA specific for various late mRNA families was
isolated by hybridization to cloned Ad2 DNA restriction fragments immobilizedon
diazoben-zyloxymethyl-paper. RNAs were denatured
withglyoxal, resolvedon1.2%agarosegels, and visualized by autoradiography.
Figure 1 shows a comparison of selected RNAs from four ofthe five 3'-coterminal mRNA families pulse-labeled for 2 h with 32p (lanes
labeled P) or pulse-labeled for 2 h and then
chased for an additional 8 h in
phosphate-con-taining medium(lanes labeled C). Aunique set
of RNA species was selected by each
family-specific DNA fragment. The size of the Ad2
RNAscorresponded welltothatexpectedfrom
heteroduplex analyses of Ad2 mRNAs (8, 11).
Cell-free translation of RNA selected by these
DNAfragmentsalso resulted in the synthesisof
theexpectedpolypeptides.
Table 1 summarizes the size and
probable
identification of the RNA species detected in
Fig. 1. RNA species isolated from
32P-pulse-labeled cells migrated slower than the
corre-spondingspecies isolated afterachase,
indicat-ing that eachRNA species hadbeen shortened by approximately 100 nucleotides with the ex-ception of the mRNA for fiber (Fig. 1.EcoRI-E, lanes P and C), which decreased by approxi-mately 150 to 250 nucleotides. Also, in contrast to the other mRNAs examined, fiber mRNA appeared in the cytoplasm as a heterogeneous species in pulse-labeled RNA but as two discrete bands after the 8-h chase period (see Fig. 1,
2,
and 3). The faster migrating of these twospecies islikely to be the form of fiber mRNA composed
solely ofthe 5'-tripartite leader and the main
[image:3.496.58.245.69.208.2]body of the fiber gene. The slower migrating of the two is likely to be the form possessing a fourth leader segment encoded at 78.5 m.u. and which has been previously reported and se-quenced(8, 38). These proposed structureswere confirmed in laterexperiments.
TABLE 1. Size of selected Ad2 cytoplasmic RNAs
DNAfrag- Approximate size of
mentused annealed RNA Approxi- Probable for hybrid- speciesa mate change polypeptide
ization (nucleotides) in size coding (m.u.) Pulse Chase (nucleotides) assignment
40.9-50.1 3,700 3,600 100 III
2,400 2,300 100 pVII
1,850 1,750 100 V
56.8-64.5 4,300 4,200 100 pVI
3,600 3,500 100 II
75.9-83.4 4,200 4,100 100 100K
2,300 2,200 100 pVIII
2,050 1,950 100
1,800 1,700 100
83.4-89.7 2,150-2,300 2,000 150-300 IV
aEstimated from the migration of RNA species observed in Fig. 1, relative to 28S and 18S rRNAs. HeLa28S and 18S RNAs were taken to be 4,400 and 1,975 nucleotides, respectively; these values were determined by comparison of their migration with numerousDNAandRNAstandards of known size in denaturing gels (John Rogers, personal communica-tion).
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[image:3.496.256.450.435.606.2]A B C D E
FIG. 2. Sizeanalysisofprecursoi isolated from total cellular RNA. T(
wereisolatedfrom infectedcellspul with [3H]uridine or pulse-labeled f
chased forvarious times inglucosami uridine. Fiber mRNA-specific speci by annealingtoimmobilizedEcoRI-: denaturedwithglyoxalandresolved( gel.Thefigureshowsafluorograph(
(A)cytoplasmicfiber mRNAfromc
for 3 h with 32p; (B) whole-cell fibe
fromcells pulse-labeledfor 1 h witt D, E, F,andG)sameasBexcept wii
hofchase, respectively; (H)sameas
hchase.
The large decrease in size a species compared to the others
consistent with thepossibility th mRNA is generated in the cyt
splicing of species with additioi
ments.Analternativeexplanatioi ent decrease in size of fiber
cytoplasm is that the nuclear pi
way that gives rise to the matur
mRNA may be slower than the
results in cytoplasmic fiber mRl leader segments. Thus, the first
pearinthecytoplasm would beti
leadersegments,followedsometi appearance of the mature form, sion of a decrease in size of tI
mRNA. Ifthiswerethecase, thi precursor to the mature form c
should be observed in the nucle time that the species possessin segments are present in the cyt(
dress thispossibilityweexamines
F G appearance of label into fiber-related RNA
spe-cies isolated from total cellular RNA during a
[3H]uridine pulse, glucosamine-uridine chase experiment. This labeling method was chosen to follow the appearance and fate offiber mRNA precursors during an effective chase, which is not possible with
32Pi
as alabel.Infected cells were pulse-labeled for 1 h with [3H]uridine and chased for various lengths of time with glucosamine and cold uridine. Poly(A)-containing fiber-specific RNAs were isolated from cellular nucleic acids and analyzed on a1.2%agarosegel. A fluorograph of thisgel is shown in Fig. 2. Fiber-specific RNA isolated
aftera1-h pulse (lane B) consisted of a complex
population of heterogeneous high-molecular-weight RNA species with a number of prominent
discrete species also evident. The largest of
these was approximately 25kilobasesin length, the predicted size of a primary transcript from 16.4 to 91.3 m.u. (the 3' terminus of fiber mRNA). Aconsiderable portion of the radioac-tivity lay in a somewhat heterogeneous species rs to fiber mRNA which comigrated with the 32P-labeled cytoplas-otal nucleic acids mic fiber mRNA (lane A). After 1 h of chase se-labeled for1 h (lane C), much of the high-molecular-weight
oneplus
unlabeled
heterogeneous
fiber-specific
RNAdisappeared
ies
were selected and most of the radioactivity comigrated with DDNA and then pulse-labeled cytoplasmicfibermRNA (lane A). n a 1.2% agarose During subsequent chases of 2, 3, 5, and 8 h of the gel. Lanes: (lanesD, E, F,andG, respectively)the conver-ells pulse-labeled sion of thisheterogeneous species to two promi-r mRNA species nent discrete species was observed. The more[3
H]uridine;
(C,
abundant ofthese species comigrated with theth
1, 2, 3,5,and 8 mature form of32P-labeled
cytoplasmic mRNA(A)
but withan8- (lane H). (The less abundant and slowermigrat-ing ofthe two species is not reproduced well in
the figure, butcomigrated with the minor fiber
if fiber mRNA mRNA species possessingan additional leader
'iral mRNAs is segment.) This experiment demonstrated that
iatmature fiber there is no precursor to the rapidly migrating
:oplasm by the fiber mRNA species present afteran 8-h chase
nal leader seg- other than thecytoplasmic species labeled in a
1-nfor theappar- to 2-h pulse.
mRNA in the Localization of regions on the Ad2 genome
rocessing path- specifying leader segments on newly synthesized
re form of fiber fiber mRNA. To identify regions of the Ad2
pathway that genome which
specify
extra leader segments on4As with extra fiber mRNA, 2P-labeled cytoplasmic fiber
species to ap- mRNA, eitherlabeledfor 3 h orlabeled for 3 h hosewith extra and chased for 8 h, was isolated by preparative imelater by the hybridization to cloned Ad2 EcoRI-E DNA
im-giving the illu- mobilized ondiazobenzyloxymethyl-paper. The
he cytoplasmic RNA was then hybridized a second time to
en aprominent various cloned Ad2 DNA fragments
represent-Af fiber mRNA ing regions of the Ad2 genome between the
us at the same promotor at 16.4m.u. and the 5' terminus of the ig extra leader main body ofthe fiber gene at 86.3 m.u. RNA
Dplasm. To ad- isolated afterthe second hybridization was
ana-d thekinetics of lyzedon a1.2%agarose gel. The two right-hand
VOL.44, 1982
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[image:4.496.60.224.80.304.2]Ka.r S-ma A
- U ( 'r. P U . U
IglF<f§ "
ts
_"
as4h_ W
FIG. 3. Identification of regions of the Ad2 genome specifying leader sequences present on fiber mRNA.32p_ labeled cytoplasmic fiber mRNA from infected cells pulse-labeled for 3 h (lanes P) or pulse-labeled for 3 h and chased for an additional 8 h (lanes C) was isolated by preparative hybridization to cloned EcoRI-E DNA, immobilized on paper. This RNA was hybridized to other immobilized DNA fragments from different regions of thegenome. Selected RNA was eluted, denatured with glyoxal, and analyzed on a1.2%agarose gel.Lanes from left to right: 14C-labeledHeLacell rRNAs;32P-labeledfiber mRNA selected by Bal-E DNA, Bal-D DNA, Bam-D DNA, Bam-C DNA, Sma-A DNA, and EcoRI-D DNA;[32P]RNA selected on EcoRI-E DNA, which was the starting material for the other hybridizations.
lanes in Fig. 3 (EcoRI-E) show 32P-pulse-labeled
fiber mRNA (lane P) and pulse-labeled and
chased fiber mRNA (lane C), which was the
starting
materialfor
theexperiment.
Thesespe-cies also
hybridized
toBal-E DNA(14.7
to21.5m.u.) and Bal-D DNA (21.5 to 28.5 m.u.); this
wasexpected because the first and second
lead-ersegmentsand thirdsegment,
respectively,
arespecified by
DNAwithin
theseregions.
NofibermRNA-related sequences annealed to DNA
fragments
derived
from 29.0to76.8m.u.(Bam-D,Bam-C, and Sma-A).
(The
higher-molecular-weight
species
selectedby
thesefragments
aremRNAs which were
originally
present in thestarting materialas
nonspecific contaminants.)
Aconsiderable amount of
pulse-labeled
fibermRNA annealed to DNA within the EcoRI-D
fragment (75.0to84.0m.u.); however,
only
theslower-migrating portion of the heterogeneous
fibermRNA
species
wasselected(compare
Fig.
3, EcoRI-E and EcoRI-D, lanes P).
Likewise,
the
faster-migrating
andmoreprominent species
ofthe
pulse-labeled
and chasedfibermRNAdidnot hybridize to 75.9 to 84.0 m.u. DNA
(com-pare Fig. 3 EcoRI-E and EcoRI-D, lanes
C);
however, the
slower-migrating species
didhy-bridizetothis
region.
Thus, themajor
fractionoffibermRNAlabeledina
pulse
orafteralengthy
chase
hybridizes only
tosequencesderived fromDNA known to
specify
the mainbody
ofthemRNA or from DNA known to
specify
thethree-segment leader sequence. A minor
frac-tion offiber mRNA is also
composed
ofthesesequences plus an additional segment derived
from between 75.9 and 84.0m.u.
Sizechangeoffiber mRNA in the cytoplasm is due tometabolism of poly(A). Becausethe major
fraction of
pulse-labeled fiber mRNA failed tohybridize to regions of the genome known to
encode additional leader segments, we
investi-gated the
possibility
that the large size changeobserved
for fiber
mRNAsimply
resulted fromthe metabolism of poly(A). This was done by
analyzing
the sizeof
molecules from whichpoly(A)
hadbeen removed by annealingtopoly-deoxythymidylic
acid anddigesting
with RNaseH.mRNAwasisolatedfrom cells pulse-labeled
with
32p
or pulse-labeled and chased for 8 hinphosphate-containing
medium. Fiber mRNAwas isolated by hybridization to immobilized
DNA from 84.0to 89.7 m.u., and L4 mRNAs
(1OOK
andpVIII)wereisolated byhybridizationto immobilizedDNA from 72.6 to 76.9 m.u. A
sample of each mRNA wasdenaturedand
ana-lyzed directly by gel
electrophoresis,
and asecond sample was analyzed after removal of
the poly(A) tail. Figure 4 shows an
autoradio-gram ofthe gel. The lOOK and pVIII mRNAs
decreased in sizeby
approximately
100nucleo-tides during the chase period,and fibermRNA
decreased in sizeby
approximately
200nucleo-tidesasobservedpreviously. However,after the
removal of poly (A), all species migrated more
rapidly, and there was nodifference in the size
of pulse-labeled or pulse-labeled and chased
species, indicating that the entire decrease in
'.,.1 E.. 7
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[image:5.496.113.397.79.239.2]231
-RNase H +RNase H
z 72.6-76.9 83.4-89.772.6-76.9 83 4-89.7
P C P C P C P C
28S-FIG. 4. Analysis of lateregion4 mRNA mRNA after removal of poly(A) tails. C poly(A)-containing RNA was isolated fro fected HeLa cellspulse-labeledfor 2 hwiti P) orpulse-labeledand then chased for 8 h Lateregion4-specific RNA species (100K mRNAs)wereisolatedbypreparativehybri Ad2 72.6-to-76.9-m.u. DNA, and fiber mr selected byhybridization to 83.4-to-89.7-n RNAs were incubated withorwithout RN polydeoxythymidylate toremovepoly(A)t scribed in the text, denatured withglyoxal lyzedona1.2% agarosegel.
size for the late mRNAsis due to theml
of
poly(A).
Inparticular,
there was eno difference in the patterns of fibe
species
after removalofpoly(A). Thus,
decrease in size of fiber mRNA in thec
is
apparently
dueto a morerapid
metapoly(A)
thaninthe other latemRNAsSynthesis
of fibermRNA in ceils infetheAd2-SV40hybridvirus
Ad2+ND5.
iis a nondefective
hybrid
virus whoseconsists of Ad2 DNA withadeletiono
to 85.5m.u.
region
andaninsertion ofof the
early
region
of SV40 from 0.3m.u. (19). Included in the deleted seq
the
region
of Ad2 DNA which encodes extra("y")
leader segment which issteady-state fiber
mRNAisolated
fr
infected with thewild-type virus.Zain
have shown that the
predominant
forrmRNA present in cells infected with
has noextraleadersegment, demonstr.,
anintermediate
possessing
theysegmrequired for the
synthesis
of the mattHowever, it is
possible
thataninterme(an
alternate
leadersegmentserves as amic precursor in the absence of the
segment. Additional leader segments
plasmicmRNA encodedat
75,
77,
anchave been
observed
in cellsinfected
with Ad2(8) andAd2+ND4 (35). If this is the case, then
we would expect to observe a significant
frac-tion of pulse-labeled fiber mRNA possessing
theseleadersegments.
!-28S
Poly(A)-containing
cytoplasmic RNA wasiso-_V lated from cells infected with Ad2or
Ad2+ND5,
pulse-labeled for2hwith 32porpulse-labeled for
2 h and chased for 8 h in
phosphate-containing
medium. RNA samples were hybridized to
im--18S
mobilized SV40 DNAorAd2 DNA specificforfiber mRNA. Selected RNAs were denatured
with glyoxal and analyzed by agarose gel
elec-trophoresis. An autoradiogram of the gel is
shown in Fig. 5. No RNA species from
Ad2-infected cells were selected by SV40 DNA, as
expected. Two major species (4,900 and 3,200
bases) isolated from Ad2+ND5-infected cells
sandfiber were selected by SV40 DNA. These species
ytoplasmic were alsoselected by Ad2L4-specific DNAand
im Ad2-in- probably represent
100K
and pVIII mRNAs, h2P
(lanes which are 3'coterminal at the SV40 poly(A) site(lanesC). (J. Ramsey,unpublished data).
dizationto A single discrete species was selected by
iRNA
was
fiber-specific
DNA(83.4
to 89.7m.u.)
fromn.u. DNA.
RNA
isolated frompulse-labeled
cells infectedlase H and withAd2+ND5. This isin contrast to the hetero-.ails, as de- geneous fiber-specific species isolated from J, and ana- Ad2-infected cells. In addition, the Ad2+ND5
fiber mRNA (pulse-labeledorpulse-labeled and
chased) comigrated with the most rapidly
mi-grating form of fiber mRNAisolated from
Ad2-etabolism infectedcells,suggestingthat itdoes not possess
,ssentially an additional leadersegment.
r mRNA Toconfirmthis, pulse-labeledAd2+ND5 fiber
,thelarge mRNA selectedby hybridizationtoAd2
83.4-to-:ytoplasm
89.7-m.u. DNAwashybridized
asecond timetobolismof
ctedwith
kd2+ND5
- genomeof the 78.7
aportion
9 to 0.11
uences is
themajor
s seen in
rom cells
et al.(38)
n of fiber this virus
atingthat
ent is not ure form.
diatewith
cytoplas-y leader
in
cyto-d 85 m.u.
ac
2e-
28S-
18S--28S
-18S
[image:6.496.50.242.80.254.2]SV40 DNA Ad2 83.4-89.7 m.u.
FIG. 5. Size analysis of fiber mRNA isolated from Ad2+ND5-infected cells. Cytoplasmic poly(A)-con-taining RNA was isolated from HeLa cells infected withAd2 orAd2+ND5 which had been pulse-labeled for 2 h with 32P or pulse-labeled and chased for 8 h. SV40-specificRNAs and fibermRNA were isolated by hybridization to immobilized SV40 DNA and Ad2 83.4-to-89.7-m.u. DNA,respectively. Selected RNAs were denatured with glyoxal and analyzed on an agarose gel.
on November 10, 2019 by guest
http://jvi.asm.org/
[image:6.496.255.446.472.589.2]supported by
kinetic and structural data whichshow that the size change in the cytoplasmic
fiber mRNAsis due exclusivelyto metabolism
of the3'-poly(A)tail and thatspecies containing
j9,u. extra leader
segments
are present atapproxi-mately the samerelative frequencies aftera2-h
pulse or aftera 2-h pulse andan 8-h chase. The
roughly
equivalent
cytoplasmic
stabilitiesof theA% ; " , multiple fiber mRNAsargueagainsta
precursor-product relationship, and the observation that
theabsence ofmostof thesemorecomplexfiber
mRNAsin HeLa cells infected with Ad2+ND5
a:4;
>Y is not grossly deleterious to viral replication FIG. 6. Hybridization ofAd2+ND5fiber mRNA to supports this conclusion.variousregions of the Ad2 genome.
32P-pulse-labeled
Other investigators have examined the possi-fiber mRNA from Ad2+ND5- or Ad2-infected cells bility of cytoplasmic splicing in a number of was isolated by preparative hybridizationto immobi-systems.
Nevins(30)
has shown that all of the lized Ad2 83.4-to-89.7-m.u. DNA. Selected RNAwas late Ad2 mRNAs of the 38-to-51-m.u. mRNA thenhybridized a second time to DNA from various family are formed in the nucleus and that the regions of the Ad2 genome as indicated, and RNA largermembers of the family are not converted selectedby thissecondhybridizationwasanalyzedon to the smallermembers by splicing in thecyto-anagarose gel. plasm. Our results (Fig. 1) also support this
conclusion; in
fact,
we have not detected anyother potential
cytoplasmic
splicing
eventsDNA spanning the Ad2 genome from 21.5 to among the Ad2 late mRNAs. Piper et
al.
(33)83.4m.u.,andselectedRNAswere
analyzed by
have investigated the synthesis and processing agarosegel
electrophoresis
(Fig.
6).
Hybridiza-
of
the three polyoma late mRNAs and havetion was
only
observed withDNAfrom21.5to likewiseconcluded that each of these mRNAs is28.5 m.u., the region which encodes the third formed in the nucleus. An early report that the common leader segment. The
slower-migrating
SV40
late19S
mRNA is processed in thecyto-fraction of
pulse-labeled
Ad2 fiber mRNA spe- plasm to formthe16S
species(4) has notsubse-cies
hybridized
to 75.9-to-83.4-m.u.DNA,
as quently been substantiated. Melton et al. (29)observed
previously. Thus,
we can find noevi- haveexamined the synthesis of yeasttRNAtYrindence in cells infected with Ad2 ND5ofa
major
Xenopuslaevis oocytes and concluded that thepulse-labeled
fiber mRNA thatcontainsaleader tRNAprecursor is spliced in the nucleus. segment in addition to the commonthree-seg-
The role ofalternate forms of viral mRNAs (ifmentleader. any) in viral replication is still of interest. Such
species may in fact be nuclearintermediates in
DISCUSSION RNA processing which are
transported
to theDISCUSSION
cytoplasm before completion. The fact thatThe presence of
multiple
fibermRNAs inthe thesespecies
representonly
a small subset ofcytoplasm
ofAd2-infected HeLacells was first nuclearprocessing
intermediates wouldsuggest
described
by
Chow and Broker(8).
The exis- that thistransport
is notrandom; i.e.,
thesetence of
heterogeneity
in thespliced
5'-leader intermediates have a structurethatmakesthem segments isnecessarily
due tothe utilization of competent for transport which other nuclearalternate
pathways
in thesplicing
oftheprimary
species
do not possess. Infact,
we haveob-transcript
of themajor
latetranscription
unit. servedasimilar selectiveprematuretransport
ofChow and Broker
(8)
anid Kilpatrick
etal.(20)
precursors to cellular mRNA in Ad2-infectedsuggested
thatcytoplasmic
adenoviral mRNAs cells(C. Lawrence,
unpublished
data).
possessing
extraleader segments may representSpecies
withextrasegmentscould also betrueintermediates in the formation of mature
processing
endpoints
which resultfromhaving
mRNAs. Because thesespecies
are found on alternateprocessing
pathways
forasingle
tran-polysomes
andthereforeareclearly
ofcytoplas-
script.
If the variouscytoplasmic
forms of a micorigin,
finalprocessing
ofthesespecies
may mRNAaretrueendpoints
they
might
be expect-occurin thecytoplasm.
ed tobefunctionally
distinct. The additional y Our results indicate thatalthough
fiber leader on fiber mRNA has no AUG codon tomRNAs
possessing
extra leader segments are initiateprotein
synthesis (12)
and is translated inreadily
detected in thecytoplasm,
there is no vitro with the sameefficiency
as mature fiberevidence to suggest that
they
serve as precur-mRNA,
suggesting
that there is no obvioussors to mature fiber mRNA. This conclusion is functional
significance
to the presence ofthe yon November 10, 2019 by guest
http://jvi.asm.org/
[image:7.496.58.256.82.209.2]Ad2 FIBER mRNA SYNTHESIS
leader. On the otherhand,the "i"leader, which is present on a large fraction of late region 1
mRNA synthesized earlyininfection, probably
encodesa14Kpolypeptide (1).Determination of
the sequences of other mRNA variants will at
leastindicate theirpotentialforencoding
differ-entpolypeptides.
ACKNOWLEDGMENTS
We thank SueBergetandJohn Wilson forprovidingvarious clonedDNAs used in thisstudy,andKathyJackson forexpert technicalassistance.
This workwassupported byPublic Health Servicegrant AI-16484from theNational Institutes of Health.
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