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A Comparison of Nuclear and Cytoplasmic Viral RNAs Synthesized Early in Productive Infection with Adenovirus 2
A Comparison of Nuclear and Cytoplasmic Viral RNAs Synthesized Early in Productive Infection with Adenovirus 2 !
HESCHELJ. RASKASAND ELIZABETH A. CRAIG* Department of Pathology Wa.yhington University School of Medicine St. Louis, Missouri
During the early phase of infection with adenoviruses, prior to the onset of viral DNA synthesis, a limited portion of the genome is expressed as cytoplasmic mRNA. The genome sites coding for the adenovirus 2 mRNAs have been determined by hybridization of viral RNA with specific viral DNA fragments ( 1 5 ) .The segments of the genome coding for individual viral mRNA species are shown in Fig. 1; this figure also shows the set of viral DNA fragments produced by cleavage with endo R.Eco R1 ( 6 ) . Four regions of the viral genome code for early messenger RNA, two regions from each strand. The DNA strand that generates rightward transcripts is designated the r strand. The extreme left-hand end of this strand is transcribed to give three RNAs, two 13 S and one 11 S species. Two mRNAs, one 19 S and one 13 S, are products of the region corresponding to Eco R1 fragments D and E. The 1 strand yields mRNAs from regions of the genome included in Eco R1 fragments C and B. A 20 S RNA is specified by the sequences in Eco R1-C, and 19 S and 11 S RNAs are detected by hybridization with Eco R1-B. To compare the nuclcar and cytoplasmic RNAs transcribed from early genes, nuclear RNA was prepared from cultures labeled with [ 'Hluridine, and cytoplasmic RNA was obtained from cultures incubated in the presence of ?LP. Since the cytoplasmic viral RNAs are polyadenylvlated (7, 8), the poly( A)-containing molecules were isolated from each these preparations. The nuclear and cytoplasmic RNAs were mixed and subjected to electrophoresis in gels containing 98%formamide. The RNA from each gel slice was eluted and then hybridized to specific DNA fragments. This procedure allows a direct comparison of the size dis-
of
* Present address: Department of Microbiology, University of California, San Francisco, California 94101. 293
294
I-IESCHEL J. RASKAS AND ELIZABETH A. CRAIG 135 115 1%
195 135
+ ++
3'
5'
----m*
A
1.
I
B -+ 1%
j F j D i E /
11s
C
-
5' r
3' I
205
FIG.1. Genome location of cytoplasmic adenovirus 2 RNAs synthesized early in productive infection. The genome sites coding for cytoplasmic viral RNA species synthesized early in infection were established by hybridization of size-fractionated RNA to specific adenovinis 2 DNA fragments (3-5, 9 ) . The five adenovirus 2 DNA sites cleaved by endo R*Eco R1 are indicated by the dashed vertical lines ( 6 ) ; the six fragments produced have the genome order A, B, F, D, E, C ( 1 3 ) . The space hetween each pair of small vertical lines represents 10%of the genome, and the lcngth of the RNAs is proportional to their molecular weights as calculated from electrophoretic mobility. The polarity of the strands was determined by analysis of the DNA termini ( 1 ).
tribution of nuclear and cytoplasmic viral RNAs transcribed from specific genes. In one experiment two adjacent fragments, Eco R1-R and F, were used as hybridization probes ( Fig. 2 ) . Hybridization with Eco R1-B DNA revealed the 19 S and 11 S cytoplasmic viral HNAs specified by this fragment. There was no detectable hybridization of cytoplasmic RNA to Eco R1-F DNA; this fragment codes exclusively for late genes. The profiles of hybridizable nuclear RNA werc considerably different. Nuclear sequences specified by Eco H1-H included two peaks that were larger than the cytoplasmic viral RNAs, one migrating as 25 S RNA and a smaller but reproducible peak migrating at the rate expected for 28 S RNA. Additional molecules migrntcd between the 11 S and 19 S cytoplasmic RNAs. The profile of Eco H1-F transcripts included two peaks that have the same migration rate as the 28 S and 25 S RNAs that hybridized to Eco RI-B DNA. From this and other observations ( 4 ) , we suggest that the 28 S nuclear HNAs detected by hybridization to Eco R1 €3 and F DNAs are actually a single molecular species that includes transcripts from both these fragments. To compare nuclear and cytoplasmic transcripts from a different rcgion of the genome, Eco R1 fragments D and E were utilized (Fig. 3 ) . At early times in infection these two adjacent fragments code for 19 S and 13 S mRNAs, which are transcribed from the T strand. The 13 S RNA that hybridizes to Eco R1-D and E is a single species transcribed from segments of both of these contiguous fragments. Hybridization of the iiuclear HNA revealed a discrete peak larger than the cytoplasmic mRNAs;
RNAS
295
FROM ADENOVIRUS-2 INFECTION 285
185
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- 4
-
T
'?
0 3 X
I '
E
8
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E
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a z
or3
n
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L2
' A
1
SLICE NUMBER
FIG.2.
10
20
SLICE NUMBER
FIG.3.
FIG. 2. Hybridization of p l y ( A )-containing nuclear and cytoplasmic RNAs to Eco R1-B and F DNA. Cytoplasmic ['"P]RNA and nuclear ['HIRNA were fractionated by electrophoresis, and the RNA eluted from gel slices was hybridized to 0.5 pg equivalents of Eco R l - B (upper panel) and F (lower panel) DNA. For experimental details see Craig and Raskas ( 9 ) . FIC:.3. Hybridization of poly ( A)-containing nuclear and cytoplasmic RNAs to Eco R1-D and E DNA. Cytoplasmic ["'PIRNA and nuclear 13H]RNA were fractionated by electrophoresis and thc RNA from gel slices was hybridized to 0.5-pg equivalents of Eco R1-D (upper panel) and E (lower panel) DNA. For experimental details see Craig and Raskas ( 9 ) .
25 S RNA hybridized to both Eco R1-D and Eco R1-E. In this instance also, it seems likely that a single 25 S molecule includes transcripts from the two adjacent fragments, D and E. For each of the four regions of the genome that code for early mRNA, we havc detected nuclear traiiscripts that are larger than the cytoplasmic mRNAs ( 9 ) . To determine the sequence relationship between the larger nuclear RNAs and cytoplasmic mRNA, hybridization-inhibition experiments were perfornicd. In such experiments the relatedness of two RNA prcparntions can be detcrniined by the ability of an unlabeled RNA to prevent the hybridization of n second RNA ( radioactive ) preparation. The procedure used for thcsc. cxperimcnts is outlined in Fig. 4.
296
HESCIIEL J . llASKAS AND ELIZABETH A. CRAIG
Nuclear RNA was labeled with [ {Hluridine and the poly( A)-containing molecules wcre purified and subjected to electrophoresis in formainide gels. The RNA eluted from each gel slice was then hybridized to three membranes, c d i containing the same DNA fragmcnt. Each of thcsc membranes was incubated with a large excess of nonradioactive HNA, either nuclear HNA from infected cc~lls, cytoplasmic RNA from infected cells, or RNA from uninfected cells, Thus, if a nuclear peak contains sequence$ identical to those prescnt in cytoplasmic RNA, its hybridization shoulcl be prevented by the prehybridization with nonradioactive cytoplasmic RNA. I285 185
Poly(A)+ Nuclear [3H] RNA
I
/Electrophoresis /Elute RNA
Cytoplasm R N A , Infected Cells 1 Tube From Eoch Gel Slice
1
g ~
~
1 1,-/ 2
F1c. 4.
'
u
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FIG.5.
FIG.4. Procedure for hybridization-inhibition studies with ['HIRNA fractionated by size. Following electrophoresis, the ['IIIRNA was eluted from each gel slice and then annealed to three filters. These filters contained the same DNA, but had been incubated with saturating ainounts of three different nonradioactive RNA preparations, as shown. For the actual experiments, the ['HIRNA from each gel slice was divided into three aliquots, and each aliquot was hybridized with one of the previously incubated iiiembranes ( 4, 9 ) . FIG.5 . Ability of early cytoplasmic RNA to inhibit hybridization of nuclear RNA transcribed from Snia I-E DNA. Poly( A)-containing nuclear ["FIIRNA was fractionated by electrophoresis, and the RNA eluted from each gel slice was analyzed by hybridization-inhibition stildies as ontlined in Fig. 4. One aliquot was hybridized to filters previously hybridized with whole-cell RNA from uninfected cells (O--O); a second aliquot was incubated with a filter similarly hybridized with nuclear RNA from cultures harvested 6 hours after infection (.--a); the third aliquot was hybridized to filters already annealed with early cytoplasmic RNA ( 0-0).
RNAS
FROM
ADENOVIRUS-2
INFECTION
297
An example of this type of experiment is presented in Fig. 5. These hybridizations were perfornicd with an adenovirus 2 DNA fragment (Sma 1-E) that includes sequences present in the left-hand 3-11%of the genome ( Mulder, Green and Delius, in preparation), These sequences code for two early cytoplasmic RNAs that migrate in electrophoresis as 13 S and 11 S RNAs (see Fig. 1).Polyadenylylated nuclear RNA transcribed from Sma I-E includes 22 S HNA as well as 11-13 S molecules. When nuclear ['HIRNA was hybridized with Sma I-E DNA which had been preincubated with RNA from uninfected cells, the profile was identical to that seen in the absencc of prehybridization. Hybridization to filters annealed in advance with nuclear RNA was reduced about 90%,as expected when the filters are pretreated with a homologous RNA. The filters presaturated with cytoplasmic RNA yielded a very different profile of hybridization. Hybridization of the 11-13 S molecules was reduced approximately 7540%. Hybridization of the larger 22 S peak was inhibited about 50%. This result demonstrates that the 22 S peak contains sequences transcribed from the same strand and the same region of the strand as the early cytoplasmic RNA. That the inhibition is only 50% suggests that the 22 S RNA contains sequences present in the nuclear transcripts but not transported to the cytoplasm. Similar results were obtained when hybridization-inhibition experiments were performed to analyze the larger nuclear RNAs transcribed from the other regions of the genome coding for early mRNA (Craig, Sayavedra and Raskas, in preparation). These findings are compatible with previous studies demonstrating the existence of nuclear-specific transcripts early in infection ( 1,3,10), Our rcsults to date are summarized in Fig. 6. The upper part of the figure shows the relevant cleavage sites for the three restriction endonucleascs used in these studies. The lower part of the figure compares the early cytoplasmic RNAs with the larger nuclear RNAs. Each region of the genome coding for early mRNA also specifies a polyadenylylated nuclear RNA large enough to include the sequences in the mRNAs as well as additional sequences. By comparing molecular weights and also from the results of the hybridization-inhibition studies, we can calculate the percent of the nucleotides in these RNAs that are nuclear-specific; for example, only 15%of the large nuclear transcript from Eco Rl-C DNA appears to be restricted to the nucleus whereas nearly 50% of the large RNA species from Eco R1-B is nuclear specific. From the 2 strand there is a 22 S nuclear RNA specified by Eco R1-C DNA as compared to the 20 S cytoplasmic species and a 28 S nuclear RNA from Eco R1 fragment B and F as compared to the 19 S and 11 S cytoplasmic RNAs. The r strand of Eco R1-D and E fragments specifies 25 S nuclear RNA and 19 S and 13 S cytoplasmic RNAs. Transcripts from the left-hand end of the
HESCIIEL J. RASKAS AND ELIZABETH A . CRAIG
913 r
E
JI
3.0
1
A
58.5 70.7 75.9 83.4 89.7 I 1 8 1 F I D I E I C ;
IG
iK 92.0 98.2
11.1
255
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& RI h
I
___)
Nucleus
-+
Cytoplosm
19s 135
22s
Cytoplosm
Nucleus
r strond
I
strond
FIG.6. A comparison of nuclear and cytoplasmic RNAs transcribed from regions
of the genonie containing early adenovirus 2 genes. The upper part of the figure
illustrates the five genome sites cleaved by endo R.Eco R 1 as well as one of the endo R . Hin dIII sites ( R. J. Roberts and collaborators, personal communication) and four of the endo R.Sona I sites (Mulder, Greene and Delius, in preparation). The cleavage sites are identified as the percent of the distance from the left-hand end of the genome; the calculated sites of cleavage were deduced from the molecular weights of fragment DNAs and therefore are subject to the uncertainties in molccufar-weight deterniinations. Fragnients prepared hy cleavage at these sites have been used in the analysis of nuclear RNA synthesized early in adenovirus 2 infection. The lower part of the figurc has been prepared as described for Fig. 1. The nuclear RNAs shown in this figure correspond to the largest moleciiles that have been detected as transcripts from specific D N A fragmcnts. When the same size class of RNA is hybridized to two adjacent fragments, we have assumed that a single transcript overlaps the cleavage sites.
genonie have been studied using Stna I fragments. Using Sma I-E DNA, 22 S and 11 S-13 S nuclear RNAs were idcntified as compared to the 11 S-13 S cytoplasmic RNAs. We have not yet detected a larger nuclear RNA transcribed from the extreme left-hand region, the first 3%of the genome (Smu I-J). The larger nuclcar RNAs we have identified may be precursors of early adenovirus mRNA. Certainly the structural analyses of these RNAs is compatible with the possibility, for they are transcribed from the “sense” strand of the DNA and include sequences present in cytoplasmic niRNA. Metabolic studies to investigate the possibility of a precursorproduct relationship are now in progress. However, in proposing models for the transcription and processing of early adenovirus RNA, three categories of transcripts need to be considered. In the present studies, we have compared the cytoplasmic mRNAs and discrete size classes of nuelcar RNA that are polyadenylylated, like mRNA, but that are larger
RNAs
r
I
FROM
--
ADENOVIRUS-2
299
INFECTION
A
MODEL 1
B
MODEL 2
-
C ~.
~~
... .
. Cf
~
c
FIG.7. Two possible models for the synthesis and processing of early adenovirus RNA. The schematic drawings A, B and C represent three groups of viral RNAs present early in infection. A represents the self-complementary RNAs transcribed from much, if not all, of the genome ( I , I 1 ) (Zimnier, Craig, Carlson and Raskas, in preparation). B illustrates the larger nuclear RNAs transcribed from the early genes. C is a representation of the early mRNAs transcribed from the adenovirus 2 genome.
than the mRNAs. Nuclei harvested early in infection contain a third category of RNA, complementary transcripts of large regions of the genome (I, I 1 ). Taking into account these three categories of RNA, two possible models for the synthesis of early mRNA are presented in Fig. 7. Primary transcription at early times may encompass most, if not all, of the genome (Model 1, A ) ; after rapid processing, relatively stable intermediates may accumulate ( B ) , followed by slower processing to yield the functional mRNAs ( C ) . Labeling for as short an interval as 10 minutes, we have not succeeded in isolating viral RNAs that migrate more slowly than 28 S RNA in formamide gels. Preliminary analysis of the nonpolyadenylylated viral RNAs has not revealed RNAs larger than 28 S. However this evidence is not compelling, and viral RNAs larger than those we have detected may be the primary transcripts. An alternative model (Model 2 ) assumes that the complementary viral RNAs present in low concentrations may be unrelated to mRNA formation. For example, these RNAs might be transcribed from viral genomes integrated into ccllular chromosomes (12). If the complementary RNAs are not precursors of mRNA, there may indeed be only four discrete pri-
300
IIESCHEL J. RASKAS AND ELIZABETH A . CRAIG
innry transcripts from the four regions of the genome coding for early mKNA ( I3 ), and these transcripts may be processed subsequently to yield the functional mRNAs ( C ) .
REFERENCES I . P. A. Sharp, P. H. Gallirriore and J. Flint, CSHSVB 39, 457 (1975). 2. L. Philipson, U. Pettersson, U. Lindberg, C. Tibbetts, B. Vennstrtim and T. Persson, C S H S Q B 39, 447 (1975). 3. E. A. Craig, J. Tal, T. Nishimoto, S. Zimmer, M. McGrogan and 11. J. Raskas, C S H S Q B 39, 483 ( 1975 ). 4. E. A. Craig, S. Zinnner and H. J. Raskas, J. Virol. 15, 1202 ( 1975). 5. E. A. Craig, M. McGrogan, C. Mulder and H. J. Raskas, J. Virol. 16, 905 (1975). 6. U. Pettersson, C. Mulder, H. Delius and P. Sharp, PNAS 70, 200 (197,3). 7. U. Lindhcrg, T. Persson and L. Philipson, J. Virol. 10, 909-919 ( 1972). 8. E. A. Craig and H. J. Raskas, J. Virol. 14, 751 ( 1974). 9. E. A. Craig and 13. J. Raskas, Cell 8, 205 ( 1976). 10. R. Wall, L. Philipson and J. E. Darnell, Virology 50, 27 (1972). I I . S. Zimmer and H. J. Raskas, Vi~ology70, 118 (1976). 12. H. Burger and W. Doerflcr, J. Virol. 13, 975 (1972). 13. C. Mulder, J. R. Arrand, H. Delius, W. Keller, U. Pettersson, R. J. Roberts and 1’. A. Sharp, CSHS@B 39,397 ( 1975).
HESCHELJ. RASKASAND ELIZABETH A. CRAIG* Department of Pathology Wa.yhington University School of Medicine St. Louis, Missouri
During the early phase of infection with adenoviruses, prior to the onset of viral DNA synthesis, a limited portion of the genome is expressed as cytoplasmic mRNA. The genome sites coding for the adenovirus 2 mRNAs have been determined by hybridization of viral RNA with specific viral DNA fragments ( 1 5 ) .The segments of the genome coding for individual viral mRNA species are shown in Fig. 1; this figure also shows the set of viral DNA fragments produced by cleavage with endo R.Eco R1 ( 6 ) . Four regions of the viral genome code for early messenger RNA, two regions from each strand. The DNA strand that generates rightward transcripts is designated the r strand. The extreme left-hand end of this strand is transcribed to give three RNAs, two 13 S and one 11 S species. Two mRNAs, one 19 S and one 13 S, are products of the region corresponding to Eco R1 fragments D and E. The 1 strand yields mRNAs from regions of the genome included in Eco R1 fragments C and B. A 20 S RNA is specified by the sequences in Eco R1-C, and 19 S and 11 S RNAs are detected by hybridization with Eco R1-B. To compare the nuclcar and cytoplasmic RNAs transcribed from early genes, nuclear RNA was prepared from cultures labeled with [ 'Hluridine, and cytoplasmic RNA was obtained from cultures incubated in the presence of ?LP. Since the cytoplasmic viral RNAs are polyadenylvlated (7, 8), the poly( A)-containing molecules were isolated from each these preparations. The nuclear and cytoplasmic RNAs were mixed and subjected to electrophoresis in gels containing 98%formamide. The RNA from each gel slice was eluted and then hybridized to specific DNA fragments. This procedure allows a direct comparison of the size dis-
of
* Present address: Department of Microbiology, University of California, San Francisco, California 94101. 293
294
I-IESCHEL J. RASKAS AND ELIZABETH A. CRAIG 135 115 1%
195 135
+ ++
3'
5'
----m*
A
1.
I
B -+ 1%
j F j D i E /
11s
C
-
5' r
3' I
205
FIG.1. Genome location of cytoplasmic adenovirus 2 RNAs synthesized early in productive infection. The genome sites coding for cytoplasmic viral RNA species synthesized early in infection were established by hybridization of size-fractionated RNA to specific adenovinis 2 DNA fragments (3-5, 9 ) . The five adenovirus 2 DNA sites cleaved by endo R*Eco R1 are indicated by the dashed vertical lines ( 6 ) ; the six fragments produced have the genome order A, B, F, D, E, C ( 1 3 ) . The space hetween each pair of small vertical lines represents 10%of the genome, and the lcngth of the RNAs is proportional to their molecular weights as calculated from electrophoretic mobility. The polarity of the strands was determined by analysis of the DNA termini ( 1 ).
tribution of nuclear and cytoplasmic viral RNAs transcribed from specific genes. In one experiment two adjacent fragments, Eco R1-R and F, were used as hybridization probes ( Fig. 2 ) . Hybridization with Eco R1-B DNA revealed the 19 S and 11 S cytoplasmic viral HNAs specified by this fragment. There was no detectable hybridization of cytoplasmic RNA to Eco R1-F DNA; this fragment codes exclusively for late genes. The profiles of hybridizable nuclear RNA werc considerably different. Nuclear sequences specified by Eco H1-H included two peaks that were larger than the cytoplasmic viral RNAs, one migrating as 25 S RNA and a smaller but reproducible peak migrating at the rate expected for 28 S RNA. Additional molecules migrntcd between the 11 S and 19 S cytoplasmic RNAs. The profile of Eco H1-F transcripts included two peaks that have the same migration rate as the 28 S and 25 S RNAs that hybridized to Eco RI-B DNA. From this and other observations ( 4 ) , we suggest that the 28 S nuclear HNAs detected by hybridization to Eco R1 €3 and F DNAs are actually a single molecular species that includes transcripts from both these fragments. To compare nuclear and cytoplasmic transcripts from a different rcgion of the genome, Eco R1 fragments D and E were utilized (Fig. 3 ) . At early times in infection these two adjacent fragments code for 19 S and 13 S mRNAs, which are transcribed from the T strand. The 13 S RNA that hybridizes to Eco R1-D and E is a single species transcribed from segments of both of these contiguous fragments. Hybridization of the iiuclear HNA revealed a discrete peak larger than the cytoplasmic mRNAs;
RNAS
295
FROM ADENOVIRUS-2 INFECTION 285
185
I ?I 4 I D
- 4
-
T
'?
0 3 X
I '
E
8
- 2
E
N 01 or
m >I
a z
or3
n
I
L2
' A
1
SLICE NUMBER
FIG.2.
10
20
SLICE NUMBER
FIG.3.
FIG. 2. Hybridization of p l y ( A )-containing nuclear and cytoplasmic RNAs to Eco R1-B and F DNA. Cytoplasmic ['"P]RNA and nuclear ['HIRNA were fractionated by electrophoresis, and the RNA eluted from gel slices was hybridized to 0.5 pg equivalents of Eco R l - B (upper panel) and F (lower panel) DNA. For experimental details see Craig and Raskas ( 9 ) . FIC:.3. Hybridization of poly ( A)-containing nuclear and cytoplasmic RNAs to Eco R1-D and E DNA. Cytoplasmic ["'PIRNA and nuclear 13H]RNA were fractionated by electrophoresis and thc RNA from gel slices was hybridized to 0.5-pg equivalents of Eco R1-D (upper panel) and E (lower panel) DNA. For experimental details see Craig and Raskas ( 9 ) .
25 S RNA hybridized to both Eco R1-D and Eco R1-E. In this instance also, it seems likely that a single 25 S molecule includes transcripts from the two adjacent fragments, D and E. For each of the four regions of the genome that code for early mRNA, we havc detected nuclear traiiscripts that are larger than the cytoplasmic mRNAs ( 9 ) . To determine the sequence relationship between the larger nuclear RNAs and cytoplasmic mRNA, hybridization-inhibition experiments were perfornicd. In such experiments the relatedness of two RNA prcparntions can be detcrniined by the ability of an unlabeled RNA to prevent the hybridization of n second RNA ( radioactive ) preparation. The procedure used for thcsc. cxperimcnts is outlined in Fig. 4.
296
HESCIIEL J . llASKAS AND ELIZABETH A. CRAIG
Nuclear RNA was labeled with [ {Hluridine and the poly( A)-containing molecules wcre purified and subjected to electrophoresis in formainide gels. The RNA eluted from each gel slice was then hybridized to three membranes, c d i containing the same DNA fragmcnt. Each of thcsc membranes was incubated with a large excess of nonradioactive HNA, either nuclear HNA from infected cc~lls, cytoplasmic RNA from infected cells, or RNA from uninfected cells, Thus, if a nuclear peak contains sequence$ identical to those prescnt in cytoplasmic RNA, its hybridization shoulcl be prevented by the prehybridization with nonradioactive cytoplasmic RNA. I285 185
Poly(A)+ Nuclear [3H] RNA
I
/Electrophoresis /Elute RNA
Cytoplasm R N A , Infected Cells 1 Tube From Eoch Gel Slice
1
g ~
~
1 1,-/ 2
F1c. 4.
'
u
L\lecbd Cells
FIG.5.
FIG.4. Procedure for hybridization-inhibition studies with ['HIRNA fractionated by size. Following electrophoresis, the ['IIIRNA was eluted from each gel slice and then annealed to three filters. These filters contained the same DNA, but had been incubated with saturating ainounts of three different nonradioactive RNA preparations, as shown. For the actual experiments, the ['HIRNA from each gel slice was divided into three aliquots, and each aliquot was hybridized with one of the previously incubated iiiembranes ( 4, 9 ) . FIG.5 . Ability of early cytoplasmic RNA to inhibit hybridization of nuclear RNA transcribed from Snia I-E DNA. Poly( A)-containing nuclear ["FIIRNA was fractionated by electrophoresis, and the RNA eluted from each gel slice was analyzed by hybridization-inhibition stildies as ontlined in Fig. 4. One aliquot was hybridized to filters previously hybridized with whole-cell RNA from uninfected cells (O--O); a second aliquot was incubated with a filter similarly hybridized with nuclear RNA from cultures harvested 6 hours after infection (.--a); the third aliquot was hybridized to filters already annealed with early cytoplasmic RNA ( 0-0).
RNAS
FROM
ADENOVIRUS-2
INFECTION
297
An example of this type of experiment is presented in Fig. 5. These hybridizations were perfornicd with an adenovirus 2 DNA fragment (Sma 1-E) that includes sequences present in the left-hand 3-11%of the genome ( Mulder, Green and Delius, in preparation), These sequences code for two early cytoplasmic RNAs that migrate in electrophoresis as 13 S and 11 S RNAs (see Fig. 1).Polyadenylylated nuclear RNA transcribed from Sma I-E includes 22 S HNA as well as 11-13 S molecules. When nuclear ['HIRNA was hybridized with Sma I-E DNA which had been preincubated with RNA from uninfected cells, the profile was identical to that seen in the absencc of prehybridization. Hybridization to filters annealed in advance with nuclear RNA was reduced about 90%,as expected when the filters are pretreated with a homologous RNA. The filters presaturated with cytoplasmic RNA yielded a very different profile of hybridization. Hybridization of the 11-13 S molecules was reduced approximately 7540%. Hybridization of the larger 22 S peak was inhibited about 50%. This result demonstrates that the 22 S peak contains sequences transcribed from the same strand and the same region of the strand as the early cytoplasmic RNA. That the inhibition is only 50% suggests that the 22 S RNA contains sequences present in the nuclear transcripts but not transported to the cytoplasm. Similar results were obtained when hybridization-inhibition experiments were performed to analyze the larger nuclear RNAs transcribed from the other regions of the genome coding for early mRNA (Craig, Sayavedra and Raskas, in preparation). These findings are compatible with previous studies demonstrating the existence of nuclear-specific transcripts early in infection ( 1,3,10), Our rcsults to date are summarized in Fig. 6. The upper part of the figure shows the relevant cleavage sites for the three restriction endonucleascs used in these studies. The lower part of the figure compares the early cytoplasmic RNAs with the larger nuclear RNAs. Each region of the genome coding for early mRNA also specifies a polyadenylylated nuclear RNA large enough to include the sequences in the mRNAs as well as additional sequences. By comparing molecular weights and also from the results of the hybridization-inhibition studies, we can calculate the percent of the nucleotides in these RNAs that are nuclear-specific; for example, only 15%of the large nuclear transcript from Eco Rl-C DNA appears to be restricted to the nucleus whereas nearly 50% of the large RNA species from Eco R1-B is nuclear specific. From the 2 strand there is a 22 S nuclear RNA specified by Eco R1-C DNA as compared to the 20 S cytoplasmic species and a 28 S nuclear RNA from Eco R1 fragment B and F as compared to the 19 S and 11 S cytoplasmic RNAs. The r strand of Eco R1-D and E fragments specifies 25 S nuclear RNA and 19 S and 13 S cytoplasmic RNAs. Transcripts from the left-hand end of the
HESCIIEL J. RASKAS AND ELIZABETH A . CRAIG
913 r
E
JI
3.0
1
A
58.5 70.7 75.9 83.4 89.7 I 1 8 1 F I D I E I C ;
IG
iK 92.0 98.2
11.1
255
&dIU
& RI h
I
___)
Nucleus
-+
Cytoplosm
19s 135
22s
Cytoplosm
Nucleus
r strond
I
strond
FIG.6. A comparison of nuclear and cytoplasmic RNAs transcribed from regions
of the genonie containing early adenovirus 2 genes. The upper part of the figure
illustrates the five genome sites cleaved by endo R.Eco R 1 as well as one of the endo R . Hin dIII sites ( R. J. Roberts and collaborators, personal communication) and four of the endo R.Sona I sites (Mulder, Greene and Delius, in preparation). The cleavage sites are identified as the percent of the distance from the left-hand end of the genome; the calculated sites of cleavage were deduced from the molecular weights of fragment DNAs and therefore are subject to the uncertainties in molccufar-weight deterniinations. Fragnients prepared hy cleavage at these sites have been used in the analysis of nuclear RNA synthesized early in adenovirus 2 infection. The lower part of the figurc has been prepared as described for Fig. 1. The nuclear RNAs shown in this figure correspond to the largest moleciiles that have been detected as transcripts from specific D N A fragmcnts. When the same size class of RNA is hybridized to two adjacent fragments, we have assumed that a single transcript overlaps the cleavage sites.
genonie have been studied using Stna I fragments. Using Sma I-E DNA, 22 S and 11 S-13 S nuclear RNAs were idcntified as compared to the 11 S-13 S cytoplasmic RNAs. We have not yet detected a larger nuclear RNA transcribed from the extreme left-hand region, the first 3%of the genome (Smu I-J). The larger nuclcar RNAs we have identified may be precursors of early adenovirus mRNA. Certainly the structural analyses of these RNAs is compatible with the possibility, for they are transcribed from the “sense” strand of the DNA and include sequences present in cytoplasmic niRNA. Metabolic studies to investigate the possibility of a precursorproduct relationship are now in progress. However, in proposing models for the transcription and processing of early adenovirus RNA, three categories of transcripts need to be considered. In the present studies, we have compared the cytoplasmic mRNAs and discrete size classes of nuelcar RNA that are polyadenylylated, like mRNA, but that are larger
RNAs
r
I
FROM
--
ADENOVIRUS-2
299
INFECTION
A
MODEL 1
B
MODEL 2
-
C ~.
~~
... .
. Cf
~
c
FIG.7. Two possible models for the synthesis and processing of early adenovirus RNA. The schematic drawings A, B and C represent three groups of viral RNAs present early in infection. A represents the self-complementary RNAs transcribed from much, if not all, of the genome ( I , I 1 ) (Zimnier, Craig, Carlson and Raskas, in preparation). B illustrates the larger nuclear RNAs transcribed from the early genes. C is a representation of the early mRNAs transcribed from the adenovirus 2 genome.
than the mRNAs. Nuclei harvested early in infection contain a third category of RNA, complementary transcripts of large regions of the genome (I, I 1 ). Taking into account these three categories of RNA, two possible models for the synthesis of early mRNA are presented in Fig. 7. Primary transcription at early times may encompass most, if not all, of the genome (Model 1, A ) ; after rapid processing, relatively stable intermediates may accumulate ( B ) , followed by slower processing to yield the functional mRNAs ( C ) . Labeling for as short an interval as 10 minutes, we have not succeeded in isolating viral RNAs that migrate more slowly than 28 S RNA in formamide gels. Preliminary analysis of the nonpolyadenylylated viral RNAs has not revealed RNAs larger than 28 S. However this evidence is not compelling, and viral RNAs larger than those we have detected may be the primary transcripts. An alternative model (Model 2 ) assumes that the complementary viral RNAs present in low concentrations may be unrelated to mRNA formation. For example, these RNAs might be transcribed from viral genomes integrated into ccllular chromosomes (12). If the complementary RNAs are not precursors of mRNA, there may indeed be only four discrete pri-
300
IIESCHEL J. RASKAS AND ELIZABETH A . CRAIG
innry transcripts from the four regions of the genome coding for early mKNA ( I3 ), and these transcripts may be processed subsequently to yield the functional mRNAs ( C ) .
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