Cell, Vol. 11, 533-544,

July

1977, Copyright

0 1977 by MIT

One Predominant S-Undecanucleotide Adenovirus 2 Late Messenger RNAs Richard E. Gelinas and Richard J. Roberts Cold Spring Harbor Laboratory Cold Spring Harbor, New York 11724

Summary Oligonucleotides containing the 5’ termini of adenovirus 2 mRNA are selectively retained on columns of dihydroxyboryl cellulose. When total late adenovirus 2 mRNA was treated with RNAase Tl, a single 5’ terminal oligonucleotide was isolated, although in several states of methylation. This oligonuclec$ide has the general structure m7G5’ppp5’AmCmU(C,,U,)G. Since at least twelve individual species of mRNA must be present late after infection, this finding was unexpected and its significance is discussed.

Introduction Adenovirus 2 (Ad2) messenger RNA may be divided into two classes, early and late, based upon whether the mRNA is synthesized before or after the onset of viral DNA synthesis during lytic infection of a permissive human cell line such as HeLa or KB (Green et al., 1970; Sharp and Flint, 1976). During lytic infection, about 20% of the viral genome is expressed both early and late (Fujinaga and Green, 1970; Tal, Craig and Raskas, 1975), whereas the remaining 80% of the viral genome is only expressed at late times (Sharp, Gallimore and Flint, 1974). Among the mRNAs which are produced late are those coding for the main structural proteins of the virion such as hexon, fiber and penton (Lewis et al., 1975). The observed expression of Ad2 genes thus implies that a control system exists which is capable of discriminating between at least two classes of viral genes. We are interested in determining the sequences present at the 5’ ends of Ad2 mRNAs to map precisely their coding regions within the Ad2 genome. These data, combined with the nucleotide sequence of small DNA fragments coding for these 5’ ends, will then allow the determination of sequences immediately preceding those transcribed. Such DNA sequences are presumably responsible for the control of the transcription process and may be either promoters or RNA processing sites. The mRNAs of most eucaryotes and their viruses contain a characteristic “capped” structure at their 5’ ends (Furuichi et al., 1975a; Adams and Cory, 1975) and Ad2 mRNAs are unexceptional in this regard (Moss and Koczot, 1976; Wold, Green and Munns, 1976; Sommer et al., 1976; Hashimoto and Green, 1976). The key feature of this structure is

in

the presence of a 7-methylguanosine residue linked via a 5’,5’-triphosphate bridge to the 5’ terminal residue of the mRNA chain. The unusual linkage of the 7-methylguanosine residue provided a way of isolating 5’ terminal oligonucleotides containing these structures. These oligonucleotides are unique in that they contain a 3’-phosphate at one terminus (for example, after RNAase Tl cleavage, they contain 3’-Gp) and a 2’,3’-cis diol from the 7-methylguanosine at the other terminus. Oligonucleotides containing a 2’,3’-cis diol have previously been isolated on affinity columns of dihydroxyboryl cellulose (DBAE-cellulose) (Weith, Wiebers and Gilham, 1970), and this method has been of value for the isolation of 3’ terminal oligonucleotides from ribosomal RNAs (Rosenberg, 1974) and capped structures generated by RNAase T2 cleavage of avian sarcoma virus mRNA (Furuichi et al., 1975b). We have extended their approach and used this column to isolate capped 5’ terminal oligonucleotides produced by RNAase Tl cleavage of Ad2 mRNAs. Surprisingly, although several independent studies have demonstrated that at least twelve different late mRNAs must be present based on estimates of the translational diversity of different mRNA size classes (Anderson et al., 1974), transcription mapping by analysis of hybridization kinetics (Flint and Sharp, 1976) and the ability to form RNA displacement loops (Westphal, Meyer and Maizel, 1976; Chow et al., 1977), we detect only one predominant 5’ terminal Tl oligonucleotide present in late Ad2 mRNAs. This’5’ terminal oligonucleotide isolated from late mRNAs differs from that isolated from early mRNAs. Results One Principal 5’ Terminal Oligonucfeotide in Ad2 mRNA Oligonucleotides from the 5’ termini of Ad2 mRNA were isolated by the scheme outlined in Figure 1. 32P-labeled RNA was digested with RNAase Tl, and oligonucleotides containing poly(A) which arose from the 3’ end of the mRNA (5’-hydroxyl, 3’-hydroxyl) were partially removed by binding to oligo(dT)-cellulose. Oligonucleotides from the 5’ termini of mRNA (3’-hydroxyl, 3’-phosphate) which are “capped” with m7G in a 5’,5’-triphosphate link, were then separated from internal oligonucleotides (5’-hydroxyl, 3’-phosphate) by affinity chromatography on DBAE-cellulose which selectively binds oligonucleotides containing 2’,3’ cis diols. The solution of these 5’ terminal oligonucleotides was desalted, concentrated and analyzed by electrophoresis on cellulose acetate at pH 3.5 in the first dimension and homochromatography in the second dimension (Brownlee and Sanger, 1969).

Cell 534

The pattern of oligonucteotides isolated from high molecular weight Ad2 mRNA purified by hybridization to Ad2 DNA is shown in Figure 2A. In this experiment, Ad2 RNA was labeled with aaP from 2 hr post-infection through 24 hr post-infection, and the 5’ terminal oligonucleotides were isolated as outlined in Figure 1. 5’ termini of Ad2 mRNA which are “capped’‘-that is, which contain m7G in a 5’,5’-triphosphate linkage followed by one or two residues with P’-O-methyl groupsshould have 5 or 6 phosphate residues which are resistant to RNAase T2 (a nuclease which degrades RNA free of 2’-O-methyl modifications to 3’ nucleoside monophosphates). Other oligonucleotides which come from internal or 3’ terminal positions should be degraded to mononucleotides by RNAase T2. To locate capped 11 oligonucleotides, the Tl digestion products of Figure 2A were redigested with RNAase T2, and the products were fractionated by electrophoresis on DEAE-cellulose paper at pH 3.5 (see Figure 3, slots 1 and 2). In this system, mononucleotides migrate faster than the blue dye (xylene cyanol FF), whereas the capped structures m7G5’pppS’NmN or m7G5’ pppNmNmN resistant to digestion with RNAase T2 migrate much more slowly than the blue dye. Capped structures were detected in oligonucleotides A and B only (Figure 3, slot 2). The relative yields of these two oligonucleotides varied among different experiments, suggesting that they were related one to another (see below). During lytic infection, Ad2-specific RNA accumu-

T,-RNAse

f HOm7G5’PPP5’N cme----GP

oligo

+ -P

+ ----AA$)H

(dT) cellulose

-AOH

I,,m7G5’ppp5’N

Pme--+P

DBAE-cell ulose

+ ---+P

----GP

j_ HOm7G5'~p~5'Nome~~ Figure 1. Scheme for the Isolation of 5’ Terminal tides from mRNA on DBAE-Cellulose Figure

2A, B, C. DBAE-Cellulose-Selected

Tl

Tl Oligonucleo-

Oligonucleotides

from

Ad2 and Mock-Infected

KB Cells

Polysomal RNA was prepared by magnesium precipitation from Ad2 or mock-infected KB cells which were labeled with 32P from 2-24 hr post-infection. (A) Tl oligonucleotides from Ad2 hybridized RNA: 1 x IO9 dpm (1 mg) of polysomal RNA from Ad2-infected cells were hybridized to 112 +g Ad2 DNA in solution. The hybridized RNA (5 x 10’ dpm) was digested with RNAase Tl and fractionated on DBAE-cellulose as described in Experimental Procedures. The bound fraction (1.5 x lo5 dpm) was analyzed by homochromatography with homomix C (Barrell, 1971). All oligonucleotides with four or more phosphates were recovered as individual spots and redigested with RNAase T2. The only oligonucleotides which had redigestion patterns characteristic of capped mRNA 5’ termini are marked A and B. The RNAase T2 digestion of the oligonucleotide marked A is shown in Figure 3, slot 2. The material at the origin after digestion with RNAase T2 yielded mononucleotides enriched in AMP and presumably represents residual poly(A). (B) Tl oligonucleotides from total cytoplasmic RNA from AdP-infected cells labeled from 2-24 hr post-infection: 3 x lo9 dpm (3 mg) of cytoplasmic RNA from AdP-infected cells were digested with Tl RNAase and bound to DBAE-cellulose. The bound fraction (2.6 x IO6 dpm) was fractionated by homochromatography as described above. All oligonucleotides were recovered and redigested with RNAase T2. The only Tl oligonucleotide with a redigestion pattern characteristic of a capped 5’ terminus is marked “A.” (C) Tl oligonucleotides from polysomal RNA from mock-infected cells: 1.3 x lo9 dpm (1.2 mg) of 32P polysomal RNA from uninfected KB cells (labeled for 12 hr) were digested with RNase Tl and selected on DBAE-cellulose. Oligonucleotideswhich migrated to a position similar to that of the principal Ad2 5’ Tl oligonucleotide shown in (A) and (B) above were redigested with RNAase T2 and liberated only mononucleotides. Figure 2D, E, F. One Principal 5’ Terminal Tl Oligonucleotide in RNA Bound to Poly(U)-Sepharose Polysomal RNA (1 mg; 4.5 x 10’ dpm) labeled from 15-24 hr post-infection was fractionated on poly(U)-sepharose. (D) The RNA which did not bind was digested with RNAase Tl, and 5’ terminal oligonucleotides were selected on DBAE-cellulose and finger-printed as described in Figure 2. (E) The RNA which bound to poly(U)-sepharose was digested with RNAase Tl and again fractionated on poly(U)-sepharose to separate 5’ termini from 3’ termini containing poly(A). The RNA which did not bind to the second poly(U)-sepharose column was selected on DBAEcellulose and fingerprinted as described above. (F) RNA which bound to the second poly(U)-sepharose column was selected on DBAE-cellulose and fingerprinted as described above. Capped oligonucleotides were located by redigestion analysis with RNAase T2 as described above and are indicated by the arrows in (D) and (E). All other oligonucleotides were digested to mononucleotides with RNAase T2. The oligonucleotide marked with an arrow in (D) was contaminated with noncapped oligonucleotides, since redigestion with RNAase T2 gave ~0.2 molar equivalents of the five- and sixphosphate caps. The RNA at the origin of the second dimension (homochromatography) of (D) and (F) was >90% polyadenylic acid.

5’ Termini 535

of Adenovirus

2 mRNA

Figure

2. A-F

Cell 536

lates in the cell, and at late times it may account for more than 90% of the newly transcribed RNA (Lindberg, Persson and Philipson, 1972). The AdP-specific 5’ terminal Tl oligonucleotide could be detected (Figure 29) by subjecting total cytoplasmic RNA, labeled throughout viral infection, to the scheme outlined in Figure 1. In contrast, this oligonucleotide was not detected in cytoplasmic RNA from mock-infected cells (Figure 2C). Only the oligonucleotides identified with arrows in Figures 2A and 29 were putative mRNA 5’ termini as judged by their partial resistance to RNAase T2. The pattern of oligonucleotides shown in Figures 29 and 2C is complex because the 3’ terminal oligonucleotides from most RNAs (for example, tRNA, rRNA) present in the cell are selected by this scheme. The intense oligonucleotide marked 18s in Figures 29 and 2C was eluted from the fingerprint of Figure 2C, and redigested with RNAase A and U2. The results were consistent with the sequence (G)AUCAUUA&, which has been identified as the 3’ terminal Tl oligonucleotide of the 18s ribosomal RNA of Drosophila melanogaster, Saccharomyces cerevisiae (Shine and Dalgarno, 1974) and rabbit reticulocytes (Hunt, 1970). When larger amounts of either 32P Ad2-specific RNA (Figure 59) or RNA from lytically infected cells were analyzed directly, other oligonucleotides with moieties resistant to digestion with RNAase T2 were sometimes detected. These minor oligonucleotides never represented more than a small percentage of the total RNAase T2-resistant material from any one fingerprint. They migrated to positions of similar charge in the first dimension, but in the second dimension migrated as if they were of shorter chain length than the principal 5’ terminal Tl oligonucleotide (A and B of Figure 2A). Composition of the Ad2 mRNA 5’ Terminus The composition of the principal AdP-specific mRNA 5’ terminus was determined by redigestion with a variety of nucleases (Figure 3 and Table 1). Although two 5’ terminal oligonucleotides were present in some fingerprints (Figure 2A, spots A and B), their relative yields were variable with A usually predominating. Redigestion of these oligonucleotides with RNAase A failed to reveal any significant differences, suggesting that they represent the same oligonucleotide but with varying extents of methylation at N6 of the adenosine residue of the cap (see Moss and Koczot, 1976). Another possibility would be that one of the oligonucleotides contains the ring-opened form of 7-methylguanosine, but we have not pursued this matter further. All the analyses described below were performed on oligonucleotide A. The oligonucleotide corresponding to A of Fig-

ure 2A was isolated and redigested with RNAase T2, RNAase A (which is pyrimidine-specific) and snake venom phosphodiesterase (an exonuclease which releases 5’-nucleoside monophosphates) as shown in Figure 3. About one half of the radioactivity in the oligonucleotide is liberated as 3’-UMP, 3’-CMP and 3’-GMP; the remainder is resistant to RNAase T2 digestion and migrates as two shorter oligonucleotides containing 5 and 6 phosphate residues as shown in Figure 3, slot 2. Mild digestion of oligonucleotide A with snake venom phosphodiesterase gave 1 mole of 5’-m7GMP per mole of oligonucleotide (Figure 3, slot 4). Redigestion with RNAase A produced 4 moles of 3’-CMP, 3 moles of 3’-UMP, 1 mole of 3’-GMP and 1 mole total of the two short oligonucleotides which contain five phosphates (0.3 mole) and six phosphates (0.7 moles; Figure 3, slot 5, and Table 1). Oligonucleotide A is resistant to RNAase U2 (specific for purines free of 2’-O-methyl modifications) and indeed was routinely repurified after elution from the first fingerprint (for example, Figure 2A) by redigestion with RNAase U2, which degraded similarly migrating contaminants [usually derived from the poly(A) background]. The five and six phosphate-containing structures prepared by affinity chromatography on DBAE-cellulose were identical with those obtained by direct RNAase T2 digestion of Ad2 mRNA using a procedure similar to that of Adams and Cory (1975) (data not shown). Other investigators (Moss and Koczot, 1976; Sommer et al., 1976) have also isolated these structures from Ad2 mRNA and have idfntified the predomina$ components as m;G5’pppAmpNp and m7GS’pppS’AmpCmpNp, where A is a mixture conisting of adenosine residues containing 0, 1 or 2 methyl groups at N6. Our re-analysis (Figure 3) of these spots is consistent with their results, with the additional observations that the six-phosphate cap contains U at the third position and the five-phosphate cap contains C at the second positioniWe have not rigorously established the nature of A. Our results, together with the nucleoside analyses of Ad2 mRNA cap structures by Moss and Koczot (1976) and Sommer et al. (1976), are consistent with the following structure for the principal $d2 mRNA 5’ oligonucleotide: m7G5’ppp5’AmC(m)U (C,, U&G. We have shown that the six phosphate-containing capped structure has the composition m7G5’pppS’iA)(Cm)Up, but have not established the order of Am and Cm. One 5’ Terminus in Ad2 mRNA Selected on Poly(U)-Sepharose An experiment was designed to test whether Ad2 mRNA purified by binding to poly(U)-sepharose contained the 5’ terminal oligonucleotide previ-

5’ Tsrmini 537

Figure

of Adenovirus

3. Composition

2 mRNA

of the Principal

Ad2 5’ Terminal

Tl Oligonucleotide

The principal Ad2 mRNA 5’ Tl Oligonucleotide (oligonucleotide A) was prepared as described in Figure 2A and redigested as indicated. Fractionation was by electrophoresis on DEAE-cellulose paper at pH 3.5 (slots l-5) or Whatman 540 paper at pH 3.5 (slots 6-8). Slot 1: RNAase T2 redigestion products of a typical oligonucleotide eluted from the fingerprints of Figures 2A, 28 or 2C which liberates the four 3’ nucleoside monophosphates. RNAase T2 digestion was performed as described by Barrel1 (1971). Slot 2: RNAase T2 redigestion of oligonucleotide A from Figure 2A. Slot 3: 32P-labeled marker 7-methylguanosine 5’-phosphate prepared from 7-methylguanosine by the method of Simsek et al. (1973). The slower migrating contaminant arises by degradation of 7-methylguanylic acid. Slot 4: Venom phosphodiesterase redigestion of oligonucleotide A. Slot 5: RNAase A redigestion of oligonucleotide A. “Cap 1” and “Cap 2” are RNAase A-resistant oligonucleotides with structures of the form

Slot

pm7GppplmpCp and pm7GpppimpCmpUp, respectively. 6: Markers prepared by penicillium nuclease (Pl) digestion of uniformly Slot 7: Pl nuclease redigestion of the cap 1 structure from slot 5. Slot 8: Pi nuclease redigestion of the cap 2 s?ructure from slot 5.

ously found in total cytoplasmic RNA and Ad2 RNA purified by hybridization. Polysomal RNA from lytitally infected cells labeled late after infection was fractionated on poly(U)-sepharose. The RNA which did not bind (51% of the input) was digested with RNAase Tl, and the resulting oligonucleotides were selected on DBAE-cellulose and subjected to fingerprint analysis (Figure 2D). The RNA which bound (44% of the input) was digested with RNAase Tl and again fractionated on poly(U)-sepharose into unbound and poly(A)-containing portions. Both these samples were also fractionated on DBAE-cellulose, and the oligonucleotides which bound were fingerprinted. Figure 2E (arrow) shows

labeled

RNA,

that only one 5’ terminal Tl oligonucleotide was found in Ad2 mRNA which originally bound to poly(U)-sepharose. No other capped oligonucleotides were found on the fingerprint shown in Figure 2E by redigestion analysis. The oligonucleotide identified with the arrow had the same composition as the eleven-residue 5’ terminal Tl oligonucleotide identified in Figure 2 from total cytoplasmic RNA or RNA hybridized to Ad2 DNA. Figure 2D shows that this eleven-residue oligonucleotide is also present in the RNA which did not bind to poly(U)-sepharose. This is probably due to incomplete binding to poly(U)-sepharose and the presence of fragmented mRNA molecules in the

Cell 538

Table 1. Quantitation Oligonucleotides

of RNAase

A Digestturr

Experiment 1

of Ad2 mR&W

5” I-1,

Nwmbe.r 3

2

f&?an

Up + U Cyclic

p

3.5

2.5

3.6

32

Cp + C Cyclic

p

4.1

4.4

4.4

45

1 .o

1 .o

1 .o

Ia

6.4

5.0

6.3

6.2

GP pm7GpppimpCmpUp vW-w~mpCp 3zP dpm/mole

Phosphate

242

110

926

The Ad2 5’ terminal Tl oligonucleotide was redigested RNAase A as described for Figure 3, slot 5. and regions chromatogram corresponding to the indicated structures quantitated by liquid scintillation counting.

with of the were

original RNA preparation. The material at the origin of Figure 2D was redigested with RNAase T2 and found to be enriched in poly(A). Similarly, the RNA which bound to the second poly(U)-sepharose column did not migrate off the origin during homochromatography as shown in Figure 2F. This RNA represents the 3’ termini of mRNA molecules and was >QO% poly(A) as judged by redigestion experiments (data not shown). The mRNA 5’ Terminus in Ad2 RNA from Different Parts of the Genome Because many different Ad2 mRNAs are present late after infection, the finding of a single 5’ terminal Tl oligonucleotide was unexpected. We anticipated one for each mRNA species. To check that our methods were not leading merely to the isolation of a single mRNA species (the most abundant), we attempted to isolate Ad2 mRNAs by hybridization to different regions of the Ad2 genome, including those regions which code for three of the most common late mRNAs: hexon, fiber and 100K. Ad2 RNA labeled late in infection was hybridized to various restriction fragments of Ad2 DNA in solution or to DNA immobilized on nitrocellulose filters. Hybridized RNA was recovered after extensive washing without nuclease, and the 5’ terminal oligonucleotides were isolated as described for Fig-

ure 2A. The principal mRNA 5’ terminus was found in RNA which hybridized to each of the Barn HI fragments of Ad2 DNA (Figure 6), as well as to fragments of the genome believed to code for hexon, fiber and 100K mRNAs (data not shown). The 5’ terminal oligonu,cleotide was not found in RNA which bound to lambda DNA. Unfortunately, upon checking the specificity of the primary hybrids by rehybridization to Ad2 DNA fragments immobilized o’n ni~trocellulose membranes (Southern, 1975), the ratio of specific to nonspecific hybrids varied from 3:l to 8:l. Thus although these RNAs were enriched for certain sequences, we cannot rigorously conclude that the 5’ terminal oligonucleotide is present on mRNAs coded by all parts of the Ad2 genome. Since limited nuclease digestion of the RNA:DNA hybrids led to the loss of the capped 5’ terminal oligonucleotide (see below), we were unable to reduce the background of nonspecifically hybridized RNA by this standard method. Sensitivity of the Ad2 mRNA 5’ Termini to Mild Nuclease Digestion of Hybridized RNA During the course of experiments designed to map the 5’ termini of specific late mRNAs, it was observed that the 5’ terminal oligonucleotide was sensitive to mild nuclease digestion of the mRNADNA hybrids. Late RNA was hybridized in solution to restriction endonuclease fragment Eco RIB, which contains sequences complementary to the 5’ end of the mRNA for the Ad2 1OOK protein, and Eco RIF, which codes for the internal portion of that protein (Lewis et al., 1975; Figure 6). The RNA-DNA hybrids were briefly exposed to RNAase Tl to trim off any unhybridized RNA, and the nuclease-resistant hybrids were separated from released oligonucleotides by gel filtration on Sephadex G-25. The hybrids were then denatured, the RNA was digested with RNAase Tl, and the mRNA 5’ termini were isolated and fingerprinted as described above. Figures 4C and 4D show that the 5’ terminal oligonucleotide is not found after mild nuclease digestion of the hybrids formed with either restriction fragment, although it is found in the hybridized RNA not exposed to RNAase Tl (Figures 4A and 4B). The Ad2 mRNA 5’ terminus is also released when RNA hybridized to filter-immobilized DNA is

Figure 4. Sensitivity of the Ad2 mRNA 5’ Terminal Oligonucleotide to Mild Nuclease Digestion of Hybridized RNA Aliquots of Ad2 polysomal RNA (1.5 x 10s dpm; 1 mg) were hybridized in solution to Eco RIB or Eco RIF DNA. Half were digested with RNAase Ti (1 fig Ti per 20 pg hybrids) for 30 min at 37°C in 0.01 M Tris-HCI (pH 7.9, 0.001 nulcease-resistant material was isolated by gel filtration on columns (5 cm x 0.6 cm diameter) of Sephadex G-25. Both control samples of the RNA-DNA hybrids were denatured, digested with RNAase Tl and selected on DBAE-cellulose. (A) RNA selected by Eco RIB DNA (1 x lo6 dpm); (B) RNA selected by Eco RIF DNA (3.6 x lo5 dpm); (C) Eco RIB DNA:RNA hybrids resistant to mild Tl RNAase digestion (8.0 x lo5 dpm); (D) Eco RIF DNA-RNA hybrids resistant to mild Ti RNAase digestion (2 X lo5 dpm). Ad2 mRNA 5’ ends are marked oligonucleotides on all four homochromatograms were redigested with RNAase T2 and gave only mononucleotides. mobility in the second dimension of A and B is probably caused by differences in the two homoplates.

the RNA-DNA hybrids M Naz EDTA, and the nuclease-treated and

with

arrows. All other The variation of

5’ Termini 539

of Adenovirus

2 mRNA

Figure

4. A-D

Cell 540

subjected to mild RNAase digestion (and can be recovered from the released RNA by selection on DBAE-cellulose; data not shown). 5’ Terminus in Ad2 mRNA Denatured with Glyoxal In view of the sensitivity of the capped Tl oligonucleotide to mild digestion with Tl RNAase, we were concerned that it might arise not from bona fide mRNAs, but rather from some small RNA which was adventitiously associated with the mRNA. We therefore attempted to denature completely our mRNA preparation before analysis to try to separate any such RNA from the mRNA. We chose glyoxal denaturation for this purpose and checked its effectiveness by electron microscopic examination of the RNA. 32P-labeled RNA was prepared from the polysomes of Ad2-infected cells and reacted with 1.4 M glyoxal at 50°C by a modification of the method of Hsu, Kung and Davidson (1973). By this treatment, no hairpin or double-stranded structures were visible in any molecule examined, including the rRNA molecules which were the predominant species in the sample. The RNA was fractionated on a sucrose gradient containing 0.1 M glyoxal, and RNA size classes corresponding to 26S, 24s and 19s were collected. Electron microscopic examination indicated that all molecules in these size classes were still denatured. The RNA was digested with RNAase Tl, and oligonucleotides containing 2’,3’ cis diols were isolated and fingerprinted as described for Figure 2. The fingerprints of these three size classes of RNA were very similar to that shown in Figure 2B, and contained only one oligonucleotide with a substantial RNAase T2-resistant moiety and which migrated in the position expected for the Ad2 mRNA 5’ end (data not shown). mRNA 5’ Termini in Early RNA At least one Ad2 mRNA which is transcribed early after viral infection does not contain the predominant eleven-residue 5’ terminus found in late Ad2 mRNA. 32P-RNA from Ad2-infected KB cells labeled early (2-10 hr post-infection) was hybridized to a nitrocellulose filter-bound fragment of Ad2 consisting of the leftmost 3% of the genome produced by cleavage of Ad2 DNA with Sma I (Figure 6). After hybridization, the filter was washed extensively and incubated with RNAase Tl at 37°C to degrade nonhybridized RNA. The nuclease-resistant RNA was eluted, digested with Tl RNAase and fingerprinted directly. No attempt was made to select or enrich for mRNA 5’ termini (Figure 5A). The oligonucleotide shown by the arrow in Figure 5A contained RNAase T2-resistant material as expected for a oligonucleotide and, furthermore, re“capped” leased 7-methylguanylic acid upon treatment with venom phosphodiesterase. More than 98% of the

RNA eluted from the filter specifically rehybridized to the Sma I fragment (Sma J) which was used to select it (data not shown). RNAase Tl oligonucleotides similar to that identified in Figure 5A as mRNA 5’ termini which migrate close to the yellow dye during homochromatography can also be identified along with the principal 5’ Tl oligonucleotide (indicated by the lowest arrow) derived from late mRNA when very large amounts of Ad2 RNA are selected by DBAE-cellulose (Figure 5B). This finding is not unexpected, since it is known that early mRNAs are capped (Hashimoto and Green, 1976) and some continue to be synthesized throughout viral infection. It should also be noted that the early capped 5’ terminal Tl oligonucleotide shown in Figure 5A was present despite the use of RNAase Tl to wash the hybrids during hybridization of the mRNA. This contrasts with the sensitivity of the late 5’ terminal Tl oligonucleotide. Discussion We have devised a scheme which would be expected to isolate selectively the 5’ terminal Tl oligonucleotides of Ad2 mRNA. When applied to mRNAs present late in infection, which contain at least twelve distinct species of mRNA, we find a single predominant oligonucleotide which accounts for >90% of all capped Tl oligonucleotides present. This oligonucleotide is frequently isolated as a doublet; however, no differences could be detected between the two forms either in chain length or upon redigestion with a variety of nucleases. Since it has already been reported that the capped structures as isolated possess varying degrees of methylation (Moss and Koczot, 1976; Sommer et al., 1976), we believe that this is likely to be responsible for the variable yield of these two spots. Although the principal Tl* oligonucleotide has the composition m7G5’pppS’AmCmU(C,,U,)G, we have not sequenced the pyrimidine block and thus cannot rigorously eliminate the possibility that sequence isomers are present. The five- and six-phosphate cap structures prepared by either the standard procedure of Adams and Cory (1975), or the procedure of Furuichi et al. (1975b) (data not shown) or RNAase T2 digestion of the 5’ terminal undecanucleotide are qualitatively identical and have compositions which agree well with results previously reported (Moss and Koczot, 1976; Sommer et al., 1976). We have not been able to quantitate rigorously the molar yield of this Tl oligonucleotide with respect to the quantity of mRNA present because some losses are encountered during the manipulation involved with the DBAE method, and because this calculation also depends upon a knowledge of mRNA chain length

5’ Termini 541

Figure

of Adenovirus

5. Comparison

2 mRNA

of mRNA

Termini

in Early

and Late Ad2 mRNA

(A) 32P polysomal RNA selected by hybridization to Sma I J. 5 x IO9 dpm (4 mg) of Ad2 cytoplasmic RNA were hybridized to 20 pg of the Sma I J fragment (leftmost 3% of the Ad2 genome) bound to a nitrocellulose filter. The filter-bound hybrids were washed extensively in 2 x SSC at 68°C and then with 2 x SSC containing 50 pg RNAase Ti per ml for 1 hr at 37°C as described in Experimental Procedures. The RNA was eluted from the filter, digested with RNAase Tl and fingerprinted directly (1 x IO4 dpm) with homomix C. All circled oligonucleotides were redigested with RNAase T2. Only the oligonucleotide marked with the arrow had an RNAase T2 redigestion pattern characteristic of a capped mRNA 5’ terminus. The yellow dye is marked by the circle of dots. (6) Ad2 polysomal RNA (5 x 10gdpm; 5 mg) was hybridized to 500 pg Ad2 DNA in solution as described for Figure 2A. The hybridized RNA was digested with RNAase Tl and selected by DBAE-cellulose as described for Figure 2. All oligonucleotides were digested with RNAase T2. The principal Ad2 mRNA 5’ end is marked by the lowest arrow. The arrows above it indicate the only other oligonucleotides with RNAase T2resistant portions. The yellow dye is marked by the circle of dots.

and abundance. Nevertheless, the two methods involving use of the DBAE column (Furuichi et al., 1975b; this paper) give yields of the five- and sixphosphate caps which are qualitatively the same and quantitatively similar. It thus appears that the cap structures previously assigned by other investigators (Moss and Koczot, 1976; Sommer et al., 1976) as being at the 5’ ends of Ad2 mRNAs account for most, if not all, of the cap structures present in our 5’ Tl oligonucleotide.

In addition to the unexpected finding of a single 5’ Tl oligonucleotide, we also observed the sensitivity of this oligonucleotide to digestion by RNAase Tl when hybrids of mRNA and Ad2 DNA were the substrate. This prompted us to examine closely the possibility that RNA molecules possessing this 5’ terminal oligonucleotide were not true mRNA molecules, but rather were passenger molecules associated with mRNA, by RNA-RNA hybridization. Two experiments render this possibility improbable.

Cell 542

29.1

B

40.9

59.0

Di

J

C

A

+

Barn

58.5

A

2.9

J+

t 0

Figure

M 11.3 10.7

L18.5 18.1

35.4

B

EhF{

70.7

+

38.6

414

51.1

D

B

75.9

+F+

56.1

83.4

D

89.7

+Et

C

A

iH+

+

C

‘i

I

I

I

I

I

I

I

I

I

20

30

40

50

60

70

80

90

6. Endonuclease

Cleavage

Maps

Eco RI

76.8

IO

The map of Eco RI on Ad2 DNA is taken and R. Greene.

HI

G ‘:K

Sma

I

I

100

of Ad2 DNA from

Mulder

et al. (1974).

First, the oligonucleotide is found on RNA preselected on each of the four Barn HI fragments. Second, after rigorous denaturation as indicated by electron microscopy, this oligonucleotide is still present on several different size classes of RNA. Finally, it should be stressed that if this capped Tl oligonucleotide is not derived from the 5’ terminus of late Ad2 mRNAs, then we must conclude that Ad2 mRNAs are not capped. The observation that translation of most Ad2 mRNA is inhibited by 7methylguanosine (N. Wills, J. Broach and R. F. Gesteland, unpublished data) argues against this possibility. Our data do not allow the conclusion that a// Ad2 mRNAs have a common eleven-nucleotide sequence at their 5’ terminus. Rather, we conclude that all major mRNA species present late after infection contain this sequence and suspect that it may prove true for all late mRNAs. In contrast, at least one early mRNA, from the extreme left end of the genome, has a different 5’ terminal Tl oligonucleotide. In addition, Hashimoto and Green (1976) reported that cap structures in early mRNA are different from those described here for late mRNA. This raises the possibility that the presence or absence of this undecanucleotide is associated with the control mechanism that defines the switch from early to late transcription. A simple model by which this might be accomplished could involve the specific recognition of all or part of this common sequence present at many sites within a large nuclear transcript, followed by processing to give the discrete cytoplasmic mRNA species. Throughout these studies, we have been sur-

The maps of Barn HI and Sma I are the unpublished

results

of C. Mulder

prised at the sensitivity to nuclease digestion of these 5’ terminal sequences when present in DNARNA hybrids. Our initial reaction was to suppose that the unusual triphosphate linkage at the 5’ end created ionic repulsions which allowed breathing of these hybrids at the RNA termini. The finding that a shorter capped Tl oligonucleotide, present on early mRNA, could be protected against nuclease digestion by hybrid formation argues against this possibility. Since our experiments suggest that this oligonucleotide is covalently bound to mRNA, we have considered other explanations. One possibility would be that this eleven-nucleotide stretch of the mRNA, or its complementary DNA, is able to form intramolecular base pairs which compete with true hybrid formation under our experimental conditions. Alternatively, we have considered post-transcriptional addition of this sequence or models involving RNA primers for the initiation of transcription (Dickson and Robertson, 1976). Experiments are in progress to determine the full length of the common sequence. One consequence will be the production of longer capped oligonucleotides which may be used as hybridization probes to specific fragments of Ad2 DNA bound to nitrocellulose by the method of Southern (1975). In this way, we will be able to show directly which restriction endonuclease fragments of the Ad2 genome contain sequences complementary to the 5’ ends of Ad2 mRNA. It has recently been shown that the capped undecanucleotide described in this paper is present in two different highly purified mRNAs coding for a major Ad2 structural protein fiber and another pro-

5’ Termini 543

of Adenovirus

2 mRNA

tein produced in large amounts during lytic infection, 1OOK (D. Klessig, unpublished data). The purification procedure involved hybridization to specific restriction endonuclease fragments of Ad2 DNA, followed by electrophoresis on a polyacrylamide gel containing 98% formamide, and yielded RNAs of >95% purity as judged by rehybridization to restriction endonuclease fragments of Ad2 DNA immobilized on a nitrocellulose membrane (Southern, 1975). These observations support the conclusion that this oligonucleotide is derived from mRNA, rather than from some ubiquitous contaminant. Experimental

Procedures

Isolation of Viral DNA and RNA DNA was prepared from Ad2 virions grown on HeLa or KB cells in suspension cultures as described by Pettersson and Sambrook (1973) and Pettersson et al. (1973). Fragments of the Ad2 genome were produced by digestion with the restriction endonucleases Bal I, Sma I, Eco RI and Barn HI. DNA fragments were fractionated by agarose slab gel electrophoresis (Sugden et al., 1975) and recovered from the agarose by chromatography on hydroxyapatite (Lewis et al., 1975) followed by phenol extraction. To prepare 3ZP-labeled RNA, HeLa or KB cells were suspended in phosphate-free F13 medium (Gibco) supplemented with 20 mM HEPES-K (pH 7.55) and 2% dialyzed fetal calf serum at a titer of 35 X lo5 cells per ml. After infection with 100 plaque forming units of Ad2 per cell, 3ZP-orthophosphate was added (100 &i/ml) for the times indicated in the text. RNA was prepared from Ad2infected or mock-infected cells by a modification of the magnesium precipitation method of Palmiter (1974). Cells from 1 liter cultures were lysed in 10 ml of 25 mM Tris-HCI (pH 7.5), 25 mM NaCl, 5 mM MgCI,, 2% Triton X-100 (lysis buffer) containing 250 mM sucrose at o”C, and nuclei and cellular debris were removed by centrifugation at 10,000 x g. The supernatant was mixed with 10 ml of the lysis buffer but containing 0.2 M MgCI, and 1 mg/ml heparin. and the mixture was centrifuged for 3 hr at 100,000 x g. RNA was purified from the polysomal pellet as described by Palmiter (1974) and concentrated by ethanol precipitation. In some experiments, RNA was prepared from the total cytoplasm of infected cells by the method of Anderson et al. (1974). Fractionation of Polysomal RNA on Poly(U)-Sepharose Up to 1 mg of polysomal RNA in 0.5 M NaCI, 0.01 M Tris-HCI (pH 7.5) was heated to 60°C for 3 min, cooled to 25°C and passed over a column (2 cm x 0.6 cm diameter) of poly(U)-sepharose (prepared as described by Jelinek et al., 1973). The column was washed with 3 column volumes of 0.5 M NaGI, 0.01 M Tris-HCI (pH 7.5), and the RNA which did not bind was recovered by ethanol precipitation. The RNA which bound to the column was eluted with 3 column volumes of 0.01 M Tris-HCI (pH 7.5), followed by 3 column volumes of formamide. This RNA was also concentrated by ethanol precipitation before digestion with RNAase Ti. RNA-DNA Hybridization RNA complementary to 100 pg of Ad2 DNA (or to fragments of the genome derived from an equivalent amount of DNA) was selected by hybridization in solution as described by Lewis et al. (1975). Hybridized RNA was denatured at 100°C for 90 set prior to digestion with RNAase Tl. Alternatively, RNA from AdP-infected cells was hybridized to endonuclease fragments of Ad2 DNA immobilized on nitrocellulose filters essentially as described by Gillespie (1968) for preparative experiments, or as described by Southern (1975) for some

control experiments. Both DNA-impregnated and blank filters were incubated with 32P polysomal RNA from Ad2infected cells in 1.0 M NaCI, 0.01 M Tris-HCI (pH 7.5), 0.01 M Na, EDTA, 0.2% sodium dodecylsulfate and 100 pglml polyadenylic acid for lo-15 hr at 68°C. Some hybridizations were carried out with the given buffer containing 50% formamide for lo-15 hr at 37°C. After hybridization, filters were washed with several changes of 2 x SSC (1 x SSC is 0.15 M NaCI, 0.015 M Na citrate) for 3 hr at either 68°C or 37°C. In some cases, filters were incubated with RNAase Tl (50 pg/ml) in 2 x SSC for 1 hr at 37°C and washed again for 3 hr at 68°C or 37°C in 2 x SSC. Hybridized RNA was eluted by heating the filters for 2 min at 100°C twice with 1 ml aliquots of 0.01 M Tris-HCI (pH 7.5), 0.001 M Na, EDTA and IO pg/ml tRNA. Reaction with Glyoxal Polysomal RNA (3 mg, 6 x lo6 dpm) from Ad2infected cells was denatured by incubation in 1.4 M glyoxal, 0.02 M Hepes-KC (pH 8.5) for 1 hr at 51°C and finally for 10 min at 61°C by a modification of the method of Hsu et al. (1973). A small amount of the RNA was examined in the electron microscope to measure the extent of denaturation, and the remainder of the sample was layered on a 35 ml, 520% (w/v) sucrose gradient prepared in 0.01 M potassium phosphate, 0.05 M NaCl, 0.001 M Na, EDTA and 0.02 M Na borate (pH 8.0), and centrifuged for 20 hr at 25,000 rpm, 20°C in the SW27 rotor. Fractions of the gradient corresponding to 26S, 24s and 19s were pooled and dialyzed against 20 mM Hepes-K’ (pH 8.5) for 36 hr at 25”C, and a small sample of RNA from each size class was again examined in the electron microscope. The remaining RNA in each size class was digested with Tl RNAase, selected on DBAE-cellulose and displayed by fingerprint analysis. Nuclease Digestion Nuclease digestions of polysomal RNA, RNA selected by hybridization or oligonucleotides eluted from homochromatography plates or DEAE paper were performed as described by Barrel1 (1971). 5’-m’GMP was liberated from capped oligonucleotides eluted from DEAE paper by digestion with 0.1 mg/ml snake venom phosphodiesterase (Sigma) in 0.05 M Tris-HCI (pH 8.9), 0.01 M MgCI,, 1 .O mg/ml tRNA for 30 min at 37°C. PI nuclease (Yamasa Shoyu Co.) was used at 100 Kg/ml in IO mM Na acetate (pH 6.0). Isolation of Oligonucleotides on DBAE-Cellulose RNAase Tl digests of polysomal RNA (up to 3 mg) were depleted of poly(A)-containing sequences by binding to poly(U)-sepharose or to oligo(dT)-cellulose (Collaborative Research) in 0.5 M KCI, 0.005 M Tris-HCI (pH 7.5). Fractions with RNA which did not bind were adjusted to 0.6 M KCI, 0.05 M N-methylmorpholine (pH 8.5), 20% ethanol (v/v) and loaded onto a column (2 cm x 0.6 cm diameter) of dihydroxyboryl-cellulose (Collaborative Research) at 2°C as described by McCutchan, Gilham and Soll (1975). The column was washed with at least 20 column volumes of the loading buffer. Oligonucleotides with 2’, 3’ ci.s diols were eluted with the loading buffer containing 0.1 M sorbitol or a buffer containing 0.2 M NaCI, 0.05 M sodium acetate (pH 5.0). The eluted oligonucleotides were diluted 12 fold with water, the pH was corrected to 7-8, 50 pg tRNA were added and the sample was bound to a column (1 .O cm x 0.6 cm diameter) of DEAE-cellulose (Whatman, DE-52). The DEAE-cellulose column was washed with 10 column volumes of water, and the oligonucleotides were eluted with 1 ml triethylammonium bicarbonate (TEC), dried and desalted on a column (2 cm x 0.6 cm diameter) of Sephadex GlO (equilibrated with 0.01 M TEC), dried and displayed by fingerprint analysis. Acknowledgments We wish to thank Dr. Tom McCutchan for help in the proper use of DBAE-cellulose, Drs. Louise Chow and Tom Broker who performed the electron microscope analysis of glyoxal denatured RNA, and our colleagues at Cold Spring Harbor including Drs.

Cell 544

Ray Gesteland and Dan Klessig for stimulating discussions. Supported by grants from the National Cancer Institute, the NSF and a National Cancer Institute postdoctoral fellowship to R.E.G. Received

March

4, 1977; revised

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14, 1977

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