Nu- cleotide pyrophosphatase and nuclease Pl were pur- chased from Sigma and Boehringer-Mannheim Corp., respectively. Alkaline phosphatase (2.35 U/ml), ...
Vol. 45, No. 1
JOURNAL OF VIROLOGY, Jan. 1983, p. 264-274
0022-538X/83/010264-11$02.00/0 Copyright © 1983, American Society for Microbiology
Characterization of the 5' Termini of Purified Nascent Simian Virus 40 Late Transcripts DEBORAH E. LYCAN AND KATHLEEN J. DANNA* Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309 Received 2 August 1982/Accepted 7 October 1982
The primary transcripts of simian virus 40 are extensively processed in the nuclei of infected monkey cells before they are transported to the cytoplasm as mature mRNAs. To investigate the early steps in this process, in particular, to determine which events occur on nascent chains before the termination of transcription, we have developed a procedure for the purification of nascent viral transcripts. This technique involves the in vitro incorporation of mercurated residues into the growing 3' ends of pre-initiated nascent chains, allowing their specific purification by sulfhydrylcellulose affinity chromatography. We further selected viral specific transcripts by hybridization to simian virus 40 DNAcellulose. We describe here our analysis of the 5' termini of purified nascent simian virus 40 transcripts. This analysis revealed various cap structures, providing direct evidence that primary viral transcripts are capped before chain completion. The various cap structures exhibited a full range of methylation states. Completely unmethylated GpppA cap cores were identified, as well as caps methylated at the penultimate position only. The presence of GpppAm and GpppmAm caps indicates that, in BSC-1 cells, the penultimate nucleotide can be methylated before 7-methyl-G formation. Furthermore, the proportions of the various intermediates suggest that, in contrast to the viral capping enzymes of vaccinia virus and reovirus, the cellular enzymes methylate in the following order: GpppA -- GpppAm -- GpppmAm -* 7mGpppmAm. In addition to capped ends, we also detected some unprocessed pppA ends. To our knowledge, this is the first time uncapped termini have been identified on RNAs known to be polymerase II products. In eucaryotic cells the formation of a translatable mRNA from an RNA polymerase II (pollI) primary transcript is a complex process. The pre-mRNA must be capped, spliced, methylated, and polyadenylated before transport. Whether these modifications occur on the growing, nascent RNA transcript before its termination and release from the template is not known. However, the analysis of ribosomal transcriptional arrays from Dictyostelium by electron microscopy has revealed that cleavage and processing of pre-rRNA primary transcripts do occur on the nascent RNA molecule (16). More recently, similar kinds of simultaneous transcription and processing have been observed for Drosophila heterogeneous nuclear RNA (hnRNA) transcription units (3). Exactly what these "processing" events are, especially for the hnRNA transcription units, is still unclear, but it is thought that they probably do not represent splicing events. Simian virus 40 (SV40) provides an excellent 264
model system with which to study mRNA processing in mammalian cells. It is a simple virus (containing only two primary transcription units), and because it codes for no processing enzymes of its own, it must depend entirely on the mammalian host cell machinery for all of its RNA processing. Furthermore, the viral system has a decided advantage over cellular systems in
that the selection of virus-specific transcripts automatically yields an RNA population containing only polIl pre-mRNA transcripts. In contrast, cellular hnRNA populations are complex, and any analysis of mRNA processing in such a preparation is complicated by the unavoidable presence of other kinds of RNA, such as small nuclear transcripts and RNA polymerase III products. We therefore chose to use SV40-infected monkey cells to investigate whether processing of pre-mRNA primary transcripts occurs while the chain is nascent. Nascent RNA, by definition, is RNA that is still attached to the template
VOL. 45, 1983
5' TERMINI OF SV40 LATE TRANSCRIPTS
and is in the process of being elongated. The problem with any analysis of nascent RNA is the difficulty of biochemically distinguishing nascent chains from finished transcripts once the RNA is extracted and purified for analysis. Previously, most investigators have equated nascent RNA with either pulse-labeled RNA or with short, promoter-proximal transcripts. However, simply because an RNA is both short and homologous to the promoter region of the genome does not necessarily indicate that it is a nascent transcript. Primary transcripts are very labile, and RNA of low molecular weight isolated on a sucrose gradient is likely to contain many RNA fragments derived, either by degradation or by processing, from finished, released transcripts. Some of these fragments will be derived from the 5' ends of completed RNAs and will therefore hybridize to the promoter region of the genome. In addition to these contaminants, the prematurely terminated, abortive transcripts, which are known to accumulate in adenovirus-infected cells (12) and which can be detected in CHO cells (29), would also be both short and promoter proximal, but not nascent. Pulse-labeling experiments are also difficult to interpret because labeled nascent RNAs cannot be distinguished from chains that were completed and released from the template during the labeling period. To equate pulse-labeled RNA with nascent RNA, one must expose cells to the labeled nucleoside for a time much shorter than that required to complete the synthesis of a given primary transcript. Since chain elongation has been estimated to proceed in vivo at a rate of 50 to 100 nucleotides per second (9, 17, 31), either the pulse must be very short or the transcription unit must be very long. For short transcription units such as the late region of SV40, the required pulse period is so short as to be technically impractical, and in experiments where the size of the transcription unit is unknown, as, for example, in studies of cellular hnRNA, it is basically impossible to determine whether the pulse-labeled RNA is nascent. We therefore decided to use a new approach to study nascent SV40 transcripts. This procedure involves pulse-labeling RNA in vivo and then elongating pre-initiated nascent transcripts in vitro in the presence of mercurated CTP (6). The incorporation of mercurated residues into the growing 3' ends of nascent chains allows their specific purification by sulfhydrylcellulose affinity chromatography. Since only elongating transcripts incorporate the mercurated triphosphates, we were able to physically separate nascent chains from the inactive finished transcripts with this technique. Since several investigators had already suggested that, in other systems, capping was an
265
early processing event (1, 18, 29), we decided to investigate whether the 5' ends of purified SV40 nascent transcripts were capped as a first step in our analysis of nascent RNA processing. In this work, we describe the biochemical analysis of nascent viral transcripts purified from productively infected BSC-1 monkey cells. We found that nascent transcripts do have processed 5' ends, indicating that modification of the 5' terminus does occur before the termination of transcription. The most abundant terminus we detected was 7mGpppmAm, which is the major cap found on mature SV40 cytoplasmic mRNAs. We also detected low levels of unprocessed triphosphate pppA ends associated with SV40 nascent transcripts. To our knowledge, this is the first time uncapped termini have been detected on purified RNA polll transcripts (1, 22, 29). We detected several cap structures at intermediate stages of methylation. Completely unmethylated cap cores were identified, indicating that the guanylyltransferase activity is not coupled in vivo to the methyltransferases that modify the cap. In addition, the fact that we detected major amounts of GpppAm and GpppmAm caps indicates that, in BSC-1 cells in vivo, the penultimate nucleotide can be methylated before 7methyl-G formation. This is in contrast to the situation with vaccinia virus and reovirus, where 2'0 methylation of the cap seems to occur only after methylation of the blocking guanosine (13, 26). MATERIALS AND METHODS Cells and virus. BSC-1 monkey cells were grown in Dulbecco modified Eagle medium supplemented with 7% calf serum. Confluent monolayers of BSC-1 cells were infected with SV40 strain 776 at a multiplicity of 50 to 100 PFU/cell. Materials. Mercurated CTP (HgCTP) was prepared by a modification of the procedure of Dale et al. (8) as described by Feist and Danna (10). We prepared SV40 DNA-cellulose by the protocol of Noyes and Stark (27), except that we substituted 0.2 M sodium acetate buffer (pH 4) for the borate buffer. SV40 DNA, digested to completion with restriction endonuclease EcoRI, was covalently linked to activated cellulose at a concentration of 30 to 40 1Lg of linearized DNA per mg of cellulose. Markers for various 5' cap structures were purchased from PL Biochemicals. Thin-layer, polyethyleneimine (PEI)-cellulose plates (with UV indicator) were obtained from Machery and Nagel (Duren, Germany). The plates were prewashed by ascending chromatography with a wick in a 10% LiCl solution overnight. We removed the LiCl from the plates by washing them twice for 10 min in anhydrous methanol. Enzymes. DNase I (Worthington Diagnostics; RNase-free grade), repurified by affinity chromatography to remove RNase (25), was the generous gift of S. Beckmann. A stock of sodium dodecyl sulfate (SDS)pronase was prepared by dissolving 5 mg of Protease
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IV (Sigma Chemical Co.) in 1 ml of 1% SDS-0.2 M NaCl-40 mM Tris-hydrochloride (pH 7.5)-10 mM EDTA and incubating it at 37°C for 2 h before it was used in order to degrade endogenous nucleases. Nucleotide pyrophosphatase and nuclease Pl were purchased from Sigma and Boehringer-Mannheim Corp., respectively. Alkaline phosphatase (2.35 U/ml), purchased from Boehringer-Mannheim, was diluted 1:150 in 5 mM Tris-hydrochloride (pH 7.9) just before use. Pulse-label. At 48 to 55 h postinfection, infected monolayers were starved in phosphate-free medium for 1 h and then pulse-labeled for 35 min with carrierfree 32p, (4 mCi/ml, 4 mCi per 100-mm dish). RNA synthesis in vitro. We extracted viral transcriptional complexes from pulse-labeled, infected cells by using Sarkosyl and high salt as described previously (11, 14). Sarkosyl extracts were incubated at 26°C for 15 min in a transcription cocktail containing 0.6 mM HgCTP, 0.6 mM GTP, 1.8 mM ATP, 0.08 mM UTP, 0.12 M (NH4)2SO4, 4 mM KCl, 5 mM CaC12, 6 mM MnCl2, 14 mM 2-mercaptoethanol, and 0.5 mM aurintricarboxylic acid. Aurintricarboxylic acid was added to inhibit RNases in the extract (19). The transcription reaction was terminated by the addition of 1/5 volume of SDS-pronase, and the mixture was incubated at 37°C for 30 min and then phenol extracted. Unincorporated nucleotides were removed by G-50 Sephadex chromatography, and the nucleic acids were precipitated with ethanol. The preparation was digested with DNase I for 40 min at 37°C in the presence of 0.5 mM aurintricarboxylic acid, and the RNA was recovered by Sephadex G-50 chromatography and precipitated with ethanol. Purification of nascent RNA by sulfliydrylcellulose chromatography. The ethanol pellet was dissolved in 50% dimethyl sulfoxide and heated at 50°C for 10 min to disrupt RNA aggregates. The sample was then diluted to 5% dimethyl sulfoxide with 0.1 M NaCl-50 mM Tris-hydrochloride (pH 7.9)-i mM EDTA and loaded onto a 0.5-ml column of activated sulfhydrylcellulose. The column was washed extensively with 0.5 M NaCl-50 mM Tris-hydrochloride (pH 7.9)-i mM EDTA and then with 50% dimethyl sulfoxide-5 mM Tris-hydrochloride (pH 7.9) (prewarmed to 50°C). The dimethyl sulfoxide wash was included to eliminate any nonspecific retention of nonmercurated RNA due to RNA aggregation (21). Finally mercurated RNA was eluted with 0.5 M J3-mercaptoethanol-0.5 M NaCl-50 mM Tris-hydrochloride (pH 7.9)-I mM EDTA and was ethanol precipitated. The preparation and assay of the sulfhydrylcellulose as well as the details of this procedure have been described previously (10). Selection of virus-specific nascent RNA. SV40-specific transcripts were selected by hybridization of the purified nascent RNA to SV40 DNA, covalently linked to cellulose (27). The pellet of nascent RNA was dissolved in hybridization buffer (50% [vol/vol] formamide, 0.1% SDS, 1 mM EDTA, 0.1 M Tris-hydrochloride [pH 7.4], 0.6 M NaCl) and mixed with SV40 DNAcellulose (10 to 15 ,ug of DNA). The suspension was mixed thoroughly and hybridized for 4 h at 37°C. The cellulose was then pelleted from the hybridization reaction by spinning in a microfuge for 2 min, the supernatant was carefully removed, and the cellulose pellet was washed five times with 200 p.1 of 40% formamide (40% [vol/vol] formamide, 0.1% SDS, 2 mM EDTA, 0.1 M Tris-hydrochloride [pH 7.4], 0.6 M
J. VIROL.
NaCI) and four times with ice-cold wash buffer (0.3 M NaCl, 0.03 M sodium citrate). SV40-specific RNA was eluted by suspending the cellulose in 100 p.1 of 99% formamide-0.1% SDS and heating at 60°C for 3 min. This elution step was repeated twice more. The combined eluates were diluted to 50% formamide with 0.2 M sodium acetate (pH 6) and ethanol precipitated. Digestion of SV40 nascent RNA and analysis of the 5' termini. SV40 nascent RNA was digested with 2 p.g of P1 nuclease in a 10-plI reaction volume of a 14 mM sodium acetate-0.4 mM ZnSO4 (pH 5.3) buffer containing S p.g of ATP to compete for any phosphatase activity in the enzyme preparation. Digestion was for 3 h at 37°C; 2 p.g of additional enzyme was added after the first 2 h of incubation. Cap core markers and purine triphosphate markers were then added to the digest, and the mixture was spotted onto a 20- by 20cm PEI thin-layer plate and dried. The products of P1 digestion were resolved by two-dimensional ascending chromatography. The first dimension was developed in 0.2 M LiCl-5 mM EDTA (pH 6.5) for 2 h (a wick was used). The plate was dried and then washed with anhydrous methanol to remove the LiCl. The second dimension was developed for 1 min (from the time the solvent front reached the sample) in 0.2 M LiCl-5 mM EDTA (pH 6.5), for 5 min in 1.0 M LiCl-5 mM EDTA (pH 6.5), and finally in 2.0 M LiCl-5 mM EDTA (pH 6.5) until the solvent front reached the top of the plate. After drying the plate, we determined the position of the markers by their absorption of UV light. Radioactive products were visualized by autoradiography at -70°C with XAR-5 film and Lightning-Plus intensifying screens (Du Pont Co.). Typically, films were exposed for 14 days. Further analysis of 5' termini. Because of the short half-life of 32P and because the recovery of label in 5' end structures is so low, any Pl products that required further analysis had to be eluted from the two-dimensional PEI plates immediately, without the normal 2week exposure period. After chromatography, we dried the plates and circled the position of the added markers in pencil. We then washed the PEI plates twice in methanol to remove the LiCl. The encircled areas of the plate containing the Pl products of interest were then scraped off, and the nucleotide products were eluted from the cellulose with 3.0 M triethylammonium carbonate (pH 9.7) as described by Volckaert et al. (34). Samples eluted with triethylammonium carbonate were evaporated to dryness, redissolved in water, and
evaporated repeatedly to remove volatile salts. For digestion with alkaline phosphatase, we suspended the washed residue in 10 p.1 of 5 mM Tris-hydrochloride (pH 7.9) and digested for 30 min at 37°C with 5 U of enzyme per ml. Samples to be digested with nucleotide pyrophosphatase were dissolved in a 10-p.l reaction volume containing 5 mM Tris-hydrochloride (pH 7.5), 2 mM MgCl2, and 0.96 U of enzyme per ml and incubated for 3 h at 37°C. The products of these digestions were resolved by one-dimensional thin-layer chromatography. The digests were spotted onto PEI plates and dried. Appropriate markers were then spotted on top of each digest, and the mixture was developed in one dimension as described above for the second dimension. Again, markers were visualized by UV absorption and radioactive products by autoradiography.
VOL. 45, 1983
5' TERMINI OF SV40 LATE TRANSCRIPTS
RESULTS Purification of SV40 nascent RNA: experimental strategy. Because we were interested in determining whether the 5' ends of nascent premRNA chains are processed before termination of transcription, our first objective was to purify nascent RNA transcripts. Our experimental strategy is outlined in Fig. 1. Late in infection, we prepared extracts of 32P pulse-labeled SV40infected cells by using Sarkosyl and high salt (11, 14, 32). Sarkosyl extracts are crude nuclear lysates from which the host chromatin has been removed by centrifugation. They contain viral transcriptional complexes with attached, preinitiated nascent chains that can be elongated in vitro under conditions in which neither reinitiation nor post-transcriptional processing occurs (4, 11). When we incubated these extracts in an in vitro transcription cocktail containing HgCTP as described by Chikaraishi and Danna (6), we could specifically add mercurated residues onto the growing 3' ends of nascent RNA chains. We then purified these nascent RNAs away from all of the mature RNAs in the extract, using sulfhydrylcellulose affinity chromatography to select only those molecules with mercurated "tails." We next selected SV40-specific molecules from the total nascent RNA pool by hybridization to SV40 DNA-cellulose. This purified preparation of SV40 nascent RNA was then analyzed biochemically for 5'-terminal modifications. Fractionation of the 5' termini. The initial step in our analysis of 5' ends was to digest the purified SV40 nascent RNA with nuclease P1. Pl hydrolyzes every phosphodiester bond, regardless of 2'O methylation, yielding 5' monophosphates from all internal positions and various resistant groups from the 5' ends. The products of Pl digestion were resolved by twodimensional chromatography on PEI plates and detected by autoradiography. Nonradioactive markers, which were visualized by their UV absorption, were added to the digests before chromatography. We have detected six different 5' termini that comigrated with known reference markers, as well as one product for which we had no marker (Fig. 2). The most abundant P1 product, aside from the internal monophosphates, comigrated with 7mGpppmAm, which is the major cap found on mature, cytoplasmic SV40 mRNAs. We also detected large amounts of two other P1 products, one that comigrated with GpppAm and another that migrated just ahead of GpppAm in both dimensions. Lesser amounts of a product that comigrated with the GpppA marker were also clearly visible. In some experiments, we detected small amounts of two other Pl products that comigrated with 7mGpppAm and 7mGpppA, respectively (data not shown). Final-
267
Iy, we reproducibly detected variable amounts of a minor Pl product that comigrated with the ATP marker. In contrast, we did not detect any radioactivity comigrating with either the GpppG or the GTP marker. Composition of the major 5' termini. Six of the seven Pl products that we could detect by autoradiography comigrated in two dimensions with known reference markers. To verify our tentative identification of these (Table 1) and to determine the structure of the unknown product, we eluted each product from the PEI plate and further characterized it by digestion with either alkaline phosphatase or nucleotide pyrophosphatase. To determine which of the seven P1 products were cap core structures, we digested each with alkaline phosphatase. Capped termini would be resistant to alkaline phosphatase, whereas any nucleoside di- or triphosphate ends or any pXpY oligomers generated by incomplete P1 digestion would release labeled phosphate upon alkaline phosphatase digestion. The results of digesting each of the eluted PI products with alkaline phosphatase are shown in
SV40 hIfected Cells (32pL prelabeled) Extraction of VTCs In Vitro Elongation of Nascent Chains with HgCTP
Purification of Total RNA
Sulfhydrycelulose Chromatography (3% Nascent) Hybridization to SV40-DNA Cellulose (90% SV40 -specific)
FIG. 1. General method for the purification of nascent SV40-specific RNA. SV40-infected BSC-1 cells were pulse-labeled with 32Pi at 48 h postinfection. Typically, we labeled five dishes (100 mm) of infected cells with a total of 20 mCi of 32Pi in 5 ml of Dulbecco modified Eagle medium without phosphate for 35 min. We then removed the labeling media and reused it on a second set of five dishes. Both sets were harvested, and Sarkosyl extracts containing viral transcriptional complexes (VTCs) were prepared. Labeled nascent chains were elongated in vitro for 15 min with HgCTP, total RNA was extracted, and mercurated, nascent chains were selected by sulfhydrylcellulose affinity chromatography. In our experiments, we found that 3 to 4% of the pulse-labeled RNA would bind specifically to the sulfhydrylcellulose column. Finally, we selected virus-specific nascent transcripts by hybridization of the purified nascent RNA to SV40 DNAcellulose. About 80 to 90% of the RNA elongated in Sarkosyl extracts was SV40 specific.
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LYCAN AND DANNA
J. VIROL.
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A
FIG. 2. Analysis of SV40 nascent RNA digested with nuclease P1. Nascent SV40 RNA from 32P,-pulselabeled, SV40-infected cells was digested with nuclease P1 and analyzed by two-dimensional, thin-layer chromatography on PEI plates. Optical density markers were cochromatographed and detected by UV illumination. (A) Autoradiogram of fingerprinted Pl digestion products. The photograph shown is a composite of two autoradiograms: a preliminary 16-h exposure and a 14-day exposure. The short exposure revealed the position of the abundant monophosphates released by Pl from all the internal positions of the nascent RNA. This area of the PEI plate was then cut out and removed before the second, longer exposure was done. This was necessary to detect the P1-resistant 5' termini. Typically we observed seven different 5' termini, which are numbered 1 through 7. For reference, the numbers 2 and 3 were placed near the position of two Pl products normally detected, even though they were not visible in this particular experiment, due to lower yields. (B) Drawing of the positions of the optical density markers. The numbers refer to the radioactive spots detected in (A).
Fig. 3 and 4. We found that most of the P1 products, except the putative ATP which was converted to 32Pi as expected (data not shown), were resistant to phosphatase. The exceptions were products 2 and 6, the putative 7mGpppAm and the putative GpppA cap structures, respectively. Both of these samples released some 32p;
digestion. However, much of the radioactivity in each remained resistant to alkaline phosphatase and still comigrated with the appropriate cap core marker, in spite of the fact that alkaline phosphatase digestion was shown to be complete under these conditions. The possibility that spots 2 or 6 could be oligomers (i.e., upon
TABLE 1. Analysis and relative abundance of 5' termini detected on nascent SV40 transcripts Alkaline Spot no. Tentative Nucleotide pyroDeduced Relative identification' (Fig. 2) phosphatase phosphatase products structure abundanceb 1
2 3
Resistant 7mGpppmAm 7mGpppAm Partially resistant Resistant 7mGpppA or 7mGpppGmc
7mGMP, ppmAm, 7mGpppmAm
None detected None detected
7mGpppmAm 7mGpppAm
10
