Major Late Transcription Unit Research Articles

Analysis of RNase-A-resistant regions of adenovirus 2 major late precursor-mRNA in splicing extracts reveals an ordered interaction of nuclear components with the substrate RNA

An RNase A protection assay was employed to investigate the interaction of nuclear components with a precursor-mRNA derived from the adenovirus 2 major late transcription unit in a splicing extract from HeLa cells. Upon incubation in the extract, two regions in the precursor-RNA become resistant to digestion with RNase A. After short incubation times (5 min) at 30 °C, fragments mapping upstream from the branch point in the intron are obtained. After ten minutes or more, additional oligonucleotides, derived from the 5′ splice site, are protected. RNase A protection of different RNA substrates demonstrates that a 5′ splice site is not required for the binding of components to the branch point region. For interaction with this site, the polypyrimidine stretch just upstream from the 3′ splice site is essential. Binding to the 5′ splice site occurs only in the presence of an intact 3′ end of the intron. Preincubation of the extract with excess unlabelled RNA containing only a 3′ splice site leads to efficient competition of binding, both in the branch point region and at the 5′ splice site, whereas an RNA that contains only 5′-splice-site sequences has no effect on the interaction with the mRNA precursor. This indicates that stable association with the 5′ splice site requires prior binding of components in the branch point region. When splicing complexes are digested with RNase A, it becomes apparent that only the branch point region is sequestered into a ribonucleoprotein (RNP) structure in the 35 S complex. The 5′ splice site becomes resistant to RNase A only when a 50 S splicing complex has been assembled. Degradation of specific regions in U1, U2 and U4 RNA with complementary oligodeoxynucleotides and RNase H has been used to analyse involvement of the U small nuclear RNPs (snRNPs) in the protection reaction. The 5′ end of U2 RNA is essential for protection of the branch point region. RNA sequences in a loop of U2 RNA (nucleotides 65 to 78) are required for the formation of an RNase-A-resistant structure at the 5′ splice site. Taken together, these results suggest that U2 snRNP participates in the formation of a pre-splicing complex, the 5′ end of its RNA being involved in the observed binding. Conversion to a 50 S splicing complex is obtained after the binding of Ul and U4/U6 snRNPs, which also requires sequences in a loop of U2 RNA. Possible interactions between the individual snRNPs and between snRNPs and precursor-mRNA are discussed.

The adenovirus E1B-55K transforming polypeptide modulates transport or cytoplasmic stabilization of viral and host cell mRNAs.

The adenovirus type 5 mutant H5dl338 lacks 524 base pairs within early region 1B. The mutation removed a portion of the region encoding the related E1B-55K and -17K polypeptides but did not disturb the E1B-21K coding region. The virus can be propagated in 293 cells which contain and express the adenovirus type 5 E1A and E1B regions, but it is defective for growth in HeLa cells, in which its final yield is reduced about 100-fold compared with the wild-type virus. The mutant also fails to transform rat cells at normal efficiency. The site of the dl338 defect was studied in HeLa cells. Early gene expression and DNA replication appeared normal. Late after infection, mRNAs coded by the major late transcription unit accumulated to reduced levels. At a time when transcription rates and steady-state nuclear RNA species were normal, the rate at which late mRNA accumulated in the cytoplasm was markedly reduced. Furthermore, in contrast to the case with the wild type, transport and accumulation of cellular mRNAs continued late after infection with dl338. Thus, the E1B product appears to facilitate transport and accumulation of viral mRNAs late after infection while blocking the same processes for cellular mRNAs.

The adenovirus E1B-55K transforming polypeptide modulates transport or cytoplasmic stabilization of viral and host cell mRNAs.

The adenovirus type 5 mutant H5dl338 lacks 524 base pairs within early region 1B. The mutation removed a portion of the region encoding the related E1B-55K and -17K polypeptides but did not disturb the E1B-21K coding region. The virus can be propagated in 293 cells which contain and express the adenovirus type 5 E1A and E1B regions, but it is defective for growth in HeLa cells, in which its final yield is reduced about 100-fold compared with the wild-type virus. The mutant also fails to transform rat cells at normal efficiency. The site of the dl338 defect was studied in HeLa cells. Early gene expression and DNA replication appeared normal. Late after infection, mRNAs coded by the major late transcription unit accumulated to reduced levels. At a time when transcription rates and steady-state nuclear RNA species were normal, the rate at which late mRNA accumulated in the cytoplasm was markedly reduced. Furthermore, in contrast to the case with the wild type, transport and accumulation of cellular mRNAs continued late after infection with dl338. Thus, the E1B product appears to facilitate transport and accumulation of viral mRNAs late after infection while blocking the same processes for cellular mRNAs.

Functions of purified E1A protein microinjected into mammalian cells.

We have purified the human type C adenovirus E1A 13S mRNA gene product, expressed in Escherichia coli, and demonstrate that the protein exhibits genuine viral functions upon microinjection into mammalian cells. We show that the E1A protein activates expression of the adenovirus E2A gene and induces expression from the major late transcription unit of the adenovirus E1A deletion mutant, H5dl312. We use this functional assay to examine the stability of E1A protein microinjected into cells and find that E1A exhibits full function for at least 18 hours after its injection. In addition, the purified E1A protein was used to generate a high-titer monospecific rabbit antiserum. This antiserum was used to detect and localize E1A proteins within adenovirus-infected cells as well as within microinjected cells. The E1A protein is found to rapidly and quantitatively localize to the cell nucleus following microinjection into the cell cytoplasm. Thus, nuclear localization is an intrinsic property of the E1A polypeptide. The ability of the E1A protein to localize to the cell nucleus and to induce expression from the H5dl312 major late transcription unit is shown to be highly heat stable.

Premature termination by human RNA polymerase II occurs temporally in the adenovirus major late transcriptional unit.

We have recently demonstrated pausing and premature termination of transcription by eucaryotic RNA polymerase II at specific sites in the major late transcriptional unit of adenovirus type 2 in vivo and in vitro. In further developing this as a system for studying eucaryotic termination control, we found that prematurely terminated transcripts of 175 and 120 nucleotides also occur in adenovirus type 5-infected cells. In both cases, premature termination occurs temporally, being found only during late times of infection, not at early times before DNA replication or immediately after the onset of DNA replication when late gene expression has begun (intermediate times). To examine the phenomenon of premature termination further, a temperature-sensitive mutant virus, adenovirus type 5 ts107, was used to uncouple DNA replication and transcription. DNA replication is defective in this mutant at restrictive temperatures. We found that premature termination is inducible at intermediate times by shifting from a permissive temperature to a restrictive temperature, allowing continuous transcription in the absence of continuous DNA replication. No premature termination occurs when the temperature is shifted up at early times before DNA replication. Our data suggest that premature termination of transcription is dependent on both prior synthesis of new templates and cumulative late gene transcription but does not require continuous DNA replication.

Premature termination by human RNA polymerase II occurs temporally in the adenovirus major late transcriptional unit.

We have recently demonstrated pausing and premature termination of transcription by eucaryotic RNA polymerase II at specific sites in the major late transcriptional unit of adenovirus type 2 in vivo and in vitro. In further developing this as a system for studying eucaryotic termination control, we found that prematurely terminated transcripts of 175 and 120 nucleotides also occur in adenovirus type 5-infected cells. In both cases, premature termination occurs temporally, being found only during late times of infection, not at early times before DNA replication or immediately after the onset of DNA replication when late gene expression has begun (intermediate times). To examine the phenomenon of premature termination further, a temperature-sensitive mutant virus, adenovirus type 5 ts107, was used to uncouple DNA replication and transcription. DNA replication is defective in this mutant at restrictive temperatures. We found that premature termination is inducible at intermediate times by shifting from a permissive temperature to a restrictive temperature, allowing continuous transcription in the absence of continuous DNA replication. No premature termination occurs when the temperature is shifted up at early times before DNA replication. Our data suggest that premature termination of transcription is dependent on both prior synthesis of new templates and cumulative late gene transcription but does not require continuous DNA replication.

Pausing and premature termination of human RNA polymerase II during transcription of adenovirus in vivo and in vitro.

The major late transcriptional unit of adenovirus type 2 has served as a model for studying transcription in eukaryotes. We report that pausing and premature termination are intrinsic to the transcription of this transcriptional unit by RNA polymerase II. In vivo and in isolated nuclei, transcription pauses at discrete sites proximal to the initiation site and can prematurely terminate at nucleotide 175 and possibly also at nucleotide 120. The prematurely terminated RNAs are not associated with the transcription complexes and accumulate in the cell nucleus in vivo, whereas paused RNAs remain associated with the transcription complexes and elongate into full-length transcripts. Pausing is also reproduced in the transcription complexes in a soluble system. 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole enhances pausing but not premature termination, and its action is reversible. The proposed premature termination site at nucleotide 175 in adenovirus type 2 bears sequence homology to the tR1 site in coliphage lambda.

A model for the excision of introns 1 and 2 from adenoviral major late pre-messenger RNAs

Total steady-state RNA was extracted from nuclei of HeLa cells late after infection with adenovirus serotype 2. Most of the nuclear RNA is transcribed from the major late transcription unit (16.2 to 100.0 map units). To study the cleavage reactions involved in the splicing of leaders 1 and 2, we have used the S 1 nuclease mapping technique with restriction fragments located in the region of intron 1 as DNA probes. The S 1 mapping data showed that in total nuclear RNA, RNA species accumulate from which the 5′ part of intron 1 has been excised, but which still contain the 3′ part of the intron. This indicates that intron 1 can be removed in a stepwise fashion following the 5′ to 3′ direction. We have compared the nucleotide sequences from the ends of the putative processing intermediates. The internal cleavage sites do not resemble the consensus 5′ or 3′ splice site sequences. However, they show considerable homology to the sequence 5′ A-T-G-A-T-G-G-C-A-T 3′, which may act as a signal for internal cleavage. The intermediates are present in both the poly(A) + and poly(A) − RNA fractions, although with different relative intensities. Primer extension experiments have been performed in which a primer, located with its left end in leader 2, is extended into intron 1. The results show that there may be a cleavage site as short as 35 nucleotides before the 3′ splice site. Cleavage at the 3′ splice site seems to be rapidly followed by ligation of leader 1 to leader 2. A model for RNA splicing based on these findings and data from the literature is presented.

Accumulation of early and intermediate mRNA species during subgroup C adenovirus productive infections

The cytoplasmic, poly(A)-containing RNA species complementary to all regions of the adenovirus type 2 (Ad2) genome expressed before the onset of viral DNA synthesis have been examined by “Northern” blotting. HeLa cells infected with low multiplicities of Ad2 were harvested at 2-hr intervals until late in the infectious cycle to establish the temporal patterns of expression of each viral gene. Under these conditions, such late mRNA products as those of the L3 and L5 families were not detected until 14 hr after infection. Although individual mRNA species complementary to several genes showed different patterns of expression, the order of appearance in the cytoplasm of substantial concentrations of adenoviral mRNA species was E1A, E3, and E4 (4 to 6 hr), E2A and E2B (8 hr), 3.7- and 4.1-kb L1 mRNA species (10–12 hr), IX and IVa 2 mRNAs (12 hr), and those encoded in the major late transcriptional unit, such as members of the L3 and L5 families (14 hr). The mRNA species encoding polypeptides IX and IVa 2 were not produced when viral DNA synthesis was blocked, whereas the larger L1 mRNA species was made under these conditions. Two E2B mRNA species, some 5.0 and 7 kb, were observed at low concentrations at 8 hr after infection and their concentration increased until 24 hr after infection, as did that of the E2A mRNA species: the products of the E2 transcription unit appeared to be expressed coordinately and at a constant ratio throughout infection. Few of the early mRNA species decreased in concentration after the onset of the late phase of infection. Examination of the viral mRNA produced when protein synthesis was inhibited by 10 μM anisomycin added 3 hr after infection suggested that processing of certain viral, early transcripts was altered in the presence of the drug.

Altered mRNA splicing in monkey cells abortively infected with human adenovirus may be responsible for inefficient synthesis of the virion fiber polypeptide.

Messenger RNA encoding the fiber protein of the human adenovirus serotype 2 (Ad2) capsid is inefficiently translated in abortively infected African green monkey kidney cells. The amount of fiber mRNA present in the cytoplasm of abortively infected monkey cells is less than that in productively infected cells by a factor of 5-10 but synthesis of the fiber polypeptide is reduced by a factor of more than 100. Evidence from a variety of experiments indicates that the defect does not lie in the translational apparatus of the monkey cell but may best be explained by differences in the fiber messages made in abortively versus productively infected cells. Here we report that fiber mRNA isolated from abortively infected monkey cells is processed differently than that made in productively infected cells. Primer extension analysis of the 5' ends of fiber messages from several different productive and abortive infections shows a direct correlation between synthesis of the fiber polypeptide in vivo and the presence of the "x" and/or "y" ancillary leaders on messages encoding the fiber polypeptide. Of all the mRNAs encoded by the major late transcriptional unit of Ad2 only the fiber message can contain the x and y leaders, and the fiber protein is the only late Ad2 protein reported to be glycosylated. We speculate that these leader sequences play a role in the synthesis of this glycoprotein, as well as that of the Ad2 19-kilodalton glycoprotein encoded by early region 3, whose mRNA also contains the x and y leaders.

Messenger RNA splicing in vitro: An excised intervening sequence and a potential intermediate

Four RNA products have been characterized from a soluble system that accurately splices purified substrate RNA. The labeled substrate RNA contained the first and second exons of the major late transcription unit of adenovirus 2 and a shortened form of the first intervening sequence. One of these RNA products was the excised intervening sequence which accumulated quantitatively with the level of splicing. This RNA species has an unusual structure and is modified at internal sites. A potential intermediate in the splicing pathway which is cleaved at the 5′ splice site of the first exon has been isolated. This RNA species is also modified at sites within the first intervening sequence. These results suggest that the splicing of mRNA precursors may involve sites in the intervening sequence, cleavage at the 5′ splice site, cleavage at the 3′ splice site, and ligation of the two exons.

Polyadenylic Acid Addition Sites in the Adenovirus Type 2 Major Late Transcription Unit

Polyadenylic Acid Addition Sites in the Adenovirus Type 2 Major Late Transcription Unit

The adenovirus major late promoter TATA box and initiation site are both necessary for transcription in vitro.

Mutagenized DNA templates and HeLa whole cell extracts were used to study the effects of promoter-specific base changes on in vitro transcription. DNA templates where the initiating adenine (+1) was changed to thymidine (AT+1) in the adenovirus 2 major late transcription unit were transcribed with 50% efficiency of the unaltered template. We have described a mutant at the TATA box, where the A at position -28 was changed to a C (AC-28). Transcription efficiency was reduced to less than 20% of control in the AC-28 mutant (Concino et al., 1983, J. Biol. Chem. 258: 8493-8496). Primer extension analysis revealed increased 5' end heterogeneity for in vitro transcripts derived from AC-28 and AT+1 DNA templates. Specific transcription was completely abolished from AT+1 DNA templates when a second change was introduced within the TATA sequence, creating a double mutant (AC-28 . AT+1). Neither the AC-28 nor the AT+1 change alone had such an effect, suggesting a coordinated interaction in transcription initiation involving both the TATA box and the initiation site.

Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts

Using a whole cell extract of HeLa cells, we synthesized unspliced RNAs containing the first two leaders and the first intervening sequence of the adenovirus 2 major late transcription unit. Upon incubation of these pre-mRNAs in reaction mixtures containing a nuclear extract from HeLa cells or combinations of the nuclear extract and a postnuclear fraction (S100), removal of the first intervening sequence and concomitant joining of the first leader to the second leader was observed. This splicing reaction requires proteins, Mg 2+ ions, and ATP. The S100 fraction alone has no splicing activity but stimulates splicing when added to the nuclear extract. Upon fractionation of the postnuclear S100 by chromatography on ion exchange and gel filtration columns, the stimulatory activity copurifies with small ribonucleoprotein particles.

Polyadenylic acid addition sites in the adenovirus type 2 major late transcription unit.

The cytoplasmic mRNAs which are transcribed from the major late adenovirus promoter can be arranged into five 3'-coterminal families, L1 to L5. We have defined the polyadenylation sites of the mRNAs that belong to the five families at the nucleotide level. From the results, the following conclusions can be made. (i) The hexanucleotide sequence AAUAAA is present at the 3' end of all late adenovirus type 2 mRNAs and precedes the site of polyadenylation by 12 to 30 nucleotides. (ii) Between one and three A residues are present in the genomic sequence at the polyadenylation site. (iii) A sequence with the composition (T)n (A)p (T)q (n, p, q greater than or equal to 1) is found 4 to 24 nucleotides beyond all the adenovirus-specific polyadenylation sites except the 3'-coterminal family L4. This sequence is also found beyond many cellular polyadenylation sites. (iv) The L1 and L2 polyadenylation sites are very similar in structure. The other polyadenylation sites show no apparent sequence relationship, except for the hexanucleotide sequence.

Alternative splicing pathways exist in the formation of adenoviral late messenger RNAs

Total rapidly labeled RNA was extracted from nuclei of HeLa cells late after infection with adenovirus type 2. Most of this nuclear RNA is transcribed from the major late transcription unit (16·2 to 100·0 map units (m.u.)). To study the cleavage reactions that are involved in RNA splicing, we used the S 1 nuclease mapping technique with HinIII B (16·8 to 31·5 m.u.) and XhoI F (15·5 to 22·4 m.u.) restriction fragments as DNA probes. The S 1 mapping data showed that both intron 1 (16·3 to 19·1 m.u.) and intron 2 (19·4 to 26·2 m.u.) can be excised in more than one step. Kinetic labeling and pulse-chase experiments demonstrated that certain cleavage sites in the RNA are used within three minutes after the start of transcription, while other cleavages occur only after a significant time-lag. The use of 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole enabled us to label the nuclear RNA exclusively from the 5′ end. Such an approach showed that the first cleavages are introduced in the nascent RNA before the RNA polymerase has passed more than 2000 nucleotides beyond the cleavage site. The chronology of the appearance of processing intermediates that results from cleavage of the RNA indicates that preferentially intron 1 is removed before intron 2. This is in agreement with the finding that leader 1 is ligated to leader 2 before ligation of leader 2 to leader 3 takes place. However, we have found that alternative splicing pathways exist in the excision of introns 1 and 2, demonstrating that cleavage in intron 2 may occur before intron 1 is attacked by the splicing enzymes.

Sequence arrangement and protein coding capacity of the adenovirus type 2 "i" leader

The adenovirus type 2 (Ad-2) "i" leader is an RNA segment which is preserved in some mRNA species from the Ad-2 late transcription unit. It maps between the second and third segments of the standard tripartite leader. We located the boundaries of the i leader in genomic Ad-2 DNA and determined its nucleotide sequence. The leader contains an ATG initiator near its 5' boundary, followed by a reading frame which is open for translation. We suggest that the i leader constitutes an Ad-2 coding sequence whose novel position within the leader of major late transcription unit messengers allows it to be translated in preference to coding sequences in mRNA main bodies. The i leader potentially contributes to the coding sequences of a family of proteins. Also, a Northern blot analysis of late mRNAs containing the i leader suggests that it may be retained in the leaders of many different late transcription unit mRNAs. We compare the i leader to the simian virus 40 agnogene.

Poly(A) sites of adenovirus serotype 2 transcription units

During productive infection of human cells, adenovirus serotype 2 transcription proceeds through early and late stages of transcription, which are separated by the onset of DNA replication. In this paper we analyze the 3′-terminal sequences of messenger RNAs from five early transcription units, and messenger RNA from the L2, L3 and L4 families of the major late transcription unit. By correlating RNAase T 1 oligonucleotides near poly(A) with the DNA sequence of Ad-2 ∥ ∥ Abbreviation used: Ad-2, adenovirus serotype 2. we have defined the poly(A) acceptor sequences of these messengers. Our principal conclusions are as follows. 1. (1)The A-A-U-A-A-A hexanucleotide homology common to many cellular mRNA 3′ ends is present near poly(A) of all but one of the mRNAs studied. Notably, it is present within the major late transcription unit at the L2, L3 and L4 coterminal family poly(A) sites, and potentially at all five poly(A) sites of the late transcription unit. Because the latter mRNA 3′ ends are formed by cleavage, the hexanucleotide probably does not specify a RNA polymerase II termination site. 2. (2) An early region III poly(A) site apparently lacks the A-A-U-A-A-A homology and may be an exception to the above generalizations. 3. (3) Region EIb and protein IX mRNA share a common poly(A) site, although their precursors are initiated at separate promoters. Also, the same poly(A) sites at the left end function during the early and late stages of infection. Thus, these poly(A) sites are not regulated in a stage-specific manner, and may function for precursors initiated at more than one promoter. 4. (4) Residues near poly(A) of mRNA belonging to the 3′ coterminal families of the late transcription unit form ribonuclease-resistant hybrids to Ad-2 DNA which encode mRNA main body sequences. Thus late mRNA 3′ ends are locally encoded. We present a model for sequence recognition during formation of 3′ ends, and discuss autonomy of poly(A) site function.

Mild nuclease treatment as a probe for a non-random distribution of adenovirus-specific RNA sequences and of cellular RNA in nuclear ribonucleoprotein fibrils

At the late period of productive infection, the RNA of hnRNP † † Abbreviations used: hnRNA, heterogeneous nuclear RNA: premRNA, premessenger RNA; RNP, ribonucleoprotein; hnRNP, ribonucleoprotein containing hnRNA; Ad2, adenovirus type 2; EGTA. ethyleneglycol-bis(aminoethyl ether) tetraacetic acid. from adenovirus-infected HeLa cells contains primarily cellular RNA and the partially processed transcripts from the major late transcription unit. Total cellular and viral RNA, seven viral RNA fragments corresponding to messenger and non-messenger RNA sequences, were determined in hnRNP. A mild nuclease treatment of 30 to 200 S native hnRNP, sufficient for the separation of 25 to 55 S monoparticles from larger (100 to 200 S) RNP, served as a probe for hnRNP structure and allowed the study of sequence distribution in these two classes of hnRNP units. The main observations were as follows: (1) 90% of the total viral RNA initially present in large hnRNP was found in the large RNP after nuclease treatment, whereas 85% of the nuclear-restricted cellular RNA was released with monoparticles; (2) viral poly(A) + RNA was enriched in large RNP indicating an enrichment in viral premessenger RNA; (3) the viral-specific sequences were distributed non-randomly between the hnRNP units. This distribution did not depend on their nucleotide sequence (messenger or non-messenger nature). Rather, the sequences from the 5′ half of the transcription unit were enriched in large RNP and those from the 3′ half in monoparticles. This may be related to the presence, in addition to authentic premessenger RNA, of nuclear-restricted hnRNA synthesized from the 3′ part of the transcription unit after the polyadenylation sites (Nevins & Darnell, 1978). The preferential localization of premessenger RNA in the large RNP isolated after mild nuclease treatment suggested that these structures are the sites of splicing. The monoparticles that are enriched in nuclear-restricted sequences might be structures where degradation starts.

Controls of RNA splicing and termination in the major late adenovirus transcription unit.

The major late adenovirus promoter is active early after infection, selectively producing messenger RNAs coding for polypeptides with molecular weights of 55,000, 52,000 and 14,000. This selective expression suggests that a differential splicing pattern occurs at the transition from early to late viral gene expression. Activation of the late promoter and splicing of the 55, 52K mRNAs does not require newly synthesized virus polypeptides.