Isolation and characterization of poly(A)-containing intranuclear polyoma-specific "giant" RNAs

Rosenthal, Leonard J.; Salomon, Consuelo; Weil, Roger

doi:10.1093/nar/3.5.1167

Cited by 16 publications

(18 citation statements)

References 17 publications

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“…Two previous reports have suggested that only a small proportion of late polyomavirus RNA in the nucleus is polyadenylated (7,30). However, in neither report was it demonstrated that the methods used for assay of polyadenylated viral RNA were quantitative.…”

Section: Discussionmentioning

confidence: 88%

“…Only a single mRNA body sequence per precursor RNA molecule is conserved during RNA splicing (8,15,21); the remaining mRNA sequences are presumably discarded in the nucleus. In addition, two reports have indicated that only a fraction of polyomavirus RNA in the nucleus becomes polyadenylated during the late phase (7,30); since viral mRNAs in the cytoplasm are polyadenylated (16, 29), presumably only precursor molecules in the nucleus containing polyadenylic acid [poly(A)]…”

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confidence: 99%

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Kinetics and efficiency of polyadenylation of late polyomavirus nuclear RNA: generation of oligomeric polyadenylated RNAs and their processing into mRNA.

Acheson¹

1984

Mol. Cell. Biol.

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The rate and efficiency of polyadenylation of late polyomavirus RNA in the nucleus of productively infected mouse kidney cells were determined by measuring incorporation of [3H]uridine into total and polyadenylated viral RNAs fractionated by oligodeoxythymidylic acid-cellulose chromatography. Polyadenylation is rapid: the average delay between synthesis and polyadenylation of viral RNA in the nucleus is 1 to 2 min. However, only 10 to 25% of viral RNA molecules become polyadenylated. Polyadenylated RNAs in the nucleus are a family of molecules which differ in size by an integral number of viral genome lengths (5.3 kilobases). These RNAs are generated by repeated passage of RNA polymerase around the circular viral DNA, accompanied by addition of polyadenylic acid to a unique 3' end situated 2.2 + n(5.3) kilobases from the 5' end of the RNAs (n can be an integer from 0 to at least 3). Between 30 and 50% of the sequences in nuclear polyadenylated RNA are conserved during processing and transport to the cytoplasm as mRNA. This is consistent with the molar ratios of nuclear polyadenylated RNAs in the different size classes, and it suggests that most polyadenylated nuclear RNA is efficiently processed to mRNA. Thus, the low overall conservation of viral RNA sequences between nucleus and cytoplasm is explained by (i) low efficiency of polyadenylation of nuclear RNA and (ii) removal of substantial parts of polyadenylated RNAs during splicing. The correlation between inefficient termination of transcription and inefficient polyadenylation of transcripts suggests that these two events may be causally linked.During the late phase of infection of mouse cells by polyomavirus, only about 5% of the viral RNA synthesized in the nucleus is successfully processed and transported to the cytoplasm as mRNA (2). This low efficiency of conservation of viral RNA during processing can be explained partly by the fact that RNA polymerase II may traverse the circular viral DNA genome (5,292 nucleotide pairs [11,35]) several times before terminating transcription (1,3). This leads to production of giant RNAs containing several tandemly repeated copies of the entire nucleotide sequence of one of the strands of the viral DNA. Only a single mRNA body sequence per precursor RNA molecule is conserved during RNA splicing (8,15,21) I have confirmed that only a small fraction (10 to 25%) of nuclear viral RNA is polyadenylated, that it is polyadenylated within less than 2 min of its synthesis, and that the remaining 75 to 90% of nuclear viral RNA is apparently never polyadenylated and cannot therefore serve as a precursor to viral mRNA in the cytoplasm. Nuclear viral RNA which is polyadenylated exists as molecules of discrete lengths of about 2.2 + n(5.3) kilobases (kb). These RNAs represent a series which have the same 5' and 3' ends but differ in the number of genome traverses made by RNA polymerase II before terminating. These results are consis-722 tent with the idea that most polyadenylated nuclear RNAs are successfully processed into mature mR...

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Section: Discussionmentioning

confidence: 88%

mentioning

confidence: 99%

Kinetics and efficiency of polyadenylation of late polyomavirus nuclear RNA: generation of oligomeric polyadenylated RNAs and their processing into mRNA.

Acheson¹

1984

Mol. Cell. Biol.

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“…The presence of late RNAs with large heterogeneous sizes late in infection has been previously described. They are thought to be generated by continuous transcription of the circular genome template, without termination, generating head-to-tail tandem repeats of the entire viral genome (1,38). However, little information has been generated in the case of similar early giant transcripts.…”

Section: Discussionmentioning

confidence: 99%

“…A polyadenylated subset is processed by leader to leader splicing, generating mature transcripts with a single mRNA body sequence, and multiple copies of a 57-nt leader (3,5,10,13,28,32,34,38,44). Leader-to-leader splicing, and hence the synthesis of giant late transcripts, plays an important role in the generation of a stable mature 16S VP1 mRNA (4,5).…”

Section: Discussionmentioning

confidence: 99%

Kinetic Analysis of the Steps of the Polyomavirus Lytic Cycle

Chen

Fluck

2001

J Virol

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Kinetic studies of the accumulation of early and late transcripts, early and late proteins, genomes, and live virus, during the lytic cycle of murine polyomavirus wild-type A2, were carried out in synchronized NIH 3T3 cells released from G 0 by the addition of serum after infection. This first-time simultaneous analysis of all parameters of the virus life cycle led to new insights concerning the transcriptional control at the early-to-late transition. During the early phase, early transcripts were synthesized at very low levels, detectable only by reverse transcription-PCR, from 6 h postinfection (hpi). Large T protein could be detected by 8 hpi (while infected cells were in the G 1 phase). The level of expression of the middle T and small T proteins was lower than that of large T at all times, due, at least in part, to a splicing preference for the large-T 5 splice site at nucleotide 411. A large increase in the level of both early and late transcripts coincided closely with the detection in mid-S phase of viral genome amplification. Thereafter, both classes of transcripts continued to further accumulate up to the end of the experiments (48 hpi). In addition, during the late phase, "giant" multigenomic transcripts were synthesized from the early as well as the late promoter. Thus, a major type of transcriptional control appears to be applied similarly to the transcription of both early and late genes. This view differs from that in the literature, which highlights the enhancement of late transcription and the repression of early transcription. However, despite this parallel transcriptional control, additional regulations are applied which result in higher levels of late compared to early transcripts, as previously described. In the accompanying article, a key role for middle T and/or small T in this late-phase enhancement of early and late transcription is demonstrated (16). Other novel findings, e.g., the synthesis of a very abundant short early promoter proximal RNA, are also described.Polyomaviruses have served as important model systems for the understanding of various aspects of cellular organization and function, such as chromatin structure, the role of enhancers in gene expression, splicing mechanism, and the composition of the DNA replication machinery, to name a few. This is in part a consequence of the DNA nature and small size of the genomes of this virus family, which render viral replication highly dependent on, and hence compatible with, cellular processes. As a result, and with the added interest in their capacity to induce neoplastic transformation, these viruses have been the subjects of extensive studies.Surprisingly at this late stage of analysis, there has been no comprehensive study of the temporal relationships between all the various steps of the lytic cycle. Current references to the timing of these steps invariably quote the 1981 review by Acheson (2), which offers a thorough integration of the studies available at the time. However, these studies were done at different times, in differ...

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“…1) gives rise to "giant" RNA molecules up to 3-4 times the size of the viral genome (2)(3)(4)(5). These giant polyoma-specific RNAs are limited to the nucleus (6) where transcription takes place; viral mRNAs in the cytoplasm are one-quarter to one-half the size of the viral DNA (6)(7)(8).…”

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confidence: 99%

Polyoma virus giant RNAs contain tandem repeats of the nucleotide sequence of the entire viral genome.

Acheson¹

1978

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACr The bulk of late virusspecific RNA synthesized in polyoma virus-infected mouse cells is larger than a single strand of polyoma DNA. The arrangement of viral nucleotide sequences in these giant polyoma RNAs was studied by electron microscopy of hybrids between purified high molecular weight viral RNA and the Hindu-i fragment of polyoma DNA, which contains 91% of the viral genome. Hybridimolecules containing a short single-stranded gap (corresponding to the 9% of viral sequences not present in Hind]-i), flanked by double-stranded regions, were photographed and measured. The majority of hybrid molecules contained no single-stranded loops or branches, showing that all viral sequences are transcribed contiguously and that no nonviral sequences are present in the RNA. Hybrid molecules, containing RNA up to 3.5 times the genome length, had a repeating structure of single-stranded gaps 8% of genome length interspersed with double-stranded regions 89% of genome length, showing that giant polyoma RNAs contain tandem repeats of the nucleotide sequence of the entire viral DNA. A small proportion of hybrid molecules contained single-stranded branches or deletion loops in characteristic positions, indicating that RNA "splicing" may occur on high molecular weight nuclear polyoma RNA.Transcription of polyoma DNA during the late phase of productive infection (for review, see ref. 1) gives rise to "giant" RNA molecules up to 3-4 times the size of the viral genome (2-5). These giant polyoma-specific RNAs are limited to the nucleus (6) where transcription takes place; viral mRNAs in the cytoplasm are one-quarter to one-half the size of the viral DNA (6-8). Most late nuclear polyoma-specific RNA is transcribed from the L strand of viral DNA (9); all sequences of the L strand are represented in late nuclear transcripts (10,11).To try to understand the mechanism by which these giant RNA molecules are synthesized, I have hybridized this RNA with a fragment (HindII-l) of polyoma DNA consisting of 91% of the genome (12,13) (Amersham,25 Ci/mmol) per culture dish in 1 ml of reinforced Eagle's medium. Cells were scraped into cold 10 mM triethanolamine, pH 8.5/10 mM NaCl/1.5 mM MgCl2 and lysed by addition of Nonidet P40 (Shell) to 0.5% final concentration. Nuclei were pelleted and resuspended in the same buffer. RNA was extracted by addition of 1 vol of 10 mM triethanolamine (pH 8.5)/100 mM NaCl/10 mM EDTA/4% triisopropylnaphthylenesulfonate (Serva) (14, 15) and 2 vol of phenol/chloroform/isoamyl alcohol, 50:50:1 (vol/vol). After three successive extractions at room temperature, nucleic acids in the aqueous phase were precipitated three times in succession with ethanol and then incubated at 00 for 20 min with DNase I (RNase-free; Worthington; 10,Mg/ml) in 10 mM Na acetate, pH 5.1/100 mM NaCI/2 mM MnCI2. RNA was extracted with phenol/chloroform in the presence of 1% sodium dodecyl sulfate (NaDodSO4) and ethanol-precipitated two times.Preparative Hybridization of RNA to Polyoma DNA. Polyoma form I DNA was denatured and bound to nitr...

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Isolation and characterization of poly(A)-containing intranuclear polyoma-specific "giant" RNAs

Cited by 16 publications

References 17 publications

Kinetics and efficiency of polyadenylation of late polyomavirus nuclear RNA: generation of oligomeric polyadenylated RNAs and their processing into mRNA.

Kinetics and efficiency of polyadenylation of late polyomavirus nuclear RNA: generation of oligomeric polyadenylated RNAs and their processing into mRNA.

Kinetic Analysis of the Steps of the Polyomavirus Lytic Cycle

Polyoma virus giant RNAs contain tandem repeats of the nucleotide sequence of the entire viral genome.

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