Use of a novel S1 nuclease RNA-mapping technique to measure efficiency of transcription termination on polyomavirus DNA.

Tseng, Richard; Acheson, Nicholas H.

doi:10.1128/mcb.6.5.1624

Cited by 21 publications

(23 citation statements)

References 29 publications

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“…Using this method, it was determined that about 70% of RNA polymerase II molecules terminate each time around the polyoma genome (40). This is in good agreement with the predicted distribution based on direct measurements of late transcription termination using 3H-uridine pulse labeling (43). The RT-PCR assay we use also gives the same quantitative results as RNAse protection assays for leader-leader splicing (data not shown).…”

Section: Introductionsupporting

confidence: 71%

Polyadenylation and transcription termination in gene constructs containing multiple tandem polyadenylation signals

1994

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The processes of pre-mRNA 3'-end cleavage and polyadenylation have been closely linked to transcription termination by RNA polymerase II. We have studied the relationship between polyadenylation and transcription termination in gene constructs containing tandem poly(A) signals, at least one of which is the inefficient polyomavirus late poly(A) site. When identical tandem viral signals were separated by fewer than 400 bp, they competed for polyadenylation. The upstream site was always chosen preferentially, but relative site choice was influenced by the distance between the signals. All of these constructs showed the same low level of transcription termination as wild type polyomavirus, which contains a single late poly(A) site. When tandem poly(A) signals were not identical, a stronger downstream signal could outcompete a weaker upstream signal for polyadenylation without altering the efficiency of transcription termination characteristic for use of the upstream signal. Thus, if a weak polyoma virus late poly(A) signal (associated with inefficient transcription termination) preceded a strong rabbit 3-globin signal (associated with efficient transcription termination), termination remained inefficient, but the distal signal was most often chosen for polyadenylation. These results are consistent with independent regulation of polyadenylation and transcription termination in this system and are discussed in light of current models for the dependence of transcription termination on a functional poly(A) site.

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Section: Introductionsupporting

confidence: 71%

Polyadenylation and transcription termination in gene constructs containing multiple tandem polyadenylation signals

1994

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“…The late leader is a 57-base exon encoded only once in the viral genome but found in multiple tandem copies on polyoma late mRNAs (26). These repeats are generated by leader-to-leader splicing of giant primary transcripts produced as a result of inefficient termination (27). Recent studies on L-RNA splicing in our laboratory have indicated that before efficient mVP1 splicing is possible, leader-to-leader splicing must occur.…”

Section: Discussionmentioning

confidence: 99%

Polyomavirus early-late switch is not regulated at the level of transcription initiation and is associated with changes in RNA processing.

Hyde-DeRuyscher

Carmichael²

1988

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

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Polyoma gene expression is temporally regulated during productive infection of mouse cells. Early genes are expressed throughout the viral life cycle, but late mRNAs are not detected until after the onset ofDNA replication. At late times, late-strand transcripts represent the great majority of viral-specific RNA in the cell. To learn more about the mechanism by which the early-late switch is regulated, we have carried out a detailed analysis of polyomavirus transcription in mouse NIH 3T6 cells. Nuclei were isolated from cells infected for 6, 12, 18, or 24 hr, and run-on assays were performed. The resulting RNAs were then hybridized to a number of immobilized early-and late-strand-specific probes, which represent the entire polyoma genome. Results indicate that the late promoter is always on, even in the absence of DNA replication. Even though the early-late switch is characterized by a >300-fold difference in the ratio ofsteady-state early-and late-strand RNAs, there is only a 2-fold effect at the level of transcription initiation. Furthermore, the efficiency of termination for late transcripts is very high at early times during infection (>90%) but drops drastically at late times (<40%). In other experiments, we have found an increase in splicing efficiency of late pre-mRNA molecules that parallels the decrease in termination efficiency. These results, taken together with other studies from our laboratory, have led us to propose two possible models for the temporal control of polyomavirus late gene expression.Polyoma is a small, double-stranded, circular DNA virus whose genome contains two transcription units (early and late) and an intergenic control region (Fig.

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“…Clearly, RNA polymerase II must be designed to transcribe these genes without interruption until the functional end of the gene has been reached. Since this functional end is the site where cleavage and polyadenylation take place, it has occurred to us (and others: Acheson, 1984;Citron et al, 1984;Lanoix et al, 1986;Tseng and Acheson, 1986;Whitelaw and Proudfoot, 1986) that polyadenylation signals may themselves transmit to RNA polymerase II the information required to terminate transcription. Falck-Pedersen et al (1985) showed that insertion of a 1.6-kb fragment of the mouse 3-globin gene, including the polyadenylation signal, within the adenovirus ElA gene caused efficient termination during the early phase of adenovirus infection.…”

Section: Introductionmentioning

confidence: 99%

A rabbit beta-globin polyadenylation signal directs efficient termination of transcription of polyomavirus DNA.

Lanoix

Acheson

1988

The EMBO Journal

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We constructed a viable insertion mutant (ins 5) of polyomavirus which contains, upstream of the L‐strand polyadenylation signal, a 94‐nt fragment of rabbit beta‐globin DNA. Included in this fragment are all of the sequence elements required for efficient cleavage and polyadenylation of rabbit beta‐globin RNA. The beta‐globin signal was efficiently recognized by the cleavage/polyadenylation machinery in mouse 3T6 cells infected with ins 5, signalling greater than 90% of the polyadenylation events on L‐strand RNAs. Furthermore, the presence of this efficient polyadenylation signal resulted in a 1.4‐ to 2.5‐fold increase in the fraction of virus‐specific RNAs that were polyadenylated. Most importantly, termination of transcription by RNA polymerase II on ins 5 DNA was also increased compared with wild‐type virus; nearly 100% of polymerases terminated per traverse of the ins 5 genome. These findings demonstrate that the rabbit beta‐globin insert, which contains a strong polyadenylation signal, also contains at least part of a signal for termination of transcription by RNA polymerase II. These results also show that the multiple, spliced leaders on polyomavirus L‐strand mRNAs, which arise as a result of inefficient termination and polyadenylation, are not necessary for efficient virus replication.

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Use of a novel S1 nuclease RNA-mapping technique to measure efficiency of transcription termination on polyomavirus DNA.

Cited by 21 publications

References 29 publications

Polyadenylation and transcription termination in gene constructs containing multiple tandem polyadenylation signals

Polyadenylation and transcription termination in gene constructs containing multiple tandem polyadenylation signals

Polyomavirus early-late switch is not regulated at the level of transcription initiation and is associated with changes in RNA processing.

A rabbit beta-globin polyadenylation signal directs efficient termination of transcription of polyomavirus DNA.

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