Synthesis of an unspliced cytoplasmic message by an adenovirus 5 deletion mutant

Carlock, Leon; Jones, Nicholas

doi:10.1038/294572a0

Cited by 32 publications

(26 citation statements)

References 27 publications

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“…Large deletions that interfere with correct splicing have been shown to have differing effects on RNA stability (38)(39)(40). The magnitude of the deletions compared to the wild type controls used in these studies, coupled with the variable results, makes interpretation of these data difficult.…”

Section: Discussiqnmentioning

confidence: 71%

The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability

Adami¹,

Carmichael

1987

Nucl Acids Res

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ABSTRACT'Polyoma virus late RNA processing provides a convenient model system in which to study the mechanics of splicing in vivo. In order to understand further the role of the untranslated "late leader" unit in late RNA processing we have constructed a group of polyoma viruses with deletions and substitutions in the leader exon. This has allowed us to determine that there is a minimum exon size required for both pre-mRNA splicing and stability in this system. We show here that the non-viability of a mutant (ALM) with a 9 base late leader unit is due to a general defect in late RNA splicing. In addition, ALM-infected cells show at least 40-fold depression in the accumulation of late nuclear RNA (spliced or unspliced). The ALM late promoter, however, functions nearly normally. Substituted leader variants with 51-to 96-base long exons of unrelated sequence are viable (G. Adami and G. Carmichael, J. Virol. 58, 417-425, 1986). We show here that late RNA from one of these substituted leader mutants (containing a 51-base leader exon) is spliced at wild type levels, with virtually no defect in accumulation. Thus, in the polyoma system, splice sites separated by only 9 bases can inhibit each others usage, presumably by steric interference. We suggest that this type of inhibition leads to extreme RNA instability. INTRODUCTION Much work in recent years has been directed towards further understanding the mechanism of splicing of mRNA precursors. Analyses of intron sequence requirements for in vivo mRNA splicing in higher eukaryotes originally indicated the importance of the 5' splice site, including the highly conserved GU dinucleotide at the 5' exon-intron boundary, and the 3' splice site, which consists of a similarly highly conserved dinucleotide, AG, at the 3' boundary, but which additionally includes an upstream stretch of pyrimidines (1-7). More recently, a third site which shows some sequence requirements for splicing in vivo has been identified -the branch point,

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Section: Discussiqnmentioning

confidence: 71%

The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability

Adami¹,

Carmichael

1987

Nucl Acids Res

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“…Genes which contain intervening sequences may also produce stable cytoplasmic RNA retaining intact introns, as is the case for the unspliced late 19S mRNA of simian virus 40 (SV40; 11,12). A deletion in adenovirus early region Ela which removes a splice acceptor site also results in the synthesis of stable unspliced mRNA (7).…”

mentioning

confidence: 99%

Synthesis of predominantly unspliced cytoplasmic RNAs by chimeric herpes simplex virus type 1 thymidine kinase-human beta-globin genes.

Greenspan¹,

Weissman²

1985

Mol. Cell. Biol.

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The herpes simplex virus type 1 thymidine kinase (tk) gene lacks introns and produces stable mRNA in the absence of splicing. We have prepared a hybrid gene by placing the first exon, first intron (first intervening sequence, designated IVS1), and most of the second exon of the normal human (-globin gene into the 3' untranslated region of the tk gene. Although this hybrid gene contains all globin sequences presumed necessary for the splicing of IVS1, predominantly, unspliced stable cytoplasmic RNA is produced in both long-and short-term expression assays. Moreover, stable unspliced cytoplasmic RNA is detected whether the intron is situated in a sense or an antisense orientation. Efficient splicing of IVS1 is obtained either by deleting the majority of tk coding sequences or by relocating the globin sequences from the 3' to the 5' untranslated region of the tk gene.Several lines of evidence have indicated splicing to be an essential function in the biogenesis of certain mRNAs (13,15). The globin genes have been studied extensively in this regard, and it has been noted that globin transcripts in the sense orientation driven from viral promoters are spliced and form stable mRNA, whereas transcripts containing the intron in the antisense orientation do not form stable cytoplasmic or nuclear (14) RNA species. This difference was considered evidence for the requirement of splicing in the expression of globin mRNAs. Further evidence was derived from the study of altered globin genes produced both by in vitro mutagenesis (38) and by spontaneous mutations as in the human thalassemias (8). A number of mutations that affect the splice junction sequences in either the large or small intron have been found to decrease the amount of stable RNA formed. Cytoplasmic RNAs that were formed used cryptic alternative splice sites, and in no case was a stable unspliced globin mRNA formed. Other studies have indicated that unspliced globin RNA (5) and unspliced RNA of other genes (24) may be selectively retained in the nucleus.In contrast to the above results, a number of genes including those for herpes simplex virus (HSV) type 1 (HSV-1) thymidine kinase (25, 36), histone (17), interferon (28), and a variety of polymerase III transcription products such as 5S RNA (18) lack intervening sequences and yet produce stable mRNAs which are exported from the nucleus. Other unspliced mRNAs have been noted. For example, cDNA corresponding to an early gene of polyoma is expressed as stable RNA in the absence of splicing (34). Genes which contain intervening sequences may also produce stable cytoplasmic RNA retaining intact introns, as is the case for the unspliced late 19S mRNA of simian virus 40 (SV40; 11,12). A deletion in adenovirus early region Ela which removes a splice acceptor site also results in the synthesis of stable unspliced mRNA (7).The role of splicing events in the production and export of stable mRNA remains puzzling. To examine this role in a new way, we have introduced an intron and splice junctions * Corresponding author.into...

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“…It was reported that the sequence complexity of heterogeneous nuclear RNA is measurably greater than that of cytoplasmic RNA (31). It could be argued, however, that this may be because transcription runs well past the polyadenylation site and that subsequent cleavage and polyadenylation could result in the excision and degradation of downstream sequences, thereby accounting for the different complexities observed.It seems unlikely, however, that such an explanation could account for the transport behavior of several constructed and naturally occurring deletion mutations in viral and cellular genes (7,8,12,16,(18)(19)(20)(21)(22)30; R. T. White, Ph.D. thesis, Stanford University, Stanford, Calif., 1980). There are now numerous examples of genes deleted downstream from promoters and upstream from polyadenylation sites that do not code for the production of stable mRNA.…”

mentioning

confidence: 99%

“…It seems unlikely, however, that such an explanation could account for the transport behavior of several constructed and naturally occurring deletion mutations in viral and cellular genes (7,8,12,16,(18)(19)(20)(21)(22)30; R. T. White, Ph.D.…”

mentioning

confidence: 99%

“…The relation between splicing and transport, however, is complicated. There are, for example, deletion mutants which efficiently transport unspliced RNA (8,15,30; White, thesis), indicating that splicing per se need not occur to allow transport. Along these lines, a hypothesis that the intervening sequences were directly involved in the retention within the nucleus of nuclear RNA was proposed and tested (28).…”

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confidence: 99%

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A splice junction deletion deficient in the transport of RNA does not polyadenylate nuclear RNA.

Villarreal¹,

White²

1983

Mol. Cell. Biol.

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A late region deletion mutant of simian virus 40 (dl5) was previously shown to be deficient in the transport of nuclear RNA. This is a splice junction deletion that has lost the 3' end of an RNA leader, an intervening sequence, and the 5' end of the splice acceptor site on the body of the mRNA. In this report, we analyzed the steady-state structure of the untransported nuclear RNA. The 5' ends of this RNA are heterogeneous but contain a prominent 5' end at the normal position (nucleotide 325) in addition to several other prominent 5' ends not seen in wildtype RNA. The 3' end of this RNA does not occur at the usual position (nucleotide 2674) of polyadenylation; instead, this RNA is non-polyadenylated, with the 3' end occurring either downstream or upstream of the normal position.Eucaryotic mRNA production occurs via a surprisingly complicated pathway. The sequence of events leading to stable mRNA includes promotion (which in itself may be multifunctional), 5' capping, 3' polyadenylation, splicing, and finally transport from the nucleus to the cytoplasm (for review, see references 9 and 14). Of these post-transcriptional events, the least investigated is the question of how nuclear transcripts are transported into the cytoplasm. Not all nuclear RNA sequences are destined to become cytoplasmic mRNA. It was reported that the sequence complexity of heterogeneous nuclear RNA is measurably greater than that of cytoplasmic RNA (31). It could be argued, however, that this may be because transcription runs well past the polyadenylation site and that subsequent cleavage and polyadenylation could result in the excision and degradation of downstream sequences, thereby accounting for the different complexities observed.It seems unlikely, however, that such an explanation could account for the transport behavior of several constructed and naturally occurring deletion mutations in viral and cellular genes (7,8,12,16,(18)(19)(20)(21)(22)30; R. T. White, Ph.D. thesis, Stanford University, Stanford, Calif., 1980). There are now numerous examples of genes deleted downstream from promoters and upstream from polyadenylation sites that do not code for the production of stable mRNA. These deletions generally affect splice junctions. An early example of a transport deficiency mutation was constructed in the late region of simian virus 40 (SV40), in which an intervening sequence was precisely deleted (18, 19). The relation between splicing and transport, however, is complicated. There are, for example, deletion mutants which efficiently transport unspliced RNA (8,15,30; White, thesis), indicating that splicing per se need not occur to allow transport. Along these lines, a hypothesis that the intervening sequences were directly involved in the retention within the nucleus of nuclear RNA was proposed and tested (28). The results of those experiments indicated that retention of nuclear RNA is probably not simply the result of the presence of intervening sequences, as transcripts containing intronic sequences were efficiently transported...

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Synthesis of an unspliced cytoplasmic message by an adenovirus 5 deletion mutant

Cited by 32 publications

References 27 publications

The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability

The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability

Synthesis of predominantly unspliced cytoplasmic RNAs by chimeric herpes simplex virus type 1 thymidine kinase-human beta-globin genes.

A splice junction deletion deficient in the transport of RNA does not polyadenylate nuclear RNA.

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