Synthesis and Processing of Avian Sarcoma Retrovirus RNA

Cm, Stoltzfus

doi:10.1016/s0065-3527(08)60707-1

Cited by 50 publications

(35 citation statements)

References 161 publications

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“…The introduction of nonsense codons within the coding region of a number of eukaryotic cellular genes has also been associated with decreased mRNA levels. In the cases of dihydrofolate reductase (dhfr) (22), triosephosphate isomerase (7), and ,-globin (5) RNAs, it appears that prematurely terminated RNA may be degraded either in the nucleus or during transport from the nucleus to the cytoplasm.The primary RNA transcript of RSV (5' gag-pol-env-src 3') is transcribed and processed by cellular machinery (9,20). This RNA is polyadenylated; however, only a fraction of it is spliced to generate env and src mRNAs.…”

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confidence: 99%

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Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction.

Barker

Beemon

1994

Mol. Cell. Biol.

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The intracellular accumulation of the unspliced RNA of Rous sarcoma virus was decreased when translation was prematurely terminated by the introduction of nonsense codons within its 5' proximal gene, the gag gene. Subcellular fractionation of transfected cells suggested that nonsense codon-mediated instability occurred in the cytoplasm. Analysis of constructs containing an in-frame deletion in the nucleocapsid domain ofgag, which prevents interaction between the Gag protein and viral RNA, showed that an open reading frame extending to approximately 30 nucleotides from the natural gag termination codon was needed for RNA stability. Sequences at the gag-pol junction necessary for ribosomal frameshifting were not required for RNA stability; however, sequences located 100 to 200 nucleotides downstream of the natural gag termination codon were found to be necessary for stable RNA. The stability of RNAs lacking this downstream sequence was not markedly affected by premature termination codons. We propose that this downstream RNA sequence may interact with ribosomes translating gag to stabilize the RNA.High levels of retroviral RNAs are generated by efficient transcription from regulatory sequences in the viral long terminal repeat (LTR) and maintained as a result of their high intracellular stability (reviewed in references 9 and 20). We are studying the mechanism of stabilization of Rous sarcoma virus (RSV) RNA. Premature termination of translation within the 5' proximal gag gene of RSV was previously shown to lead to reduced accumulation of unspliced RSV RNA because of its increased degradation rate (2, 4). In contrast, the level of spliced RSV mRNAs was not affected by nonsense codons within the gag gene (4). The introduction of nonsense codons within the coding region of a number of eukaryotic cellular genes has also been associated with decreased mRNA levels. In the cases of dihydrofolate reductase (dhfr) (22), triosephosphate isomerase (7), and ,-globin (5) RNAs, it appears that prematurely terminated RNA may be degraded either in the nucleus or during transport from the nucleus to the cytoplasm.The primary RNA transcript of RSV (5' gag-pol-env-src 3') is transcribed and processed by cellular machinery (9,20). This RNA is polyadenylated; however, only a fraction of it is spliced to generate env and src mRNAs. Maintenance and nucleocytoplasmic transport of a portion of the unspliced RNA is critical to viral replication; the unspliced RNA is the form of the viral genome that is packaged into virions, and it is also the mRNA for Gag and Gag-Pol polyproteins. The major translation product of the unspliced RSV RNA is the 76-kDa Gag precursor polyprotein, which is subsequently processed into several virion structural proteins and a protease. Approximately 5% of ribosomes progress beyond the normal gag termination codon via a translational frameshift (12, 13), producing a 180-kDa Gag-Pol polyprotein, which is processed to generate the viral reverse transcriptase and integrase. We have proposed that a cis-acting interac...

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confidence: 99%

“…The primary RNA transcript of RSV (5' gag-pol-env-src 3') is transcribed and processed by cellular machinery (9,20). This RNA is polyadenylated; however, only a fraction of it is spliced to generate env and src mRNAs.…”

mentioning

confidence: 99%

Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction.

Barker

Beemon

1994

Mol. Cell. Biol.

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“…Retrovirus RNA transcripts are subject to multiple pathways of RNA processing and transport (for a review, see Stoltzfus 1989). The primary full-length RNA po7lI transcript of -7-10 kb functions as genomic RNA for progeny virions and mRNA for structural proteins and enzymes.…”

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confidence: 99%

“…An understanding of the mechanisms involved in this splicing may reveal both general and novel features of the splice site selection process. Several models for maintaining the balance of spliced and unspliced forms of retrovirus RNA have been proposed (Coffin 1985;Katz et al 1988;Stoltzfus 1989). They include (I) rapid and selective transport of a portion of the unspliced viral RNA from the nucleus, (2) negative regulatory sequences that inhibit splicing in cis (Arrigo and Beemon 1988), (3) regulation of RNA processing or transport by viral proteins in trans, and (4) suboptimal cis-acting signals involved in splice site selection (Katz and Skalka 1990).…”

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The role of branchpoint and 3'-exon sequences in the control of balanced splicing of avian retrovirus RNA.

Fu¹,

Katz²,

Skalka³

et al. 1991

Genes Dev.

110

128

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We previously described an avian sarcoma-leukosis virus (ASLV) insertion mutation that causes a decrease in the ratio of unspliced to spliced RNA in vivo, resulting in a replication defect. Pseudorevertant viruses containing cis-acting suppressor mutations that restored the normal ratio were isolated. One class of the suppressor mutations consists of single-base changes or small deletions near the 3' splice site, while another consists of deletions in the 3' exon. In this paper we report results from an in vitro analysis of wild-type, mutant, and pseudorevertant pre-mRNA splicing. We find that wild-type RNA is spliced inefficiently in vitro, and that the insertion mutation and suppressors act directly at the level of splicing. Characterization of splicing intermediates reveals that the insertion mutation and suppressor mutations located within the intron alter the pattern of lariat formation. In contrast, suppressor mutations consisting of 3' exon deletions act at an earlier step in the splicing pathway. Thus, the efficiency of splicing at the env 3' splice site can be affected at the level of spliceosome assembly, lariat formation, or cleavage at the 3' splice site and exon ligation.[Key Words: Retrovirus; splicing; splice site selection] Received September 18, 1990; revised version accepted December 13, 1990.Retrovirus RNA transcripts are subject to multiple pathways of RNA processing and transport (for a review, see Stoltzfus 1989). The primary full-length RNA po7lI transcript of -7-10 kb functions as genomic RNA for progeny virions and mRNA for structural proteins and enzymes. Both of these functions require transport of an intact viral RNA to the cytoplasm. In addition, a fraction of the full-length transcripts is spliced in the nucleus to generate subgenomic mRNAs that encode other viral proteins (Fig. lA). Thus, a balance between full-length RNA and spliced viral mRNAs is required for replication of retroviruses.Multiple CIS-acting sequences have been shown to play a role in splice site selection in cellular pre-mRNAs (for review, see Krainer and Maniatis 1988). These sequences include the 5' and 3' splice sites, the branchpoint sequence (BPS), and sequences within the flanking exons. Interaction of these cis-acting elements with small nuclear ribonuclear proteins (snRNPs) and many protein splicing factors leads to the assembly of spliceosomes in which the splicing reaction takes place (see Krainer and Maniatis 1988;Steitz et al. 1988; for recent reviews, see Bindereif and Green 1990).Retroviral transcripts are presumably processed by the same mechanisms as cellular pre-mRNAs. However, unlike most cellular pre-mRNAs, a portion of viral RNA transcripts escape splicing entirely. Balanced splicing of retroviral RNA is therefore a novel form of alternative splicing. An understanding of the mechanisms involved in this splicing may reveal both general and novel features of the splice site selection process. Several models for maintaining the balance of spliced and unspliced forms of retrovirus RNA have been pro...

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“…Nearly all eucaryotic transcripts either are spliced to completion, so that no unspliced RNA is detectable in the cytoplasm, or do not contain introns and so require no splicing (reviewed in references 14 and 27). Retroviruses, on the other hand, produce a single primary transcript which is utilized in both spliced and unspliced forms; unspliced RNAs serve as genomic RNA and also as mRNA for the gag and pol genes, while spliced mRNAs are translated to form env products (32,36). In the case of the avian transforming retrovirus Rous sarcoma virus (RSV), alternative splicing of the primary transcript from a common splice donor also generates the src mRNA (32,36).…”

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Regulation of Rous sarcoma virus RNA splicing and stability.

Arrigo¹,

Beemon²

1988

Mol. Cell. Biol.

132

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Only a fraction of retroviral primary transcripts are spliced to subgenomic mRNAs; the unspliced transcripts are transported to the cytoplasm for packaging into virions and for translation of the gag and pol genes. We identified cis-acting sequences within the gag gene of Rous sarcoma virus (RSV) which negatively regulate splicing in vivo. Mutations were generated downstream of the splice donor (base 397) in the intron of a proviral clone of RSV. Deletion of bases 708 to 800 or 874 to 987 resulted in a large increase in the level of spliced RSV RNA relative to unspliced RSV RNA. This negative regulator of splicing (nrs) also inhibited splicing of a heterologous splice donor and acceptor pair when inserted into the intron. The nrs element did not affect the level of spliced RNA by increasing the rate of transport of the unspliced RNA to the cytoplasm but interfered more directly with splicing. To investigate the possible role of gag proteins in splicing, we studied constructs carrying frameshift mutations in the gag gene. While these mutations, which caused premature termination of gag translation, did not affect the level of spliced RSV RNA, they resulted in a large decrease in the accumulation of unspliced RNA in the cytoplasm.Retroviral pre-mRNA splicing differs dramatically from that of cellular mRNAs. Nearly all eucaryotic transcripts either are spliced to completion, so that no unspliced RNA is detectable in the cytoplasm, or do not contain introns and so require no splicing (reviewed in references 14 and 27). Retroviruses, on the other hand, produce a single primary transcript which is utilized in both spliced and unspliced forms; unspliced RNAs serve as genomic RNA and also as mRNA for the gag and pol genes, while spliced mRNAs are translated to form env products (32,36). In the case of the avian transforming retrovirus Rous sarcoma virus (RSV), alternative splicing of the primary transcript from a common splice donor also generates the src mRNA (32, 36). Retroviral RNA must be spliced to an intermediate degree rather than to completion, so that the proper balance of unspliced and spliced RNAs reaches the cytoplasm. Since retroviruses must use the same splicing machinery as the eucaryotic cells they infect, the retroviral transcripts need to be partially spliced by a machinery which seems geared to all-or-none splicing.There may be regulatory functions within retroviruses which mediate incomplete splicing (reviewed in references 10 and 32). One possible mechanism is that the retroviral splice donors and acceptors may be recognized inefficiently by splicing factors. Alternatively, cis-acting sequences elsewhere in the viral genome may directly interfere with splicing processes or may facilitate transport of the unspliced precursor RNA away from the splicing machinery, indirectly resulting in a decrease in the level of spliced RNA. A third possibility is that viral proteins may be involved in regulating the level of splicing. A final possibility is that the regulation occurs at the level of RNA stability as...

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Synthesis and Processing of Avian Sarcoma Retrovirus RNA

Cited by 50 publications

References 161 publications

Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction.

Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction.

The role of branchpoint and 3'-exon sequences in the control of balanced splicing of avian retrovirus RNA.

Regulation of Rous sarcoma virus RNA splicing and stability.

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