Characterization of an RNP Complex That Assembles on the Rous Sarcoma Virus Negative Regulator of Splicing Element

Cook, Craig R.; McNally, Mark

doi:10.1093/nar/24.24.4962

Cited by 8 publications

(9 citation statements)

References 34 publications

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“…Alternatively, with the full-length construct, enhancer factors bound to the 5Ј region might interact with and be preoccupied by factors bound to NRS3Ј (e.g., U11 or U1 snRNP), which might make them unavailable for interactions that result in enhancement of the dsx 3Ј splice site. This is supported by the in vitro formation of a large RNP complex on the NRS (the NRS complex) but not on NRS3Ј alone (5).…”

Section: Discussionmentioning

confidence: 94%

An RNA Splicing Enhancer-Like Sequence Is a Component of a Splicing Inhibitor Element from Rous Sarcoma Virus

McNally

1998

Molecular and Cellular Biology

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The accumulation in infected cells of large amounts of unspliced viral RNA for use as mRNA and genomic RNA is a hallmark of retrovirus replication. The negative regulator of splicing (NRS) is a long cis-acting RNA element in Rous sarcoma virus that contributes to unspliced RNA accumulation through splicing inhibition. One of two critical sequences located in the NRS 3 region resembles a minor class 5 splice site and is required for U11 small nuclear ribonucleoprotein (snRNP) binding to the NRS. The second is a purine-rich region in the 5 half that interacts with the splicing factor SF2/ASF. In this study we investigated the possibility that this purine-rich region provides an RNA splicing enhancer function required for splicing inhibition. In vitro, the NRS acted as a potent, orientation-dependent enhancer of Drosophila doublesex pre-mRNA splicing, and enhancer activity mapped to the purine-rich domain. Analysis of a number of site-directed and deletion mutants indicated that enhancer activity was diffusely located throughout a 60-nucleotide area but only the activity associated with a short region previously shown to bind SF2/ASF correlated with efficient splicing inhibition. The significance of the enhancer activity to splicing inhibition was demonstrated by using chimeras in which two authentic enhancers (ASLV and FP) were substituted for the native NRS purine region. In each case, splicing inhibition in transfected cells was restored to levels approaching that observed for the NRS. The observation that a nonfunctional version of the FP enhancer (FPD) that does not bind SF2/ASF also fails to block splicing when paired with the NRS 3 region supports the notion that SF2/ASF binding to the NRS is relevant, but other SR proteins may substitute if an appropriate binding site is supplied. Our results are consistent with a role for the purine region in facilitated snRNP binding to the NRS via SF2/ASF. Retrovirus replication requires a substantial level of unspliced RNA for structural-protein production and for use as genomes in progeny virions (4). The unspliced RNA also serves as a substrate for RNA splicing in the nucleus to produce subgenomic RNAs such as env, the mRNA encoding the envelope protein. Efficient replication requires a precise balance between the unspliced and spliced RNA, which in the simple virus Rous sarcoma virus (RSV) is approximately 80% unspliced RNA. The RNA ratio is not influenced by viral proteins but, rather, results from the interaction of the host splicing machinery with regulatory or control elements within the viral RNA. A number of cis elements within the RSV primary transcripts contribute to the observed unspliced-tospliced-RNA ratio, and two of these are represented by the env and src 3Ј splice sites themselves, which have either a suboptimal branch point sequence (12, 22) or a pyrimidine tract (58). Mutation of these sequences toward the consensus splicing signals results in an oversplicing phenotype and a replication defect that is probably due to a shortage of unspliced RNA. When...

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Section: Discussionmentioning

confidence: 94%

An RNA Splicing Enhancer-Like Sequence Is a Component of a Splicing Inhibitor Element from Rous Sarcoma Virus

McNally

1998

Molecular and Cellular Biology

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“…There is also strong evidence that binding of the SR protein SF2/ASF is required for inhibition (12,19). These observations suggested that the NRS may be recognized as a 5Ј ss; in support of this proposal, we recently showed that the NRS assembles an ATP-independent complex, called NRS-C, that resembles the U1 E5Ј complex (7). NRS-C assembly requires ASF/SF2, U1 snRNP, and the U11 binding site, and based on a correlation between U11 binding and function (12), we speculated that NRS-C may represent a U11 E5Ј complex (8).…”

mentioning

confidence: 65%

“…pAd3Ј was made by cutting pAdHS with HindIII and BstEII to remove exon 1 and part of the intron, blunting, and religating. p3ZBB, used to generate NRS RNA, was described previously (7). pAdmutPyr, in which the pyrimidine tract is changed from TTTTTTTT to GTG ATCAC, was made by U.S.E.…”

Section: Methodsmentioning

confidence: 99%

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Interaction between the Negative Regulator of Splicing Element and a 3′ Splice Site: Requirement for U1 Small Nuclear Ribonucleoprotein and the 3′ Splice Site Branch Point/Pyrimidine Tract

Cook

McNally

1999

J Virol

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The negative regulator of splicing (NRS) from Rous sarcoma virus suppresses viral RNA splicing and is one of several ciselements that account for the accumulation of large amounts of unspliced RNA for use as gag-pol mRNA and progeny virion genomic RNA. The NRS can also inhibit splicing of heterologous introns in vivo and in vitro. Previous data showed that the splicing factors SF2/ASF and U1, U2, and U11 small nuclear ribonucleoproteins (snRNPs) bind the NRS, and a correlation was established between SF2/ASF and U11 binding and activity, suggesting that these factors are important for function. These observations, and the finding that a large spliceosome-like complex (NRS-C) assembles on NRS RNA in nuclear extract, led to the proposal that the NRS is recognized as a minor-class 5′ splice site. One model to explain NRS splicing inhibition holds that the NRS interacts nonproductively with and sequesters U2-dependent 3′ splice sites. In this study, we provide evidence that the NRS interacts with an adenovirus 3′ splice site. The interaction was dependent on the integrity of the branch point and pyrimidine tract of the 3′ splice site, and it was sensitive to a mutation that was previously shown to abolish U11 snRNP binding and NRS function. However, further mutational analyses of NRS sequences have identified a U1 binding site that overlaps the U11 site, and the interaction with the 3′ splice site correlated with U1, not U11, binding. These results show that the NRS can interact with a 3′ splice site and suggest that U1 is of primary importance for NRS splicing inhibition.

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“…Using gel filtration chromatography, it was demonstrated that the NRS alone assembles into a complex that is indistinguishable from the early (E′) complex that forms on isolated 5′ splice sites (45,46). Furthermore, the complex appeared functionally relevant since there was a correlation between the sequence requirements for assembly in vitro and splicing inhibition in vivo, and assembly of the complex required SR proteins, the U1 binding site, and U1 snRNP (47).…”

Section: 33mentioning

confidence: 99%

RNA processing control in avian retroviruses

McNally¹

2008

Front Biosci

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Upon integration into the host chromosome, retroviral gene expression requires transcription by the host RNA polymerase II, and viral messages are subject RNA processing events including 5′-end capping, pre-mRNA splicing, and polyadenylation. At a minimum, RNA splicing is required to generate the env mRNA, but viral replication requires substantial amounts of unspliced RNA to serve as mRNA and for incorporation into progeny virions as genomic RNA. Therefore, splicing has to be controlled to preserve the large unspliced RNA pool. Considering the current view that splicing and polyadenylation are coupled, the question arises as to how genome-length viral RNA is efficiently polyadenylated in the absence of splicing. Polyadenylation of many retroviral mRNAs is inefficient; in avian retroviruses, ∼15% of viral transcripts extend into and are polyadenylated at downstream host genes, which often has profound biological consequences. Retroviruses have served as important models to study RNA processing and this review summarizes a body of work using avian retroviruses that has led to the discovery of novel RNA splicing and polyadenylation control mechanisms. Keywordsretroviruses; RNA splicing; Spliceosome; snRNP; SR protein; hnRNP H; Polyadenylation; Review INTRODUCTION Retroviruses and RNA processingRetroviruses employ a unique replication scheme in which a long, single-stranded RNA genome is converted into a double-stranded DNA molecule that is inserted into and becomes a permanent resident of the host genome (reviewed in (1,2)). From the chromosomal position, the integrated viral DNA (the provirus) is transcribed by the host RNA polymerase II (pol II) to generate genome-length viral RNA that has the same modifications as typical host mRNAs (a 5′ cap and a 3′ poly(A) tail). A portion of this full-length viral RNA is packaged into progeny virions, and an additional pool is translated into structural and enzymatic proteins that compose the virus particles. However, some viral proteins are synthesized from spliced transcripts, so the primary transcript also serves as a substrate for RNA splicing. The number of spliced mRNA species can be quite large, as is the case for complex retroviruses like human immunodeficiency virus (HIV) (see (3) and a review by M. McLaren, K. Marsh, and A. Cochrane in this series). Clearly, the extent of splicing must necessarily be controlled to preserve the genome-length RNA, which typically represents ∼50% or greater of the total. Another issue raised by the recent appreciation that splicing and polyadenylation are coupled is how the full-length viral RNA is efficiently polyadenylated in the absence of splicing. This

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Characterization of an RNP Complex That Assembles on the Rous Sarcoma Virus Negative Regulator of Splicing Element

Cited by 8 publications

References 34 publications

An RNA Splicing Enhancer-Like Sequence Is a Component of a Splicing Inhibitor Element from Rous Sarcoma Virus

An RNA Splicing Enhancer-Like Sequence Is a Component of a Splicing Inhibitor Element from Rous Sarcoma Virus

Interaction between the Negative Regulator of Splicing Element and a 3′ Splice Site: Requirement for U1 Small Nuclear Ribonucleoprotein and the 3′ Splice Site Branch Point/Pyrimidine Tract

RNA processing control in avian retroviruses

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