CA- and Purine-Rich Elements Form a Novel Bipartite Exon Enhancer Which Governs Inclusion of the Minute Virus of Mice NS2-Specific Exon in Both Singly and Doubly Spliced mRNAs

Gersappe, Anand; Pintel, David J.

doi:10.1128/mcb.19.1.364

Cited by 22 publications

(24 citation statements)

References 61 publications

(86 reference statements)

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“…Samples were run on a 6% acrylamide-urea gel. WT (ϪRT) is a control reaction utilizing WT RNA but excluding reverse transcriptase; p⌬D1/2, a mutation in which both the donors of the downstream small intron were deleted and which results in almost uniform skipping of the NS2-specific exon (18,48), was used as a control for amplification of the ES product. An RNase protection analysis, using probe B, of RNA generated by WT was used as a marker (sizes of the marker bands are shown on the left in the left-hand panel and on the right in the right-hand panel) for the sizes of the RT-PCR amplified bands.…”

Section: Resultsmentioning

confidence: 99%

“…Construction of p⌬D1/2, p⌬SX, p⌬XP, p⌬PH, p⌬HS, p⌬SXϩ⌬HS pCSD⌬SXϩ⌬HS, p4T⌬SXϩ⌬HS, and p2018 TAA has been previously described (18,32,48).…”

Section: Methodsmentioning

confidence: 99%

“…1A). Efficient inclusion of the NS2-specific exon as an internal exon in vivo, and consequent excision of the upstream large intron from P4-generated pre-mRNA to generate R2, requires an internally redundant, bipartite exon splicing enhancer (ESE) comprised of 5Ј and 3Ј elements within the NS2-specific exon (18). We have previously shown that PTCs in either the first exon (NS1/NS2 common exon) or second exon (the NS2-specific exon) of R2 (Fig.…”

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confidence: 99%

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A Premature Termination Codon Interferes with the Nuclear Function of an Exon Splicing Enhancer in an Open Reading Frame-Dependent Manner

Gersappe

Pintel

1999

Molecular and Cellular Biology

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Premature translation termination codon (PTC)-mediated effects on nuclear RNA processing have been shown to be associated with a number of human genetic diseases; however, how these PTCs mediate such effects in the nucleus is unclear. A PTC at nucleotide (nt) 2018 that lies adjacent to the 5 element of a bipartite exon splicing enhancer within the NS2-specific exon of minute virus of mice P4 promoter-generated pre-mRNA caused a decrease in the accumulated levels of P4-generated R2 mRNA relative to P4-generated R1 mRNA, although the total accumulated levels of P4 product remained the same. This effect was seen in nuclear RNA and was independent of RNA stability. The 5 and 3 elements of the bipartite NS2-specific exon enhancer are redundant in function, and when the 2018 PTC was combined with a deletion of the 3 enhancer element, the exon was skipped in the majority of the viral P4-generated product. Such exon skipping in response to a PTC, but not a missense mutation at nt 2018, could be suppressed by frame shift mutations in either exon of NS2 which reopened the NS2 open reading frame, as well as by improvement of the upstream intron 3 splice site. These results suggest that a PTC can interfere with the function of an exon splicing enhancer in an open reading frame-dependent manner and that the PTC is recognized in the nucleus.Premature termination codons (PTCs) decrease the accumulated levels of most known mRNAs in which they reside (reviewed in reference 28). In the yeast Saccharomyces cerevisiae, the UPF genes are involved in the degradation of PTCcontaining RNAs in the cytoplasm (review in reference 37). Similarly, the SMG proteins of Caenorhabditis elegans are required for the rapid decay of PTC-containing unc-54 myosin heavy-chain mRNAs (reviewed in reference 41).PTC-mediated effects on nuclear RNA processing have been shown to be associated with a number of human genetic diseases (reviewed in references 28 and 29); however, how PTCs mediate such effects in the nucleus is still unclear. In mammalian cells, PTCs have been shown to decrease the levels of nucleus-associated mRNAs by a posttranscriptional mechanism, often attributed to mRNA decay (3,6,9,28,45). Experiments done with PTCs in the human TPI gene suggest that nonsense-mediated decay (NMD) occurs on a fully spliced nucleus-associated mRNA molecule, possibly during mRNA export to the cytoplasm (4, 5, 9, 28).There is also increasing evidence that PTCs may affect mammalian RNA processing events other than decay. For example, several instances of PTC-associated exon skipping have been described (15,19,28,33). Perhaps the best-studied example is skipping of the 66-nucleotide (nt) exon 51 of fibrillin FBN1 RNA, which was detected when nonsense but not missense mutations were present within this exon, which was independent of protein synthesis, and for which normal splicing was restored when the nonsense codon was shifted out of frame with the initiation codon (14, 23). Retention of introns upstream of PTC-containing exons has also been reported for P4-ge...

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Section: Resultsmentioning

confidence: 99%

“…Construction of p⌬D1/2, p⌬SX, p⌬XP, p⌬PH, p⌬HS, p⌬SXϩ⌬HS pCSD⌬SXϩ⌬HS, p4T⌬SXϩ⌬HS, and p2018 TAA has been previously described (18,32,48).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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A Premature Termination Codon Interferes with the Nuclear Function of an Exon Splicing Enhancer in an Open Reading Frame-Dependent Manner

Gersappe

Pintel

1999

Molecular and Cellular Biology

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“…Therefore, mutations in either of these regions may have effects on the levels of NS2 and hence ssDNA production. The 5Ј end of the NS2-specific exon, adjacent to the large-intron 3Ј splice site, contains both an amino acid motif important for the NS2 interaction with Crm1 (5) and a nucleotide motif that functions as an exon splicing enhancer that plays a critical role in upstream large-intron excision (13). The six-amino-acid NS2-CRM1 interaction mutant we previously characterized, which is deficient in ssDNA production (17), shows no decrease in excision of the large intron and makes at least wild-type levels of mutant NS2 (data not shown), and so the phenotype of this mutant is related to the loss of NS2 function rather than a decrease in protein abundance.…”

Section: Discussionmentioning

confidence: 99%

Replication of Minute Virus of Mice DNA Is Critically Dependent on Accumulated Levels of NS2

Choi

Newman

Burger

et al. 2005

J Virol

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Following transfection of murine fibroblasts, the lymphotropic strain of minute virus of mice (MVMi) does not efficiently produce progeny single-strand DNA (ssDNA). However, changing a single nucleotide in the MVMi 3 splice site to that found in the fibrotropic strain MVMp enabled full DNA replication and production of ssDNA. This change enhanced excision of the large intron and the production of NS2, likely by improving interaction, in fibroblasts with the branch point-binding U2 snRNA. One function of NS2 involves interaction with the nuclear export protein Crm1. The defect in production of MVMi ssDNA in fibroblasts can also be overcome by introducing a mutation in MVMi NS2 that enhances its interaction with Crm1. Although MVMi contains a 3 splice site that performs poorly in fibroblasts, MVMi generated at least as much R2 and NS2 in murine lymphocytes as did MVMp in fibroblasts. Therefore, it appears that MVMp has acquired a mutation that improves the excision of the large intron, as it adapted to fibroblasts to accommodate the need for NS2 for replication in these cells, and that the ratio of NS1 to NS2 may play a larger role in the host range of MVM than previously appreciated.Two strains of minute virus of mice, fibrotropic strain MVMp and lymphotropic strain MVMi, are reciprocally restricted for growth in murine cells (2,10,15,23,25,26). MVMp productively infects murine fibroblasts but not murine lymphocytes, while MVMi does the opposite. The major viral determinant of this host range resides within the capsid. The primary block to restrictive infections is intracellular, following binding to cells but before deposition of viral DNA in the nucleus (1,3,4,11,12,20).Characterization of MVMp and MVMi has also led to the identification of another determinant that affects viral replication in a host cell-dependent manner. Analysis of MVMi virus selected for growth on fibroblasts and analysis of the replication of engineered MVMi and MVMp chimeras on murine lymphocytes have demonstrated that a region encompassing the large-intron 3Ј splice site and P38 TATA box also plays a role in the efficiency of viral DNA replication in these two cell types (7, 12). The role that this region plays in replication has not been well characterized, but in both cases, differences in the ratio of mRNA R1 to R2, which encode NS1 and NS2, respectively, have been observed (4, 7, 11). The importance of the NS region in cell type-specific replication has also been noted for cells of neuronal origin (22).Previous studies in our lab have shown that even minor alterations of the MVM large-intron 3Ј splice site can have significant effects on the steady-state ratio of R1 to R2 and, hence, on the ratio of the viral nonstructural proteins NS1 and NS2 (19,28). Differences between MVMi and MVMp in this area have been previously noted, and ways in which these differences might affect RNA processing in the two strains have been discussed previously (19). Recently, two reports have In this study we have shown that, following transfection of mur...

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“…In particular, a bipartite enhancer consisting of CA-and purine-rich elements in the NS-2 specific exon mediate proper levels of this exon into the mRNA (Gersappe and Pintel, 1999). Although parvovirus alternative splicing does not seem to be regulated in a temporal fashion during infection, it may be regulated in a cell-type specific way and so have an impact on the tropism of parvoviruses.…”

Section: Parvovirus Infection and Replicationmentioning

confidence: 99%

Molecular and Structural Basis of the Evolution of Parvovirus Tropism

Tijssen¹

1999

Acta Veterinaria Hungarica

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CA- and Purine-Rich Elements Form a Novel Bipartite Exon Enhancer Which Governs Inclusion of the Minute Virus of Mice NS2-Specific Exon in Both Singly and Doubly Spliced mRNAs

Cited by 22 publications

References 61 publications

A Premature Termination Codon Interferes with the Nuclear Function of an Exon Splicing Enhancer in an Open Reading Frame-Dependent Manner

A Premature Termination Codon Interferes with the Nuclear Function of an Exon Splicing Enhancer in an Open Reading Frame-Dependent Manner

Replication of Minute Virus of Mice DNA Is Critically Dependent on Accumulated Levels of NS2

Molecular and Structural Basis of the Evolution of Parvovirus Tropism

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