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Impairment of alternative splice sites defining a novel gammaretroviral exon within gag modifies the oncogenic properties of Akv murine leukemia virus.

Sørensen AB, Lund AH, Kunder S, Quintanilla-Martinez L, Schmidt J, Wang B, Wabl M, Pedersen FS - Retrovirology (2007)

Bottom Line: Interestingly, a broader spectrum of diagnoses was made from the two single splice-site mutants than from as well the wild-type as the double splice-site mutant.Both single- and double-spliced transcripts are produced in vivo using the SA' and/or the SD' sites, but the mechanisms underlying the observed effects on oncogenesis remain to be clarified.Likewise, analyses of provirus integration sites in tumor tissues, which identified 111 novel RISs (retroviral integration sites) and 35 novel CISs (common integration sites), did not clearly point to specific target genes or pathways to be associated with specific tumor diagnoses or individual viral mutants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, University of Aarhus, Denmark. abs@statsbiblioteket.dk

ABSTRACT

Background: Mutations of an alternative splice donor site located within the gag region has previously been shown to broaden the pathogenic potential of the T-lymphomagenic gammaretrovirus Moloney murine leukemia virus, while the equivalent mutations in the erythroleukemia inducing Friend murine leukemia virus seem to have no influence on the disease-inducing potential of this virus. In the present study we investigate the splice pattern as well as the possible effects of mutating the alternative splice sites on the oncogenic properties of the B-lymphomagenic Akv murine leukemia virus.

Results: By exon-trapping procedures we have identified a novel gammaretroviral exon, resulting from usage of alternative splice acceptor (SA') and splice donor (SD') sites located in the capsid region of gag of the B-cell lymphomagenic Akv murine leukemia virus. To analyze possible effects in vivo of this novel exon, three different alternative splice site mutant viruses, mutated in either the SA', in the SD', or in both sites, respectively, were constructed and injected into newborn inbred NMRI mice. Most of the infected mice (about 90%) developed hematopoietic neoplasms within 250 days, and histological examination of the tumors showed that the introduced synonymous gag mutations have a significant influence on the phenotype of the induced tumors, changing the distribution of the different types as well as generating tumors of additional specificities such as de novo diffuse large B cell lymphoma (DLBCL) and histiocytic sarcoma. Interestingly, a broader spectrum of diagnoses was made from the two single splice-site mutants than from as well the wild-type as the double splice-site mutant. Both single- and double-spliced transcripts are produced in vivo using the SA' and/or the SD' sites, but the mechanisms underlying the observed effects on oncogenesis remain to be clarified. Likewise, analyses of provirus integration sites in tumor tissues, which identified 111 novel RISs (retroviral integration sites) and 35 novel CISs (common integration sites), did not clearly point to specific target genes or pathways to be associated with specific tumor diagnoses or individual viral mutants.

Conclusion: We present here the first example of a doubly spliced transcript within the group of gammaretroviruses, and we show that mutation of the alternative splice sites that define this novel RNA product change the oncogenic potential of Akv murine leukemia virus.

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Related in: MedlinePlus

Location of the trapped exon. Upper panel shows the structure of proviral Akv MLV DNA with the positions of the splice sites indicated (SD; splice donor, SA; splice acceptor). Arrows signify the PCR primers used to verify the stability of the introduced mutations. Lower panel shows the positions and types of the introduced mutations, marked by asterisks and underlined. The SA'/SD'-delineated exon is indicated by the box. The boldfaced A in the sequence indicates the presumed branch point.
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Figure 1: Location of the trapped exon. Upper panel shows the structure of proviral Akv MLV DNA with the positions of the splice sites indicated (SD; splice donor, SA; splice acceptor). Arrows signify the PCR primers used to verify the stability of the introduced mutations. Lower panel shows the positions and types of the introduced mutations, marked by asterisks and underlined. The SA'/SD'-delineated exon is indicated by the box. The boldfaced A in the sequence indicates the presumed branch point.

Mentions: In order to identify potential alternative splice donor and splice acceptor sites in Akv MLV, exon-trapping was performed using the exon-trapping vector pSPL3 (see Materials and Methods). In short, an exon resulting from usage of the alternative splice acceptor (SA') and either one of two alternative splice donor (SD' or SD*) sites located in the capsid region of gag (Fig. 1), was isolated and verified by RT-PCR analyses of RNA isolated from Akv MLV infected cells (data not shown). The size of the exon is 235 bp or 180 bp, depending on the splice donor site used.


Impairment of alternative splice sites defining a novel gammaretroviral exon within gag modifies the oncogenic properties of Akv murine leukemia virus.

Sørensen AB, Lund AH, Kunder S, Quintanilla-Martinez L, Schmidt J, Wang B, Wabl M, Pedersen FS - Retrovirology (2007)

Location of the trapped exon. Upper panel shows the structure of proviral Akv MLV DNA with the positions of the splice sites indicated (SD; splice donor, SA; splice acceptor). Arrows signify the PCR primers used to verify the stability of the introduced mutations. Lower panel shows the positions and types of the introduced mutations, marked by asterisks and underlined. The SA'/SD'-delineated exon is indicated by the box. The boldfaced A in the sequence indicates the presumed branch point.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1936429&req=5

Figure 1: Location of the trapped exon. Upper panel shows the structure of proviral Akv MLV DNA with the positions of the splice sites indicated (SD; splice donor, SA; splice acceptor). Arrows signify the PCR primers used to verify the stability of the introduced mutations. Lower panel shows the positions and types of the introduced mutations, marked by asterisks and underlined. The SA'/SD'-delineated exon is indicated by the box. The boldfaced A in the sequence indicates the presumed branch point.
Mentions: In order to identify potential alternative splice donor and splice acceptor sites in Akv MLV, exon-trapping was performed using the exon-trapping vector pSPL3 (see Materials and Methods). In short, an exon resulting from usage of the alternative splice acceptor (SA') and either one of two alternative splice donor (SD' or SD*) sites located in the capsid region of gag (Fig. 1), was isolated and verified by RT-PCR analyses of RNA isolated from Akv MLV infected cells (data not shown). The size of the exon is 235 bp or 180 bp, depending on the splice donor site used.

Bottom Line: Interestingly, a broader spectrum of diagnoses was made from the two single splice-site mutants than from as well the wild-type as the double splice-site mutant.Both single- and double-spliced transcripts are produced in vivo using the SA' and/or the SD' sites, but the mechanisms underlying the observed effects on oncogenesis remain to be clarified.Likewise, analyses of provirus integration sites in tumor tissues, which identified 111 novel RISs (retroviral integration sites) and 35 novel CISs (common integration sites), did not clearly point to specific target genes or pathways to be associated with specific tumor diagnoses or individual viral mutants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, University of Aarhus, Denmark. abs@statsbiblioteket.dk

ABSTRACT

Background: Mutations of an alternative splice donor site located within the gag region has previously been shown to broaden the pathogenic potential of the T-lymphomagenic gammaretrovirus Moloney murine leukemia virus, while the equivalent mutations in the erythroleukemia inducing Friend murine leukemia virus seem to have no influence on the disease-inducing potential of this virus. In the present study we investigate the splice pattern as well as the possible effects of mutating the alternative splice sites on the oncogenic properties of the B-lymphomagenic Akv murine leukemia virus.

Results: By exon-trapping procedures we have identified a novel gammaretroviral exon, resulting from usage of alternative splice acceptor (SA') and splice donor (SD') sites located in the capsid region of gag of the B-cell lymphomagenic Akv murine leukemia virus. To analyze possible effects in vivo of this novel exon, three different alternative splice site mutant viruses, mutated in either the SA', in the SD', or in both sites, respectively, were constructed and injected into newborn inbred NMRI mice. Most of the infected mice (about 90%) developed hematopoietic neoplasms within 250 days, and histological examination of the tumors showed that the introduced synonymous gag mutations have a significant influence on the phenotype of the induced tumors, changing the distribution of the different types as well as generating tumors of additional specificities such as de novo diffuse large B cell lymphoma (DLBCL) and histiocytic sarcoma. Interestingly, a broader spectrum of diagnoses was made from the two single splice-site mutants than from as well the wild-type as the double splice-site mutant. Both single- and double-spliced transcripts are produced in vivo using the SA' and/or the SD' sites, but the mechanisms underlying the observed effects on oncogenesis remain to be clarified. Likewise, analyses of provirus integration sites in tumor tissues, which identified 111 novel RISs (retroviral integration sites) and 35 novel CISs (common integration sites), did not clearly point to specific target genes or pathways to be associated with specific tumor diagnoses or individual viral mutants.

Conclusion: We present here the first example of a doubly spliced transcript within the group of gammaretroviruses, and we show that mutation of the alternative splice sites that define this novel RNA product change the oncogenic potential of Akv murine leukemia virus.

Show MeSH
Related in: MedlinePlus