Limits...
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.

Show MeSH

Related in: MedlinePlus

RT-PCR analyses of splice products generated in vivo. (A) The structures of the potential splice products A to D are illustrated at the top, with the positions and orientations of the PCR primers (see Materials and Methods) from the four primer sets depicted below. The predicted origins and sizes of the amplified fragments are given at the right. (B) Shown are examples from each series of amplified RT-PCR products visualized on ethidium bromide-stained agarose gels. The employed primer sets (#1 to #4) are listed above the lanes. Size markers are indicated at the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1936429&req=5

Figure 4: RT-PCR analyses of splice products generated in vivo. (A) The structures of the potential splice products A to D are illustrated at the top, with the positions and orientations of the PCR primers (see Materials and Methods) from the four primer sets depicted below. The predicted origins and sizes of the amplified fragments are given at the right. (B) Shown are examples from each series of amplified RT-PCR products visualized on ethidium bromide-stained agarose gels. The employed primer sets (#1 to #4) are listed above the lanes. Size markers are indicated at the left.

Mentions: The observed effect of the mutated splice sites on the oncogenic properties advocates that RNA splicing by means of the alternative SA' and SD' sites does indeed take place in vivo. To clarify and confirm the identity of the produced transcripts, the splice pattern in tumor tissues (and for comparison in NIH 3T3 cells infected with the same four viruses) was analyzed. RNA from the individual end-stage tumors (or from virally infected cells) was isolated, and conventional RT-PCRs were performed with primers designed in such a way that it should be possible to identify all four potential splice products using 4 different primer sets as shown in Fig. 4A.


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)

RT-PCR analyses of splice products generated in vivo. (A) The structures of the potential splice products A to D are illustrated at the top, with the positions and orientations of the PCR primers (see Materials and Methods) from the four primer sets depicted below. The predicted origins and sizes of the amplified fragments are given at the right. (B) Shown are examples from each series of amplified RT-PCR products visualized on ethidium bromide-stained agarose gels. The employed primer sets (#1 to #4) are listed above the lanes. Size markers are indicated at the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: RT-PCR analyses of splice products generated in vivo. (A) The structures of the potential splice products A to D are illustrated at the top, with the positions and orientations of the PCR primers (see Materials and Methods) from the four primer sets depicted below. The predicted origins and sizes of the amplified fragments are given at the right. (B) Shown are examples from each series of amplified RT-PCR products visualized on ethidium bromide-stained agarose gels. The employed primer sets (#1 to #4) are listed above the lanes. Size markers are indicated at the left.
Mentions: The observed effect of the mutated splice sites on the oncogenic properties advocates that RNA splicing by means of the alternative SA' and SD' sites does indeed take place in vivo. To clarify and confirm the identity of the produced transcripts, the splice pattern in tumor tissues (and for comparison in NIH 3T3 cells infected with the same four viruses) was analyzed. RNA from the individual end-stage tumors (or from virally infected cells) was isolated, and conventional RT-PCRs were performed with primers designed in such a way that it should be possible to identify all four potential splice products using 4 different primer sets as shown in Fig. 4A.

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