Aberrant splicing events that are induced by proviral integration: implications for myb oncogene activation.

Abstract

Activation of the mouse c-myb oncogene in Abelson virus-induced plasmacytoid lymphosarcomas was studied using cDNA cloning and nucleotide sequence analysis. The results presented here show that viral integration in the myb locus generates splicing errors at the 5' and 3' regions. Viral integration results in transcriptional initiation within the viral long terminal repeat and generation of a chimeric mRNA that lacks the rst three coding exons. The alterations at the 3' end are caused by an aberrant splicing event in which additional splice-donor and -acceptor sequences within intronic sequences are used to splice an additional 363 nucleotides into the myb transcripts. The resulting insertion of 121 amino acids is in a region of the protein where other activated forms of the myb gene product have deletions. These results suggest that alterations in the 3' end of the myb gene play a crucial role in the activation of this gene.A comparative study of retroviral oncogenes and their cellular homologues has given us invaluable information regarding the structural changes that protooncogenes often undergo to acquire transforming potential. A comparison of the c-myb cDNA sequence (1) with the v-myb sequence of avian myeloblastosis virus (AMV) (2, 3) and E26 leukemia virus (4) revealed that large stretches of coding sequences from the 5' and 3' ends have been deleted during the transduction of this gene into the viral genome. The AMV myb gene is derived from seven of the internal coding exons of the c-myb gene (3, 5) and has undergone deletions of 142 amino acids at its N-terminal end and 192 amino acids at its C-terminal end (1). Similar deletions have occurred in the E26 virus, in which myb is coded as part of a fusion polypeptide of three distinct elements, the viral gag sequences, the viral myb, and a third element derived from the protooncogene ets (4). Deletions of similar stretches of sequences in two independent viral isolates suggest that these deletions are important for the oncogenic activation of the myb gene.Another model system that has allowed us to study the activation of the myb gene is the Abelson leukemia virusinduced plasmacytoid lymphosarcomas (ABPL tumors) of the mouse (6). Detailed analysis ofthese tumors revealed that they do not contain the integrated Abelson leukemia virus genome, but instead have undergone rearrangements in their myb locus as a result of Moloney murine leukemia virus (Mo-MuLV) integration (5,7,8).To study the effect of the viral integration on the transcription of the rearranged myb locus in ABPL tumors, we have undertaken the cDNA cloning of myb mRNA from one of the ABPL tumors, ABPL-2, and determined its sequence. Our results show that the viral integration results in aberrant splicing events at both the 5' and 3' ends of the mRNA transcripts producing alterations in similar positions to those seen in AMV and E26 virus.

Alternative splicing of RNAs transcribed from the human c-myb gene.

“…The 4.4-kb mRNA species in the other cell types was detected in the autoradiograph after 18 h of exposure as a faint smear just above the 4.0-kb band. This is consistent with the previous finding that the steady-state level of E6A-containing myb transcripts was much lower than that of the major c-myb transcript in mouse cells (34,39 expressed in Jurkat cells, suggesting that other myb-related transcripts are present in these cells. No E7A sequence was detected in the murine NFS467 cells.…”

“…An additional in-frame exon, termed E6A, was first identified in cDNA clones isolated from c-myb transcripts derived from one of the murine myeloid tumors that carries a provirus-disrupted c-myb allele (34). It was initially proposed that the alternative exon was used as a consequence of upstream proviral insertion, but subsequent analyses showed that the E6A exon is also present in myb transcripts expressed in normal and tumor cells which lack c-myb rearrangements (39).…”

Molecular Mechanisms Associated with the Regulation of Apoptosis by the Two Alternatively Spliced Products of c-Myb

“…This 89-kDa protein is translated from an alternatively spliced mRNA encoded by the c-myb gene, which results in the addition of 363 bp between exons 9 and 10. This region has been designated exon 9A (50,55).…”

Hematopoietic lineage-specific heterogeneity in the 5'-terminal region of the chicken proto-myb transcript.

“…Alternative splicing further downstream between exons 6 and 7 of the murine c-myb transcript has been observed in normal thymus and spleen cells (34) as well as in myeloid tumor cells induced by proviral integration within the 5'-terminal region of the c-myb gene (31).…”

“…Also, the chicken and murine c-myb genes are targets for proviral insertions that lead to hematopoietic malignancies of myeloid or B-cell origin (15,19,31,35).…”