Cryptic splice site activation during RNA processing of MLL/AF4 chimeric transcripts in infants with t(4;11) positive ALL.
Co-expression of multiple variants of the MLL/AF4 fusion transcript is a common phenomenon in patients with acute lymphoblastic leukemia (ALL) with t(4;11)(q21;q23). Different transcriptional and post-transcriptional mechanisms were found to contribute to the heterogeneity of the chimeric transcripts. Multiple splice variants are generated by utilizing alternative splice sites that result in the joining of different MLL-exons within the breakpoint cluster region to one of three exons in the AF4 fusion partner. To address the question of how splice site selection occurs during RNA processing, we investigated der(11) transcripts in 10 infants with t(4;11) positive ALL. Specific RT-PCR products were analyzed by Southern blot hybridization, SSCP, endonuclease digestion, cloning and sequencing. In patients co-expressing as many as six different chimeric mRNA species, activation of cryptic splice sites has been detected in MLL-exons 8 and 10. This led to the formation of four novel transcript variants, three of which maintained open reading frames (ORFs). Patients with cryptic donor site activation in MLL-exon 8 did not have any MLL-exon 8/AF4 transcripts using the authentic 5' splice site, although this site is 100% homologous to the consensus sequence. However, since MLL-exon 8 does not end in-phase, the use of the authentic splice site would result in loss of the ORF of the fusion message. The activated cryptic splicing sites are located in the vicinity of the polypurine stretches present in MLL-exons 8 and 10, which are known to function as splicing enhancers recognized by SR proteins. We postulate that both the nonsense-mediated decay eliminating correctly spliced MLL-exon 8/AF4 mRNAs and activation of suboptimal splicing sites contribute to the diversity of MLL/AF4 RNA species.
Divoky
V
V
Children's Cancer Research Institute (CCRI), St. Anna Children's Hospital, Kinderspitalgasse 6, A-1090, Wien, Austria.
Trka
J M
JM
Watzinger
F
F
Lion
T
T
D017398:Alternative Splicing / Q000235:genetics*; D001665:Binding Sites; D019457:Chromosome Breakage; D002880:Chromosomes, Human, Pair 11 / Q000235:genetics*; D002894:Chromosomes, Human, Pair 4 / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D005091:Exons; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D018740:Genetic Heterogeneity; D014644:Genetic Variation; D011495:Histone-Lysine N-Methyltransferase; D006801:Humans; D007223:Infant; D007438:Introns; D009154:Mutation; D051788:Myeloid-Lymphoid Leukemia Protein; D009363:Neoplasm Proteins / Q000235:genetics; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D016366:Open Reading Frames; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics*; D011519:Proto-Oncogenes; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors; D014158:Transcription, Genetic; D014178:Translocation, Genetic; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C497422:KMT2A protein, human; C113855:MLL-AF4 fusion protein, human; D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; D012333:RNA, Messenger; D014157:Transcription Factors; D051788:Myeloid-Lymphoid Leukemia Protein; D011495:Histone-Lysine N-Methyltransferase