Journal of Heredity Advance Access originally published online on May 19, 2006
Journal of Heredity 2006 97(3):253-260; doi:10.1093/jhered/esj037
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Noncanonical RNAs From Transcripts of the Drosophila muscleblind Gene
From the Department of Genetics, University of Valencia, Doctor Moliner 50, 46100 Burjasot, Valencia, Spain (Garcia-Casado, Pascual, Paricio, and Artero); and Institute of Biomedical and Life Sciences, University of Glasgow, Anderson College Complex, 56 Dumbarton Road, Glasgow G11 6NU, UK (Houseley, O'Dell, and Monckton). Jonathan M. Houseley is now at The Wellcome Trust Centre for Cell Biology, Michael Swann Building, Mayfield Road, Edinburgh EH9 3JR, UK; and Zaida Garcia-Casado is now at the Department of Haematology, Hospital Universitario La Fe, Valencia, Spain
Address correspondence to R. D. Artero at the address above, or e-mail: ruben.artero{at}uv.es.
It has become increasingly evident that eukaryotic cells produce RNA molecules from coding genes with constitutions other than those of typically spliced mRNA transcripts. Here we describe new cDNAs from the Drosophila melanogaster muscleblind (mbl) locus that identify two such atypical RNA molecules: RNAs containing an incomplete exon 2 tandem repetition (mblE2E2') or having exons with a different order compared to the corresponding genomic DNA (mblE2E3'E2'; exon scrambling). The existence of exon duplications and rearrangements in the genomic locus that might explain such cDNAs was ruled out by genomic Southern blotting and in silico analysis of the Drosophila genome sequence. The incomplete exon 2 tandem repetition was confirmed by sequencing reverse transcriptase-polymerase chain reaction (RT-PCR) products, rapid amplification of cDNA ends, and detection of a band consistent with cDNA sizes in total RNA northern blots. RT-PCRs with exon-specific primers downstream of exon 2 were unable to amplify products other than those expected from canonical mbl isoforms, thus indicating that no other exons were efficiently spliced downstream of exon 2. Moreover, mblE2E2' transcripts seem to be poorly polyadenylated, if at all, and behave aberrantly in a polyacrylamide gel electrophoresis (PAGE) mobility assay. Taken together, lack of polyadenylation, lack of downstream splicing events, small size of mblE2E2', and PAGE behavior all suggest that these noncanonical transcripts may be circular RNAs. The functional implications for these noncanonical transcripts are unclear. A developmental expression profile of mblE2E2' revealed an almost constant expression except during early embryogenesis and early adulthood. The protein putatively encoded is unlikely to be functional because an in-frame stop codon occurs almost immediately after the splice site. Such noncanonical transcripts have previously been observed in vertebrates, and these data provide the first experimental evidence for similar phenomena in invertebrates.