Micro-processing events in mRNAs identified by DHPLC analysis.
Post-transcriptional processes such as alternative splicing and RNA editing have a huge impact on the diversity of the proteome. Detecting alternatively spliced transcripts is difficult when they are rare. In addition, edited transcripts often differ from the genomic sequence by only a few nucleotides. Denaturing high performance liquid chromatography (DHPLC) is routinely used for single nucleotide polymorphism detection and we used this method to detect alternatively spliced or edited transcripts. As the sites of RNA editing appear to be conserved within gene families, we investigated whether editing sites are conserved in the murine homologue of the Drosophila cacophony transcript encoding the alpha1 subunit of a voltage-gated calcium channel that is edited at 10 independent positions. Although DHPLC analysis detects RNA editing at as low as 3% in control transcripts, no evidence of RNA editing was found in the analysed murine transcript. However an alternative exon was identified at the 3' end of the mouse Cacna1alpha transcript and an alternative micro-exon encoding only two amino acids (NP) was found in the extracellular loop before the IVS4 helix in the same transcript. In the homologous Drosophila transcript a micro-exon also encoding two amino acids was found at the same position before the IVS4 helix.
Gallo
Angela
A
MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK.
Thomson
Emma
E
Brindle
James
J
O'Connell
Mary A
MA
Keegan
Liam P
LP
D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015220:Calcium Channels / Q000235:genetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004330:Drosophila / Q000235:genetics; D005091:Exons / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D017393:RNA Editing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D015220:Calcium Channels; D012333:RNA, Messenger