RT–PCR analysis of 5′ to 3′-end-ligated mRNAs identifies the extremities of cox2 transcripts in pea mitochondria

Gene expression in plant mitochondria is still inadequately analyzed. To learn more about transcription and RNA processing in plant mitochondria, the 5′- and 3′-RNA extremities and the promoters of the cytochrome oxidase gene (cox2) were analyzed in pea. Both 5′ and 3′ ends of cox2 transcripts were examined by RT–PCR across the ligation site of circularized mitochondrial RNA as template. This approach identified 5′ ends a few nucleotides shorter than three major 5′ ends mapped by primer extension analysis. Presumably, only monophosphate 5′ ends derived from processing can be ligated. In vitro transcription assays using a homologous mitochondrial protein extract from pea strongly suggest the major 5′ ends to derive from transcription initiation. The cDNA analysis of the head-to-tail ligated cox2 mRNA identified 3′ ends within a thymidine stretch ∼300 nt downstream of the reading frame in a sequence segment that was not present in the previous investigation of this gene. Nuclease S1 protection experiments confirmed this newly identified 3′ terminus and corroborated the validity of this technique in mRNA end analysis. The general use of the circularized RNA (CR)–RT–PCR approach for the simultaneous analysis of the 5′ and 3′ extremities of mRNA molecules is discussed.