Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

In plant mitochondria, some steady-state transcripts have 5' triphosphate derived from transcription initiation (primary transcripts), while the others contain 5' monophosphate generated post-transcriptionally (processed transcripts). To discriminate between the two types of transcripts, several strategies have been developed, and most of them depend on presence/absence of 5' triphosphate. However, the triphosphate at primary 5' termini is unstable, and it hinders a clear discrimination of the two types of transcripts. To systematically differentiate and map the primary and processed transcripts stably accumulated in maize mitochondrion, we have developed a circular RT-PCR (cRT-PCR)-based strategy by combining cRT-PCR, RNA 5' polyphoshpatase treatment, quantitative RT-PCR (RT-qPCR), and Northern blot. As an improvement, this strategy includes an RNA normalization step to minimize the influence of unstable 5' triphosphate.In this protocol, the enriched mitochondrial RNA is pre-treated by RNA 5' polyphosphatase, which converts 5' triphsophate to monophosphate. After circularization and reverse transcription, the two cDNAs derived from 5' polyphosphatase-treated and non-treated RNAs are normalized by maize 26S mature rRNA, which has a processed 5' end and is insensitive to 5' polyphosphatase. After normalization, the primary and processed transcripts are discriminated by comparing cRT-PCR and RT-qPCR products obtained from the treated and non-treated RNAs. The transcript termini are determined by cloning and sequencing of the cRT-PCR products, and then verified by Northern blot.By using this strategy, most steady-state transcripts in maize mitochondrion have been determined. Due to the complicated transcript pattern of some mitochondrial genes, a few steady-state transcripts were not differentiated and/or mapped, though they were detected in a Northern blot. We are not sure whether this strategy is suitable to discriminate and map the steady-state transcripts in other plant mitochondria or in plastids.