RNA processing and multiple transcription initiation sites result in transcript size heterogeneity in maize mitochondria

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Abstract
Variation in the length of the 5′ non-coding region of mitochondrial gene transcripts could result from multiple transcription initiation sites or post-transcriptional processing events. To distinguish between these possibilities, we have utilized the in vitro capping reaction catalyzed by guanylyl transferase to specifically label the 5′ end of primary, unprocessed transcripts. Hybridization of in vitro capped mtRNA to immobilized DNA from the 5′ flanking regions of 26 S, 18 S and 5 S rRNA genes and two proteincoding genes, ATP synthase subunit 9 (atp9) and apocytochrome b (cob), identified regions where transcription initiates. Single-strand specific RNase treatment of in vitro capped RNA hybridized to immobilized DNA containing the 5′ flanking sequences from cob and atp9 suggests that these genes have multiple transcription initiation sites. Direct mapping of transcription initiation sites for the rRNA genes indicated that single major transcription initiation sites exist at approximately 180 and 230 nucleotides upstream from the mature 26 S and 18+5 S rRNA genes, respectively. Labeling of processed transcripts bearing a 5′ hydroxyl moiety with T4 polynucleotide kinase and subsequent hybridization to the rRNA genes indicated that the mature forms of the rRNA are processed.