Major contribution of transcription initiation to 5’-end formation of mitochondrial steady-state transcripts in maize

Abstract

ABSTRACTIn plant mitochondria, some steady-state transcripts contain primary 5ʹ ends derived from transcription initiation, while the others have processed 5ʹ termini generated by post-transcriptional processing. Differentiation and mapping of the primary and processed transcripts are important for unraveling the molecular mechanism(s) underlying transcription and transcript end maturation. However, previous efforts to systematically differentiate these two types of transcripts in plant mitochondria failed. At present, it is considered that the majority of mature mRNAs may have processed 5ʹ ends in Arabidopsis. Here, by combination of circular RT-PCR, quantitative RT-PCR, RNA 5ʹ-polyphosphatase treatment and Northern blot, we successfully discriminated and mapped the primary and processed transcripts in maize mitochondria. Among the thirty-five mature and eight precursor RNAs analyzed in this study, about one half (21/43) were found to have multiple isoforms. In total, seventy-seven steady-state transcripts were determined, and forty-seven of them had primary 5ʹ ends. Most transcription initiation sites (126/167) were downstream of a crTA-motif. These data suggested a major contribution of transcription initiation to 5ʹ-end formation of steady-state transcripts in maize mitochondria. Moreover, the mapping results revealed that mature RNA termini had largely been formed before trans-splicing, and C→U RNA editing was accompanied with trans-splicing and transcript end formation in maize mitochondria.