An efficient transcript end mapping tool, Hiten, uncovers the functions of Z m RN ase II and Z m PPR 67 in organellar RNA processing and stability in maize

RNA end maturation and stabilization are crucial for plant organellar gene expression, yet the mechanisms remain elusive, partially due to the lack of efficient RNA end mapping methods. We developed a high-throughput transcript end mapping tool (Hiten) by integrating in vitro RNA circularization, next-generation sequencing, and circRNA identification algorithm MeCi. Using Hiten, we systematically mapped 5' and 3' ends of organellar mRNAs and noncoding RNAs (ncRNAs) and characterized organellar polyadenosine tails in maize (Zea mays). Combining RNA 5'-polyphosphatase treatment with Hiten demonstrates that transcription initiation plays a major role in 5'-end formation of mRNAs and ncRNAs in chloroplasts and mitochondria. Furthermore, Hiten was used to identify the RNA substrates of chloroplast- and mitochondrion- dual-localized ZmRNase II and mitochondria-targeted ZmPPR67. The results show that almost all chloroplast mRNAs and ncRNAs, and mitochondrial atp1 and atp4 mRNAs carry short 3'-extensions when ZmRNase II is mutated. In the Zmppr67 mutant, 5' end-truncated atp9 mRNAs are accumulated, accompanied by a significant reduction in mature atp9 mRNA levels. This study introduces an efficient tool for mapping organellar RNA ends and screening organellar RNA substrates and reveals that ZmRNase II predominantly functions in chloroplast RNA 3'-end maturation, whereas ZmPPR67 stabilizes mitochondrial atp9 mRNA by protecting its 5' end.