High transcript abundance, RNA editing, and small RNAs in intergenic regions within the massive mitochondrial genome of the angiosperm Silene noctiflora

Zhiqiang Wu, James D. Stone, Helena Štorchová, Daniel B. Sloan

Species within the angiosperm genus Silene contain the largest mitochondrial genomes ever identified. The enormity of these genomes (up to 11 Mb in size) appears to be the result of increased non-coding DNA, which represents >99 % of the genome content. These genomes are also fragmented into dozens of circular-mapping chromosomes, some of which contain no identifiable genes, raising questions about if and how these 'empty' chromosomes are maintained by selection. To assess the possibility that they contain novel and unannotated functional elements, we have performed RNA-seq to analyze the mitochondrial transcriptome of Silene noctiflora. We identified regions of high transcript abundance in almost every chromosome in the mitochondrial genome including those that lack any annotated genes. In some cases, these transcribed regions exhibited higher expression levels than some core mitochondrial protein-coding genes. We also identified RNA editing sites throughout the genome, including 97 sites that were outside of protein-coding gene sequences and found in pseudogenes, introns, UTRs, and transcribed intergenic regions. Unlike in protein-coding sequences, however, most of these RNA editing sites were only edited at intermediate frequencies. Finally, analysis of mitochondrial small RNAs indicated that most were likely degradation products from longer transcripts, but we did identify candidates for functional small RNAs that mapped to intergenic regions and were not associated with longer RNA transcripts. Our findings demonstrate transcriptional activity in many localized regions within the extensive intergenic sequence content in the S. noctiflora mitochondrial genome, supporting the possibility that the genome contains previously unidentified functional elements. However, transcription by itself is not proof of functional importance, and we discuss evidence that some of the observed transcription and post-transcriptional modifications are non-adaptive. Therefore, further investigations are required to determine whether any of the identified transcribed regions have played a functional role in the proliferation and maintenance of the enormous non-coding regions in Silene mitochondrial genomes.