Fig. 2: miR-805 is an mtDNA-encoded non-coding RNA.

a Alignment of the predicted miR-805 to the mouse mitochondrial genome. The last row depicts the sequence obtained by RNA-sequencing analysis. b, c MLE12 cells were exposed or not to 10% CSE; cytosolic and mitochondrial extracts were generated. Fractions were analyzed for b cytosolic protein lactate dehydrogenase A (LDHA) and mitochondrial protein succinate dehydrogenase subunit A (SDHA) and c the expression levels of miR-805. d Schematic representation of the mito-ncR-805 genomic location. The circular mtDNA with the heavy (H) strand in dark purple, the light (L) strand in light purple, and the LSP indicated by the black arrow. A portion of the mtDNA control region near the LSP is shown with the H-strand nucleotide sequence. The LSP transcription initiation start site is indicated. The 5′-end of mito-ncR-805 (blue) maps one nucleotide downstream of the LSP transcription initiation site and the 3′-end maps within the conserved sequence block (CSB) III (orange box). mito-ncR-805 appears to be a product of the LSP promoter transcription. CSB II with a G-quartet important for transcription pausing and R-loop formation is also indicated (orange box). e Control (non-targeting), Ago2-, and Dicer-specific shRNA MLE12 cells, as well as rho⁰ MLE12 cells were treated with 10% CSE for the indicated times. Small RNA-enriched fractions were resolved on a 15% urea-gel and hybridized with probes complementary to miR-805 and to U6 (loading control). f Transcription profile of mitochondrial transcripts generated from 16,000–16,210 region of mtDNA. g Summary of frequency of reads of the 70-bp transcript. h Expression levels of mito-ncR-805 using two sets of primers, directed against the 5′ end (first 20 bp of mito-ncR-805), or directed against the 3′-end of mito-ncR-805 (30–55 bp of mito-ncR-805). i Alignment of all the sequence reads against the entire mouse nuclear and mitochondrial genomes. j–k rho⁰ MLE12 cells were obtained by growing cells in the presence of ethidium bromide added fresh daily for 2 passages. i DNA was extracted and mitochondrial copy number was determined (Control 100%, rho⁰ 4%). k mito-ncR-805 expression levels in MLE12 control and rho⁰ cells.