miRNAs in mtDNA-less cell mitochondria

Abstract

The novel regulation mechanism in mtDNA-less cells was investigated. Very low mtDNA copy in mtDNA-less 206 ρ° cells was identified. But no 13 mitochondria-specific proteins were translated in 206 ρ° cells. Their mitochondrial respiration complexes V, III and II were 86.5, 29.4 and 49.6% of 143B cells, respectively. Complexes I and IV completely lack in 206 ρ° cells. Non-mitochondrial respiration to generate ATP in 206 ρ° cells was discovered. The expression levels of some mitochondrial RNAs including 12S rRNA, COX1, COX2, COX3, ND4 and ND5 were low. However, ND1, ND3 and Cyto b were not expressed in 206 ρ° cells. Unequal transcription of mitochondrial RNAs indicated the post-transcriptional cleavage and processing mechanisms in the regulation of mitochondrial gene expression in 206 ρ° cells. MicroRNAs (miRNAs) may modulate these mitochondrial RNA expression in these cells. RNA-induced silencing complex indeed within 206 ρ° cell mitochondria indicated miRNAs in 206 ρ° cell mitochondria. miRNA profile in mtDNA-less 206 ρ° cells was studied by next-generation sequencing of small RNAs. Several mitochondria-enriched miRNAs such as miR-181c-5p and miR-146a-5p were identified in 206 ρ° cell mitochondria. miR-181c-5p and miR-146a-5p had 23 and 19 potential targets on mitochondrial RNAs respectively, and these two miRNAs had multiple targets on mitochondria-associated messenger RNAs encoded by nuclear genes. These data provided the first direct evidence that miRNAs were imported into mitochondria and regulated mitochondrial RNA expressions.

Figure 1

The assay of mitochondrial RNA expression efficiency by RT-PCR. Left bands are from 143B mt-R RNA cDNA and right bands are from 143B-206 ρ° mt-R cDNA.

Figure 2

Mitochondrial function assay of 143B and 143B-206 ρ° cells by Seahorse XF 24 analyzer. The bioenergetic profiles of 143B and 143B-206 ρ° cells were generated by injecting electron transport chain inhibitors: oligomycin, FCCP and antimycin A to assess mitochondrial respiration. The results indicated mitochondrial dysfunction in 143B-206 ρ° cells. Non-mitochondrial respiration was kept in 143B-206 ρ° cells.

Figure 3

Pure mitochondria, Ago2 within mitochondria. (a) Northern blotting was performed by using mt tRNA Asn probe and cytosolic tRNA Asn probe to check C-p, mt and mt-R, which indicated that mitochondria were pure, and cyto tRNA was not present in mt-R. (b) Western blot results confirmed the presence of Ago2 within 143B and 206 ρ° mitochondria (mt and mt-R samples).

Figure 4

(a) Hotmap of mitochondria-enriched miRNAs. 30=143B mt-R RNA, 28=206 ρ° mt-R RNA, 29=143B C-p RNA, 27=206 ρ° C-p RNA and 35=206 ρ° mt RNA. (b) RT-qPCR results confirmed miR-181c-5p (miR-181c), miR-146a-5p were highly enriched in 206 ρ° mitochondria (mt-R), the control miR-423-5p was not enriched in 206 ρ° mitochondria. 5S was as an internal control for quantification.