Cannon Matthew V, Irwin Michael H, Pinkert Carl A
Department of Pathobiology, Auburn University, Auburn, AL, USA.
Drug Dev Res. 2015 Mar;76(2):61-71. doi: 10.1002/ddr.21241. Epub 2015 Apr 3.
The import of nuclear transcribed RNAs into mitochondria is an emerging area that presents a tremendous opportunity to develop human metabolic therapeutics. However, our knowledge base is quite limited. Much remains to be discovered regarding specific RNA localization and mechanisms of import. To identify novel RNAs imported into mitochondria, all RNAs within the mitochondria were characterized using next generation sequencing technology. Several nuclear transcribed RNAs were found within mitochondrial RNA (mtRNA) samples, including nuclear ribosomal RNAs, gamma satellite RNA and VL30 retroelement RNA. The presence of these RNAs within mitochondria coupled with RNA sequencing data from other laboratories investigating mtRNA processing, lead us to hypothesize that nuclease treatment of mitoplasts is insufficient for removing contaminating cytoplasmic RNAs. In contrast to traditional methodology, mitochondrial import was evaluated by qRT-PCR after stepwise removal of the outer mitochondrial membrane and subsequent lysis of mitochondria. This allowed identification of RNAs lost from the mitochondria with the same kinetics as mitochondrial DNA-transcribed RNAs. This approach provided an improved evaluation of nuclear RNA enrichment within mitochondrial membranes to characterize nuclease protection and mitochondrial import and identify false-positive detection errors. qRT-PCR results confirmed the presence of VL30 retroelement RNA within mitochondria and question the hypothesis that the RNA component of RNase P is imported. These results illustrate a reliable approach for evaluating the presence of RNAs within mitochondria and open new avenues of investigation relating to mtRNA biology and in targeting mitochondrial based therapeutics.
