Controversially, the application of CRISPR/Cas9 for manipulation of mammalian mtDNA in human cells has been reported.

Although present in lower metazoans, the weight of evidence against an efficient endogenous RNA import mechanism in mammalian mitochondria is considerable.

Engineering of mammalian mtDNA has been hampered by an inability to import nucleic acids into mitochondria.

In recent years mitochondrial DNA (mtDNA) has transitioned to greater prominence across diverse areas of biology and medicine. The recognition of mitochondria as a major biochemical hub, contributions of mitochondrial dysfunction to various diseases, and several high-profile attempts to prevent hereditary mtDNA disease through mitochondrial replacement therapy have roused interest in the organellar genome. Subsequently, attempts to manipulate mtDNA have been galvanized, although with few robust advances and much controversy. Re-engineered protein-only nucleases such as mtZFN and mitoTALEN function effectively in mammalian mitochondria, although efficient delivery of nucleic acids into the organelle remains elusive. Such an achievement, in concert with a mitochondria-adapted CRISPR/Cas9 platform, could prompt a revolution in mitochondrial genome engineering and biological understanding. However, the existence of an endogenous mechanism for nucleic acid import into mammalian mitochondria, a prerequisite for mitochondrial CRISPR/Cas9 gene editing, remains controversial.

Biological understanding of complex organisms in the modern era relies heavily on reverse genetics. As an area of interest for many, a robust method for directed genetic manipulation of mammalian mitochondria has been sought for several decades. More recently, efforts to this end have largely focused on the search for treatments of mitochondrial disease. Incurable and largely intractable, mitochondrial diseases caused by mutation of the mitochondrial genome affect approximately one in 5000 and represent a substantial disease burden []. The dawn of the genome-editing era augurs well for both basic and clinical mitochondrial research, and the CRISPR/Cas9 revolution in particular seems to bring a paradigm shift within our grasp. However, fundamental questions regarding the capacity of mammalian mitochondria to import the(gRNA; see Glossary ) molecules needed for a viable CRISPR/Cas9 system cast doubt upon such an enterprise. Over recent years evidence against the notion of endogenous import of nucleus-encoded RNA into mammalian mitochondria has accrued. In this article we discuss the mitochondrial genetic system, evidence for and against endogenous RNA import into, and proposed functions within, mammalian mitochondria, and recent efforts towards genetic manipulation of mitochondria, including the controversial report of a mitochondrial CRISPR/Cas9 system.

Mammalian mitochondrial DNA (mtDNA) is a multi-copy, circular, double-stranded DNA molecule encoding 13 essential membrane-bound polypeptide subunits of the respiratory chain complexes I, III, IV, and ATP synthase, 22 tRNAs, and two ribosomal RNAs (rRNAs). At ∼16.5 kb, mammalian mtDNAs are relatively small and genetically compact, containing very little non-coding sequence and two overlapping genes []. The mitochondrial genome is packaged into individual nucleoids that consist principally of the mitochondrial transcription factor A (TFAM) [], but likely also contain other factors [], and these nucleoids are tightly associated with the IMM within the matrix. The mechanism by which mtDNA is replicated has, over the years, been no small matter of debate [], with recent data pointing towards the originally proposed strand-displacement mechanism []. Transcription of mtDNA occurs from thepromoter (HSP) and thepromoter (LSP), resulting in polycistronic transcripts that undergo substantial processing to yield the mature mRNA, tRNA, and rRNA molecules that are required for translation by mitochondrial ribosomes (mitoribosomes) []. A diverse array of DNA repair pathways exist in mammalian mitochondria [], with the notable absence of efficient DNA double-strand break (DSB) repair [], and either inefficient or absent homologous recombination (HR) []. The mitochondrial genome is, in mammals, strictly maternally inherited, demonstrating a more stochastic mode of transmission than Mendelian genetics as a consequence of the mtDNA]. Diseases arising from mutations in mtDNA most often present in astate, where a substantial proportion of mtDNA molecules bear a pathogenic mutation that is partially rescued by the presence of wild-type molecules in the same cell [].

Mutations causing mitochondrial disease: what is new and what challenges remain?.

No recombination of mtDNA after heteroplasmy for 50 generations in the mouse maternal germline.

Replication of mitochondrial DNA in mouse L cells and their thymidine kinase derivatives: displacement replication on a covalently-closed circular template.

Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.

From the initial alphaproteobacterial engulfment, that formed the first eukaryote through endosymbiosis, to the present-day organelle residing in mammalian cells, the relationship between mitochondria and their hosts has evolved substantially. Where once mitochondria-like symbionts were advantageous principally for their capacity to harness redox chemistries, the role of mitochondria in diverged eukaryotes, such as mammals, is much more intricately embedded in essential organismal function. Facilitation of these functions relies upon an electrochemical disequilibrium potential across the inner mitochondrial membrane (IMM) that is generated through proton pumping by respiratory chain complexes I, III, and IV. Taken together, the respiratory chain and ATP synthase consist of ∼90 protein subunits, forming IMM-bound protein complexes. The vast majority of these proteins are encoded in and expressed from the nuclear genome; however, a subset is encoded within a spatially and heritably separate genome – the mitochondrial genome.

A Role for Endogenous RNA Import in Mammalian Mitochondria

22 Suzuki T.

et al. Human mitochondrial tRNAs: biogenesis, function, structural aspects, and diseases. Figure 1 54 Borowski L.S.

et al. Human mitochondrial RNA decay mediated by PNPase-hSuv3 complex takes place in distinct foci. 61 Matilainen S.

et al. Defective mitochondrial RNA processing due to PNPT1 variants causes Leigh syndrome. 49 Brown A.

et al. Structure of the large ribosomal subunit from human mitochondria. 98 Greber B.J.

et al. Architecture of the large subunit of the mammalian mitochondrial ribosome. 31 Holzmann J.

et al. RNase P without RNA: identification and functional reconstitution of the human mitochondrial tRNA processing enzyme. 37 Jacobson M.R.

et al. Dynamic localization of RNase MRP RNA in the nucleolus observed by fluorescent RNA cytochemistry in living cells. 38 Kiss T.

Filipowicz W. Evidence against a mitochondrial location of the 7-2/MRP RNA in mammalian cells. 65 Mercer T.R.

et al. The human mitochondrial transcriptome. 66 Lightowlers R.N.

et al. Mitochondrial protein synthesis: figuring the fundamentals, complexities and complications, of mammalian mitochondrial translation. 99 Gold V.A.

et al. Visualization of cytosolic ribosomes on the surface of mitochondria by electron cryo-tomography. Overview of Putative RNA Import into Mitochondria. (A) An overview of historically proposed mechanisms and functional roles of endogenous RNAs imported into mammalian mitochondria. Nucleus-encoded RNA is suggested to enter the mitochondrial matrix in complex with polynucleotide phosphorylase (PNPase), via the mitochondrial protein translocase of outer membrane (TOM) and translocase of inner membrane (TIM), as well as by other undescribed and undefined mechanisms of transport. Endogenous RNA species with previously proposed functional roles in mammalian mitochondria are H1 RNA (RNase P), 7-2 RNA (RNase MRP), and 5S rRNA (mt-LSU). (B) A revised overview of the proposed mechanisms and functional roles of endogenous RNAs imported into mammalian mitochondria, modified to reflect findings from recent papers concerning (i) the function of PNPase as a key constituent of the mtRNA degradasome [ It is well-established that 11 protein-coding mRNAs, encoding 13 polypeptides of respiratory chain complexes and ATP synthase, are transcribed from the mitochondrial genome and translated by mitoribosomes. In placental mammals a full complement of 22 functional tRNA species capable of recognizing 60 sense codons, and two rRNAs that are required for translation by mitoribosomes, are also encoded in mtDNA. Considering the substantial structural differences between mitochondrial and cytosolic tRNAs, the divergence and incompatibility of codon usage between mitochondrial and nuclear mRNAs, the lack of unassigned codons in mitochondrial open reading frames (ORFs) [], and that all other mitochondrial proteins are encoded and expressed from the nuclear genome, any mRNA-decoding function for RNA imported into mitochondria is not immediately apparent. However, various other roles for endogenous, nuclear-encoded RNAs imported into mitochondria have been debated ( Figure 1 A,B).

23 Ojala D.

et al. tRNA punctuation model of RNA processing in human mitochondria. mitochondrial RNase P (mtRNase P). Both nuclear and mitochondrial RNases P liberate the 5′ ends of immature tRNA transcripts through structure-guided endonucleolytic processing. RNase P is an ancient enzyme, initially identified in bacteria, followed by eukaryotic nuclei and yeast mitochondria [ 24 Robertson H.D.

et al. Purification and properties of a specific Escherichia coli ribonuclease which cleaves a tyrosine transfer ribonucleic acid presursor. 25 Koski R.A.

et al. Identification of a ribonuclease P-like activity from human KB cells. 26 Hollingsworth M.J.

Martin N.C. RNase P activity in the mitochondria of Saccharomyces cerevisiae depends on both mitochondrion and nucleus-encoded components. H1 RNA) that is necessary for catalytic function [ 27 Guerrier-Takada C.

et al. The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. 28 Gold H.A.

Altman S. Reconstitution of RNAase P activity using inactive subunits from E. coli and HeLa cells. 29 Puranam R.S.

Attardi G. The RNase P associated with HeLa cell mitochondria contains an essential RNA component identical in sequence to that of the nuclear RNase P. 30 Rossmanith W.

Potuschak T. Difference between mitochondrial RNase P and nuclear RNase P. 31 Holzmann J.

et al. RNase P without RNA: identification and functional reconstitution of the human mitochondrial tRNA processing enzyme. 1R methyltransferase, TRMT10C (MRPP1), a member of the short-chain dehydrogenase/reductase (SDR) family, SDR5C1 (HSD17B10, MRPP2), and a protein with homology to PiIT N terminus (PIN) domain-like metallonucleases, PRORP (MRPP3) [ 31 Holzmann J.

et al. RNase P without RNA: identification and functional reconstitution of the human mitochondrial tRNA processing enzyme. 32 Vilardo E.

et al. A subcomplex of human mitochondrial RNase P is a bifunctional methyltransferase – extensive moonlighting in mitochondrial tRNA biogenesis. proteinaceous RNase P (PRORP) has since been identified in most eukaryal lineages ([ 33 Rossmanith W. Of P and Z: mitochondrial tRNA processing enzymes. 34 Lechner M.

et al. Distribution of ribonucleoprotein and protein-only RNase P in Eukarya. An unusual, bacteria-like feature of mammalian mitochondrial gene expression is the near-unit length polycistronic transcripts produced through transcription of mtDNA. Within the polycistrons, most gene products are punctuated by one or more tRNAs, which require endonucleolytic processing at both 5′ and 3′ ends to release individual transcripts, a concept termed ‘tRNA punctuation’ []. Essential to this process is(mtRNase P). Both nuclear and mitochondrial RNases P liberate the 5′ ends of immature tRNA transcripts through structure-guided endonucleolytic processing. RNase P is an ancient enzyme, initially identified in bacteria, followed by eukaryotic nuclei and yeast mitochondria []. Among several protein subunits, the nuclear (n)RNase P holoenzyme contains a single RNA subunit () that is necessary for catalytic function []. It was shown that mtDNA of some fungi and protists encode RNase P RNA, therefore it was assumed, and later controversially reported, that mammalian mtRNase P would require a catalytic RNA subunit to function []. However, it has since emerged that human mtRNase P, a product of convergent evolution, bears no relation to nRNase P []. Human mtRNase P consists of three protein subunits: a mitochondrially targeted tRNA mR methyltransferase, TRMT10C (MRPP1), a member of the short-chain dehydrogenase/reductase (SDR) family, SDR5C1 (HSD17B10, MRPP2), and a protein with homology to(PIN) domain-like metallonucleases, PRORP (MRPP3) []. Importantly, the human mtRNase P was shown specifically not to contain any trans-acting RNA. This paradigm-shifting subclass of(PRORP) has since been identified in most eukaryal lineages ([] for an in-depth review on the discovery and evolution of PRORPs).

RNase MRP). Similarly to nRNase P, RNase MRP possesses a RNA subunit (termed 7-2 RNA in the early literature) and several protein components, most of which are shared with nRNase P [ 35 Rosenblad M.A.

et al. Inventory and analysis of the protein subunits of the ribonucleases P and MRP provides further evidence of homology between the yeast and human enzymes. 36 Chang D.D.

Clayton D.A. A mammalian mitochondrial RNA processing activity contains nucleus-encoded RNA. 37 Jacobson M.R.

et al. Dynamic localization of RNase MRP RNA in the nucleolus observed by fluorescent RNA cytochemistry in living cells. 38 Kiss T.

Filipowicz W. Evidence against a mitochondrial location of the 7-2/MRP RNA in mammalian cells. 39 Goldfarb K.C.

Cech T.R. Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing. 40 Wanrooij P.H.

et al. G-quadruplex structures in RNA stimulate mitochondrial transcription termination and primer formation. A further controversy concerning mitochondrial RNA processing by imported endogenous RNAs concerns the nRNase P-related endonuclease, the mitochondrial RNA processing ribonuclease (). Similarly to nRNase P, RNase MRP possesses a RNA subunit (termedin the early literature) and several protein components, most of which are shared with nRNase P []. RNase MRP was first described as a ribonucleoprotein complex present in mitochondria that is involved in the formation of a RNA primer during initiation of mammalian mtDNA replication []. However, subsequent studies have provided compelling evidence against a mitochondrial localization of 7-2 RNA in mammalian cells, arguing that RNase MRP, like nRNase P, is found mainly in the nucleolus [] where it plays an essential role in pre-ribosomal RNA processing []. In addition, in vitro reconstitution experiments have suggested an RNase MRP-independent mechanism for primer processing in mtDNA replication, where the 3'-end of the RNA primer is generated by site-specific termination of transcription owing to G-quadruplex formation in nascent RNA, rather than cleavage by RNase MRP []. These findings point away from the requirement for non-mtDNA transcribed RNA to be present in mitochondria for RNA processing, suggesting that endogenous RNA import into mammalian mitochondria is not required for normal cellular functions.

5S rRNA that is found in ribosomes from other cellular compartments and organisms. Several groups have argued in favor of 5S rRNA being present in mammalian mitochondria [ 41 Yoshionari S.

et al. Existence of nuclear-encoded 5S-rRNA in bovine mitochondria. 42 Magalhaes P.J.

et al. Evidence for the presence of 5S rRNA in mammalian mitochondria. 43 Entelis N.S.

et al. 5S rRNA and tRNA import into human mitochondria. Comparison of in vitro requirements. 44 Smirnov A.

et al. Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria. 45 Smirnov A.

et al. Mitochondrial enzyme rhodanese is essential for 5S ribosomal RNA import into human mitochondria. 46 Smirnov A.V.

et al. Specific features of 5S rRNA structure – its interactions with macromolecules and possible functions. 47 Smirnov A.

et al. Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18. Phe or mt-tRNAVal, is embedded in the large subunit of the mammalian mitoribosome (mt-LSU), to the exclusion of any 5S rRNA molecule, and that would require substantial remodeling of the mitoribosomal central protuberance to accommodate 5S rRNA [ 48 Amunts A.

et al. The structure of the human mitochondrial ribosome. 49 Brown A.

et al. Structure of the large ribosomal subunit from human mitochondria. 50 Greber B.J.

et al. The complete structure of the 55S mammalian mitochondrial ribosome. 51 Greber B.J.

et al. The complete structure of the large subunit of the mammalian mitochondrial ribosome. Val leads to destabilization of this tRNA and a switch in the structural RNA content of mt-LSU from mt-tRNAVal to mt-tRNAPhe [ 52 Rorbach J.

et al. Human mitochondrial ribosomes can switch their structural RNA composition. 53 Chrzanowska-Lightowlers Z.

et al. Human mitochondrial ribosomes can switch structural tRNAs – but when and why?. Another area of debate concerning mitochondrial import of endogenous RNA in mammals focuses on the RNA content of mitoribosomes, specifically the existence of a minor structural rRNA species analogous to thethat is found in ribosomes from other cellular compartments and organisms. Several groups have argued in favor of 5S rRNA being present in mammalian mitochondria []. Key determinants of efficient 5S rRNA import are suggested to include specific RNA structural folds and protein cofactors [], and incorporation of 5S rRNA into the mitoribosome through interactions with proposed mitoribosomal protein MRPL18 has been described []. However, the notion of 5S rRNA incorporation within the mitoribosome has been categorically disregarded since publication of high-resolution structures of porcine and human mitoribosomes demonstrating that a mtDNA-encoded tRNA (mt-tRNA), either mt-tRNAor mt-tRNA, is embedded in the large subunit of the mammalian mitoribosome (mt-LSU), to the exclusion of any 5S rRNA molecule, and that would require substantial remodeling of the mitoribosomal central protuberance to accommodate 5S rRNA []. Further, it has been reported that a homoplasmic disease-causative point mutation in mt-tRNAleads to destabilization of this tRNA and a switch in the structural RNA content of mt-LSU from mt-tRNAto mt-tRNA]. These data raise questions regarding a physiological role for imported RNAs in mammalian mitoribosomes.

polynucleotide phosphorylase (PNPase). PNPase is a homotrimeric 3′–5′ exoribonuclease which, together with mitochondrial RNA-specific helicase, hSUV3, forms the RNA degradasome in the mitochondrial matrix [ 54 Borowski L.S.

et al. Human mitochondrial RNA decay mediated by PNPase-hSuv3 complex takes place in distinct foci. 55 Chujo T.

et al. LRPPRC/SLIRP suppresses PNPase-mediated mRNA decay and promotes polyadenylation in human mitochondria. 56 Wang G.

et al. PNPASE regulates RNA import into mitochondria. 57 Shepherd D.L.

et al. Exploring the mitochondrial microRNA import pathway through polynucleotide phosphorylase (PNPase). 54 Borowski L.S.

et al. Human mitochondrial RNA decay mediated by PNPase-hSuv3 complex takes place in distinct foci. 58 Piwowarski J.

et al. Human polynucleotide phosphorylase, hPNPase, is localized in mitochondria. 59 Antonicka H.

Shoubridge E.A. Mitochondrial RNA granules are centers for posttranscriptional RNA processing and ribosome biogenesis. 60 Rhee H.W.

et al. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. 61 Matilainen S.

et al. Defective mitochondrial RNA processing due to PNPT1 variants causes Leigh syndrome. 100 Rhee H.W.

et al. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. A factor suggested to directly facilitate endogenous RNA import into mammalian mitochondria is a component of the mitochondrial RNA degradation machinery,(PNPase). PNPase is a homotrimeric 3′–5′ exoribonuclease which, together with mitochondrial RNA-specific helicase, hSUV3, forms the RNA degradasome in the mitochondrial matrix []. However, an alternative function and localization of PNPase has been proposed. Detection of PNPase in the mitochondrial intermembrane space (IMS), rather than in the matrix, has led to suggestions that it could mediate mitochondrial matrix translocation of 5S rRNA, H1 RNA, 7-2 RNA, and more recently also microRNAs (miRNAs) by an uncharacterized mechanism []. This was surprising because PNPases, an ancient family of enzymes, had previously been found to reside in the matrix and to be involved in degradation of RNA, rather than in transport []. Interestingly, pathogenic compound heterozygous mutations in the PNPase gene (PNPT1), that were predicted to disrupt the homotrimer and therefore abolish any catalytic or transport function of PNPase, led to an accumulation of aberrantly processed mitochondrial RNA species within mitochondria, in line with the expectation of a role for PNPase in degradation of mitochondrial RNA []. Notably, the accumulated RNA intermediates were correctly processed at 5′ tRNA junctions, strongly suggesting that any mitochondrial import of H1 RNA by PNPase is dispensable for function of mitochondrial RNase P, as previously discussed. Given the consensus localization of PNPase in the mitochondrial matrix [], its well-described role in mitochondrial RNA degradation, the lack of a well-understood RNA import mechanism, and the likely dispensable role of RNAs it is alleged to transport, PNPase-mediated RNA import into mammalian mitochondria is not widely accepted, and requires further exploration and confirmation.

62 Zhang X.

et al. MicroRNA directly enhances mitochondrial translation during muscle differentiation. 63 Dietrich A.

et al. Organellar non-coding RNAs: emerging regulation mechanisms. 64 Rubio M.A.

et al. Mammalian mitochondria have the innate ability to import tRNAs by a mechanism distinct from protein import. In addition to the research concerning import of endogenous RNAs into mammalian mitochondria, discussed above, there also exists a less well interrogated literature suggesting both import and export of miRNAs [], long non-coding RNAs (lncRNAs) [], and tRNAs [] into and from mammalian mitochondria, which will not be discussed here because we believe this requires validation by independent studies.