*6.2. mtDNA Editing*

As mitochondria are organelles separated with double membranes that harbor strict import machinery, for a long time, it has seemed impossible to stably edit mtDNA in vertebrates. While proteins can e fficiently be targeted and delivered into mitochondria, nucleic acid uptake remains controversial. As such, import of the nucleic acid component for mtDNA transformation and editing appeared to be a hurdle [176]. This di fficulty can be overcome by the use of protein-only nucleases that can e fficiently be engineered to target the mitochondrial matrix and induce sequence-specific double-strand breaks in mtDNA molecules [177]. This strategy takes advantage of mtDNA heteroplasmy and is based on the cleavage of mtDNA molecules harboring certain sequences (for example, pathological mutations). As a double-strand break repair pathway is not present in mitochondria, cleaved mtDNA molecules are rapidly degraded [178–180]. Remaining, noncleaved mtDNA molecules with normal sequences can then be replicated to reconstitute mtDNA content [181–184]. Two DNA editing platforms were introduced into mitochondria: transcription activator-like e ffector nucleases (TALEN) [185] and zing-finger nucleases (ZFN) [183]; these platforms enabled e fficient heteroplasmy shift. In 2018, two breakthrough studies published back-to-back by the Minczuk and Moraes labs showed successful mtDNA editing in vivo [186,187]. In a mouse model of heteroplasmic mitochondrial disease, mitochondrially targeted ZFN [186] and TALENs [187] delivered by the adeno-associated virus led to the successful elimination of mutated mtDNA and subsequent reversion of the pathological phenotype [186,187]. These studies may open a new era for the treatment of heteroplasmic mitochondrial diseases.

#### *6.3. RNA Import into Mitochondria*

As mentioned earlier, nucleic acid import into mammalian mitochondria arouses controversy. First, it is not fully clear which RNA molecules can be transported into mitochondria; second, the machinery responsible for this process is debated. PNPase, a component of the mitochondrial degradosome complex, was proposed to play a role in RNA import into mitochondria [96,188]. Indeed, a pool of PNPase is found in the mitochondrial intermembrane space [94,96,189], where it could take part in importing RNA. Nevertheless, the exact mechanism of this process is not known, and it is not certain whether PNPase plays this role alone or cooperates with other proteins. While degradation of mtRNAs is a widely accepted function of PNPase, it is unclear how the enzyme mediates the transport of RNA instead of its destruction. Another suggested pathway of RNA import concerns mitochondrial protein translocase complexes TOM and TIM [190]; however, this has not been confirmed in mammalian cells. Another controversy refers to the RNA species that could potentially be imported into mitochondria. A few studies have indicated the mitochondrial import of recombinant heterologous tRNAs comprising the introduction of motifs derived from yeas<sup>t</sup> tRNA [191–193]. Of note, tRNAs were shown to be imported into mitochondria of trypanosomes and plants [194,195]. Nevertheless, import of endogenous RNAs H1 RNA (as an RNA component of the canonical RNaseP), 7-2 RNA (as an RNA component of the RNase MRP) and 5S rRNA (as a constituent of the mitoribosome) was recently challenged [176], as those RNAs were suggested to be dispensable in human mitochondria. Indeed, it was shown that mitochondrial RNaseP does not possess an RNA moiety [88], RNase MRP localizes predominantly to the nucleolus [196–198] and 5S rRNA is replaced by tRNA in the mitochondrial ribosome [199–201]. Establishing the molecular mechanism of mitochondrial RNA import would be of grea<sup>t</sup> value, as it could enable the import of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system constituents for mtDNA manipulation. Although obviously revolutionary and beneficial, introduction of this system in mitochondria seems to be challenging, if not impossible [176].

#### *6.4. Small RNAs in Mitochondrial Biology*

Transcription of mtDNA may be a source of distinctive mtRNA species. Recent studies reported that a various range of small ncRNAs may emerge form mitochondrial transcription. A study engaging high-throughput sequencing of small RNA (sRNA) revealed that around 3% of whole-cell sRNAs maps uniquely to the mitochondrial genome in 143B human cells [90]. Interestingly, most of reported unique small RNA sequences were derived from tRNA genes [90]. The functional role of these RNA species is not known and whether they can be a part of RNA interference pathway as it was shown for processed nuclear tRNAs is an open question [90,202,203]. Other reported mitochondrial sRNAs comprise small non-coding transcripts called mitosRNAs (mitochondrial genome-encoded small RNAs) [204] and mitochondrial microRNAs (also called mitomiRs) [205]. Notably, whether the novel sRNA species are just a consequence of mtRNA processing and whether they possess any biological function is currently debated. Small non-coding RNAs appear as a novel class of transcripts that may have regulatory functions in mitochondria, nevertheless their exact mode of action and some aspects of their biogenesis are far from well established and evoke controversies. A canonical function of cytoplasmic microRNA (miRNA) is sequence-specific gene silencing in RNA interference pathway [206]. Several reports suggested presence of microRNAs in mitochondria [207–210] proposed to be either products of mtDNA transcription or an e ffect of import of cytoplasmic transcripts inside mitochondria [205]. Nevertheless, miRNA biogenesis encompasses multiple steps of processing and require specialized machinery and it is not known which proteins could be engaged in these processes in mitochondria. Moreover, it is not clear how miRNAs could enter mitochondria. These questions need to be answered in order to establish functional potential of mitochondrial small ncRNAs.

Future studies will show whether identified short RNAs have functional importance or they are merely by-products of mtRNA processing and decay. For example, a recently described short RNA called tRNA-like accumulates strongly upon inhibition of mtRNA degradation by the mitochondrial degradosome [106]. This may imply that most, if not all, tRNA-like molecules are just by-products of mtRNA processing which are removed by the degradosome. Or this transcript has functional importance, but its levels need to be strictly controlled.

#### *6.5. 37 or More?*

Early studies of the human mitochondrial genome led to the mapping of mtDNA-encoded genes [21]. For a long time, human mtDNA was considered to encode 37 genes; however, a few recent reports suggested that there may be additional coding sequences within human mtDNA. Human mitochondrial genome may harbor nested small open reading frames (sORF) yielding short peptides derived from polycistronic mt-mRNAs. For example, human 16S rRNA gene was suggested to contain sORF encoding 24 amino acid peptide called humanin, which may have cytoprotective properties [211,212]. It is not known, however, whether humanin is translated inside mitochondria or in the cytoplasm and in the latter case, how humanin transcript could be exported from mitochondria and recognized by cytoplasmic translation apparatus [213]. Other mitochondria-derived peptides (MDPs) were proposed to arise from mitochondrial sORFs and were suggested to have regulatory functions within the cell. Among them are mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) and small humanin-like peptides 1–6 (SHLP1-6) located within 12S rRNA and 16S rRNA genes, respectively [214–216]. However, synthesis of MOTS-c would require export of corresponding encoding RNA to the cytoplasm, a process of unknown mechanism. Mitochondrial sORF and sRNAs are intriguing in concept, nonetheless, initial reports of them require further substantiation. It cannot be excluded that mitochondrial sRNAs and MDPs are by-products of mitochondrial gene expression. Therefore, proposed mitochondrial sORF and sRNAs require further studies to be fully embraced by the field.

**Author Contributions:** Conceptualization, R.J.S. and A.V.K., supervision R.J.S., A.V.K. drafted the manuscript and prepared the figures. R.J.S. reviewed and edited the manuscript. A.V.K. and R.J.S. prepared the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the POIR.04.04.00-00-5E63/18-00 project (to R.J.S.) carried out within the First TEAM programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund and by the National Science Centre, Poland, gran<sup>t</sup> number UMO-2014/12/W/NZ1/00463 (to R.J.S.).

**Conflicts of Interest:** The authors declare no conflict of interest.
