*4.3. mtRNA Modifications*

Mitochondrial RNAs undergo diverse modifications. One of the most common is adenylation of the 3' end. Human mt-mRNAs, with the exception of mt-ND6, are adenylated at the 3' ends, and this reaction is catalyzed by a noncanonical mitochondrial poly(A) polymerase (MTPAP) [123]. The role of mt-mRNAs' adenylation is not fully understood. For some mt-mRNAs, the addition of adenine residues at the 3' end is necessary to create a complete termination codon, as it is not encoded in the genome [21]. Initial studies reported some contradictory findings about the role of mt-mRNA polyadenylation, showing that changes in poly(A) tails had diverse e ffects on mt-mRNAs. Some of the transcripts were up-regulated, some were una ffected, and others were down-regulated [123–126]. This draws a speculation that individual transcripts may be di fferentially controlled, as it seems that adenylation may stabilize some transcripts while it may also direct other transcripts for degradation [20]. Notably, the polyadenylation pattern may vary within a cell type, and the same mt-mRNA can be adenylated to di fferent extents in various cell types [127]. This individualized regulation is most likely achieved by transcript-specific protein-RNA interactions. In yeast, it was shown that each mt-mRNA has specific translation coactivators; moreover, it was proposed that they can function as a part of feedback control loops regulating translation e fficiency according to current cell demands [128]. Similarly, the presence of the specific translation activator of cytochrome c oxidase I (TACO1) [129] was also detected in human cells. In addition to 3'-end adenylation, it was recently shown that human mt-mRNAs may also undergo other modifications, such as methylation [130,131] and pseudouridylation [132,133], unraveling an additional layer of mitochondrial gene expression regulation.

Mitochondrial tRNAs seem to be the most extensively modified among mtRNAs. Precursor tRNAs are modified at the 3' end by tRNA nucleotidyl transferase 1 (TRNT1), which adds the CCA sequence not encoded in the genome [134]. They also undergo several other modifications, which are essential for their stability and proper function [135]. Similarly, mt-rRNAs require several chemical modifications for proper folding, stability and correct mitoribosome assembly, underscoring the important role of RNA modifications in orchestrating various mitochondrial processes [24].

The mitochondrial RNA-binding proteome has been intensely studied, and novel members of this group have been continuously discovered. Among them are mtRNA modifying enzymes, mtRNA processing and mitoribosome assembly factors. Here, we delineated only selected aspects of mtRBP-associated regulation of mitochondrial gene expression, mostly related to mtRNA degradation. For further reading on other aspects of mitochondrial RBP function and detailed insight into mt-tRNA and mt-rRNA editing as well as the mitochondrial epitranscriptome, we guide interested readers to other reviews [136–138].

#### **5. Mitochondrial Non-Coding Transcripts and Their Implications on Human Health**

#### *5.1. Mitochondrial Double-Stranded RNA (mt-dsRNA) and the Innate Immune Response*

Convergent transcription, synthesis of antisense RNA or expression of hairpin-containing RNAs can result in the formation of double-stranded RNA (dsRNA) [139]. RNA:RNA molecules seem to have important signaling or regulatory roles [140–142], and the function of a particular dsRNA depends on its source and form and the proteins that interacts with it. It is known that the presence of dsRNA can induce the antiviral response and that properly processed dsRNA can regulate gene expression [140–142].

The expression of the human mitochondrial genome is especially prone to produce dsRNA molecules, as this genome is an extraordinary example of convergen<sup>t</sup> transcription. This results in the synthesis of complementary RNAs that can subsequently hybridize and form dsRNA. Under normal conditions, most L-strand transcripts are quickly removed [91,94], which prevents the formation of intermolecular dsRNA. Nevertheless, even in physiological conditions, mt-dsRNAs can be detected [143]. We and others have found that mtDNA transcription is a significant source of dsRNA in humans and have shown that mt-dsRNA can play a signaling role and trigger the interferon response [143,144].

The interferon response pathway is a complex process that is part of the innate immunity response, which together with the adaptive system, provides protection from pathogens. This pathway is far from being fully understood; nevertheless, it can be divided into three major steps: (1) detection of a pathogen, (2) induction of interferon (IFN) expression, and (3) up-regulation of IFN-stimulated genes. Consequently, the cell starts producing anti-pathogen agents and reshapes already active processes to combat a pathogen, or at least hampers its spread [145].

The detection step involves recognition of "pathogen-associated molecular patterns" (PAMPs), which are viral or bacterial nuclei acids, or other molecules specific for microorganisms [146]. The recognition is performed by several families of host sensors collectively called pattern recognition receptors (PRRs), such as interferon induced helicase C domain-containing protein 1 (MDA5) and RNA helicase RIG-I. The binding of immunogenic dsRNA by MDA5 and RIG-I initiates an orchestrated cascade of events that results in the up-regulation of interferon- and interferon-stimulated genes [147]. Normally, this pathway is kept silent due to the mechanisms that help to discriminate between host and foreign nucleic acids, remove potentially immunogenic host-produced nucleic acids or keep them away from PAMP receptors. For example, dsRNA, which can activate MDA5 or RIG-I, is localized in the nucleus and mitochondria, precluding its interaction with MDA5 and RIG-I, which are localized in the cytoplasm.

Recently it was found that mt-dsRNA can be released from mitochondria and, once localized to the cytoplasm, can activate interferon-dependent cellular pathways. Notably degradosome constituents SUV3 and PNPase were established as major actors in the regulation of mt-dsRNA [144]. A crucial role was assigned to PNPase in preventing the induction of the interferon response by mt-dsRNA. PNPase controls mt-dsRNA at two di fferent levels that are spatially separated. First, as a component of the mitochondrial degradosome, it prevents the accumulation of dsRNA by degrading mitochondrial antisense transcripts. The mechanism that determines specificity of PNPase towards antisense transcripts has not been fully uncovered. This may result from targeting of PNPase-SUV3 to G4 mtRNAs by GRSF1. Alternatively, but not mutually exclusive, sense transcripts are protected by mtRBPs, while unprotected antisense mtRNAs are exposed to PNPase-SUV3. This function of PNPase requires cooperation with SUV3 and takes place in the mitochondrial matrix. A second function of PNPase in controlling mt-dsRNAs takes place in the intermembrane space, where a fraction of PNPase prevents the release of mt-dsRNA into the cytosol. This function is SUV3-independent because the helicase is absent from the intermembrane space [97]. The dysfunction of SUV3 thus results in the accumulation of mt-dsRNA exclusively in the matrix but does not lead to the release of mt-dsRNA into the cytosol due to the protective activity of PNPase in the intermembrane space. Therefore, silencing of SUV3 does not stimulate interferon expression, while an increase in interferon levels is observed in PNPase-depleted cells. Since inactivation of the degradosome components causes a vast increase in non-coding L-strand transcripts [91,94,106], it is most likely that the mt-dsRNA that accumulates upon degradosome dysfunction results from intermolecular RNA-RNA interactions. The mechanism by which mt-dsRNA exits mitochondria is unknown, and its identification will be an important step in deciphering a role of mt-dsRNA in cell biology.

The protective role of PNPase is of grea<sup>t</sup> importance, as revealed by the fact that mutations in *PNPT1* (PNPase encoding gene) cause pathogenesis associated with the up-regulation of mt-dsRNA and interferon response [144,148]. A comparison of the type I interferon response across species, which involved 10 mammals and one bird, revealed PNPase as one of 62 core vertebrate interferon-stimulated genes [149]. Moreover, the PNPase protein level was observed to be higher in human cells infected by human respiratory syncytial virus (HRSV), a cause of serious infections in infants [150]. Taken together with the results on the protective role of PNPase in mt-dsRNA release, these data sugges<sup>t</sup> that PNPase can be involved in the suppression of the IFN response. In fact, IFN-desensitization is an important process that enables cells to recover from IFN signaling [145]. Otherwise, prolonged activation of IFN-dependent pathways can lead to permanent dysregulation of cellular homeostasis. At the organismal level, this is manifested by the development of pathologies called type I interferonopathies. An example of such a disease is Aicardi–Goutières syndrome [151].

Interestingly, the contribution of mt-dsRNA to innate immunity was also observed in other studies. It was shown that the immune response can be modulated by mt-dsRNA via protein kinase RNA-activated (PKR). The canonical PKR-induced response comprises sensing of viral dsRNA by PKR following phosphorylation of the enzyme and altering cellular signaling pathways to promote an antiviral defense. Recent findings revealed that PKR can also be induced by endogenous RNAs, primarily by mt-dsRNAs [143]. PKR was proposed to associate with mitochondria, and mt-dsRNA sensing was suggested to take part in the mitochondrial matrix [143]. However, the proposed mechanism assumes that PKR needs to be exported to the cytoplasm to induce downstream signaling pathways. How PKR translocation to and from mitochondria is maintained remains unclear. It cannot be excluded that PKR induction may take place during mt-dsRNA escape into the cytoplasm, for example, upon aberrant PNPase function.

Notably, the role of mt-dsRNA in the modulation of innate immunity is not restricted to humans, as similar responses were reported in other species, suggesting that mt-dsRNAs are important regulators of the immune response across species. A study in a fly model demonstrated that disrupted mtRNA turnover leads to mt-dsRNA accumulation and altered immune response once mt-dsRNAs are released into the cytoplasm [152]. A study conducted in a murine model showed that the lack of p53 protein results in the activation of innate immunity by dsRNA of mitochondrial origin [153]. Consistent with previous findings on human cells [144], the immune response was shown to be dependent on MDA5 and RIG-I activation [153]. Altogether, these studies established mt-dsRNAs as important regulators of the immune response and revealed a new RNA-dependent mechanism by which mitochondria regulate cell fate.

#### *5.2. Mitochondrial Long Non-Coding RNAs (mt-lncRNAs)*

While most non-coding transcripts are swiftly degraded after synthesis, a pool of antisense mtRNAs exhibits some stability, resulting in their detection under normal conditions. These transcripts, called mirror RNAs, such as mirror ND2 [91,94], or long non-coding RNAs (ncND5, ncND6, and ncCytB) [154], are complementary to sense transcripts. Whether these mt-lncRNAs play any role remains to be elucidated; nevertheless, di fferential tissue-specific expression of these RNA species [154] suggests their potential regulatory function. One can imagine that by hybridizing to their coding counterparts, mt-lncRNAs could regulate the stability or translation e fficiency of corresponding mtRNAs.

Other examples of lncRNAs possibly originating from mitochondrial transcription were proposed [155,156]. An example is a 2374 nt-long transcript containing an antisense 16S mt-rRNA sequence (inverted repeat) linked to the 5 end of the 16S mt-rRNA forming a long double-stranded structure called SncmtRNA (sense non-coding mitochondrial RNA). Interestingly, this transcript was reported to be overexpressed in proliferating but not resting tumor cells [155]. Further studies from the same group reported the presence of 2 other lncRNAs containing inverted repeats linked to the 5 end of the antisense 16S mt-rRNA, called ASncmtRNAs (antisense non-coding mitochondrial RNAs). These transcripts were found in normal proliferating cells and were reported to be down-regulated in tumor cells [156]. Detected transcripts were postulated to be hallmarks of proliferating tumor cells vs nontumor cells, which may play a role in regulating tumor progression [156,157].

A group of lncRNAs mapped to mtDNA was detected in patients with heart disease [158], and these include circulating mitochondria-derived lncRNA LIPCAR (long intergenic non-coding RNA predicting cardiac remodeling) [159]. The 5 end of this 781nt long chimeric lncRNA maps to the antisense of the mt-Cytb gene, while its 3 end maps to the antisense of the mt-COX2 gene. The LIPCAR transcript was shown to be up-regulated in the late stages after myocardial infarction and to be elevated in patients with chronic heart failure, serving as a potential biomarker of cardiac remodeling and cardiovascular mortality [159,160].

As presented here, ncRNAs encompass a group of potentially important molecules that can act in various ways to influence mitochondrial function and cell physiology (Figure 2). Their putative functions involve the regulation of mitochondrial translation and mtRNA stability. They may also function as sponges occupying mtRBPs. Moreover, their ability to form mt-dsRNA together with sense transcripts predisposes them to act as signaling molecules modulating the immune response. Altogether, mitochondrial non-coding RNAs extend the repertoire of potential mechanisms by which mitochondrial gene expression is regulated and influences cell physiology. Thus far, antisense transcripts were mostly studied or linked with non-physiological conditions. It remains to be seen whether this class of RNAs has any roles under normal, nonperturbed conditions.

**Figure 2.** Cellular functions of mitochondria-derived transcripts. MtDNA may be a source and a target for non-coding RNA. Question mark indicates that the process or mechanism is currently undefined. Nucleus-encoded miRNAs (mitomiRs) are suggested to enter mitochondria and regulate mtDNA expression, nevertheless their exact import pathway is currently unexplained. Transcription of mtDNA may result in formation of small non-coding RNAs (mitosRNAs) which may interplay with mt-mRNAs stability. Mechanism of action of mitochondrial small non-coding RNAs is currently undefined. Long non-coding RNAs (mt-lncRNAs) may arise from mtDNA transcription and may regulate stability and transcription of mt-mRNAs or may serve as baits for mitochondrial RNA-binding proteins (mtRBPs). Convergent transcription of mtDNA may result in formation of double-stranded RNA (mt-dsRNA). Under normal conditions, L-strand derived transcripts are swiftly degraded which prevents formation of mt-dsRNAs. Dysfunction of PNPase causes accumulation of mt-dsRNA and its release into cytoplasm. Once released into the cytoplasm, mt-dsRNAs can induce type I interferon response via activation of MDA5 receptors.

#### **6. Emerging Aspects of mtDNA Maintenance and Expression**

#### *6.1. mtDNA Polymerases and mtDNA Repair*

POLG, responsible for replication of mtDNA, was long believed to be the only DNA polymerase present in mitochondria [161]. In contrast, recent reports sugges<sup>t</sup> that there may be more than one DNA polymerase operating in mammalian mitochondria. Recent studies proposed several additional DNA polymerases to be implicated in mtDNA metabolism; nevertheless, some of those findings require further validation [162]. The most well-documented novel mtDNA polymerase, primase and DNA directed polymerase (PrimPol), takes part both in nuclear and mitochondrial DNA maintenance [163]. PrimPol may act as a primase forming DNA and RNA primers or as a DNA polymerase to extend the DNA primers and it is also able to perform translesion synthesis [164]. It seems that in human mitochondria, the main role of PrimPol is not to prime mtDNA replication but rather to rescue stalled replication forks by bypassing lesions or repriming DNA synthesis at the site of DNA damage. Further polymerases suggested to take part in mtDNA metabolism comprise polymerases Beta (POLB), Zeta (POLZ) and Theta (POLQ) [162]. These polymerases were proposed to participate in mtDNA repair (POLB) [165] (reviewed by Kaufman and Van Houten [166], mtDNA stability (POLZ) [167] and mtDNA maintenance (POLQ) [168].

Importantly, the discovery of novel mtDNA polymerases broadens the horizon for prospective mtDNA repair pathways. Mitochondria are thought to possess a limited DNA repair system comprising mainly base excision repair (BER). In mitochondrial BER, gap filling is driven by POLG assisted by several other proteins engaged in recognition and cleavage of a damaged DNA base, elimination of the abasic site, DNA 5-end processing to generate substrate for POLG and, finally, ligation [169–174]. Other mtDNA repair pathways are being debated; nucleotide excision repair (NER) is thought to be absent from mitochondria, and only a few studies have reported mismatch repair to be present in mitochondria [52]. It is thus possible that novel enzymes could be engaged in these processes, expanding the repertoire of possible mtDNA repair pathways. This is especially important as mtDNA damage may have significant implications for human health [175].
