**3. Mitochondrial Transcription**

MtDNA is composed of heavy (H-strand) and light (L-strand) strands (Figure 1) that can be distinguished by different sedimentation attributes in buoyant density ultracentrifugation due to the uneven distribution of guanines between DNA strands [21]. In humans, the G-rich H-strand serves as a template for the transcription of most mitochondrially encoded genes, while the transcription of the complementary L-strand results in the formation of mostly non-coding RNA (ncRNA) [53]. Only one protein-coding gene and 8 tRNAs are transcribed from the L-strand [53] (Figure 1). The exceptional ~1 kb, non-coding regulatory region (NCR) plays an important role both in mtDNA replication and transcription. The NCR plays an important role as a site of H-strand synthesis initiation [49,54]; moreover, transcription initiation start sites for both mtDNA strands (ITL, light-strand transcription initiation site, ITH, heavy-strand transcription initiation site) are also located within the NCR. The synthesis of RNA starts in both directions within the NCR and leads to the production of long, polycistronic transcripts (Figure 1) [55].

The human transcription apparatus appears to be simple and composed of a monomeric RNA polymerase (POLRMT) that is homologous to bacteriophage polymerases and only a few co-factors [19]. In addition to POLRMT, basic factors participating in the mitochondrial transcription process include TFAM, mitochondrial transcription factor B2 (TFB2M) and mitochondrial transcription elongation factor (TEFM) (Figure 1) [19,56]. Initial in vitro experiments showed that transcription can proceed with the presence of only two proteins: POLRMT and TFB2M. Nevertheless, the e fficiency of this transcription apparatus is rather low [57]. Thanks to recent structural studies, the detailed step-by-step initiation of mitochondrial transcription was revealed [56]. It was shown that mitochondrial transcription is initiated by the binding of TFAM to the promoter region and the following recruitment of POLRMT to TFAM-bound mtDNA near the transcription start site [58,59]. Next, TFB2M is recruited to induce and stabilize the open conformation of mtDNA. After initiation of RNA synthesis, TFAM and TFB2M are subsequently released, and the elongation factor TEFM is recruited to enable the transition into transcription elongation [56]. TEFM was proposed to increase the processivity of POLRMT and to enable nearly whole-genome transcription [60–62]. Additionally, TEFM was shown to participate in the regulation of a replication/transcription switch [62–64]. Recently, TEFM was also proposed to play a role in mitochondrial RNA (mtRNA) processing [65].

Termination of mtDNA transcription is far less understood than its initiation. Mitochondrial transcription termination factor 1 (MTERF1) plays an important role in the termination of mtDNA transcription initiated from the ITL; however, it is not clear which factors take part in the termination of mtDNA transcription initiated from the ITH [56]. MTERF1 binds specific sequences within the tRNALeu gene, causing DNA unwinding and base flipping (i.e., rotation of the nucleotide base outside the DNA double helix), leading to transcription termination [66,67]. MTERF1 was also suggested to prevent interference of the transcription complexes operating in opposite directions [68] and to prevent collision of transcription and replication machineries [69]. Several other MTERF proteins are conserved in vertebrates and plants [70]. Despite playing a role in mitochondrial gene expression, none of the MTERF2-4 proteins were shown to act as a transcription terminator in mammals [71–73]. This raises the question whether there are other termination factors ye<sup>t</sup> to be discovered in the human mitochondria.

Among mitochondrial transcription machinery constituents, only TFAM mutations are well established to contribute to human disease [17]. TFAM binds DNA in both sequence-specific and nonspecific manners. The former enables the initiation of mitochondrial transcription [58], and the latter enables the compaction of the mitochondrial genome [74,75]. As mtDNA transcription by POLRMT may serve as a source of RNA primers for mtDNA replication [76], it seems that both sequence-specific and nonspecific manners of TFAM DNA-binding contribute to the maintenance of the mtDNA copy number (reviewed in [77]). TFAM knockout in mice causes severe depletion of mtDNA and is embryonically lethal [78]. In humans, mutations in the TFAM gene were shown to cause a decreased mtDNA copy number and impaired cellular respiration underlying progressive liver failure with neonatal onset [79]. Altered TFAM levels and related changes in the mtDNA copy number were also proposed to be associated with neurodegeneration [80].

Recently, some novel factors participating in mitochondrial transcription regulation were reported. Among them, an interesting example pertains to mitochondrial transcription rescue factor 1 (MTRES1), which was shown to interact with POLRMT and TFAM and to prevent stress-induced loss of mtRNAs by acting at the mitochondrial transcription initiation level [81]. Another example refers to mitochondrial ribosomal protein L7/L12 (MRPL12), whose mutations may lead to respiratory chain deficiency that manifest as growth retardation and neurological deterioration [82]. In addition to being a constituent of the mitochondrial ribosome, MRPL12 also exists in a "free", ribosome-unrelated matrix pool [83]. MRPL12 was shown to interact with POLRMT to regulate transcription [84]. It was proposed that MRPL12 may serve to coordinate transcription, ribosome biogenesis and/or protein synthesis processes [83,84]. Di fferent roles of MRPL12 in mitochondrial gene regulation may be connected with the presence of two forms, the short and long forms, generated by proteolytic cleavage upon being imported into mitochondria, which may have distinct properties [85]. Several other factors, such as hormones, nuclear transcription factors and chromatin remodeling enzymes, were proposed to regulate mitochondrial transcription either by direct binding to mtDNA or by indirect regulation (reviewed in [86]).

#### **4. Post-Transcriptional Regulation of mtRNAs**

Mitochondrial transcription spans almost the entire mitochondrial genome, leading to the formation of three polycistronic transcripts (Figure 1) [19,55]. Two of them, resulting from transcription of either the H- or L-strand, encompass almost the entire genome and carry sequences corresponding to mRNAs, tRNAs, rRNAs and ncRNAs. The third transcript covers genes for two tRNAs (Phe and Val) and both rRNAs [19]. Further steps of cleavage and processing are required to obtain mature, functional RNAs [87].

In nascent precursor transcripts, most mitochondrial mRNAs and rRNAs are punctuated by tRNAs. The first stage of primary RNA processing is the excision of tRNA molecules from the polycistronic transcript, which leads to the formation of immature mRNAs and rRNAs [19,20]. This is an endonucleolytic cleavage mediated by RNAse P and elaC ribonuclease Z 2 (ELAC2), which act at the 5 and 3 ends of tRNAs, respectively, to release individual RNAs from polycistronic precursors [88,89]). Mitochondrial RNAse P, unlike the canonical RNase P present in the nucleus, is a complex of three proteins tRNA methyltransferase 10C (TRMT10C), hydroxysteroid 17-beta dehydrogenase 10 (HSD17B10) and protein-only RNase P catalytic subunit (PRORP) and does not contain an RNA component [88]. Released, immature transcripts undergo further processing steps or, as in the case of the majority of ncRNAs, are rapidly removed [20].

Due to the structural and transcriptional organization of the mitochondrial genome, most genes on the same strand are transcribed with equal e fficiency, which leads to the formation of equal amounts of their precursors. Nevertheless, the levels of mature RNAs can di ffer significantly [90]. Although transcription initiated from the L-strand is more frequent than that of the H-strand [55], emerging non-coding RNAs are barely detectable [91]. These two examples show that post-transcriptional processes, especially mtRNA decay, play an important role in controlling mitochondrial gene expression to regulate steady-state levels of specific transcripts [90,92,93].

#### *4.1. Degradation of mtRNAs*

The machinery responsible for RNA decay in human mitochondria has remained unknown for many years. Studies performed within the last several years established that the mitochondrial degradosome, a complex of ATP-dependent RNA helicase SUPV3L1 (SUV3) and polynucleotide phosphorylase (PNPase also known as PNPT1), is a key player in mtRNA degradation [91,94]. The importance of mtRNA decay in maintaining mitochondrial homeostasis is underscored by the fact that the disruption of the SUV3 or PNPase gene is embryonically lethal in mice [95,96].

While SUV3 is a helicase that catalyzes the unwinding of RNA duplexes, an activity dependent on ATP hydrolysis by SUV3 [97], PNPase is a phosphorolytic 3-5 exoribonuclease, which catalyzes degradation of phosphodiester bonds in RNA [98]. In vitro experiments showed that PNPase is unable to degrade dsRNA substrates unless it forms a complex with SUV3 that unwinds the substrate for degradation [99]. The interaction between SUV3 and PNPase is a prerequisite for mtRNA degradation in vivo and occurs locally in D-foci [94]. The discovery of these structures shows that the RNA decay process in human mitochondria is spatially organized. Notably, components of the mitochondrial degradosome di ffer markedly in the submitochondrial localization. While SUV3 localizes only to the mitochondrial matrix [97], most of the PNPase is found in the mitochondrial intermembrane space [94,96]. Thus, only a fraction of the PNPase localizes to the mitochondrial matrix and cooperates with SUV3 in mtRNA degradation [94], whereas the rest of the PNPase functions in an SUV3-independent manner. Crystal structures of both proteins revealed the presence of some peculiarities [98,100]. Human PNPase, such as PNPases from other organisms, forms a trimer but has an untypical arrangemen<sup>t</sup> of RNA-binding domains [98]. Human SUV3 also has some distinctive features in terms of substrate binding, and it was even suggested that Suv3-like proteins may constitute a separate subfamily of helicases [100].

The main role of the degradosome appears to be clearing of non-coding mtRNA species, which arise mostly from transcription of the L-strand. Radiolabeling studies showed that under normal

conditions, these RNAs are swiftly degraded [101]. Consequently, their steady-state levels are very low, and it is only when the degradosome function is impaired that they become readily detectable [91,94]. The degradosome complex was also found to be important in mt-mRNA turnover [94,102], 16S rRNA decay [103] and the exonucleolytic processing of the ND6 mRNA precursor [104]. The final products of the degradosome-mediated RNA decay are several nucleotides in length. These short RNA degradation intermediates are probably removed by RNA exonuclease 2 (REXO2), which is a postulated mitochondrial oligoribonuclease [105]. The activity of the mitochondrial degradosome is modulated by mtRNA binding proteins. While the complex of leucine rich pentatricopeptide repeat containing and SRA stem-loop interacting RNA binding proteins (LRPPRC-SLIRP complex) was suggested to suppress the degradosome-mediated decay of mitochondrial protein-coding RNAs [102]. G-rich RNA sequence binding factor 1 (GRSF1) was recently found to enhance the degradosome activity towards mtRNAs containing a G-quadruplex (G4), which are mostly non-coding mtRNAs [106].

#### *4.2. Mitochondrial RNA-Binding Proteins (mtRBPs)*

LRPPRC and SLIRP are among the best characterized noncatalytic mitochondrial RNA-binding proteins containing known RNA-interacting domains, pentatricopeptide repeats (PPR) and RRM domain, respectively. LRPPRC and SLIRP form a complex involved in the regulation of mtRNA stability, and the levels of both proteins are mutually dependent; silencing of LRPPRC results in the depletion of SLIRP and vice versa [107,108]. A recent study using RNA UV crosslinking and RNase footprinting procedures revealed that the LRPPRC-SLIRP complex modulates the secondary structures of mitochondrial transcripts, suggesting that this complex may serve as a chaperone for mtRNAs [109]. The presence of LRPPRC was shown to be important for the existence of a nontranslated, mitoribosome-unbound pool of mt-mRNAs [110]. In addition, LRPPRC was shown to be required for efficient polyadenylation of mt-mRNAs [108,110]. In mice, LRPPRC knockout is embryonically lethal [110], and in humans, mutations in the LRPPRC gene underlie Leigh syndrome, French Canadian type [111]. Recent studies sugges<sup>t</sup> that LRPPRC may play a role in other various pathological states in humans, such as tumors or neurodegeneration (reviewed in detail by Cui et al. [112], emphasizing the very important role of this protein. SLIRP was shown to regulate the translation process by mediating the association of mtRNAs with mitoribosomes [113]. Surprisingly, although SLIRP knockout in mice results in extensive loss of mtRNAs, it is manifested only as a minor weight loss of the animals without any other observable phenotypes [113].

Another important RNA-binding protein, GRSF1, a member of the quasi-RRM (qRRM) family of RNA-binding proteins, was originally identified as a cytoplasmic poly(A)+ mRNA binding protein interacting with G-rich sequences [114]. Later, it was found that GRSF1 is targeted to mitochondria where it localizes to RNA-containing granules [115,116]. It was postulated that GRSF1 participates in the initial stages of polycistronic mtRNA precursor processing [116] and in the translation of some mt-mRNAs [115]. Recent findings, however, reported that GRSF1 takes part in the RNA surveillance pathway, showing that GRSF1 cooperates with the mitochondrial degradosome to regulate mtRNAs that contain G4s [106]. Vertebrates' mitochondrial genomes have exceptional GC skews, i.e., high guanine content on one strand. As a result, transcripts that are produced by transcription of the G-poor template (i.e., L-strand) are G-rich RNAs; thus, they are prone to form G4s structures. Since G4s are stable, their presence in RNA can hinder its degradation; nevertheless, steady-state levels of mt-ncRNAs that can form G4s are extremely low. GRSF1 was found to positively regulate the degradosome-dependent decay of G4-containing mitochondrial non-coding transcripts by binding and melting G4 structures, which in turn facilitates their degradation [106,117].

Recently, two studies reported novel mtRBP engaged in the regulation of mitochondrial gene expression [81,118]. The level of C6orf203/MTRES1 protein was found to be elevated in cells under stress, and this up-regulation of MTRES1 (mitochondrial transcription rescue factor 1) was shown to prevent mitochondrial transcript loss under perturbed mitochondrial gene expression [81]. MTRES1 associates with the mitochondrial transcription machinery and acts by increasing the mitochondrial

transcription without influencing the stability of mitochondrial transcripts [81]. The protective function of MTRES1 depends on its RNA-binding ability since the mutated version incapable of RNA-binding does not prevent a decrease in the mitochondrial RNA [81]. MTRES1 was also shown to associate with a large subunit of the mitochondrial ribosome and to influence mitochondrial translation [118]. Interestingly, silencing of MTRES1 causes the down-regulation of transcripts originating only from NCR without influencing other transcripts [81], which cannot explain the decrease in mitochondrial translation observed in MTRES1 knockout [118]. In contrast, it was reported that the depletion of MTRES1 leads to alterations of the mt-mRNAs' association with the mitoribosome without influencing mitoribosome stability [118]. MTRES1 is an exciting example of mitochondrial RBP that can play a role in the regulation of mitochondrial gene expression at multiple levels. It is tempting to speculate that MTRES1 could serve as a regulatory factor coupling mitochondrial transcription and translation processes. It is possible that MTRES1 could act by interacting with mitochondrial transcription machinery and to facilitate loading of nascent mt-mRNAs on the mitoribosome. It cannot be excluded that MTRES1 could be a key regulator of mitochondrial transcription/translation coupling, especially under stress conditions. Importantly, another mtRBP, MRPL12, was shown to perform double functions in mitochondrial transcription and translation [83,84], highlighting the possible roles of MTRES1.

Another example of important players in the mitochondrial gene expression regulation concerns members of the FASTK family. Fas activated serine/threonine kinase (FASTK) and its homologs FASTKD1-5 are mitochondrially targeted RNA-binding proteins that play various roles in mtRNA metabolism as processing, translation and mitoribosome assembly proteins [104,112,119–121]. The FASTK family was reviewed in detail by Jourdain et al. [122]; therefore, we will not focus on these proteins here.
