**Mitochondrial Gene Expression and Beyond—Novel Aspects of Cellular Physiology**

#### **Anna V. Kotrys and Roman J. Szczesny \***

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland; akotrys@ibb.waw.pl

**\*** Correspondence: rszczesny@ibb.waw.pl; Tel.: +48-22-592-30-23

Received: 28 November 2019; Accepted: 17 December 2019; Published: 19 December 2019

**Abstract:** Mitochondria are peculiar organelles whose proper function depends on the crosstalk between two genomes, mitochondrial and nuclear. The human mitochondrial genome (mtDNA) encodes only 13 proteins; nevertheless, its proper expression is essential for cellular homeostasis, as mtDNA-encoded proteins are constituents of mitochondrial respiratory complexes. In addition, mtDNA expression results in the production of RNA molecules, which influence cell physiology once released from the mitochondria into the cytoplasm. As a result, dysfunctions of mtDNA expression may lead to pathologies in humans. Here, we review the mechanisms of mitochondrial gene expression with a focus on recent findings in the field. We summarize the complex turnover of mitochondrial transcripts and present an increasing body of evidence indicating new functions of mitochondrial transcripts. We discuss mitochondrial gene regulation in different cellular contexts, focusing on stress conditions. Finally, we highlight the importance of emerging aspects of mitochondrial gene regulation in human health and disease.

**Keywords:** mitochondria; mitochondrial gene expression; mtDNA; mtDNA transcription; mtRNA; post-transcriptional mtRNA processing; dsRNA; innate immunity; interferon response

#### **1. Introduction: The Role of Mitochondria within the Cell**

Mitochondria are present in the majority of eukaryotic cells, where they play a central role in many processes. They are hubs of energy production as sites of oxidative phosphorylation [1]. They are also engaged in maintaining an appropriate redox state and recycling oxidized electron carriers that are important for cell proliferation [2,3]. Moreover, mitochondria play a key role in cellular signaling, buffering calcium ions and regulating apoptosis processes [4,5]. Mitochondria are also the source of most of the cellular reactive oxygen species (ROS), which may impact on various cellular process [6,7]. Mitochondria take part in response to external stimuli, e.g., viral infection [4], and they are also the place where many basic processes related to innate immune intersect [8,9]. Mitochondrial malfunction is related to numerous pathological states in humans, such as cancer and neurodegeneration [10–12]. Furthermore, many human hereditary diseases are caused by mitochondrial dysfunction. Mutations in the mitochondrial genome or nuclear genes that encode mitochondrial proteins lead to primary and secondary mitochondrial diseases connected with improper mitochondrial function [13,14]. In this review, we will focus on the emerging aspects of mitochondrial biology and its implications in human health and we guide interested readers to detailed reviews on mitochondrial disorders [15–17].

Mitochondrial biogenesis is far more complex in comparison to other cellular organelles since they possess their own genome, which requires dedicated gene expression machinery [18–20]. As a consequence, mitochondrial protein production must be coordinated with the nucleocytoplasmic compartment for proper organelle homeostasis. Many nucleus-encoded proteins are needed for mitochondrial transcription, RNA processing, degradation and translation; all to produce just over a

dozen proteins that are parts of the oxidative phosphorylation complexes encoded by the mitochondrial genome [18,19]. Recently, new findings deciphering the link between mitochondrial gene expression and cellular homeostasis were reported. Here, we review the basis of mitochondrial gene regulation with a focus on recent findings, highlighting new RNA-driven mechanisms by which mitochondria contribute to the regulation of cell biology in human health and disease.

#### **2. Mitochondrial Genome—A Simple Molecule with Unorthodox Organization**

Mitochondria are semiautonomous organelles possessing their own genome. Human mitochondrial DNA (mtDNA) is a ~16 kb circular molecule composed of double-stranded DNA [21]. Human mtDNA contains only a few genes, but all of them are essential for proper cell function, and mutations in mtDNA have dire consequences [13]. Although human mitochondria contain more than 1100 proteins [22], only 13 of them are encoded by mtDNA. The remaining 24 of the 37 genes present in human mtDNA encode RNAs required for the mitochondrial translational apparatus (22 tRNAs and 2 rRNAs) [19] (Figure 1). The rest of the proteins necessary for mitochondrial function are encoded in the nuclear genome and are imported into mitochondria upon synthesis in the cytoplasm [23]. These proteins are key factors in mtDNA replication, transcription and translation processes [23,24]. The presence of a distinct mitochondrial genome is linked to the endosymbiotic origin of this organelle [25,26]. Recent studies of the mitochondrial genome indicated that mitochondria evolved from a proteobacterial lineage [27]. The human mitochondrial genome was first described in the early 1960s and was one of the first genomes to be fully sequenced [28,29]. Since then, the molecular mechanisms of mtDNA expression have been intensively investigated; nevertheless, our knowledge about these processes is still not complete.

The human mitochondrial genome is tightly packed in nucleoprotein complexes called nucleoids [30]. The application of super-resolution microscopy and mass spectrometry-based techniques significantly contributed to the determination of the nucleoid characteristics [31–34]. It was shown that each nucleoid contains one or several copies of mtDNA [33,34] and is spatially organized by DNA-binding proteins [31]. More than 20 proteins were identified to copurify with mitochondrial nucleoids [32], among which the most abundant protein mitochondrial transcription factor A (TFAM) that may serve as a mtDNA copy number regulator [35]. Discrete nucleoid complexes are dispersed among the mitochondrial network in the cell [33]. An estimated number of mtDNA molecules per mitochondrion may vary greatly between tissues and range from less than a hundred to thousands [36–38]. Each mtDNA molecule can be independently segregated in daughter cells, as nucleoids are exchanged within the mitochondrial network upon fusion/fission processes [39,40]. More than one mtDNA sequence can be present within an individual, leading to so-called mitochondrial heteroplasmy. This can be caused by somatic mutations, heteroplasmy of the oocyte or paternal leakage of mtDNA [41,42]. For a long time, mtDNA was considered to be strictly maternally inherited [43]; nevertheless, few reports sugges<sup>t</sup> paternal contribution in the inheritance of the mitochondrial genome [44,45].

There are several models describing the mechanism of mtDNA replication. Notably, the proposed models are not mutually exclusive. Instead, they were proposed to operate in a complementary mode depending on the tissue, cellular state or energy demand [46–48]. For a detailed review of mtDNA replication, please see Holt and Reyes [49] and Gustafsson et al. [50].

Notably, mutations in nuclear-encoded mitochondrial proteins that participate in mtDNA maintenance are implicated in mitochondrial disorders [51]. To date, nearly 300 pathogenic point mutations have been reported in the DNA polymerase gamma (POLG) gene encoding mtDNA replicase, and POLG mutations are one of the main causes of inherited mitochondrial disorders [51,52].

*Cells* **2020**, *9*, 17

**Figure 1.** Schematic of human mtDNA and basic steps of mtRNA metabolism. Human mtDNA is a circular double-stranded molecule. Marked are template heavy (H-strand) and light (L-strand) strands of mtDNA. Marked are genes encoding subunits of Complex I (blue), III (orange), IV (violet) and subunits of ATP Synthase (pink). ND4/ND4L and ATP6/ATP8 open reading frames are overlapping and are included in bicistronic mRNAs. Genes encoding rRNAs are colored purple. Genes encoding tRNAs are represented as yellow dots with single letter code depicting their aminoacids. Mitochondrial transcription is initiated from L- and H- strand promoters (ITL and ITH, respectively) located within non-coding regulatory region (NCR). Transcription is driven by DNA-dependent RNA polymerase (POLRMT) with help of its co-factors: TFAM, TFB2M, TEFM and MTRES1. Mitochondrial transcription leads to the formation of three polycistronic transcripts that undergo further processing. Most of mitochondrial mRNAs and rRNAs are punctuated by tRNAs which are excised from the nascent RNA precursors by RNAse P and Z. Arisen transcripts may be subjected to various subsequent processes as maturation, stabilization, degradation, and modification. Selected factors engaged in these processes are presented.
