Leber’s Hereditary Optic Neuropathy: the roles of mitochondrial transfer RNA variants

Mitochondrial biology and genetics

Mitochondria originated from within the bacterial phylum α-Proteobacteria and became established via an endosymbiotic event (Lane & Martin, 2010). Mitochondria are critical organelles that perform a remarkably diverse set of cellular functions. The most important of these is the generation of ATP via OXPHOS, but mitochondria also play critical roles in the regulation of apoptosis, maintenance of cellular redox homeostasis and intracellular calcium signaling (Duchen, 2004; Tait & Green, 2010; Sena & Chandel, 2012; Rizzuto et al., 2012).

The human mitochondrial genomes (mitogenomes) are circular, 16,569-bp in length, and contain 37 genes encoding 13 proteins required for OXPHOS and the electron transport chain (ETC) (Fig. 1) (Andrews et al., 1999). mtDNA also encodes RNAs, which are involved in the translation of ETC proteins (Luo et al., 2018). Owing to its location within the mitochondrial matrix, lack of protective histone wrapping, as well as a comparatively limited repair mechanism, mtDNA is more vulnerable to oxidative modifications which accumulate over time (Yakes & Van Houten, 1997). Indeed, it has been shown that mtDNA has a significantly higher mutation rate than nuclear DNA (Neckelmann et al., 1987).

Unlike nuclear DNA, in which there are only two copies of each gene per cell, thousands of copies of mtDNA are presented in every cell. Typically, individuals harbor only one mtDNA genotype, and all mitogenomes are genetically identical, a condition called homoplasmy. This contrasts with heteroplasmy, which involves the presence of a mixture of mutant and wild-type mtDNA genomes within a cell. Through somatic mutagenesis and ongoing replication of mtDNA, mutations can clonally expand through either random drift or selective processes, and become present at varying proportions or degrees of heteroplasmy with cells (Elson et al., 2001). Among families affected by LHON, 85%–90% of carriers are homoplasmic for mtDNA mutation. However, some studies have indicated that mtDNA heteroplasmy may be a factor determining the penetrance of LHON (Li et al., 2019; Finsterer & Zarrouk-Mahjoub, 2018)). In certain families, rapid segregation of the mitochondrial genotype toward mutant-type homoplasmy of either 11778G>A (Bolhuis et al., 1990) or 3460G>A mutation in blood (Black et al., 1996) has been shown to be associated with the development of LHON in later generations. It has been suggested that the risk of disease conversion is low if the mutational load is below the threshold of 60% (Chinnery et al., 2001). Although it is not possible to accurately predict whether an LHON carrier will eventually lose vision, individuals can be counseled based on the two major identifiable risk factors in this disorder: age and sex.

Three LHON-associated primary mutations

The majority of patients with LHON harbor one of three primary mtDNA point mutations: 3460G>A (Howell et al., 1991; Huoponen et al., 1991), 11778G>A (Wallace et al., 1988), and 14484T>C (Johns, Neufeld & Park, 1992; Mackey & Howell, 1992). They are found exclusively in families affected by LHON and never in control subjects. The G to A transition at position 11778 converts a conserved arginine to histidine, has been associated with poor visual outcome and prognosis (Newman, Lott & Wallace, 1991). Meanwhile, the 3460G>A mutation causes the alteration of a highly conserved alanine to threonine, which is present in around 15% of LHON families (Howell, et al. 1991). Moreover, the T to C transition at nucleotide 14484 in ND6 (methionine to valine) has been shown to be tightly linked to the LHON phenotype (Johns, Neufeld & Park, 1992). Interestingly, younger age at onset (<15 years) and mutation type appear to dictate visual outcome; patients with the 14484T>C mutation have a better visual prognosis with 60% attaining some visual improvement compared with only 5% of those carrying the 11778G>A mutation.

The incomplete penetrance, high male to female ratio, and existence of LHON plus cases strongly suggest the involvement of modifying factors such as genetic or environmental ones (Tońska, Kodroń & Bartnik, 2010; Caporali et al., 2017). In particular, genetic factors such as mt-tRNA variants can play active roles in the phenotypic manifestation of LHON-associated primary mutations.

Nuclear genes

Although the mitochondrial proteome consists of over 1000 proteins, only 14 of them are encoded by mtDNA. Thus, the nuclear genome encodes >90% of peptides involved in the OXPHOS system (Leigh-Brown, Enriquez & Odom, 2010). Moreover, incomplete penetrance and male bias in patients with LHON suggest that an X-linked modified gene is necessary for the disease expression (Bu & Rotter, 1991). A recent genome-wide study of 1281 Chinese probands with LHON identified a novel LHON susceptibility allele (c.157C>T, p. Arg53Trp) in the PRICKLE3 gene, which links to ATPase biogenesis manifested LHON (Yu et al., 2020). Moreover, a missense mutation in YARS2 (c.572G>T, p. Gly191Val) was shown to interact with the 11778G>A mutation to cause visual failure (Jiang et al., 2016).

mt-tRNA structure and function

mt-tRNA is a short, non-coding RNA that constitutes approximately 4∼10% of all cellular RNAs (Kirchner & Ignatova, 2015). In fact, most mt-tRNAs from all domains of life have a highly conserved cloverleaf structure, consisting of an acceptor arm, D-arm, anticodon stem, variable region, and TψC loop, with an average length of 73 nucleotides (nts). However, mt-tRNA genes encode transcripts that show considerable deviation of this standard, having a reduced D-arm or TψC loop or even completely lacking one of these elements (e.g., tRNA^{Ser(AGY )}), resulting in tRNAs as small as 66 nts (Hanada et al., 2001). In addition, mt-tRNA^Ser(UCN) has several distinct structural features, including only one base (A9) between the acceptor arm and D-arm, a short D-loop, a variable region, and an extended anticodon stem with 6-bp (Watanabe et al., 1994).

As adapter molecules to convert the information stored in amino acid (AA) sequences, tRNAs play a central role in protein synthesis (Stowe & Camara, 2009). Although tRNAs comprise only around 10% of the total coding capacity of the mitogenomes , more than half of mtDNA mutations causing diseases are located in mt-tRNA genes (https://www.mitomap.org/MITOMAP) (Taanman, 1999), emphasizing the importance of tRNAs for mitochondrial function.

tRNA end processing

The excision of tRNAs from primary polycistronic mitochondrial transcripts is catalyzed by two specialized enzymes, RNase P and tRNase Z (Fig. 2). RNase P is an endonuclease that catalyzes the cleavage of the 5′leader sequence from pre-tRNA transcripts (Rossmanith et al., 1995). Human mitochondrial RNase P (mtRNase P) is a RNase P complex consisted of three proteins, called MRPP1; MRPP2 and MRPP3 (Holzmann et al., 2008). In fact, human mtRNase P cleaves a wide range of tRNA precursors in vitro (Rossmanith, 1997; Rossmanith et al., 1995), and its activity is detectable even in crude mitochondrial extracts and thereby appears to be relatively abundant (Rossmanith et al., 1995).

At the other end, 3′ trailers are removed by the endonuclease tRNase Z (Phizicky & Hopper, 2010; Hartmann et al., 2009; Maraia & Lamichhane, 2011). Both short and long forms of tRNase Z are present in eukaryotes, designated tRNase ZS (280 to 360 AAs) and tRNase ZL (750 to 930 AAs) respectively (Ishii et al., 2005; Li de la Sierra-Gallay, Pellegrini & Condon, 2005). The C-terminal part of tRNase ZL has sequence homology with tRNase ZS. However, in contrast to the single mechanism of 5′ leader removal, 3′ trailers can also be removed by one or more 3′ exoribonucleases (Rex1p, and perhaps others) (Copela et al., 2008; Ozanick et al., 2009). The 5′-before-3′ end processing appears to apply most clearly when tRNase Z is used for 3′ processing.

tRNA post-transcriptional modifications

For mt-tRNA maturation, post-transcriptional modifications, together with the 5′and 3′nucleolytic excision from precursor RNAs are required. Certain modifications are necessary for maintenance of mt-tRNA structure and steady-state level, as well as ensuring the efficiency of protein synthesis during mitochondrial translation. Up to date, more than 30 modified mt-tRNA positions have been reported (Suzuki, Nagao & Suzuki, 2011) (Fig. 3). Modifications cluster occurs at two main regions of tRNA molecule: the structural core and the anticodon stem. Chemical modifications in the structural region are relatively simple, for instance, methylation, pseudouridylation and dihydrouridylation. Furthermore, modifications in the anticodon stem of mt-tRNAs include methylation and pseudouridylation, in several cases, with more complex additions, specially the modifications at positions 34 and 37 (El Yacoubi, Bailly & Crécy-Lagard, 2012). Four types of modified nucleotides were found at the wobble positions of 10 tRNA species that correspond to two codon sets. The modifications consisted of 5-formylcytidine at the wobble position of tRNA^Met (Bilbille et al., 2011), queuosine at the wobble positions of four tRNA^Tyr, tRNA^His, tRNA^Asn and tRNA^Asp (Iwata-Reuyl, 2008). In addition, five tRNAs were found to have taurine-containing uridines 5-taurinomethyluridine was identified in the tRNA^Leu(UUR) and tRNA^Trp, and 5-taurinomethyl-2-thiouridine in tRNA^Lys, tRNA^Glu and tRNA^Gln (Nagao et al., 2009; Suzuki et al., 2002).

tRNA aminoacylation

Aminoacyl-tRNA synthetases (aaRSs), encoded by nuclear genes, play essential roles in protein synthesis. To start this process, aaRSs must catalyze the attachment of AAs to the corresponding tRNAs (Yao & Fox, 2013). This biochemical reaction requires the following steps: 1. AA+ATP →aminoacyl-AMP+PPi; 2. aminoacyl-AMP + tRNA →aminoacyl-tRNA+AMP. Today, nineteen species of aaRS genes were annotated in the human genome database (Antonellis & Green, 2008). Mammalian mitochondria had no enzyme corresponding to glutaminyl-tRNA synthetase (GlnRS) (Nagao et al., 2009). Mitochondrial LysRS and GlyRS were encoded by the same genes as the cytoplasmic LysRS and GlyRS, respectively, whereas the other aaRSs were encoded by genes different from the cytoplasmic ones (Ling, Reynolds & Ibba, 2009).

Secondary mt-tRNA variants

Although most LHON cases are caused by one of three pathogenic mtDNA mutations, no primary mutations are identified in a minority of LHON patients, these other homoplasmic mtDNA are considered as secondary variants that can be responsible for disease phenotype variation and different penetrance having synergistic effects with the primary mtDNA mutations (Bosley & Abu-Amero, 2010; Zhang et al., 2009) (Fig. 4 and Tables 1 and 2).

mt-tRNA variants enhance the phenotypic expression of the primary mtDNA mutations

Other reported mt-tRNA variants