The m.3243A>G transition mutation in MT-TL1 was first described by Goto et al. (1990) using PCR techniques to sequence mitochondrial tRNA genes in 26 patients with mitochondrial encephalopathy lactic acidosis and stroke-like episodes (MELAS) and one with CPEO. This mutation affects nucleotides A14 and U8 in a highly conserved region of the dihydrouridine loop (D-loop) of mt-tRNALeu(UUR) (Figure 3.1). Early studies using transmitochondrial cybrid
cells confirmed the presence of the m.3243A>G mutation resulted in compromised protein synthesis and respiratory chain activity (Chomyn et al., 1992; King et al., 1992). A number of pathogenic mechanisms underlying the cellular and clinical phenotypes associated with the m.3243A>G mutation have been suggested and are described below.
The earliest proposed mechanism was based upon altered transcription termination observed in vitro by Hess et al. (1991). The mTERF binding site spans a 28bp region of MT-
TL1 within which the m.3243A>G mutation lies (Kruse et al., 1989). In the presence of the
m.3243A>G mutation, Hess et al. (1991) report that binding of human mitochondrial transcription termination factor (mTERF) is compromised, possibly leading to altered synthesis of 16S and 12S rRNA. However, studies in vivo have not observed a defect in transcription termination, possibly due to a compensatory over-expression of mTERF or increase in transcription rate buffering the effect of reduced mTERF binding (Chomyn et al., 1992; Koga et al., 1993).
It has also been suggested that a reduction in the availability of mt-tRNALeu(UUR) could
contribute to the pathogenicity of the m.3243A>G mutation. Börner et al. (2000) evaluated total and mutant populations of mtDNA, free mt-tRNALeu(UUR) and aminoacylated mt-
tRNALeu(UUR) in 8 patients with the m.3243A>G mutation and MELAS/related symptoms
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Figure 3.1: Cloverleaf structure of mt-tRNALeu(UUR). The A14 residue of the D-loop (shown in
red) is affected by the m.3243A>G mutation, in the presence of which it is substituted for a G- residue. The uridine residue situated at the wobble position (shown in blue) is subject to post-transcriptional modification to become 5-taurinomethyluridine (τm5U). Adapted from
Wittenhagen and Kelley (2002).
mutant tRNALeu(UUR) in the total tRNALeu(UUR) pool was decreased, compared to the proportion
of mutant mtDNA present in the tissue. This suggested a reduction in expression of the mutant tRNALeu(UUR) in half of the patients studied, and was not related to mtDNA
heteroplasmy level. Furthermore, six patients showed a decrease in the proportion of aminoacylated mutant tRNALeu(UUR) when compared to the mutant mtDNA level. Only one
patient sample maintained equal proportions of mutant in mtDNA, tRNALeu(UUR) and
aminoacylated tRNALeu(UUR). As such, the authors concluded that the mutation had a variable
impact on the expression of mt-tRNALeu(UUR) and the availability of the aminoacylated tRNA
molecule across patients, alluding to the involvement of additional factors contributing to pathogenicity.
The effect of the m.3243A>G mutation on post-transcriptional modification of the mt-
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in transmitochondrial cybrid cells that the presence of this mutation prevents the taurine modification of uridine (5-taurinomethyluridine (τm5U)) at the wobble position (Yasukawa et al., 2000; Suzuki et al., 2002). This modification reinforces the tertiary, L-shaped structure of
mt- tRNALeu(UUR) (Helm et al., 1998), and is required to facilitate effective recognition of
codons by the tRNA. Indeed, Kirino et al. (2004) have shown that tRNALeu(UUR) molecules
lacking the τm5U modification resulted in a severe and specific reduction in UUG translation
in transmitochondrial cybrid cells. This indicated that the m.3243A>G mutation amplifies the translation defect, possibly due to the de-stabilisation of the tertiary structure of the tRNA. Of further interest, Kirino et al. (2004) note the high abundance of UUG codons in the MT-
ND6 gene, pertinent to the observation that complex I activity is often reduced in patients
with m.3243A>G mutation (Goto et al., 1992). However, others have reported no relationship between the translation of UUR codons and the synthesis of these mitochondrial proteins (Chomyn et al., 1992).
Several mutations in the MT-TL1 gene (m.3243A>G, m.3244G>A, m.3258T>C, m.3271T>C and m.3291T>C) associated with the MELAS phenotype have been shown to prevent the τm5U modification (Kirino et al., 2005). This study also reported normal τm5U modification in
patient tissue or transmitochondrial cybrid lines harbouring alternative MT-TL1 mutations associated with non-MELAS phenotypes (m.3242G>A, m.3250T>C, m.3254C>T and
m.3280A>G). This would suggest a strong correlation between the clinical phenotype and the presence of the τm5U modification. While this can be said for the MELAS phenotype
studied by Kirino et al. (2005), it may not be the case for other phenotypes associated with the m.3243A>G mutation.
The final, and most recently proposed mechanism of pathogenicity is based on reports of tRNA dimerisation induced by the presence of the m.3243A>G mutation (Wittenhagen and Kelley, 2002). The mutation introduces a self-complementary hexanucleotide (5’GGGCCC; G= mutation site) into the conserved D-loop, disrupting the native tertiary structure and
facilitating dimerisation of the tRNA. To characterise the molecular effects of the
dimerisation, in vitro tests using tRNA constructs found a reduction in the aminoacylation activity in the presence of the mutation. This was partially rescued by the addition of a second mutation, designed to disrupt the dimerisation process. As aminoacylation was not
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fully restored in the absence of the tRNA dimer, it was concluded that this mechanism was not exclusively the cause of m.3243A>G pathogenicity (Wittenhagen and Kelley, 2002).