Preferential Selection of the 5′-Terminal Start Codon on Leaderless mRNAs by Mammalian Mitochondrial Ribosomes

Abstract

Mammalian mitochondrial mRNAs are basically leaderless, having few or no untranslated nucleotides prior to the 5-start codon. We demonstrate here that mammalian mitochondrial 55 S ribosomes preferentially form initiation complexes at a 5-terminal AUG codon over an internal AUG. The preferential use of the 5-start codon is also seen on mitochondrial 28 S small subunits, which suggests that mitochondrial translation initiation on leaderless mRNAs does not require the large ribosomal subunit. The selection of the 5-AUG is dependent on the presence of fMet-tRNA and is enhanced by the presence of the mitochondrial initiation factor IF2 mt . In prokaryotes, IF3 is believed to antagonize initiation on leaderless mRNAs. However, IF3 mt stimulates initiation complex formation on leaderless mRNAs when tested with 55 S ribosomes. The addition of even a few nucleotides 5 to the AUG codon significantly reduces the efficiency of initiation, highlighting the importance of the leaderless or nearly leaderless nature of mitochondrial mRNAs. In addition, very few initiation complexes could form on a hybrid mRNA construct consisting of tRNA Met attached at the 5-end of a mitochondrial protein-coding sequence. This observation demonstrates that post-transcriptional processing must occur prior to translation in mammalian mitochondria.In contrast to other genomes, the mammalian mitochondrial genome is very compact, containing ϳ16 kilobase pairs of DNA with very few noncoding nucleotides. This DNA encodes 2 rRNAs, 13 proteins, and 22 tRNAs (1). The 13 proteins are all essential components of the electron transport chain and ATP synthase. They are synthesized by the mitochondrial translational machinery from nine monocistronic mRNAs and two dicistronic mRNAs with overlapping reading frames. Mitochondrial mRNAs contain few or no noncoding nucleotides prior to the 5Ј-terminal start codon and are largely unstructured at their 5Ј-ends (2, 3). The exact number of nucleotides prior to the 5Ј-start codon in mature mitochondrial mRNAs has been determined in both humans and fruit flies. Direct analysis of the 5Ј-ends of the 11 open reading frames located at the 5Ј-ends of the human mitochondrial mRNAs demonstrated that post-transcriptional processing completely eliminates the 5Ј-leader in all but three mRNAs (4). These three mRNAs have one, two, and three nucleotides 5Ј to the start codon. None of the 5Ј-cistrons of Drosophila melanogaster mitochondrial mRNAs begin with noncoding nucleotides (5).Translation of mitochondrial mRNAs is accomplished by mitochondrial ribosomes, which sediment as 55 S particles and dissociate into 28 S and 39 S subunits. The translation process in mitochondria occurs with the help of two initiation factors: IF2 mt promotes the binding of fMet-tRNA to the 28 S small ribosomal subunit, and IF3 mt stimulates initiation complex formation by facilitating the dissociation of mitochondrial 55 S ribosomes. No factor homologous to prokaryotic IF1 has been found in mitochondria, and the role of this factor is thought to be carr...

Using mitoribosomal profiling to investigate human mitochondrial translation

“…Here, the footprints on MTCO1 and ATP8 showed a similar distribution with the majority of protected sequences, 86% and 62% respectively, covering the non-coding nucleotides ( Figure 2C). This difference in mitoribosome behaviour is consistent with the in vitro observation that initiation is affected by nucleotides upstream of the start codon 31 . Do these minimal 5’UTRs have an even greater impact on the interaction with aberrant mitoribosomes containing mt-tRNA Phe ?…”

“…Previous in vitro studies by Spremulli and colleagues investigated the interaction of the 55S monosome and the 28S mt-SSU with RNA templates that had AUG start codons with differing numbers of nucleotides upstream 31 . They were able to show that the mitoribosome binds the mt-mRNAs with a strong preference for start codons at the very 5’-terminus.…”

“…The first 151 nucleotides of this transcript was used as a template by Spremulli and colleagues. Their analyses showed that the addition of even a single nucleotide ahead of the start codon for MTCO1 reduced binding, and 3 nucleotides decreased formation of an initiation complex by more than 40% 31 . We, therefore, aimed to identify if the absence or presence of nucleotides preceding the initiating codon affected the mitoribosome residency over the initiation site in these transcripts in mitoribosome profiles from live cells.…”

Four translation initiation pathways employed by the leaderless mRNA in eukaryotes

“…In the case of the leaderless mRNA, the initial IF2 binding is mandatory for the 30S-mediated pathway, and the elevated IF2 level selectively stimulates its translation both in vitro and in vivo 7. Stimulation of the leaderless mRNA translation by IF2 orthologs is also revealed for archaeal and mitochondrial systems918.…”

“…Although these molecules lack any special nucleotide sequences at their 5′-termini, except for the AUG itself, they can efficiently direct protein synthesis in bacterial, archaeal, or mitochondria systems in vitro as well as in vivo 6789. The structural peculiarity of the leaderless mRNA imparts some unusual translational properties.…”