Using mitoribosomal profiling to investigate human mitochondrial translation

Abstract

Background: Gene expression in human mitochondria has various idiosyncratic features. One of these was recently revealed as the unprecedented recruitment of a mitochondrially-encoded tRNA as a structural component of the large mitoribosomal subunit. In porcine particles this is mt-tRNA Phe whilst in humans it is mt-tRNA Val. We have previously shown that when a mutation in mt-tRNA Val causes very low steady state levels, there is preferential recruitment of mt-tRNA Phe. We have investigated whether this altered mitoribosome affects intra-organellar protein synthesis. Methods: By using mitoribosomal profiling we have revealed aspects of mitoribosome behaviour with its template mt-mRNA under both normal conditions as well as those where the mitoribosome has incorporated mt-tRNA Phe. Results: Analysis of the mitoribosome residency on transcripts under control conditions reveals that although mitochondria employ only 22 mt-tRNAs for protein synthesis, the use of non-canonical wobble base pairs at codon position 3 does not cause any measurable difference in mitoribosome occupancy irrespective of the codon. Comparison of the profile of aberrant mt-tRNA Phe containing mitoribosomes with those of controls that integrate mt-tRNA Val revealed that the impaired translation seen in the latter was not due to stalling on triplets encoding either of these amino acids. The alterations in mitoribosome interactions with start codons was not directly attributable to the either the use of non-cognate initiation codons or the presence or absence of 5’ leader sequences, except in the two bicistronic RNA units, RNA7 and RNA14 where the initiation sites are internal. Conclusions: These data report the power of mitoribosomal profiling in helping to understand the subtleties of mammalian mitochondrial protein synthesis. Analysis of profiles from the mutant mt-tRNA Val cell line suggest that despite mt-tRNA Phe being preferred in the porcine mitoribosome, its integration into the human counterpart results in a suboptimal structure that modifies its interaction with mt-mRNAs.

Balanced mitochondrial and cytosolic translatomes underlie the biogenesis of human respiratory complexes

“…For mRNAs with a concise (1–3 nt) leader or no (0-nt) leader, the plots reveal an enrichment of 15-nt half-size RPFs increasing in size to full-length, at which point the mRNA channel is fully occupied. These shorter half-footprints have not been previously detected ( 30 ) and indicate that mitoribosomes load at the ends of messages with the P site on the start codon. Translation initiation occurs from the canonical AUG but also AUA and AUU.…”

The complexity and dynamics ofin organellotranslation assessed by high-resolution mitochondrial ribosome profiling

“…The sucrose density gradient with ultracentrifugation has been applied to enrich mitoribosomes and their footprints and deplete cytosolic ribosome (cytoribosome) footprints in order to survey mitochondrial translation by Ribo-Seq 8, [10][11][12][13][14][15][16] . A drawback of this strategy is in its limitation in handling many samples (i.e., the maximum number of samples is limited to the 6 tubes that the ultracentrifuge rotor holds) as well as its timeconsuming nature (~1 d).…”

“…A variety of techniques have been developed to investigate translation in mitochondria 8 . Among them, mitochondria-optimized ribosome profiling (or Ribo-Seq), which is based on deep sequencing of ribosome-protected RNA fragments remaining after RNase digestion 9 , has been a powerful tool (MitoRibo-Seq) [10][11][12][13][14][15][16] . Technically, MitoRibo-Seq requires the depletion of cytosolic ribosome (cytoribosome) footprints; otherwise, they occupy large sequencing space.…”

The Mitochondrial Genome–on Selective Constraints and Signatures at the Organism, Cell, and Single Mitochondrion Levels

“…Accordingly, experiments in Drosophila suggested that, in some cases, tRNA synthetase undergoes coordinated changes alongside their corresponding tRNAs to maintain function (Meiklejohn et al, 2013;Holmbeck et al, 2015). The availability of ribosome profiling as a highly quantitative approach to assess rates of translation, and its recent adaptation to the mtDNA (Rooijers et al, 2013;Gao et al, 2017), serves as a promising future experimental approach to compare rates of mtDNA translation between samples differing in synonymous mtDNA mutations.…”