Natural pyrrolysine-biased translation of stop codons in mitochondrial peptides entirely coded by expanded codons.

During the noncanonical deletion transcription, k nucleotides are systematically skipped/deleted after each transcribed trinucleotide producing deletion-RNAs (delRNAs). Peptides matching delRNAs either result from (a) canonical translation of delRNAs; or (b) noncanonical translation of regular transcripts along expanded codons. Only along frame "0" (start site) (a) and (b) produce identical peptides. Here, mitochondrial mass spectrometry data analyses assume expanded codon/del-transcription with 3 + k (k from 0 to 12) nucleotides. Detected peptides map preferentially on previously identified delRNAs. More peptides were detected for k (1-12) when del-transcriptional and expanded codon translations start sites coincide (i.e. the 0th frame) than for frames +1 or +2. Hence, both (a) and (b) produced peptides identified here. Biases for frame 0 decrease for k > 2, reflecting codon/anticodon expansion limits. Further analyses find preferential pyrrolysine insertion at stop codons, suggesting Pyl-specific mitochondrial suppressor tRNAs loaded by Pyl-specific tRNA synthetases with unknown origins. Pyl biases at stops are stronger for regular than expanded codons suggesting that Pyl-tRNAs are less competitive with near-cognate tRNAs in expanded codon contexts. Statistical biases for these findings exclude that detected peptides are experimental and/or bioinformatic artefacts implying both del-transcription and expanded codons translation occur in human mitochondria.

Chimeric Translation for Mitochondrial Peptides: Regular and Expanded Codons.

Frameshifting protein translation occasionally results from insertion of amino acids at isolated mono- or dinucleotide-expanded codons by tRNAs with expanded anticodons. Previous analyses of two different types of human mitochondrial MS proteomic data (Fisher and Waters technologies) detect peptides entirely corresponding to expanded codon translation. Here, these proteomic data are reanalyzed searching for peptides consisting of at least eight consecutive amino acids translated according to regular tricodons, and at least eight adjacent consecutive amino acids translated according to expanded codons. Both datasets include chimerically translated peptides (mono- and dinucleotide expansions, 42 and 37, respectively). The regular tricodon-encoded part of some chimeric peptides corresponds to standard human mitochondrial proteins (mono- and dinucleotide expansions, six (AT6, CytB, ND1, 2xND2, ND5) and one (ND1), respectively). Chimeric translation probably increases the diversity of mitogenome-encoded proteins, putatively producing functional proteins. These might result from translation by tRNAs with expanded anticodons, or from regular tricodon translation of RNAs where transcription/posttranscriptional edition systematically deleted mono- or dinucleotides after each trinucleotide. The pairwise matched combination of adjacent peptide parts translated from regular and expanded codons strengthens the hypothesis that translation of stretches of consecutive expanded codons occurs. Results indicate statistical translation producing distributions of alternative proteins. Genetic engineering should account for potential unexpected, unwanted secondary products.