Unbiased Mitoproteome Analyses Confirm Non-canonical RNA, Expanded Codon Translations

Abstract

Proteomic MS/MS mass spectrometry detections are usually biased towards peptides cleaved by experimentally added digestion enzyme(s). Hence peptides resulting from spontaneous degradation and natural proteolysis usually remain undetected. Previous analyses of tryptic human proteome data (cleavage after K, R) detected non-canonical tryptic peptides translated according to tetra- and pentacodons (codons expanded by silent mono- and dinucleotides), and from transcripts systematically (a) deleting mono-, dinucleotides after trinucleotides (delRNAs), (b) exchanging nucleotides according to 23 bijective transformations. Nine symmetric and fourteen asymmetric nucleotide exchanges (X ↔ Y, e.g. A ↔ C; and X → Y → Z → X, e.g. A → C → G → A) produce swinger RNAs. Here unbiased reanalyses of these proteomic data detect preferentially non-canonical tryptic peptides despite assuming random cleavage. Unbiased analyses couldn't reconstruct experimental tryptic digestion if most detected non-canonical peptides were false positives. Detected non-tryptic non-canonical peptides map preferentially on corresponding, previously described non-canonical transcripts, as for tryptic non-canonical peptides. Hence unbiased analyses independently confirm previous trypsin-biased analyses that showed translations of del- and swinger RNA and expanded codons. Accounting for natural proteolysis completes trypsin-biased mitopeptidome analyses, independently confirms non-canonical transcriptions and translations.

Directed Mutations Recode Mitochondrial Genes: From Regular to Stopless Genetic Codes

“…Indeed, analyses of mitochondrial mass spectra searching for peptides matching translations assuming tetra-and pentacodons, codons expanded by one or two silent nucleotides, detected numerous tetra-and pentacoded peptides [38][39][40][41][42][43].…”

“…It is important to understand in this context that the genetic code's discovery, among the greatest fundamental discoveries, is not over, but only in process. Indeed, coding sequences include much more information than generally believed, even beyond RNA editing (RDD [91]), systematic transformations during replication [44][45][46] and transcription [39,[47][48][49][50][51], and translation along expanded codons [32][33][34][35][36][37][38][39][40][41][42][43]. Cryptic codes [92,93] such as the well-developed theory of the natural circular code [94][95][96][97][98][99][100][101][102][103][104][105][106][107][108][109][110][111][112] regulate the ribosomal translation frame [113][114]…”

Systematic Nucleotide Exchange Analysis of ESTs From the Human Cancer Genome Project Report: Origins of 347 Unknown ESTs Indicate Putative Transcription of Non-Coding Genomic Regions

“…The swinger transcriptome of the amoeban-hosted Mimivirus was also confirmed by two different sequencing techniques (SOLID and 454, Seligmann and Raoult, 2018). Detected peptides matching translation of the swingertransformed mitogenome tend to map on detected swinger RNAs (Seligmann, 2016a;Seligmann, 2016b;Seligmann, 2016c;Seligmann, 2017a). These findings were further confirmed in purified mitochondrial transcriptomes (Warthi and Seligmann, 2018), rejecting the possibility of cytoplasmic contamination.…”

“…These represent 0.23% of the 149,500 unidentified ESTs from that project. Note that other types of systematic transformations apparently produce noncanonical RNAs, such as systematic deletions, which produce so-called delRNAs (Seligmann, 2015c;Seligmann, 2016f;El Houmami and Seligmann, 2017;Seligmann, 2017b; and corresponding peptides (Seligmann, 2015c;Seligmann, 2016a;Seligmann, 2016b;Seligmann, 2016c;Seligmann, 2016d;Seligmann, 2016e), including chimeric peptides (Seligmann and Warthi, 2019). This underlines a general approach to identify unknown sequences generated by various sequencing methods.…”

Swinger RNAs in the Human Mitochondrial Transcriptome

“…Hence, swinger RNAs result from canonical transcription of swinger-transformed DNA or swinger transcription of regular DNA [22]. Some mass spectra match predicted peptides translated from del-and swinger-transformed RNA [39][40][41][42]. Detection of chimeric RNAs, consisting of part regular, and part swinger-transformed contiguous sequences suggests that regular canonical and swinger-transformed RNA result from single polymerisation events, probably by the same polymerase [43].…”