Mitochondrial translation often exhibits departures from the standard genetic code, but the full spectrum of these changes has certainly not yet been described and the molecular mechanisms behind the changes in codon meaning are rarely studied. Here we report a detailed analysis of the mitochondrial genetic code in the stramenopile group Labyrinthulea (Labyrinthulomycetes) and their relatives. In the genus Aplanochytrium, UAG is not a termination codon but encodes tyrosine, in contrast to the unaffected meaning of the UAA codon. This change is evolutionarily independent of the reassignment of both UAG and UAA as tyrosine codons recently reported from two uncultivated labyrinthuleans (S2 and S4), which we show are not thraustochytrids as proposed before, but represent the clade LAB14 previously recognised in environmental 18S rRNA gene surveys. We provide rigorous evidence that the UUA codon in the mitochondria of all labyrinthuleans serves as a termination codon instead of encoding leucine, and propose that a sense-to-stop reassignment has also affected the AGG and AGA codons in the LAB14 clade. The distribution of the different forms of sense-to-stop and stop-to-sense reassignments correlates with specific modifications of the mitochondrial release factor mtRF2a in different subsets of labyrinthuleans, and with the unprecedented loss of mtRF1a in Aplanochytrium and perhaps also in the LAB14 clade, pointing towards a possible mechanistic basis of the code changes observed. Curiously, we show that labyrinthulean mitochondria also exhibit a sense-to-sense codon reassignment, manifested as AUA encoding methionine instead of isoleucine. Furthermore, we show that this change evolved independently in the uncultivated stramenopile lineage MAST8b, together with the reassignment of the AGR codons from arginine to serine. Altogether, our study has uncovered novel variants of the mitochondrial genetic code and previously unknown modifications of the mitochondrial translation machinery, further enriching our understanding of the rules governing the evolution of one of the central molecular process in the cell.

• Keywords
• Codon reassignment, Evolution, Genetic code, Labyrinthuleans, Mitogenomes, Release factor,
• MeSH
• Phylogeny MeSH
• Genetic Code * MeSH
• Stramenopiles classification   genetics   MeSH
• Codon MeSH
• Mitochondria genetics   MeSH
• Evolution, Molecular * MeSH
• Protein Biosynthesis genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Codon MeSH

Recent years have seen a great expansion in our understandings of how silent mutations can drive a disease and that mRNAs are not only mere messengers between the genome and the encoded proteins but also encompass regulatory activities. This review focuses on how silent mutations within open reading frames can affect the functional properties of the encoded protein. We describe how mRNAs exert control of cell biological processes governed by the encoded proteins via translation kinetics, protein folding, mRNA stability, spatio-temporal protein expression and by direct interactions with cellular factors. These examples illustrate how additional levels of information lie within the coding sequences and that the degenerative genetic code is not redundant and have co-evolved with the encoded proteins. Hence, so called synonymous mutations are not always silent but 'whisper'.

• MeSH
• Genetic Code genetics   MeSH
• Codon genetics   MeSH
• Humans MeSH
• RNA, Messenger chemistry   genetics   MeSH
• Models, Genetic MeSH
• Mutation * MeSH
• Open Reading Frames genetics   MeSH
• Proteins chemistry   genetics   metabolism   MeSH
• Protein Biosynthesis genetics   MeSH
• Protein Folding MeSH
• RNA Folding MeSH
• RNA Stability genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Codon MeSH
• RNA, Messenger MeSH
• Proteins MeSH

Blastocrithidia nonstop is a protist with a highly unusual nuclear genetic code, in which all three standard stop codons are reassigned to encode amino acids, with UAA also serving as a sole termination codon. In this study, we demonstrate that this parasitic flagellate is amenable to genetic manipulation, enabling gene ablation and protein tagging. Using preassembled Cas9 ribonucleoprotein complexes, we successfully disrupted and tagged the non-essential gene encoding catalase. These advances establish this single-celled eukaryote as a model organism for investigating the malleability and evolution of the genetic code in eukaryotes.

• Keywords
• CRISPR‐Cas9, codon reassignment, genetic code, model organism, trypanosomatids,
• MeSH
• Genetic Code * genetics   MeSH
• Catalase genetics   MeSH
• Protozoan Proteins genetics   MeSH
• Codon, Terminator genetics   MeSH
• Trypanosomatina * genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Catalase MeSH
• Protozoan Proteins MeSH
• Codon, Terminator MeSH

UNLABELLED: Trypanosomatids are among the most extensively studied protists due to their parasitic interactions with insects, vertebrates, and plants. Recently, Blastocrithidia nonstop was found to depart from the canonical genetic code, with all three stop codons reassigned to encode amino acids (UAR for glutamate and UGA for tryptophan), and UAA having dual meaning also as a termination signal (glutamate and stop). To explore features linked to this phenomenon, we analyzed the genomes of four Blastocrithidia and four Obscuromonas species, the latter representing a sister group employing the canonical genetic code. We found that all Blastocrithidia species encode cognate tRNAs for UAR codons, possess a distinct 4 bp anticodon stem tRNATrpCCA decoding UGA, and utilize UAA as the only stop codon. The distribution of in-frame reassigned codons is consistently non-random, suggesting a translational burden avoided in highly expressed genes. Frame-specific enrichment of UAA codons immediately following the genuine UAA stop codon, not observed in Obscuromonas, points to a specific mode of termination. All Blastocrithidia species possess specific mutations in eukaryotic release factor 1 and a unique acidic region following the prion-like N-terminus of eukaryotic release factor 3 that may be associated with stop codon readthrough. We infer that the common ancestor of the genus Blastocrithidia already exhibited a GC-poor genome with the non-canonical genetic code. Our comparative analysis highlights features associated with this extensive stop codon reassignment. This cascade of mutually dependent adaptations, driven by increasing AU-richness in transcripts and frequent emergence of in-frame stops, underscores the dynamic interplay between genome composition and genetic code plasticity to maintain vital functionality. IMPORTANCE: The genetic code, assigning amino acids to codons, is almost universal, yet an increasing number of its alterations keep emerging, mostly in organelles and unicellular eukaryotes. One such case is the trypanosomatid genus Blastocrithidia, where all three stop codons were reassigned to amino acids, with UAA also serving as a sole termination signal. We conducted a comparative analysis of four Blastocrithidia species, all with the same non-canonical genetic code, and their close relatives of the genus Obscuromonas, which retain the canonical code. This across-genome comparison allowed the identification of key traits associated with genetic code reassignment in Blastocrithidia. This work provides insight into the evolutionary steps, facilitating an extensive departure from the canonical genetic code that occurred independently in several eukaryotic lineages.

• Keywords
• AT-rich genomes, eukaryotic release factors, nuclear genetic code, reassigned codon, tRNA structure, termination of translation,
• MeSH
• Cell Nucleus * genetics   MeSH
• Phylogeny MeSH
• Genetic Code * MeSH
• Genome, Protozoan * MeSH
• Genomics MeSH
• Evolution, Molecular MeSH
• RNA, Transfer genetics   MeSH
• Codon, Terminator genetics   MeSH
• Trypanosomatina * genetics   classification   MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• RNA, Transfer MeSH
• Codon, Terminator MeSH

BACKGROUND: Almost all extant organisms use the same, so-called canonical, genetic code with departures from it being very rare. Even more exceptional are the instances when a eukaryote with non-canonical code can be easily cultivated and has its whole genome and transcriptome sequenced. This is the case of Blastocrithidia nonstop, a trypanosomatid flagellate that reassigned all three stop codons to encode amino acids. RESULTS: We in silico predicted the metabolism of B. nonstop and compared it with that of the well-studied human parasites Trypanosoma brucei and Leishmania major. The mapped mitochondrial, glycosomal and cytosolic metabolism contains all typical features of these diverse and important parasites. We also provided experimental validation for some of the predicted observations, concerning, specifically presence of glycosomes, cellular respiration, and assembly of the respiratory complexes. CONCLUSIONS: In an unusual comparison of metabolism between a parasitic protist with a massively altered genetic code and its close relatives that rely on a canonical code we showed that the dramatic differences on the level of nucleic acids do not seem to be reflected in the metabolisms. Moreover, although the genome of B. nonstop is extremely AT-rich, we could not find any alterations of its pyrimidine synthesis pathway when compared to other trypanosomatids. Hence, we conclude that the dramatic alteration of the genetic code of B. nonstop has no significant repercussions on the metabolism of this flagellate.

• Keywords
• Blastocrithidia, In silico, Metabolic predictions, Non-canonical genetic code, Trypanosomatid,
• MeSH
• Eukaryota genetics   MeSH
• Genetic Code MeSH
• Parasites * genetics   MeSH
• Codon, Terminator MeSH
• Trypanosoma brucei brucei * genetics   MeSH
• Trypanosomatina * genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Codon, Terminator MeSH

A limited number of non-canonical genetic codes have been described in eukaryotic nuclear genomes. Most involve reassignment of one or two termination codons as sense ones [1-4], but no code variant is known that would have reassigned all three termination codons. Here, we describe such a variant that we discovered in a clade of trypanosomatids comprising nominal Blastocrithidia species. In these protists, UGA has been reassigned to encode tryptophan, while UAG and UAA (UAR) have become glutamate encoding. Strikingly, UAA and, less frequently, UAG also serve as bona fide termination codons. The release factor eRF1 in Blastocrithidia contains a substitution of a conserved serine residue predicted to decrease its affinity to UGA, which explains why this triplet can be read as a sense codon. However, the molecular basis for the dual interpretation of UAR codons remains elusive. Our findings expand the limits of comprehension of one of the fundamental processes in molecular biology.

• Keywords
• Blastocrithidia, evolution, genetic code, phylogeny, protists, translation, trypanosomatids,
• MeSH
• Cell Nucleus genetics   MeSH
• Phylogeny MeSH
• Genetic Code genetics   MeSH
• Codon chemistry   genetics   MeSH
• Protozoan Proteins chemistry   genetics   MeSH
• Amino Acid Sequence MeSH
• Codon, Terminator chemistry   genetics   MeSH
• Trypanosomatina genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Codon MeSH
• Protozoan Proteins MeSH
• Codon, Terminator MeSH

Mitochondria of diverse eukaryotes have evolved various departures from the standard genetic code, but the breadth of possible modifications and their phylogenetic distribution are known only incompletely. Furthermore, it is possible that some codon reassignments in previously sequenced mitogenomes have been missed, resulting in inaccurate protein sequences in databases. Here we show, considering the distribution of codons at conserved amino acid positions in mitogenome-encoded proteins, that mitochondria of the green algal order Sphaeropleales exhibit a diversity of codon reassignments, including previously missed ones and some that are unprecedented in any translation system examined so far, necessitating redefinition of existing translation tables and creating at least seven new ones. We resolve a previous controversy concerning the meaning the UAG codon in Hydrodictyaceae, which beyond any doubt encodes alanine. We further demonstrate that AGG, sometimes together with AGA, encodes alanine instead of arginine in diverse sphaeroplealeans. Further newly detected changes include Arg-to-Met reassignment of the AGG codon and Arg-to-Leu reassignment of the CGG codon in particular species. Analysis of tRNAs specified by sphaeroplealean mitogenomes provides direct support for and molecular underpinning of the proposed reassignments. Furthermore, we point to unique mutations in the mitochondrial release factor mtRF1a that correlate with changes in the use of termination codons in Sphaeropleales, including the two independent stop-to-sense UAG reassignments, the reintroduction of UGA in some Scenedesmaceae, and the sense-to-stop reassignment of UCA widespread in the group. Codon disappearance seems to be the main drive of the dynamic evolution of the mitochondrial genetic code in Sphaeropleales.

• Keywords
• Sphaeropleales, codon reassignments, genetic code, green algae, mitogenomes, release factor,
• MeSH
• Chlorophyta genetics   MeSH
• Genome, Mitochondrial MeSH
• Codon * MeSH
• Mitochondrial Proteins chemistry   genetics   MeSH
• Mitochondria genetics   MeSH
• Evolution, Molecular * MeSH
• Peptide Termination Factors chemistry   genetics   MeSH
• RNA, Transfer genetics   MeSH
• Codon, Terminator MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Codon * MeSH
• Mitochondrial Proteins MeSH
• Peptide Termination Factors MeSH
• RNA, Transfer MeSH
• Codon, Terminator MeSH

BACKGROUND: Departures from the standard genetic code in eukaryotic nuclear genomes are known for only a handful of lineages and only a few genetic code variants seem to exist outside the ciliates, the most creative group in this regard. Most frequent code modifications entail reassignment of the UAG and UAA codons, with evidence for at least 13 independent cases of a coordinated change in the meaning of both codons. However, no change affecting each of the two codons separately has been documented, suggesting the existence of underlying evolutionary or mechanistic constraints. RESULTS: Here, we present the discovery of two new variants of the nuclear genetic code, in which UAG is translated as an amino acid while UAA is kept as a termination codon (along with UGA). The first variant occurs in an organism noticed in a (meta)transcriptome from the heteropteran Lygus hesperus and demonstrated to be a novel insect-dwelling member of Rhizaria (specifically Sainouroidea). This first documented case of a rhizarian with a non-canonical genetic code employs UAG to encode leucine and represents an unprecedented change among nuclear codon reassignments. The second code variant was found in the recently described anaerobic flagellate Iotanema spirale (Metamonada: Fornicata). Analyses of transcriptomic data revealed that I. spirale uses UAG to encode glutamine, similarly to the most common variant of a non-canonical code known from several unrelated eukaryotic groups, including hexamitin diplomonads (also a lineage of fornicates). However, in these organisms, UAA also encodes glutamine, whereas it is the primary termination codon in I. spirale. Along with phylogenetic evidence for distant relationship of I. spirale and hexamitins, this indicates two independent genetic code changes in fornicates. CONCLUSIONS: Our study documents, for the first time, that evolutionary changes of the meaning of UAG and UAA codons in nuclear genomes can be decoupled and that the interpretation of the two codons by the cytoplasmic translation apparatus is mechanistically separable. The latter conclusion has interesting implications for possibilities of genetic code engineering in eukaryotes. We also present a newly developed generally applicable phylogeny-informed method for inferring the meaning of reassigned codons.

• Keywords
• Codon reassignment, Evolution, Evolutionary constraint, Fornicata, Genetic code, Iotanema spirale, Lygus hesperus, Protists, Rhizaria, Transcriptome,
• MeSH
• Cell Nucleus genetics   MeSH
• Ciliophora genetics   MeSH
• Phylogeny MeSH
• Genetic Code * MeSH
• Glutamine genetics   MeSH
• Insecta parasitology   MeSH
• Codon genetics   MeSH
• Leucine genetics   MeSH
• Evolution, Molecular MeSH
• Open Reading Frames genetics   MeSH
• Rhizaria genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glutamine MeSH
• Codon MeSH
• Leucine MeSH

Regulation of translation is essential for the diverse biological processes involved in development. Particularly, mammalian oocyte development requires the precisely controlled translation of maternal transcripts to coordinate meiotic and early embryo progression while transcription is silent. It has been recently reported that key components of mRNA translation control are short and long noncoding RNAs (ncRNAs). We found that the ncRNABrain cytoplasmic 1 (BC1) has a role in the fully grown germinal vesicle (GV) mouse oocyte, where is highly expressed in the cytoplasm associated with polysomes. Overexpression of BC1 in GV oocyte leads to a minute decrease in global translation with a significant reduction of specific mRNA translation via interaction with the Fragile X Mental Retardation Protein (FMRP). BC1 performs a repressive role in translation only in the GV stage oocyte without forming FMRP or Poly(A) granules. In conclusion, BC1 acts as the translational repressor of specific mRNAs in the GV stage via its binding to a subset of mRNAs and physical interaction with FMRP. The results reported herein contribute to the understanding of the molecular mechanisms of developmental events connected with maternal mRNA translation.

• Keywords
• Non-coding RNA, development, embryo, oocyte, translation,
• MeSH
• Cytoplasm genetics   metabolism   MeSH
• Mice, Inbred ICR MeSH
• Mice MeSH
• RNA, Untranslated genetics   MeSH
• Oocytes cytology   physiology   MeSH
• Oogenesis * MeSH
• Polyribosomes genetics   metabolism   MeSH
• Protein Biosynthesis * MeSH
• RNA, Small Cytoplasmic genetics   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Untranslated MeSH
• RNA, Small Cytoplasmic MeSH

Protein production must be strictly controlled at its beginning and end to synthesize a polypeptide that faithfully copies genetic information carried in the encoding mRNA. In contrast to viruses and prokaryotes, the majority of mRNAs in eukaryotes contain only one coding sequence, resulting in production of a single protein. There are, however, many exceptional mRNAs that either carry short open reading frames upstream of the main coding sequence (uORFs) or even contain multiple long ORFs. A wide variety of mechanisms have evolved in microbes and higher eukaryotes to prevent recycling of some or all translational components upon termination of the first translated ORF in such mRNAs and thereby enable subsequent translation of the next uORF or downstream coding sequence. These specialized reinitiation mechanisms are often regulated to couple translation of the downstream ORF to various stimuli. Here we review all known instances of both short uORF-mediated and long ORF-mediated reinitiation and present our current understanding of the underlying molecular mechanisms of these intriguing modes of translational control.

• MeSH
• Bacteria genetics   metabolism   MeSH
• Eukaryota genetics   MeSH
• Humans MeSH
• Open Reading Frames genetics   MeSH
• Protein Biosynthesis genetics   physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Intramural MeSH