Arbitrariness in the genetic code is one of the main reasons for a linguistic approach to molecular biology: the genetic code is usually understood as an arbitrary relation between amino acids and nucleobases. However, from a semiotic point of view, arbitrariness should not be the only condition for definition of a code, consequently it is not completely correct to talk about "code" in this case. Yet we suppose that there exist a code in the process of protein synthesis, but on a higher level than the nucleic bases chains. Semiotically, a code should be always associated with a function and we propose to define the genetic code not only relationally (in basis of relation between nucleobases and amino acids) but also in terms of function (function of a protein as meaning of the code). Even if the functional definition of meaning in the genetic code has been discussed in the field of biosemiotics, its further implications have not been considered. In fact, if the function of a protein represents the meaning of the genetic code (the sign's object), then it is crucial to reconsider the notion of its expression (the sign) as well. In our contribution, we will show that the actual model of the genetic code is not the only possible and we will propose a more appropriate model from a semiotic point of view.

• Klíčová slova
• Arbitrariness, Biosemiotics, Genetic code, Protein function, Semiotics,
• MeSH
• aminokyseliny chemie   MeSH
• bodová mutace MeSH
• DNA chemie   MeSH
• genetický kód * MeSH
• histony chemie   MeSH
• modely genetické MeSH
• nukleotidy genetika   MeSH
• RNA chemie   MeSH
• sbalování proteinů MeSH
• teoretické modely MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminokyseliny MeSH
• DNA MeSH
• histony MeSH
• nukleotidy MeSH
• RNA MeSH

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.

• Klíčová slova
• Codon reassignment, Evolution, Genetic code, Labyrinthuleans, Mitogenomes, Release factor,
• MeSH
• fylogeneze MeSH
• genetický kód * MeSH
• Heterokontophyta klasifikace   genetika   MeSH
• kodon MeSH
• mitochondrie genetika   MeSH
• molekulární evoluce * MeSH
• proteosyntéza genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kodon 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.

• Klíčová slova
• CRISPR‐Cas9, codon reassignment, genetic code, model organism, trypanosomatids,
• MeSH
• genetický kód * genetika   MeSH
• katalasa genetika   MeSH
• protozoální proteiny genetika   MeSH
• terminační kodon genetika   MeSH
• Trypanosomatina * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• katalasa MeSH
• protozoální proteiny MeSH
• terminační kodon 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.

• Klíčová slova
• Blastocrithidia, In silico, Metabolic predictions, Non-canonical genetic code, Trypanosomatid,
• MeSH
• Eukaryota genetika   MeSH
• genetický kód MeSH
• paraziti * genetika   MeSH
• terminační kodon MeSH
• Trypanosoma brucei brucei * genetika   MeSH
• Trypanosomatina * genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• terminační kodon 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
• genetický kód genetika   MeSH
• kodon genetika   MeSH
• lidé MeSH
• messenger RNA chemie   genetika   MeSH
• modely genetické MeSH
• mutace * MeSH
• otevřené čtecí rámce genetika   MeSH
• proteiny chemie   genetika   metabolismus   MeSH
• proteosyntéza genetika   MeSH
• sbalování proteinů MeSH
• sbalování RNA MeSH
• stabilita RNA genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• kodon MeSH
• messenger RNA MeSH
• proteiny 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.

• Klíčová slova
• AT-rich genomes, eukaryotic release factors, nuclear genetic code, reassigned codon, tRNA structure, termination of translation,
• MeSH
• buněčné jádro * genetika   MeSH
• fylogeneze MeSH
• genetický kód * MeSH
• genom protozoální * MeSH
• genomika MeSH
• molekulární evoluce MeSH
• RNA transferová genetika   MeSH
• terminační kodon genetika   MeSH
• Trypanosomatina * genetika   klasifikace   MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• RNA transferová MeSH
• terminační kodon 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.

• Klíčová slova
• Blastocrithidia, evolution, genetic code, phylogeny, protists, translation, trypanosomatids,
• MeSH
• buněčné jádro genetika   MeSH
• fylogeneze MeSH
• genetický kód genetika   MeSH
• kodon chemie   genetika   MeSH
• protozoální proteiny chemie   genetika   MeSH
• sekvence aminokyselin MeSH
• terminační kodon chemie   genetika   MeSH
• Trypanosomatina genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kodon MeSH
• protozoální proteiny MeSH
• terminační kodon MeSH

The site-specific chemical modification of proteins through incorporation of noncanonical amino acids enables diverse applications, such as imaging, probing, and expanding protein functions, as well as to precisely engineer therapeutics. Here we report a general strategy that allows the incorporation of noncanonical amino acids into target proteins using the amber suppression method and their efficient secretion in the biotechnological relevant expression host Bacillus subtilis. This facilitates efficient purification of target proteins directly from the supernatant, followed by their functionalization using click chemistry. We used this strategy to site-specifically introduce norbornene lysine into a single chain antibody and functionalize it with fluorophores for the detection of human target proteins.

• MeSH
• Bacillus subtilis genetika   metabolismus   MeSH
• click chemie MeSH
• CRISPR-Cas systémy MeSH
• ELISA MeSH
• genetické vektory MeSH
• genetický kód MeSH
• isopropylthiogalaktosid farmakologie   MeSH
• kreatinkinasa, forma MM metabolismus   MeSH
• lidé MeSH
• lysin chemie   MeSH
• norbornany chemie   MeSH
• proteinové inženýrství metody   MeSH
• regulace genové exprese u bakterií účinky léků   MeSH
• rekombinantní proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2-norbornene MeSH Prohlížeč
• isopropylthiogalaktosid MeSH
• kreatinkinasa, forma MM MeSH
• lysin MeSH
• norbornany MeSH
• rekombinantní proteiny MeSH
• zelené fluorescenční proteiny MeSH

Whether we emphasize the notion of 'sign' or the notion of 'code', either way the main interest of biosemiotics and Code Biology is the same, and we argue that the idea of the lower threshold is what still unifies these two groups. Code Biology concentrates on the notion of code: living organisms are defined as code-users or code-makers, and so it may be called the 'lower coding threshold' in this case. The semiotic threshold on the other hand is a concept without a specific definition. There are many possible ways of understanding this term. In order to maintain the lower threshold as the unifying concept between Code Biology and biosemiotics, it is important to be very clear about where this threshold is located and how it is defined. We focus on establishing the lower semiotic threshold at protein biosynthesis, and we propose basing the semiotic understanding of the lowest life forms on the following criteria: arbitrariness, representation, repetition, historicity and self-replication. We also offer that this definition of the lower threshold need not include the notion of interpretation, in the hope that this newly specified common principle of the lower threshold be accepted as a way forward in the conversation between Code Biology and biosemiotics.

• Klíčová slova
• Arbitrariness, Code biology, Evolutionarity, Protein synthesis, Repetition, Representation, Semiosis, Semiotic threshold,
• MeSH
• genetický kód fyziologie   MeSH
• lidé MeSH
• molekulární evoluce * MeSH
• robotika metody   trendy   MeSH
• systémová biologie metody   trendy   MeSH
• umělá inteligence trendy   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy 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.

• Klíčová slova
• Sphaeropleales, codon reassignments, genetic code, green algae, mitogenomes, release factor,
• MeSH
• Chlorophyta genetika   MeSH
• genom mitochondriální MeSH
• kodon * MeSH
• mitochondriální proteiny chemie   genetika   MeSH
• mitochondrie genetika   MeSH
• molekulární evoluce * MeSH
• peptidy - faktory ukončení chemie   genetika   MeSH
• RNA transferová genetika   MeSH
• terminační kodon MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kodon * MeSH
• mitochondriální proteiny MeSH
• peptidy - faktory ukončení MeSH
• RNA transferová MeSH
• terminační kodon MeSH