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

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.

• Klíčová slova
• Codon reassignment, Evolution, Evolutionary constraint, Fornicata, Genetic code, Iotanema spirale, Lygus hesperus, Protists, Rhizaria, Transcriptome,
• MeSH
• buněčné jádro genetika   MeSH
• Ciliophora genetika   MeSH
• fylogeneze MeSH
• genetický kód * MeSH
• glutamin genetika   MeSH
• hmyz parazitologie   MeSH
• kodon genetika   MeSH
• leucin genetika   MeSH
• molekulární evoluce MeSH
• otevřené čtecí rámce genetika   MeSH
• Rhizaria genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glutamin MeSH
• kodon MeSH
• leucin 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

The phylum Heterolobosea Page and Blanton, 1985 is a group of eukaryotes that contains heterotrophic flagellates, amoebae, and amoeboflagellates, including the infamous brain-eating amoeba Naegleria fowleri. In this study, we investigate the deep evolutionary history of Heterolobosea by generating and analyzing transcriptome data from 16 diverse isolates and combine this with previously published data in a comprehensive phylogenomic analysis. This dataset has representation of all but one of the major lineages classified here as orders. Our phylogenomic analyses recovered a robustly supported phylogeny of Heterolobosea providing a phylogenetic framework for understanding their evolutionary history. Based on the newly recovered relationships, we revised the classification of Heterolobosea to the family level. We describe two new classes (Eutetramitea cl. nov. and Selenaionea cl. nov) and one new order (Naegleriida ord. nov.), and provide a new delimitation of the largest family of Heterolobosea, Vahlkampfiidae Jollos, 1917. Unexpectedly, we unveiled the first two cases of genetic code alterations in the group: UAG as a glutamine codon in the nuclear genome of Dactylomonas venusta and UGA encoding tryptophan in the mitochondrial genome of Neovahlkampfia damariscottae. In addition, analysis of the genome of the latter species confirmed its inability to make flagella, whereas we identified hallmark flagellum-specific genes in most other heteroloboseans not previously observed to form flagellates, suggesting that the loss of flagella in Heterolobosea is much rarer than generally thought. Finally, we define the first autapomorphy of the subphylum Pharyngomonada, represented by a fusion of two key genes for peroxisomal β-oxidation enzymes.

• Klíčová slova
• Alternative genetic code, Cryptic flagella, Mitochondrial genome, Molecular phylogenomics, Naegleria,
• MeSH
• Bayesova věta MeSH
• Eukaryota * genetika   klasifikace   MeSH
• flagella genetika   MeSH
• fylogeneze * MeSH
• genetický kód * MeSH
• molekulární evoluce MeSH
• transkriptom MeSH
• Publikační typ
• časopisecké články MeSH

The canonical stop codons of the nuclear genome of the trypanosomatid Blastocrithidia nonstop are recoded. Here, we investigated the effect of this recoding on the mitochondrial genome and gene expression. Trypanosomatids possess a single mitochondrion and protein-coding transcripts of this genome require RNA editing in order to generate open reading frames of many transcripts encoded as 'cryptogenes'. Small RNAs that can number in the hundreds direct editing and produce a mitochondrial transcriptome of unusual complexity. We find B. nonstop to have a typical trypanosomatid mitochondrial genetic code, which presumably requires the mitochondrion to disable utilization of the two nucleus-encoded suppressor tRNAs, which appear to be imported into the organelle. Alterations of the protein factors responsible for mRNA editing were also documented, but they have likely originated from sources other than B. nonstop nuclear genome recoding. The population of guide RNAs directing editing is minimal, yet virtually all genes for the plethora of known editing factors are still present. Most intriguingly, despite lacking complex I cryptogene guide RNAs, these cryptogene transcripts are stochastically edited to high levels.

• MeSH
• buněčné jádro * genetika   metabolismus   MeSH
• editace RNA * MeSH
• genetický kód MeSH
• genom mitochondriální * MeSH
• guide RNA, Kinetoplastida genetika   metabolismus   MeSH
• kodon genetika   MeSH
• messenger RNA genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• otevřené čtecí rámce genetika   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• RNA transferová * genetika   metabolismus   MeSH
• terminační kodon genetika   MeSH
• Trypanosomatina genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• guide RNA, Kinetoplastida MeSH
• kodon MeSH
• messenger RNA MeSH
• protozoální proteiny MeSH
• RNA transferová * MeSH
• terminační kodon MeSH

Anaerobes have emerged in several major lineages of ciliates, but the number of independent transitions to anaerobiosis among ciliates is unknown. The APM clade (Armophorea, Muranotrichea, Parablepharismea) represents the largest clade of obligate anaerobes among ciliates and contains free-living marine and freshwater representatives as well as gut endobionts of animals. The evolution of APM group has only recently started getting attention, and our knowledge on its phylogeny and genetics is still limited to a fraction of taxa. While ciliates portray a wide array of alternatives to the standard genetic code across numerous classes, the APM ciliates were considered to be the largest group using exclusively standard nuclear genetic code. In this study, we present a pan-ciliate phylogenomic analysis with emphasis on the APM clade, bringing the first phylogenomic analysis of the family Tropidoatractidae (Armophorea) and confirming the position of Armophorida within Armophorea. We include five newly sequenced single cell transcriptomes from marine, freshwater, and endobiotic APM ciliates - Palmarella salina, Anteclevelandella constricta, Nyctotherus sp., Caenomorpha medusula, and Thigmothrix strigosa. We report the first discovery of an alternative nuclear genetic code among APM ciliates, used by Palmarella salina (Tropidoatractidae, Armophorea), but not by its close relative, Tropidoatractus sp., and provide a comparative analysis of stop codon identity and frequency indicating the precedency to the UAG codon loss/reassignment over the UAA codon reassignment in the specific ancestor of Palmarella. Comparative genomic and proteomic studies of this group may help explain the constraints that underlie UAR stop-to-sense reassignment, the most frequent type of alternative nuclear genetic code, not only in ciliates, but eukaryotes in general.

• Klíčová slova
• Alternative genetic code, Anaerobic ciliates, Caenomorpha, Phylogenomics, Single cell transcriptomes, Tropidoatractidae,
• MeSH
• Ciliophora * genetika   MeSH
• fylogeneze MeSH
• genetický kód MeSH
• proteomika * MeSH
• stanovení celkové genové exprese MeSH
• terminační kodon MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• terminační kodon MeSH

RNA polymerase II (RNA pol II) is not only the fundamental enzyme for gene expression but also the central coordinator of co-transcriptional processing. RNA pol II associates with a large number of enzymes and protein/RNA-binding factors through its C-terminal domain (CTD) that consists of tandem repeats of the heptapeptide consensus Y(1)S(2)P(3) T(4)S(5)P(6)S(7). The CTD is posttranslationally modified, yielding specific patterns (often called the CTD code) that are recognized by appropriate factors in coordination with the transcription cycle. Serine phosphorylations are currently the best characterized elements of the CTD code; however, the roles of the proline isomerization and other modifications of the CTD remain poorly understood. The dynamic remodeling of the CTD modifications by kinases, phosphatases, isomerases, and other enzymes introduce changes in the CTD structure and dynamics. These changes serve as structural switches that spatially and temporally regulate the binding of processing factors. Recent structural studies of the CTD bound to various proteins have revealed the basic rules that govern the recognition of these switches and shed light on the roles of these protein factors in the assemblies of the processing machineries.

• MeSH
• genetická transkripce MeSH
• methyltransferasy metabolismus   MeSH
• peptidylprolylisomerasa Pin1 MeSH
• peptidylprolylisomerasa metabolismus   MeSH
• posttranslační úpravy proteinů * MeSH
• prolin metabolismus   MeSH
• proteinfosfatasy MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• RNA-polymerasa II * chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• Saccharomyces cerevisiae enzymologie   genetika   MeSH
• sekvence aminokyselin MeSH
• terciární struktura proteinů MeSH
• transportní proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• ESS1 protein, S cerevisiae MeSH Prohlížeč
• methyltransferasy MeSH
• mRNA (nucleoside-O(2'))-methyltransferase MeSH Prohlížeč
• peptidylprolylisomerasa Pin1 MeSH
• peptidylprolylisomerasa MeSH
• PIN1 protein, human MeSH Prohlížeč
• prolin MeSH
• proteinfosfatasy MeSH
• proteiny vázající RNA MeSH
• RNA-polymerasa II * MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• SSU72 protein, human MeSH Prohlížeč
• transportní proteiny MeSH

Non-coding RNA polymerase II transcripts are processed by the poly(A)-independent termination pathway that requires the Nrd1 complex. The Nrd1 complex includes two RNA-binding proteins, the nuclear polyadenylated RNA-binding (Nab) 3 and the nuclear pre-mRNA down-regulation (Nrd) 1 that bind their specific termination elements. Here we report the solution structure of the RNA-recognition motif (RRM) of Nab3 in complex with a UCUU oligonucleotide, representing the Nab3 termination element. The structure shows that the first three nucleotides of UCUU are accommodated on the β-sheet surface of Nab3 RRM, but reveals a sequence-specific recognition only for the central cytidine and uridine. The specific contacts we identified are important for binding affinity in vitro as well as for yeast viability. Furthermore, we show that both RNA-binding motifs of Nab3 and Nrd1 alone bind their termination elements with a weak affinity. Interestingly, when Nab3 and Nrd1 form a heterodimer, the affinity to RNA is significantly increased due to the cooperative binding. These findings are in accordance with the model of their function in the poly(A) independent termination, in which binding to the combined and/or repetitive termination elements elicits efficient termination.

• MeSH
• genetická transkripce * MeSH
• jaderné proteiny chemie   genetika   metabolismus   MeSH
• konformace proteinů MeSH
• magnetická rezonanční spektroskopie MeSH
• multimerizace proteinu MeSH
• oligonukleotidy chemie   metabolismus   MeSH
• proteiny vázající RNA chemie   genetika   metabolismus   MeSH
• roztoky MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• sekvence nukleotidů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• jaderné proteiny MeSH
• NAB3 protein, S cerevisiae MeSH Prohlížeč
• NRD1 protein, S cerevisiae MeSH Prohlížeč
• oligonukleotidy MeSH
• proteiny vázající RNA MeSH
• roztoky MeSH
• Saccharomyces cerevisiae - proteiny MeSH

The escape of DNA from mitochondria into the nuclear genome (nuclear mitochondrial DNA, NUMT) is an ongoing process. Although pervasively observed in eukaryotic genomes, their evolutionary trajectories in a mammal-wide context are poorly understood. The main challenge lies in the orthology assignment of NUMTs across species due to their fast evolution and chromosomal rearrangements over the past 200 million years. To address this issue, we systematically investigated the characteristics of NUMT insertions in 45 mammalian genomes and established a novel, synteny-based method to accurately predict orthologous NUMTs and ascertain their evolution across mammals. With a series of comparative analyses across taxa, we revealed that NUMTs may originate from nonrandom regions in mtDNA, are likely found in transposon-rich and intergenic regions, and unlikely code for functional proteins. Using our synteny-based approach, we leveraged 630 pairwise comparisons of genome-wide microsynteny and predicted the NUMT orthology relationships across 36 mammals. With the phylogenetic patterns of NUMT presence-and-absence across taxa, we constructed the ancestral state of NUMTs given the mammal tree using a coalescent method. We found support on the ancestral node of Fereuungulata within Laurasiatheria, whose subordinal relationships are still controversial. This study broadens our knowledge on NUMT insertion and evolution in mammalian genomes and highlights the merit of NUMTs as alternative genetic markers in phylogenetic inference.

• Klíčová slova
• evolution, genome microsynteny, mammal, nuclear mitochondrial DNA segment (NUMT),
• MeSH
• buněčné jádro genetika   MeSH
• fylogeneze MeSH
• genom mitochondriální * MeSH
• genomika * MeSH
• mitochondriální DNA genetika   MeSH
• mitochondrie genetika   MeSH
• molekulární evoluce MeSH
• savci genetika   MeSH
• sekvenční analýza DNA MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mitochondriální DNA MeSH