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
• Cell Nucleus * genetics   metabolism   MeSH
• RNA Editing * MeSH
• Genetic Code MeSH
• Genome, Mitochondrial * MeSH
• RNA, Guide, Kinetoplastida genetics   metabolism   MeSH
• Codon genetics   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Mitochondria genetics   metabolism   MeSH
• Open Reading Frames genetics   MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• RNA, Transfer * genetics   metabolism   MeSH
• Codon, Terminator genetics   MeSH
• Trypanosomatina genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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.

• 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

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.

• 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

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.

• 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

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.

• 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