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

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

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
• Transcription, Genetic MeSH
• Nuclear Proteins chemistry   genetics   metabolism   MeSH
• Protein Conformation MeSH
• Magnetic Resonance Spectroscopy MeSH
• Protein Multimerization MeSH
• Oligonucleotides chemistry   metabolism   MeSH
• RNA-Binding Proteins chemistry   genetics   metabolism   MeSH
• Solutions MeSH
• Saccharomyces cerevisiae Proteins chemistry   genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   MeSH
• Base Sequence MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Several diseases (atherosclerosis, diabetes mellitus, chronic renal failure) are associated with oxidative and carbonyl stress, microinflammation and eventually autoimmune reaction. Both oxidative and carbonyl stress cause damage to important biological structures-proteins, carbohydrates, lipids and nucleic acids and may enhance inflammatory response. New compounds and modified structures are formed, among them advanced oxidation protein products (AOPP), advanced glycation end products (AGEs-e.g. pentosidine, carboxymethyllysine) and advanced lipoperoxidation end products (ALEs). Accumulation of glycoxidation products, upregulation of protective mechanisms like glyoxalase I as well as enhanced transcription of genes coding for cytokines, growth factors and adhesive molecules via AGE-RAGE (receptor for AGEs) interaction and subsequent increase of classical acute phase reactants (e.g. CRP-C-reactive protein or orosomucoid) can be observed in a variety of chronic diseases. Additionally, several RAGE gene polymorphisms have shown association with some pathological states-diabetic complications, vascular damage, inflammatory response or antioxidant status. Recent advances in understanding the pathogenesis of chronic diseases provide new possibilities for diagnostics and monitoring of severely ill patients, however, further studies are still required to establish efficient therapeutical strategies.

• MeSH
• Chronic Disease MeSH
• Kidney Failure, Chronic complications   metabolism   MeSH
• Diabetes Mellitus genetics   metabolism   MeSH
• Financing, Organized MeSH
• Chemistry, Clinical methods   MeSH
• Lactoylglutathione Lyase genetics   metabolism   MeSH
• Humans MeSH
• Mitogen-Activated Protein Kinases genetics   metabolism   MeSH
• Nucleotides metabolism   MeSH
• Oxidation-Reduction MeSH
• Oxidative Stress MeSH
• Polymorphism, Genetic MeSH
• Glycation End Products, Advanced analysis   metabolism   MeSH
• Receptors, Cytoplasmic and Nuclear metabolism   MeSH
• Inflammation etiology   MeSH
• Check Tag
• Humans MeSH

Kabuki syndrome is mainly caused by dominant de-novo pathogenic variants in the KMT2D and KDM6A genes. The clinical features of this syndrome are highly variable, making the diagnosis of Kabuki-like phenotypes difficult, even for experienced clinical geneticists. Herein we present molecular genetic findings of causal genetic variation using array comparative genome hybridization and a Mendeliome analysis, utilizing targeted exome analysis focusing on regions harboring rare disease-causing variants in Kabuki-like patients which remained KMT2D/KDM6A-negative. The aCGH analysis revealed a pathogenic CNV in the 14q11.2 region, while targeted exome sequencing revealed pathogenic variants in genes associated with intellectual disability (HUWE1, GRIN1), including a gene coding for mandibulofacial dysostosis with microcephaly (EFTUD2). Lower values of the MLL2-Kabuki phenotypic score are indicative of Kabuki-like phenotype (rather than true Kabuki syndrome), where aCGH and Mendeliome analyses have high diagnostic yield. Based on our findings we conclude that for new patients with Kabuki-like phenotypes it is possible to choose a specific molecular testing approach that has the highest detection rate for a given MLL2-Kabuki score, thus fostering more precise patient diagnosis and improved management in these genetically- and phenotypically heterogeneous clinical entities.

• MeSH
• Child MeSH
• DNA-Binding Proteins genetics   MeSH
• Peptide Elongation Factors genetics   MeSH
• Exome MeSH
• Phenotype * MeSH
• Genetic Heterogeneity * MeSH
• Genotype * MeSH
• Histone Demethylases genetics   MeSH
• Nuclear Proteins genetics   MeSH
• Hematologic Diseases diagnosis   genetics   physiopathology   MeSH
• Humans MeSH
• Chromosomes, Human, Pair 14 MeSH
• Ribonucleoprotein, U5 Small Nuclear genetics   MeSH
• Mandibulofacial Dysostosis genetics   MeSH
• Intellectual Disability genetics   MeSH
• Microcephaly genetics   MeSH
• Abnormalities, Multiple diagnosis   genetics   physiopathology   MeSH
• Neoplasm Proteins genetics   MeSH
• Tumor Suppressor Proteins genetics   MeSH
• Face abnormalities   physiopathology   MeSH
• Child, Preschool MeSH
• Nerve Tissue Proteins genetics   MeSH
• Receptors, N-Methyl-D-Aspartate genetics   MeSH
• Comparative Genomic Hybridization MeSH
• Ubiquitin-Protein Ligases genetics   MeSH
• Vestibular Diseases diagnosis   genetics   physiopathology   MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH

• MeSH
• Cell Biology MeSH
• Cell Cycle MeSH
• Cells MeSH
• DNA MeSH
• Genetic Code MeSH
• Genome MeSH
• Molecular Biology MeSH

• Conspectus
• Biochemie. Molekulární biologie. Biofyzika
• NML Fields
• biologie

An extraordinary variation in mitochondrial DNA sequence exists in angiosperm Silene vulgaris. The atp1 gene is flanked by very variable regions, as deduced from four completely sequenced mitochondrial genomes of this species. This diversity contributed to a highly variable transcript profile of this gene observed across S. vulgaris populations. We examined the atp1 transcript in the KOV mitochondrial genome and found three 5' ends, created most likely by the combination of transcription initiation and RNA processing. Most atp1 transcripts terminated about 70 bp upstream of the translation stop codon, which was present in only 10 % of them. Controlled crosses between a KOV mother and a geographically distant pollen donor (Krasnoyarsk, Russia) showed that nuclear background also affected atp1 transcription. The distant pollen donor introduced the factor(s) preventing the formation of a long 2,100 nt-transcript, because this long atp1 transcript reappeared in the progeny from self-crosses. The highly rearranged mitochondrial genomes with a variation in gene flanking regions make S. vulgaris an excellent model for the study of mitochondrial gene expression in plants.

• MeSH
• 5' Untranslated Regions genetics   MeSH
• Cell Nucleus genetics   MeSH
• Transcription, Genetic * MeSH
• Genome, Mitochondrial genetics   MeSH
• Genome, Plant genetics   MeSH
• Gene Rearrangement genetics   MeSH
• Crosses, Genetic MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Molecular Sequence Data MeSH
• Blotting, Northern MeSH
• Open Reading Frames genetics   MeSH
• Pollen genetics   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Proteins genetics   metabolism   MeSH
• Base Sequence MeSH
• Sequence Alignment MeSH
• Silene genetics   MeSH
• Gene Expression Profiling MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Aim. This study was designed to examine whether the class I and class IIa histone deacetylase (HDAC) inhibitors, sodium butyrate and sodium valproate alter the expression of human NCOR1 and/or NCOR2 genes coding for N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone receptors), respectively. Methods. Human leukemia HL-60 cells were treated for 24 h with 0.5 and 1 mM sodium butyrate, 1 to 3 mM sodium valproate, 1 mcM all-trans retinoic acid (ATRA) or cotreated with 1 mcM ATRA and 0.5 mM sodium butyrate. The acetylation of histones H3 and H4 was analysed by western blotting. The levels of NCOR1 and NCOR2 mRNA were determined by quantitative real-time PCR. Expression of NCF2 gene coding for the NADPH oxidase subunit p67phox was evaluated as a marker of myeloid differentiation. Results. Both butyrate and valproate increased the acetylation of histone H3 at Lys9 and/or Lys14 as well as histone H4 at Lys12. Both HDAC inhibitors caused a significant increase in NCF2 mRNA levels without affecting NCOR1 or NCOR2 mRNA levels. Similarly, ATRA alone or in combination with butyrate induced NCF2 gene expression without any significant influence on the expression of NCOR1 or NCOR2 genes. Conclusion. We conclude that inhibitors of class I and class IIa HDACs do not alter the expression of human NCOR1 or NCOR2 genes and that the onset of myeloid differentiation is not accompanied by induction or repression of these genes in HL-60 cells.

• MeSH
• Transcriptional Activation genetics   drug effects   MeSH
• Butyrates antagonists & inhibitors   metabolism   MeSH
• Financing, Organized MeSH
• Genetic Techniques utilization   MeSH
• Histone Deacetylase 1 pharmacokinetics   MeSH
• Histone Deacetylase 2 pharmacokinetics   MeSH
• HL-60 Cells immunology   metabolism   MeSH
• Co-Repressor Proteins genetics   immunology   metabolism   MeSH
• Valproic Acid analogs & derivatives   antagonists & inhibitors   metabolism   MeSH
• Receptors, Cytoplasmic and Nuclear pharmacokinetics   MeSH

In mammals, the noncoding Xist RNA triggers transcriptional silencing of one of the two X chromosomes in female cells. Here, we report a genetic screen for silencing factors in X chromosome inactivation using haploid mouse embryonic stem cells (ESCs) that carry an engineered selectable reporter system. This system was able to identify several candidate factors that are genetically required for chromosomal repression by Xist. Among the list of candidates, we identify the RNA-binding protein Spen, the homolog of split ends. Independent validation through gene deletion in ESCs confirms that Spen is required for gene repression by Xist. However, Spen is not required for Xist RNA localization and the recruitment of chromatin modifications, including Polycomb protein Ezh2. The identification of Spen opens avenues for further investigation into the gene-silencing pathway of Xist and shows the usefulness of haploid ESCs for genetic screening of epigenetic pathways.

• MeSH
• Embryonic Stem Cells metabolism   MeSH
• Haploidy MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Cells, Cultured MeSH
• Mice MeSH
• Polycomb Repressive Complex 2 genetics   metabolism   MeSH
• RNA, Long Noncoding genetics   MeSH
• Gene Silencing * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH