Cell type-specific analysis by single-cell profiling identifies a stable mammalian tRNA-mRNA interface and increased translation efficiency in neurons

The correlation between codon and anticodon pools influences the efficiency of translation, but whether differences exist in these pools across individual cells is unknown. We determined that codon usage and amino acid demand are highly stable across different cell types using available mouse and human single-cell RNA sequencing atlases. After demonstrating the robustness of ATAC-seq measurements for the analysis of tRNA gene usage, we quantified anticodon usage and amino acid supply in both mouse and human single-cell ATAC-seq atlases. We found that tRNA gene usage is overall coordinated across cell types, except in neurons, which clustered separately from other cell types. Integration of these datasets revealed a strong and statistically significant correlation between amino acid supply and demand across almost all cell types. Neurons have an enhanced translation efficiency over other cell types, driven by an increased supply of tRNAAla (AGC) anticodons. This results in faster decoding of the Ala-GCC codon, as determined by cell type-specific ribosome profiling, suggesting that the reduction of tRNAAla (AGC) anticodon pools may be implicated in neurological pathologies. This study, the first such examination of codon usage, anticodon usage, and translation efficiency resolved at the cell type level with single-cell information, identifies a conserved landscape of translation elongation across mammalian cellular diversity and evolution.

Proteogenomics analysis of CUG codon translation in the human pathogen Candida albicans

Background Yeasts of the CTG-clade lineage, which includes the human-infecting Candida albicans, Candida parapsilosis and Candida tropicalis species, are characterized by an altered genetic code. Instead of translating CUG codons as leucine, as happens in most eukaryotes, these yeasts, whose ancestors are thought to have lost the relevant leucine-tRNA gene, translate CUG codons as serine using a serine-tRNA with a mutated anticodon, $$ {\mathrm{tRNA}}_{\mathrm{CAG}}^{\mathrm{Ser}} $$ tRNA CAG Ser . Previously reported experiments have suggested that 3–5% of the CTG-clade CUG codons are mistranslated as leucine due to mischarging of the $$ {\mathrm{tRNA}}_{\mathrm{CAG}}^{\mathrm{Ser}} $$ tRNA CAG Ser . The mistranslation was suggested to result in variable surface proteins explaining fast host adaptation and pathogenicity. Results In this study, we reassess this potential mistranslation by high-resolution mass spectrometry-based proteogenomics of multiple CTG-clade yeasts, including various C. albicans strains, isolated from colonized and from infected human body sites, and C. albicans grown in yeast and hyphal forms. Our data do not support a bias towards CUG codon mistranslation as leucine. Instead, our data suggest that (i) CUG codons are mistranslated at a frequency corresponding to the normal extent of ribosomal mistranslation with no preference for specific amino acids, (ii) CUG codons are as unambiguous (or ambiguous) as the related CUU leucine and UCC serine codons, (iii) tRNA anticodon loop variation across the CTG-clade yeasts does not result in any difference of the mistranslation level, and (iv) CUG codon unambiguity is independent of C. albicans’ strain pathogenicity or growth form. Conclusions Our findings imply that C. albicans does not decode CUG ambiguously. This suggests that the proposed misleucylation of the $$ {\mathrm{tRNA}}_{\mathrm{CAG}}^{\mathrm{Ser}} $$ tRNA CAG Ser might be as prevalent as every other misacylation or mistranslation event and, if at all, be just one of many reasons causing phenotypic diversity.

Structural basis of Ty3 retrotransposon integration at RNA Polymerase III-transcribed genes

Retrotransposons are endogenous elements that have the ability to mobilise their DNA between different locations in the host genome. The Ty3 retrotransposon integrates with an exquisite specificity in a narrow window upstream of RNA Polymerase (Pol) III-transcribed genes, representing a paradigm for harmless targeted integration. Here we present the cryo-EM reconstruction at 4.0 Å of an active Ty3 strand transfer complex bound to TFIIIB transcription factor and a tRNA gene. The structure unravels the molecular mechanisms underlying Ty3 targeting specificity at Pol III-transcribed genes and sheds light into the architecture of retrotransposon machinery during integration. Ty3 intasome contacts a region of TBP, a subunit of TFIIIB, which is blocked by NC2 transcription regulator in RNA Pol II-transcribed genes. A newly-identified chromodomain on Ty3 integrase interacts with TFIIIB and the tRNA gene, defining with extreme precision the integration site position.

Rapid Shifts in Mitochondrial tRNA Import in a Plant Lineage with Extensive Mitochondrial tRNA Gene Loss

In most eukaryotes, transfer RNAs (tRNAs) are one of the very few classes of genes remaining in the mitochondrial genome, but some mitochondria have lost these vestiges of their prokaryotic ancestry. Sequencing of mitogenomes from the flowering plant genus Silene previously revealed a large range in tRNA gene content, suggesting rapid and ongoing gene loss/replacement. Here, we use this system to test longstanding hypotheses about how mitochondrial tRNA genes are replaced by importing nuclear-encoded tRNAs. We traced the evolutionary history of these gene loss events by sequencing mitochondrial genomes from key outgroups (Agrostemma githago and Silene [=Lychnis] chalcedonica). We then performed the first global sequencing of purified plant mitochondrial tRNA populations to characterize the expression of mitochondrial-encoded tRNAs and the identity of imported nuclear-encoded tRNAs. We also confirmed the utility of high-throughput sequencing methods for the detection of tRNA import by sequencing mitochondrial tRNA populations in a species (Solanum tuberosum) with known tRNA trafficking patterns. Mitochondrial tRNA sequencing in Silene revealed substantial shifts in the abundance of some nuclear-encoded tRNAs in conjunction with their recent history of mt-tRNA gene loss and surprising cases where tRNAs with anticodons still encoded in the mitochondrial genome also appeared to be imported. These data suggest that nuclear-encoded counterparts are likely replacing mitochondrial tRNAs even in systems with recent mitochondrial tRNA gene loss, and the redundant import of a nuclear-encoded tRNA may provide a mechanism for functional replacement between translation systems separated by billions of years of evolutionary divergence.

Complete Genome Sequence of Mycobacteriophage Joy99

Joy99 is a siphoviral mycobacteriophage with a 59,837-base pair double-stranded DNA genome and is predicted to contain 97 protein-coding genes and a single tRNA gene. Joy99 was isolated in Saint Louis, MO, and annotated by students at Bluff Dale High School in community engagement with Tarleton State University.

Frequent tRNA gene translocation towards the boundaries with control regions contributes to the highly dynamic mitochondrial genome organization of the parasitic lice of mammals

Background The typical single-chromosome mitochondrial (mt) genome of animals has fragmented into multiple minichromosomes in the lineage Mitodivisia, which contains most of the parasitic lice of eutherian mammals. These parasitic lice differ from each other even among congeneric species in mt karyotype, i.e. the number of minichromosomes, and the gene content and gene order in each minichromosome, which is in stark contrast to the extremely conserved single-chromosome mt genomes across most animal lineages. How fragmented mt genomes evolved is still poorly understood. We use Polyplax sucking lice as a model to investigate how tRNA gene translocation shapes the dynamic mt karyotypes. Results We sequenced the full mt genome of the Asian grey shrew louse, Polyplax reclinata. We then inferred the ancestral mt karyotype for Polyplax lice and compared it with the mt karyotypes of the three Polyplax species sequenced to date. We found that tRNA genes were entirely responsible for mt karyotype variation among these three species of Polyplax lice. Furthermore, tRNA gene translocation observed in Polyplax lice was only between different types of minichromosomes and towards the boundaries with the control region. A similar pattern of tRNA gene translocation can also been seen in other sucking lice with fragmented mt genomes. Conclusions We conclude that inter-minichromosomal tRNA gene translocation orientated towards the boundaries with the control region is a major contributing factor to the highly dynamic mitochondrial genome organization in the parasitic lice of mammals.

Structural basis of Ty3 retrotransposon integration at RNA Polymerase III-transcribed genes

Retrotransposons are endogenous elements that have the ability to mobilise their DNA and integrate at different locations in the host genome. In budding yeast, the Ty3 retrotransposon integrates with an exquisite specificity in a narrow window upstream of RNA Polymerase III-transcribed genes, such as the genes of transfer RNAs, representing a paradigm for specific targeted integration. Here we present the cryo-EM reconstruction at 4.0 Å-resolution of an active Ty3 strand-transfer complex (Ty3 intasome) caught in the act of integrating onto a specific tRNA gene bound to the RNA Polymerase III general transcription factor TFIIIB, which is required for Ty3 specific targeting. The structure unravels the molecular mechanisms underlying Ty3 integration specificity at RNA Polymerase III-transcribed genes and sheds light into the architecture of a retrotransposon integration machinery during the process of strand transfer at a genomic locus. The Ty3 intasome establishes contacts with a region of the TATA-binding protein (TBP), a subunit of TFIIIB, which is blocked by the ubiquitous transcription regulator negative cofactor 2 (NC2) in RNA Pol II-transcribed genes. A previously unrecognised chromodomain of the Ty3 integrase mediates non-canonical interactions with TFIIIB and the tRNA gene itself, defining with extreme precision the position of the integration site. Surprisingly, Ty3 retrotransposon tethering to TFIIIB topologically resembles LEDGF/p75 transcription factor targeting by HIV retrovirus, highlighting mechanisms of convergent evolution by unrelated mobile elements and host organisms. The Ty3 intasome-TFIIIB-tRNA promoter complex presented here represents a detailed molecular snapshot of a general transcription factor's co-option by a mobile element, resulting in harmless integration into the host genome.

Rapid shifts in mitochondrial tRNA import in a plant lineage with extensive mitochondrial tRNA gene loss

In most eukaryotes, transfer RNAs (tRNAs) are one of the very few classes of genes remaining in the mitochondrial genome, but some mitochondria have lost these vestiges of their prokaryotic ancestry. Sequencing of mitogenomes from the flowering plant genus Silene previously revealed a large range in tRNA gene content, suggesting rapid and ongoing gene loss/replacement. Here, we use this system to test longstanding hypotheses about how mitochondrial tRNA genes are replaced by importing nuclear-encoded tRNAs. We traced the evolutionary history of these gene loss events by sequencing mitochondrial genomes from key outgroups (Agrostemma githago and Silene [=Lychnis] chalcedonica). We then performed the first global sequencing of purified plant mitochondrial tRNA populations to characterize the expression of mitochondrial-encoded tRNAs and the identity of imported nuclear-encoded tRNAs. We also confirmed the utility of high-throughput sequencing methods for the detection of tRNA import by sequencing mitochondrial tRNA populations in a species (Solanum tuberosum) with known tRNA trafficking patterns. Mitochondrial tRNA sequencing in Silene revealed substantial shifts in the abundance of some nuclear-encoded tRNAs in conjunction with their recent history of mt-tRNA gene loss and surprising cases where tRNAs with anticodons still encoded in the mitochondrial genome also appeared to be imported. These data suggest that nuclear-encoded counterparts are likely replacing mitochondrial tRNAs even in systems with recent mitochondrial tRNA gene loss, and the redundant import of a nuclear-encoded tRNA may provide a mechanism for functional replacement between translation systems separated by billions of years of evolutionary divergence.