Structure Of Ribosome Research Articles

Transcriptome analysis reveals the effect of high-grain pelleted and non-pelleted diets on ruminal epithelium of Hu-lamb

High-grain non-pelleted (HG) and high-grain pelleted (HP) diets are becoming prevalent for ruminant feeding in intensive farms. However, rare information is about their effect on sheep and the comparison between these two kinds of diets. The current study investigated how HG and HP diets affected the transcriptome profiles of rumen epithelium in Hu-lamb. Fifteen male Hu-lambs were assigned randomly to three groups (n = 5 for each group). Lambs in the control (CON), HG, and HP groups were fed with low-grain non-pelleted diet (30% grain), HG diet (70% grain), and HP diet (70% grain), respectively, for 42 days. All these lambs were slaughtered to collect ruminal epithelium samples for transcriptome analysis. Results showed both HG and HP diets obviously changed the transcriptome profiles, and 192, 319, and three differentially expressed genes (DEGs) were identified for CON_HG, CON_HP, and HG_HP comparisons, respectively. Clusters of orthologous group functional classification of CON_HG and CON_HP DEG datasets both showed the enrichments of DEGs in pathways involved in protein biogenesis and modification as well as energy production and conversion. Kyoto encyclopedia of genes and genomes pathway analysis of CON_HG and CON_HP DEG datasets both displayed the enrichments of DEGs in ribosome and oxidative phosphorylation. Almost all these DEGs involved in translation and ribosomal structure and biogenesis as well as oxidative phosphorylation were downregulated in the HG and HP groups compared to the CON group. Furthermore, CON_HP comparison demonstrated more DEGs related to these two pathways than CON_HG comparison. In conclusion, both HG and HP diets inhibited energy production and conversion as well as protein synthesis and modification in ruminal epithelium. HP diet showed lower growth benefits, induced severer rumen acidosis, and more seriously inhibited energy production and protein synthesis as compared to HG diet.

Abstract P-30: Structure of the 80S Ribosome From Candida Albicans Revealed by Integrative Structural Biology Approach

Background: The fungus Candida albicans is one of the most common fungal human pathogens, causing numerous mucocutaneous infections. About 80% of the world population is colonized with this pathogen, and in many cases, it does not reveal its pathogenic properties. However in the events when the suppression of normal bacterial flora occurs, e.g. as a consequence of chemotherapy or antibiotics treatment, multi-resistant strains of Candida albicans can rapidly colonize the host organism and prevent the normal flora recovery, which in turn can trigger many other diseases especially in the case of immunocompromised patients. Methods: We used an integrated structural biology approach based on the single-particle cryo-EM reconstruction and macromolecule X-ray crystallography. Results: One of the most promising targets for antibiotic action is a protein synthesis apparatus and ribosomes in the cell. In this study, we characterized structurally the protein synthesis machinery of Candida albicans using the state-of-the-art techniques of single-particle Cryo-electron microscopy and macromolecular X-ray crystallography. Conclusion: We obtained the 2.4 Å resolution structure of the 80S ribosome from Candida albicans and the 4.2 Å resolution structure of the vacant C. albicans ribosome by X-ray crystallography. We believe that this study will shed light on mechanisms of antimicrobial resistance in C. albicans and improve candidiasis treatment in the future.

Abstract P-20: Sub 3 Resolution Cryo-EM Structure of Eukaryotic Small (40S) Ribosomal Subunit

Background: The ribosome is a molecular machine that translates mRNAs into proteins. In eukaryotes, ribosome consists of small (40S) and large (60S) subunits. Translation in eukaryotes is a complicated molecular process that involves the formation of various molecular complexes consisting of ribosomal subunits and protein factors. Cryo-EM approaches such as single particle analysis are widely used for structural analysis of components and intermediates of the translation machinery. However, the process of translation in plants is still poorly characterized at a structural level. Here, we present the structure of Triticum aestium small ribosomal subunit obtained at sub 3 resolution that can be used for further structural studies of the translation process in plants. Methods: The structures of the 40S subunits purified from wheat germ extract were obtained using high-resolution single particle cryo-EM. For cryo-EM sample preparation were used Quantifoil R 1.2/1.3 grids coated with an additional 2 nm amorphous carbon film were glow-discharged for 30 seconds at 15 mA using PELCO easiGlow (Ted Pella). 3 µL of the sample were applied onto the grids, blotted for 3 sec at 10°C and 100% humidity, and plunge-frozen in liquid ethane using Vitrobot Mark IV (Thermo Fisher). Cryo-EM data were collected using a Cs-corrected Titan Krios (Thermo Fisher) transmission electron microscope, equipped with a Falcon II direct electron detector. Data were acquired with defocus range of -0.6 to -2.0 at a nominal magnification of 75,000x, giving a calibrated pixel size of 0.86 Å/pixel. The micrographs were recorded as movie stacks. The exposure time for each stack was 1.6 s, corresponding to a total electron dose of ~84 e-/Å2 fractionated into 32 frames (~2.6 e-/Å2 per single frame). A total of 5521 movie stacks was collected. Raw cryo-EM data preprocessing was performed with Warp software (Tegunov et al., 2019). All further data processing steps were performed using the cryoSPARC v3.2.0 software (Punjani et al., 2017). Results: For final cryo-EM map refinement, 140,000 particles were used resulting in 2.7 Å resolution estimated using an FSC=0.143 gold-standard threshold. The obtained structural data clearly demonstrate the peculiarities of the spatial organization of the 40S ribosomal subunit, like the motility of the head relative to the body revealed by 3D variability analysis. Conclusion: The resulting structure was solved at a significantly higher resolution compared to the previously published structure of a plant ribosome (Armache et al., 2010) and will be used as a reference for further studies of translation initiation in plants.

The holobiont transcriptome of teneral tsetse fly species of varying vector competence

BackgroundTsetse flies are the obligate vectors of African trypanosomes, which cause Human and Animal African Trypanosomiasis. Teneral flies (newly eclosed adults) are especially susceptible to parasite establishment and development, yet our understanding of why remains fragmentary. The tsetse gut microbiome is dominated by two Gammaproteobacteria, an essential and ancient mutualist Wigglesworthia glossinidia and a commensal Sodalis glossinidius. Here, we characterize and compare the metatranscriptome of teneral Glossina morsitans to that of G. brevipalpis and describe unique immunological, physiological, and metabolic landscapes that may impact vector competence differences between these two species.ResultsAn active expression profile was observed for Wigglesworthia immediately following host adult metamorphosis. Specifically, ‘translation, ribosomal structure and biogenesis’ followed by ‘coenzyme transport and metabolism’ were the most enriched clusters of orthologous genes (COGs), highlighting the importance of nutrient transport and metabolism even following host species diversification. Despite the significantly smaller Wigglesworthia genome more differentially expressed genes (DEGs) were identified between interspecific isolates (n = 326, ~ 55% of protein coding genes) than between the corresponding Sodalis isolates (n = 235, ~ 5% of protein coding genes) likely reflecting distinctions in host co-evolution and adaptation. DEGs between Sodalis isolates included genes involved in chitin degradation that may contribute towards trypanosome susceptibility by compromising the immunological protection provided by the peritrophic matrix. Lastly, G. brevipalpis tenerals demonstrate a more immunologically robust background with significant upregulation of IMD and melanization pathways.ConclusionsThese transcriptomic differences may collectively contribute to vector competence differences between tsetse species and offers translational relevance towards the design of novel vector control strategies.

Computational Studies of the Structural Basis of Human RPS19 Mutations Associated With Diamond-Blackfan Anemia.

Diamond-Blackfan Anemia (DBA) is an inherited rare disease characterized with severe pure red cell aplasia, and it is caused by the defective ribosome biogenesis stemming from the impairment of ribosomal proteins. Among all DBA-associated ribosomal proteins, RPS19 affects most patients and carries most DBA mutations. Revealing how these mutations lead to the impairment of RPS19 is highly demanded for understanding the pathogenesis of DBA, but a systematic study is currently lacking. In this work, based on the complex structure of human ribosome, we comprehensively studied the structural basis of DBA mutations of RPS19 by using computational methods. Main structure elements and five conserved surface patches involved in RPS19-18S rRNA interaction were identified. We further revealed that DBA mutations would destabilize RPS19 through disrupting the hydrophobic core or breaking the helix, or perturb the RPS19-18S rRNA interaction through destroying hydrogen bonds, introducing steric hindrance effect, or altering surface electrostatic property at the interface. Moreover, we trained a machine-learning model to predict the pathogenicity of all possible RPS19 mutations. Our work has laid a foundation for revealing the pathogenesis of DBA from the structural perspective.

In Vivo and In Vitro Genome-Wide Profiling of RNA Secondary Structures Reveals Key Regulatory Features in Plasmodium falciparum.

It is widely accepted that the structure of RNA plays important roles in a number of biological processes, such as polyadenylation, splicing, and catalytic functions. Dynamic changes in RNA structure are able to regulate the gene expression programme and can be used as a highly specific and subtle mechanism for governing cellular processes. However, the nature of most RNA secondary structures in Plasmodium falciparum has not been determined. To investigate the genome-wide RNA secondary structural features at single-nucleotide resolution in P. falciparum, we applied a novel high-throughput method utilizing the chemical modification of RNA structures to characterize these structures. Structural data from parasites are in close agreement with the known 18S ribosomal RNA secondary structures of P. falciparum and can help to predict the in vivo RNA secondary structure of a total of 3,396 transcripts in the ring-stage and trophozoite-stage developmental cycles. By parallel analysis of RNA structures in vivo and in vitro during the Plasmodium parasite ring-stage and trophozoite-stage intraerythrocytic developmental cycles, we identified some key regulatory features. Recent studies have established that the RNA structure is a ubiquitous and fundamental regulator of gene expression. Our study indicate that there is a critical connection between RNA secondary structure and mRNA abundance during the complex biological programme of P. falciparum. This work presents a useful framework and important results, which may facilitate further research investigating the interactions between RNA secondary structure and the complex biological programme in P. falciparum. The RNA secondary structure characterized in this study has potential applications and important implications regarding the identification of RNA structural elements, which are important for parasite infection and elucidating host-parasite interactions and parasites in the environment.

Conserved Heterodimeric GTPase Rbg1/Tma46 Promotes Efficient Translation in Eukaryotic Cells

Conserved developmentally-regulated GTP-binding (Drg) proteins and their binding partner Dfrp proteins are known to be important for embryonic development, cellular growth control, differentiation and proliferation. Here, we report that the yeast Drg1/Dfrp1 ortholog, Rbg1/Tma46, facilitates translational initiation, elongation and termination via suppressing prolonged ribosome pausing. Consistent with the genome-wide observation, Rbg1 reverses the growth defect resulting from translation stalling and stabilizes mRNAs against no-go decay. Furthermore, we provide a cryoEM structure of the 80S ribosome bound with Rbg1/Tma46 that reveals the molecular interactions responsible for Rbg1/Tma46 function. The Rbg1 subunit binds to the GTPase association center of the ribosome and the A-tRNA, and the N-terminal zinc finger domain of Tma46 subunit binds to the 40S, establishing an interaction critical for the complex ribosomal association. Our results answer the fundamental question on how a paused ribosome resumes translation and show that Drg1/Dfrp1 proteins play a critical role in ensuring orderly translation.

Development of a cell-free protein synthesis system for practical use.

Conventional cell-free protein synthesis systems had been the major platform to study the mechanism behind translating genetic information into proteins, as proven in the central dogma of molecular biology. Albeit being powerful research tools, most of the in vitro methods at the time failed to produce enough protein for practical use. Tremendous efforts were being made to overcome the limitations of in vitro translation systems, though mostly with limited success. While great knowledge was accumulated on the translation mechanism and ribosome structure, researchers rationalized that it may be impossible to fully reconstitute such a complex molecular process in a test tube. This review will examine how we have solved the difficulties holding back progress. Our newly developed cell-free protein synthesis system is based on wheat embryos and has many excellent characteristics, in addition to its high translation activity and robustness. Combined with other novel elementary technologies, we have established cell-free protein synthesis systems for practical use in research and applied sciences.

Differential proteomics of tobacco seedling roots at high and low potassium concentrations

The effects of high potassium and normal potassium treatments on protein expression in roots of flue-cured tobacco plant HKDN-5 at the seedling stage were analyzed by an unlabeled protein quantification technique. The results showed that 555 proteins were differentially expressed (245 proteins were down-regulated and 310 proteins were up-regulated) in high potassium treatment compared with normal potassium treatment. Differentially expressed proteins were involved in 96 metabolic pathways (42 metabolic pathways, 21 synthetic pathways as well as catabolic pathways, including fatty acid metabolism, phenylpropane biosynthesis, ketone body synthesis and degradation, and butyric acid metabolism. Root processing of high potassium concentrations leads to increases in the synthesis of peroxidase, superoxide dismutase and acyl-coenzyme-A synthetase. Additional proteomic differences observed in tobacco roots grown in high potassium include proteins involved with genetic information processing as well as environmental sensing. Examples include RNA helicase, ABC transporters and large subunit GTPases. These up-regulated differentially expressed proteins function mainly in protein translation, ribosome structure and protein synthesis. This indicates that under high potassium treatment, root protein synthetic processes are accelerated and substance metabolism pathways are enhanced; thus, providing the material and energetic basis for root growth.

Study on the mechanism of resistance to cypermethrin in Culex pipiens pallens using proteomics

To compare the differentially expressed proteins between cypermethrin-resistant and -sensitive Culex pipiens pallens, so as to unravel the mechanism underlying the resistance to cypermethrin in Cx. p. pallens. A quantitative proteomic analysis was performed among cypermethrin-sensitive and -resistant isolates of Cx. p. pallens using isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography with tandem mass spectrometry (LC-MS/MS). A total of 164 differentially expressed proteins were identified between cypermethrin-sensitive and -resistant isolates of Cx. p. pallens, including 54 up-regulated proteins and 110 down-regulated proteins. A large number of cuticular proteins, larval cuticular proteins, pupal cuticular proteins and cuticular structural constituent proteins, which are associated with cytoskeletal structure and components, were differentially expressed between cypermethrin-sensitive and -resistant isolates of Cx. p. pallens. Thirteen proteins, which were involved in energy production and conversion, translation, ribosomal structure and biogenesis, lipid transport and metabolism, post-translational modification, protein turnover, chaperones, cytoskeleton and intracellular transportation, were validated to be differentially expressed between cypermethrin-sensitive and -resistant isolates of Cx. p. pallens, which may serve as potential markers of cypermethrin resistance. Multiple insecticide resistance mechanisms contribute to the resistance to cypermethrin in Cx. p. pallens, including cuticular resistance and metabolic resistance, and the cuticular protein genes and cytochrome P450 enzymes may play an important role in the resistance of Cx. p. pallens to cypermethrin.

Binding and Action of Triphenylphosphonium Analog of Chloramphenicol upon the Bacterial Ribosome.

Chloramphenicol (CHL) is a ribosome-targeting antibiotic that binds to the peptidyl transferase center (PTC) of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving the properties of this inhibitor, we explored ribosome binding and inhibitory properties of a semi-synthetic triphenylphosphonium analog of CHL—CAM-C4-TPP. Our data demonstrate that this compound exhibits a ~5-fold stronger affinity for the bacterial ribosome and higher potency as an in vitro protein synthesis inhibitor compared to CHL. The X-ray crystal structure of the Thermus thermophilus 70S ribosome in complex with CAM-C4-TPP reveals that, while its amphenicol moiety binds at the PTC in a fashion identical to CHL, the C4-TPP tail adopts an extended propeller-like conformation within the ribosome exit tunnel where it establishes multiple hydrophobic Van der Waals interactions with the rRNA. The synthesized compound represents a promising chemical scaffold for further development by medicinal chemists because it simultaneously targets the two key functional centers of the bacterial ribosome—PTC and peptide exit tunnel.

Proteome-Wide Analysis of Protein Lysine N-Homocysteinylation in Saccharomyces cerevisiae.

Protein N-homocysteinylation by a homocysteine (Hcy) metabolite, Hcy-thiolactone, is an emerging post-translational modification (PTM) that occurs in all tested organisms and has been linked to human diseases. The yeast Saccharomyces cerevisiae is widely used as a model eukaryotic organism in biomedical research, including studies of protein PTMs. However, patterns of global protein N-homocysteinylation in yeast are not known. Here, we identified 68 in vivo and 197 in vitro N-homocysteinylation sites at protein lysine residues (N-Hcy-Lys). Some of the N-homocysteinylation sites overlap with other previously identified PTM sites. Protein N-homocysteinylation in vivo, induced by supplementation of yeast cultures with Hcy, which elevates Hcy-thiolactone levels, was accompanied by significant changes in the levels of 70 yeast proteins (38 up-regulated and 32 down-regulated) involved in the ribosomal structure, amino acid biosynthesis, and basic cellular pathways. Our study provides the first global survey of N-homocysteinylation and accompanying changes in the yeast proteome caused by elevated Hcy level. These findings suggest that protein N-homocysteinylation and dysregulation of cellular proteostasis may contribute to the toxicity of Hcy in yeast. Homologous proteins and N-homocysteinylation sites are likely to be involved in Hcy-related pathophysiology in humans and experimental animals. Data are available via ProteomeXchange with identifier PXD020821.

A comprehensive examination of the lysine acetylation targets in paper mulberry based on proteomics analyses.

Rocky desertification is a bottleneck that reduces ecological and environmental security in karst areas. Paper mulberry, a unique deciduous tree, shows good performance in rocky desertification areas. Its resistance mechanisms are therefore of high interest. In this study, a lysine acetylation proteomics analysis of paper mulberry seedling leaves was conducted in combination with the purification of acetylated protein by high-precision nano LC-MS/MS. We identified a total of 7130 acetylation sites in 3179 proteins. Analysis of the modified sites showed a predominance of nine motifs. Six positively charged residues: lysine (K), arginine (R), and histidine (H), serine (S), threonine (T), and tyrosine (Y) occurred most frequently at the +1 position, phenylalanine (F) was both detected both upstream and downstream of the acetylated lysines; and the sequence logos showed a strong preference for lysine and arginine around acetylated lysines. Functional annotation revealed that the identified enzymes were mainly involved in translation, transcription, ribosomal structure and biological processes, showing that lysine acetylation can regulate various aspects of primary carbon and nitrogen metabolism and secondary metabolism. Acetylated proteins were enriched in the chloroplast, cytoplasm, and nucleus, and many stress response-related proteins were also discovered to be acetylated, including PAL, HSP70, and ERF. HSP70, an important protein involved in plant abiotic and disease stress responses, was identified in paper mulberry, although it is rarely found in woody plants. This may be further examined in research in other plants and could explain the good adaptation of paper mulberry to the karst environment. However, these hypotheses require further verification. Our data can provide a new starting point for the further analysis of the acetylation function in paper mulberry and other plants.

First comprehensive proteomics analysis of lysine crotonylation in leaves of peanut (Arachis hypogaea L.).

Lysine crotonylation is an important post-translational modification process. Most research in this area has been carried out on mammals and yeast, but there has been little research on it in plants. In the current study, large-scale lysine crotonylome analysis was performed by a combination of affinity enrichment and high-resolution LC-MS/MS analysis. Altogether, 6051 lysine crotonylation sites were identified in 2508 protein groups. Bioinformatics analysis showed that lysine-crotonylated proteins were involved in many biological processes, such as carbon fixation in photosynthetic organisms, biosynthesis of amino acids, ribosomes structure and function. In particular, subcellular localization analysis showed that 43% of the crotonylated proteins were located in the chloroplast. Twenty-nine crotonylation proteins were associated with photosynthesis and functional enrichment that these proteins were associated with the reaction center, photosynthetic electron transport, and ATP synthesis. Based on these results, further studies to expand on the lysine crotonylome analysis were suggested.

A high-throughput RNA-Seq approach to elucidate the transcriptional response of Piriformospora indica to high salt stress

Piriformospora indica, a root endophytic fungus, augments plant nutrition and productivity as well as protects plants against pathogens and abiotic stresses. High salinity is a major problem faced by plants as well as by microbes. Until now, the precise mechanism of salt stress tolerance in P. indica has remained elusive. In this study, the transcriptomes of control and salt-treated (0.5 M NaCl) P. indica were sequenced via the RNA-seq approach. A total of 30,567 transcripts and 15,410 unigenes for P. indica were obtained from 7.3 Gb clean reads. Overall 661 differentially expressed genes (DEGs) between control and treated samples were retrieved. Gene ontology (GO) and EuKaryotic Orthologous Groups (KOG) enrichments revealed that DEGs were specifically involved in metabolic and molecular processes, such as “response to salt stress”, “oxidoreductase activity”, “ADP binding”, “translation, ribosomal structure and biogenesis”, “cytoskeleton”, and others. The unigenes involved in “cell wall integrity”, “sterol biosynthesis”, and “oxidative stress” such as Rho-type GTPase, hydroxymethylglutaryl-CoA synthase, and thioredoxin peroxidase were up-regulated in P. indica subjected to salt stress. The salt-responsive DEGs have shown that they might have a potential role in salt stress regulation. Our study on the salt-responsive DEGs established a foundation for the elucidation of molecular mechanisms related to P. indica stress adaptation and a future reference for comparative functional genomics studies of biotechnologically important fungal species.

The First Whole Genome Sequence and Characterisation of Avian Nephritis Virus Genotype 3.

Avian nephritis virus (ANV) is classified in the Avastroviridae family with disease associations with nephritis, uneven flock growth and runting stunting syndrome (RSS) in chicken and turkey flocks, and other avian species. The whole genome of ANV genotype 3 (ANV-3) of 6959 nucleotides including the untranslated 5′ and 3′ regions and polyadenylated tail was detected in a metagenomic virome investigation of RSS-affected chicken broiler flocks. This report characterises the ANV-3 genome, identifying partially overlapping open reading frames (ORFs), ORF1a and ORF1b, and an opposing secondary pseudoknot prior to a ribosomal frameshift stemloop structure, with a separate ORF2, whilst observing conserved astrovirus motifs. Phylogenetic analysis of the Avastroviridae whole genome and ORF2 capsid polyprotein classified the first complete whole genome of ANV-3 within Avastroviridae genogroup 2.

Ribosome-targeting antibacterial agents: Advances, challenges, and opportunities.

Ribosomes, which synthesize proteins, are critical organelles for the survival and growth of bacteria. About 60% of approved antibiotics discovered so far combat pathogenic bacteria by targeting ribosomes. However, several issues, such as drug resistance and toxicity, have impeded the clinical use of ribosome-targeting antibiotics. Moreover, the complexity of the bacteria ribosome structure has retarded the discovery of new ribosome-targeting agents that are considered as the key to the drug-resistance and toxicity. To deal with these challenges, efforts such as medicinal chemistry optimization, combination treatment, and new drug delivery system have been developed. But not enough, the development of structural biology and new screening methods bring powerful tools, such as cryo-electron microscopy technology, advanced computer-aided drug design, and cell-free in vitro transcription/translation systems, for the discovery of novel ribosome-targeting antibiotics. Thus, in this paper, we overview the research on different aspects of bacterial ribosomes, especially focus on discussing the challenges in the discovery of ribosome-targeting antibacterial drugs and advances made to address issues such as drug-resistance and selectivity, which, we believe, provide perspectives for the discovery of novel antibiotics.

Structure of Erm-modified 70S ribosome reveals the mechanism of macrolide resistance.

Many antibiotics inhibit bacterial growth by binding to the ribosome and interfering with protein biosynthesis. Macrolides represent one of the most successful classes of ribosome-targeting antibiotics. The main clinically-relevant mechanism of resistance to macrolides is dimethylation of the 23S rRNA nucleotide A2058 located in the drug binding site, a reaction catalyzed by the Erm-type rRNA-methyltransferases. Here, we present the crystal structure of the Erm-dimethylated 70S ribosome at 2.4Å resolution together with the structures of unmethylated 70S ribosome functional complexes alone and in combination with macrolides. Altogether, our structural data do not support the previous models and, instead, suggest a principally new explanation of how A2058-dimethylation confers resistance to macrolides. Moreover, high-resolution structures of two macrolide antibiotics bound to the unmodified ribosome revealed a previously unknown role of desosamine moiety in drug binding, laying a foundation for the rational knowledge-based design of macrolides that can overcome Erm-mediated resistance.

142 Nulliparity alters gene expression in inner cell mass and trophoblast of equine blastocysts in old mares

An increased incidence in early embryo loss has been observed in aged mares. Moreover, the first foal born to a mare is lighter than her subsequent foals, with reported impaired placental function at term. Because trophoblast function may be affected from the embryo stage, the aim of this project was to determine the effect of parity in aged mares on gene expression in Day-8.5 embryos. Middle-aged (13.5±2.2 years) nulliparous (never foaled) (ON) or multiparous (1.8±1.6 foals) (OM) Saddlebred, non-nursing mares were inseminated with the semen of one unique stallion. At 8 days post-ovulation (10 days post-hCG), embryos were recovered by uterine flushing and bisected to obtain samples of pure (trophectoderm, TE) or inner cell mass enriched (ICM) trophoblast. Paired end, non-oriented RNA sequencing was performed with Illumina (NextSEqn 500) on 5 and 6 TE and ICM collected from ON and OM, respectively. Differential expression was analysed with DESEqn 2. Embryo size was included in the model and a P<0.05 cutoff was used after false discovery rate correction. Gene set enrichment analysis (GSEA) was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases. Out of the 13 007 and 12 706 genes expressed in ICM and TE, respectively, only 8 in ICM and 6 in TE were differentially expressed, with 2 genes in common. Nevertheless, 19 gene sets were enriched and 6 depleted in the ICM of ON, whereas 2 gene sets were enriched and 8 depleted in the TE of ON compared with OM. Gene sets involved in ribosomal activity and structure, proteasome, integral component of plasma membrane, and immune response were enriched in ICM from ON embryos, and gene sets linked to sphingolipid metabolism, nucleosome, and constituents of the extracellular matrix (ECM) were depleted. In TE from ON mares, enriched gene sets were involved with ribosomes and depleted gene sets were linked to extracellular matrix, focal adhesion, myosin complex, and sequence-specific DNA binding. Overall, 1 enriched (linked to extracellular matrix) and 1 depleted gene set (involved in ribosomal structure) were common to ICM and TE. Thus, embryos from aged nulliparous mares seem to have higher protein turnover and higher immune response compared with those of OM, whereas the depletion of gene sets associated with extracellular matrix and membrane may indicate differences in cellular organisation into lineages. More work is ongoing to study effects on subsequent development.

Theoretical Ribosomal Protein Mass Fingerprint Database

Ribosomes are highly conserved macromolecular machines whose critical function is protein synthesis. However, existence of unique molecular mass of the same type of ribosomal protein for individual species in the same domain of life raises the interesting question concerning the interaction between natural selection forces and the conservation of structure and function of ribosomal proteins. Thus, given differentiated molecular mass and sequence of ribosomal proteins across species, the structures of ribosomes are correspondingly differentiated even though the general structure and function of the macromolecular machine is conserved across species in the same domain of life. The collection of molecular mass of all ribosomal proteins in the large and small ribosome subunits can be understood as the ribosomal protein mass fingerprint of the species useful for gaining fundamental knowledge of ribosomal proteins, as well as serving as tools for species identification through comparison of ribosomal protein mass spectra. This preprint introduces the Theoretical Ribosomal Protein Mass Fingerprint database that comprises the theoretical molecular mass of all ribosomal proteins of a species calculated based on available amino acid sequence information of the ribosomal proteins. Using amino acid sequence information from the Ribosomal Protein Gene Database, the Theoretical Ribosomal Protein Mass Fingerprint database (https://ngwenfa.wordpress.com/database/) spans species from cyanobacteria, fungus, bacteria, archaea, nematodes, diatoms, micro-algae, and various model organisms. The database should be useful as a resource for gaining fundamental understanding of the mass distribution of ribosomal proteins of a species, or serving as a limited reference database for identifying species based on comparing experimental ribosomal protein mass fingerprint of unknown species against theoretically calculated ones of known species. Future expansion of the database will aim to catalogue the theoretical ribosomal protein mass fingerprint of more microbial species using amino acid sequence information from UniProt