Bacteria use several means to survive under stress conditions such as nutrient depletion. One such response is the formation of hibernating 100S ribosomes, which are translationally inactive 70S dimers. In Gammaproteobacteria (Enterobacterales), 100S ribosome formation requires ribosome modulation factor (RMF) and short hibernation promoting factor (HPF), whereas it is mediated by only long HPF in the majority of bacteria. Here, we investigated the role of HPFs of Comamonas testosteroni, which belongs to the Betaproteobacteria with common ancestor to the Gammaproteobacteria. C. testosteroni has two genes of HPF homologs of differing length (CtHPF-125 and CtHPF-119). CtHPF-125 was induced in the stationary phase, whereas CtHPF-119 conserved in many other Betaproteobacteria was not expressed in the culture conditions used here. Unlike short HPF and RMF, and long HPF, CtHPF-125 could not form 100S ribosome. We first constructed the deletion mutant of Cthpf-125 gene. When the deletion mutant grows in the stationary phase, 70S particles were degraded faster than in the wild strain. CtHPF-125 contributes to stabilizing the 70S ribosome. CtHPF-125 and CtHPF-119 both inhibited protein synthesis by transcription-translation in vitro. Our findings suggest that CtHPF-125 binds to ribosome, and stabilizes 70S ribosomes, inhibits translation without forming 100S ribosomes and supports prolonging life.

Transfer RNAs (tRNAs) are the most highly modified cellular RNAs, both with respect to the proportion of nucleotides that are modified within the tRNA sequence and with respect to the extraordinary diversity in tRNA modification chemistry. However, the functions of many different tRNA modifications are only beginning to emerge. tRNAs have two general clusters of modifications. The first cluster is within the anticodon stem-loop including several modifications essential for protein translation. The second cluster of modifications is within the tRNA elbow, and roles for these modifications are less clear. In general, tRNA elbow modifications are typically not essential for cell growth, but nonetheless several tRNA elbow modifications have been highly conserved throughout all domains of life. In addition to forming modifications, many tRNA modifying enzymes have been demonstrated or hypothesized to additionally play an important role in folding tRNA acting as tRNA chaperones. In this review, we summarize the known functions of tRNA modifying enzymes throughout the lifecycle of a tRNA molecule, from transcription to degradation. Thereby, we describe how tRNA modification and folding by tRNA modifying enzymes enhance tRNA maturation, tRNA aminoacylation, and tRNA function during protein synthesis, ultimately impacting cellular phenotypes and disease.

The antibiotic fusidic acid (FA) is used to treat Staphylococcus aureus infections. It inhibits protein synthesis by binding to elongation factor G (EF-G) and preventing its release from the ribosome after translocation. While FA, due to permeability issues, is only effective against gram-positive bacteria, the available structures of FA-inhibited complexes are from gram-negative model organisms. To fill this knowledge gap, we solved cryo-EM structures of the S. aureus ribosome in complex with mRNA, tRNA, EF-G and FA to 2.5 Å resolution and the corresponding complex structures with the recently developed FA derivative FA-cyclopentane (FA-CP) to 2.0 Å resolution. With both FA variants, the majority of the ribosomal particles are observed in chimeric state and only a minor population in post-translocational state. As expected, FA binds in a pocket between domains I, II and III of EF-G and the sarcin-ricin loop of 23S rRNA. FA-CP binds in an identical position, but its cyclopentane moiety provides additional contacts to EF-G and 23S rRNA, suggesting that its improved resistance profile towards mutations in EF-G is due to higher-affinity binding. These high-resolution structures reveal new details about the S. aureus ribosome, including confirmation of many rRNA modifications, and provide an optimal starting point for future structure-based drug discovery on an important clinical drug target.

The heterotrimeric eIF2 complex consists of a core eIF2γ subunit to which binds eIF2α and eIF2β subunits and plays an important role in delivering the Met-tRNAiMet to the 40S ribosome and start codon selection. The intricacies of eIF2β-γ interaction in promoting Met-tRNAiMet binding are not clearly understood. Previously, the zinc-binding domain (ZBD) eIF2βS264Y mutation was reported to cause Met-tRNAiMet binding defect due to the intrinsic GTPase activity. We showed that the eIF2βS264Y mutation has eIF2β-γ interaction defect. Consistently, the eIF2βT238A intragenic suppressor mutation restored the eIF2β-γ and Met-tRNAiMet binding defect. The eIF2β-ZBD residues Asn252Asp and Arg253Ala mutation caused Met-tRNAiMet binding defect that was partially rescued by the eIF2βT238A mutation, suggesting the eIF2β-ZBD modulates Met-tRNAiMet binding. The suppressor mutation rescued the translation initiation fidelity defect of the eIF2γN135D SW-I mutation and eIF2βF217A/Q221A double mutation in the HTH domain. The eIF2βT238A suppressor mutation could not rescue the eIF2β binding defect of the eIF2γV281K mutation, however, combining the eIF2βS264Y mutation with the eIF2γV281K mutation was lethal. In addition to the previously known interaction of eIF2β with the eIF2γ subunit via its α1-helix, the eIF2β-ZBD also interacts with the eIF2γ subunit via guanine nucleotide-binding interface; thus, the eIF2β-γ interacts via two distinct binding sites.

BACKGROUND: Factors contributing to recurrent pregnancy loss (RPL) in more than half of the cases are still unknown. The incidence and societal impact of this condition requires urgent elucidation of the mechanisms behind it, which could aid in significant improvement of clinical management.
METHODS: Using a highly efficient in-solution digestion method and label-free data-independent LC-MS/MS acquisition with ion mobility, we performed comparative proteomics analysis of the decidua tissues from 19 RPL patients and 10 controls. Differentially abundant proteins (DAPs) were compared and correlated with 3 publicly available transcriptomic datasets and the expression of selected biomarkers was tested by qPCR in decidua and chorionic villi from an extended cohort.
RESULTS: From 1952 proteins identified based on ≥2 peptides, the statistically significant difference in abundance (Anova p ≤ 0.05) and fold change ≥1.2 showed 85 proteins. Pathway analysis using Reactome, KEGG and Wiki pathways identified enrichment of \"Signaling by ROBO receptors\", \"RNA degradation\" and \"Cytoplasmic Ribosomal Proteins\". The correlation between protein and gene expression in decidua revealed that the down-regulated ribosomal proteins in our dataset (RPS15, RPS17, RPL27A, RPL35A and RPL18) showed the same regulation trend at the mRNA level, which was later confirmed for transcripts of RPS15 and RPL18 in our cohort.
CONCLUSIONS: Our data suggests that the potential causes of RPL from the maternal side could be associated with impaired RNA processing machinery. Furthermore, the list of DAPs in RPL opens future investigations in terms of screening novel gene variants predisposing to pregnancy failure and developing biomarkers for RPL risk.

BACKGROUND: Whether there are invasive components in pure ground glass nodules(pGGNs) in the lungs is still a huge challenge to forecast. The objective of our study is to investigate and identify the potential biomarker genes for pure ground glass nodule(pGGN) based on the method of bioinformatics analysis.
METHODS: To investigate differentially expressed genes (DEGs), firstly the data obtained from the gene expression omnibus (GEO) database was used.Next Weighted gene co-expression network analysis (WGCNA) investigate the co-expression network of DEGs. The black key module was chosen as the key one in correlation with pGGN. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyses were done. Then STRING was uesd to create a protein-protein interaction (PPI) network, and the chosen module genes were analyzed by Cytoscape software.In addition the polymerase chain reaction (PCR) was used to evaluate the value of these hub genes in pGGN patients\' tumor tissues compared to controls.
RESULTS: A total of 4475 DEGs were screened out from GSE193725, then 225 DEGs were identified in black key module, which were found to be enriched for various functions and pathways, such as extracellular exosome, vesicle, ribosome and so on. Among these DEGs, 6 overlapped hub genes with high degrees of stress method were selected. These hub genes include RPL4, RPL8, RPLP0, RPS16, RPS2 and CCT3.At last relative expression levels of CCT3 and RPL8 mRNA were both regulated in pGGN patients\' tumor tissues compared to controls.
CONCLUSIONS: To summarize, the determined DEGs, pathways, modules, and overlapped hub genes can throw light on the potential molecular mechanisms of pGGN.

The Picornaviridae is a family of icosahedral viruses with single-stranded, highly diverse positive-sense RNA genomes. Virions consist of a capsid, without envelope, surrounding a core of RNA genome. A typical genome of picornavirus harbors a well-conserved and highly structured RNA element known as the internal ribosome entry site (IRES), functionally essential for viral replication and protein translation. Based on differences in their structures and mechanisms of action, picornaviral IRESs have been categorized into five types: type I, II, III, IV, and V. Compared with the type IV IRES, the others not only are structurally complicated, but also involve multiple initiation factors for triggering protein translation. The type IV IRES, often referred to as hepatitis C virus (HCV)-like IRES due to its structural resemblance to the HCV IRES, exhibits a simpler and more compact structure than those of the other four. The increasing identification of picornaviruses with the type IV IRES suggests that this IRES type seems to reveal strong retention and adaptation in terms of viral evolution. Here, we systematically reviewed structural features and biological functions of the type IV IRES in picornaviruses. A comprehensive understanding of the roles of type IV IRESs will contribute to elucidating the replication mechanism and pathogenesis of picornaviruses.

Age-related dopamine (DA) neuron loss is a primary feature of Parkinson\'s disease. However, it remains unclear whether similar biological processes occur during healthy aging, albeit to a lesser degree. We therefore determined whether midbrain DA neurons degenerate during aging in mice and humans. In mice, we identified no changes in midbrain neuron numbers throughout aging. Despite this, we found age-related decreases in midbrain mRNA expression of tyrosine hydroxylase (Th), the rate limiting enzyme of DA synthesis. Among midbrain glutamatergic cells, we similarly identified age-related declines in vesicular glutamate transporter 2 (Vglut2) mRNA expression. In co-transmitting Th +/Vglut2 + neurons, Th and Vglut2 transcripts decreased with aging. Importantly, striatal Th and Vglut2 protein expression remained unchanged. In translating our findings to humans, we found no midbrain neurodegeneration during aging and identified age-related decreases in TH and VGLUT2 mRNA expression similar to mouse. Unlike mice, we discovered diminished density of striatal TH+ dopaminergic terminals in aged human subjects. However, TH and VGLUT2 protein expression were unchanged in the remaining striatal boutons. Finally, in contrast to Th and Vglut2 mRNA, expression of most ribosomal genes in Th + neurons was either maintained or even upregulated during aging. This suggests a homeostatic mechanism where age-related declines in transcriptional efficiency are overcome by ongoing ribosomal translation. Overall, we demonstrate species-conserved transcriptional effects of aging in midbrain dopaminergic and glutamatergic neurons that are not accompanied by marked cell death or lower striatal protein expression. This opens the door to novel therapeutic approaches to maintain neurotransmission and bolster neuronal resilience.

Gonorrhea, which is caused by Neisseria gonorrhoeae, is the second most prevalent sexually transmitted infection worldwide. The increasing appearance of isolates that are resistant to approved therapeutics raises the concern that gonorrhea may become untreatable. Here, we serendipitously identified oxydifficidin as a potent N. gonorrhoeae antibiotic through the observation of a Bacillus amyloliquefaciens contaminant in a lawn of N. gonorrhoeae. Oxydifficidin is active against both wild-type and multidrug-resistant N. gonorrhoeae. It\'s potent activity results from a combination of DedA-assisted uptake into the cytoplasm and the presence of an oxydifficidin-sensitive ribosomal protein L7/L12 (RplL). Our data indicates that oxydifficidin binds to the ribosome at a site that is distinct from other antibiotics and that L7/L12 is uniquely associated with its mode of action. This study opens a potential new avenue for addressing antibiotic resistant gonorrhea and underscores the possibility of identifying overlooked natural products from cultured bacteria, particularly those with activity against previously understudied pathogens.

Although alternative splicing (AS) is a major mechanism that adds diversity to gene expression patterns, its precise role in generating variability in ribosomal proteins, known as ribosomal heterogeneity, remains unclear. The ribosomal protein S24 (RPS24) gene, encoding a ribosomal component, undergoes AS; however, in-depth studies have been challenging because of three microexons between exons 4 and 6. We conducted a detailed analysis of RPS24 AS isoforms using a direct approach to investigate the splicing junctions related to these microexons, focusing on four AS isoforms. Each of these isoforms showed tissue specificity and relative differences in expression among cancer types. Significant differences in the proportions of these RPS24 AS isoforms between cancerous and normal tissues across diverse cancer types were also observed. Our study highlighted a significant correlation between the expression levels of a specific RPS24 AS isoform and the epithelial-mesenchymal transition process in lung and breast cancers. Our research contributes to a better understanding of the intricate regulatory mechanisms governing AS of ribosomal protein genes and highlights the biological implications of RPS24 AS isoforms in tissue development and tumorigenesis.