Large Ribosomal Subunit Protein Research Articles

Abstract 3100: BRCA1-RpL17 Interaction Regulates Angiogenesis

Background: Angiogenesis, forming new blood vessels from existing ones, is crucial in development, diabetes, and cancer. We found that Large Ribosomal subunit protein 17 (RpL17) played a key role in endothelial cell (EC) proliferation and angiogenesis. Reduced RpL17 (KD) promoted angiogenesis in human ECs and RpL17-deficient mouse retinas. RNA-sequencing and bioinformatics suggested a role for Early growth response 1(EGR1) transcription factor. Bioinformatics revealed Breast Cancer gene 1 (BRCA1) as a potential upstream regulator of RpL17, suggesting a novel pathway for angiogenesis. Hypothesis: BRCA1 interacts with RpL17 and upregulates EGR1 to stimulate vessel elongation in angiogenesis. Goal: Determine the role of BRCA1-RpL17 interaction in angiogenesis and their potential feedback loop via EGR1. Methods/Approach: RNA-sequencing of mouse ECs, whole retina mount staining (comparing wild-type and RpL17+/- mice). Tube formation assays with HUVECs to investigate angiogenesis after RpL17, BRCA1, and EGR1 knockdowns. Bioinformatics analysis with Python for RNA-seq data. Immunoblotting and RT-qPCR to measure protein and DNA expression. Immunofluorescence (IF) staining of HUVECs to observe BRCA1, RpL17, and EGR1 expression and localization. Preliminary Results: Whole retina mounts showed increased angiogenesis in RpL17-deficient mouse retinas compared to wildtype (p<0.009). HUVECs with RpL17 KD had increased junctions and longer branches than control HUVECs (p<0.0002). RNA-seq identified increased EGR1 expression (q<2.5x10 -8 ) in RpL17 KD ECs, confirmed with RT-qPCR (p<0.002). Bioinformatics predicted BRCA1 as a regulator of RpL17. HUVEC IF staining showed decreased RpL17 expression after BRCA1 KD. Angiogenesis assays of BRCA1 KD ECs exhibited an angiogenic phenotype like RpL17 KD, rescued following double BRCA1/RpL17 KD. Conclusion: BRCA1-RpL17 interaction stimulates angiogenesis and may be regulated by an EGR1 feedback loop, likely by EC proliferation. Detailed understanding of this pathway will provide new therapies to regulate angiogenesis.

Dynamic protein composition of Saccharomyces cerevisiae ribosomes in response to multiple stress conditions reflects alterations in translation activity

Ribosomes, intercellular macromolecules responsible for translation in the cell, are composed of RNAs and proteins. While rRNA makes the scaffold of the ribosome and directs the catalytic steps of protein synthesis, ribosomal proteins play a role in the assembly of the subunits and are essential for the proper structure and function of the ribosome. To date researchers identified heterogeneous ribosomes in different developmental and growth stages. We hypothesized that under stress conditions the heterogeneity of the ribosomes may provide means to prepare the cells for quick recovery. Therefore the aim of the study was the identification of heterogeneity of ribosomal proteins within the ribosomes in response to eleven stress conditions in Saccharomyces cerevisiae, by means of a liquid chromatography/high resolution mass spectrometry (LC-HRMS) and translation activity tests. Out of the total of 74 distinct ribosomal proteins identified in the study 14 small ribosomal subunit (RPS) and 8 large ribosomal subunit (RPL) proteins displayed statistically significant differential abundances within the ribosomes under stress. Additionally, significant alterations in the ratios of 7 ribosomal paralog proteins were observed. Accordingly, the translational activity of yeast ribosomes was altered after UV exposure, during sugar starvation, cold shock, high salt, anaerobic conditions, and amino acid starvation.

The zinc finger motif in the mitochondrial large ribosomal subunit protein bL36m is essential for optimal yeast mitoribosome assembly and function

Ribosomes across species contain subsets of zinc finger proteins that play structural roles by binding to rRNA. While the majority of these zinc fingers belong to the C2-C2 type, the large subunit protein L36 in bacteria and mitochondria exhibits an atypical C2-CH motif. To comprehend the contribution of each coordinating residue in S. cerevisiae bL36m to mitoribosome assembly and function, we engineered and characterized strains carrying single and double mutations in the zinc coordinating residues. Our findings reveal that although all four residues markedly influence protein stability, C to A mutations in C66 and/or C69 have a more pronounced effect compared to those at C82 and H88. Importantly, protein stability directly correlates with the assembly and function of the mitoribosome and the growth rate of yeast in respiratory conditions. Mass spectrometry analysis of large subunit particles indicates that strains deleted for bL36m or expressing mutant variants have defective assembly of the L7/L12 stalk base, limiting their functional competence. Furthermore, we employed a synthetic bL36m protein collection, including both wild-type and mutant proteins, to elucidate their ability to bind zinc. Our data indicate that mutations in C82 and, particularly, H88 allow for some zinc binding albeit inefficient or unstable, explaining the residual accumulation and activity in mitochondria of bL36m variants carrying mutations in these residues. In conclusion, stable zinc binding by bL36m is essential for optimal mitoribosome assembly and function. MS data are available via ProteomeXchange with identifierPXD046465.

Mirror-image trypsin digestion and sequencing of D-proteins.

The development of mirror-image biology systems and related applications is hindered by the lack of effective methods to sequence mirror-image (D-) proteins. Although natural-chirality (L-) proteins can be sequenced by bottom-up liquid chromatography-tandem mass spectrometry (LC-MS/MS), the sequencing of long D-peptides and D-proteins with the same strategy requires digestion by a site-specific D-protease before mass analysis. Here we apply solid-phase peptide synthesis and native chemical ligation to chemically synthesize a mirror-image version of trypsin, a widely used protease for site-specific protein digestion. Using mirror-image trypsin digestion and LC-MS/MS, we sequence a mirror-image large subunit ribosomal protein (L25) and a mirror-image Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), and distinguish between different mutants of D-Dpo4. We also perform writing and reading of digital information in a long D-peptide of 50 amino acids. Thus, mirror-image trypsin digestion in conjunction with LC-MS/MS may facilitate practical applications of D-peptides and D-proteins as potential therapeutic and informational tools.

Extracellular Proteins Isolated from L. acidophilus as an Osteomicrobiological Therapeutic Agent to Reduce Pathogenic Biofilm Formation, Regulate Chronic Inflammation, and Augment Bone Formation In Vitro.

Periprosthetic joint infection (PJI) is a challenging complication that can occur following joint replacement surgery. Efficacious strategies to prevent and treat PJI and its recurrence remain elusive. Commensal bacteria within the gut convey beneficial effects through a defense strategy named "colonization resistance" thereby preventing pathogenic infection along the intestinal surface. This blueprint may be applicable to PJI. The aim is to investigate Lactobacillus acidophilus spp. and their isolated extracellular-derived proteins (LaEPs) on PJI-relevant Staphylococcus aureus, methicillin-resistant S. aureus, and Escherichia coli planktonic growth and biofilm formation in vitro. The effect of LaEPs on cultured macrophages and osteogenic, and adipogenic human bone marrow-derived mesenchymal stem cell differentiation is analyzed. Data show electrostatically-induced probiotic-pathogen species co-aggregation and pathogenic growth inhibition together with LaEP-induced biofilm prevention. LaEPs primemacrophages for enhanced microbial phagocytosis via cathepsin K, reduce lipopolysaccharide-induced DNA damage and receptor activator nuclear factor-kappa B ligand expression, and promote a reparative M2 macrophage morphology under chronic inflammatory conditions. LaEPs also significantly augment bone deposition while abating adipogenesis thus holding promise as a potential multimodal therapeutic strategy. Proteomic analyses highlight high abundance of lysyl endopeptidase, and urocanate reductase. Further, in vivo analyses are warranted to elucidate their role in the prevention and treatment of PJIs.

Functional characterization of differentially expressed proteins coming from unisexual and bisexual infected Schistosoma japonicum female worms

Schistosomiasis is an important zoonotic disease affecting up to 40 kinds of animals and is responsible for ∼250 million human cases per year. Due to the extensive use of praziquantel for the treatment of parasitic diseases, drug resistance has been reported. Consequently, novel drugs and effective vaccines are urgently needed for sustained control of schistosomiasis. Targeting reproductive development of Schistosoma japonicum could contribute to the control of schistosomiasis. In this study, five highly expressed proteins (S. japonicum large subunit ribosomal protein L7e, S. japonicum glutathione S-transferase class-mu 26 kDa isozyme, S. japonicum UDP-galactose-4-epimerase and two hypothetical proteins SjCAX70849 and SjCAX72486) in 18, 21, 23, and 25-day mature female worms compared to single-sex infected female worms were selected based on our previous proteomic analysis. Quantitative real-time polymerase chain reaction analysis and long-term interference with small interfering RNA were performed to identify the biological functions of these five proteins. The transcriptional profiles suggested that all five proteins participated in the maturation of S. japonicum. RNA interference against these proteins resulted in morphological changes to S. japonicum. The results of an immunoprotection assay revealed that immunization of mice with recombinant SjUL-30 and SjCAX72486 upregulated production of immunoglobulin G-specific antibodies. Collectively, the results demonstrated that these five differentially expressed proteins were vital to reproduction of S. japonicum and, thus, are potential candidate antigens for immune protection against schistosomiasis.

Dynamic RNA-Seq Study Reveals the Potential Regulators of Seed Germination in Paris polyphylla var. yunnanensis.

Paris polyphylla var. yunnanensis is an important traditional Chinese medicine, but poor seed germination limits its large-scale artificial cultivation. Thus, it is crucial to understand the regulators of seed germination to obtain clues about how to improve the artificial cultivation of Paris polyphylla. In this study, the seeds at three germination stages, including ungerminated seeds (stage 1), germinated seeds with a 0.5 cm radicel length (stage 2), and germinated seeds with a 2.0 cm radicel length (stage 3) after warm stratification (20 °C) for 90 days were used for RNA sequencing. Approximately 220 million clean reads and 447,314 annotated unigenes were obtained during seed germination, of which a total of 4454, 5150, and 1770 differentially expressed genes (DEGs) were identified at stage 1 to stage 2, stage 1 to stage 3, and stage 2 to stage 3, respectively. Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the DEGs were significantly enriched in carbohydrate metabolism, lipid metabolism, signal transduction, and translation. Of them, several genes encoding the glutamate decarboxylase, glutamine synthetase, alpha-galactosidase, auxin-responsive protein IAA30, abscisic-acid-responsive element binding factor, mitogen-activated protein kinase kinase 9/18, and small and large subunit ribosomal proteins were identified as potentially involved in seed germination. The identified genes provide a valuable resource to study the molecular basis of seed germination in Paris polyphylla var. yunnanensis.

Abstract 106: Ribosomal Protein Rpl17 Controls Endothelial Cell Translation: A Key Mediator Of Intima Growth And Vascular Disease

Background: Carotid intima-media thickening (IMT) is a significant risk factor for cardiovascular disease (CVD). Using genetic analysis of the partial carotid ligation (PCL) model of disturbed flow induced IMT, we identified a strong association between decreased expression of the large ribosomal subunit protein 17 (RpL17) and increased IMT. RpL17 is located at the exit tunnel of the ribosome, making it critical for processing of newly translated proteins. To define the mechanism by which RpL17 inhibits IMT, we generated EC-restricted (Cdh5-Cre) conditional RpL17 heterozygous mice (EC-RpL17). Results: 1) PCL of EC-RpL17 increased IMT vs wild type (Control) littermates. 2) EM analysis revealed decreases in total ribosome number and polysomes in EC-RpL17, consistent with decreased global translation. 3) RNA-seq showed major changes in two pathways: Eukaryotic Initiation Factor 2 (eIF2α) that promotes adaptive translational responses to stress, and oxidative phosphorylation (OXPHOS), the electron transport metabolic pathway that produces ATP. 4) Profiling of mRNAs bound to polysomes in RpL17-EC revealed increased translational efficiency (TE) of selective redox regulators, pro-inflammatory mediators, matrix metalloproteases, and endothelial to mesenchymal transition (endMT) factors; consistent with processes that promote IMT. 5) Increased levels of RNA binding proteins (RBPs) in EC-RpL17, such as QKI, HuR, and hn-RNP, correlated with enhanced stability of specific mRNAs and their TE. 6) Increased TE of glycolytic enzymes and the lactate transporter was consistent with increased EC glycolysis and OXPHOS dysfunction. 7) Increased glycolysis correlated with greater cell proliferation, suggesting RpL17 is a growth suppressor. Conclusion: These data define a new mechanism for control of EC function via selective changes in mRNA stability and ribosome profile and number that decreased OXPHOS and global protein translation, leading to EC-proliferation and endMT, which enhanced IMT in the PCL model of disturbed flow.

Evolutionarily conserved inhibitory uORFs sensitize Hox mRNA translation to start codon selection stringency

Translation start site selection in eukaryotes is influenced by context nucleotides flanking the AUG codon and by levels of the eukaryotic translation initiation factors eIF1 and eIF5. In a search of mammalian genes, we identified five homeobox (Hox) gene paralogs initiated by AUG codons in conserved suboptimal context as well as 13 Hox genes that contain evolutionarily conserved upstream open reading frames (uORFs) that initiate at AUG codons in poor sequence context. An analysis of published cap analysis of gene expression sequencing (CAGE-seq) data and generated CAGE-seq data for messenger RNAs (mRNAs) from mouse somites revealed that the 5' leaders of Hox mRNAs of interest contain conserved uORFs, are generally much shorter than reported, and lack previously proposed internal ribosome entry site elements. We show that the conserved uORFs inhibit Hox reporter expression and that altering the stringency of start codon selection by overexpressing eIF1 or eIF5 modulates the expression of Hox reporters. We also show that modifying ribosome homeostasis by depleting a large ribosomal subunit protein or treating cells with sublethal concentrations of puromycin leads to lower stringency of start codon selection. Thus, altering global translation can confer gene-specific effects through altered start codon selection stringency.

Trap-TRAP, a Versatile Tool for Tissue-Specific Translatomics in Zebrafish.

Developmental and physiological processes depend on the transcriptional and translational activity of heterogeneous cell populations. A main challenge in gene expression studies is dealing with this intrinsic complexity while keeping sequencing efficiency. Translating ribosome affinity purification (TRAP) methods have allowed cell-specific recovery of polyribosome-associated RNAs by genetic tagging of ribosomes in selected cell populations. Here we combined the TRAP approach with adapted enhancer trap methods (trap-TRAP) to systematically generate zebrafish transgenic lines suitable for tissue-specific translatome interrogation. Through the random integration of a GFP-tagged version of the large subunit ribosomal protein L10a (EGFP-Rpl10a), we have generated stable lines driving expression in a variety of tissues, including the retina, skeletal muscle, lateral line primordia, rhombomeres, or jaws. To increase the range of applications, a UAS:TRAP transgenic line compatible with available Gal4 lines was also generated and tested. The resulting collection of lines and applications constitutes a resource for the zebrafish community in developmental genetics, organ physiology and disease modelling.

RplI interacts with 5' UTR of exsA to repress its translation and type III secretion system in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important opportunistic pathogen capable of causing variety of infections in humans. The type III secretion system (T3SS) is a critical virulence determinant of P. aeruginosa in the host infections. Expression of the T3SS is regulated by ExsA, a master regulator that activates the expression of all known T3SS genes. Expression of the exsA gene is controlled at both transcriptional and posttranscriptional levels. Here, we screened a P. aeruginosa transposon (Tn5) insertional mutant library and found rplI, a gene coding for the ribosomal large subunit protein L9, to be a repressor for the T3SS gene expression. Combining real-time quantitative PCR (qPCR), western blotting and lacZ fusion assays, we show that RplI controls the expression of exsA at the posttranscriptional level. Further genetic experiments demonstrated that RplI mediated control of the exsA translation involves 5’ untranslated region (5’ UTR). A ribosome immunoprecipitation assay and qPCR revealed higher amounts of a 24 nt fragment from exsA mRNA being associated with ribosomes in the ΔrplI mutant. An interaction between RplI and exsA mRNA harboring its 24 nt, but not 12 nt, 5’ UTR was confirmed by RNA Gel Mobility Shift and Microscale Thermophoresis assays. Overall, this study identifies the ribosomal large subunit protein L9 as a novel T3SS repressor that inhibits ExsA translation in P. aeruginosa.

Ionizing Radiation-Induced RPL23a Reduction Regulates Apoptosis via RPL11-MDM2-p53 Pathway in Mouse Spermatogonia

ObjectiveThe expression patterns of ribosomal large subunit protein 23a (RPL23a) in mouse testes and GC-1 cells were analyzed to investigate the potential relationship between RPL23a expression and spermatogonia apoptosis upon exposure to X-ray. MethodsMale mice and GC-1 cells were irradiated with X-ray, terminal dUTP nick end-labelling (TUNEL) was performed to detect apoptotic spermatogonia in vivo. Apoptotic rate and cell cycle phase of GC-1 cells were analyzed with flow cytometry. Protein interactions were detected by Immunoprecipitation and protein localization as studied by immunofluorescence. Immunoblotting and real-time PCR were applied to analyze to protein and gene expression. ResultsIonizing radiation (IR) increased spermatogonia apoptosis, the expression of RPL11, MDM2 and p53, and decreased RPL23a expression in mice spermatogonia in vivo and in vitro. RPL23a knockdown weakened the interaction between RPL23a and RPL11, leading to p53 accumulation. Moreover, knockdown and IR decreased RPL23a that induces spermatogonia apoptosis via RPL23a-RPL11-MDM2-p53 pathway in GC-1 cells. ConclusionThese results suggested that IR reduced RPL23a expression, leading to weakened the RPL23a-RPL11 interactions, which may have activated p53, resulting in spermatogonia apoptosis. These results provide insights into environmental and clinical risks of radiotherapy following exposure to IR in male fertility.The graphical abstract was available in the web of www.besjournal.com.

Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors.

In yeast and human cells many of the ribosomal proteins (r-proteins) are required for the stabilisation and productive processing of rRNA precursors. Functional coupling of r-protein assembly with the stabilisation and maturation of subunit precursors potentially promotes the production of ribosomes with defined composition. To further decipher mechanisms of such an intrinsic quality control pathway we analysed here the contribution of three yeast large ribosomal subunit r-proteins rpL2 (uL2), rpL25 (uL23) and rpL34 (eL34) for intermediate nuclear subunit folding steps. Structure models obtained from single particle cryo-electron microscopy analyses provided evidence for specific and hierarchic effects on the stable positioning and remodelling of large ribosomal subunit domains. Based on these structural and previous biochemical data we discuss possible mechanisms of r-protein dependent hierarchic domain arrangement and the resulting impact on the stability of misassembled subunits.

Proteostasis regulated by testis-specific ribosomal protein RPL39L maintains mouse spermatogenesis

SummaryMaintaining proteostasis is important for animal development. How proteostasis influences spermatogenesis that generates male gametes, spermatozoa, is not clear. We show that testis-specific paralog of ribosomal large subunit protein RPL39, RPL39L, is required for mouse spermatogenesis. Deletion of Rpl39l in mouse caused reduced proliferation of spermatogonial stem cells, malformed sperm mitochondria and flagella, leading to sub-fertility in males. Biochemical analyses revealed that lack of RPL39L deteriorated protein synthesis and protein quality control in spermatogenic cells, partly due to reduced biogenesis of ribosomal subunits and ribosome homeostasis. RPL39/RPL39L is likely assembled into ribosomes via H/ACA domain containing NOP10 complex early in ribosome biogenesis pathway. Furthermore, Rpl39l null mice exhibited compromised regenerative spermatogenesis after chemical insult and early degenerative spermatogenesis in aging mice. These data demonstrate that maintaining proteostasis is important for spermatogenesis, of which ribosome homeostasis maintained by ribosomal proteins coordinates translation machinery to the regulation of cellular growth.

COVID-19: Mechanisms of the Antiviral Activities of Selective Antibiotics Targeting the Human 80S Ribosome

Background: The majority of scientists, physicians, and healthcare professionals were trained with the paradigm: “antibiotics are for bacteria only !”, because they misunderstood the definition of the ribosome targeting antibiotics. In the context of the current worldwide COVID-19 pandemic, it might be useful to recall as precisely as possible the definition of the word antibiotic and provide evidence that some classes of antibiotics could offer excellent means to counteract viral infections via specific mechanisms. Methods: Molecular modeling and docking studies were used, as well as the tRNAox labeling reaction of the ribosomal protein eL42 in situ on human 80S ribosomes to demonstrate that cycloheximide and its thiosemicarbazone analogues bind to the catalytic Lys-53 residue of the human large subunit ribosomal protein eL42. Results: Comparison of the binding sites for Cycloheximide (CHX) and Sparsomycin (SPS) on the evolutionarily conserved E. coli bL12 and S. cerevisiae eL42 by means of molecular modeling and docking studies showed that: (i) SPS binds in proximity to the catalytic Lys-65 residue of the GANK motif of rp bL12 and to the catalytic Lys-55 residue of the GGQTKP motif of rp eL42; (ii) CHX failed to bind to the GANK motif, while the glutarimide moiety of SPS and CHX was found to make contact with Lys-55 of the GGQTKP motif of rp eL42. Conclusion: In this report, we demonstrate that cycloheximide and its thiosemicarbazone analogues are capable of inhibiting the human 80S ribosomes selectively through their binding to the ε-amino group of the side chain of Lys-53. As a consequence, these small-molecule inhibitors of translation are susceptible to exhibit antiviral activities by preventing the human ribosomes of the SARS-CoV-2 infected cells from synthesizing the viral proteins and enzymes.

Dimerization underlies the aggregation propensity of intrinsically disordered coiled-coil domain-containing 124.

Coiled-coil domain-containing 124 (CCDC124) is a recently discovered ribosome-binding protein conserved in eukaryotes. CCDC124 has regulatory functions on the mediation of reversible ribosomal hibernation and translational recovery by direct attachment to large subunit ribosomal protein uL5, 25S rRNA backbone, and tRNA-binding P/A-site major groove. Moreover, it independently mediates cell division and cellular stress response by facilitating cytokinetic abscission and disulfide stress-dependent transcriptional regulation, respectively. However, the structural characterization and intracellular physiological status of CCDC124 remain unknown. In this study, we employed advanced in silico protein modeling and characterization tools to generate a native-like tertiary structure of CCDC124 and examine the disorder, low sequence complexity, and aggregation propensities, as well as high-order dimeric/oligomeric states. Subsequently, dimerization of CCDC124 was investigated with co-immunoprecipitation (CO-IP) analysis, immunostaining, and a recent live-cell protein-protein interaction method, bimolecular fluorescence complementation (BiFC). Results revealed CCDC124 as a highly disordered protein consisting of low complexity regions at the N-terminus and an aggregation sequence (151-IAVLSV-156) located in the middle region. Molecular docking and post-docking binding free energy analyses highlighted a potential involvement of V153 residue on the generation of high-order dimeric/oligomeric structures. Co-IP, immunostaining, and BiFC analyses were used to further confirm the dimeric state of CCDC124 predominantly localized at the cytoplasm. In conclusion, our findings revealed in silico structural characterization and in vivo subcellular physiological state of CCDC124, suggesting low-complexity regions located at the N-terminus of disordered CCDC124 may regulate the formation of aggregates or high-order dimeric/oligomeric states.

Innate immunity to yeast prions: Btn2p and Cur1p curing of the [URE3] prion is prevented by 60S ribosomal protein deficiency or ubiquitin/proteasome system overactivity.

[URE3] is an amyloid-based prion of Ure2p, a negative regulator of poor nitrogen source catabolism in Saccharomyces cerevisiae. Overproduced Btn2p or its paralog Cur1p, in processes requiring Hsp42, cure the [URE3] prion. Btn2p cures by collecting Ure2p amyloid filaments at one place in the cell. We find that rpl4aΔ, rpl21aΔ, rpl21bΔ, rpl11bΔ, and rpl16bΔ (large ribosomal subunit proteins) or ubr2Δ (ubiquitin ligase targeting Rpn4p, an activator of proteasome genes) reduce curing by overproduced Btn2p or Cur1p. Impaired curing in ubr2Δ or rpl21bΔ is restored by an rpn4Δ mutation. No effect of rps14aΔ or rps30bΔ on curing was observed, indicating that 60S subunit deficiency specifically impairs curing. Levels of Hsp42p, Sis1p, or Btn3p are unchanged in rpl4aΔ, rpl21bΔ, or ubr2Δ mutants. Overproduction of Cur1p or Btn2p was enhanced in rpn4Δ and hsp42Δ mutants, lower in ubr2Δ strains, and restored to above wild-type levels in rpn4Δ ubr2Δ strains. As in the wild-type, Ure2N-GFP colocalizes with Btn2-RFP in rpl4aΔ, rpl21bΔ, or ubr2Δ strains, but not in hsp42Δ. Btn2p/Cur1p overproduction cures [URE3] variants with low seed number, but seed number is not increased in rpl4aΔ, rpl21bΔ or ubr2Δ mutants. Knockouts of genes required for the protein sorting function of Btn2p did not affect curing of [URE3], nor did inactivation of the Hsp104 prion-curing activity. Overactivity of the ubiquitin/proteasome system, resulting from 60S subunit deficiency or ubr2Δ, may impair Cur1p and Btn2p curing of [URE3] by degrading Cur1p, Btn2p or another component of these curing systems.

Cryo-electron microscopy structure of the 70S ribosome from Enterococcus faecalis

Enterococcus faecalis is a gram-positive organism responsible for serious infections in humans, but as with many bacterial pathogens, resistance has rendered a number of commonly used antibiotics ineffective. Here, we report the cryo-EM structure of the E. faecalis 70S ribosome to a global resolution of 2.8 Å. Structural differences are clustered in peripheral and solvent exposed regions when compared with Escherichia coli, whereas functional centres, including antibiotic binding sites, are similar to other bacterial ribosomes. Comparison of intersubunit conformations among five classes obtained after three-dimensional classification identifies several rotated states. Large ribosomal subunit protein bL31, which forms intersubunit bridges to the small ribosomal subunit, assumes different conformations in the five classes, revealing how contacts to the small subunit are maintained throughout intersubunit rotation. A tRNA observed in one of the five classes is positioned in a chimeric pe/E position in a rotated ribosomal state. The 70S ribosome structure of E. faecalis now extends our knowledge of bacterial ribosome structures and may serve as a basis for the development of novel antibiotic compounds effective against this pathogen.

Heavy ion radiation-induced DNA damage mediates apoptosis via the Rpl27a-Rpl5-MDM2-p53/E2F1 signaling pathway in mouse spermatogonia.

The risk of exposure to ionizing radiation (IR) environments has increased with the development of nuclear technology. IR exposure induces excessive apoptosis of the spermatogonia, which leads to male infertility. Spermatogonia apoptosis may be involved in ribosomal stress triggered by DNA damage following exposure to IR because ribosomal proteins (RPs) directly interact with mouse double minute 2 homolog (MDM2) to induce apoptosis. This study aimed to use comparative proteomics and transcriptomics approach to screen the differential RPs and ribosomal mRNAs in mouse testes following high linear energy transfer (LET) carbon ion radiation (CIR). The expression of ribosomal large subunit protein 27a (Rpl27a) decreased at both protein and mRNA levels in the spermatogonia in vivo. After 6h of CIR, the immunofluorescence signal of 8-oxo-dG and phosphorylated ataxia-telangiectasia-mutated protein (ATM)/histone H2Ax increased, but that of Rpl27a decreased in the spermatogonia of p53 wild-type and knockout mouse testes. Moreover, the nucleolin was scattered throughout the nucleoplasm after CIR. These results suggested that CIR-induced DNA damage might trigger ribosomal stress, and the reduction in the expression of Rpl27a was associated with DNA damage in the spermatogonia. Similarly, in vitro, the immunofluorescence signal of 8-oxo-dG increased in the GC-1cells after CIR. Moreover, the expression of Rpl27a was regulated by DNA damage because the co-transfection of ATM and Rpl27a or inhibition of ATM-treated CIR could restore the expression of Rpl27a. Furthermore, the reduction in the expression of Rpl27a led to weakened binding of E2F transcription factor 1 (E2F1) and p53 to MDM2, causing p53 activation and E2F1 degradation in p53 wild-type and knockdown GC-1cells. This study proposed that heavy ion radiation-induced DNA damage mediated spermatogonia apoptosis via the Rpl27a-Rpl5-MDM2-p53/E2F1 signaling pathway. The results provided the underlying molecular mechanisms of spermatogonia apoptosis following exposure to high LET radiation.

Post-transcriptional regulation of C-C motif chemokine ligand 2 expression by ribosomal protein L22 during LPS-mediated inflammation.

Monocyte infiltration to the site of pathogenic invasion is critical for inflammatory response and host defence. However, this process demands precise regulation as uncontrolled migration of monocytes to the site delays resolution of inflammation and ultimately promotes chronic inflammation. C-C motif chemokine ligand 2 (CCL2) plays a key role in monocyte migration, and hence, its expression should be tightly regulated. Here, we report a post-transcriptional regulation of CCL2 involving the large ribosomal subunit protein L22 (RPL22) in LPS-activated, differentiated THP-1 cells. Early events following LPS treatment include transcriptional upregulation of RPL22 and its nuclear accumulation. The protein binds to the first 20 nt sequence of the 5'UTR of ccl2 mRNA. Simultaneous nuclear translocation of up-frameshift-1 protein and its interaction with RPL22 results in cytoplasmic degradation of the ccl2 mRNA at a later stage. Removal of RPL22 from cells results in increased expression of CCL2 in response to LPS causing disproportionate migration of monocytes. We propose that post-transcriptional regulation of CCL2 by RPL22 fine-tunes monocyte infiltration during a pathogenic insult and maintains homeostasis of the immune response critical to resolution of inflammation. DATABASES: Microarray data are available in NCBI GEO database (Accession No GSE126525).