40S Ribosomal Subunit Research Articles

Toeprint Assays for Detecting RNA Structure and Protein-RNA Interactions.

Toeprint assays are primer extension inhibition assays that can detect the 3' end of an RNA secondary structure, the position of a bound RNA binding protein, as well as the position of a bound 30S ribosomal subunit or a stalled ribosome. Here we describe how this assay was used to identify an RNA hairpin that sequesters a Shine-Dalgarno sequence, how the RNA-binding protein CsrA can alter RNA structure and affect 30S ribosomal subunit binding, and how the macrolide antibiotic tylosin can induce ribosome stalling.

Eukaryotic Ribosome Biogenesis: The 60S Subunit.

Ribosome biogenesis is consecutive coordinated maturation of ribosomal precursors in the nucleolus, nucleoplasm, and cytoplasm. The formation of mature ribosomal subunits involves hundreds of ribosomal biogenesis factors that ensure ribosomal RNA processing, tertiary structure, and interaction with ribosomal proteins. Although the main features and stages of ribosome biogenesis are conservative among different groups of eukaryotes, this process in human cells has become more complicated due to the larger size of the ribosomes and pre-ribosomes and intricate regulatory pathways affecting their assembly and function. Many of the factors involved in the biogenesis of human ribosomes have been identified using genome-wide screening based on RNA interference. A previous part of this review summarized recent data on the processing of the primary rRNA transcript and compared the maturation of the small 40S subunit in yeast and human cells. This part of the review focuses on the biogenesis of the large 60S subunit of eukaryotic ribosomes.

Evaluation of partial 12S rRNA, 16S rRNA, COI and Cytb gene sequence datasets for potential single DNA barcode for hylids (Anura: Hylidae)

We evaluated the extent of intraspecific and interspecific genetic distances and the effectiveness of predefined threshold values using the main genes for estimates of biodiversity and specimens' identification in anurans. Partial sequences of the mitochondrial genes for small (12S) and large (16S) ribosomal subunits, cytochrome c oxidase subunit I (COI) and Cytochrome b (Cytb) of the family Hylidae were downloaded from GenBank and curated for length, coverage, and potential contaminations. We performed analyses for all sequences of each gene and the same species present in these datasets by distance and tree (monophyly)-based evaluations. We also evaluated the ability to identify specimens using these datasets applying "nearest neighbor" (NN), "best close match" (BCM) and "BOLD ID" tests. Genetic distance thresholds were generated by the function 'threshVal' and "localMinima" from SPIDER package and traditional threshold values (1%, 3%, 6% and 10%) were also evaluated. Coding genes, especially COI, had a better identification capacity than non-coding genes on barcoding gap and monophyly analysis and NN, BCM, BOLD ID tests. Considering the multiple factors involved in global DNA barcoding evaluations, we present a critical assessment of the use of these genes for biodiversity estimation and specimens' identification in anurans (e.g. hylids).

RPS3-induced antiviral cytokines inhibit the proliferation of classical swine fever virus.

Classical swine fever virus (CSFV) infection results in serious economic losses to the pig industry. This positive-sense RNA virus hijacks cell host proteins for its own replication. Although previous studies have shown that RPS3, a40S ribosomal subunit protein, is mainly required for DNA repair, apoptosis and inflammation, the effect of RPS3 on CSFV replication remains uncertain. Thus, we investigated the potential role of RPS3 in CSFV infection in RPS3-knockdown and -overexpressing cell lines using real-time fluorescence quantitative PCR (RT-qPCR) and indirect immunofluorescence assays. Results showed that knockdown of RPS3 by lentiviruses enhanced CSFV replication, whereas overexpression of RPS3 by lentiviruses inhibited CSFV replication. These findings indicated the antiviral role of RPS3 in CSFV infection. Subsequent experiments revealed that CSFV replication was inhibited in cells cultured with the supernatants of RPS3-overexpressing cell, suggesting that the RPS3-mediated inflammatory response was involved in CSFV infection. Furthermore, enzyme-linked immunosorbent assay (ELISA) revealed that the secretion of antiviral cytokines (IL-8 and INF-β) was increased in cells with sufficient RPS3 expression but decreased in cells lacking RPS3 expression. RT-qPCR and immunofluorescence assays revealed that CSFV infection inhibited RPS3-mediated antiviral cytokine secretion. Taken together, these findings reveal that RPS3 is anovel antiviral factor that inhibits CSFV proliferation by increasing antiviral cytokine secretion. Keywords: classical swine fever virus; ribosome protein S3; IL-8; INF-β.

넙치(Paralichthys olivaceus)조직에서 lincomycin의 약동학 분석을 위한 액체크로마토그래피-질량분석법 개발

Lincomycin is a drug known to be effective in treating streptococcus aureus as it inhibits the protein synthesis by binding to 50S ribosomal subunits. However, in the test method registered in Korean Food Code the Maximum Residue Limit (MRL) is set to be 0.1 mg/kg regardless of the kinds or body areas of fishes. In addition, only analysis using high performance liquid chromatography mass spectrometry (HPLC-MS/MS) was presented, so we developed analytical methods to detect trace concentrations of serum and muscle in the olive flounder using HPLC-MS/MS. The analyte was quantified in the positive ion mode using multiple reaction monitoring (MRM). Samples were extracted with methanol and then Hydrphilic-Liphophilic Balance (HLB) was used for purification. Matrix-matched calibration (MMC) presented linearity with the correlation coefficient (r²)>0.99, analyzed at 0.5~50 ng/g concentration. The limit of quantitation (LOQ) of muscle was 0.5 ng/g and recovery was 89.33%(n=9, Intra-day). The LOQ of serum was 0.5 ng/g and recovery was 97.40%(n=9, Intra-day) which were suitable for CODEX guideline. This analysis would be appropriate for the study of lincomycin residues and pharmacokinetics in olive flounder.

Functional analysis of the 1p34.3 risk locus implicates GNL2 in high-grade serous ovarian cancer

The high-grade serous ovarian cancer (HGSOC) risk locus at chromosome 1p34.3 resides within a frequently amplified genomic region signifying the presence of an oncogene. Here, we integrate in silico variant-to-function analysis with functional studies to characterize the oncogenic potential of candidate genes in the 1p34.3 locus. Fine mapping of genome-wide association statistics identified candidate causal SNPs local to H3K27ac-demarcated enhancer regions that exhibit allele-specific binding for CTCF in HGSOC and normal fallopian tube secretory epithelium cells (FTSECs). SNP risk associations colocalized with eQTL for six genes (DNALI1, GNL2, RSPO1, SNIP1, MEAF6, and LINC01137) that are more highly expressed in carriers of the risk allele, and three (DNALI1, GNL2, and RSPO1) were upregulated in HGSOC compared to normal ovarian surface epithelium cells and/or FTSECs. Increased expression of GNL2 and MEAF6 was associated with shorter survival in HGSOC with 1p34.3 amplifications. Despite its activation of β-catenin signaling, RSPO1 overexpression exerted no effects on proliferation or colony formation in our study of ovarian cancer and FTSECs. Instead, GNL2, MEAF6, and SNIP1 silencing impaired in vitro ovarian cancer cell growth. Additionally, GNL2 silencing diminished xenograft tumor formation, whereas overexpression stimulated proliferation and colony formation in FTSECs. GNL2 influences 60S ribosomal subunit maturation and global protein synthesis in ovarian cancer and FTSECs, providing a potential mechanism of how GNL2 upregulation might promote ovarian cancer development and mediate genetic susceptibility of HGSOC.

Members of the ribosomal protein S6 (RPS6) family act as pro-viral factor for tomato spotted wilt orthotospovirus infectivity in Nicotiana benthamiana.

To identify host factors for tomato spotted wilt orthotospovirus (TSWV), a virus‐induced gene silencing (VIGS) screen using tobacco rattle virus (TRV) was performed on Nicotiana benthamiana for TSWV susceptibility. To rule out any negative effect on the plants’ performance due to a double viral infection, the method was optimized to allow screening of hundreds of clones in a standardized fashion. To normalize the results obtained in and between experiments, a set of controls was developed to evaluate in a consist manner both VIGS efficacy and the level of TSWV resistance. Using this method, 4532 random clones of an N. benthamiana cDNA library were tested, resulting in five TRV clones that provided nearly complete resistance against TSWV. Here we report on one of these clones, of which the insert targets a small gene family coding for the ribosomal protein S6 (RPS6) that is part of the 40S ribosomal subunit. This RPS6 family is represented by three gene clades in the genome of Solanaceae family members, which were jointly important for TSWV susceptibility. Interestingly, RPS6 is a known host factor implicated in the replication of different plant RNA viruses, including the negative‐stranded TSWV and the positive‐stranded potato virus X.

Oxazolidinone-containing Hybrids with Antibacterial Activity against Methicillin-resistant Staphylococcus aureus (MRSA): A Mini-review.

The increasing danger of methicillin-resistant Staphylococcus aureus (MRSA) and the limited therapeutic options for invasive MRSA infections make an urgent demand for the development of novel anti-MRSA agents. Oxazolidinone derivatives could inhibit protein synthesis by acting on the ribosomal 50S subunit of the bacteria and prevent the formation of a functional 70S initiation complex, so oxazolidinones are a novel class of antimicrobial agents with potential activity against a wide range of clinically significant multidrug-resistant Gram-positive pathogens. However, oxazolidinones such as linezolid are associated with significant adverse events, and myelosuppression represents the main unfavorable side effects. Moreover, MRSA isolates that are resistant to oxazolidinones have already emerged. Hybridization of oxazolidinone with other antibacterial pharmacophores has the potential to interact with multiple targets or to counterbalance the known side effects associated with each pharmacophore. Thus, oxazolidinone-containing hybrids are useful scaffolds for the development of novel anti-MRSA agents. This review covers the recent advances of oxazolidinonecontaining hybrids with anti-MRSA activity developed in the last decade to set up the direction for the design and development of oxazolidinone-containing hybrids with high efficiency and low toxicity.

Computational investigation of potent inhibitors against YsxC: structure-based pharmacophore modeling, molecular docking, molecular dynamics, and binding free energy

In S. aureus, ribosome biogenesis GTP-binding (YsxC), a GTPase interacts with 50S subunit and 30S subunit of ribosome, and β’ subunit of RNA polymerase and played an important role in protein synthesis. For the identification of potent lead molecules, we have conducted pharmacophore modeling by consideration of pharmacophore features of GTP among YsxC-GTP complex. Virtual screening and molecular docking results displayed that five pharmacokinetic and ADMET filtered molecules—ZINC000006424138, ZINC000095502032, ZINC000225415132, ZINC000095475800, and ZINC000012990761—had higher binding affinities than GTP with YsxC. All the identified molecules shared similar pharmacophore features of GTP and were stabilized via hydrogen bonds and hydrophobic interactions with YsxC. Molecular dynamics analysis revealed that YsxC-inhibitor(s) complexes were lesser dynamics and higher stable than YsxC-GTP complex. Molecular Mechanics/Poisson-Boltzmann Surface Area (MMPBSA) results confirmed that identified molecules bound at the active site (Arg33, Ser34, Asn35, Val36, Lys38, Ser39, Thr40, Thr54, Ser55, Pro58, Lys60, Thr61, Thr144, Lys145, Ser178, and Ile179) of YsxC and formed the lower energy (–190.32 ± 3.46 to –217.03 ± 2.55 kJ/mol) complexes than YsxC-GTP (–157.16 ± 2.89 kJ/mol) complex. The identified molecules in this study can be further tested and utilized to design novel antimicrobial agents for S. aureus. Communicated by Ramaswamy H. Sarma

The mitochondrial genome sequence analysis of Ophidascaris baylisi from the Burmese python (Python molurus bivittatus).

Ophidascaris species are parasitic roundworms that inhabit the python gut, resulting in severe granulomatous lesions or even death. However, the classification and nomenclature of these roundworms are still controversial. Our study aims to identify a snake roundworm from the Burmese python (Python molurus bivittatus) and analyze the mitochondrial genome. We identified this roundworm as Ophidascaris baylisi based on the morphology and cytochrome c oxidase subunit I (cox1) sequence. Ophidascaris baylisi complete mitochondrial genome was 14,784bp in length, consisting of two non-coding regions and 36 mitochondrial genes (12 protein-coding genes, 22 tRNA genes, and two rRNA genes). The protein-coding genes used TTG, ATG, ATT, or TTA as start codons and TAG, TAA, or T as stop codons. All tRNA genes showed a TV-loop structure, except trnS1AGN and trnS2UCN revealed a D-loop structure. The mitochondrial large ribosomal subunit 16S (rrnL) and small ribosomal subunit 12S (rrnS) were 956bp and 700bp long, respectively. Phylogenetic analysis based on O. baylisi mitochondrial protein-coding genes demonstrated that O. baylisi clustered with the family Ascarididae members and was most closely related to Ophidascaris wangi. These results may enhance the nematode mitochondrial genome database and provide valuable molecular markers for further research on the taxonomy, phylogeny, and genetic relationships of Ophidascaris nematodes.

Conformational exploration of RbgA using molecular dynamics: Possible implications in ribosome maturation and GTPase activity in different nucleotide bound states

Ribosome biogenesis GTPase A (RbgA) is involved in the late steps of the 50S ribosomal subunit maturation by binding into the 45S pre-ribosomal subunit. The association of RbgA to the 45S intermediate subunit depends on its bound nucleotide (GTP/GDP), probably because of the conformational shifts that occur between the GTP and GDP bound states. However, the available crystal structures of Staphylococcus aureus RbgA (SaRbgA) do not show any significant variations between different nucleotide bound states. Therefore, conformational exploration of SaRbgA in different nucleotide bound states was carried out using all-atom molecular dynamics (MD) simulations. Exploration of conformational distribution using cluster analysis and principal component analysis (PCA) revealed that GDP and pppGpp bound systems exhibit a larger distribution. This is majorly due to the fluctuations of the C-terminal tail (C-tail) as a result of the unwinding of α-helical secondary conformations into loop conformations which are observed from RMSF and DSSP analyses. Further investigation of the network of interactions revealed that the GTP and GMPPNP bound systems hold the C-tail in an α-helical form through stronger interactions between the active-site and C-tail. We also find that the presence of Mg2+ positions Sw-I loop away from the bound nucleotide and stabilizes the active-site water molecules. This seems to assist SaRbgA GTPase activity. In addition, mutations at the C-terminal and Sw-II conserved residues exhibit a larger conformational distribution majorly due to the C-tail fluctuations suggesting that the C-tail of SaRbgA probably interacts with the rRNA or rprotein in the process of ribosome biogenesis.

Hippocampal neurons’ cytosolic and membrane-bound ribosomal transcript profiles are differentially regulated by learning and subsequent sleep

The hippocampus is essential for consolidating transient experiences into long-lasting memories. Memory consolidation is facilitated by postlearning sleep, although the underlying cellular mechanisms are largely unknown. We took an unbiased approach to this question by using a mouse model of hippocampally mediated, sleep-dependent memory consolidation (contextual fear memory). Because synaptic plasticity is associated with changes to both neuronal cell membranes (e.g., receptors) and cytosol (e.g., cytoskeletal elements), we characterized how these cell compartments are affected by learning and subsequent sleep or sleep deprivation (SD). Translating ribosome affinity purification was used to profile ribosome-associated RNAs in different subcellular compartments (cytosol and membrane) and in different cell populations (whole hippocampus, Camk2a+ neurons, or highly active neurons with phosphorylated ribosomal subunit S6 [pS6+]). We examined how transcript profiles change as a function of sleep versus SD and prior learning (contextual fear conditioning; CFC). While sleep loss altered many cytosolic ribosomal transcripts, CFC altered almost none, and CFC-driven changes were occluded by subsequent SD. In striking contrast, SD altered few transcripts on membrane-bound (MB) ribosomes, while learning altered many more (including long non-coding RNAs [lncRNAs]). The cellular pathways most affected by CFC were involved in structural remodeling. Comparisons of post-CFC MB transcript profiles between sleeping and SD mice implicated changes in cellular metabolism in Camk2a+ neurons and protein synthesis in highly active pS6+ (putative "engram") neurons as biological processes disrupted by SD. These findings provide insights into how learning affects hippocampal neurons and suggest that the effects of SD on memory consolidation are cell type and subcellular compartment specific.

Gingival Crevicular Fluid as Biomarker’s Source for Alzheimer’s Disease

Periodontitis is a low-grade inflammatory disease caused by a subgingival dysbiotic microbiota. Multiple studies have determined the higher prevalence of tooth loss and poor oral hygiene in patients with Alzheimer’s disease (AD). However, the periodontal diagnosis, periodontal bacteria or mediators has not been measured to date. Aim: To determine the periodontal status, the pro-inflammatory mediators, Porphyromonas gingivalis load, and Apoliporpotein E (ApoE) in patients with AD. A complete dental examination was performed on 30 patients, and cognitive status was determined by the Montreal Cognitive Assessment (MoCA). Subgingival microbiota and GCF samples were then taken from all patients from the deepest sites. Total DNA was isolated from the microbiota samples for the quantification of the 16S ribosomal subunit. Pro-inflammatory mediators and ApoE were quantified from the gingival crevicular fluid (GCF). Patients with AD had periodontitis stage III-IV in 80%, a higher concentration of pro-inflammatory and ApoE mediators, and a higher P. gingivalis load compared to healthy subjects. The pro-inflammatory mediators, P. gingivalis load had a negative correlation with the MoCA test scores. Finally, a ROC curve was performed to assess the specificity and sensitivity of ApoE levels, detecting an area of 84.9%. In AD patients, we found a more severe periodontitis, a higher levels of pro-inflammatory mediators, and higher bacterial load. In addition, there is an increase in ApoE that allows to clearly determine patients with health, periodontitis and periodontitis and AD.

Evaluation of Polymerase Chain Reaction in the Identification and Quantification of Clinically Relevant Bacterial Species in Lower Extremity Wound Infections

Identification of bacteria by polymerase chain reaction (PCR) is known to be more sensitive than culture, which brings to question the clinical applicability of the results. In this study, we evaluate the ability of PCR to detect clinically relevant bacterial species in lower extremity wound infections requiring operative debridement, as well as the quantitative change in biodiversity and bacterial load reflected by PCR during the course of treatment. Thirty-four infected lower extremity were examined by analysis of 16S ribosomal RNA subunit and by culture. McNemar's test was used to measure the concordance of clinically relevant bacterial species identified by PCR compared to culture during each debridement. Change in wound biodiversity from initial presentation to final closure was evaluated by Wilcoxon signed-rank test. Kaplan-Meier survival curve was used to characterize change in measured bacterial load over the course of operative debridement. A total of 15 and 12 clinically relevant bacterial species were identified by PCR and culture, respectively. The most common bacterial species identified were Coagulase-negative Staphylococcus, Staphylococcus aureus, and Enterococcus spp. PCR was less likely to detect Enterococcus spp. on initial debridement and Coagulase-negative Staphylococcus on closure in this study population. A significant decrease in mean number of clinically relevant species detected from initial debridement to closure was reflected by culture (p=.0188) but not by PCR (p=.1848). Both PCR (p=.0128) and culture (p=.0001) depicted significant reduction in mean bacterial load from initial debridement to closure. PCR is able to identify common clinically relevant bacterial species in lower extremity surgical wound infections. PCR displays increased sensitivity compared to culture with relation to detection of biodiversity, rather than bacterial load. Molecular diagnostics and conventional culture may serve a joint purpose to assist with rendering clinical judgment in complex wound infections.

Nsp1 of SARS-CoV-2 stimulates host translation termination

ABSTRACT Nsp1 of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of the Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1, eRF1 and ABCE1. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates peptide release and formation of termination complexes. Detailed analysis of Nsp1 activity during translation termination stages reveals that Nsp1 facilitates stop codon recognition. We demonstrate that Nsp1 stimulation targets eRF1 and does not affect eRF3. Moreover, Nsp1 increases amount of the termination complexes at all three stop codons. The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that the biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and remove them from the pool of the active ribosomes.

A Survey of Spontaneous Antibiotic-Resistant Mutants of the Halophilic, Thermophilic Bacterium Rhodothermus marinus.

Rhodothermus marinus is a halophilic extreme thermophile, with potential as a model organism for studies of the structural basis of antibiotic resistance. In order to facilitate genetic studies of this organism, we have surveyed the antibiotic sensitivity spectrum of R. marinus and identified spontaneous antibiotic-resistant mutants. R. marinus is naturally insensitive to aminoglycosides, aminocylitols and tuberactinomycins that target the 30S ribosomal subunit, but is sensitive to all 50S ribosomal subunit-targeting antibiotics examined, including macrolides, lincosamides, streptogramin B, chloramphenicol, and thiostrepton. It is also sensitive to kirromycin and fusidic acid, which target protein synthesis factors. It is sensitive to rifampicin (RNA polymerase inhibitor) and to the fluoroquinolones ofloxacin and ciprofloxacin (DNA gyrase inhibitors), but insensitive to nalidixic acid. Drug-resistant mutants were identified using rifampicin, thiostrepton, erythromycin, spiramycin, tylosin, lincomycin, and chloramphenicol. The majority of these were found to have mutations that are similar or identical to those previously found in other species, while several novel mutations were identified. This study provides potential selectable markers for genetic manipulations and demonstrates the feasibility of using R. marinus as a model system for studies of ribosome and RNA polymerase structure, function, and evolution.

Analysis between Linagliptin and Azithromycin: In vitro and In vivo Interaction Study.

Linagliptin is prescribed as a dipeptidyl peptidase-4 (DPP-4) inhibitor. Azithromycin is specified as an antibiotic that binds with 23s rRNA of the 50s ribosomal subunit, obstructing the microbial protein synthesis. Our study focuses on the drug-drug interactions of these drugs. The purpose of the study is to understand the bioavailability and physicochemical approaches of Linagliptin and Azithromycin interaction mediated through the strength and nature of the complexation. TheIn vitro assessment of drug interaction was conducted using Ultraviolet-visible spectroscopy (UV/VIS), Ultra-Performance Liquid Chromatography (UPLC), Fourier transform infrared spectroscopy (FT-IR), and Differential scanning calorimetry (DSC), while the Oral Glucose Tolerance Test (OGTT) was performed for theIn vivo assessment of drug interaction in a mouse model. Mild variation in interaction was observed at different pH values at a specific temperature by Job's and Ardon's equations. In UPLC, the drug mixture assessment showed that the area of Linagliptin was 2013793, and the area of Azithromycin was 54631 in 50 mg/l. The height of Linagliptin in the drug mixture was 579234, and that of Azithromycin was 11442. For Azithromycin, the wavelength of 731.02 cm-1, 993.34 cm-1, 1379.10 cm-1, and 1718.58 cm-1 decreased in the mixture. Also, for Linagliptin, the wavelength 1363.67 cm-1, 1473.62 cm-1, and 1718.58 cm-1 decreased in the drug mixture. The melting endotherm was 125.55°C, melting normalized energy was -3.0 W/mg, and 225.75°C with melting normalized energy of -5.5 W/mg of the drug mixture as indicated by DSC. In the OGTT test, the blood glucose level of Linagliptin decreased in the drug mixture by 13.58 % and 57.25%. Hence, the concomitant administration of Linagliptin and Azithromycin simultaneously might reduce the therapeutic effect of the drug complex.

Rational Design of an Antimicrobial Peptide Based on Structural Insight into the Interaction of Pseudomonas aeruginosa Initiation Factor 1 with Its Cognate 30S Ribosomal Subunit.

Bacterial infections continue to represent a major worldwide health hazard following the emergence of drug-resistant pathogenic strains. Pseudomonas aeruginosa is an opportunistic pathogen causing nosocomial infections with increased morbidity and mortality. The increasing antibiotic resistance in P. aeruginosa has led to an unmet need for discovery of new antibiotic candidates. Bacterial protein synthesis is an essential metabolic process and a validated target for antibiotic development; however, the precise structural mechanism in P. aeruginosa remains unknown. In this work, the interaction of P. aeruginosa initiation factor 1 (IF1) with the 30S ribosomal subunit was studied by NMR, which enabled us to construct a structure of IF1-bound 30S complex. A short α-helix in IF1 was found to be critical for IF1 ribosomal binding and function. A peptide derived from this α-helix was tested and displayed a high ability to inhibit bacterial growth. These results provide a clue for rational design of new antimicrobials.

A Multi-Perspective Proximity View on the Dynamic Head Region of the Ribosomal 40S Subunit.

A comparison of overlapping proximity captures at the head region of the ribosomal 40S subunit (hr40S) in Saccharomyces cerevisiae from four adjacent perspectives, namely Asc1/RACK1, Rps2/uS5, Rps3/uS3, and Rps20/uS10, corroborates dynamic co-localization of proteins that control activity and fate of both ribosomes and mRNA. Co-locating factors that associate with the hr40S are involved in (i) (de)ubiquitination of ribosomal proteins (Hel2, Bre5-Ubp3), (ii) clamping of inactive ribosomal subunits (Stm1), (iii) mRNA surveillance and vesicular transport (Smy2, Syh1), (iv) degradation of mRNA (endo- and exonucleases Ypl199c and Xrn1, respectively), (v) autophagy (Psp2, Vps30, Ykt6), and (vi) kinase signaling (Ste20). Additionally, they must be harmonized with translation initiation factors (eIF3, cap-binding protein Cdc33, eIF2A) and mRNA-binding/ribosome-charging proteins (Scp160, Sro9). The Rps/uS-BioID perspectives revealed substantial Asc1/RACK1-dependent hr40S configuration indicating a function of the β-propeller in context-specific spatial organization of this microenvironment. Toward resolving context-specific constellations, a Split-TurboID analysis emphasized the ubiquitin-associated factors Def1 and Lsm12 as neighbors of Bre5 at hr40S. These shuttling proteins indicate a common regulatory axis for the fate of polymerizing machineries for the biosynthesis of proteins in the cytoplasm and RNA/DNA in the nucleus.

Association of snR190 snoRNA chaperone with early pre-60S particles is regulated by the RNA helicase Dbp7 in yeast

Synthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit.