The incorporation of extracellular vesicle markers varies among vesicles with distinct surface charges.

Extracellular vesicles (EVs) are important mediators of intercellular communication. However, the methods available for distinguishing the heterogeneity of secreted EVs and isolating and purifying them are limited. This study introduced a HiBiT-tag to detect various EV markers, including CD63, CD9, Epidermal Growth Factor Receptor (EGFR), Flotilin1, and Syndecan-1, and investigated whether these marker-containing vesicles were capable of binding to differently charged column carriers. Four column carriers, Diethylaminoethyl (DEAE), Capto Adhere, Blue and Heparin, showed affinity for CD63 containing EVs, but their elution patterns varied. Furthermore, we observed that the elution patterns of the EV markers differed among vesicles with distinct surface charges when a DEAE column was used. This suggests that the incorporation of EV markers varied between these vesicles. The markers showed different subcellular localizations, indicating that the site of vesicle formation may contribute to the production of vesicles with varying charges and marker incorporation. These findings may have implications for the development of methods to purify homogeneous EVs, which could be useful in EV-mediated drug delivery systems.

A leaderless mRNA including tRNA-like sequence encodes a small peptide that regulates the expression of GcvB small RNA in Escherichia coli.

A tRNA-like sequence conserved in the genomes of all Escherichia coli strains was found. The sequence resembles arginine-tRNA, which is present in E. coli pathogenic islands and phages. Expression experiments revealed that this sequence is a part of a leaderless mRNA encoding a short peptide (60 amino acids: XtpA). A deletion mutant of this gene is more sensitive than wild-type cell to several aminoglycoside antibiotics at low concentrations. Further analyses indicated that XtpA positively regulates the expression of GcvB small RNA, which is involved in the intrinsic resistance to aminoblycosides in E. coli.

Identification of a short form of a Caenorhabditis elegans Y RNA homolog Cel7 RNA.

Cel7 RNA is a member of the Caenorhabditis elegans stem-bulge RNAs (sbRNAs) that are classified into the Y RNA family based on their structural similarity. We identified a 15-nucleotide-shorter form of Cel7 RNA and designated it Cel7s RNA. Both Cel7 and Cel7s RNAs increased during the development of worms from L1 to adult. Cel7s RNA was notably more abundant in embryos than in L1 to L3 larvae. Cel7 RNA in embryo was less than those in L2 to adult. The ratio of cellular level of Cel7 RNA to that of Cel7s RNA was higher in L1 to L4, but reversed in embryos and adults. In rop-1 mutants, in which the gene for the C. elegans Ro60 homolog, ROP-1, was disrupted, Cel7s RNA decreased similar to CeY RNA, another C. elegans Y RNA homolog. Surprisingly, Cel7 RNA, existed stably in the absence of ROP-1, unlike Cel7s and CeY RNAs. Gel-shift assays demonstrated that Cel7 and Cel7s RNAs bound to ROP-1 in a similar manner, which was much weaker than CeY RNA. The 5'-terminal 15-nt of Cel7 RNA could be folded into a short stem-loop structure, probably contributing to the stability of Cel7 RNA in vivo and the distinct expression patterns of the 2 RNAs.

Involvement of GcvB small RNA in intrinsic resistance to multiple aminoglycoside antibiotics in Escherichia coli.

Deleting the gene for small RNA GcvB in Escherichia coli was found to increase the sensitivity to several aminoglycoside antibiotics, such as neomycin, streptomycin, kanamycin, kasugamycin and spectinomycin, at low concentrations. GcvB, conserved in gram-negative enteric bacteria, is known to negatively control the expression of many genes for amino acid incorporation systems, especially the periplasmic ABC-transporter proteins. Deletions of several amino acid transporter genes in ΔgcvB cells decreased the antibiotic sensitivity to the wild-type level, suggesting that those genes are involved in uptake of aminoglycosides into the cell. Since GcvB is constitutively synthesized in growing cells, repressing synthesis of amino acid transporters, it contributes to the intrinsic resistance to several aminoglycoside antibiotics.

Split Nano Luciferase-based Assay to Measure Assembly of Japanese Encephalitis Virus.

Cells infected with flavivirus release various forms of infectious and non-infectious particles as products and by-products. Comprehensive profiling of the released particles by density gradient centrifugation is informative for understanding viral particle assembly. However, it is difficult to detect low-abundance minor particles in such analyses. We developed a method for viral particle analysis that integrates a high-sensitivity split luciferase system and density gradient centrifugation. This protocol enables high-resolution profiling of particles produced by cells expressing Japanese encephalitis virus factors.

Functional Correlation between Subcellular Localizations of Japanese Encephalitis Virus Capsid Protein and Virus Production.

The flavivirus capsid protein is considered to be essential for the formation of nucleocapsid complexes with viral genomic RNA at the viral replication organelle that appears on the endoplasmic reticulum (ER), as well as for incorporation into virus particles. However, this protein is also detected at the lipid droplet (LD) and nucleolus, and physiological roles of these off-site localizations are still unclear. In this study, we made a series of alanine substitution mutants of Japanese encephalitis virus (JEV) capsid protein that cover all polar and hydrophobic amino acid residues to identify the molecular surfaces required for virus particle formation and for localization at the LD and nucleolus. Five mutants exhibited a defect in the formation of infectious particles, and two of these mutants failed to be incorporated into the subviral particles (SVP). Three mutants lost the ability to localize to the nucleolus, and only a single mutant, with mutations at α2, was unable to localize to the LD. Unlike the cytoplasmic capsid protein, the nucleolar capsid protein was resistant to detergent treatment, and the α2 mutant was hypersensitive to detergent treatment. To scrutinize the relationship between these localizations and viral particle formation, we made eight additional alanine substitution mutants and found that all the mutants that did not localize at the LD or nucleolus failed to form normal viral particles. These results support the functional correlation between LD or nucleolus localization of the flaviviral capsid protein and the formation of infectious viral particles.IMPORTANCE This study is the first to report the comprehensive mutagenesis of a flavivirus capsid protein. We assessed the requirement of each molecular surface for infectious viral particle formation as well as for LD and nucleolar localization and found functional relationships between the subcellular localization of the virus capsid protein and infectious virus particle formation. We developed a system to independently assess the packaging of viral RNA and that of the capsid protein and found a molecular surface of the capsid protein that is crucial for packaging of viral RNA but not for packaging of the capsid protein itself. We also characterized the biochemical properties of capsid protein mutants and found that the capsid protein localizes at the nucleolus in a different manner than for its localization to the LD. Our comprehensive alanine-scanning mutagenesis study will aid in the development of antiflavivirus small molecules that can target the flavivirus capsid protein.

RsgA couples the maturation state of the 30S ribosomal decoding center to activation of its GTPase pocket.

During 30S ribosomal subunit biogenesis, assembly factors are believed to prevent accumulation of misfolded intermediate states of low free energy that slowly convert into mature 30S subunits, namely, kinetically trapped particles. Among the assembly factors, the circularly permuted GTPase, RsgA, plays a crucial role in the maturation of the 30S decoding center. Here, directed hydroxyl radical probing and single particle cryo-EM are employed to elucidate RsgA΄s mechanism of action. Our results show that RsgA destabilizes the 30S structure, including late binding r-proteins, providing a structural basis for avoiding kinetically trapped assembly intermediates. Moreover, RsgA exploits its distinct GTPase pocket and specific interactions with the 30S to coordinate GTPase activation with the maturation state of the 30S subunit. This coordination validates the architecture of the decoding center and facilitates the timely release of RsgA to control the progression of 30S biogenesis.

(p)ppGpp-dependent and -independent pathways for salt tolerance in Escherichia coli.

Addition of some kinds of translation inhibitors targeting the ribosome such as kasugamycin to the culture medium as well as removal of a ribosome maturation factor or a ribosomal protein provides Escherichia coli cells with tolerance to high salt stress. Here, we found that another kind of translation inhibitor, serine hydroxamate (SHX), which induces amino acid starvation leading to (p)ppGpp production, also has a similar effect, but via a different pathway. Unlike kasugamycin, SHX was not effective in (p)ppGpp-null mutant cells. SHX and depletion of RsgA, a ribosome maturation factor, had an additive effect on salt tolerance, while kasugamycin or depletion of RsgA did not. These results indicate the presence of two distinct pathways, (p)ppGpp-dependent and -independent pathways, for salt tolerance of E. coli cell. Both pathways operate even in the absence of σ(S), an alternative sigma factor involved in the stationary phase or stress response. Hastened activation of the exocytoplasmic stress-specific sigma factor, σ(E), after salt shock was observed in the cells treated with SHX, as has been observed in the cells treated with a translation inhibitor or depleted of a ribosome maturation factor.

Structural insights into the assembly of the 30S ribosomal subunit in vivo: functional role of S5 and location of the 17S rRNA precursor sequence.

The in vivo assembly of ribosomal subunits is a highly complex process, with a tight coordination between protein assembly and rRNA maturation events, such as folding and processing of rRNA precursors, as well as modifications of selected bases. In the cell, a large number of factors are required to ensure the efficiency and fidelity of subunit production. Here we characterize the immature 30S subunits accumulated in a factor-null Escherichia coli strain (∆rsgA∆rbfA). The immature 30S subunits isolated with varying salt concentrations in the buffer system show interesting differences on both protein composition and structure. Specifically, intermediates derived under the two contrasting salt conditions (high and low) likely reflect two distinctive assembly stages, the relatively early and late stages of the 3' domain assembly, respectively. Detailed structural analysis demonstrates a mechanistic coupling between the maturation of the 5' end of the 17S rRNA and the assembly of the 30S head domain, and attributes a unique role of S5 in coordinating these two events. Furthermore, our structural results likely reveal the location of the unprocessed terminal sequences of the 17S rRNA, and suggest that the maturation events of the 17S rRNA could be employed as quality control mechanisms on subunit production and protein translation.

GTPases involved in bacterial ribosome maturation.

The ribosome is an RNA- and protein-based macromolecule having multiple functional domains to facilitate protein synthesis, and it is synthesized through multiple steps including transcription, stepwise cleavages of the primary transcript, modifications of ribosomal proteins and RNAs and assemblies of ribosomal proteins with rRNAs. This process requires dozens of trans-acting factors including GTP- and ATP-binding proteins to overcome several energy-consuming steps. Despite accumulation of genetic, biochemical and structural data, the entire process of bacterial ribosome synthesis remains elusive. Here, we review GTPases involved in bacterial ribosome maturation.

Dissecting the in vivo assembly of the 30S ribosomal subunit reveals the role of RimM and general features of the assembly process.

Ribosome biogenesis is a tightly regulated, multi-stepped process. The assembly of ribosomal subunits is a central step of the complex biogenesis process, involving nearly 30 protein factors in vivo in bacteria. Although the assembly process has been extensively studied in vitro for over 40 years, very limited information is known for the in vivo process and specific roles of assembly factors. Such an example is ribosome maturation factor M (RimM), a factor involved in the late-stage assembly of the 30S subunit. Here, we combined quantitative mass spectrometry and cryo-electron microscopy to characterize the in vivo 30S assembly intermediates isolated from mutant Escherichia coli strains with genes for assembly factors deleted. Our compositional and structural data show that the assembly of the 3'-domain of the 30S subunit is severely delayed in these intermediates, featured with highly underrepresented 3'-domain proteins and large conformational difference compared with the mature 30S subunit. Further analysis indicates that RimM functions not only to promote the assembly of a few 3'-domain proteins but also to stabilize the rRNA tertiary structure. More importantly, this study reveals intriguing similarities and dissimilarities between the in vitro and the in vivo assembly pathways, suggesting that they are in general similar but with subtle differences.

RsgA releases RbfA from 30S ribosome during a late stage of ribosome biosynthesis.

RsgA is a 30S ribosomal subunit-binding GTPase with an unknown function, shortage of which impairs maturation of the 30S subunit. We identified multiple gain-of-function mutants of Escherichia coli rbfA, the gene for a ribosome-binding factor, that suppress defects in growth and maturation of the 30S subunit of an rsgA-null strain. These mutations promote spontaneous release of RbfA from the 30S subunit, indicating that cellular disorders upon depletion of RsgA are due to prolonged retention of RbfA on the 30S subunit. We also found that RsgA enhances release of RbfA from the mature 30S subunit in a GTP-dependent manner but not from a precursor form of the 30S subunit. These findings indicate that the function of RsgA is to release RbfA from the 30S subunit during a late stage of ribosome biosynthesis. This is the first example of the action of a GTPase on the bacterial ribosome assembly described at the molecular level.

A novel GTPase activated by the small subunit of ribosome.

The GTPase activity of Escherichia coli YjeQ, here named RsgA (ribosome small subunit-dependent GTPase A), has been shown to be significantly enhanced by ribosome or its small subunit. The enhancement of GTPase activity was inhibited by several aminoglycosides bound at the A site of the small subunit, but not by a P site-specific antibiotic. RsgA stably bound the small subunit in the presence of GDPNP, but not in the presence of GTP or GDP, to dissociate ribosome into subunits. Disruption of the gene for RsgA from the genome affected the growth of the cells, which predominantly contained the dissociated subunits having only a weak activation activity of RsgA. We also found that 17S RNA, a putative precursor of 16S rRNA, was contained in the small subunit of the ribosome from the RsgA-deletion strain. RsgA is a novel GTPase that might provide a new insight into the function of ribosome.

tRNA2Thr complements temperature sensitivity caused by null mutations in the htrB gene in Escherichia coli.

According to the wobble rule, tRNA2Thr is nonessential for protein synthesis, because the codon (ACG) that is recognized by tRNA2Thr is also recognized by tRNA4Thr. In order to investigate the reason that this nonessential tRNA nevertheless exists in Escherichia coli, we attempted to isolate tRNA2Thr-requiring mutants. Using strain JM101F(-), which lacks the gene for tRNA2Thr, we succeeded in isolating two temperature-sensitive mutants whose temperature sensitivity was complemented by introduction of the gene for tRNA2Thr. These mutants had a mutation in the htrB gene, whose product is an enzyme involved in lipid A biosynthesis. Although it is known that some null mutations in the htrB gene give a temperature-sensitive phenotype, our mutants exhibited tighter temperature sensitivity. We discuss a possible mechanism for the requirement for tRNA2Thr.

HemK, a class of protein methyl transferase with similarity to DNA methyl transferases, methylates polypeptide chain release factors, and hemK knockout induces defects in translational termination.

HemK, a universally conserved protein of unknown function, has high amino acid similarity with DNA-(adenine-N6) methyl transferases (MTases). A certain mutation in hemK gene rescues the photosensitive phenotype of a ferrochelatase-deficient (hemH) mutant in Escherichia coli. A hemK knockout strain of E. coli not only suffered severe growth defects, but also showed a global shift in gene expression to anaerobic respiration, as determined by microarray analysis, and this shift may lead to the abrogation of photosensitivity by reducing the oxidative stress. Suppressor mutations that abrogated the growth defects of the hemK knockout strain were isolated and shown to be caused by a threonine to alanine change at codon 246 of polypeptide chain release factor (RF) 2, indicating that hemK plays a role in translational termination. Consistent with such a role, the hemK knockout strain showed an enhanced rate of read-through of nonsense codons and induction of transfer-mRNA-mediated tagging of proteins within the cell. By analysis of the methylation of RF1 and RF2 in vivo and in vitro, we showed that HemK methylates RF1 and RF2 in vitro within the tryptic fragment containing the conserved GGQ motif, and that hemK is required for the methylation within the same fragment of, at least, RF1 in vivo. This is an example of a protein MTase containing the DNA MTase motif and also a protein-(glutamine-N5) MTase.