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.

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.

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.

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.

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.

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.

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.

(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.

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).

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.

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.

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.

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.

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.

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.