Depletion of S-adenosylmethionine impacts on ribosome biogenesis through hypomodification of a single rRNA methylation.

S-adenosylmethionine (SAM) is an essential metabolite and a methyl group donor in all living organisms. The intracellular SAM concentration is tightly regulated, and depletion causes hypomethylation of substrates, growth defects and pathological consequences. In the emerging field of epitranscriptomics, SAM-dependent RNA methylations play a critical role in gene expression. Herein, we analyzed the methylation status of ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) in Escherichia coli Δmtn strain in which cellular SAM was down-regulated, and found hypomodification of several methylation sites, including 2'-O-methylation at position 2552 (Um2552) of 23S rRNA. We observed severe growth defect of the Δmtn strain with significant accumulation of 45S ribosomal precursor harboring 23S rRNA with hypomodified Um2552. Strikingly, the growth defect was partially restored by overexpression of rlmE encoding the SAM-dependent methyltransferase responsible for Um2552. Although SAM is involved not only in rRNA methylation but also in various cellular processes, effects on ribosome biogenesis contribute substantially to the observed defects on cell proliferation.

Sensing Framework for the Internet of Actors in the Value Co-Creation Process with a Beacon-Attachable Indoor Positioning System.

To evaluate and improve the value of a service, it is important to measure not only the outcomes, but also the process of the service. Value co-creation (VCC) is not limited to outcomes, especially in interpersonal services based on interactions between actors. In this paper, a sensing framework for a VCC process in retail stores is proposed by improving an environment recognition based indoor positioning system with high positioning performance in a metal shelf environment. The conventional indoor positioning systems use radio waves; therefore, errors are caused by reflection, absorption, and interference from metal shelves. An improvement in positioning performance was achieved in the proposed method by using an IR (infrared) slit and IR light, which avoids such errors. The system was designed to recognize many and unspecified people based on the environment recognition method that the receivers had installed, in the service environment. In addition, sensor networking was also conducted by adding a function to transmit payload and identification simultaneously to the beacons that were attached to positioning objects. The effectiveness of the proposed method was verified by installing it not only in an experimental environment with ideal conditions, but posteriorly, the system was tested in real conditions, in a retail store. In our experimental setup, in a comparison with equal element numbers, positioning identification was possible within an error of 96.2 mm in a static environment in contrast to the radio wave based method where an average positioning error of approximately 648 mm was measured using the radio wave based method (Bluetooth low-energy fingerprinting technique). Moreover, when multiple beacons were used simultaneously in our system within the measurement range of one receiver, the appropriate setting of the pulse interval and jitter rate was implemented by simulation. Additionally, it was confirmed that, in a real scenario, it is possible to measure the changes in movement and positional relationships between people. This result shows the feasibility of measuring and evaluating the VCC process in retail stores, although it was difficult to measure the interaction between actors.

Single methylation of 23S rRNA triggers late steps of 50S ribosomal subunit assembly.

Ribosome biogenesis requires multiple assembly factors. In Escherichia coli, deletion of RlmE, the methyltransferase responsible for the 2'-O-methyluridine modification at position 2552 (Um2552) in helix 92 of the 23S rRNA, results in slow growth and accumulation of the 45S particle. We demonstrate that the 45S particle that accumulates in ΔrlmE is a genuine precursor that can be assembled into the 50S subunit. Indeed, 50S formation from the 45S precursor could be promoted by RlmE-mediated Um2552 formation in vitro. Ribosomal protein L36 (encoded by rpmJ) was completely absent from the 45S precursor in ΔrlmE, and we observed a strong genetic interaction between rlmE and rpmJ. Structural probing of 23S rRNA and high-salt stripping of 45S components revealed that RlmE-mediated methylation promotes interdomain interactions via the association between helices 92 and 71, stabilized by the single 2'-O-methylation of Um2552, in concert with the incorporation of L36, triggering late steps of 50S subunit assembly.

Structural basis for nonribosomal peptide synthesis by an aminoacyl-tRNA synthetase paralog.

Cyclodipeptides are secondary metabolites biosynthesized by many bacteria and exhibit a wide array of biological activities. Recently, a new class of small proteins, named cyclodipeptide synthases (CDPS), which are unrelated to the typical nonribosomal peptide synthetases, was shown to generate several cyclodipeptides, using aminoacyl-tRNAs as substrates. The Mycobacterium tuberculosis CDPS, Rv2275, was found to generate cyclodityrosine through the formation of an aminoacyl-enzyme intermediate and to have a structure and oligomeric state similar to those of the class Ic aminoacyl-tRNA synthetases (aaRSs). However, the poor sequence conservation among CDPSs has raised questions about the architecture and catalytic mechanism of the identified homologs. Here we report the crystal structures of Bacillus licheniformis CDPS YvmC-Blic, in the apo form and complexed with substrate mimics, at 1.7-2.4-Å resolutions. The YvmC-Blic structure also exhibits similarity to the class Ic aaRSs catalytic domain. Our mutational analysis confirmed the importance of a set of residues for cyclodileucine formation among the conserved residues localized in the catalytic pocket. Our biochemical data indicated that YvmC-Blic binds tRNA and generates cyclodileucine as a monomer. We were also able to detect the presence of an aminoacyl-enzyme reaction intermediate, but not a dipeptide tRNA intermediate, whose existence was postulated for Rv2275. Instead, our results support a sequential catalytic mechanism for YvmC-Blic, with the successive attachment of two leucine residues on the enzyme via a conserved serine residue. Altogether, our findings suggest that all CDPS enzymes share a common aaRS-like architecture and a catalytic mechanism involving the formation of an enzyme-bound intermediate.

MicroRNA-8073: Tumor suppressor and potential therapeutic treatment.

The comprehensive screening of intracellular and extracellular microRNAs was performed to identify novel tumor suppressors. We found that miR-8073 was present in exosome and predominantly exported from colorectal cancer cells. Treatment with a synthetic miR-8073 mimic resulted in a dramatic decrease in the proliferation of various types of cancer cells, which was not observed in similarly treated normal cells. As little is known about the biological functions of miR-8073, its target mRNAs were analyzed by both mRNA expression and in silico sequence analyses, leading to five probable target candidates (FOXM1, MBD3, CCND1, KLK10, and CASP2) that enhance survival during the regulation of the cell cycle, cell proliferation, and apoptosis. We experimentally confirmed that miR-8073 binds the 3'-UTR of each of these mRNA target candidates and that the introduction of a synthetic miR-8073 mimic into cancer cells reduced levels of protein expression. Finally, the antiproliferative effects of miR-8073 were validated in vivo: the subcutaneous injection of a synthetic miR-8073 mimic suppressed colorectal tumor volume to 43% in tumor-bearing xenografted mice. These results suggest that because miR-8073 binds, and thus reduces the levels of, these oncogenic targets, cancer cells must actively downregulate miR-8073 as a survival mechanism. The introduction of miR-8073 into tumors could thus inhibit tumor growth, indicating its great potential for cancer therapeutics.