Effect of RIP Overexpression on Abiotic Stress Tolerance and Development of Rice

Ribosome-inactivating proteins (RIPs) are a class of cytotoxic enzymes that can inhibit protein translation by depurinating rRNA. Most plant RIPs are synthesized with a leader sequence that sequesters the proteins to a cell compartment away from the host ribosomes. However, several rice RIPs lack these signal peptides suggesting they reside in the cytosol in close proximity to the plant ribosomes. This paper aims to elucidate the physiological function of two nucleocytoplasmic RIPs from rice, in particular, the type 1 RIP referred to as OsRIP1 and a presumed type 3 RIP called nuRIP. Transgenic rice lines overexpressing these RIPs were constructed and studied for developmental effects resulting from this overexpression under greenhouse conditions. In addition, the performance of transgenic seedlings in response to drought, salt, abscisic acid and methyl jasmonate treatment was investigated. Results suggest that both RIPs can affect methyl jasmonate mediated stress responses.

Transcriptome-based design of antisense inhibitors potentiates carbapenem efficacy in CREEscherichia coli

In recent years, the prevalence of carbapenem-resistantEnterobacteriaceae(CRE) has risen substantially, and the study of CRE resistance mechanisms has become increasingly important for antibiotic development. Although much research has focused on genomic resistance factors, relatively few studies have examined CRE pathogens through changes in gene expression. In this study, we examined the gene expression profile of a CREEscherichia coliclinical isolate that is sensitive to meropenem but resistant to ertapenem to explore transcriptomic contributions to resistance and to identify gene knockdown targets for carbapenem potentiation. We sequenced total and short RNA to analyze the gene expression response to ertapenem or meropenem treatment and found significant expression changes in genes related to motility, maltodextrin metabolism, the formate hydrogenlyase complex, and the general stress response. To validate these findings, we used our laboratory’s Facile Accelerated Specific Therapeutic (FAST) platform to create antisense peptide nucleic acids (PNAs), gene-specific molecules designed to inhibit protein translation. PNAs were designed to inhibit the pathways identified in our transcriptomic analysis, and each PNA was then tested in combination with each carbapenem to assess its effect on the antibiotics’ minimum inhibitory concentrations. We observed significant PNA–antibiotic interaction with five different PNAs across six combinations. Inhibition of the geneshycA,dsrB, andbolApotentiated carbapenem efficacy in CREE. coli, whereas inhibition of the genesflhCandygaCconferred added resistance. Our results identify resistance factors and demonstrate that transcriptomic analysis is a potent tool for designing antibiotic PNA.

Structure and Activity of a Cytosolic Ribosome-Inactivating Protein from Rice

Ribosome-inactivating proteins (RIPs) are cytotoxic enzymes that inhibit protein translation by depurinating ribosomal RNA. Although most plant RIPs are synthesized with leader sequences that sequester them away from the host ribosomes, several RIPs from cereals lack these signal peptides and therefore probably reside in the cytosol near the plant ribosomes. More than 30 RIP genes have been identified in the rice (Oryza sativa spp. japonica) genome, many of them lacking a signal peptide. This paper focuses on a presumed cytosolic type-1 RIP from rice, referred to as OsRIP1. Using 3D modeling it is shown that OsRIP1 structurally resembles other cereal RIPs and has an active site that meets the requirements for activity. Furthermore, localization studies indicate that OsRIP1-eGFP fusion proteins reside in the nucleocytoplasmic space when expressed in epidermal cells of Nicotiana benthamiana or Arabidopsis thaliana suspension cells. Finally, OsRIP1 was recombinantly produced in Escherichia coli and was demonstrated to possess catalytic activity. Interestingly, this recombinant RIP inactivates wheat ribosomes far less efficiently than rabbit ribosomes in an in vitro system. These findings raise some interesting questions concerning the mode of action and physiological role of OsRIP1. This is the first time a RIP from rice is investigated at protein level and is shown to possess biological activity.

A highly conserved G-rich consensus sequence in hepatitis C virus core gene represents a new anti–hepatitis C target

G-quadruplex (G4) is one of the most important secondary structures in nucleic acids. Until recently, G4 RNAs have not been reported in any ribovirus, such as the hepatitis C virus. Our bioinformatics analysis reveals highly conserved guanine-rich consensus sequences within the core gene of hepatitis C despite the high genetic variability of this ribovirus; we further show using various methods that such consensus sequences can fold into unimolecular G4 RNA structures, both in vitro and under physiological conditions. Furthermore, we provide direct evidences that small molecules specifically targeting G4 can stabilize this structure to reduce RNA replication and inhibit protein translation of intracellular hepatitis C. Ultimately, the stabilization of G4 RNA in the genome of hepatitis C represents a promising new strategy for anti–hepatitis C drug development.

Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine

Malaria inflicts an enormous burden on global human health. The emergence of parasite resistance to front-line drugs has prompted a renewed focus on the repositioning of clinically approved drugs as potential anti-malarial therapies. Antibiotics that inhibit protein translation are promising candidates for repositioning. We have solved the cryo-EM structure of the cytoplasmic ribosome from the human malaria parasite, Plasmodium falciparum, in complex with emetine at 3.2 Å resolution. Emetine is an anti-protozoan drug used in the treatment of ameobiasis that also displays potent anti-malarial activity. Emetine interacts with the E-site of the ribosomal small subunit and shares a similar binding site with the antibiotic pactamycin, thereby delivering its therapeutic effect by blocking mRNA/tRNA translocation. As the first cryo-EM structure that visualizes an antibiotic bound to any ribosome at atomic resolution, this establishes cryo-EM as a powerful tool for screening and guiding the design of drugs that target parasite translation machinery.