Structural basis of rotavirus RNA chaperone displacement and RNA annealing

Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA–RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo–electron microscopy reconstructions of an NSP2–RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling.

Structural insight into Marburg virus nucleoprotein-RNA complex formation

The nucleoprotein (NP) of Marburg virus (MARV), a close relative of Ebola virus (EBOV), encapsidates the single-stranded, negative-sense viral genomic RNA (vRNA) to form the helical NP-RNA complex. The NP-RNA complex serves as a scaffold for the assembly of the nucleocapsid that is responsible for viral RNA synthesis. Although appropriate interactions among NPs and RNA are required for the formation of nucleocapsid, the structural basis of the helical assembly remains largely elusive. Here, we show the structure of the MARV NP-RNA complex determined using cryo-electron microscopy at a resolution of 3.1 angstrom. The structures of the asymmetric unit, a complex of an NP and six RNA nucleotides, was very similar to that of EBOV, suggesting that both viruses share common mechanisms for the nucleocapsid formation. Structure-based mutational analysis of both MARV and EBOV NPs identified key residues for the viral RNA synthesis as well as the helical assembly. Importantly, most of the residues identified were conserved in both viruses. These findings provide a structural basis for understanding the nucleocapsid formation and contribute to the development of novel antivirals against MARV and EBOV.

Diversity and prevalence of ANTAR RNAs across actinobacteria

Background Computational approaches are often used to predict regulatory RNAs in bacteria, but their success is limited to RNAs that are highly conserved across phyla, in sequence and structure. The ANTAR regulatory system consists of a family of RNAs (the ANTAR-target RNAs) that selectively recruit ANTAR proteins. This protein-RNA complex together regulates genes at the level of translation or transcriptional elongation. Despite the widespread distribution of ANTAR proteins in bacteria, their target RNAs haven’t been identified in certain bacterial phyla such as actinobacteria. Results Here, by using a computational search model that is tuned to actinobacterial genomes, we comprehensively identify ANTAR-target RNAs in actinobacteria. These RNA motifs lie in select transcripts, often overlapping with the ribosome binding site or start codon, to regulate translation. Transcripts harboring ANTAR-target RNAs majorly encode proteins involved in the transport and metabolism of cellular metabolites like sugars, amino acids and ions; or encode transcription factors that in turn regulate diverse genes. Conclusion In this report, we substantially diversify and expand the family of ANTAR RNAs across bacteria. These findings now provide a starting point to investigate the actinobacterial processes that are regulated by ANTAR.

Iron enhances the binding rates and translational efficiency of iron responsive elements (IREs) mRNA with initiation factor eIF4F

Interaction of iron responsive elements (IRE) mRNA with the translational machinery is an early step critical in the initiation of protein synthesis. To investigate the binding specificity of IRE mRNA for eIF4F, kinetic rates for the eIF4F·IRE RNA interactions were determined and correlated with the translational efficiency. The observed rate of eIF4F·FRT IRE RNA interactions was 2-fold greater as compared to eIF4F·ACO2 IRE RNA binding. Addition of iron enhanced the association rates and lowered the dissociation rates for the eIF4F binding to both IRE RNAs, with having higher preferential binding to the FRT IRE RNA. The binding rates of both eIF4F·IRE RNA complexes correlated with the enhancement of protein synthesis in vitro. Presence of iron and eIF4F in the depleted WGE significantly enhanced translation for both IRE RNAs. This suggests that iron promotes translation by enhancing the binding rates of the eIF4F∙IRE RNA complex. eIF4F·IRE RNA binding is temperature-dependent; raising the temperature from 5 to 25°C, enhanced the binding rates of eIF4F·FRT IRE (4-fold) and eIF4F·ACO2 IRE (5-fold). Presence of Fe2+ caused reduction in the activation energy for the binding of FRT IRE and ACO2 IRE to eIF4F, suggesting a more stable platform for initiating protein synthesis. In the presence of iron, lowered energy barrier has leads to the faster association rate and slower rate of dissociation for the protein-RNA complex, thus favoring efficient protein synthesis. Our results correlate well with the observed translational efficiency of IRE RNA, thereby suggesting that the presence of iron leads to a rapid, favorable, and stable complex formation that directs regulatory system to respond efficiently to cellular iron levels.

Photophysics of DFHBI bound to RNA aptamer Baby Spinach

The discovery of the GFP-type dye DFHBI that becomes fluorescent upon binding to an RNA aptamer, termed Spinach, led to the development of a variety of fluorogenic RNA systems that enable genetic encoding of living cells. In view of increasing interest in small RNA aptamers and the scarcity of their photophysical characterisation, this paper is a model study on Baby Spinach, a truncated Spinach aptamer with half its sequence. Fluorescence and fluorescence excitation spectra of DFHBI complexes of Spinach and Baby Spinach are known to be similar. Surprisingly, a significant divergence between absorption and fluorescence excitation spectra of the DFHBI/RNA complex was observed on conditions of saturation at large excess of RNA over DFHBI. Since absorption spectra were not reported for any Spinach-type aptamer, this effect is new. Quantitative modelling of the absorption spectrum based on competing dark and fluorescent binding sites could explain it. However, following reasoning of fluorescence lifetimes of bound DFHBI, femtosecond-fluorescence lifetime profiles would be more supportive of the notion that the abnormal absorption spectrum is largely caused by trans-isomers formed within the cis-bound DFHBI/RNA complex. Independent of the origin, the unexpected discrepancy between absorption and fluorescence excitation spectra allows for easily accessed screening and insight into the efficiency of a fluorogenic dye/RNA system.

Design and transfer of OsSWEET14-editing T-DNA construct to bac thom 7 rice cultivar

Bac thom 7 rice (BT7 rice) is one of the major elite rice cultivars in Vietnam with superior productivity and quality but very susceptible to bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae. The gene OsSWEET14, belonging to the OsSWEET family which encodes sugar transport proteins, is considered to be a susceptible gene involved in the virulence of Xoo. At least three cis-elements (EBE - Effector-binding element), including Tal5, PthXo3 and AvrXa7 on BT7 OsSWEET14 promoter, bind specifically to well-known transcription activator–like effectors (TALEs) of many Asian Xoo bacterium strains. In this study, a T-DNA construct which expressed the protein-RNA complex CRISPR/Cas for editing three EBEs position on the OsSWEET14 promoter was designed. The recombinant binary vector was tested by PCR, restriction enzyme and finally sequencing and then successfully transferred to Bac thom 7 rice through Agrobacterium tumefaciens. 28 of 30 hygromycin-resistant regenerated rice lines that grew and developed normally under nethouse conditions were selected by PCR with specific primers. Among these, twelve transgenic rice lines were identified carrying one copy of the T-DNA construct. The presence of CRISPR/Cas9-induced mutations of the targeted promoter in the transgenic plants were confirmed by T7EI assay. These results provide the basis to determine the role of OsSWEET14 in the susceptibility of Bac thom 7 rice to Xanthomonas oryzae pv. oryzae -caused disease, towards the further goal of generating an improved Bac thom 7 rice variety with broad-spectrum bacterial leaf blight resistance using CRISPR/Cas9 technology.