The indodicarbocyanine fluorophores Cy3 and Cy5 are extensively used as donor-acceptor pairs in fluorescence resonance energy transfer experiments, especially those involving single molecules. When terminally attached to double-stranded nucleic acids via the 5′ phosphate group these fluorophores stack onto the ends of the molecule. Knowledge of the positions of the fluorophores is critical to the interpretation of fluorescence resonance energy transfer data. The positions have been demonstrated for double-stranded (ds) DNA using NMR spectroscopy. Here, we have used x-ray crystallography to analyze the location of Cy3 and Cy5 on dsRNA, using complexes of an RNA stem-loop bound to L5 protein determined at 2.4 Å resolution. This confirms the tendency of both fluorophores to stack on the free end of RNA, with the long axis of the fluorophores approximately parallel to that of the terminal basepair. However, the manner of interaction of both Cy3 and Cy5 with the terminus of the dsRNA is significantly different from that deduced for dsDNA using NMR. The fluorophores are stacked on the terminal basepair such that their indole nitrogen atoms lie on the major groove side, and thus their pendant methyl groups are on the minor groove side. EFRET) as the interfluorophore distance changes. Transfer of electronic excitation energy is due to coupling between the donor emission dipole vector and the excitation dipole vector of the acceptor. The scalar product of these vectors is a function of the angle between them, and this complicates the extraction of absolute distance information from FRET data (Fluorescence resonance energy transfer (FRET) has been extensively used in single-molecule studies of nucleic acid structure and dynamics , 2, 3. MRET data , 5, 6. I3 or C5 polymethine linker connects two indolenine ring systems  and quantum yields of 0.15 and 0.3, respectively. They are also reasonably photostable, especially in the presence of oxygen-scavenging systems and triplet state quenching. Although the spectroscopic properties are favorable, when terminally attached to double-stranded nucleic acids they are at least partially oriented. Therefore, the transition moment orientation factor κ2 cannot be assumed to be a constant, such as taking the value of 2/3, which would be appropriate for fluorophores that undergo rapid reorientation during the lifetime of the excited state  DNA via a Ctroscopy . We substroscopy . In bothtroscopy , 12.2 would be expected to vary sinusoidally with helix length, with two maxima and two minima per helical turn. This was performed for dsDNA of 10–24 bp, and the results were in excellent agreement with that predicted for a B-form helix -TEV (tobacco etch virus) cleavage site-L5 protein fusion was inserted into the pET28a vector (Novagen). After confirmation by sequencing, the plasmid containing the correct gene was transformed into fresh BL21(DE3) Rosetta competent cells  for expression. A single colony was picked and incubated in 1 L Luria broth cell culture at 37°C for 8 h, and isopropyl β-D-1-thiogalactopyranoside added to a final concentration of 1 mM to induce protein expression at 37°C for 4 h. Cells were harvested and resuspended in 2× phosphate-buffered saline, 10 mM imidazole buffer, and frozen at −20°C. After thawing at room temperature, the cells were sonicated, and the lysate containing recombinant protein was purified using a Ni-nitrilotriacetic acid gravity column . After elution using phosphate-buffered saline and 200 mM imidazole buffer, the protein was incubated with TEV protease to cleave the His6-MBP tag. The cleaved protein was then applied to heparin and size exclusion columns  for further purification. The purified L5 protein was concentrated to 0.1 mM and kept on ice.A gene encoding tert-butyldimethyl-silyl protection (μL of anhydrous DMSO and 125 μL triethylaminetrihyrofluoride (Sigma-Aldrich) to remove 2′O-tert-butyldimethyl-silyl groups, and agitated at 65°C in the dark for 2.5 h before butanol precipitation. RNA was purified by electrophoresis in 12% polyacrylamide under denaturing conditions in 90 mM Tris.borate (pH 8.3), 10 mM EDTA (Tris-borate EDTA buffer), and 7 M urea. RNA was electroeluted from excised gel slices using an Elutrap system  in 0.5 × Tris-borate EDTA buffer overnight at 4°C, recovered by ethanol precipitation, and redissolved in 5 mM HEPES (pH 7.0) and 20 mM NaCl.RNA was synthesized using UltraMILD ribonucleotidephosphoramidites with 2′O-otection , 19 , and 50 mM KF. This was sealed with 0.5 mL mother liquid at 4°C in hanging-drop mode and crystals of 20 × 20 × 200 1MJI in the PHENIX suite  to create a gene expressing (from the N- to C-termini) His6-MBP gene-TEV cleavage site-L5 protein. The fusion was expressed in in Escherichia coli BL21(DE3) and purified by affinity chromatography on Ni-nitrilotriacetic acid. After cleavage of the fusion with TEV protease, the free L5 protein was purified by ion exchange and gel filtration chromatography.A synthetic gene encoding 3 methylene linker.Thirty-three nucleotide RNA species designed to form flush-ended stem-loop structures  were che1212 with closely similar cell dimensions and diffracted to a resolution of 2.4 Å  and are related by a noncrystallographic two-fold axis between the two terminal loops. The conformation of the RNA is closely similar to the structure of Perederina et al. , with ovβ3 (88–94) and basic side chains extend from the extreme N-terminal end of α3 (R95 and R96) toward the backbone, together with K67 from the α2-β2 linking section. The distal end of the RNA is far removed from the directly bound L5 protein.In each complex, the protein interacts with the minor groove face of the terminal loop . The staRp nonbridging phosphate oxygen atom of C47 of the other RNA. The axes of the two RNA molecules are inclined with an included angle of 120°. Within the dimer, the minor groove of the open end of the RNA helix is juxtaposed with the open planar face of the β-sheet of the protein that is bound to the loop of the other RNA in the dimer. However, we observe no protein electron density at the end of the helix, at the position where the fluorophores are located.The asymmetric unit comprises two RNA-protein complexes in which the RNA stem-loops are associated at the loop end , with a 1212) from the original published structure (that was P21)  , so crysα3 and β4 (residues 113–120) forms an arch over the end of the RNA, leaving a cavity in which the fluorophore is located  is well defined for the whole fluorophore, including the C5 polymethine section. The electron density map shows that Cy5 is stacked upon the terminal basepair, with both rings of the distal indole stacked over the guanine nucleobase (G58) (average spacing 3.9 Å). The C5 polymethine section sits over the cytosine nucleobase (C26) and the proximal indole is directed out into the solvent. The long axis of the fluorophore is rotated 10° about the helical axis relative to that of the terminal C-G basepair. Both the polymethylene linkers are well defined in the omit map, showing that the Cy5 fluorophore is stacked on the face that places its indole nitrogen atoms on the major groove side of the C-G basepair. Electron density for the Cy5 at the RNA-RNA interface is much more poorly defined (C), although there is some density indicating the position of the indole ring stacked over G58. Average B-factors for this Cy5 were high , with an excellent overlap between the six-membered rings at a mean distance of 3.4 Å. By contrast, the five-membered ring of the proximal indole lies above the ribose of U26, with the six-membered ring directed out into the solvent, and the C3 polymethine section lies above its pyrimidine nucleobase. The centers of the regions of density corresponding to the two indole ring systems shows that the long axis of the Cy3 is close to parallel with that of the terminal U-A basepair. In addition, the density for the polymethylene chain unambiguously demonstrates that the indole nitrogen atoms of the fluorophore are located on the major groove side of the terminal U-A basepair.In general, the electron density for Cy3 was weaker than that observed for the Cy5 fluorophore, and average B-factors were higher . No elecT. thermophilus L5 protein-RNA complex that creates two different environments for the free end of the dsRNA. This system could be of general utility for the study of molecules interacting with the end of RNA species.Here we present, to our knowledge, the first x-ray crystallographic structural analysis of cyanine fluorophores attached to dsRNA tethered through the terminal 5′ phosphate. This was carried out using a EFRET exhibited a sinusoidal variation with helix length consistent with terminal stacking of both fluorophores.Our crystallographic studies confirm the predisposition of Cy3 and Cy5 to stack on the final basepair of double-stranded nucleic acids. We have noted that this is true irrespective of the identity of the sequence of the terminal basepair, although we present only the best-defined structures for each fluorophore here, i.e., Cy3 on U-A and Cy5 on C-G. Terminal stacking is consistent with earlier data measuring FRET efficiency between Cy3 and Cy5 tethered to the two 5′-termini of double stranded DNA and RNA helices , 11. In EFRET on helix length indicated a lateral motion of significant amplitude  stack on RNA such that the indole nitrogen atoms lie on the major groove side of the terminal basepair, placing the pendant methyl groups on the minor groove side. This is well defined by the data, because the electron density of the polymethylene chains is extremely clear in both structures (B and B). By contrast, in the DNA structures (exemplified by C), the fluorophore is rotated 180° about its long axis, so that the indole nitrogen atoms are now on the minor groove side of the terminal basepair , finding the expected modulation with helix length (The conformation we observe in the crystal is consistent with experimental FRET data. Furthermore, we used the crystal structures to create models of dsRNA with terminally attached Cy3 and Cy5 exactly as observed in the crystal, generating a series of duplexes with lengths from 10 to 22 bp. We calculated x length . These pThe average position of the cyanine fluorophores terminally attached to dsRNA in the crystal structures is unambiguous. This information will be important in the interpretation of distance and angular information in FRET experiments with RNA species. When the position and orientation of the fluorophores are known, then potentially both angular and distance information within the attached macromolecule can be deduced.X-ray crystallography shows the strong tendency of the indodicarbocyanine fluorophores Cy3 and Cy5 terminally attached to double-stranded RNA to stack onto the ends of the molecule. The fluorophores are stacked on the terminal basepair such that their indole nitrogen atoms lie on the major groove side.Y.L. performed experimental work and crystallographic analysis. Y.L. and D.M.J.L. planned experiments and analyzed data. D.M.J.L. wrote the article.


RNA molecules have been known to play a variety of significant roles in cells. In principle, the functions of RNAs are largely determined by their three-dimensional (3D) structures. As more and more RNA 3D structures are available in the Protein Data Bank (PDB), a bioinformatics tool, which is able to rapidly and accurately search the PDB database for similar RNA 3D structures or substructures, is helpful to understand the structural and functional relationships of RNAs.Since its first release in 2011, R3D-BLAST has become a useful tool for searching the PDB database for similar RNA 3D structures and substructures. It was implemented by a structural-alphabet (SA)-based method, which utilizes an SA with 23 structural letters to encode RNA 3D structures into one-dimensional (1D) structural sequences and applies BLAST to the resulting structural sequences for searching similar substructures of RNAs. In this study, we have upgraded R3D-BLAST to develop a new web server named R3D-BLAST2 based on a higher quality SA newly constructed from a representative and sufficiently non-redundant list of RNA 3D structures. In addition, we have modified the kernel program in R3D-BLAST2 so that it can accept an RNA structure in the mmCIF format as an input. The results of our experiments on a benchmark dataset have demonstrated that R3D-BLAST2 indeed performs very well in comparison to its earlier version R3D-BLAST and other similar tools RNA FRABASE, FASTR3D and RAG-3D by searching a larger number of RNA 3D substructures resembling those of the input RNA.http://genome.cs.nthu.edu.tw/R3D-BLAST2/.R3D-BLAST2 is a valuable BLAST-like search tool that can more accurately scan the PDB database for similar RNA 3D substructures. It is publicly available at  Besides being involved in protein synthesis, RNAs have been found to perform other diverse functions in the cell, such as processing and modification of RNAs, regulation of gene expression, and degradation and translocation of proteins . In prinIn principle, both RNA FRABASE and FASTR3D use pattern-based approaches to search the PDB database for RNAs that have exactly the same secondary (2D) structure as that of the query RNA. As for R3D-BLAST, it reduces RNA 3D structures into one-dimensional (1D) structural sequences according to some local structure features in the nucleotide backbone conformation and applies BLAST to the resulting 1D structural sequences for searching similar RNA 3D substructures. RAG-3D exploits a coarse-grained graph representation to describe RNA 3D structures as simplified 3D graphs and searches for RNA 3D substructures with the same graph topology (i.e. pattern of vertex connectivity) as the query RNA substructure.η and θ), which are dihedral angles defined based on C4 ′ and P atoms from consecutive bases, are adequate to represent the backbone conformation of an RNA nucleotide. Therefore, the SA mentioned above was previously constructed from a collection of 117 RNA 3D structures  using the η and θ values of their nucleotide backbones. Since the public release of R3D-BLAST in 2011, however, several hundreds of new RNA 3D structures have been experimentally determined and also deposited in the PDB database. Therefore, it can be expected that these newly determined RNA 3D structures should allow us to construct a new and sufficiently high-quality SA that can be used to further improve the search performance of R3D-BLAST.The above method we used to implement R3D-BLAST  is the sAnother reason to upgrade our R3D-BLAST is that the PDB data files used by R3D-BLAST to retrieve their RNA 3D structures or uploaded by the user to run R3D-BLAST were in the PDB format only. However, the PDB format now is a legacy format, because the size of a structure represented in a single PDB formatted file was limited to 99,999 atoms and the relationships among their data items were implicit . The mmCIn this study, we have upgraded our RNA structural search tool R3D-BLAST to develop a new web server named R3D-BLAST2 (meaning R3D-BLAST version 2) based on a totally new SA that is constructed from a representative and sufficiently non-redundant list of 876 atomic-resolution RNA 3D structures . In addition, we have modified the kernel program in R3D-BLAST2 so that it can retrieve RNA 3D structures from the PDB data files in the mmCIF format and also allows the user to upload an mmCIF formatted file to search for similar RNA 3D substructures. For validation, we have used a benchmark dataset of RNA 3D structures to test R3D-BLAST2 and compare its search performance with its previous version R3D-BLAST and other similar RNA structural search tools, such as RNA FRABASE, FASTR3D and RAG-3D. Our experimental results have finally shown that R3D-BLAST2 indeed outperforms R3D-BLAST, as well as RNA FRABASE, FASTR3D and RAG-3D, by searching a larger number of RNA 3D substructures resembling those of the query RNA.η- θ plot) from a representative and sufficiently non-redundant list of 876 RNA 3D structures determined by X-ray crystallography and electron microscopy (resolution ≤4 Å) from the RNA 3D Hub [η and θ values. In principle, the AP algorithm is a message-passing method that operates by simultaneously considering all nucleotides as possible exemplars (i.e. centers) and recursively exchanging two types of real-valued messages, called responsibility and availability, between nucleotides until a collection of suitable exemplars and corresponding clusters appears. The responsibility, denoted by r, is sent from nucleotide i to candidate exemplar k and reflects the accumulated evidence for how well k can serve as the exemplar for i with taking into account other potential exemplars. The availability, denoted by a, is sent from candidate exemplar k to nucleotide i and reflects the accumulated evidence for how appropriate it would be for i to select k as its exemplar with taking into account the support from other nucleotides that k should be an exemplar. Note that the AP algorithm has been shown to perform better than K-centers clustering algorithm, which tends to fall into local optimality and is susceptive to noise and outliers [We used the following SA-based algorithm to implement R3D-BLAST2. First, we collected 63 402 non-terminal nucleotides , structural alignment score (SAS) and/or percentage of structural identity (PSI), where SAS equals to 100×RMSD/ [Inevitably, R3D-BLAST2 may return some RNA 3D substructures that actually do not resemble any query RNA substructure. In fact, the esidues)  and PSI esidues) . For thehttp://genome.cs.nthu.edu.tw/R3D-BLAST2/ by an easy-to-operate interface  in the ‘Advanced Parameters’ section. After that, R3D-BLAST2 quickly shows the details of each RNA substructure hit, such as PDB ID, chain ID, molecular function, structure determination method and structural resolution, as well as the information about its structural alignment against the query RNA, such as E-value, alignment range and coverage in both the query and subject RNAs, calculated PSI, SAS and RMSD (if the filter in the ‘Advanced Parameter’ section is selected) and a JSmol window displaying the superposition of aligned structures.R3D-BLAST2 can be accessed at see Fig. . It acceWe utilized a benchmark dataset consisting of eight different kinds of RNAs (see Table Consequently, as shown in Table In addition, except pseudoknot and ribozyme in the benchmark dataset, R3D-BLAST2 still identified more RNA structure hits with 100% query coverage whose entire 3D structures are highly resemble the query RNA when comparing with RNA FRABASE and FASTR3D. Recall that RNA FRABASE and FASTR3D were both developed to search for RNAs that have the same 2D structure as the query RNA without any insertions and deletions. As demonstrated in our experimental results, therefore, they could inevitably miss those RNAs that possess the same overall 3D structures but different 2D structures and/or lengths. When queried with the pseudoknot and ribozyme in the benchmark dataset, both RNA FRABASE and FASTR3D returned more RNA structure hits as compared to the search result of R3D-BLASTS2 with 100% query coverage. In fact, some regions around these RNA 3D structures identified by RNA FRABASE or FASTR3D are not quite similar to the query RNA and as a result, they were removed from those RNA structure hits returned by R3D-BLAST2. In other words, all the pseudoknots and ribozymes returned by both RNA FRABASE and FASTR3D still can be found by R3D-BLAST2, but with a query coverage less than 100%. On the other hand, R3D-BLAST2 returned a large number of other structurally similar RNA substructures that actually are missed by both RNA FRABASE and FASTR3D.When compared to RAG-3D, R3D-BLAST2 was able to find many more RNA substructure hits that are quite structurally similar to the query RNA substructures. It should be noted that some of RNA substructures returned by RAG-3D may be structurally different from those of the query RNA, as shown in Fig. Figure Table In this study, we upgraded our previous RNA structural search tool R3D-BLAST to develop a new web server named R3D-BLAST2 based on a newly constructed structural alphabet with a higher capability of representing the most common backbone conformations of RNA nucleotides. In contrast to the previous version, R3D-BLAST2 now can retrieve RNA 3D structures from the PDB data files in the mmCIF format and also allow the user to upload an mmCIF formatted file as an input to search for its similar RNA 3D substructures. According to our experimental results on a benchmark dataset, R3D-BLAST2 indeed outperforms its previous version R3D-BLAST and other similar RNA structural search tools RNA FRABASE, FASTR3D and RAG-3D by searching a larger collection of RNA 3D substructures resembling a query RNA substructure. It can be expected that R3D-BLAST2 will become a valuable tool to annotate RNA structures and functions because RNA molecules with the same function usually share similar 3D substructures.


Positive-strand RNA viruses have evolved multiple strategies to not only circumvent the hostile decay machinery but to trick it into being a priceless collaborator supporting viral RNA translation and replication. In this review, we describe the versatile interaction of positive-strand RNA viruses and the 5′-3′ mRNA decay machinery with a focus on the viral subversion of decapping activators. This highly conserved viral trickery is exemplified with the plant Brome mosaic virus, the animal Flock house virus and the human hepatitis C virus. Viruses maintain a constant duel with their infected host cells. Not only do they evolve strategies to circumvent deleterious cellular responses, but they also take advantage of the rich pools of host factors as controllable resources. Hijacking these resources is essential for the expansion of viruses as their gene-coding capacity is limited. One exquisite example of such viral manipulation is the use of the cellular mRNA decay machinery by a group of positive-strand RNA ((+)RNA) viruses. They use different strategies to turn the mRNA decay proteins into allies that support their replication and expansion.The (+)RNA virus group includes numerous plant, animal and human pathogens such as the hepatitis C virus (HCV) and the emerging mosquito-borne Zika virus (ZIKV), Dengue virus (DENV), West Nile virus (WNV) and Chikungunya virus (CHIKV). Despite their diversity in terms of genome organization or virion morphology, the replication cycle of (+)RNA viruses is highly conserved . Upon enCytoplasmic mRNA decay occurs via two major pathways—the deadenylation-dependent 5′-3′ decay and the exonucleolytic 3′-5′ decay pathways—that are conserved in all eukaryotes  [3]. In 7GDP) and a 5′ monophosphate mRNA. To be fully active, Dcp2 requires a conformational change mediated by the Dcp1 protein. The other enzyme is the 5′-3′ exonuclease Xrn1 that degrades the mRNA after decapping. In addition, there are other factors, named decapping activators, that assist and enhance the efficacy of the pathway. They include Sm-like proteins 1–7 (Lsm1–7), DNA topoisomerase 2-associated protein (Pat1), DExD/H-box ATP-dependent RNA helicase 1 (Dhh1), enhancer of mRNA-decapping protein 1–3 (Edc1–3) and the Suppressor of Clathrin Deficiency (Scd6). From these, Lsm1–7, Pat1 and Dhh1 are the most characterized ones. The Lsm1–7 ring is constituted by seven Lsm proteins that belong to the conserved Sm family of proteins and acts as an RNA chaperone facilitating a variety of RNA-RNA and RNA-protein interactions [The deadenylation-dependent 5′-3′ decay pathway is the main cytoplasmic decay pathway RNA viruses have developed a myriad of strategies to shield their RNA from degradation by Xrn1, often by directly suppressing or degrading the cellular decay machinery. For example, picornaviruses use an aggressive mechanism to combat decay by inducing the rapid degradation of Xrn1 and Dcp1 ,23. WNV Saccharomyces cerevisiae (reviewed in [A fruitful model system to study (+)RNA virus–host interactions is the replication of the plant BMV in iewed in ,32). Theiewed in ). RNA1 aStudies with the BMV/yeast model system led to the identification and characterization of hundreds of host factors required for different steps in the BMV life cycle ,35. ThreLSM1 gene, a key component of the Lsm1–7 ring, affect differentially BMV RNA translation and recruitment [cis-elements in the viral RNA, thereby remodeling mRNA secondary structures and promoting its circularization and translation. Since poly(A)-tails in cellular mRNAs mediate 5′-3′ circularization via the binding of the poly(A) binding protein, binding of Lsm1–7, Pat1 and Dhh1 to the 5′ and 3′ UTRs structure and to initiation factors at the 5′ UTR would establish such circularization in BMV RNAs. During recruitment, circularization would be disrupted as the Lsm1–7/Pat1 complex would now bind to the viral 1a protein, driving the viral RNA from translation to replication.The mechanisms by which the Lsm1–7/Pat1 complex promotes translation and recruitment of BMV RNAs are different. Mutations in the ruitment . Importaruitment . The molDrosophila, Caenorhabditis elegans, plants, mammals and Saccharomyces cerevisiae. Accordingly, the host factors hijacked by FHV to replicate are highly conserved [The FHV (reviewed in ) is an ionserved ,45,46,47onserved .The FHV bipartite genome consists of two capped but non-polyadenylated RNA segments. RNA1 encodes protein A, the only FHV protein required for replication, and RNA2 encodes the capsid precursor. During replication, RNA1 also produces sub-genomic RNA3 that encodes protein B1, of unknown function, and protein B2, required to suppress RNA silencing in infected hosts. RNA3 corresponds to the 3′ end of RNA1 and it is synthesized during RNA1 replication. In addition to its coding function, RNA3 coordinates the production of appropriate levels of RNA1 and RNA2 ,50,51. Tcis-elements in RNA1 [Lsm1–7, Pat1 and Dhh1 control RNA3 synthesis . Differe in RNA1 . These b in RNA1  and RNA3 in RNA1 , Lsm1–7, in RNA1 . Interes in RNA1 .cis-sequences in the DENV RNA genome and it co-localizes with the DENV replication complex [Since the 5′-3′ decay pathway is strongly conserved from yeast to humans, human (+)RNA viruses might as well subvert it to favor viral replication. Indeed, the human counterparts of Lsm1–7, Pat1 and Dhh1—namely Lsm1–7, PatL1 and DEAD-Box Helicase 6 (DDX6)—are required for the expansion of the flaviviruses HCV, DENV and WNV. Depletion of Lsm1–7, PatL1 or DDX6 directly and specifically inhibits HCV RNA translation and replication ,54. More complex .Flaviviridae family, the conserved use of decapping factors by (+)strand RNA viruses that infect plants (BMV), insects (FHV) and even bacteria (Qβ [Flaviviridae family. Depriving (+)RNA viruses of this highly conserved strategy by targeting these decay factors with drugs would therefore appear to be a promising strategy to generate broad-spectrum antiviral drugs. The fact that the individual, transient knock-down of Lsm1–7, PatL1 or DDX6 proteins in human cells is not toxic and that the respective yeast knockout strains are viable, stresses the feasibility of such an approach for the future.Although all the above described examples of (+)RNA viruses belong to the eria (Qβ ) underlicis-acting signals in the viral RNA genome. The role of these decapping proteins as catalyzers of mRNP transitions that direct cellular mRNAs from translation to decay suggests that they act similarly on the highly structured viral (+)RNA genomes, directing them to translation or replication.What are the common features of the aforementioned activities of Lsm1–7, Pat1/PatL1 and Dhh1/DDX6 in (+)RNA viral life cycles? In all cases, the decapping activators interact with viral replication proteins, and/or with specific and structured regulatory Lsm1–7, PatL1 and DDX6 accumulate in P-bodies ,60. P-boThe obvious question to ask is whether P-body disruption is required for viral infection or whether it is just a consequence. The data currently available suggest that it is a mere consequence, as P-body formation is not required for mRNA decay  and deplViruses are masters in converting hostile conditions into a paradise for their own replication. The sequestering of the cellular decay machinery by (+)RNA viruses is a remarkable example of this that goes beyond using the decay components themselves towards altering the whole transcriptional/translational landscape of the host. The strong conservation of this viral strategy across species pinpoints a weak spot that can be exploited for the development of broad-spectrum antiviral drugs. Yet, some essential questions remain open. For example, how are viral infection, the host transcriptome/translatome and pathogenesis linked? How do viruses regulate host transcription to favor their own replication? The basic nature of these questions highlights that scientists in this area still have a long path to go.


Aging-related neurodegenerative diseases are progressive and fatal neurological diseases that are characterized by irreversible neuron loss and gliosis. With a growing population of aging individuals, there is a pressing need to better understand the basic biology underlying these diseases. Although diverse disease mechanisms have been implicated in neurodegeneration, a common theme of altered RNA processing has emerged as a unifying contributing factor to neurodegenerative disease. RNA processing includes a series of distinct processes, including RNA splicing, transport and stability, as well as the biogenesis of non-coding RNAs. Here, we highlight how some of these mechanisms are altered in neurodegenerative disease, including the mislocalization of RNA-binding proteins and their sequestration induced by microsatellite repeats, microRNA biogenesis alterations and defective tRNA biogenesis, as well as changes to long-intergenic non-coding RNAs. We also highlight potential therapeutic interventions for each of these mechanisms. Summary: In this At a Glance review, Edward Lee and co-authors provide an overview of RNA metabolism defects, including mislocalization of RNA-binding proteins and microRNA biogenesis alterations, that contribute to neurodegenerative disease pathology. Aging-related neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal degeneration (FTD) and amyotrophic lateral sclerosis (ALS), among others, are relentlessly progressive and uniformly fatal neurological diseases that are characterized by irreversible neuron loss and gliosis. Although dementia prevalence as a percentage of the elderly has declined in developed countries, the absolute number of dementia cases is growing as a result of an increase in the aging population . Thus, iAlthough much emphasis has been placed on the role of protein aggregates in neurodegenerative diseases, multiple lines of evidence also converge on altered RNA processing as a contributing factor in the pathogenesis of these diseases . Defectsposter panel A). Two notable examples of RBP defects occur in familial and sporadic cases of ALS and FTD. ALS is a neurodegenerative disease that leads to the loss of upper and lower motor neurons from the motor cortex and spinal cord, respectively, whereas FTD is associated with neuronal loss in the temporal and frontal cortex. Despite different areas of neuronal atrophy, a common link between ALS and FTD patients is a nuclear RBP called TAR DNA-binding protein-43 (TDP-43). Post-mortem brains from human ALS and FTD patients show a characteristic mislocalization of TDP-43 from the nucleus into phosphorylated, ubiquitylated cytoplasmic TDP-43 aggregates (see poster panel A)  , were identified in a subset of individuals with ALS, and FUS was revealed to be mislocalized to the cytoplasm (HNRNPA1) and A2B1 (HNRNPA2B1) contribute to disease. MSP is characterized by the progressive degeneration of muscle, brain, motor neurons and bone, which sometimes manifests as ALS or FTD (VCP), a triple ATPase protein involved in many cellular functions including endolysosomal degradation, autophagy, and the ubiquitin proteasome system, also causes MSP  . Indeed,ause ALS , althougause ALS , stabiliause ALS  and tranause ALS . Shortlyytoplasm . Similarytoplasm . An addiS or FTD . Mutatiouses MSP .Tardbp depleted TDP-43, show altered global RNA expression affecting 601 genes, and specifically neuronal genes with long introns  and nuclear paraspeckle assembly transcript 1 (Neat1) (poster panel A). Given that the depletion of these RBPs alters the expression of thousands of genes, it is likely that some, or even all of these changes contribute to disease pathogenesis.It is thought that the loss of RBPs through their nuclear mislocalization and/or the toxicity caused by their cytoplasmic aggregation can lead to disease, but their relative contributions to disease remain unclear. Mouse models where antisense oligonucleotides (ASOs) against  introns . To bett introns . Some of (Neat1) . Thus, iposter).The formation of cytoplasmic RNA granules that lead to cytoplasmic aggregates has also been proposed to be pathogenic. When cells are stressed, cytoplasmic RNA granules that contain stalled translational complexes are formed. TDP-43, FUS, and other RBPs, such as hnRNPA1 and A2B1, localize to stress granules . Indeed,TARDBP or mutant FUS bearing ALS-associated mutations are overexpressed, develop cytoplasmic aggregation and exhibit features of ALS and FTD, including cortical and hippocampal neuronal loss and motor deficits  activating the heat shock response; (2) using heat shock protein (Hsp)104 ‘disaggregases’; or (3) by modulating the ubiquitin proteasome system and autophagy. HSPs function as molecular chaperones and are involved in protein folding, protein trafficking and in coping with denatured proteins . Prior wmutation . Until tposter panel B) (Microsatellite repeat expansion disorders are a class of neurodegenerative diseases caused by repetitive DNA elements that form long expansions within gene bodies or in untranslated regions. Over 25 human genes that contain repeat expansions have been identified to date . Some nepanel B) .DMPK, which leads to myotonic dystrophy (DM)  . This exphy (DM)  and recrphy (DM) . By sequet genes .FMR1 is transcribed into RNA and interacts with hnRNPs, MBNL1 and other RBPs  also sequester RBPs. In FXTAS, a short repeat expansion (<200 repeats) in the untranslated region of the gene 4C2) expansion in the first intron of the gene C9orf72 is bidirectionally transcribed into mutant RNA that forms aggregates in the nucleus (poster panel B). Additionally, the repeat expansion, which normally regulates vesicle trafficking and autophagy . An alternative to targeting the repeat-containing transcript for degradation is to target proteins that are responsible for transcribing the repeat expansion. For instance, knockdown of the transcription elongation factor SUPT4H1 selectively decreases the repeat-containing RNA in C9orf72 expansion fibroblasts derived from human carriers . miRNAs are initially transcribed by RNA polymerase II and then undergo sequential cleavage, first by Drosha and DGCR8 in the nucleus – to generate pre-miRNAs from pri-miRNAs – and then by Dicer after being exported into the cytoplasm, to generate a miRNA duplex . The miRTARDBP and FUS in a cell-free dicing activity assay, Dicer function was shown to be altered resulting in an inhibition of miRNA biogenesis (poster panel C). Conversely, activation of Dicer with enoxacin in an ALS mouse model that carries a mutation in the gene Cu/Zn superoxide dismustase 1 (SOD1) reverses miRNA downregulation and neuromuscular defects . There are notable examples in Alzheimer's disease (AD) and ALS. AD is the most common form of dementia and is characterized by progressive memory loss, impaired cognitive function, and the inability to perform daily tasks. Pathologically, AD is defined by the presence of extracellular amyloid-β (Aβ) plaques and of intracellular hyper-phosphorylated neurofibrillary tangles composed of tau , increases BDNF levels and improves memory function (SOD1. A locked nucleic acid (modified RNA nucleotide) anti-miR-155 reduces miR-155 levels in this mouse  that differ by disease stage see  Cogswel. Furtherfunction . Anotheris mouse , therebyis mouse . Becauseposter panel D). Additionally, during cellular stress, angiogenin (ANG) cleaves tRNAs into fragments that might inhibit translation or target specific mRNAs for degradation as a cellular protective mechanism (CLP1 (R140H) is a cause of progressive brain atrophy and microcephaly in humans . Mutations in ANG that might contribute to the loss of ANG function also occur in ALS RNAs are important for gene expression , it is pechanism . Indeed,echanism . It is uechanism . A mutatn humans . CLP1 R1r in ALS . Alterednslation . These sposter panel E). These RNAs are longer than 200 nucleotides and fulfill various functions, including acting as scaffolds for chromatin modifiers and nuclear paraspeckles, as transcriptional co-regulators, and even as decoys for other RNAs (BACE1-antisense (AS) and lnc-SCA7 . BACE1-AS is increased in AD brains and competes with miRNA-545-5p binding to stabilize BACE1 mRNA. This is associated with increased BACE1 expression and with the formation of the Aβ peptides that contribute to AD pathology (see poster panel E)   to repress gene expression (poster). Thus, lncRNAs are likely to influence gene expression post-transcriptionally to contribute to neurodegenerative disease.lncRNAs have also been implicated in neurodegenerative diseases (see her RNAs . Alteratpanel E) . In spinoduction . Other eormation . Both of and FUS . MALAT1 panel E) . Additioe stress . Other Hpression   blocking the interaction of the antisense and sense mRNA by degrading the antisense strand, which leads to the transcriptional repression of the gene; (2) using aptamers to bind and inhibit lncRNA structures and prevent their activity; and (3) employing small molecules that inhibit lncRNA interactions . Althougduced Aβ , this fiNeurodegenerative diseases occur by different means and present with various pathologies. However, it is becoming increasingly clear that altered or defective RNA metabolism, including mislocalized RBPs and aberrant ncRNA biogenesis or expression, can contribute to neurodegeneration. Because numerous disease-associated pathways are perturbed in these neurodegenerative diseases (as summarized in the accompanying poster), it is unlikely that targeting only one of these modalities will lead to a complete cure. Nonetheless, reversing some of these RNA aberrations could prove to be effective in modifying the incessantly downward disease trajectory associated with these diseases. Perhaps by modifying disease progression, such approaches could provide a significant benefit for those afflicted by neurodegenerative disease.


Pseudomonas aeruginosa. During carbon catabolite repression (CCR) Hfq together with the catabolite repression control protein Crc can act as a translational repressor of catabolic genes. Upon relief of CCR, the level of the Hfq-titrating RNA CrcZ is increasing, which in turn abrogates Hfq-mediated translational repression. As the interdependence of Hfq-mediated and RNA based control mechanisms is poorly understood, we explored the possibility whether the regulatory RNA CrcZ can interfere with riboregulation. We first substantiate that the P. aeruginosa Hfq is proficient and required for riboregulation of the transcriptional activator gene antR by the small RNA PrrF1-2. Our studies further revealed that CrcZ can interfere with PrrF1-2/Hfq-mediated regulation of antR. The competition for Hfq can be rationalized by the higher affinity of Hfq for CrcZ than for antR mRNA.The RNA chaperone Hfq regulates virulence and metabolism in the opportunistic pathogen  E. coli, and it is now well established that Hfq fulfills several functions in post-transcriptional regulation. It can stabilize small regulatory RNAs (sRNAs) and facilitate annealing between sRNAs and their target mRNAs. The latter mode of action may result either in translational repression accompanied by degradation of both RNAs or in translational activation and stabilization of the mRNA. In addition, it may stimulate polyadenylation of mRNAs, which can trigger 3´ to 5´directional decay -ATP  and polynucleotide kinase (Thermo Scientific). The labelled RNAs were gel-purified following standard procedures, eluted and kept in diethylpyrocarbonate- (DEPC) treated water. Labeled RNA (0.1 pmol) dissolved in DEPC water was incubated with increasing amounts of HfqPae, HfqPaeY25DFlag and HfqPaeK56A proteins  immediately before loading and separated on 4% polyacrylamide gels using Tris-borate buffer. The radioactively labeled bands were visualized with a PhosphorImager (Molecular Dynamics) and quantified with ImageQuant software 5.2.The proteins  or unlabproteins  and S5 iantR(-95–+67) to Hfq, respectively, was determined by microscale thermophoresis (MST) ; y = fraction annealed, kobs = observed annealing reaction constant, A = maximum reaction amplitude. The reaction rate kobs was calculated from the average of three independent experiments.RNA annealing activities of Hfq(TIF)Click here for additional data file.S5 Fig·Hfq and PrrF2·Hfq·antR(-95–+67) complexes, respectively. The plus symbol denotes the PrrF2·antR(-95–+67) complex. Right panel: Western-blot of the gel shown at the left probed with anti-Hfq antibodies. The presence of Hfq in the PrrF2·Hfq and PrrF2·Hfq·antR(-95–+67) complexes is indicated by single and double circles, respectively. (B) Hfq accelerates PrrF-antR duplex formation. 10 nM radioactively labeled antR(-95–+67) RNA was incubated alone (lane 1), with HfqPae (lane 6) or with increasing amounts of PrrF2  in the absence (lanes 2–5) or presence (lanes 6–10) of HfqPae (the molar ratio of HfqPae-hexamer to antR(-95–+67) RNA to was 2:1), and the resulting complexes were analyzed on a 4% native polyacrylamide gel. Single and double circles denote the antR(-95–+67)·Hfq and PrrF2 Hfq·antR(-95–+67) complexes, respectively. The plus symbol denotes the PrrF2·antR(-95–+67) complex.(A) Left panel: The samples corresponding to those shown in (TIF)Click here for additional data file.


Furthermore, a total of 90 editing sites were detected on transcripts of six mitochondrial genes  in both Huhan-1A and Huhan-1B on the 0, 1st, and 3rd DAT. Forty-eight sites were furthermore determined as stress-responsive sites (SRS). Generally, in response to oxidative stress, SRS in Huhan-1A increased the resulting editing efficiencies, while SRS in Huhan-1B decreased the resulting editing efficiencies. In addition, 33 and 22 sites at ccmB and ccmC were differentially edited between Huhan-1A and Huhan-1B, respectively, on the 0, 1st, and 3rd DAT. Editing efficiencies of ccmB and ccmC were generally lower in Huhan-1A  than those in Huhan-1B . Deficiencies of RNA editing in Huhan-1A at ccmB and ccmC could lead to the loss of transmembrane domains in their protein structures. Consequently, differences in RNA editing at ccmB and ccmC between the WA-CMS line and its maintainer line partially explained their different performances under stress. Moreover, we detected differences in expressions of pentatricopeptide repeat (PPR) genes between both lines, as well as significant correlations with RNA editing. Our study indicated potential associations of RNA editing and PPR genes in rice tolerance to abiotic stresses. However, the underlying molecular mechanisms of stress-adaptation, which are attributed to RNA editing on transcripts of mitochondrial genes, require further investigation.RNA editing of mitochondrial gene transcripts plays a central role during plant development and evolutionary adaptation. RNA editing has previously been reported to differ between the rice cytoplasmic male sterile (CMS) line and its maintainer line, which has been suggested as a cause for their different performances under environmental stress. To specifically test this hypothesis, a wild abortive (WA) CMS line (Huhan-1A) and its maintainer line (Huhan-1B) were utilized to investigate performances in response to oxidative stress, as well as RNA editing efficiencies on transcripts of six selected mitochondrial genes. Compared to the maintainer line, Huhan-1A represented both lower plant height and total antioxidant capacity, possessed higher total soluble protein and chlorophyll contents, accumulated less H RNA editing is a post-transcriptional mechanism that alters the nucleotide sequence of an RNA molecule after its transcription  describes the maternally inherited inability to produce functional pollen in plants. The successful hybrid rice breeding in China is largely attributed to the utilization of CMS resources and their comparatively high rates of out-crossing  and its maintainer line (Huhan-1B) were used for our study. Huhan-1B has been developed from an upland rice line (EAIC139-55-1-23) by the International Rice Research Institute (IRRI) via selfing and purifying. Huhan-1A was developed via backcrossing between Zhanshan97A  × Huhan-1B for at least seven generations, during which Huhan-1B was used as recurrent pollen donor Luo, . Huhan-12O2 in the final concentration. A further four replicates were kept in normal solution as controls (CK). Normal and H2O2-added nutrient solutions were renewed once every 3 days.Seedlings of WA-CMS and its maintainer line were transplanted to a 96-well plate 3 days after germination and cultivated in a nutrient solution , the specific activity of superoxide dismutase (SOD), and the specific activity of catalase (CAT). GPX activity was measured with the Glutathione Peroxidase Cellular Activity Assay Kit . TSP (Product# A045-2), AOC (Product# A015), H2O2 content (Product# A064), SOD activity (Product# A001-1), and CAT activity (Product# A007-1) were measured following the manufacturer's instructions of their corresponding test kits from Nanjing JianCheng Bioengineering Institute, China (http://elder.njjcbio.com/html_en/search.php). Since the degradation of chlorophyll is one of the most noticeable physiological responses of rice to oxidative stress, well-maintained chlorophyll levels could be a key parameter to describe the tolerance to oxidative stress. Consequently, chlorophyll was ethanol-extracted from leaf tissues and quantified via spectrophotometry. Briefly, 0.05 g fresh leaves from seedlings were crushed and put into 20 ml 95% ethanol for 24 h in the dark. The 250 ul supernatant was then measured at both 649 nm and 665 nm. The content of chlorophyll-a (Ca) (mg/L) was calculated via the empirical formula: 13.95*A665–6.88*A649. The content of chlorophyll-b (Cb) (mg/L) was calculated via the empirical formula: Cb = 24.96*A649-7.32*A665. The chlorophyll content was calculated as (Ca + Cb) and its unit was converted to mg/g of fresh leaf.The above ground parts (including both stem and leaf tissue) of three individual seedlings per plate were mixed sampled used to measure stress-related physiological traits on 0 (before treatment), 1st, and 3rd day after treatment (DAT) for the oxidative-stressed plates. Each treatment had four biological repeats (four plates). Six oxidative stresses related to physiological traits were measured, including the total soluble protein (TSP) content, the total antioxidant capacity (AOC), the HTo evaluate rice growth during the stress, plant height was measured from two individual seedlings per plate for both CK and oxidative-stressed plates on the 0, 1st, and 3rd DAT using four biological repeats (four plates). These stress-exposed plates were re-cultivated in normal nutrient solution on the 5th DAT and the survival ratio was measured 2 days later from the remaining seedlings (~24 seedlings per plate). Furthermore, another three individual seedlings were mixed sampled per plate on the 0, 1st, and 3rd DAT to extract their RNA for testing RNA editing and gene expression, using three biological repeats (three plates). The last plate was used to extract DNA for gene cloning. All seedling samples were immediately frozen in liquid nitrogen and kept at −80°C until further use.atp9, nad2, nad7, nad9, ccmB, and ccmC) related to ETC were selected to test RNA editing on their transcripts. atp9 encodes the ATP synthase subunit nine. It is an indispensable part of the electron transport chain of respiration. Efficiencies of RNA editing at sites of atp9 have already been determined previously in a different WA-CMS line (Zhengshan97A) and its maintainer line   could lead to ROS over-production  with at least 10 M of clean data per sample. Three replicates were utilized for the RNA editing testing. Quality-controlled reads were mapped to the reference gene sequences of Huhan-1A and Huhan-1B that originated from the DNA sequencing above  or edited type (T/A) of bases could then be counted at this particular site. The editing efficiency at one site could then be calculated as: counts of edited bases (T and A)/total counts of bases * 100%. The code of an editing site was named by its position after the first base of the translation start site.Samples of all other three stressed plates were used to extract RNA to test both RNA editing and gene expressions. Total RNA was extracted via the TRNzol-A+ Total RNA Reagent , using entire seedlings (containing both stems and leaf tissues) that were harvested on the 0, 1st, and 3rd DAT, respectively. cDNA was obtained via reverse transcription of total RNA using the PrimeScript® RT reagent Kit  and following the manufacturer's instructions. The target regions of these selected mitochondrial genes were amplified, using corresponding primers Table . The tare Figure . Since Rogr1, emp5, and mpr25) in response to the oxidative stress of Huhan-1A and Huhan-1B and conducted correlation analyses between their respective expressions and RNA editing efficiencies. These three PPR genes have been reported to play essential roles in RNA editing of mitochondrial genes in rice  and emp5 (LOC_Os01g72930) differed in their sequences between Huhan-1A and Huhan-1B, while mpr25 (LOC_Os04g51350) did not (unpublished data). Real-time PCR was conducted using Hard-Shell® 96-Well PCR Plates , utilizing the CFX96TM Real-Time System . The utilized reaction system contained 10 μl of 2x SYBR Premix Ex TaqTM , 20 ng cDNA, and 0.1 μM of gene-specific primers . One-way ANOVA (SNK method) was conducted to detect differences in the measured physiological traits, RNA editing efficiencies, as well as gene expressions among the 0, 1st, and 3rd DAT, respectively, in Huhan-1A and Huhan-1B. Since one editing site may have contributed in either a minor or major way to the normal function of a particular mitochondrial protein, we could not estimate the significance of each editing site for a mitochondrial gene. We therefore calculated a general editing efficiency, which enabled us to describe a general tendency (increasing or decreasing) of RNA editing in response to oxidative stress (one-way ANOVA). Furthermore, we detected general differences in RNA editing between Huhan-1A and Huhan-1B (via paired t-test) for each mitochondrial gene. This was calculated as the means of editing efficiencies at all editing sites of a particular mitochondrial gene, and represented general differences either in RNA editing between Huhan-1A and Huhan-1B, or among time points. In addition, general differences in editing efficiencies at stress-responsive sites (SRS) between Huhan-1A and Huhan-1B were calculated as the means of differences of editing efficiencies of all SRSs between Huhan-1B and Huhan-1A. Paired t-test conducted to test for any differences among the 0, 1st, and 3rd DAT. Correlation analyses were conducted between gene expressions and editing efficiencies to test for any impacts caused by mitochondrial and PPR genes on RNA editing. Since the general editing efficiency was low for Huhan-1A and high for Huhan-1B, we defined the fully edited forms of ccmB and ccmC as Huhan-1B type, while defining non-edited forms as Huhan-1A type. Transmembrane helices in ccmB and ccmC proteins of fully edited (Huhan-1B type) or non-edited (Huhan-1A type) forms were predicted via TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) using default settings   and during (1st and 3rd DAT) the oxidative stress Table . DNA seq9) Table . It was 9) Table .atp9  had decreased editing efficiencies in Huhan-1B in response to the oxidative stress, while no SRS were detected in Huhan-1A. Furthermore, four sites  and three sites  could be defined as DESs of nad2 between Huhan-1A and Huhan-1B on 0 DAT and the 1st DAT, respectively  were determined as DESs between Huhan-1A and Huhan-1B on the 1st and 3rd DAT, respectively    at only one site (site331 of ccmC).The relative expression level of A Figure . Oxidati) Figure  and nad2) Figure  in HuhanC Figure  and nad22 Figure  on the 19 Figure  and nad77 Figure  were up-7 Figure . OxidatiB Figure . Its expB Figure . The ediorg1 remained at the same level on the 0, 1st, and 3rd DAT in Huhan-1A, while in Huhan-1B, it was up-regulated on the 1st DAT and then recovered to the original level. However, its expressions were similar in Huhan-1A and Huhan-1B during all three time points  with the editing efficiency on site2657 of nad7, while it was positively correlated (r = 0.697) with the editing efficiency on site82 of atp9. The expression of rice emp5 was negatively correlated (r = −0.693) with the editing efficiency on site3000 of nad2.The expression of s Figure . The expB Figure . The expT Figure . The exp2O2 treatment between a HL-CMS line (Yuetai A) and its maintainer line (Yuetai B)  and its maintainer line (Huhan-1B). However, the maintainer line retained a slight growth during the oxidative stress due to antioxidant enzyme induced activation, resulting in a significantly higher plant height than that of the CMS line on the 3rd DAT. This observation is consistent with previous reports on the performance of rice in 50 mmol HWA352 was at the steady state in rice leaf tissues since its transcript did not accumulate in contrast to what has been reported by a previously published study      in the WA-CMS line were mainly detected in ccmB  and ccmC  under both normal and/or stress conditions. Due to different editing efficiencies between Huhan-1A and Huan-1B for both mitochondrial genes, secondary structures of ccmB and ccmC proteins of WA-CMS types (non-edited forms) differed greatly from those of maintainer types (fully-edited forms), which was indicated by the loss of transmembrane domains. Although we did not exactly test whether lower efficiencies of RNA editing on transcripts of ccmB and ccmC in Huhan-1A could lead to the observed differences in performance of Huhan-1B under stress, loss or decrease of RNA editing on transcripts of numerous other mitochondrial genes in mutants of PPR genes can cause morphological alterations under both normal or stress conditions . Mutations of ogr1 and emp5 have been shown to exert negative influences on plant development and pollen/seed formation   have differences in the DNA sequence between the WA-CMS and its maintainer line (unpublished data). We suggest that many more PPR genes may express differently between the WA-CMS and its maintainer line, similarly to what we observed from mpr25 and emp5 in this study. In addition, a PPR gene with a different form  in the WA-CMS line could also profoundly influence its efficiency in RNA editing, rather than its expression level  whether differences in RNA editing between the CMS and its maintainer line are responsible for their differences in mitochondrial genome or whether they are responsible for their differences in PPR genes. (2) How would RNA editing perform under stress in hybrid rice bred from the WA-CMS line × a restore line? Investigating associations among RNA editing, PPR genes, and rice stress adaptation can provide revealing cues for rice breeding of better tolerances to abiotic-stress.Differences in the stress-response of RNA editing efficiency between WA-CMS and its maintainer line should not be closely related to expressions of mitochondrial genes, as revealed by their poor correlations. In contrast, two out of three selected PPR genes showed significant differences in expressions between the WA-CMS line and its maintainer line. They were also activated in response to oxidative stress and a significant number of genes belong to the PPR family  within PPR genes with differences in RNA editing between the WA-CMS and its maintainer line. The underlying molecular mechanisms of stress-adaptation that can be attributed to RNA editing at ccmB and ccmC  should also provide a valuable investigation. Our findings confirmed the important roles played by mitochondria associated with RNA editing in the rice adaptation to oxidative stress. Stress-adaptation associated with RNA editing at mitochondrial genes should also have significance for hybrid rice breeding, which requires further investigation.The WA-CMS line and its maintainer line performed differently in response to oxidative stress. The WA-CMS line completely inhibited its growth under oxidative stress to ensure a higher survival ratio, while the maintainer line maintained a slight growth, which resulted in a lower survival ratio. Responses of RNA editing to oxidative stress were observed at sites of many mitochondrial genes. We also detected large differences in efficiencies of RNA editing between WA-CMS and its maintainer line at transcripts of JX and TT conducted most of the experiments and participated in data analyses. JX also participated in the drafting of the manuscript. SY and ZL participated in the experiments. YL, GL, XY, and LL bred and provided the WA-CMS and its maintainer line used for this study. HX designed the experiment, analyzed the data, and drafted the manuscript. LL was involved in the design of the experiment and the drafting of the manuscript. All authors have critically revised this manuscript before submission and agreed to all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In vitro binding studies indicated that the NLS region of M protein and the amino acids 336–433 of importin β1 that belonged to the RanGTP binding region were important for binding. Importantly, a recombinant virus with M/NLS mutation resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chicken fibroblasts and SPF chickens. In agreement with the binding data, nuclear import of NDV M protein in digitonin-permeabilized HeLa cells required both importin β1 and RanGTP. Interestingly, importin α5 was verified to interact with M protein through binding importin β1. However, importin β1 or importin α5 depletion by siRNA resulted in different results, which showed the obviously cytoplasmic or nuclear accumulation of M protein and the remarkably decreased or increased replication ability and pathogenicity of NDV in chicken fibroblasts, respectively. Our findings therefore demonstrate for the first time the nuclear import mechanism of NDV M protein and the negative regulation role of importin α5 in importin β1-mediated nuclear import of M protein and the replication and pathogenicity of a paramyxovirus.The matrix (M) protein of Newcastle disease virus (NDV) is demonstrated to localize in the nucleus via intrinsic nuclear localization signal (NLS), but cellular proteins involved in the nuclear import of NDV M protein and the role of M's nuclear localization in the replication and pathogenicity of NDV remain unclear. In this study, importin β1 was screened to interact with NDV M protein by yeast two-hybrid screening. This interaction was subsequently confirmed by co-immunoprecipitation and pull-down assays.  Avulavirus in the family Paramyxoviridae, is an important avian pathogen that causes substantial economic losses to the poultry industry worldwide [Newcastle disease virus (NDV), a member of the genus orldwide ,2. The gorldwide . Of all orldwide . Like moorldwide . In addiorldwide , the NDVorldwide . The nucorldwide  and vesiorldwide , and alsorldwide . Numerouorldwide . Our recorldwide . Previouorldwide , but celIt has been demonstrated that nuclear transport of proteins carrying NLS through the nuclear envelope-embedded nuclear pore complexes (NPCs) is mediated by members of the importin superfamily including importin α and importin β . The claMany recent studies have proven that nuclear localization of viral proteins is crucial for viral replication and propagation . For exaIn this study, importin β1 was identified to be the nuclear transport receptor of NDV M protein and mediate the nuclear import of NDV M protein by binding its NLS region via the RanGTP-dependent pathway. Further studies showed that NLS mutation in the M protein disrupted its nuclear localization and reduced viral replication in chicken fibroblasts and attenuated viral replication and pathogenicity in SPF chickens. Interestingly, importin α5 was demonstrated to be a negative regulator in importin β1-mediated nuclear import of M protein and the replication and pathogenicity of NDV, as importin α5 deletion remarkably increased the nuclear accumulation of M protein and the replication ability and pathogenicity of NDV in chicken fibroblasts. Our studies will provide deep insights into understanding the more functions of M's nuclear localization in the life cycle and pathogenesis of NDV.To define cellular proteins that interact with NDV M protein, a yeast two-hybrid screening strategy was employed. The NDV M protein was used as the bait for screening the cDNA library generated from DF-1 cells. The results showed that six colonies (No. 3 to 8) could grow on the auxotrophic medium SD/-Ade/-His/-Trp/-Leu and turn blue in the presence of X-α-gal, which had the same presentation as the positive control transformed with pGBKT7-53 and pGADT7-T (No. 2)  and B. Hin vitro binding assay of the fusion protein GST-M to the purified His-importin β1 protein showed that His-importin β1 was pulled-down by GST-M protein but not by GST . These results suggest that that NDV M protein physically interacts with importin β1 protein in vivo and in vitro.To verify the interaction between NDV M protein and importin β1, we first performed co-immunoprecipitation assay with DF-1 cells transiently transfected with plasmid encoding Myc-importin β1 and infected with NDV. Expression of the fusion protein Myc-importin β1 and viral M protein was confirmed with anti-Myc and anti-M antibodies, respectively  -positive allantoic fluid was directly detected in the parental virus rSS1GFP, but three extra egg passages were required for the M/NLS mutant virus rSS1GFP-M/NLSm to be detected by HA test, indicating that the growth of the mutant virus in chicken eggs was slowed down. To determine the stability of M gene mutant virus, the rescued virus rSS1GFP-M/NLSm was plaque purified and passaged five times in 10-day-old specific pathogen free (SPF) chicken eggs. Sequence analysis of the whole-genome of the mutant virus after five passages showed that the introduced M/NLS mutation was unaltered , and no additional mutations were observed in the M gene and other viral genes (data not shown). The immunofluorescence results showed that NLS mutation absolutely disrupted the nuclear localization of M protein in virus-infected cells . MeanwhiP<0.01) . MeanwhiP<0.01) , which sin vivo pathogenesis assessment of M/NLS mutant virus rSS1GFP-M/NLSm in 4-week-old SPF chickens was then evaluated. The resulting survival curves were shown in The 46 that are specific for the transportin-1 pathway or the importin α1, α3, α6 and α7 pathways, respectively, RanGTP mutant (Ran/Q69L) [Numerous studies have shown that importin β1 together with RanGDP and/or RanGTP participate in the nuclear import of many cargo proteins . To idenan/Q69L) , which ian/Q69L) ,49, whicIn vitro nuclear import assay was then performed to further confirm that importin β1 and RanGTP are sufficient for the nuclear import pathway of NDV M protein. The results showed that the fusion protein GST-M-GFP was efficiently imported into the nucleus of digitonin-permeabilized HeLa cells in the presence of exogenous cytosol  were transfected into DF-1 cells respectively, and importin β1 RNAi#3 or importin α5 RNAi#2 could effectively lower the expression level of importin β1 or importin α5 without causing discernable changes in cell morphology  and B. IP < 0.001)   and F. TParamyxoviruses are a diverse group of enveloped viruses with non-segmented negative-sense single-stranded RNA genomes that include a number of important human and animal pathogens . To dateNumerous studies have revealed that NLS-mediated nuclear localization of viral proteins is crucial for the replication and pathogenicity of most viruses . We prevIn recent years, cellular nuclear transport receptor proteins-mediated nuclear import of viral proteins has been always the spotlight. In the present study, importin β1 was identified to mediate the nuclear import of NDV M protein by binding its NLS region via the RanGTP-dependent pathway. Numerous studies have shown that Arg/Lys-rich NLSs within cargo proteins are the binding sites for the recognition and binding of importin α or importin β . GeneralPrevious study has indicated that importin β1 contains two major domains, including importin-β N-terminal (IBN_N) domain at the N-terminus and multiple “HEAT repeat” regions that mostly occupy the C-terminus . Of whicAlthough importin α/β1 heterodimer-mediated nuclear import is thought to be widely used in cells , but somIn summary, we demonstrated for the first time the nuclear import mechanism of NDV M protein and the negative regulation role of importin α5 in importin β1-mediated nuclear import of M protein and the replication and pathogenicity of NDV. Our results will provide a better understanding of the exact role of M's nuclear localization in NDV life cycle and aid in understanding the poorly understood NDV pathogenesis.et al. [The chicken embryonic fibroblast cell line (DF-1) and HeLa cells were purchased from cell resource center of Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences. BHK-21 cells (clone BSR-T7/5), generated by Buchholz et al. , were a All enzymes used for cloning procedures were purchased from Thermo Scientific Company. The ORF of M gene was amplified from the cDNA derived from the virulent genotype Ⅶd NDV strain Sheldrake duck/China/Guizhou/SS1/2014 (SS1) (GenBank no.KP742770) and then subcloned into pGBKT7, pEGFP-C1, pGEX-6p-1, pCMV-HA, pGEX-GFP to generate pGBKT7-M, pEGFP-M, pGEX-6p-M, pCMV-HA-M and pGEX-M-GFP, respectively. The chicken importin β1 or importin α5 ORF was amplified from the cDNA derived from DF-1 cells and then used to construct the plasmids pET-32a-importin β1, pCMV-Myc-importin β1, pGEX-6p-importin β1 and pET-32a-importin α5, respectively. Annealed oligonucleotides encoding the M9M  or Bimax247AAGAAVIFDKIEEKIAA263) of M protein, the fragment containing the restriction enzyme sties AgeⅠ and BstZ17Ⅰwas amplified by two pairs of specific primers, which were used to introduce multiple amino acid substitutions in the M protein, to generate two overlapping PCR fragments. The two PCR fragments were joined in a second PCR and the obtained fragment was digested with AgeⅠ and BstZ17Ⅰ to replace the corresponding region in the full-length cDNA clone pNDV/SS1GFP  [To generate the recombinant infectious clones harboring alanine (A) substitution targeting basic amino acid residues in the NLS motif  . The resThe yeast AH109 containing bait plasmid pGBKT7-M was grown on SD/-Trp/X-α-gal, SD/-Trp/-His/X-α-gal and SD/-Trp/-Ade/X-α-gal to exclude the autonomous transcriptional activity. Then the transformed AH109 mated with yeast Y187 containing pGADT7-Rec with the cDNA library of DF-1 cells for 24 h. The screening was performed according to the manufacturer's instructions (Matchmaker™ GAL4 Two-Hybrid System 3) and as previously described . In β-ga2. For the transfection experiments, 4 × 105 DF-1 cells were grown to 80% confluence in 35-mm-diameter dishes and then double or single transfected with a total of 3 μg plasmid using the FuGENE HD Transfection Reagent (Roche) according to the manufacturer's recommendations. Twenty-four hours after transfection, cells expressing the fluorescence-fused proteins were rinsed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.25% Triton X-100 in PBS for 5 min, and then counterstained with DAPI (Sigma) to detect the nuclei. Fluorescent images were obtained under a Nikon fluorescence microscope (Japan). Analysis and merging of the images were done with Adobe Photoshop 7.0 software.DF-1 cells were cultured in Dulbecco's modified Eagle's medium  containing 10% fetal bovine serum  supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C under an atmosphere with 5% CO5 DF-1 cells grown in 35-mm-diameter dishes were transfected with plasmid pCMV-Myc-importin β1 for 24 h and then infected with NDV strain rSS1GFP at a multiplicity of infection (MOI) of 0.1. On the other hand, DF-1 cells cultured in 35 mm dishes were co-transfected with pCMV-HA-M and the indicated plasmids. At 24 h post-infection or post-transfection, cells were washed and lysed with immunoprecipitation buffer. After centrifugation, the supernatants were collected and incubated with the corresponding antibodies overnight at 4°C. The immune complexes were recovered by adsorption to protein A+G-Sepharose (Sigma) for 3 h at 4°C. After three washes in immunoprecipitation buffer, the immunoprecipitates were detected by Western blotting.For co-immunoprecipitation assay, 4 × 10E. coli BL21 (DE3) (4 h induction with 1.0 mM IPTG at 30°C), and the soluble His-tagged proteins were purified on Ni-NTA His Bind Resin. GST-M and GST-M/NLSm (4 h induction with 0.5 mM IPTG at 28°C), and GST-importin β1 (4 h induction with 1.0 mM IPTG at 30°C) were expressed in E. coli BL21 (DE3), respectively, and the soluble fusion proteins were purified on Glutathione-Sepharose beads. In the GST pull-down experiments, the purified GST-M or GST-M/NLSm or GST-importin β1 protein was immobilized on Gluthatione-Sepharose beads (3 μg protein/10 μL beads). After washing with transport buffer, the immobilized proteins were incubated with the purified His-importin β1 or His-importin β1(△336–433) or His-importin α5  for 2 h at 4°C. The beads were then washed three times with transport buffer and the bound proteins were eluted from the beads and used for SDS-PAGE followed by Coomassie blue staining or Western blot analysis. In the His pull-down experiments, His*Bind Resin-binded His-importin β1 or His-importin β1(△336-433) (3 μg protein/10 μL resins) was incubated with purified GST-M (3 μg) for 2 h at 4°C. The resins were dealt with as described above, and the target protein GST-M was detected by Western blotting.For pull-down assays, the His-importin β1 or His-importin β1(△336–433) or His-importin α5 fusion protein was expressed in In vitro nuclear import assays in digitonin-permeabilized cells were performed as previously described [escribed . BrieflyFor the virus rescue, BSR-T7/5 cells were grown in DMEM medium containing 10% FBS and 1 mg/ml geneticin G418 for five generation before transfection. Cells at 70% confluence in 35 mm dishes were transfected with the full-length cDNA clone (pNDV/SS1GFP or pNDV/SS1GFP-M/NLSm) together with three SS1-derived helper plasmids at a total of 3 μg . At 60 h5.0 EID50/100 μL per bird, or with 100 μL PBS as the negative control. The birds were monitored for clinical signs daily for 10 dpi. Three birds were euthanized daily from 4–5 dpi for gross lesion observation, and samples of the heart, liver, spleen, lung, kidney, brain, trachea, duodenum, bursa of Fabricius and thymus were collected to detect virus titration in DF-1 cells at 5 dpi. The virus titers were determined as the TCID50 per gram (log10TCID50 g−1 tissue) using the endpoint method of Reed and Muench. The part of collected lymphoid tissues  was fixed in 10% neutral formalin, routinely sectioned and stained with hematoxylin-eosin, and then examined for lesions using Nikon light microscopy.The pathogenicity assessment of the rescued viruses rSS1GFP and rSS1GFP-M/NLSm was determined in 4-week-old SPF chickens. Forty-eight chickens were assigned randomly into three experimental groups, consisting of rSS1GFP- (n = 16), rSS1GFP-M/NLSm- (n = 16) and mock-  infected groups. For each group, 6 birds and 10 birds were used for sampling and clinical observation, respectively. Chickens were inoculated via the eye drop/intranasal route with each virus at a dose of 10DF-1 cells grown in 12-well plates were treated with the drug ivermectin or DMSO as described previously . Cells w50) in DF-1 cells [The sequences of three pairs of siRNA designed to knockdown importin β1 or importin α5 in DF-1 cells were shown in supplemental Table S1. Negative siRNA control (Cat. No.12935-400) and siRNA transfection reagent were purchased from Invitrogen. For transfection with the siRNA against importin β1 or importin α5, low-passage DF-1 cells were transfected with the indicated siRNAs at a confluence of 80% on 35 mm dishes, and the knockdown efficiency was checked by immunoblot analysis at 48 h post-transfection. To study the effect of importin β1 or importin α5 knockdown on the subcellular localization of M protein and the replication of NDV, the NDV strain rSS1GFP was used to infect importin β1 or importin α5 siRNA-treated DF-1 cells at an MOI of 1. The subcellular localization of M protein was examined by immunofluorescence assay and immunoblotting analysis and at 6 hpi and 12 hpi, respectively. In addition, the cell culture supernatants and cell pellets were collected at the indicated time points , and the virus titers were determined as 50% tissue culture infective dose . For quantification of the mRNA of NDV M gene, a SYBR green-based real-time PCR method (TaKaRa) was used, and GAPDH mRNA was quantified to normalize the total RNA concentration between different samples. The primers for detecting the M gene and GAPDH gene were designed with reference to previous publication . The reap-values between identified samples were generated using unpaired two-tailed Student's t-test. All experiments were repeated at least three times and the results were presented as the mean ± standard deviation (SD). The significance levels were defined as P < 0.05.Statistical analysis was performed using the GraphPad Prism 6.0 software . The


AbstractEustrashanghaiensis Song, sp. n., is described, illustrated, and distinguished from the treated congeners. New distributional data or biological notes on Eustrachinensis Bänninger, 1949, Itamuscastaneus Schmidt-Goebel, 1846, and Platyrhopalusdavidis Fairmaire, 1886 are provided.Four paussine species belonging to three different genera are discovered in Shanghai. A new species,  Paussinae Latreille, 1807 currently contains more than 30 species from China , the genus contains two species from China: E.chinensis , E.taiwanica Deuve, 2001 . In 2014, Guéorguiev described the first Chinese troglobitic Eustra species, E.petrovi from Xianrendong, Yunnan. In this paper, a new Eustra species is described from Shanghai, illustrations provideded of all Shanghainese paussines, and biological information about the habitats and behaviors of Eustrashanghaiensis sp. n., Eustrachinensis, Platyrhopalusdavidis, and Itamuscastaneus observed in nature and captivity are provided.According to the latest revision of Material used in this study is deposited in the following public and private collections:SNUC Insect Collection of Shanghai Normal University, Shanghai, China;SNHM Shanghai Natural History Museum;KUM The Kyushu University Museum, Fukuoka;MNHN The French National Museum of Natural History;cBWX private collection of Wen-Xuan Bi, Shanghai, China;cLW private collection of Wei Liu, Zhejiang, China;cJRX private collection of Ri-Xing Jiang, Shandong, China;cSXB private collection of Xiao-Bin Song, Shanghai, China;cWYX private collection of Yong-Xiang Wu, Shanghai, China;cYZZ private collection of Zhi-Zhou Yu, Shanghai, China.The following abbreviations are applied in the text:BL body length, from the anterior margin of the head to the apices of elytra;HW head width, maximum width of the head;AL length of antenna;ACL maximum length of antennal club;ACW maximum width of antennal club;PL length of the pronotum along the midline;PW maximum width of pronotum;EL length of the elytra along the suture.All measurements are in millimeters.PageBreak棒角甲亚科折缘粗角步甲族双斑粗角步甲亚族双斑粗角步甲属Taxon classificationAnimaliaColeopteraCarabidaeSongsp. n.http://zoobank.org/26391AC8-3D78-41C6-BA55-F3322740E088Holotype. ♂, (SNUC), labeled ‘CHINA: Shanghai, Pudong New District, Shanghai Binjiang Forest Park (上海滨江森林公园), 31°23'25"N, 121°22'10"E, alt. 5 m, 7.v.2017, Song, Peng, Hu, Wang & Liu leg. / HOLOTYPE [red], Eustrashanghaiensis sp. nov., Song det.2017’.Paratypes. 3♂♂, 3♀♀, (SNHM), same data as holotype; 2♂♂, 2♀♀, (KUM), ditto; 1♂, 1♀, (MNHN), ditto; 5♂♂, 5♀♀, 84 exs, (SNUC), ditto; 74 exs, (cSXB), same data as holotype, but 27.vii.2017, Song, Zhou, Wang, Wang & Zhang leg; 11 exs, (cSXB), same data as holotype, but 24.ix.2016, Zhong Peng leg; 1 ex (cSXB), labeled ‘CHINA: Shanghai, Changning District, Tianshan Park (天山公园), 31°12'45"N, 121°24'10"E, alt. 14 m, 20.iv.2008, Xiao-Bin Song leg.; 1ex, (cSXB), ditto, but 12.iv.2008; 1 ex, (cSXB), ditto, but iv.2008; 1 ex, (cBWX), labeled ‘CHINA: Shanghai, Pudong New District, nr. Zhangjianggaoke (张江高科), 31°11'84"N, 121°34'82"E, alt. 4 m, 28.iii.2006, Wen-Xuan Bi leg.; 8 exs, (cSXB), ditto, but 5.ii.2009, Song & Ding leg. / all paratypes, labeled ‘Paratype [yellow], Eustrashanghaiensis sp. nov. Song det. 2017’.Eustrashanghaiensis sp. n. is closely allied to E.hammondi Deuve, 2001 from Mindanao, Philippines in sharing similar body size, general habitus and aedeagal structure . Bänninger, 1949: 134 ; cSXB), labeled ‘CHINA: Shanghai, Xuhui District, Shanghai Normal University (上海师范大学), 31°09'48"N, 121°24'45"E , alt. 4 m, PageBreak11.V.2017, Xiao-Bin Song leg., [from colony of Ectomomyrmesjavana]’; 1♂, 1 ex, (cSXB), ditto, but 20.ix.2016, Zhong Peng leg.; 1 ex, (cJRX), ditto; 1 ex, (cSXB), labeled ‘CHINA: Shanghai, Changning District, Zhongshan Park (中山公园), 31°13'25"N, 121°25'00"E, alt. 9 m, xi.2006, Xiao-Bin Song leg.’1♂, 1♀, , labeled ‘CHINA: Zhejiang, Hangzhou City, Lin’an District, West Tianmushan (西天目山), 30°19'28"N, 119°26'54"E , alt. 380 m, 16.vii–9.viii.2017, Xiao-Bin Song leg., [F. I. T.].’Eustrachinensis is characterized by the large body size and the broad elytra. This is the only known myrmecophilous species of the genus Eustra. Adults are collected from Ectomomyrmesjavana  nests under the stone . Wendy et al. (2011) described an unidentified Eustra larva which collected during the excavation of nest of Ectomomyrmesjavana in Taiwan, based on its same special symbiotic host and the reasonable distribution, the larva should belongs to Eustrachinensis.one Fig. , and havBL, 4.03–4.30; HW, 0.94–0.96; PL, 0.65–0.68; PW, 1.21–1.25; EL, 2.32–2.61.China: Shanghai, Zhejiang , Taiwan; Japan: Yaeyama-shoto.Ectomomyrmesjavana   Figs , 7A, B.折缘粗角步甲亚族伊塔粗角步甲Taxon classificationAnimaliaColeopteraCarabidaeSchmidt-Goebel, 1846Itamuscastaneus Schmidt-Goebel, 1846: 67 ; cSXB), labeled ‘CHINA: Shanghai, Changning District, nr. Zhongshan Park (中山公园), 31°12'59"N, 121°25'17"E, alt. 11m, 28.vii.2017, Xiao-Bin Song leg.’; 3♀♀, (cSXB), ditto, but 22.viii.2017; 4 exs, (cSXB), ditto, but 25.viii.2017; 1 ex, (cSXB), ditto, but 7.ix.2017; 2 exs, (cSXB), ditto, but 27.ix.2017; 1 ex, (cWYX), labeled ‘CHINA: Shanghai, Jing’an District, nr. Jiang’an Temple (静安寺), 10.viii.2017, Yong-Xiang Wu leg.’; 1 ex, (cWYX), ditto, but vii.2017.1♂, (cSXB), labeled ‘CHINA: Yunnan, Xishuangbanna, Xishuangbanna Tropical Botanical Garden (西双版纳植物园), iv-2009, Xiao-Yu Zhu leg.’; Fujian: 1 ex, (cSXB), labeled ‘CHINA: Fujian, Nanping City, Wuyishan (武夷山), Yunvfeng (玉女峰), Zu-Qi Mai leg., under rotten wood.’; Zhejiang: 1 ex, (cLW), labeled ‘CHINA: Zhejiang, Hangzhou, Hangzhou Botanical Gardern, Taoyuanling (桃源岭), viii-2017, Wei Liu leg.’; 3 exs, (cLW), ditto, but Pujiaxincun (濮家新村), vi-2015, on Broussonetiapapyrifera trees.Yunnan: 1ex,  and dead cave cricket (Rhaphidophoridae). One specimen from Fujian was collected under decaying wood (Mai per. comm.). Three individuals from Zhejiang are collected at night on the trunks of Broussonetiapapyrifera (L.) L’Hér. ex Vent. trees (Liu per. comm.).ber Fig. , individBL, 15.62; HW, 3.61; PL, 3.22; PW, 4.28; EL, 8.90.China: Shanghai , Zhejiang , Fujian , Guangdong, Yunnan ; Myanmar; Laos; Sri Lanka; Thailand.Free living, not associated with ant.棒角甲族棒角甲亚族圆角棒角甲属Taxon classificationAnimaliaColeopteraCarabidaeFairmaire, 1886Platyrhopalusdavidis Fairmaire, 1886: 224 ; cSXB), labeled ‘上海植物园, 3-XI-2007, 毕文烜’; 1ex, ditto, but, 21-IV-2007, pinned with Pheidole ant ; 1♀, (cSXB), labeled ‘SH. Botanical Gardern Xuhui District, Shanghai City, 27-VII-2007’; 1 ex, PageBreakPageBreak(cSXB), ditto, but 25-VI-2008; 1 ex, (SNUC), labeled ‘CHINA: Shanghai, Fengxian District, Shanghai Normal Univeristy (上海师范大学), 30°50'09"N, 121°31'09"E, alt. 6 m, 15.vi.2007, Xiao-Yu Zhu leg.’; 1 ex, (SNUC), ditto, but 20.v.2008, Yu-Di Wang leg.; 1 ex, (SNUC), ditto, but 1.2008; 1♀, (cSXB), labeled ‘ CHINA: Shanghai, Fengxian District, Shanghai Institute of Technology (上海应用技术大学), 30°50'15"N, 121°30'20"E, alt. 5 m, vii.2011, De-Yao Zhou leg.’; 2 ex, (SNUC), labeled ‘CHINA: Shanghai, Chongming District, Dongtan N. R. (东滩), 7.v.2007, Jia-Yao Hu leg.’; 1♂, (SNUC), ditto, but 30.vi.2007; 1 ex, (SNUC), labeled ‘CHINA: Shanghai, Chongming District, Beihu (北湖), 1.vii.2008, Jia-Yao Hu leg.’, pinned with 6 Tetramoriumcaespitum workers; 1 ex (cYZZ), ditto, but Xitan (西滩), 15.vii.2007, Hong-Qiong Li leg.1♂, 1ex, (cSXB), labeled ‘CHINA: Anhui, Fuyang City, Yingzhou District (颍州), Qiyuhedong Vill. (七渔河村), near dam, from ant nest, nr. 32°54'31"N, 115°46'29"E, 29-VI-2013, J-B Dong leg.’; Fujian: 1 ex, PageBreak(SNUC), labeled ‘Mt. Wuyi, Fujian, Li-Zhen Li leg., 10~14-VII-2002’; Shandong: 1 ex, (cSXB), labeled ‘鲁, 莱阳, 旌旗山, 14.5.15., JRX.’; Hubei: 1 ex, (cSXB), labeled ‘湖北, 大店林场, 26.v.2016’ ; Hunan: 1 ex, (cSXB), labeled ‘湖南, 长沙, 1980.9, 灯下, 徐慧?’; Yunnan: 1 ex, (cSXB), labeled ‘CHINA: Hunan Province, Leiyang City (耒阳), vi-2011, Hao Xu leg.’; 1 ex, (cSXB), labeled ‘云南, 昭通, 黄华, 石水井—花椒地, 2007-8-13’; 1 ex, (cSXB), labeled ‘CHINA, Yunnan Prov., Yingjiang County (盈江县), Tongbiguan (铜壁关), alt. 1330 m, 23°36'N, 97°36'E , 23-V-2013, Chao Wu leg.’; Xizang: 1 ex, (cSXB), labeled ‘CHINA, Xizang, Linzhi, Motuo County, Beibeng Vill. (背崩乡), alt. 780 m, 10-viii-2010, Wen-Xuan Bi leg.’; 1 ex, (cSXB), labeled ‘CHINA, Xizang, Linzhi, Motuo County, Beibeng Vill. (背崩乡), 29.243469,95. 169677,769.01, 26-vi-2017, Jing-Song Shi leg.’.Anhui: 1ex, .nts Fig. , but oneBL, 6.84–7.55; HW, 1.41–1.56; PL, 1.37–1.46; PW, 1.59–1.80; EL, 4.45–5.00; ACL, 1.70–1.87; ACW, 1.50–1.57.China: Beijing, Shanxi, Shanghai , Jiangsu, Zhejiang , Anhui , Fujian, Jiangxi, Shandong , Henan, Hubei , Hunan, Guangdong , Sichuan, Guizhou, Yunnan , Xizang?, Shaanxi.Pheidole sp. (Figs sp. Figs , 7C, D.