RNA-RNA interaction slowly emerges as a critical component for the smooth functioning of gene expression processes, in particular in translation where the central actor is an RNA powered molecular machine. Overall, ribosome dynamic results from sequential interactions between three main RNA species: ribosomal, transfer and messenger RNA . In recent decades, special attention has been paid to the physical principles governing codon-anticodon pairing, whereas individual RNA positioning mostly relies on ribosomal RNA framework. Here, we provide a brief overview on the actual knowledge of RNA infrastructure throughout the process of translation in mammalian cells: where and how do these physical contacts occur? What are their potential roles and functions? Are they involved in disease development? What will be the main challenges ahead? Proper folding of RNA through intra- and inter-molecular interactions is essential to the workings of several cellular processes  that shape gene expression and drive cell fate. This is due to (i) the intimate relationship between RNA structure and function, and (ii) the fact that structured RNAs are central components of essential molecular machines such as the spliceosome, telomerase and ribosome. Base pairing is a major contributor for the stability of both RNA folding and RNA-RNA intermolecular interactions.RNA-RNA interaction is involved in every step of the ribosome life cycle, from its biogenesis in the nucleus to its cytoplasmic role in protein synthesis. The mature 80S ribosome is composed of two subunits: the small 40S ribosomal subunit contains the 18S ribosomal RNA (rRNA) and 33 ribosomal proteins (RP) and the large 60S ribosomal subunit comprises the 28S, 5.8S and 5S rRNAs and 47 RPs [During the process of translation, messenger RNA (mRNA) enters the ribosome between the head and the shoulder and passes through the mRNA channel. At the ribosome interface the mRNA codon is read by its cognate transfer RNA (tRNA) at the decoding center (DC). The mRNA exits the 40S mRNA channel through the mRNA exit site (between the head and the platform). The 60S subunit displays a crown shape and contains different structural landmarks known as the central protuberance, the two stalks and the sarcin–ricin loop. The peptidyl transferase center (PTC), where peptide bond formation occurs, is located on the interface side of the 60S subunit. The three tRNA binding sites  are located in the cavity between the two ribosomal subunits. The incoming aminoacyl tRNA is recruited in the A site, the binding of the peptidyl tRNA occurs in the P site, and deacetylated tRNA binds in the E site before the release of the discharged tRNA from the ribosome . The whocis-acting elements such as a 5′ cap structure , a 3′ poly(A) tail and the involvement of at least 11 eukaryotic initiation factors (eIF) [cis-acting elements and circularize the transcript. Second, the 43S initiation complex, which consists of eIF3, eIF1, eIF1A, eIF2-GTP-tRNAiMet, eIF5 and the 40S ribosomal subunit, is recruited to the m7G cap structure of transcripts. The 48S preinitiation complex thus created initiates ribosome scanning toward the start codon. Positioning of the small subunit over the start codon of the mRNA permits the base-pairing interaction between this codon and the anticodon of the initiator tRNAiMet. This junction is the trigger for a series of events which results in the recruitment of the 60S ribosomal subunit and the assembly of an active 80S with the tRNAiMet paired with the start codon in the P site. Next to it, in the A (acceptor) site, the following codon of the open reading frame (ORF) awaits binding of the cognate aminoacyl-tRNA, which will mark the start of elongation [The translation process breaks down into three stages: initiation, elongation and termination. Translation initiation is defined as the process by which the proper start position on the mRNA is identified by the translation machinery and aligned with the anticodon of initiator tRNA. In prokaryotes, the recruitment of the small ribosomal subunit onto the start codon is mediated by base pairing between the mRNA Shine-Dalgarno (SD) sequence and the 3′ end of the 16S rRNA [rs (eIF) . First, RNA-RNA interaction throughout translation process generally refers to codon/anticodon base-pairing, a vital ingredient of the decoding process. Yet, multiple RNA-RNA interactions between ribosomal RNA framework and ribosome RNA substrates, tRNA and mRNA, have been described. Several lines of evidence suggest that these interactions may play a major role in fine-tuning ribosome dynamic and gene expression.rRNA-mRNA interaction may principally intervene for recruiting and positioning ribosome during the translation initiation phase. To ascertain mRNA positioning and the fidelity of translation initiation, the start codon is usually adjacent to a characteristic pattern of bases named Kozak sequence ((A/G)CCAUGG)  that migFGF2 and Gtx [IGF1R internal ribosome entry site (IRES) and the helix 23b of the 18S rRNA  that may recruit the 40S ribosome and impact IRES translation activity [The first example of eukaryotic rRNA-mRNA interaction is the ribosomal shunting involving the interaction of the 5′ leader of the mouse Gtx homeodomain mRNA and the 18S rRNA . The aut and Gtx . In 2010activity . All of activity . FinallyiMet is located in the binding pocket of the 40S subunit where the anticodon stem loop interacts with several helices of the body, the platform and the head of 18S rRNA [iMet is re-oriented toward the P site in order to promote 60S joining [Positioning of eukaryotic tRNAs within the translating ribosome has been made possible by technological improvements in cryo-electron microscopy (cryo-EM) , X-ray c joining  and trig joining . It is s joining ,16,20. I joining . Yet, it62A1781, m62A1782 and acetylation ac4C1773 on helix 45), close to the mRNA channel (ac4C1280 on helix 34), in the tRNA binding sites (hypermodified pseudouridine m1acp3Ψ1191 and methylation m7G1575), at the ribosome interface (methylations m1A2142 and m5C2278) and the peptidyl transferase center  may impact the translation process by altering tRNA–mRNA [O-methylation sterically perturbs interactions of universally conserved monitoring bases of the small subunit  with cognate codon–anticodon helices. This results in a higher tRNA rejection during proofreading and ribosome stalling [1G9, altered the tRNA usage in translation while having no impact on tRNA stability. This discovery was made possible by the use of Ribo-tRNA-Seq technology that permits to quantify and identify intra-ribosomal tRNAs and detect some of their modifications. The development of this type of mapping technology will be essential to evaluate whether RNA modifications within individual RNA species—rRNA, tRNA and mRNA—could interplay to fine-tune RNA-RNA interaction and protein expression both in a physiological and pathological context.Recent re-discovery of post-transcriptional modification of nucleotides has significantly shifted our view of gene expression programs. To date, more than 140 different types of RNA modifications have been reported in all kingdoms of life . They coiewed in ). The reiewed in  reflectsstalling . They deLittle is known about the direct implication of either mRNA-rRNA or tRNA-rRNA interaction in disease onset and progression. Yet, where there is a potential cellular mechanism to divert, there will always be a virus to take advantage of it. Many viruses use IRES mediated cap independent translation to hijack the translational machinery of the host cells for their own translation. In the case of hepatitis C virus (HCV), many studies reported the binding of HCV IRES to ribosomal proteins as well as interaction with the 18S rRNA . This IRO-methylation (2′-O-Me) patterns, triggers changes in translational fidelity and promotes cap independent translation of IRES-containing mRNAs [O-Me plasticity occurs in specific regions involved in intermolecular interactions, such as between tRNA and the A-site [1-methyladenosine (m1A58) modification of tRNA has also been shown to promote cancer progression through translation reprogramming [1A58 (T loop) may influence large subunit recruitment during the initiation phase and/or tRNA positioning (for instance in respect to 28S rRNA) throughout the elongation phase. Overall, studying RNA-RNA interaction in disease onset and/or progression may be of relevance for understanding their functional role at the molecular level.In the same vein, rRNA modification plays a role in selective translation of specific mRNA and/or the use of alternative translation initiation sites. In breast cancer, FBL expression alters rRNA 2′-ng mRNAs . Strikine A-site . Anothere A-site ). These e A-site . X-linkee A-site . This dee A-site . N1-methgramming , though IGF1R—are an eloquent illustration of mRNA filtering. While this list is certainly not exhaustive, genetic analysis of mRNA-rRNA base pairing is hampered by the susceptibility of the ribosome to point mutations in 18S rRNA, in particular those that may render the ribosome ineffective. While RNA-RNA interactions are at the heart of ribosome function, several aspects have been largely unexplored, owing primarily to technical and/or model limitations. Yet, acquiring this knowledge is a challenge that must be met in order to comprehend how the ribosome fine-tunes gene expression control. For instance, mRNA-rRNA base pairing could well represent an essential pillar of the “ribosomal filter hypothesis” . This moOn the same trend, tRNA selection inside ribosomes may affect decoding properties of elongating ribosomes, for instance in response to environmental cues . tRNA geTo further complicate matters, the biological relevance of several mRNA chemical modifications is beginning to take shape: several nucleobase modifications profoundly affect structural principles of RNA base pairing  and impaRecent years have been marked by improvement in structural biology technologies (X-ray crystallography, cryo-EM  and mass


Dirofilaria immitis, the etiologic agent of canine heartworm disease, like several other filarial nematodes, harbors the bacterial endosymbiont Wolbachia. To investigate metabolic and functional pathways of D. immitis and Wolbachia individually, along with their interactions, the use of both transcriptomic and genome analysis has becoming increasingly popular. Although several commercial kits are available for the single extraction of either DNA or RNA, no specific protocol has been described for simultaneous extraction of DNA and RNA from such a large organism like an adult D. immitis, where female worms generally reach ∼25 cm in length. More importantly, adult worms of D. immitis can only be obtained either through necropsy of experimentally infected dogs or by minimally-invasive surgical heartworm removal of naturally infected dogs. This makes each individual worm sample extremely important. Thus, in the context of a project aimed at the evaluation of both gene expression analysis and Wolbachia population assessment following different treatments, an optimized protocol for co-extraction of DNA and RNA from a single sample of adult D. immitis has been developed.•An optimized method for DNA/RNA co-extraction from large size nematodes using TRIzol® reagent.•D. immitis.Allows maximum exploitation of unique samples as adults of  Specification TableD. immitis has been validated using TRIzol® as the main reagent. The described extraction procedure grants maximum exploitation of a single sample of adult D. immitis worm, allowing the possibility of focusing on different levels, either on the parasite itself or on its symbiont, and thus investigating different aspects of their metabolisms.A method for co-extraction of DNA and RNA from a single worm of D. immitis, respectively 6 females and 6 males, were collected during minimally-invasive surgical heartworm removal from two dogs previously diagnosed with patent D. immitis infection. Extracted worms were washed in Hank’s Balanced Salt Solution medium  at room temperature, checked for viability, counted and sexed. Three female and three male worms were individually placed into 15 mL Falcon tubes and stored at −80 °C. Single worms were used as starting material for the RNA/DNA co-extraction.Following owners’ consent, 12 adult worms of D. immitis are longer than males , with approximately double the starting weight compared to males, the following steps of extraction were performed slightly differently between the two sexes.Because female Each frozen worm was homogenized individually. The worm was placed into a precooled Petri dish with 500 μL of cold TRIzol® Reagent . With the help of a sterile scalpel, the worm was chopped into tiny pieces, thin enough so that they could be picked easily up by trimmed 1000 μL tips, and then placed it into a sterile RNase and DNase free 1.5 mL Eppendorf tube. Each worm was homogenized using an electric pellet pestle  for 3 min. Then, an additional 500 μL of TRIzol® Reagent were added to partially homogenized sample, and after mixing it thoroughly, 500 μL of it were transferred into a new sterile tube. Homogenization with the electric pestle was repeated once more per each of the two tubes, for 2 min. For females, since their initial weight was twice that of males (∼220 mg and ∼115 mg respectively), the homogenization step was repeated one more time per each of the two samples just prepared. A further 500 μL of TRIzol® Reagent was added to each of the two tubes and, after mixing the sample thoroughly, 500 μL of sample were transferred into a new tube. Each sample was homogenized again for a 1 min. All steps were performed on ice, to avoid RNA degradation.To each tube containing 500 μL of homogenized sample a further 500 μL of TRIzol® reagent were added. They were treated according the TRIzol® Reagent manufacturer protocols . Once thThe final elution of RNA samples was performed in 100 μL of DEPC water, and the rehydration of the sample was performed at 56 °C per 13 min. Some samples, due to the presence of a greater pellet, were placed in a water bath at 56 °C for a longer time, until the pellet was fully rehydrated.The final elution of DNA samples was performed in 300 μL of DEPC water, and they were then placed in a water bath at 46 °C for 15 min.After resuspension, all samples were briefly vortexed and analyzed at the spectrophotometer . For each sample, data concerning concentration (ng/μL) and quality, described as 260/280 and 260/230 values were collected.In order to guarantee RNA quality which would allow down-stream analysis, such as quantitative/relative RT-PCR, each sample required the elimination of any possible residue of genomic DNA.Therefore, a solution of 150 ng/μL of RNA in a final volume of 50 μL was prepared for each sample. To each sample, 5 μL of TURBO™ DNase buffer and 2 μL of TURBO™ DNase  were added, followed by incubation at 37 °C for 30 min. After the first incubation, a further 2 μL of TURBO™ DNase enzyme was added and incubated again at 37 °C for other 30 min.A 500 μL of TRIzol® Reagent was added to each tube and a second RNA extraction was performed following the manufacturer protocol. The final elution was performed in 35 μL of DEPC water and as performed for the first RNA extraction, pellets were rehydrated at 56 °C for 13 min. All samples were quantified at the spectrophotometer. Results obtained from both the first and the second RNA extraction as well as those obtained for DNA have been reported in To obtain a fully pure RNA sample to be used for gene expression analysis, an additional purification step was performed. RNA samples were treated with AccuRT Genomic DNA Removal Kit  according to the manufacturer protocol. Starting with 300 ng of final RNA, and followed by an incubation at room temperature of 8 min. For cDNA synthesis, the OneScript® cDNA Synthesis Kit  was used. The presence of any genomic contamination was checked by PCR reaction using GOTAQ Flexi DNA Polymerase Green G2  and 0.35 μM of specific primers designed on Dim-pgp-10 for cDNA   [To validate the efficiency of proposed protocol, RNA products obtained with the just described protocol were compared to those obtained with the use of a product specific for the optimization and simplification of RNA isolation: Phasemaker™ Tubes .D. immitis per each sex were analyzed. Thus, two samples per each female and one per each male were used for the extraction procedure with Phasemaker™ Tubes. The manufacturer’s protocol was followed. All remaining samples, respectively two samples per each female and one per each male, were processed using the proposed co-extraction protocol. Results from both RNA extractions are reported and compared in Three adult worms of When compared, results obtain from the Phasemaker™ Tubes with those obtained following the co-extraction protocol; both their quality and quantity of RNA appeared similar between the protocols. In fact, no statistical difference was observed between the two different extraction procedures when the single worms were considered separately. On the other hand, when the overall results from each sex were compared, the statistical analysis showed a marked significant difference  in female step 1 RNA and a less significant difference for final RNA . No difference was observed for all RNA extracted from males. This difference is probably related to the difference in size among the female worms used for this assay.D. immitis. To the authors’ knowledge, this is the first attempt to develop a protocol for DNA/RNA co-extraction in nematodes.Results obtained validate the possibility of using the proposed protocol as an efficient option for simultaneous extraction of good quality of both DNA and RNA from a unique sample of adult D. immitis. Most of the existing methods for molecular analysis of filarial nematodes have focused on single RNA or DNA extraction. According to previous studies, DNA is generally isolated by means of either commercial kits such as DNeasy Blood and Tissue Kit  and QIAamp® DNA Mini Kit , or of alternative protocols involving a combination lysis buffers, composed primarily of Tris-HCl and K proteinase enzyme [Until now, co-extraction has never been described for nematodes and, to the authors’ knowledge, there are no reports of this procedure from a single adult nematode sample, with a large size such as that of e enzyme . For RNAe enzyme .D. immitis. This optimization grants full exploitation of a single sample, so that both molecules could be used for specific molecular analysis, such as quantitative RT-PCR, which otherwise would be impossible to perform on a same sample. In particular, this new protocol will allow to correlate genes expression by the parasite with bacterial load; hence it may offer new insights into the effect of Wolbachia reduction on the parasite host’s biology and function, and thus into the adulticide effects of specific treatments.Thus, the purpose of this study was not to develop a novel protocol for ribonucleic acid co-extraction, but to adapt and optimize an already existing method for simultaneous DNA/RNA isolation from a single adult of The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


In the original article, there was an error. In the abstract, the sentence “While VTE at the first choice point increased with the number of repeated choices, VTE at the second choice point did not, and only increased after unexpected transitions within the task.” should read “While VTE at the first choice point decreased with the number of repeated choices, VTE at the second choice point did not, and only increased after unexpected transitions within the task.” The corrected abstract should read:“Current theories suggest that decision-making arises from multiple, competing action-selection systems. Rodent studies dissociate deliberation and procedural behavior, and find a transition from procedural to deliberative behavior with experience. However, it remains unknown how this transition from deliberative to procedural control evolves within single trials, or within blocks of repeated choices. We adapted for rats a two-step task which has been used to dissociate model-based from model-free decisions in humans. We found that a mixture of model-based and model-free algorithms was more likely to explain rat choice strategies on the task than either model-based or model-free algorithms alone. This task contained two choices per trial, which provides a more complex and non-discrete per-trial choice structure. This task structure enabled us to evaluate how deliberative and procedural behavior evolved within-trial and within blocks of repeated choice sequences. We found that vicarious trial and error (VTE), a behavioral correlate of deliberation in rodents, was correlated between the two choice points on a given lap. We also found that behavioral stereotypy, a correlate of procedural automation, increased with the number of repeated choices. While VTE at the first choice point decreased with the number of repeated choices, VTE at the second choice point did not, and only increased after unexpected transitions within the task. This suggests that deliberation at the beginning of trials may correspond to changes in choice patterns, while mid-trial deliberation may correspond to an interruption of a procedural process.”The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way.The original article has been updated.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.


The number of novel bacteriophage sequences has expanded significantly as a result of many metagenomic studies of phage populations in diverse environments. Most of these novel sequences bear little or no homology to existing databases . Also, these sequences are primarily derived from DNA-encoded bacteriophages (phages) with few RNA phages included. Despite the rapid advancements in high-throughput sequencing, few studies enrich for RNA viruses, i.e., target viral rather than cellular fraction and/or RNA rather than DNA via a reverse transcriptase step, in an attempt to capture the RNA viruses present in a microbial communities. It is timely to compile existing and relevant information about RNA phages to provide an insight into many of their important biological features, which should aid in sequence-based discovery and in their subsequent annotation. Without comprehensive studies, the biological significance of RNA phages has been largely ignored. Future bacteriophage studies should be adapted to ensure they are properly represented in phageomic studies. They iosphere . They weiosphere ,6. The fA phages . WeissmaA phages . Since tA phages ,10,11,12The RNA phage MS2, isolated by Alvin John Clark in 1961 and highly similar phage f2 , have beWhile there is a substantial amount of literature and studies involving the bacterial component of the gut microbial system, there is still relatively little known about the human virome, and in particular the bacteriophage fraction of the microbiome. In addition, most newly identified phage sequences do not have known counterparts in viral databases, and these unknown sequences are sometimes referred to as the “viral dark matter” . Phages The genomic composition of phages are extremely diverse and are composed of either DNA or RNA, which can in turn be either single-stranded (ss) or double stranded (ds). Single-stranded RNA genomes can exist in two variants; negative sense (−ve) and positive sense (+ve). This depends on their orientation and whether there is a prerequisite for transcription prior to translation. Some eukaryotic RNA viruses use reverse transcriptase to replicate their genetic material through a DNA intermediate, while no DNA stage has been observed amongst bacterial RNA phages to date. In addition, the genomes of phages are described as being “mosaic”, composed of individual modules that may appear in other phages but in an alternative arrangement ,23. WhilTypically, phage infection proceeds via adsorption, penetration, replication, assembly, and release. Briefly, phages use specialized surface receptor-binding proteins to interact with and adhere to their specific cognate host receptor. Phages then use various mechanisms to breach the cell wall of bacteria and inject their genomes into the cytoplasm of the host. Infection can then proceed via a lysogenic (temperate) or lytic (virulent) lifecycle. Virulent phages hijack the host’s cellular components to direct the replication of the phage’s genome and produce the necessary viral encoded proteins. Once the phage genome is replicated, it is packed into self-assembled viral particles. Phages induce host cell lysis and the assembled phage progeny are released into the surrounding environment for successive infections. Temperate phages that can replicate through lytic or lysogenic lifecycles are typically able to integrate into the bacterial chromosome and are subsequently replicated in tandem with the host genome. Temperate phages can also be maintained through formation of an episome within the host, which is disseminated through a population via cell division . TemperaCystoviridae (dsRNA phage) with 1 genus, Cystovirus, with 7 recognised species, and Leviviridae (ssRNA phage), with 2 genera, Levivirus and Allolevivirus, each of which contain two species [Although studies into the phage component of the microbiome have increased rapidly in recent years, databases are dominated by phages with DNA genomes. According to the latest (2017) report by the International Committee for the Taxonomy of Viruses (ICTV), viruses are separated into 134 families. The same report separated RNA phages into only two families;  species ,28.Streptomyces avermitilis, a Gram-positive bacterium, known as Streptomyces phage phi0. This phage is thought to belong to the Cystoviridae family based on RNA-dependent RNA-polymerase (RdRp) analysis. This was the first report of an RNA phage with a natural affinity for a Gram-positive host. In addition, a recent pre-print has described an RNA virus, a planarian-infecting Nidovirales, with a genome of 41.1 kb in length, significantly longer than the previous largest RNA virus genome of 30 kb [A recent examination of RNA phage populations in 2016 by Krishnamurthy and Wang, through metagenomic dataset analysis, led to the identification of 122 partial genomes of novel RNA phages . The hosThis review focuses on examining known RNA phages, both dsRNA and ssRNA, which target bacterial cells. Outlined are their mechanisms of adsorption through to the release of progeny. Future endeavors may use conserved features of RNA phages as genetic signatures to aid in prospective metagenomic exploration of RNA phages in the “dark matter” via sequence-based targeting.Cystoviridae listed in the 2017 ICTV report. The type species of Cystovirus family is phi6, which for a long time was thought to be unique as a dsRNA phage. The Cystoviridae have a tri-segmented, linear dsRNA genome, with the concatenated genome varying size from 12.7 kb (phi2954) to 15.0 kb (phi8). Individual genome segments range in size from 2.9 kb to 6.4 kb , the M-segment encodes the complex essential for host recognition (P3 and P6) and the S-segment is responsible for the shell protein of the nucleocapsid (P8 (except in phi8), P9, P12, and P5) [cis-acting RNA sequence known as the pac sequence [pac sequence is composed of 200 nucleotides located within a number of stem-loop structures. The pac sequences act in unison with other fundamental structural elements to ensure the correct packaging of the genome when required. and P5) ,40,41,42 and P5) . The nonsequence . The segCystoviridae, P3, to the viral envelope. It is this multimeric spike protein, P3, which enables the recognition of the host bacteria receptor pilin, the protein monomer making up bacterial pili, by the phi6 phage. The P3 protein of phages phi8, phi12, phi13, and phiYY have been suggested to be a single polypeptide or a multimer [Pseudomonas syringae, which then retracts to bring the phage into close proximity of the host membrane [Cystoviridae, such as phi8, phi12, phi13, and phiYY, utilize their heteromeric P3 protein to attach to the lipopolysaccharide (LPS) on the cell surface [The integral-membrane, fusogenic P6 protein is responsible for securing the receptor-binding protein of multimer . The P3 membrane ,47. Thismembrane ,40. Othe surface . The P3 The loss of viral membrane around the NC enables the muralytic (peptidoglycan-degrading) enzyme P5, located on the NC surface, to degrade the peptidoglycan layer of the bacterial cell wall ,50. The 2+) and magnesium (Mg2+) ions entering through another [Cystoviridae control transcription through an interchange of two independent mechanisms (a) plus-sense initiation sites are preferred by the polymerase and (b) initiation competent ssRNA templates have more available transcription initiation sites [The core particle is composed of P1, P2, P4, and P7. These proteins are involved in the transcription of the phi6 genome. The monomeric RdRp of the P2 gene is activated by PC entry into the cytoplasm. This enzyme catalyzes the semi-conservative transcription of polycistronic mRNAs within the core particle . Bacteri another . Initial another . The stron sites . InitiatBy directly releasing the mRNA transcripts into the cytoplasm, the dsRNA genome is never exposed to the host cytoplasm which helps the phage to avoid host defense mechanism activation. The mRNA transcripts are used as templates for translation of the necessary proteins. The early stage of infection is characterized by equal amounts of mRNA from L, M, and S segments ,62. Howepac sequences [Cystoviridae are yet to be fully described in exact detail.The large free-strand +ve ssRNA is then translated to form P1, P2, P4, and P7, which are subsequently assembled to form empty PCs . The hexequences ,63. Thisequences . Studiesequences . AlthougThe size and organization of this PC is regulated through the activity of inner capsid protein P1 and P4 ,66,67. PCystoviridae are categorized as virulent phages as they induce lysis of their host bacterium at the end of the infection cycle in order to release viral progeny, through P5 and P10 activity [Cystoviridae species phage phi6 targets the Gram-negative bacterium, Pseudomonas syringae, an important plant pathogen. This phage was first isolated in the 1970’s in the USA from Pseudomonas-infected bean straw [activity ,50. Howeactivity . Cystovian straw .Cystoviridae isolated and characterized in the 2017 ICTV report with another five requiring further analysis. Their genetic and structural similarities with phage phi6 suggest that there will be an expansion of this phage taxonomic family with further classification required. Sampling of various legumes in the USA have resulted in the isolation of additional dsRNA phages but these have not been characterized beyond their sequences [Pseudomonas phage phiNN was isolated from a freshwater sample in Finland, while Pseudomonas phage phiYY came from hospital sewage waste in China [P. aeruginosa strains, an opportunistic pathogen of immuno-compromised individuals. This suggests there may be potential to develop a phage therapy to combat Pseudomonas infections in these individuals.There have recently been six additional equences ,72,73. Ain China ,42. PhagCystoviridae from multiple environments, with only a single member infecting a Gram-positive host, that there are many more RNA phages yet to be discovered. Recently, Alphonse and Ghose (2017) examined known Cystoviridae using their encoded RdRp [Cystoviridae in metagenomic datasets using a marker such as the RdRp is complicated by the tri-segmented nature of the Cystoviridae genomes. Therefore, sequence based detection of all three genomic segments of Cystoviridae, particularly if they are divergent from sequences present in public repositories, will be challenging. Incorporation of genetic tags from each of the three segments will greatly enhance de novo efforts of finding Cystoviridae members.It is clear from the recent isolations of ded RdRp . While sded RdRp , RdRp apLeviviridae family encompass phages with a positive-sense single stranded, monopartite RNA genome of 3.3–4.3 kb in length. The nonenveloped, somewhat spherical virion capsid is composed of 178 copies of the dimeric coat protein (CP) and a single copy of the maturation protein  and BZ13-like (genogroup II) and Allolevivirus has Qß-like (genogroup III) and F1-like (genogroup IV) [There are two genera of  density . More reectively . These group IV) .Leviviridae phages that target E. coli, “coliphage”, which are male-specific, adsorb along the fertility (F) pilus, coded by the F-plasmid of Escherichia coli, or the chromosomal marker Hfr, whereas in non-coliphage species alternative pili are exploited [Leviviridae phages induce lysis of their host is a notable difference between the genera; Levivirus phages encode a separate lysis polypeptide, whereas Allolevivirus phages utilise their maturation protein in lysis mediation [E lysin from phage φX174, a ssDNA Microviridae representative [xploited . Alternaxploited . The prexploited . Phages ediation ,81. Thesentative . The lysLevivirus is the Enterobacteria phage MS2, a member of the MS2-like phages (genogroup I). Phages of the Levivirus genus infect their host targets through the initial adsorption of the virion along the sides of pili using the “maturation A-protein” (MatL) as the receptor binding protein [L binds the RNA at two distinct regions: the MatL coding region and the 3′-UTR [L-RNA complex may be injected into the cell as opposed to free RNA, suggesting that the MatL protein may have a greater biological role than originally envisaged [L-RNA complex gains entry to the host remains undescribed, but could involve a type IV secretion system (T4SS) homolog [L may also contribute to the replication process of the RNA genome.The type species of  protein . This re protein . This in protein . The frae 3′-UTR . It appenvisaged . The exa homolog . It has Levivirus genomes, there is a signature sequence of 5′-ACCACCCA-3′ [As the nucleic acid is a single copy of +ve ssRNA, it functions both as the genome template and mRNA upon infection. Thus, there is constant competition between replication and translation processes as the ribosome and replicase run in opposite directions along the template strand . The twoACCCA-3′ .Leviviruses encode a copy of RdRp that codes for the catalytic ß-subunit of the replicase. This protein associates with three host proteins: ribosomal protein S1 [For effective genome replication, otein S1  and the otein S1 , to formotein S1 . This GTotein S1 ,93. Sequotein S1 .Studies have shown that there are two internal sequences which are key to the recognition of the plus strand by the replicase, the S site and M site. . The S sLevivirus species. This protein does not interact with the polymerase machinery but instead binds directly to the 3′ terminal of the mRNA template [The RNA template is protected from cellular nuclease degradation through an unknown mechanism. There is an additional host factor required for successful translation that has been isolated but not genetically identified in the case of template . The repAs the infection cycle reaches the end-stage, the CP-dimers bind the replicase gene start site, located within a hairpin-structured operator, and act as translational repressors ,100. ThiNonetheless, the molecular information and functioning schema of such an autolytic pathway have yet to be identified . Recent Translation of the phage genes requires the ribosome to associate with the RNA through a Shine–Dalgarno sequence, the start codon and the host ribosomal S1 protein. The S1 protein can bind the S site, as mentioned above. This creates a situation whereby the S1 protein of the ribosome and replicase are competing for the same RNA binding site.There are a variety of systems that regulate protein synthesis, including: RNA secondary structure, ribosome access to the initiation codon, and folding kinetics ,108,109.L protein [Newly synthesized viral particles require only one copy of the lysis protein and the Mat protein ,112,113. protein ,114. Stu protein ,116. The protein ,118. TheL protein is only transcribed from newly synthesized genome templates [L gene is positioned. In nascent RNA strands, there is an alternate, shorter hairpin structure created that enables translation of the maturation gene in the 5′ terminal by allowing access and binding of the ribosome. This RNA-folding intermediate of newly-synthesized strands enable the ribosome access to the start codon of the A-protein.The Matemplates . The strAcinetobacter species, AP205, was found to have an unusual genome structure with the lysis gene located in the 5′ terminal [Levivirus genome map, the secondary structure and 3′-UTR follows that of Allolevivirus. This phage has yet to be approved as a Levivirus. Potential Leviviruses of Pseudomonas, phages PPR7 and PRR1, have also been isolated and characterized, both exhibit particular hallmarks of Levivirus phages [A recently isolated RNA phage for terminal . Althougs phages ,123,124.Allolevivirus is Qß, the representative of the Qß-like phages (genogroup III). Species of Allolevivirus contain a longer version of the genome with an extension of the C-terminal of the CP gene [1 (MCPA1) protein, also known as the ‘read-through protein’, is a feature unique to Allolevivirus phages [1 and the maturation A2 (MA2) proteins are essential for host attachment [Allolevivirus members were found to encode an Arg-Gly-Asp (RGD) motif, essential for host cell recognition and attachment, within their MCPA1 and/or MA2 [Levivirus phages. Similar to the signature 3′-terminal sequence of Levivirus, Allolevivirus species contain a 5′-TCCTCCCA-3′ within the 3′-terminal of their genome [The type species of  CP gene . The pres phages . Both thtachment . The majd/or MA2 . This mor genome .Levivirus with minor variations. The host factor that associates with the functional replicase has been isolated, purified and genetically characterized for Qß as the protein encoded by the host factor of Qß (hfq) gene of E. coli [The underlying translation and replication mechanisms are similar to  E. coli ,127. Thi E. coli ,129,130.Levivirus, with a slight change; the translation of the (MA2) is controlled in a temporal manner as opposed to a structural intermediate. This is dictated by the length of time it takes for the polymerase to move from the start site of the maturation gene to the complement of the Shine–Dalgarno sequence [1 protein is formed following ribosomal read-through of the leaky-stop codon (UGA) of the CP gene [1 protein. The ribosome occasionally, in approximately 5% of cases, translates past this leaky termination sequence for an additional 600 nucleotides to form a C-terminal extension of the CP [1 protein, have shown it to be unique to the small group of Allolevivirus phages [2 and the MCPA1 protein, whose exact role is unknown as of yet, are essential for successful infection of pili-positive hostsThe transition between replication and translation is similar to the above mentioned sequence ,132. Onc CP gene . It is rf the CP . This prs phages . The MA2Allolevivirus is the absence of a lysis gene in the genome. Instead, the MA2 protein has a secondary function to induce the lysis of the host cell for release of viral progeny [2 protein is referred to as an amurin as it does not destroy the peptidoglycan layer directly through muralytic activity. It is also known as an “antibiotic protein” due to the similarity in function to antibacterial agents which target cell walls [2 induces host cell lysis by inhibiting the enzymatic activity of MurA, a UDP-N-acetylglucosamine-enolpyruvyl transferase. This is an essential enzyme in the production of peptidoglycan as it catalyses the first committed step, the biosynthesis of murein precursor [Another notable difference in the infection pattern of  progeny . The MA2ll walls . It has recursor . At the Levivirus and Allolevivirus genera had strong homogeny across position of ORF, length of proteins and the catalytic ß-domains of the RdRp [Leviviridae.A study by Friedman et al. (2009), noted that the sequences of both the RdRp . The conLevivirus and Allolevivirus phages target the pilus of their hosts as receptors to initiate, the fact there is no conserved infection mechanism suggests that there may be varying mechanisms for the RNA to enter the cell. Originally thought to only affect plasmid-encoded appendages, there have been Leviviridae specific for genome-encoded pili of Gram-negative bacteria, such as Pseudomonas phage PP7 and Acinetobacter phage AP205.Although both Although there have only been a limited number of RNA phages identified to date, their “true” diversity and abundance in nature remains unknown. Current approaches used for the isolation, selection, and purification of viral particles, including precipitation by polyethylene glycol (PEG) and caesium-chloride (CsCl) gradient purification, are almost certainly biased against RNA phages . The selThe fragile nature of RNA and the widespread presence of RNases in human and animal derived samples also hinders studies involving RNA phages. The development of RNA phage-selective isolation protocols will also greatly enhance our endeavors. For example, separation of DNA and RNA fractions of samples and complete eradication of unwanted RNase is recommended. It should also be noted that the low abundance of RNA phages in databases will result in reduced “hits” for novel sequences. As research into the RNA section of the phage community is expanded, we can expect the databases to become more representative of the wider RNA phage community. Picobirnaviridae family may not be eukaryotic viruses as originally thought, but may in fact represent a novel family of RNA phages [An interesting paper recently proposed that members of the A phages . This reA phages . This tyLeviviridae and Cystoviridae families should also be used in specific studies. Contigs with homologs to both the leviviral and cystoviral RdRp gene are potential RNA phages and should be subjected to further analysis. Based on the recent studies mentioned above, homologs to the RdRp gene of picobirnaviruses should also be included [Levivirus and Allolevivirus members could be used to further classify these phages. Signature features of Cystoviridae members, such as the muralytic enzyme gene or the nucleocapsid shell protein gene, could also serve as genetic signatures when screening the databases for RNA phages [Identifiable RNA phage-specific domains, such as the RdRp gene, capsid gene, maturation protein gene, or the NTPase gene, can serve as features which one could use to mine metagenomic databases for RNA phages. However, since the RdRp gene is conserved amongst RNA viruses, unique genetic elements of included . The stuincluded . The speA phages .Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Clinical isolates of P. aeruginosa have been found to be resistant to most of the antibiotics normally used to treat this infection [Levivirus P. aeruginosa phage PP7 has been identified which targets this bacterium via a pilin-specific mechanism [A common theme of this review is the need for greater efforts to be directed towards the discovery of more RNA phages for all potential applications, such as tools for advancing molecular biology and as potential phage therapeutics. The rise in antimicrobial resistance across bacteria is not a novel problem but it is alarming. The host range of RNA phages could offer therapeutic potential against some of the World Health Organizations’ (WHO) list of deadly pathogens, including some of the Gram-negative members of the ESKAPE pathogens, such as nfection . An unclechanism . Further


Structural analysis of the NCD-UGGAA/UGGAA complex by nuclear magnetic resonance (NMR) spectroscopy clarifies the mode of binding that recognizes four guanines in the UGGAA/UGGAA pentad by hydrogen bonding with four naphthyridine moieties of two NCD molecules. Biological studies show that NCD disrupts naturally occurring RNA foci built on r(UGGAA)n repeat RNA known as nuclear stress bodies (nSBs) by interfering with RNA–protein interactions resulting in the suppression of nSB-mediated splicing events. Feeding NCD to larvae of the Drosophila model of SCA31 alleviates the disease phenotype induced by toxic r(UGGAA)n repeat RNA. These studies demonstrate that small molecules targeting toxic repeat RNAs are a promising chemical tool for studies on repeat expansion diseases.Synthetic small molecules modulating RNA structure and function have therapeutic potential for RNA diseases. Here we report our discovery that naphthyridine carbamate dimer (NCD) targets disease-causing r(UGGAA) n repeat RNAs in spinocerebellar ataxia type 31.Synthetic small molecules modulating RNA structure and function have therapeutic potential for RNA diseases. Here the authors show the mechanism by which a small molecule targets the disease-causing r(UGGAA) Expansion of >36 CAG repeats in the Huntingtin gene HTT and 200 CGG repeats in the FMR1 gene caused Huntington’s disease and Fragile X syndrome, respectively. Long CTG repeats have been identified in the DMPK gene of patients of Myotonic Dystrophy type 1 (DM1), although the healthy person has the CTG repeat within the range of 5–38. These diseases have also been classified as trinucleotide repeat diseases because the disease-causing repeat sequences consisted of three nucleotides. Besides these, diseases caused by the aberrant expansion of tetra-, penta-, and hexanucleotide repeats are also reported9.Aberrant expansion of specific repeat sequences in the human genome causes >40 neurological disorders called repeat expansion diseases14. The proposed mechanisms of onset by toxic RNAs are sequestration of RNA-binding proteins (RBPs) in nuclei and unusual translation initiation called repeat-associated non-AUG (RAN) translation16. RNA foci are the aggregates of toxic RNAs and sequester RBPs such as splicing factors in nuclei, resulting in dysfunction of these proteins. In contrast, RAN translation produces peptides with the repeated amino acid sequence with the tendency of forming aggregates in the cytoplasm. This gain-of-function of toxic RNAs can account for some characteristics of the pathogenesis of repeat expansion diseases.Recent studies on repeat expansion diseases showed that the transcript of aberrantly expanded repeat, thus, long repeat RNA termed as toxic RNAs would be a cause of most repeat expansion diseases21, small molecule microarrays23, and phenotypic assays25 have been performed to discover small molecules binding to RNAs. Towards this end, our laboratory has focused on the repositioning of previously developed mismatch-binding ligands (MBLs) that target base mismatches in double-stranded DNA (dsDNA)32. MBLs are designed to bind to the specific mismatches in dsDNA and do not always bind to the mismatches with the same sequence context in double-stranded RNAs (dsRNAs). We anticipated, however, many chances to discover molecules binding to mismatches in dsRNA by screening from our in-house MBL library. We here report that one of MBLs, naphthyridine carbamate dimer (NCD)29, binds in high affinity to the 5ʹ-UGGAA-3ʹ/5ʹ-UGGAA-3ʹ site produced in the hairpin structure of the r(UGGAA)n repeat RNA causing spinocerebellar ataxia type 31 (SCA31). SCA31 is an autosomal dominant spinocerebellar degenerative disorder, and the patients of SCA31 have 2.5–3.8 kbp multiple pentanucleotide repeats containing d(TGGAA)n, d(TAGAA)n, d(TAAAA)n and d(TAGAATAAAA)n in the genome. Among these repeats, d(TGGAA)n is identified as a pathogenic repeat, as other repeats n, d(TAAAA)n, and d(TAGAATAAAA)n) are found in unaffected individuals33. Nuclear magnetic resonance (NMR) spectra of the NCD-UGGAA/UGGAA pentad complex reveal the characteristic NCD-bound RNA structure involving the formation of hydrogen bonding pair with the naphthyridine and guanine residue, the zigzag binding orientation of two NCD molecules in the pentad site accompanied with adenine flip out, and the syn glycosidic conformation for all naphthyridine-bound guanines. In vitro pulldown studies reveal that NCD interfered with the interaction of several RBPs with r(UGGAA)n repeat RNA. NCD treatment resulted in suppression of RNA foci formation in HeLa cells with overexpressed r(UGGAA)n SCA31-related RNA and the inhibition of endogenous nuclear stress bodies (nSBs) assembly upon thermal stress exposure resulting in the disruption of nSB-dependent splicing regulation. Most significantly, feeding NCD to larvae of the Drosophila model of SCA31 results in alleviation of disease phenotype induced by toxic r(UGGAA)n repeat RNA. The observed phenotype changes are most likely due to the interference of the interaction between the r(UGGAA)n repeat RNA and RBPs by NCD as confirmed in vitro and cells. Studies described here clearly show that small molecules targeting toxic repeat RNAs have enormous potential to alleviate the toxic RNA function in vivo.Repeat DNA and RNA sequences can fold into hairpin structures involving mismatches consisting of partially hydrogen-bonded structures, and these structures are potential binding sites for small molecules. Despite the understandings of potential vital roles of small molecules in treating repeat expansion diseases, the exploration of small molecules targeting toxic RNAs remains a challenge. High-throughput screening methods including fluorescent indicator-displacement assaysn repeat identified as a SCA31-causing repeat34 produced long r(UGGAA)n repeat RNAs35, which formed foci with concomitant co-localization of 43 kDa TAR-DNA binding protein (TDP-43) in addition to >80 protein components36. Drosophila models of SCA31 expressing long r(UGGAA)n repeat RNA showed degeneration of the eye morphology, life-shortening, locomotor defects, and exhibited similar pathogenesis observed in human SCA31 such as accumulation of r(UGGAA)n-containing RNA foci. Coincidentally, the r(UGGAA)n repeat sequence is also found in the HSATIII long noncoding RNAs (lncRNAs), which are transcribed from Satellite III pericentromeric repeat upon thermal stress exposure37. HSATIII lncRNAs also sequester multiple RBPs to form RNA foci called nSBs in the nucleus. The formation of nSBs results in the suppression of pre-mRNA splicing (or promote intron retention) of ~500 introns during recovery from thermal stress38. Overexpression of motor neuron disease-associated RBPs such as TDP-43 and fused in sarcoma (FUS) in Drosophila models of SCA31 mitigated toxicity of r(UGGAA)n repeat RNA accompanied with the dispersion of RNA foci36. These results suggested that binding of these RBPs to r(UGGAA)n repeat RNA induced the release of the sequestered proteins from the RNA and, more importantly, suggest the possibility of small molecules in reducing the RNA toxicity by their binding to r(UGGAA)n repeat RNA. This hypothesis is relevant to those proposed for small molecules targeting disease-causing toxic RNAs including r(CUG)n in DM141, r(CCUG)n in DM243, r(CGG)n in fragile X-associated tremor/ataxia syndrome45, r(GGGGCC)n in ALS/FTD47, and r(AUUCU)n in SCA1048. We, therefore, started to screen our in-house chemical library to find molecules binding to r(UGGAA)n repeat RNA.Transcription of the d(TGGAA)n repeat RNAs, we have screened our in-house MBL library by surface plasmon resonance (SPR) assay, where three repeat RNAs r(UGGAA)9, r(UAGAA)9, and r(UAAAA)9 were immobilized on its surface. Non-pathogenic r(UAGAA)9 and r(UAAAA)9 repeats were used as control RNAs. Most disease-causing repeat RNAs responsible for RNA-mediated neurodegeneration can form hairpin structures involving internal loops consisting of multiple mismatched base pairs49. The r(UGGAA)n repeat RNA may fold into the hairpin structures, which include the UGGAA/UGGAA pentad containing the 5ʹ-GGA-3ʹ/3ʹ-AGG-5ʹ internal loop  9. The other three compounds were either dimeric form or a very close derivative of NCD.To explore molecules binding to r(UGGAA)oop Fig. . From ouase Fig. . Among 2SA) Fig.  with con9, we carried out structure-binding relationship studies on NCD derivatives by EMSA 9. UV melting profiles measured for the r(UGGAA)9 in the presence of NCD (20 μM) showed the melting temperature (Tm) of 73.7 °C 9 in the presence of NCD, and the r(UAAAA)9 did not show any increase in Tm by NCD  °C Fig. , whereasing Fig.  suggeste9 involved the binding to the UGGAA/UGGAA pentad in the secondary hairpin structure, Tms of the RNA duplexes containing the URRAA/URRAA pentad, where R is guanine (G) or adenine (A), were measured in the absence and presence of NCD  in the presence of NCD (red bar), whereas no increase in the Tm was observed with quinoline carbamate dimer (QCD) (blue bar). QCD did not show the band shift for r(UGGAA)9 in EMSA  in the presence of NCD. Further substitution of the guanine in 5ʹ-GGA-3ʹ/3ʹ-AGA-5ʹ with adenine to 5ʹ-AGA-3ʹ/3ʹ-AGA-5ʹ and 5ʹ-GGA-3ʹ/3ʹ-AAA-5ʹ diminished the effect of NCD on the increase in Tm. Substantial changes on CD spectra of the duplex containing the UGGAA/UGGAA pentad were also observed in the presence of NCD but not QCD , leading to an apparent dissociation constant Kd(app) of 32 nM NCD Fig. . A markeQCD Fig. . These r nM Fig. .Fig. 2BiInteractions of NCD with the SCA31 hpRNA Fig.  were anasyn conformation regarding the N-glycosidic bond; therefore, NCD bound to the UGGAA/UGGAA pentad from the minor groove. The four naphthyridine-guanine base pairs are stacked between the two A-U base pairs, and the two A residues (A8 and A24) are flipped out toward the major groove. The linker of NCD is located in the minor groove connecting G6 and G23 as well as G22 to G7. It is noted that only the current combination of naphthyridine-guanine pairs provided us acceptable converged structures. Four methyl groups of two NCD molecules are located in line to form a hydrophobic core in the major groove. These structural features of cross-connections and hydrophobic core may contribute to the stabilization of the complex. Two of four methyl groups in NCD were observed in the high field (0.7 ppm), whereas the other two methyl groups were observed at around 1.9 ppm. Naphthyridine rings opposite to G7 and G23 probably caused the ring current shifts observed for the two methyl groups of naphthyridine opposite to G6 and G22, respectively. It is important to note that the methyl group of free NCD was observed at 2.0 ppm. We also analyzed the overall quality of the structure by MolProbity50 n repeat RNA and RBPs. To investigate the effect of NCD on the RNA–protein interactions, we performed an in vitro pulldown assay using biotinylated r(UGGAA)20 with HeLa nuclear extract in the absence and presence of NCD 20 20 NCD Fig. . Among R)20 Fig. , indicat)20 Fig. , but mod76 to examine if NCD disrupts the formation of the RNA foci containing r(UGGAA)n repeat RNA by fluorescence in situ hybridization (FISH). In the HeLa cells transfected with the plasmid for expressing r(UGGAA)76, the accumulation of RNA foci was observed after incubation for 24 h in the nuclei as confirmed by DAPI staining 76 under the conditions ing Fig. ; left. Iing Fig. ; middle.ing Fig. ; right. 52. The effects of NCD on the inhibition of RNA foci assembly raised the intriguing possibility that NCD may inhibit the nSB formation and function in thermal stress-exposed cells by interfering with the sequestration of nSB proteins. We analyzed the effect of NCD on the assembly of nSBs by RNA FISH and immunofluorescence (IF). In the control and QCD-treated HeLa cells, nSBs were normally formed upon thermal stress exposure at 42 °C for 2 h followed by recovery at 37 °C for 1 h and scaffold attachment factor B (SAFB)  Fig.  and SRSFFB) Fig.  were detFB) Fig. , and SAFFB) Fig. . As compFB) Fig. . NCD mayFB) Fig. , suggestCLK1 pre-mRNA during thermal stress and subsequent stress recovery  and excised upon thermal stress exposure , which is promoted by thermal stress-dependent de-phosphorylation of SRSFs. During stress recovery, the intron-retaining CLK1 pre-mRNA is re-accumulated through CLK1-dependent re-phosphorylation of SRSFs. The re-accumulation of the intron-retaining CLK1 pre-mRNA is accelerated through the efficient re-phosphorylation within nSBs54. As shown in Fig. CLK1 pre-mRNA was detected in normal conditions  and converted to intron-excised mRNA upon thermal stress  for the control and NCD- or QCD-treated cells. In contrast, re-accumulation of intron-retaining CLK1 pre-mRNA was significantly suppressed only in the NCD-treated cells , suggesting that NCD specifically inhibits the nSB-dependent part of the auto-regulation mechanisms of CLK1 pre-mRNA splicing. Consistently, NCD showed a limited effect on thermal stress-responsive alternative splicing of pre-mRNAs of HSP105 and TNRC6a 80–100 repeats (r(UGGAA)exp) in the compound eyes and showed degenerated eye morphology with depigmentation and decrease of the eye area n and r(UAAAAUAGAA)n was used as a control . When NCD (100 μM) was fed to larvae of the Drosophila model of SCA31, the degeneration of compound eyes in adults was suppressed exp were statistically significant (UAAAAUAGAA)exp was affected neither by NCD nor QCD at all regarding morphology and eye area of compound eyes ant Fig. ; left. Iant Fig. ; left. Fyes Fig.  right.26. The molecule NCD we described here showed Kd(app) of 180 nM to the G-G mismatched base pair in dsDNA with the sequence context of 5ʹ-CGG-3ʹ/3ʹ-GGC-5ʹ  for the NCD binding to UGGAA/UGGAA pentad in dsRNA was 32 nM  for the binding to 5ʹ-CGG-3ʹ/3ʹ-GGC-5ʹ in dsDNA.As the roles of functional noncoding RNAs (ncRNAs) in biological phenomenon and diseases became apparent, much needs for the small molecules binding to biologically relevant RNAs appeared. Synthetic RNA-binding molecules increase their importance in consideration of a growing number of evidence that various functional ncRNAs are involved in biological phenomenon and diseases. Among diverse approaches to discover RNA-binding molecules, our group has taken a repositioning approach of MBLs. MBLs consisted of two heterocycles having hydrogen bonding groups located at the edge of the heterocyclic ring, and the heterocycle can form the hydrogen-bonded pair with the specific nucleotide base. The hydrogen-bonded pairs would be further stabilized by stacking with the neighboring base pairs within the interior of the DNA π-stack nM Fig. , which w49. The expanded r(UGGAA)n repeat RNA, causing SCA31 likely takes various secondary and tertiary structures, and these structures are under the dynamic equilibrium. We made use of our in-house MBL library containing heterocycles with different hydrogen bonding surface and identified NCD as the hit compound binding to the r(UGGAA)n repeat RNA. The binding site of NCD in r(UGGAA)n repeat RNA was firmly determined to be the UGGAA/UGGAA pentad in the secondary hairpin structure by NMR structural analysis, which also showed the substantial structural changes induced on the RNA. In the NCD-SCA31 RNA complex, NCD bound to the RNA from the minor groove side. Each one of four guanines in the UGGAA/UGGAA pentad formed the hydrogen bonds to the N-acyl-2-amino-1,8-naphthyridine in NCD, and the produced guanine-naphthyridine hydrogen-bonded pairs were stacked inside the π-stack in dsRNA. Remarkably, all four guanosine residues bound by naphthyridine formed a syn conformation regarding the glycosidic bond. Previously, we reported the NMR structure of the complex of CAG/CAG triad DNA with two molecules of naphthyridine-azaquinolone (NA), where the guanine-naphthyridine and adenine-azaquinolone hydrogen-bonded pairs were produced28. In the case of the NA-CAG/CAG triad complex, NA bound to DNA from the major groove side and the glycosidic bond in guanosine and adenosine bound by NA adopt the anti conformation.One of the common structural characteristics observed for the toxic RNAs is the formation of hairpin structures involving multiple mismatched base pairsn repeat RNAs and the RBPs, including TDP-43, HNRNPM, and SRSF9 that preferentially bind to the r(UGGAA)n repeat sequence. Previous studies reported that GAAUG was identified as a TDP-43-binding motif55. Furthermore, it has been reported that HNRNPM and SRSF9 preferentially bound to purine-rich sequences56. Our NMR structural analysis of the NCD-UGGAA/UGGAA complex indicated the NCD binding to 5ʹ-GGA-3ʹ/3ʹ-AGG-5ʹ internal loop through sequestration of four guanines. Notably, the recognition sequence of NCD in r(UGGAA)n is overlapped with those of TDP-43, HNRNPM, and SRSF9, supporting the inhibitory effect of NCD on the specific interaction of r(UGGAA)n with these RBPs. While a moderate decrease in pulldown efficiency was observed in FUS, it is likely due to multiple RNA-binding modes of FUS by sequence and shape recognition through two separate domains57.Our in vitro pulldown assay showed that NCD inhibited RNA–protein interaction between r(UGGAA)n using the thermal stress-induced nSBs consisting of HSATIII lncRNAs as a model system, demonstrating that NCD impaired the nSB assembly without affecting HSATIII lncRNA levels. It has been shown that the RBP(s) harboring intrinsically disordered regions such as FUS and TDP-43 play a crucial role in the assembly of specific RNA foci59. Therefore, the effect of NCD was attributed to impairment of RNA–protein interactions required for the nSB assembly that consequently leads to suppression of the nSB-dependent intron retention that we recently reported38.We further investigated the mode of action of NCD to r(UGGAA)n-expressing cells and alleviation of the disease phenotype in the Drosophila model of SCA31. It is remarkably noticeable that the difference in the effect of NCD and QCD on the SCA31 disease phenotype strongly correlates with their differences in the results of our in vitro binding, pulldown and cell-based assays. Although the elucidation of precise mechanisms alleviating the disease phenotype by NCD needs further studies, the disruption of r(UGGAA)n RNA foci in the SCA31 cell model by NCD likely occurs in a similar mode of action to that of nSBs. Preceding studies demonstrated that accumulation of r(UGGAA)n RNA foci clearly correlated with the severity of disease phenotype in Drosophila model of SCA3136. In addition, the disruption of the RNA foci by binding of TDP-43 to r(UGGAA)n alleviated the disease phenotype in Drosophila model of SCA3136. The high affinity and specific binding of NCD to SCA31 repeat RNA may prevent pathogenic functions in r(UGGAA)n such as RNA misfolding and RNA–protein interaction, eventually leading to the rescue of phenotype in Drosophila model of SCA31. We cannot exclude concerns regarding the off-target effect of NCD on other G-rich sequences existing in the human genome and transcripts because NCD was originally designed to bind to the G-rich sequences. However, the phenotypic change in Drosophila model of SCA31 by NCD is most likely due to the NCD binding to r(UGGAA)n, because of high binding affinity of NCD to the UGGAA/UGGAA pentad, specific suppression of nSB-dependent splicing event by NCD, and the structure-activity relationship between NCD and QCD in the phenotypic change of Drosophila model of SCA31. Our demonstration of the mode of NCD-action to nSBs and the phenotype changes in the Drosophila model of SCA31 by NCD provides a potential therapeutic strategy of SCA31 for targeting r(UGGAA)n RNAs by small molecules. The well-defined structures of NCD-RNA complex and bioactivity of NCD for the SCA31 disease model suggest that targeting mismatched base pairs in toxic repeat RNAs by small molecules could be a potential therapeutic strategy to treat repeat expansion diseases.Our studies on biological activity using the SCA31 models demonstrated reduction of the RNA foci in r(UGGAA)9, r(UAGAA)9 and r(UAAAA)9 repeat RNAs were immobilized on the SA sensor chip (BIAcore) that coated the surface with streptavidin. The surface of sensor chip SA was washed with 50 mM NaOH and 1 M NaCl at three times for 60 s with the flow rate of 30 µl min−1. 5ʹ-biotin-TEG repeat RNAs were immobilized to the surface under the following conditions: 200 nM repeat RNA in 10 mM HEPES (pH 7.4), 500 mM NaCl. Amount of r(UGGAA)9, r(UAGAA)9 and r(UAAAA)9 immobilized on the chip surface was 586, 658, and 529 response units (RU), respectively. SPR analysis for the binding of in-house chemical library to the repeat RNA-immobilized surfaces was performed using a BIAcore T200 SPR system  under the following condition: 500 nM compounds in HBS-EP + buffer  containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20.5ʹ-biotin-TEG r(UGGAA)Repeat RNA (200 nM) without and with ligand (2 µM) in 10 mM sodium cacodylate buffer (pH 7.0) containing 100 mM NaCl was incubated at room temperature for 15 min. The mixtures were mixed with 10× loading buffer (TAKARA) and were subjected to electrophoresis through a native polyacrylamide gel with 1× Tris/Borate/EDTA buffer at room temperature. After electrophoresis, the gels were stained for 10 min with SYBR Gold (Thermo).–1). Tm was calculated using the median method.Thermal denaturation profiles were recorded on a UV-2700 spectrophotometer (Shimadzu) equipped with the TMSPC-8 temperature controller. The absorbance of RNAs (4 µM for RNA duplexes and 2 µM for repeat RNAs) without and with ligand (20 µM) in 10 mM sodium cacodylate buffer (pH 7.0) containing 100 mM NaCl was monitored at 260 nm from 2 to 100 °C (1 °C minSamples were prepared by mixing UGGAA/UGGAA pentad-containing hairpin-RNA (10 µM) and NCD (5–40 µM) in water containing 100 mM ammonium acetate and 50% methanol. Mass spectra were obtained with JEOL JMS-T100LP AccuTOF LC-plus 4 G mass spectrometer in negative mode. Spray temperature was fixed at –10 °C.CD experiments were carried out on a J-725 CD spectrometer (JASCO) using a 10 mm path length cell. CD spectra of RNAs (4 µM for RNA duplex and 2 µM for repeat RNA) in the absence and presence of ligand (20 µM) were measured in 10 mM sodium cacodylate buffer (pH 7.0) containing 100 mM NaCl.200 calorimeter. Thermodynamic parameters were calculated from the binding curve using Microcal origin 7.0 with a binding model involving a single set of identical sites.A solution of UGGAA/UGGAA pentad-containing hairpin-RNA (2.5 µM) or CGG/CGG-containing hairpin DNA (4 µM) was titrated with NCD solution (50 µM for RNA or 80 µM for DNA) at 25 °C in 10 mM sodium cacodylate buffer (pH 7.0) containing 100 mM NaCl on a MicroCal iTC2O. The sample concentration was 75 µM. NMR spectra were measured at 288 K with Avance600 spectrometer (Bruker BioSpin). For water signal suppression, the jump-and-return pulse60 was used for the 1D imino proton spectra, and the 3-9-19 pulse61 were used for other measurements. After the NMR measurement in free form, the SCA31 hpRNA was titrated by NCD with molar ratio of 1:1.5, 1:2, 1:2.5, 1:2.75, 1:3, and 1:3.5. Then, the excess amount of NCD was removed by ultrafiltration by Vivaspin (Amicon). The solvent was replaced by 100% D2O, and NMR spectra were measured for structural determination.RNA sample for NMR measurements was purchased from GeneDesign. RNA sample was dissolved in 20 mM sodium phosphate buffer (pH 6.9) with 100 mM NaCl and 5% D62. Most of the signals for base protons and H1’ protons were assigned for RNA, and all CH protons of NCD were assigned. Structure of the RNA-NCD complex was calculated with CNS_SOLVE63. One-hundred ninety-two distance restraints including 36 hydrogen bonding restraints, 157 dihedral restraints, and 14 planarity restraints were used (Supplementary Table 64 and ANTECHAMBER65.NMR data were processed with TopSpin (Bruker Biospin) and analyzed with SparkyForty-four structures without any violation for restraints were obtained by 100 calculations. Ten structures with the lowest energy were chosen, and the averaged-minimized structure was obtained. R.m.s.d. for the ten structures for heavy atoms was 1.966 ± 0.775 and 1.069 ± 0.388 Å for all and the core region, U5-G7, A9, U21-G23, A25, and NCD, respectively.76 was delegated to Thermo Fisher Scientific. The d(TGGAA)76-containing plasmid was digested with EcoRI and XbaI, and inserted into EcoRI–XbaI site of pCMVTnT to give pCMVTnT-d(TGGAA)76 plasmid.Preparation of plasmid containing d(TGGAA)2 in Dulbecco’s modified eagle’s medium (Sigma) containing 10% fetal bovine serum , penicillin and streptomycin (Thermo). For thermal stress induction, the cells were incubated at 42 °C in an incubator with 5% CO2.HeLa cells  were cultured at 37 °C in 5% CO66. In brief, d(GGAAT)20 dsDNA was introduced into pCR-Blunt II-TOPO vector (Thermo Fisher). Then, UGGAA and the antisense repeat RNAs were synthesized by in vitro transcription using T7 and SP6 RNA polymerases, respectively with Biotin RNA labeling mix (Sigma) and purified using Centri-Sep Spin Columns (Thermo Fisher). Biotinylated r(UGGAA)20 or r(UUCCA)20 repeat RNA (1 µg) was bound to Tamavidin2-REV magnetic beads, mixed with pre-absorbed HeLa nuclear extract diluted in PBS containing 0.1% Triton X-100, 1× protease inhibitor, 1 mM PMSF and RNase inhibitor without and with NCD or QCD (2 µM), and rotated overnight at 4 °C. After washing five times with cold PBS containing 0.1% Triton X-100, the co-precipitated proteins were eluted in SDS sample buffer for 5 min at 95 °C.In vitro pulldown assay was performed as previously reportedProtein extracts in 1× SDS sample buffer were boiled for 5 min, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to a polyvinylidene fluoride membrane (Millipore) by electroblotting. After incubation with primary and HRP-conjugated secondary antibodies, the signals on the membranes were developed by a chemiluminescence reaction using the ImmunoStar Kit , detected with a ChemiDoc imaging system (BioRad), and analyzed using ImageJ software (NIH). Antibodies are as follows. Anti-HNRNPM , anti-SFPQ , anti-SRSF9 , anti-ALYREF , anti-TDP-43 , anti-FUS , anti-Mouse IgG (HRP-Linked) , anti-Rabbit IgG (HRP-Linked) .5 cells were seeded into 24-well plates containing coverslips. After a 24 h culture, the cell culture medium containing compound was added to the cells. Plasmids (500 ng) expressing r(UGGAA)76 were transfected to the cells using 2 μl of FuGENE HD Transfection Reagent (Promega). After 24 h of transfection, the cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 30 min at 4 °C. The fixed cells were washed with PBS and permeabilized with PBS containing 2% acetone pre-chilled at –20 °C for 5 min at 4 °C. Subsequently, the cells were washed with PBS and stored in 70% EtOH at –20 °C overnight. After washing with PBS and rehydration with 30% formamide in 2× saline sodium citrate (SSC) for 10 min at room temperature, the cells were pre-hybridized in hybridization buffer  for 30 min at 37 °C and hybridized in hybridization buffer containing 1 nM Alexa647-labeled (TTCCA)5 DNA/LNA probe . Anti-SAFB , anti-SRSF2 , anti-Digitonin , anti-TDP-43 , anti-Mouse IgG (H + L) (Alexa Fluor488) , anti-Rabbit IgG (H + L) (Alexa Fluor488) , anti-Rabbit IgG (H + L) (Alexa Fluor568) , anti-Goat IgG H&L (Alexa Flour 488) , anti-Mouse IgG H&L (Alexa Fluor568) . The image analysis was performed using ImageJ (NIH) software. Areas of r(UGGAA)76 RNA foci, nSBs, nuclear speckles, and nuclei were defined and measured by binarized images of r(UGGAA)76 RNA, HSATIII, SRSF2, and DAPI, respectively.A total of 1 × 1038. In brief, total RNAs were prepared using TRI Reagent , according to the manufacturer’s manual. The RNAs were treated with RQ1 RNase-free DNase (Promega) according to the manufacturer’s manual. First-strand cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kits (Thermo Fisher Scientific). For RT-qPCR, the cDNAs were amplified using KAPA SYBR FAST qPCR Master Mix (KAPA Biosystems) and monitored using the LightCycler 480 System (Roche). For semi-quantitative RT-PCR, cDNAs were amplified by PCR to unsaturated levels, separated by electrophoresis, and stained with ethidium bromide. Images were obtained with a ChemiDoc system (BioRad) and analyzed with ImageJ software (NIH). Primers for PCR are shown in Supplementary Table RT-qPCR and semi-quantitative RT-PCR were performed as previously reported4 cells/well in 96-well plates and cultured for 24 h. Small molecules were added to final concentrations of 5 µM to the medium and the cells were cultured further for 24 h. CCK-8 solution (5 µl) was added to each well and the plates were further incubated for several hours at 37 °C before the measurement of the absorbance at 450 nm by a plate reader .Effect of small molecule treatment on cell viability was determined by WST-8 assay . HeLa cells were seeded at 2 × 10Drosophila Medium Blue containing dry yeast was mixed with ultrapure water or 100 μM compound. Parent flies exp/+ for Drosophila expressing r(UGGAA)exp or GMR-Gal4/+;; and UAS-(UAGAA)(UAAAAUAGAA)exp/+ for Drosophila expressing r(UAGAA)(UAAAAUAGAA)exp) were crossed on the food without and with 100 μM compound, and the offspring were generated on the same food at 25 °C. The eye morphology of the 1–2-day-old flies was analyzed using the stereoscopic microscope model SZX10 (Olympus).Instant Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


A long-standing debate revolves around which mental codes allow humans to control behavior. The internal stimulus model (going back to Rudolf Hermann Lotze) proposes that behavior is controlled by codes of stimuli that had previously preceded corresponding motor activities. The internal effect model (going back to Emil Harleß) proposes that behavior is controlled by codes of perceptual effects that had previously resulted from corresponding motor activities. Here, we present a test of these two control models. We observed evidence for both models with stronger evidence for the internal stimulus model. We suggest that the proposed experimental setup might be a useful tool to study the relative strengths of stimulus control and effect control of behavior in various contexts. Both approaches share assumptions. They both propose that actions are generated by means of codes of perceptual events, which had become linked to motor activities by previous learning experience. They differ, though, in the type of perceptual codes doing so  stimulation  lingering codes of internal stimuli and/or internal effects can express themselves independently. We focused on a situation in which the choice of what to do was relatively free to the actor within moderate constraints, because there was neither a specific stimulus to respond to, nor a specific effect to aim at  with a nonspatial response (pressing a button once or twice). The responses contingently produced horizontally arranged effects (a square left or right to the center of screen depending on the response). Thus, a particular response led to an apparent movement of the square, and participants are expected to establish links between their behavior and the effects of that behavior.Specifically, we intermixed forced-choice trials and free-choice trials in the experiment  near the center of the screen. Participants were instructed to choose between responding with one or two key presses, irrespective of whether they saw the rectangle or not. After responding, participants placed a small rectangle where they believed it to have appeared. We expected a rather veridical location report in these free-choice trials that also served to induce participants’ confidence that barely perceptible rectangles would occur, and as a check that they complied with task instructions.Two types of free-choice trials were employed. In no rectangle was actually presented (free choice without test event). If the generation of the eventually executed action was mediated by an internally generated code of a certain stimulus, we expected the “perceived” location to resemble, and thus to be biased towards, the vertical position where the stimulus for that action would normally appear. In contrast, if the execution of the eventually chosen action was mediated by a code of a certain effect, we expected the reported location to be biased towards that horizontal position where the effect of that action would normally appear. Such biases towards highly activated perceptual representations due to top-down influences are commonly observed  for monetary compensation. All participants reported normal or corrected-to-normal vision, were naïve regarding the underlying hypotheses, and provided written informed consent prior to data collection.Stimulus presentation and response collection happened on a PC connected to a 17-inch CRT monitor . Stimuli were white squares (30 × 30 px) displayed above or below (150 px) the center of the screen  or near the center (± 70 px) of the screen . Action effects were the same white squares displayed left or right (150 px) of the center of the screen. Masks were five images of white noise displayed at the center of the screen. The background was black. The manual responses were given with a response key placed under a finger of the participants. The placement of where participants thought the stimulus appeared in the free-choice trials was controlled by the arrow keys on the keyboard.The task was either to give a predefined response (one or two button presses) to the two forced-choice stimuli or to choose freely within moderate constraints one of the two possible responses in free-choice trials . Within each block, there was an equal number (20 each) of forced-choice trials requiring one response, two responses, free-choice trials with, and free-choice trials without a test event.n = 20 participants). This is a large number of participants, and is larger than in previous studies where we used similar criteria. At present, we can only speculate that the particular choice  plays a role in this. The forced-choice stimulus–response mapping and the response–effect mapping were counterbalanced across participants.An even distribution of responses composed of one and two key presses over the course of the experiment as well as the avoidance of strategies in the free-choice responses were instructed at the beginning of the single individual test sessions of about 60 minutes. When participants responded unevenly (≥80% of responses composed of either one or two key presses), the data were discarded, and the participant was replaced by a new participant to ensure that both response options were at least chosen approximately equally often (y-axis) or the effect (x-axis), and negative values indicated a bias away from the stimulus or the effect.The dependent variables of most interest were the deviation of the participants’ stimulus placement on the vertical and the horizontal axes in pixels towards the corresponding stimulus/effect. Each free-choice trial was categorized based on whether the response-associated stimulus  would be located above or below the center and whether the response-associated effect  would be located left or right from the center. Deviations were then remapped so that positive values indicated a bias toward the stimulus  of all trial types were submitted to an ANOVA (forced choice vs. free-choice with test event vs. free-choice without test event). RTs were measured from stimulus/mask onset until the (first) key press.The central analyses were y-axis and on the x-axis. Trials were then classified according to whether the location above/below or left/right of the screen center would fit with one or two responses, respectively, according to the requirements in forced-choice trials. Then, the mean choice rate of congruent response choices was calculated per participant and evaluated with t tests against a value of μ0 = 0.5.Supplemental tests were run on free-choice trials with a test event. In these trials, the test event was positioned randomly and always had a deviation from screen center on the p values were Greenhouse–Geisser adjusted when the assumption of sphericity was violated. In these cases, the respective epsilon is reported.Trials were excluded from RT analyses as outliers if their RTs deviated more than 2.5 standard deviations from the respective cell mean . All Overall, one key press was given in 58.8% (range: 37.3%–76.2%) of the free-choice trials. In free-choice trials with a test event, the percentage was 53.0 (range: 25.4%–71.4%) and in free-choice trials without a test event, the percentage was 64.9 (range: 25.0%–85.0%). Deviations of the participants’ stimulus placement from the stimulus center  and from the screen center  are visualized in Fig. y-axis), t(31) = 1.92, p = .063, d = 0.34, nor toward the effect (x-axis), t(31) = 0.70, p = .490, d = 0.12, nor did these nonsignificant biases differ significantly from each other, t(31) = 1.55, p = .132, d = 0.27. Most importantly, for free-choice trials without a test event , t(31) = 4.99, p < .001, d = 0.88, and toward the effect (x-axis), t(31) = 2.32, p = .027, d = 0.41. The former bias was larger in size than was the latter bias, t(31) = 4.57, p < .001, d = 0.81.For free-choice trials with a test event , t1 = 1.92,-axis), t1 = 1.92,t(31) = 7.61, p < .001, d = 1.34. No comparable effect was observed for congruency with effect position (51.2%), t(31) = 1.29, p = .207, d = 0.23.Participants responded congruent with the stimulus position in 68.6% of the trials, F = 52.63, p < .001, ηp2 = .63, ε = .76. Paired t tests indicated differences between all three task types, with forced-choice trials (466 ms) having a shorter average RT than both free-choice trials with a test event (726 ms), t(31) = 9.27, p < .001, d = 2.32, and those without a test event (823 ms), t(31) = 7.98, p < .001, d = 1.99. Furthermore, free-choice trials with a test event had shorter RTs than those without a test event, t(31) = 2.95, p = .006, d = 0.74. In the forced-choice trials, the wrong number of key presses was given on average 10.5% of the time, ranging from 2.9% to 22.1%.The three task types differed in their average RTs, Which mental codes trigger freely chosen actions? Codes of stimuli or codes of effects to which the chosen actions are linked? The present study suggests: both. When people generate a motor action, they tend to report perceptual events that resemble both, the stimulus that normally requires the generated actions as well as the effect that normally results from that action. We consider this as strong evidence for the internal activation of corresponding event codes because in the crucial condition, no actual event was presented.or effect codes, but rather for both. This might be explained by the assumption that actions are generated neither by stimulus codes nor by effect codes alone, but by codes that represent the transition of a specific stimulus to a subsequent specific effect (cf. Kunde, Schmidts, Wirth, & Herbort, At first glance, it might come to a surprise that we did not observe evidence exclusively for activation of either stimulus y-axis—that is, depending on the stimulus position. This might be because such biases express themselves more strongly when judging stimuli on the vertical rather than horizontal dimension. Alternatively, stimuli might have received more attention in this particular setup, because they were clearly task relevant whereas the ensuing effects were not. The impact of action effects might become larger if they are made task relevant (see Janczyk, Yamaguchi, Proctor, & Pfister, Admittedly, the bias towards action-contingent stimuli was overall larger than the bias towards action-contingent effects regarding the placements of the test event. In addition, a priming of a particular response in free-choice trials with a test event (akin to that reported by Elsner & Hommel, One may further argue that our observation in free-choice trials without test event reflects a bias to report corresponding events without actually having a corresponding perception. Nonperceptual explanations for putatively perceptual phenomena are a notorious problem (see, e.g., Firestone & Scholl, We have intermixed forced-choice and free-choice tasks, and this may limit how we can generalize our conclusion to situations where only free-choice tasks are administered. First, the history of experienced forced-choice trials might influence choices, in particular when participants are not entirely free in their choices but need to attain a certain ratio of choices (see Gozli, externally triggered/stimulus-based actions versus self-generated/intention-based actions, which are experimentally addressed by using forced-choice and free-choice tasks (e.g., Brass & Haggard, In a broader context, it is discussed what free-choice tasks essentially are and whether they do operationalize what they are intended to operationalize. This discussion revolves mostly around the putative distinction of In sum, our experiment revealed that both stimulus codes, and, to a lesser extent, effect codes are indeed imagined when it comes to carrying out a freely chosen action in a free-choice task. Future studies may more thoroughly investigate circumstances changing the relative impact of these codes, and we believe that the present experiment provides a valuable tool to further investigate the importance of imagined stimulus codes and effect codes in action control.


To the editor: We read with interest the recent article by Reusken et al. about laboratory readiness for molecular testing of the novel coronavirus 2019, recently named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in expert laboratories in 30 European countries [In our reference institute for infectious diseases, we have been implementing since the end of January 2020 PCR detection of SARS-CoV-2 RNA using several systems, including those released at the European level . In totaThus, it is surprising to see that all the attention focused on a virus whose mortality ultimately appears to be of the same order of magnitude as that of common coronaviruses or other respiratory viruses such as influenza or respiratory syncytial virus, while the four common HCoV diagnosed go unnoticed although their incidence is high. In fact, the four common HCoV are often not even identified in routine diagnosis in most laboratories, although they are genetically very different from each other  and asso