Long non-coding RNAs (lncRNAs) are attracting widespread attention for their emerging regulatory, transcriptional, epigenetic, structural and various other functions. Comprehensive transcriptome analysis has revealed that retrotransposon elements (REs) are transcribed and enriched in lncRNA sequences. However, the functions of lncRNAs and the molecular roles of the embedded REs are largely unknown. The secondary and tertiary structures of lncRNAs and their embedded REs are likely to have essential functional roles, but experimental determination and reliable computational prediction of large RNA structures have been extremely challenging. We report here the nuclear magnetic resonance (NMR)-based secondary structure determination of the 167-nt inverted short interspersed nuclear element (SINE) B2, which is embedded in antisense Uchl1 lncRNA and upregulates the translation of sense Uchl1 mRNAs. By using NMR ‘fingerprints’ as a sensitive probe in the domain survey, we successfully divided the full-length inverted SINE B2 into minimal units made of two discrete structured domains and one dynamic domain without altering their original structures after careful boundary adjustments. This approach allowed us to identify a structured domain in nucleotides 31–119 of the inverted SINE B2. This approach will be applicable to determining the structures of other regulatory lncRNAs. Comprehensive mouse transcriptome analysis has identified a large number of long non-coding RNAs (lncRNAs) containing retrotransposon elements . Short iSINE elements that upregulate translation) are comprised of a binding domain—a complementary sequence of target mRNAs—and an effector domain consisting of the SINE B2. SINEUPs broadly enhance the translation of targeted mRNAs (Arthrobacter luteus (Alu) elements (FRAM and MIRb) enhance target mRNA translation, although the sequence identity among these RNAs is <50%  were registered in the Protein Data Bank (PDB), but only 157 structures of long RNAs (>100 nt). More than 140 000 structures are registered for proteins in the PDB (http://www.rcsb.org/). Also, with the exception of one case  lncRNA contains a 167-nt inverted SINE B2, which is an effector domain that stimulates the translation of sense Uchl1 mRNA . This fied mRNAs . Proteined mRNAs . In addi is <50% . We hypo is <50% ,19. To eone case , no struone case . Generalone case  and a feone case . Further1H NMR spectra of fragmented inverted SINE B2 with the deletion of n × 10 nt from both the 5′ end and the 3′ end. The deletion experiments suggested that inverted SINE B2 includes at least two structured domains, which we later named the C and M domains. Third, we compared our NMR findings with five secondary structures calculated by prediction programs. Two of them were consistent with the NMR experimental data and were used as reference structures. To determine the secondary structure of inverted SINE B2, we divided it into four fragments. Finally, the whole secondary structure was determined, with the assignment of most of the imino proton signals of the 167 nt of full-length inverted SINE B2. Interestingly, our NMR experiments revealed that the terminal domain (T domain) in nucleotides 1–21 and 141–167—for which all of the programs predicted a stem structure—did not form a rigid stem. The 31–119 fragment  in nucleotides 34–119), which retained 80% of the SINEUP function of the full-length inverted SINE B2, showed an identical fold to that of the corresponding region of the full-length RNA. This suggested that U118 and G119 are important for the stability of the region 43–58 stem-loop structure. We also identified a stable stem formation in the middle domain (M domain) in nucleotides 23–33 and 130–140. Thus, we identified two structured domains (C and M domains) and one dynamic domain (T domain), the structures of which deviated substantially from any of previous secondary structure predictions. Our NMR results, together with those of previous deletion experiments, suggest that the 43–58 stem-loop may include a tertiary structure and is vital to the regulatory role of SINEUPs  functional inverted SINE B2 RNA, for which there is little structural information, by using NMR. First, we prepared fragments to identify the region responsible for the structure relevant to the SINEUP function. Second, we compared the Δ5–14, Δ35–44, Δ65–74, Δ100–111, Δ100–111 Δ130–141, Δ130–141, and Δ1–30 Δ120–167 deletion mutants (fragment 31–119) were created by using molecular cloning techniques. Briefly, fragment 31–119 (Δ1–30 Δ120–167) was chemically synthesized  and swapped with the full-length inverted SINE B2 sequence in SINEUP-GFP (Δ5′–32 nt); Δ5–14, Δ35–44, Δ65–74, Δ100–111, Δ100–111 Δ130–141 and Δ130–141 deletion mutants were mutagenized by using QuikChange II site-directed mutagenesis kits  in accordance with a protocol published previously (Full-length inverted SINE B2 plasmid (SINEUP-GFP (Δ5′–32)) and the fragment 31–119 plasmid (see ‘Plasmid and cloning’ section) were transfected with pEGFP-C2 plasmid  into HEK293T/17 (human embryonic kidney) cells (ATCC: CRL-11268) in accordance with a protocol published previously .https://imagej.nih.gov/ij/), as described in references  and 0.005% phenylmethylsulfonyl fluoride (PMSF) , and then the protein lysates (10 μg) were separated by 10% polyacrylamide gel electrophoresis . Green fluorescent protein (GFP) and β-actin proteins were detected by western blotting, which was performed in accordance with a protocol published previously ,30. GFP ferences  and (30)Enhanced green fluorescent protein (EGFP) was detected by using anti-GFP rabbit antibody . β-Actin was detected by using anti-β-actin antibody . The secondary antibodies were horseradish peroxidase (HRP)-conjugated polyclonal goat anti-rabbit antibody  and HRP-conjugated polyclonal goat anti-mouse antibody .−ΔΔCT method  and Maxwell RSC Instrument in accordance with the manufacturer's protocol. Total RNA (500 ng) was reverse transcribed to cDNA by using a PrimeScript 1st strand cDNA synthesis kit  in accordance with the manufacturer's protocol. SINEUP-GFP RNA, EGFP RNA and GAPDH RNA expression were quantified by polymerase chain reaction (PCR) amplification using SYBR Premix Ex Taq (Tli RNaseH Plus) (TaKaRa Bio Inc.). Relative expression was analyzed by using the 2T method .The following qRT-PCR primers were used:hGAPDH_Fw: TCTCTGCTCCTCCTGTTChGAPDH_Rv: GCCCAATACGACCAAATCCEGFP_Fw: GCCCGACAACCACTACCTGAGEGFP_Rv: CGGCGGTCACGAACTCCAGSINEUP-GFP_Fw: CTGGTGTGTATTATCTCTTATGSINEUP-GFP_Rv: CTCCCGAGTCTCTGTAGC.All predicted secondary structures were calculated by using the user-defined parameters of the computational prediction software on the respective web servers. The temperature was set to 37°C (310.15K) for all predictions. All predictions were performed under standard parameters, including the energy model recommended by each program . For Mfold , ‘RNA sein vitro transcription was amplified from SINEUP-GFP  were purchased from Eurofins Genomics  and Hokkaido System Science Co., Ltd.  The DNA template of full-length inverted SINE B2 (CAGTGCTAGAGGAGGTCAGAAGAGGGCATTGGATCCCCCAGAACTGGAGTTATACGGTAACCTCGTGGTGGTTGTGAACCACCATGTGGATGGATATTGAGTTCCAAACACTGGTCCTGTGCAAGAGCATCCAGTGCTCTTAAGTGCTGAGCCATCTCTTTAGCTCC) for NEUP-GFP  by usingFive forward primers  and eight reverse primers  were purchased from Eurofins Genomics. A T7 promoter sequence (forward: TAATACGACTCACTATA) was added to each primer sequence. The amplified templates were purified twice by ethanol precipitation and then lyophilized.in vitro by using T7 RNA polymerase from an AmpliScribe T7-Flash Transcription Kit  and precipitated by using isopropanol with 2 mM ethylenediaminetetraacetic acid (EDTA). RNA pellets were suspended in 8 M urea, 0.1% Orange G, 0.05% xylene cyanol and 0.05% bromophenol blue running dye, and then centrifuged at 20 000 × g for 15 min at room temperature. The supernatants, which contained the RNAs, were separated in a 12.5% polyacrylamide—8 M urea gel. After the electroseparation, the appropriate bands were cut out and the RNAs were collected by using an electro-elution system . The RNAs were collected by ethanol precipitation with 2 mM EDTA twice. RNA pellets were dissolved in MilliQ water  and then lyophilized.All RNA fragments were transcribed 2O.Four buffers were tested the NMR: 20 mM sodium phosphate, 50 mM NaCl (pH 7.2 or pH 6.85); 20 mM 2-(N-morpholino)ethanesulfonic acid (MES), 50 mM NaCl (pH 6.0); and 89 mM Tris-borate (pH 8.9). The pH of the MES buffer was adjusted to 6.0 with NaOH. All buffers contained 8% D2O, with the solution then adjusted to 30–300 μM. Shigemi tubes (diameter 4 mm)  were used for the full-length inverted SINE B2 RNA and 31–119 RNA samples, and 5-mm-diameter Shigemi tubes were used for the other RNAs. It was found that samples prepared and kept in the NMR tubes showed no NMR spectral changes for at least a year without the addition of RNase inhibitors. We confirmed that the NMR spectra of the full-length inverted SINE B2 or its fragments did not change with the addition of an annealing protocol. Therefore, all of the NMR spectra used in this work were recorded from samples without annealing.For structure determination, RNAs were dissolved in 20 mM MES—NaOH (pH 6.0), 50 mM NaCl and 8% D1H NMR spectra were collected with a data size of 16 k, 512 scans and a spectral width of 25 ppm at 298K. 2D NOESY (nuclear Overhauser effect spectroscopy) experiments were performed with 2048 × 512 data points and a spectral width of 25 ppm at 288K or 293K. Water suppression was achieved with a Watergate 3–9–19 pulse sequence. Spectra were analyzed by using Sparky NMR assignment and integration software (https://www.cgl.ucsf.edu/home/sparky/) and TopSpin 3.61 NMR processing software .NMR spectra were recorded on Bruker Avance 700, 800 and 900 MHz spectrometers with cryogenic probes . 1D 1H exchange rate between the imino group and water depends on the solvent pH: exchange is minimized at pH lower than 5. Moreover, the thermodynamic stability of some functional RNAs is influenced by the solvent pH: for example, in the case of tRNALys,3, the Tm value increases by about 6°C at pH 5 compared with pH 7.2  standard protocol was followed for library preparation and RNA secondary structure analysis (3 reagent (modified) or with dimethyl sulfoxide (DMSO) (mock control). Two biological replicates for each of the NAI-N3 and DMSO libraries were made. RNA was extracted by using an RNeasy Mini Kit  and digested with DNase I to remove DNA contamination by using a TURBO DNA-free kit . Purified RNA was ribo-depleted by using a magnetic RiboZero kit , and RNA quality was checked with an Agilent RNA 6000 Nano kit . Next, 500 ng of ribo-depleted RNA was used for biotin-click reaction to label probed bases, and RNA was fragmented for 40 s by using RNA fragmentation reagents . Fragmented RNA was end-repaired, and the icSHAPE adapter ligation standard protocol was then followed. Adapter-ligated RNA was size-selected and reverse transcribed by using icSHAPE-specific barcoded RT-primer.  In the next step, truncated cDNA products were selected by using a streptavidin-based system and further size-selected . In the case of SINEUP, mapping was focused on the region starting from 1 bp upstream of SINE B2 and extending to 88 bp downstream of it to avoid normalization bias from flanking sequences and to capture reads uniquely mapping to SINE B2 only. Next, transcript abundance and RT stop numbers were calculated, and this was followed by calculation of the correlation between replicates and then replicate merging. The mean number of RT stops of the 90–95% most reactive bases was normalized to 1, and all other RT stops were scaled proportionally . Enrichment reactivity scores were then calculated by subtracting the background signal (DMSO data) and outliers were removed by 90% Winsorization. Valid enrichment reactivity scores were filtered and visualized in IGV . The icSHAPE enrichment score data were used as a soft constraint in the RNAfold program of ViennaRNA  in ref.  of the inverted SINE B2  full-length inverted SINE B2 (167 nt and GG), (B) the Δ35–44 mutant (157 nt and GG) and (C) the Δ1–30 Δ120–167 mutant (89 nt). The spectra showed numerous imino proton signals, which we used as the ‘fingerprints’ of the RNA structure. In comparing the spectra of the two mutants and the native full-length inverted SINE B2, we noticed that most of the signals showed identical chemical shifts. However, it was clear that five imino proton signals were not observed, or were substantially broadened, in the Δ35–44 mutant  of inverted SINE B2 showed SINEUP activity similar to that of full-length inverted SINE B2, whereas the Δ35–44 deletion mutant loses activity (1H NMR spectra of these fragments and the full-length RNA (1H NMR spectra of the imino proton region acts as a sensitive probe to distinguish the formation of base pairs (i.e. secondary structures). If a fragment shows different imino 1H chemical shifts from those of the full-length RNA, then the secondary structure of the fragment should differ from that of the full-length RNA. This step allows us to survey regions (or domains) that can be safely divided without altering their structures. The 1H NMR spectra of fragments 11–157 (5′Δ10nt/3′Δ10nt) and 21–147 (5′Δ20nt/3′Δ20nt) did not show any signals that were not observed from the full-length RNA  full-length inverted SINE B2 lncRNA, and fragments (b) 31–127 (5′Δ30nt/3′Δ40nt), (c) 31–119 (5′Δ30nt/3′Δ48nt), (d) 31–117 (5′Δ30nt/3′Δ50nt) and (e) 41–117 (5′Δ40nt/3′Δ50nt). Although the asymmetric deletion fragment 31–127 (5′Δ30nt/3′Δ40nt) in (b) still exhibited a signal that was not observed in the full-length RNA at 14.2 ppm in (a), to our surprise, it regained native signals  that were not observed in the symmetric deletion fragments 31–137  without substantially altering the core structural motif. The data suggested that full-length inverted SINE B2 is likely to have at least three structured regions: 21–31, 120–147 and 31–119. Fragment 21–147 did not give any artificial signals that were not observed in the full-length RNA, whereas fragment 31–137 had an artificial structure; this indicates that at least fragment 21–31 or fragment 137–147, or both, were required to maintain a native structure. Although the differences in the 1D spectra of fragments 21–147 and 31–119 in 1H-15N HMQC (heteronuclear multiple quantum coherence) and 1H-1H NOESY NMR spectra revealed that region 31–119 included one UU pair and two GU pairs. The structures predicted by RNAfold and RNAstructure were consistent with our NMR experimental results , TM , C (nucleotides 31–119) and MC2 (nucleotides 21–60 and 90–147). We then measured the 1D A Figure .Although the imino proton resonances of the TM, MC2 and C fragments Figure –E genera1H-1H NOESY spectra of full-length inverted SINE B2 and the TM, MC2 and C fragments , in the absence of other cellular components. For instance, to our surprise, our NMR experimental data indicated that the T domain and the region 91–109 stem-loop did not exhibit rigid stem structures, contrary to all of the secondary structure prediction results and the findings of previous studies .The icSHAPE data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus  and are gkaa598_Supplemental_FileClick here for additional data file.


In December 2019, the novel coronavirus Coronavirus Disease 2019 (COVID-19) outbreak started in Wuhan, the capital of Hubei province in China. Since then it has spread to many other regions, including low-income countries. Coronavirus Disease 2019 (COVID-19) outbreak started in Wuhan, the capital of Hubei province in China. Since then it has spread to many other regions, including low-income countries [1In December 2019, the novel coronavirus untries 1. Large aDuring the past 20 years, outbreak and prevalence of severe respiratory infections have been seen as a major hazard to global health. In December 2019, a series of pneumonia cases of unknown aetiology were documented in Wuhan. High-throughput sequencing from respiratory tract samples revealed this novel coronavirus strain, named SARS-CoV-2 . In respAs a global organization representing the cardiovascular community, with more than 200 cardiovascular societies and foundations, the World Heart Federation is concerned that previous studies on other coronavirus diseases such as SARS-CoV and MERS-CoV have demonstrated a relationship between cardiovascular disease (cardiac disease and hypertension), diabetes and an increased morbidity and mortality due to coronavirus disease 6. Guan eThe goal of this document is to update our members from low-income countries on important facts on COVID-19 prevention in low- and middle-income countries.Coronaviruses belong to a family of viruses that can cause mild disease such as a common cold, but also severe respiratory disease such as Middle East Respiratory Syndrome (MERS-CoV) or Severe Acute Respiratory Syndrome (SARS-CoV). The novel coronavirus epidemic Coronavirus Disease 2019 (COVID-19) is caused by a coronavirus named SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2).SARS-CoV-2 is thought to have a zoonotic origin and was first isolated from patients with pneumonia in Wuhan, China. The spread from human-to-human is mainly an aerosol transmission through contaminated respiratory droplets (coughing and sneezing). In addition, respiratory droplets containing the virus may contaminate surfaces up to 96 hours, for example screens of smart phones , multi-organ failure and death have been reported Figure .Basic recommendations to prevent COVID-19: Follow six easy rules to reduce the risk of coronavirus transmission. The rules are built on the principle of protecting yourself and protecting others Figure .People with chronic underlying disease may be at increased risk of severe COVID-19 disease and death (Table Avoid large gatherings – stay at home.Keep at least 1–2 meters distance from a person with respiratory symptoms and do not stay in the same room with this person.Vulnerable people should consider moving to relatives in rural areas and spend their time in voluntary isolation, such as a small hut, receiving food supplies via a neighbour or relative, but without direct contact.Travel should be reduced to a minimum. Use a mask if travelling in a bus, train or plane. If masks are not available or affordable, cover your nose and mouth with a cloth or similar.In case SARS-CoV-2/COVID-19 begins to spread in low-income countries at high risk of airborne diseases, as described above, containment may not be realistic and response efforts will likely need to transition to various mitigation strategies, which could include isolating ill people at home, closing schools, universities, places of religious worship and public events, which would also include attending funerals. Please follow recommendations of leading health organizations as outlined in Table


Most RNA-binding modules are small and bind few nucleotides. RNA-binding proteins typically attain the physiological specificity and affinity for their RNA targets by combining several RNA-binding modules. Here, we review how disordered linkers connecting RNA-binding modules govern the specificity and affinity of RNA–protein interactions by regulating the effective concentration of these modules and their relative orientation. RNA-binding proteins also often contain extended intrinsically disordered regions that mediate protein–protein and RNA–protein interactions with multiple partners. We discuss how these regions can connect proteins and RNA resulting in heterogeneous higher-order assemblies such as membrane-less compartments and amyloid-like structures that have the characteristics of multi-modular entities. The assembled state generates additional RNA-binding specificity and affinity properties that contribute to further the function of RNA-binding proteins within the cellular environment. RNA–protein complexes are termed ribonucleoprotein (RNPs) particles ,2. RNA-bIn this review, we discuss the modular characteristics of RBPs and how modularity is exploited to mediate interactions with the target RNA. We illustrate the crucial role of IDRs in defining RNA-binding affinity and sequence specificity. IDRs often behave as linkers connecting RNA-binding modules within a single RBP where they dictate how modules are arranged and relate to each other. The structural constraints and topologies defined by linkers define the RNA targets of a single RBP. IDRs can also function to connect structural elements between and among proteins and RNA promoting the formation of higher-order RNA–protein assemblies. We propose an analogy between intra-protein linkers and assembly-promoting IDRs. As in individual proteins where IDRs link RNA-binding modules, in higher-order assemblies, IDRs can connect proteins and RNA resulting in complexes that have the characteristics of multi-modular entities. The assembled state can result in new RNA-binding specificity and/or affinity that further the function of RBPs within the cell.2.Before we discuss how RNA-binding modules combine with one another, we first briefly review the principles underlying their characteristics. RNA-binding modules are versatile elements . Althouga). Within the β-sheet, two conserved motifs, RNP1 and RNP2, contact two contiguous nucleotides, conferring specificity for single-stranded RNA [in vitro. Target recognition involves the β-sheet binding a triplet within the pentaloop and the β2/β3 loop identifying the RNA stem through its shape [figure 1b). This module contains approximately 70 residues organized in an antiparallel β-sheet packed against three α-helices. These structural elements, together with other elements contained in the α1/α2 and β2/β3 loops, form a binding cleft contacting four single-stranded nucleotides with low affinity [The RNA-recognition motif (RRM) is a common and well-characterized RNA-binding module and here we use it as an example of how an element recognizes and binds RNA ,8. The Rfigure 1nded RNA ,18. RNP1nded RNA ,18. Thesnded RNA . For exats shape . An extrts shape ,22. Thests shape . Extensiaffinity . Extensiaffinity . The aboThe intricate contacts established between RNA and proteins often cannot be explained with a rigid lock-and-key docking model ,24. Comp3.a,b). These combinations collectively define the RNA-binding properties of the protein [c).Individual RNA-binding modules typically recognize short RNA stretches and bind them with low affinity ,7. To acfigure 2 protein . For exa protein . By cont protein  . Linkers that do not establish direct contacts with other proteins or nucleic acids in the bound state, but remain unstructured, confer a dynamic disorder connotation to the complexes [While some disordered linkers connecting RNA-binding modules do not contact the modules or the RNA, others do establish these contacts  figure b. Linkeromplexes ,36. Thesomplexes ,9. This omplexes . These lomplexes . Linker omplexes ,6,9. Theomplexes .Disordered linkers that enable molecular recognition by establishing contacts with the RNA and the RNA-binding modules allow for cooperative RNA binding ,18 and u4.c). RNA-binding IDRs exhibit distinguishing features. They are usually enriched in residues that are typically found in structured RNA-binding modules such as arginine, lysine, histidine and the order-promoting aromatic residues tyrosine and phenylalanine [RNA-binding IDRs are widespread in the proteome and can be found either alone or associated with structured RNA-binding modules ,43 figuc. RNA-bilalanine ,43,44. Mlalanine . Argininlalanine . These slalanine . Argininlalanine . Argininlalanine . One notlalanine . The higlalanine . Similarlalanine ,26,43. Blalanine ,43. Finalalanine .5.c,d). The typically expanded conformations of IDRs provide flexible and conformationally adaptable interfaces that facilitate ligand binding and enable contacts with multiple partners simultaneously [A striking characteristic of RBPs is that they are enriched for IDRs longer than 30 residues  that profigure 2aneously ,50. IDR aneously ,51. Disoaneously . IDRs caaneously . Many rianeously . IDRs caaneously . The buianeously .Some IDRs have the remarkable capability to drive the formation of higher-order assemblies with variable stoichiometry and heterogeneous conformation  figure . These ILow complexity IDRs can promote the formation of higher-order assemblies because they are multivalent, that is, they contain multiple binding moieties able to establish intramolecular and/or intermolecular, homotypic and/or heterotypic interactions ,59 figu. Multivaa). These systems have a dynamic connotation [Within the cell, higher-order assemblies containing RBPs and RNA can form membrane-less compartments that enable the local concentration of selected proteins and nucleic acids  figure . Severalfigure 3notation , as theynotation ,64.b). The network formed through these homotypic multivalent interactions results in the formation of unbranched fibrils with a typical cross-β-sheet structure [in vivo and in vitro. These fibrils may exhibit different biochemical properties and these differences could have important physiological consequences. Amyloid-like structures share a subset of amyloid-associated structural and biochemical properties such as a cross-β-sheet structure, ability to bind hydrophobic dyes and/or resistance to ionic detergents [In vitro phase-separated proteinaceous droplets can convert into amyloid-like structures [continuum [Some RNP granules contain amyloid-like structures. Amyloids are highly ordered assemblies built through the repetitive addition of multiple copies of a protein or a peptide b. The netructure ,66. The tergents . Amyloidtergents ,68,69 antergents ,66. In vructures ,70,71. Tontinuum ,64. The ontinuum . In thisontinuum .6.a). Deletion of the glutamine/asparagine-rich IDR of Lsm4, a multifunctional yeast RBP found in processing (P) bodies, causes loss of P bodies in some conditions. Replacement of the Lsm4 IDR with the glutamine/asparagine-rich IDR of the yeast protein Rnq1 rescues assembly of these RNP granules [IDRs of RBPs are key determinants of RNP granule formation and function  figure a. Deletigranules . Similargranules . These egranules .in vitro play a role in defining the identity of a phase-separated droplet by allowing the retention of specific components and excluding others [in vitro can retain other IDR-containing proteins, like hnRNP A1 and TIA-1, with different avidity, suggesting preferences for some partners over others [in vitro also exhibit RNA sequence selectivity. Here, the specificity of binding depends on the RNA-binding modules. For example, FUS hydrogels selectively retain the RNAs that are typically contained in RNP granules where FUS is found in vivo [The varied functions of RNP granules reflect a diverse composition, raising the question of how selected proteins and RNAs are correctly recruited. The weak, exchangeable, homotypic and heterotypic multivalent interactions between IDRs that drive phase separation g others ,76. One g others . FUS hyd in vivo . These e in vivo . Further in vivo ,31,47,72Ashbya gossypii RBP Whi3 contains a glutamine-rich IDR that drives the formation of Whi3 condensates in vivo and in vitro [in vitro, such as viscosity and their propensity to fuse, depend on the RNA species they contain [in vivo [Drosophila germ granules contain several species of RNA that self-assemble in distinct clusters. Because this homotypic self-assembly is sequence-independent, it has been proposed that it arises as a result of the combination of several properties such as RNA length, modifications and structures, and interacting proteins [RNA also contributes to the identity of RNP granules. RNA possesses structural and chemical features, such as multivalency and flexibility, that make it a key component and driver of these compartments ,78. The in vitro . In thisin vitro ,81. The in vitro . The phy contain . These d[in vivo ,82. A spproteins . These eproteins ,28.7.b).The ability of some RBPs to assemble into an amyloid-like structure allows these proteins to rapidly transition from a monomeric to a stable assembled state. This switch-like transition is an effective strategy for signal propagation and perpetuation ,84. Physfigure 3Aplysia californica contains a glutamine-rich IDR that behaves like a prion-like domain in yeast [Drosophila melanogaster [Drosophila neurons are surrounded by lower-resolution densities that have been interpreted as the RRMs and the ZZ domains projecting out of the axis [A. californica CPEB provide further insight as to how amyloid-like fibrils can serve as axes that coordinate the spatial orientation of RNA-binding modules. The CPEB C-terminus, which contains the RNA-binding modules, maintains flexibility when CPEB assembles into fibrils [The neuronal isoform of cytoplasmic polyadenylation element binding protein CPEB regulates synaptic protein translation . Neuronain yeast ,92. Uponin yeast . This abnogaster –96. CPEBnogaster . CPEB afnogaster . The higthe axis . Nuclear fibrils . These sAnother example of a functional amyloid-like structure comes from the yeast protein Rim4, which functions as a translational repressor. Starvation triggers Rim4 assembly, which results in translational repression of selected transcripts during meiosis –100. Upo8.Many decades of research have established that structured RNA-binding modules are critical determinants of RNA–protein interactions. However, their general low sequence specificity and binding affinity require that RBPs combine several of these modules to finely tune their binding abilities. In this modular context, the disordered intra-protein linkers that connect RNA-binding modules affect and control the binding properties of an RBP. Additionally, IDRs promoting protein–protein interactions can generate new combinations of RNA-binding modules by connecting different RBPs. Thus, the assembled state of an RBP can have different functional capabilities compared to its unassembled state. As opposed to mutations in well-structured modules, mutations within an IDR are more tolerated, resulting in the rapid evolution of these sequences . However


As an indispensable structure protein, the herpes simplex virus 1 (HSV-1) UL6 has been described to exert numerous roles in viral proliferation. However, its exact subcellular localization and subcellular transport mechanism is not well known. In the present study, by utilizing confocal fluorescent microscopy, UL6 was shown to mainly locate in the nucleus in enhanced yellow fluorescent protein or Flag tag fused expression plasmid-transfected cells or HSV-1-infected cells, whereas its predicted nuclear localization signal was nonfunctional. In addition, by exploiting dominant negative mutant and inhibitor of different nuclear import receptors, as well as co-immunoprecipitation and RNA interference assays, UL6 was established to interact with importin α1, importin α7 and transportin-1 to mediate its nuclear translocation under the help of Ran-mediated GTP hydrolysis. Accordingly, these results will advance the knowledge of UL6-mediated biological significances in HSV-1 infection cycle. Herpes simplex virus 1 (HSV-1), a large nuclear duplicating DNA virus, is an epidemic human microbe that can provoke a lytic infection in the mucosal epithelial cells but a life-long latent infection in neurons. As one of the fundamental structure proteins of HSV-1, UL6 has obtained remarkable concern by virtue of its association with numerous viral propagation processes, including establishing the portal for DNA entry into the HSV capsid, cleavage, processing and packaging of replicated viral DNA, assembling of a minor constituent of virions and capsids, and locating on the external surface of the viral capsid –10. HoweAs it is known to all, investigating the precise subcellular localization of a specific protein is a meaningful way to initially discern its detailed roles. UL6 has been previously demonstrated to target to the nuclei in chemical fixed cells , 11, 12.Protein is the executor of life activity, which need to be transported into certain cell compartments for its execution of specific biological function. UL6 was previously demonstrated to localize in the nucleus in chemical fixed cells , 11, 12.Since EYFP is a relatively considerable tag (~27 kDa), it may alter the nuclear localization of UL6. To avoid this hypothesis, plasmid encoding Flag-tagged UL6 (pCMV-Flag-UL6) was constructed and immunofluorescence assay (IFA) was performed to examine the subcellular localization of the UL6. As shown in It is well known that viral protein may show distinct subcellular localization fashions in plasmid transfected and virus infected cells. Therefore, the subcellular localization of UL6 was investigated in HSV-1 infected cells. For this sake, Vero cells were infected with HSV-1 and then IFA was carried out. As a result, UL6 also displayed dominantly nuclear localization when cells were infected at an MOI of 1 at 8 h post-infection .Accordingly, the above data showed that UL6 localized in the nucleus regardless in live cells or chemical fixed cells, as well as in plasmid transfected cells or HSV-1 infected cells. UL6 is shown to exert certain roles that are generally associated with the nucleus, such as constituting the portal for the access of DNA into the HSV capsid, installing of a minor constituent of virions and capsids, and cleavage, disposal and encasement of duplicated viral DNA , 25. ThuNuclear localization signal (NLS), predominantly possessed of basic residues, is vital for the nuclear accumulation of specific protein . BioinfoTo date, Ran GTPase is reported to be indispensable for the nuclear transport process of most nuclear target protein . To probAbout the nuclear translocation, the NLS of cargo is bound by different members of the importin family. In heterodimer importin α/β, importin α binds the NLS of a specific cargo, while importin β is responsible for the conformational alteration of importin α, to reinforce the interaction of importin α-NLS . Then, tCOS-7 cells were transfected with plasmid expressing UL6 deletion mutants 1-296, 297-676, 1–177 and 171–296 fused to the C-terminus of EYFP. 24 h post-transfection, cells were examined by confocal fluorescence microscopy.Expression plasmid of FLAG-UL6 was co-transfected with the plasmid expressing Ran-Q69L-mCherry, RFP-Bimax2, RFP-M9M, DN kα1-mCherry, DN kβ1-mCherry or mCherry into COS-7 cells. 24 h post-transfection, cells were examined for the subcellular localization of UL6 by confocal fluorescence microscopy.co-transfection of Bimax2 and M9M could efficiently diminished the nuclear import of UL6, whereas DN kα1 or DN kβ1 did not obviously lessened the nuclear trafficking of UL6. As negative control, UL6 was not relocalized by mCherry when COS-7 cells were co-transfected with pCMV-Flag-UL6 and mCherry vector . These dTo further verify the assumption mentioned above, plasmids combination of pFLAG-CMV-transportin-1/pEYFP-UL6, Flag-kα2 (importin α1)/pEYFP-UL6, Flag-kα4 (importin α3)/pEYFP-UL6, Flag-kα1/pEYFP-UL6, Flag-kα6 (importin α7)/pEYFP-UL6 or pCMV9-3×Flag-importin β1/pEYFP-UL6 were co-transfected into HEK293T cells for 24 h, then cell lysates were collected and co-immunoprecipitation (Co-IP) was implemented using anti-Flag mAb or mouse IgG. As results, UL6 was efficiently Co-IPed with transportin-1 , importi.To finally validate the nuclear import mechanism of UL6, short hairpin RNA (shRNA) expression plasmids were constructed to knock down the expression of importin α1, importin α7 and transportin-1. Compared to the shRNA control vector (shRandom), shImportin-α1, shImportin-α7 and shTransportin-1 could effectively knock down the expression of importin α1, importin α7 and transportin-1, respectively , suggestAs we known, HSV-1 encodes more than 80 structural proteins, some of which need to be transported into the nucleus for their functions execution, such as promoting viral proliferation, restraining host transcription and expression, inhibiting host innate immunity, etc. The nuclear accumulation of these proteins is mediated by one or more different nuclear import receptors, of course including importin α1, importin α7 and transportin-1. In addition, some host proteins also need to be transported into the nucleus by different nuclear import receptors, to perform their corresponding functions. Therefore, it is bound to affect the nuclear accumulation of many proteins of HSV-1 (and host) when the DN mutants of importin α 1, importin α 7 and transportin-1 are transfected into cells or these nuclear import receptors are knocked down by shRNA expression plasmid. Consequently, it is difficult for us to determine whether the reduction of DNA replication, nucleocapsid assembly and virions production of HSV-1 is the direct outcome of the inhibition of UL6 nuclear translocation.In conclusion, we had proved that UL6 was a genuine nuclear localization protein. Although the predicted NLS of UL6 was nonfunctional, it was identified to be transported into the nucleus through Ran-, transportin-1-, importin α1- and importin α7-dependent nuclear import mechanism, which was largely mediated by the later two nuclear import receptor. These results dissected the molecular determinant for the nuclear transport of UL6, and will shine light for the further study of its biological roles during HSV-1 infection.DNA polymerase KOD-Plus-Neo, restriction enzyme and T4 DNA ligase that involved in molecular cloning were supplied by TOYOBO , New England Biolabs  and Takara , respectively. The UL6 ORF of HSV-1 (F strain) was amplified from plasmid template pYEbac102  and insepRan-Q69L-mCherry, pFLAG-CMV-transportin-1, pDN kα1-mCherry and pDN kβ1-mCherry were described in our previous studies , 22, 24.Plasmid transfection and fluorescence analysis were carried out, as described in our previous studies , 41, 45.Vero cells infected with HSV-1 (MOI=1) for 8 h were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained with the anti-UL6 polyclonal antibody (pAb) . Then, coC. Then, lysis buffer was used to wash beads for three times. Finally, cell lysates and the Co-IPed proteins, were subjected to IB analysis with anti-Flag mAb and anti-YFP pAb (Santa Cruz Biotechnology). All Co-IP were duplicated at least two times, and analogous data were obtained.Co-IP and immunoblotting (IB) assays were manipulated as described previously , 47, 48.


B and nPRB), membrane progesterone receptors  and progesterone receptor membrane components (PGRMC1and PGRMC2). The human cervical cell line (C-4 I) A previous investigation showed that the endometrium normalized in women with endometrial hyperplasia after three months treatment with high dose levonorgestrel IUS (intrauterine system)  Specifications TableValue of the Data•The regulation of receptors that activate signal pathways of progesterone and gestagens are incompletely understood•The present data will be useful for scientists in the basic and clinical research of progesterone biochemistry, physiology and pharmacology•The present data represent a potential tool for scientists to characterize the complex interplay between nuclear progesterone receptors (nPRs), membrane progesterone receptors (mPRs) and progesterone receptor membrane components (PGRMCs) ,10•n vivo and in vitroThe data can lead to development of new experiments to extend the knowledge of the regulation of nuclear as well as membrane progesterone receptors under various physiological conditions and under influence of pharmacological substances i1The present data are a result of the investigation of nPR, mPR and PGRMC gene expression in human endometrium and in a human cancer cell line from the uterine cervix (C-4I) and obtained according to the flowchart in 22.12.2n = 61) was recruited to a prospective, multicenter pilot investigation to assess the efficacy of LNG-IUS 13.5 mg  for treatment of endometrial hyperplasia. Only those women (n = 49) with a completed treatment period of six months were included in the present work. In seven of the 49 women, endometrial biopsy material was insufficient for qPCR analysis. Endometrial biopsies were obtained by an endometrial suction cuvette  or dilatation and curettage or hysteroscopic transcervical resection. All endometrial tissue specimens (baseline and post therapy biopsies) were conserved in RNAlater stabilization solution  and immediately frozen and kept at-18 °C until further analysis. Details of the clinical investigations are reported earlier [Endometrial biopsies with analyzable tissue samples were obtained from 42 women. A group of women .The human cell line C-4I  was derived from a squamous carcinoma of the uterine cervix 4EDTA, , and the trypsin activity was terminated by addition of 800 μL of incubation media. The cell counts were obtained  and the cells were conserved in RNA stabilization solution , stored at 4 °C overnight and then transferred to −20 °C until further analysis.To collect the cultures for RNA extraction, media was removed by aspiration, and the cells were brought into a suspension using 200 μL 0.25% (w/v) trypsin with 0.2% (w/v) Na2.3g for 30 s and cooled on ice for 2 min. Afterwards, the samples were left at room temperature for 5 min. RNA was eluted and stored at −70 °C.The first step was RNA isolation and cDNA synthesis. Total RNA extraction from endometrial tissue samples and C-4I cells was performed using a commercial RNA extraction kit . RNA was isolated according to manufacturer's instruction manual. Tissue samples (10–30 mg) were transferred to MagNA Lyser Green Beads tubes . Lysis buffer (700 µL) from RNA isolation kit, containing 1% (v/v) β-mercaptoethanol , was added to the tubes. The samples were homogenized in a Precellys™ 24 high-throughput homogenizer  at 6000 × RNA quality was evaluated with the Experion™ electrophoresis system  using the Experion™ RNA StdSens Analysis Kit . RNA samples isolated from patient biopsies and from cultured cells were examined for concentration, purity and integrity. The concentrations and the purity of RNA were determined using NanoDrop 2000c spectrometer . The endometrial RNA samples showed A260/280 ratio of 2.1 ± 0.023 (mean ± SD) and a RNA integrity of 8.5 ± 0.9 (mean ± SD). The A260/280 ratio of the C-4I cell RNA samples was 2.1 ± 0.012 (mean ± SD) and with a RNA integrity of 9.9 ± 0.1 (mean ± SD).The concentration of RNA was measured using NanoDrop 2000c spectrophotometer . Reverse transcription was performed using iScript™ cDNA Synthesis Kit . A total of 500 ng RNA was reverse transcribed in a final volume of 20 µL. After the synthesis, cDNA was diluted 10 x with nuclease free water . The cDNA was stored at −20 °C for further use.(https://genorm.cmgg.be) was used to find the most stable gene transcript of reference genes for normalization controls. In the qPCR analyses, several reference genes belonging to different functional classes were evaluated by the geNorm software. For the endometrial biopsies five reference genes  were examined. Six genes  were evaluated as reference genes for C-4I cells. Pairwise variation (Vn/n + 1) geNorm V analysis was carried out to determine the number of reference genes required for normalization. In the case of C-4I cells, the optimal number was two reference genes, Cyc1 and ATP5B.The next step was analysis of reference gene stability, selection of primers and reaction efficiency. The most stable reference genes were chosen for normalization calculations in qBase program. Predesigned reference genes were obtained from Sigma-Aldrich  and Primerdesign Ltd . The geNorm program http://blast.ncbi.nlm.nih.gov/). The design of mPRα and mPRγ primers was performed as described by Dressing and Thomas PRA+PRB, PRB and mPRβ primers were designed using Primer-blast on the NCBI website  and SsoAdvance Universal SYBR Green Supermix  and human SYBRgreen reference gene detection kit. 900 reaction . The assay was run with 2.5 µL of 10 × diluted cDNA in a 10 µL total reaction supermix. The thermal profile for SYBR real-time qPCR included an initial heat-denaturing step at 95 °C for 30 s, 40 cycles at 95 °C for 5 s and 60 °C for 30 s. Following amplification, a melt-curve analyses of the PCR products were performed from 65 °C to 95 °C to determine the specificity of amplification. Each sample was run in duplicate. A non-template control and an inter-run calibrator were added to each run. Data acquisition and subsequent data analyses were performed using the CFX Manager software . Gene expression was analyzed with qBase software as described by Hellemans et al. None.


The human prototypical SR protein SRSF1 is an oncoprotein that contains two RRMs and plays a pivotal role in RNA metabolism. We determined the structure of the RRM1 bound to RNA and found that the domain binds preferentially to a CN motif (N is for any nucleotide). Based on this solution structure, we engineered a protein containing a single glutamate to asparagine mutation (E87N), which gains the ability to bind to uridines and thereby activates SMN exon7 inclusion, a strategy that is used to cure spinal muscular atrophy. Finally, we revealed that the flexible inter-RRM linker of SRSF1 allows RRM1 to bind RNA on both sides of RRM2 binding site. Besides revealing an unexpected bimodal mode of interaction of SRSF1 with RNA, which will be of interest to design new therapeutic strategies, this study brings a new perspective on the mode of action of SRSF1 in cells. SRSF1 is an oncoprotein that plays important roles in RNA metabolism. We reveal the structure of the human SRSF1 RRM1 bound to RNA, and propose a bimodal mode of interaction of the protein with RNA. A single mutation in RRM1 changed SRSF1 specificity for RNA and made it active on SMN2 exon7 splicing. In addition to being an oncoprotein, SRSF1 was shown to be involved in several steps of RNA metabolism, including alternative splicing regulation, mRNA transcription, stability and nuclear export, translation, nonsense-mediated mRNA decay, sumoylation, and miRNA processing4. An important example of SRSF1 involvement in alternative splicing regulation is linked to spinal muscular atrophy (SMA). SMA is characterized by the progressive degeneration of spinal motoneurons, subsequent muscle weakness, and atrophy. The disease is caused by deletion or mutation within survival of motor neuron 1 (SMN1) gene. The SMN2 gene, a nearly identical copy of SMN1 (>99.9% sequence identity), fails to prevent SMA mostly due to a critical C-to-U mutation at position +6 of exon76. This nucleotide change does not alter an amino acid but rather promotes the recruitment of two antagonist splicing regulators. It was proposed that the presence of a cytosine induces the binding of the splicing activator SRSF1 on the SMN1 exon7 ESE1 (Exonic Splicing Enhancer 1), which activates exon7 inclusion7. In SMN2 exon7, the corresponding uridine was reported to prevent the recruitment of SRSF1 and instead initiate the binding of the splicing repressor hnRNP A1, which primarily promotes the skipping of the exon8. As a consequence, the majority of SMN2 transcripts lacks exon7, which leads to a C-terminal truncated SMN protein that is unstable and gets rapidly degraded9. In addition to the recruitment of SRSF1 or hnRNP A1, additional splicing factors were shown to be involved in the splicing regulation of the exon7 and the structure adopted by the pre-mRNA also plays a role in this regulation10. Despite the fact that SMN2 exon7 splicing is altered, the gene still produces some full-length transcripts (∼10%), and hence some SMN proteins9. However, this is not sufficient to compensate for a loss of SMN1 expression. Consequently, increasing the amount of functional SMN protein by restoring a SMN1-like splicing pattern from the SMN2 gene was one of the most promising approaches to treat and possibly cure SMA. Using this strategy, an antisense oligonucleotide (ASO) that prevents the binding of hnRNP A1 to the intronic splicing silencer ISS-N1 located in intron7 was designed and produced as a therapeutic molecule12. This drug (SPINRAZA®) has recently been approved by the Food and Drug Administration as the first treatment against SMA13.SRSF1 is the first identified member of the SR protein family, which is characterized by the presence of a domain enriched in arginine and serine residues14. The cellular localization of this protein as well as its interaction with RNA and other proteins depends on the phosphorylation state of this domain15. A structural model of the N-terminal part of the SRSF1 RS domain (RS1) was determined and showed that the phosphorylation of this peptide induces a shift from a fully disordered state to a partially rigidified arch-like structure16. In addition to the RS domain, SRSF1 contains two RNA recognition motifs (RRM1 and RRM2) that are responsible for its specific interaction with RNA14. Structures of the pseudo-RRM (RRM2) in its free form18 and bound to the SRPK1 kinase19 and to RNA20 were determined. The structure of the protein–RNA complex showed that the domain recognizes a GGA motif using a non-canonical mode of interaction centered on the α1-helix of the RRM20. A more recent study also suggested that the unphosphorylated N-terminal part of the RS domain (RS1) could interact in trans with SRSF1 RRM217 and arginines contained in the inter-RRM linker were shown to be involved in SRSF1 interaction with TAP mRNA export factor18. However, no structural data are available for SRSF1 RRM1 so far. Nevertheless, the N-terminal extremity of this domain was shown to play an important role in pre-mRNA splicing21 and a SELEX experiment indicated that the protein could bind specifically to RNA22. A GGAGA-binding consensus motif was identified from CLIP-seq experiment performed with this protein23 and was consistent with previous investigations31. However, this identified targeted sequence could not explain the specific interaction of SRSF1 with the cytosine found at position +6 of SMN1 exon7.Like all members of the SR protein family, SRSF1 is characterized by the presence of a C-terminal RS domain enriched in arginine and serine residues, which is primarily involved in protein–protein interactionsHere we investigated the mode of RNA recognition of SRSF1 RRM1 and found that it binds preferentially to cytosines and recognizes the CA motif found in SMN2 exon7. By solving the solution structure of this complex, we could engineer a version of SRSF1 RRM1 containing a single glutamate to asparagine substitution (E87N) that binds to uridines. Remarkably, this protein was then able to activate the splicing of SMN2 exon7, in which the cytosine at position +6 is replaced by a uridine. Finally, we also investigated the mode of interaction with RNA of both SRSF1 RRMs linked by their natural linker and showed that RRM1 can bind RNA upstream or downstream of RRM2-binding site due to its flexible inter-RRM linker. This result explains the difficulties of traditional approaches to identify a consensus binding sequence for this protein and reveals almost 30 years after its discovery an unexpected bimodal mode of RNA recognition.20. However, this result could not explain the specific interaction of SRSF1 observed with the cytosine located at position +6 of SMN1 exon77. As a consensus sequence enriched in CG dinucleotides was previously obtained by SELEX with SRSF1 RRM1 (5’-ACGCGCA-3’)22, we investigated whether SRSF1 RRM1 could be responsible for the recognition of this +6 cytosine. Using nuclear magnetic resonance (NMR), we tested RRM1 binding to polyA, polyC, polyG, and polyU sequences  but not with the corresponding sequence in SMN2 exon7 (UUUAGA)  followed by a non-canonical RRM (RRM2) called pseudo-RRM Fig. . The solces Fig. . SurprisB52 Fig.  suggestiGA) Fig. . As the tif Fig. . Neverthtif Fig.  and was We noticed that SRSF1 RRM1 tends to aggregate at the high protein concentration required for structure determination and therefore had to mutate two solvent accessible tyrosine residues located in α-helices of RRM1 (Tyr37 and Tyr72: RRM1 YS) to serine. Importantly, these mutations did not change the mode of interaction of the protein with RNA Fig. . We calc1α1β2β3α2β4 fold that is unchanged compared to the apo form of the protein (PDB number 1X4A). The structure validates the recognition of a CA motif by the RRM using the canonical β-sheet surface. All nucleotides are in the C2’-endo sugar conformation. In good agreement with the chemical-shift perturbations observed in the N-terminal part of RRM1 , which is most likely due to an avidity effect.All the intermolecular interactions found in the solution structure were also stable in MD simulations: the stacking of Cain Fig. . In addiose Fig.  suggestiose Fig. , which i4 and the N-terminal part of the domain. Instead, our simulations indicated that it preferentially formed direct hydrogen bonds with the Arg17 and Asp46 side chains  protein interacted only with polyC  with an RNA containing an invariant GGA motif in the middle flanked by 12 degenerated nucleotides on both sides , which was similarly decreasing when the CA was mutated to UU in both RNAs   Fig. . These d20. In this study, we revealed that SRSF1 RRM1 also contributes to the specific interaction of the protein with RNA by interacting preferentially with CN motifs. In addition, we showed that the gly-rich interdomain linker allowed the binding of RRM1 to RNA either upstream or downstream of RRM2-binding site at two precise positions 31 matches well one of the two sequences identified in this study by SELEX (cytosine at position −4 from the GGA motif). Finally, the motif identified in the Krainer’s laboratory by functional SELEX as the consensus sequence required for SRSF1 activity in splicing (CcccGG/cA) also contained the GGA motif bound by RRM2 and was enriched in cytosines with a preference at position −433 indicating that our structural data are relevant in cells and for the function of SRSF1 in splicing. In addition, a re-analysis of eCLIP data of SRSF1 (obtained from ENCODE) in two human cell lines (HepG2 and K562) shows a decrease in the frequency of GGA motifs with the rank of the peak, which could well be explained by the predominant function of RRM2 in targeting SRSF1 to its RNA partners in cells  with the 20,000 lowest-confidence peaks, we found that cytosine nucleotides generally flanked the RRM2-binding GGA motifs, but their position was not as clear as in the SELEX data ts Table 24,42,43.20. The structure of SRSF1 RRM1 bound to RNA allowed us to design a single mutation (E87N) in the β4 strand of the domain, which enables the interaction of the protein with uridines in addition to cytosines. This rationally engineered SRSF1 protein was then able to substantially increase SMN2 exon7 inclusion . Surprisingly, intron7 was still present in the splicing products after E87N variant overexpression in cells  codon plus cells in minimal M9 medium containing 1 g/l 15NH4Cl and 4 g/l glucose (for 15N labeled protein) or 1 g/l 15NH4Cl and 2 g/l 13C-glucose (for 15N- and 13C-labeled protein). All proteins were purified using two consecutive nickel affinity chromatography (QIAGEN) steps, dialyzed in a 50 mM L-Glu, 50 mM L-Arg, 0.05% β-mercaptoethanol, and 20 mM Na2HPO4 at pH = 7 buffer and concentrated to 0.8 mM with a 10-kDa molecular mass cutoff Centricon device (Vivascience). GB1-SRSF1 RRM1+2 protein YS (aa 1–196 with the Y37S and Y72S mutations in RRM1) was dialyzed against a buffer that allows the solubility of the two RRM-containing protein at a concentration of 0.2 mM . WT and mutant RNA oligonucleotides were purchased from Dharmacon, deprotected according to the manufacturer’s instructions, lyophilized, and resuspended in the NMR buffer. RNA–protein complexes used to solve structures were formed in NMR buffer at an RNA:protein ratio of 1:1, at a 0.5 mM concentration.We cloned in the pET24 expression vector all RRM1 open reading frames (ORFs) corresponding to amino acids 1–97 of SRSF1, 1–94 of B52, 1–72 of SRSF4 and SRSF6, 1–78 of SRSF5, and 1–88 of SRSF9. These recombinant proteins were fused to an N-terminal GB1 solubility tag followed by 6xHis tag and overexpressed at 37 °C in http://www.cgl.ucsf.edu/home/sparky/).All NMR measurements were performed in the NMR buffer at 313 °K using Bruker AVIII-500 MHz, 600 MHz, 700 MHz, and Avance-900 spectrometers equipped with a cryoprobe. Data were processed using Topspin 3.6.2 (Bruker) and analyzed with Sparky 3.133 (1H-15N heteronuclear single quantum coherence (HSQC), 2D 1H-13C HSQC, three-dimensional (3D) HNCA, 3D HNCO, 3D CBCACONH, 3D HcCH total correlated spectroscopy (TOCSY), 3D NOESY 1H-15N HSQC, and 3D NOESY 1H-13C HSQC aliphatic . Aromatic proton assignments were performed using 2D 1H-1H TOCSY and 3D NOESY 1H-13C HSQC aromatic.Protein sequence-specific backbone and side chain assignments were achieved using two-dimensional (2D) 1H-1H TOCSY, 2D 1H-1H NOESY, 2D 13C 1F-filtered 2F-filtered NOESY48, and natural abundance 2D (13C-1H) HSQC in 100% D2O.Resonance assignments of RNA in complex with SRSF1 RRM1 were performed using 2D 1H-1H NOESY, 2D 1F-edited 2F-filtered NOESY, and 3D 13C-resolved 1F-edited 3F-filtered HSQC-NOESY49 using unlabeled RNA and 15N and 15N-13C-labeled proteins, respectively. All NOESY spectra were recorded with a mixing time of 150 ms, the 3D TOCSY spectrum with a mixing time of 23 ms, and the 2D TOCSY with a mixing time of 50 ms.Intermolecular NOEs were obtained using 2D 51 was used to generate preliminary structures and a list of automatically assigned NOE distance constraints for SRSF1 RRM1 in complex with RNA. Peak picking and NOE assignments were performed using 3D NOESY (15N- and 13C-edited) spectra. Additionally, intraprotein hydrogen bond constraints were added based on hydrogen–deuterium exchange experiments on the amide protons. For these hydrogen bonds, the oxygen acceptors were identified based on preliminary structures calculated without hydrogen bond constraints.AtnosCandid 2.1 software51 by adding the manually assigned intramolecular RNA and RNA–protein intermolecular distance restraints. For each CYANA run, 50 independent structures were calculated. These 50 structures were refined with the SANDER module of AMBER 1252 by simulated annealing run in implicit water using the ff99 force field.Seven iterations were performed and 100 independent structures were calculated at each iteration step. Structures of the protein–RNA complexes were calculated with CYANA 3.98.1353. The Ramachandran plot of the SRSF1 RRM1 in complex with RNA indicates that 77.8% of the residues are in the most favored regions, 21% in the additional allowed regions, 0.7% in the generously allowed regions, and 0.5% in the disallowed regions. All the figures showing structures were generated with MOLMOL 2K.254.The best structures based on energy and NOE violations were analyzed with PROCHECK 3.5.42, 6 mM rNTPs, 4 mM GMP, 5× dimethyl sulfoxide in TB buffer  for 3 h at 37 °C. RNA was purified on high-performance liquid chromatography, butanol extracted, and incubated with 100 µl of Ni-NTA agarose beads slurry (QIAGEN) in the absence of protein to eliminate RNA molecules having some affinity for the beads. Twenty nmoles of eluted RNA was incubated for 30 min at 4 °C with His-tagged GB1-SRSF1 RRM1+2 protein at a 1:20 RNA:protein ratio in 300 µl of buffer D  and added to 100 µl of Ni-NTA agarose bead slurry (QIAGEN) for 2 h at 4 °C. Beads were then washed 5 times with buffer D containing 0.05% of NP40 and incubated with 100 µg of proteinase K for 15 min at 37 °C. After phenol–chloroform extraction, selected RNAs were precipitated with 0.3 M sodium acetate in the presence of 1 µl of glycogen and reverse-transcribed using a reverse primer (5’-GCGCTCGAGATGGGCACTATTTATATCAAC-3’) and the reverse transcriptase Superscript III (Invitrogen) for 50 min at 50 °C.A DNA matrix containing a central GGA motif with 12 degenerated nucleotides on each side was produced by DNA pol I elongation (Biolabs) for 1 h at 37 °C using two partially complementary oligonucleotides (5’-CGCGAATTCtaatacgactcactataGCGCCGACCAACGACATT-3’ and 5’-GCGCTCGAGATGGGCACTATTTATATCAACNNNNNNNNNNNNTCCNNNNNNNNNNNNAATGTCGTTGGTCGGCGC-3’) containing a T7 RNA polymerase promoter (lower case). After sodium acetate precipitation, 10 µg of DNA was used as a template for in vitro transcriptions with 20 mM MgCl4, 0.4 mM dNTPs, and 1.25 units of Taq DNA polymerase (Biolabs). The amplified DNA fragments were then used as a new matrix for in vitro transcription and additional cycle of selection. After six cycles of amplification–selection experiments, DNA fragments were tagged and submitted to HT-sequencing.Then 30 cycles of PCR amplification were performed in the presence of two primers (5’-CGCGAATTCTAATACGACTCACTATAGCGCCGACCAACGACATT-3’ and 5’-GCGCTCGAGATGGGCACTATTTATATCAAC-3’) (50 pmol each), 4 mM MgSOITC experiments were performed on a VP-ITC instrument , calibrated according to the manufacturer’s instructions. Protein and RNA samples were dialyzed against the NMR buffer. Concentrations of proteins and RNAs were determined using optical density absorbance at 280 and 260 nm, respectively. Ten µM of each RNA were titrated with 300 µM of GB1-SRSF1 RRM1 YS protein and 10 µM of GB1-SRSF1 RRM12 YS protein was titrated with 100 µM of each RNA by 40 injections of 6 µl every 5 min at 40 °C. Raw data were integrated, normalized for the molar concentration, and analyzed using the Origin 7.0383 software according to a 1:1 RNA:protein ratio binding model.55 to generate position-specific weight matrices representing these sequences. Furthermore, to determine the positions with the most stringent selection during SELEX, we calculated the relative entropy of position-dependent nucleotide frequency distributions of foreground and background sequences selected as described above. That is, if Fi(α) is the nucleotide frequency distribution at position i in the foreground and Bi(α) the corresponding distribution in background sequences, with α ϵ , the relative entropy at position i was calculated as ΣαF(α) ∗ log(F(α)/B(α)).We sequenced a total of 14,500,301 unique sequences and 28,192,850 unique sequences obtained after 6 rounds of selection. In the input set, there were 7044 sequences that occurred in >1 copy, whereas in the SELEX-selected set, 1,605,630 sequences had >1 copy. To uncover the positions which experienced the most selection, the nucleotide composition of the 212 sequences with >100 copies in the SELEX-selected set (referred to as “foreground” sequences) relative to that of sequences with at most 10 copies in the input data set (referred to as “background”) were analyzed. Upon inspection, we found that the foreground sequences followed two main patterns: 94 of the 212 unique sequences had GGANGGA at positions 29–35, while 98 sequences had only a GGA at positions 33–35 (occurrences of the GGA motif at other positions in these sequences were not excluded but did not follow a position-specific pattern). We used the weblogo software56 were obtained from ENCODE57, in the form of BED files . There are two replicates per cell type. For each of these replicates, the peaks are sorted according to their p value. The center of each peak is used and extended by 25 nucleotides on each side, and then peaks that overlap by one or more nucleotides are merged. The p value of the merged peak is the most significant of the p values of the merged peaks. Non-overlapping sets of 1000 peaks, starting from the most to the least significant, are then used to calculate the average frequency of GGA motifs per position per peak, first calculating the number of GGAs detected in each peak divided by the peak length and then averaging over 1000 peaks at a time , the best 10,000 peaks according to their PWM Fig. . A simil3 to U3 and A4 to G4 nucleotide replacements, respectively, were prepared by molecular modeling. AMBER16 was used to prepare the initial files and to execute the simulations. We have used bsc0χOL358 and ff12SB59 force fields to describe the RNA and protein, respectively. The systems were simulated in octahedral box of explicit SPC/E60 water molecules with minimal distance of 12 Å between solute and the box border and KCl ions61 were used to neutralize the system, achieving an excess salt concentration of 0.15 M. The NMR restraints were utilized in initial part of the simulations to improve stability of the subsequent unbiased simulations62. Details of the simulation equilibration and production protocol were discussed previously63. We have performed four simulations of the WT RRM1 bound to AACAAA RNA, which corresponds to the solution structure presented in this work. We have further performed four simulations of the WT RRM1/AACGAA and two and three simulations of the E87N RRM1/AACAAA and E87N RRM1/AAUAAA systems, respectively. The length of the simulations was 1 µs. One WT RRM1/CA and one WT RRM1/CG simulation were extended to 10-µs timescale to more thoroughly examine the N-terminus/RNA interactions. VMD 1.9.2 was used to visualize MD trajectories.We have used the first frame of the structural ensemble of the SRSF1 RRM1 protein/RNA complex (this work) to start all MD simulations. The structures containing the E87N mutation and the CHuman embryonic kidney HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (GibcoBRL) supplemented with 10% fetal calf serum (GibcoBRL). The mammalian vectors expressing FLAG-tagged SRSF1 WT, Y19A, and E87N were obtained by cloning the SRSF1 ORF, amplified by PCR, in frame into the pCAG vector (Addgene). SRSF1 Y19A and E87N mutants were created by site-directed mutagenesis using specific primers.64 (WT or mutants) plasmid was cotransfected with 3 µg of pcFLAG-SRSF1 (WT or mutants) using the calcium phosphate method. Equal amounts of DNA were transfected by addition of empty vector when necessary. Forty-eight hours after transfection, total RNA was isolated. One microgram of total RNA was reversed transcribed using oligo(dT) and Superscript II (Invitrogen) or M-MuLV Reverse Transcriptase RNAseH− (Finnzyme). One-tenth of the cDNA was amplified by PCR using a vector-specific forward primer (pCI-forw: 5’-GGTGTCCACTCCCAGTTCAA) and the SMN-specific reverse primer SMNex8-rev (SMNex8-rev: 5’-GCCTCACCACCGTGCTGG). The SMNex8-rev primer was 32P 5’-end labeled and PCR reactions were terminated during the linear phase . After electrophoresis on a 4% polyacrylamide gel, the ratio of exon inclusion (SMN2-FL) to exon skipping (SMN2∆7) was determined by using AlphaView . Means and standard error of the mean were calculated from the three independent experiments.HEK293 cells were plated on six-well plates. For each transfection, 1 µg of pCI-SMN2Protein samples were separated on a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then electroblotted onto nitrocellulose membranes (Whatman Optitran BA-S 83), blocked with 5% non-fat dry milk in Tris-buffered saline + 0.1% Tween (TBS-T). Probing of the blots was done with monoclonal ANTI-FLAG M2 Antibody (mouse) from Sigma (F1804) 1 µl diluted 1000× in 5 ml of TBS-T buffer and 5% non-fat dry milk, followed by incubation with 1 µl horseradish peroxidase-conjugated anti-mouse (Sigma) for 2 h in 10 ml  of TBS-T and 5% non-fat dry milk. Protein signals were detected with chemiluminescence imaging (Amersham Imager 600RGB).Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


To identify functional cis-acting elements systematically across the HEV genome, we utilized an ORF1 transcomplementation system. Ultimately, we found two highly conserved cis-acting RNA elements within the ORF1 and ORF2 coding regions that are required for viral genome replication in a diverse panel of HEV genotypes. Synonymous mutations in the cis-acting RNA elements, not altering the ORF1 and ORF2 protein sequences, significantly impaired production of infectious viral particles. Mechanistic studies revealed that the cis-acting elements form secondary structures needed to interact with the HEV ORF1 protein to promote HEV replication. Thus, these cis-acting elements function as a scaffold, providing a specific “signal” that recruits viral and host factors to assemble the viral replication complex. Altogether, this work not only facilitates our understanding of the HEV life cycle and provides novel, RNA-directed targets for potential HEV treatments, but also sheds light on the development of HEV as a therapeutic delivery vector.There are approximately 20 million events of hepatitis E virus (HEV) infection worldwide annually. The genome of HEV is a single-strand, positive-sense RNA containing 5’ and 3’ untranslated regions and three open reading frames (ORF). HEV genome has 5’ cap and 3’ poly(A) tail to mimic host mRNA to escape the host innate immune surveillance and utilize host translational machineries for viral protein translation. The replication mechanism of HEV is poorly understood, especially how the viral polymerase distinguishes viral RNA from host mRNA to synthesize new viral genomes. We hypothesize that the HEV genome contains  Hepatitis E virus (HEV) is an underestimated pathogen, causing approximately 20 million infections worldwide annually and leading to about 60,000 deaths. There are no direct-acting antivirals for treating HEV, and although significant progress has been made to establish robust HEV cell culture models, the life cycle remains poorly characterized. A better understanding of HEV replication could facilitate the development of new drugs targeting this critical process. Our study found that RNA elements in the HEV genome interact with the HEV replicases to promote viral replication, suggesting that these RNA elements function as a scaffold for recruitment and assembly of the viral replication complex. This work furthers our understanding of HEV replication and could inform the generation of RNA-based therapeutics for treating HEV infection. Hepatitis E virus (HEV) remains a considerable health problem in both developing and developed countries, causing an estimated 60,000 deaths and 3,000 stillbirths a year , 2. In hHepeviridae family and the Orthohepevirus genus, which is subdivided into four species . The HEV strains infecting humans all belong to the Orthohepevirus A species and include genotypes 1, 2, 3, 4, and 7 [HEV is a positive-sense, single-stranded RNA virus belonging to the 4, and 7 . The HEV4, and 7 , 12, Y d4, and 7 , putativ4, and 7 –16, hype4, and 7 , 18, pol4, and 7 , X domai4, and 7 , 21, RNA4, and 7 –24, and 4, and 7 . It rema4, and 7 , 27. Rec4, and 7 , suggest4, and 7 . ORF3 is4, and 7 , 31.cis-acting RNA elements) for this complex process to be successfully completed. These RNA structures can be involved in interactions with viral and cellular proteins during viral genome translation [cis-acting RNA elements that can also extend into adjacent coding sequences [After entering hepatocytes, HEV can translate ORF1 directly from its RNA genome , 33. Furnslation , replicanslation . It has equences , 41.cis-acting RNA elements are critical for the life cycles of multiple RNA viruses [RNA structures or  viruses , 43 and  viruses –47. The  viruses , 49.in trans, providing the polymerase activity needed to successfully replicate transfected HEV RNA. This ORF1 transcomplementation system thus creates a unique opportunity to uncouple viral RNA function from protein translation. Using this tool, the promoter regulating the HEV subgenomic RNA, from which OR2 and ORF3 are translated, was identified [An HEV ORF1 transcomplementation cell culture system, where HepG2C3A hepatoma cells are lentivirally transduced with HEV ORF1, was recently established as an efficient tool for understanding HEV replication and transcription . In thisentified .cis-acting RNA elements in the ORF1 and ORF2 coding region of the KernowC1/p6 strain . These two cis-acting elements are highly conserved across HEV genotypes and required for genome replication of multiple HEV genotypes. Furthermore, mechanistic studies found that these cis-acting elements form secondary RNA structures that recruit ORF1 and subsequent assembly of the viral replication complex.In this study, we performed an unbiased screen across the entire HEV genome using this ORF1 transcomplementation system and identified two cis-acting elements in the HEV genome and highlight the interplay of RNA structure with viral replicases to dictate viral genome replication. These findings shed more light on our understanding of viral replication and could inform the generation of RNA-based therapeutics for treating HEV infection.Collectively, we describe two novel, essential cis-acting RNA elements in the viral genome required for HEV replication. We previously established an HEV ORF1-based transcomplementation cell culture model and demonstrated that ORF1 could function in trans to replicate the viral genome [cis-acting RNA elements required for HEV replication across the whole HEV genome, excluding the 5’ and 3’UTRs  [cis-acting RNA elements required for HEV replication were likely contained in these regions.First, we generated a series of truncated Kernow C1/p6-Gluc GAD (rHEV-Gluc GAD) replicon mutants harboring several hundred nucleotide (nt) deletions between positions 27nt to 4766nt and positions 5741nt to 7340nt . These m3A-ORF1) , and the3A-ORF1)  mutant. vs #19; #16 vs #25). To more accurately pinpoint the functional cis-acting RNA elements, we generated series of 10nt deletions between 92nt-141nt or 7291nt-7340nt. Deletion of 102nt-111nt, 112nt-121nt or 122nt-131nt dramatically impaired HEV replication to a degree comparable with the deletion of 92nt-141nt . Deletion of 7311nt-7320nt, 7321nt-7330nt or 7331nt-7340nt significantly decreased HEV replication more than 90%, comparable to deletion of 7291nt-7340nt . Intriguingly, we also noticed that some deletions, 92nt-101nt or 132nt-141nt, enhanced HEV replication by 3-fold (vs #27 or #31), suggesting the presence of RNA elements that negatively regulate virus replication. Collectively, these data suggest that viral cis-acting RNA elements can function as positive or negative regulators of viral genome replication. Of note, nucleotides 102–131 (within the methyltransferase domain of ORF1) and 7311–7340 (ORF2 coding region) constitute cis-acting RNA elements required for HEV replication.To accurately localize the relevant sequences, we created additional mutant rHEV-Gluc GAD replicon genomes lacking 50-nt segments between positions 27nt to 241nt or 7141nt to 7340nt . As befocis-acting RNA elements during HEV replication with an HEV subgenomic replicon. We introduced synonymous mutations every 3 or 4 amino acids in the 102nt-131nt region of the ORF1 sequence of Kernow C1/p6-Gluc (rHEV-Gluc) (in vitro-transcribed WT or synonymous mutant (SMs) viral replicon RNA were transfected into HepG2C3A cells, and Gluc activity was monitored to measure as a proxy for replication. Gluc activity was dramatically reduced in the supernatants of cells transfected with the rHEV-Gluc SMs compared to the parental sequence. Notably, #38 and #39 SM mutants harboring 3 and 5 mutations, respectively, were severely impaired, with a 95% reduction in Gluc activity compared to the WT replicon (vs.#40-#42), suggesting that G113 is critical for HEV replication. Similarly, we took the same approach in the region 7309–7340 nt of ORF2 and identified G7335 as the particularly important nucleotide for HEV replication (cis-acting RNA elements (102nt-131nt and 7311nt-7340nt) in the HEV genome  . The in replicon . To detereplicon , #40-#51lication , #60. CoV genome , with nucis-acting RNA elements in other genetically diverse HEV genotypes. First, we assessed the conservation of these regions in the other genotypes of the Orthohepevirus A species. Sequence alignments of the cis-acting elements and their proximal regions  showed that, compared to the proximal regions, the 111nt-121nt and 7330nt-7336nt are highly conserved (100%) across genotypes 1 to 8 , we introduced synonymous mutations in the ORF1 (G113C or G113T) or ORF2 (G7335A) coding sequences of SAR55-Gluc (GT1) [in vitro transcribed WT, GAD or SM replicon RNA for each of these replicons was transfected into HepG2C3A cells, and Gluc activity was measured. Consistent with previous data, Gluc activity was reduced in the supernatants of cells transfected with the SMs G113C, G113T and G7335A) at levels comparable to those of the GAD mutant  , pSHEV3-uc (GT1) , and TW6uc (GT1)  repliconD mutant . These rcis-acting elements in the context of a full-length infectious viral genome, we introduced the same SMs in the Kernow C1/p6 genome while retaining the ORF1 and ORF2 protein sequence  assay [cis-acting elements, such as 5’UTR, 3’UTR, subgenomic RNA promoter, etc. also contribute to the binding with ORF1 in the G113C or G113T mutant genome, but are not sufficient for viral replication. Notably, G113C or G113T mutations in the negative-strand HEV RNA genome had little effect on ORF1 binding compared with GAD (We hypothesized that the P) assay , 60. We with GAD . These dwith GAD .cis-acting elements formed RNA structures required for interaction with ORF1. However, we could not validate functional structures in the 92-141nt regions  replicon, which harbors the S17 insertion in the ORF1 [cis-acting elements in ORF2 [Previous studies showed that the 5’UTR and 3’UTR contain lication , 41. In [in vivo . Another[in vivo . Howeverthe ORF1 , 63, as  in ORF2  had a mocis-acting elements we identified are highly conserved across all eight HEV genotypes and were required for replication of genetically diverse HEV genomes . To ascertain that these sequence elements are functionally relevant for virus infectivity, we mutated these sequences in the context of full-length HEV genomes. Indeed, mutations within the cis-acting elements abrogated the production of infectious HEV virions. Since the mutations we introduced were synonymous, the reductions in viral replication can be directly attributed to the disruption of cis-acting element, not to any changes in ORF1 expression as demonstrated by western blot. Future structural studies will be crucial to determine how these synonymous mutations affect RNA structure, how ORF1 protein gains specificity for target sequences and a detailed picture of the entire HEV genome’s architecture and secondary structure.These cis-acting RNA elements are specific sequences the viral replicase and related host factors can recognize to initiate the replication complex [cis-acting RNA elements in the HEV genome are difficult to study since the RNA also serves as the template for translation of gene products, including those required for virus replication. As a result, we believed that our ORF1 based transcomplementation cell culture system was especially well-suited for overcoming this problem. This system was previously utilized to identify the intragenomic promoter directing HEV subgenomic RNA synthesis [cis-acting elements across the entire viral genome required for HEV replication. Our studies provide an experimental framework to identify the functional cis-acting RNA elements regulating viral replication more comprehensively and in greater detail than previously possible. Additionally, ORF1 has also been reported as the genetic determinant of HEV host tropism [Virus replication is achieved by the viral replication complex, which involves viral RNA/RNA interactions, RNA and viral factor interactions, as well as interaction with host factors . The cis complex , 66. Theynthesis , and we  tropism , and ORFcis-acting elements that reside within the region. Thus, a reliable genomic structural model is necessary to better interpret data and avoid erroneous conclusions. In addition to using reverse genetics to understand viral biology, we can also use these approach to manipulate viral genomes to serve as delivery vectors for foreign genes [cis-acting RNA elements could better inform the design of more efficient viral vectors.To dissect the function of a given viral gene, it is common to introduce mutation(s) into the gene in the infectious clone . Howevergn genes . Howevercis-acting elements in the HEV RNA genome that recruit viral factors to assemble the replication complex for viral genome replication and thus represent a novel RNA-directed drug target. Meanwhile, these cis-acting elements serve as the specific signals by which the viral replication complex distinguishes the small fraction of viral RNA from the host RNA to ensure the high-efficiency of replication. Our study not only sheds more light on the mechanisms regulating HEV replication and identifies novel RNA-directed drug targets to combat HEV infection but is also insightful for further development of HEV as a therapeutic delivery vector.In summary, we identified novel functional 2 incubator at 37°C.HEK293T cells , HepG2C3A cells  and S10-3 cells were maintained in Dulbecco’s modified Eagle medium (DMEM)  supplemented with 10% (vol/vol) fetal bovine serum (FBS), and 50 IU/ml penicillin/streptomycin in a humidified 5% (vol/vol) COcis-acting RNA elements by ClonExpress MultiS One Step Cloning Kit .The pKernow-C1 p6/Gluc was used as the template to generate the truncation or synonymous mutants for the mapping the functional Vesicular stomatitis virus G protein (VSV-G) pseudotyped lentiviruses were produced by transient cotransfection of the third-generation packaging plasmids pMD2G  and psPAX2  and the transfer vector with VigoFect DNA transfection reagent (Vigorous) into HEK293T cells. The medium was changed 12 h post transfection. Supernatants were collected at 36, 60 and 84 h after transfection, pooled, passed through a 0.45-μm filter, aliquoted, and frozen at -80°C.in vitro from linearized plasmid using HiScribe T7 antireverse cap analog (ARCA) mRNA kit  according to the manufacturer’s instructions. Viral RNA was transfected into HepG2C3A cells or S10-3 cells using TransIT-mRNA transfection reagent  according to the instructions.HEV Kernow-C1/p6, HEV Kernow-C1/p6-Gluc, and the truncated mutant plasmids were linearized by MluI. pSAR55-Gluc was linearized by BglII, pGEM-9Zf-pSHEV3-Gluc was linearized by XbaI, and pGEM-7Zf(-)-TW6196E and pGEM-7Zf(-)-TW6196E/Gluc were linearized by SpeI. Viral RNAs were transcribed Gaussia luciferase activity was measured using Renilla Luciferase Assay System . Specifically, 10 μl cell culture medium was added per well of a 96-well polystyrene microplate , followed by the addition of Renilla luciferase assay substrate and the detection of luminescence was performed using Micro plate spectrophotometer (EnVision).2O and put on ice for 30 min, further lysed by freeze-thawing 3 times. Cellular debris was removed by centrifugation at 13,000r/m for 15 min, and the supernatant was added with 1/10 volume of 10×phosphate-buffered saline (PBS). For virus infection, HepG2C3A cells were seeded into 24-well plate one day before infection at 30% confluence. Cells were infected with virus in 4% PEG8000 culture medium. The virus mixture was removed one day later, and cell culture medium containing 2% DMSO was added, followed by incubation at 34.5°C for 5 days.S10-3 cells were seeded into 6-well plate one day before transfection at 50% confluence. Cells are transfected with Kernow C1/p6 WT or mutant RNAs and incubated at 34.5°C for 7 days. For virus collection, cells were trypsinized and pelleted in a 1.5-ml tube by centrifugation, liquid was removed. Cells were lysed by adding 0.6 ml sterilized HBalb/C mice were obtained from Dashuo Biotech , and the animal protocol was reviewed and approved by the Animal Welfare Committee of Northwest A&F University. All mice were monitored at a daily basis for any clinical sign. All effort was made to minimize the suffering of mice, and mice were euthanized if a humane endpoint was reached according our protocol. Mice were immunized with 100 μg of recombinant HEV-ORF2 179 protein or MBP-fused HEV-ORF1-X-domain (Sar55 strain) with an equal volume of adjuvant for a total of 3 times with an interval of two weeks. The Freund’s Complete Adjuvant (Sigma-Aldrich) was used for the primary immunization, while Freund’s Incomplete Adjuvant (Sigma-Aldrich) was used for the rest. Mice serum was collected on a weekly basis to monitor the serum antibody level by enzyme-linked immunoprecipitation assay (ELISA). Serum collected before immunization was included as the negative control. After the final immunization, 100 μg of recombinant protein without adjuvant was used to boost the mice 4 days before the spleen collection. The mice with the highest antibody titers were used for cell fusion. Cell fusion of spleen cells with S/p20 cell was conducted as previous described [The 6-week old escribed . The selCells in plate were fixed with 4% paraformaldehyde at RT for 20 min. The cells were permeabilized in 0.5% triton X-100 for 20 minutes and blocked in 1% BSA for 60 minutes at RT. Cells were stained by ORF2 antibody (2G8) in 1% BSA and incubated at RT for 1 hour, then cells are washed with PBS three times. Goat anti-mouse IgG (H+L) secondary antibody Alexa Fluor 488 conjugate (1:1000 Invitrogen Cat# A11029) diluted in 1% BSA is used to stain cells at RT for 1 hour, then cells are washed with PBS three times. Nucleus is stained by DAPI for 5 minutes at RT, then cells are washed with PBS three times. The stained cells are observed under microscope.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting was performed as follows: After trypsinization and cell pelleting at 2,000 × g for 10 min, whole-cell lysates were harvested in RIPA lysis buffer  supplemented with protease inhibitor cocktail (Sigma). Lysates were electrophoresed in 12% polyacrylamide gels and transferred onto nitrocellulose membrane. The blots were blocked at room temperature for 0.5 h using 5% nonfat milk in 1× phosphate-buffered saline (PBS) containing 0.1% (v/v) Tween 20. The blots were exposed to primary antibodies anti-ORF1, β-Tubulin , Flag  in 5% nonfat milk in 1×PBS containing 0.1% Tween 20 for 2 h. The blots were then washed in 1×PBS containing 0.1% Tween 20. After 1h exposure to HRP-conjugated secondary antibodies and subsequent washes were performed as described for the primary antibodies. Membranes were visualized using the Luminescent image analyzer (GE).7 293T cells were transfected with 10 μg pLVX-ORF1(GAD)-Flag-zsGreen plasmids using VigoFect (Vigorous) for 72 hours and 6μg HEV replicon RNAs by TransIT-mRNA Transfection Kit (Mirus) for 24 hours. Cells were lysed in ice-cold RIP lysis buffer  for 30min with constant rotation. Add RQ1 DNase into the lysates to digest DNA, and incubate at 37°C for 10min. The lysates were cleared by centrifugation at 13000r/m and 4°C for 15 minutes. 7.5% of the cell lysate was saved as input for RT-qPCR. Cell lysates were aliquoted into two parts. One part was added with 4μg Flag antibody and the other with 4μg IgG, with incubation at 4°C overnight. 25μl equilibrated protein G beads were added for another 4 hours at 4°C. After 4×5 minutes washes by RIP200 buffer , RNA was eluted in Proteinase K digestion buffer  at 55°C for 1 hours. The RNA was extracted by TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. Reverse transcription was performed using ReverTra Ace qPCR RT Kit (TOYOBO) with random primers. Quantitative real-time PCR was performed using 2×RealStar Green Power Mixture (Genstar) according to the instruction. Primers THU-0158 (5’-TTGCCTCCGAGTTAGTCATC-3’) and THU-0159 (5’-TGCAAAGCATTACCAGACCG-3’) were used for HEV viral genome; Primers THU-0003 (5’-GAAGGTGAAGGTCGGAGTC-3’) and THU-0004 (5’-GAAGATGGTGATGGGATTTC-3’) were used for GAPDH gene amplification for qPCR.About 1×10For HEV negative strand RT-qPCR, we followed the methods published previously . The priin vitro by HiScribe T7 ARCA mRNA Kit  according to the manufacturer’s protocol. Biotin-16-UTPs (Roche) were added as 10% of UTP in the reactions. About 5 μg of biotinylated RNAs were heated for 5 min at 65°C, then cooled down to room temperature in RNA structure buffer . About 2×107 293T cells overexpressing ORF1-Flag were used for each RNA pulldown experiment. Cells were trypsin digested and re-suspended in 1 ml lysis buffer  followed by 30 min incubation on rotator. Cell lysates were centrifuged at 13,000 rpm at 4°C for 15 min, and the supernatant was transferred to a new tube. Cell lysates were pre-cleared by 30 μl M280 beads (Life technology) and 20 μg yeast RNA for 1 hour at 4°C, then incubated with 5 μg biotinylated RNA at 4°C overnight, followed by addition of 50 μl equilibrated M280 beads for additional 3 hours at 4°C. After 4×5 min washes by RIP200 buffer , proteins bound to RNA were eluted in 2% SDS sample buffer by heating at 95°C for 10 min, and then analyzed by western blot using ORF1 antibody.RNA pulldown assay was performed as described from previous study with some modifications accordingly . Brieflyt test or one-way analysis of variance (ANOVA) with Tukey’s honestly significant difference (HSD) test was used to test for statistical significance of the differences between the different group parameters. P values of less than 0.05 were considered statistically significant.Student’s S1 Fig(A-B) Replication of HEV RNA mutants in ORF1 transcomplemented HepG2C3A cells. A series of 500nt-, 200nt- or 300nt-truncated HEV RNA replicons harboring secretory Gaussia luciferase (Gluc) reporter were transfected into HepG2C3A-ORF1 cells. Cell culture supernatants from each group were collected and Gluc activity measured two days after transfection. The data are presented as the percentage of Gluc activity relative to that of the full-length rHEV-Gluc GAD. Values are means plus standard deviations (SD) (error bars) (n = 3). *, P < 0.05; **, P < 0.01; n.s., not significantly different by one-way ANOVA.(TIF)Click here for additional data file.S2 Fig(A-B) Replication of series of HEV RNA mutants in ORF1 transcomplemented HepG2C3A cells. Series of 50nt-truncated HEV RNA replicons harboring a secretory Gaussia luciferase (Gluc) reporter were transfected into HepG2C3A-ORF1 cells. Cell culture supernatants from each group were collected and Gluc activity measured two days after transfection. The data are presented as the percentage of Gluc activity relative to that of the full-length rHEV-Gluc GAD. Values are means plus standard deviations (SD) (error bars) (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significantly different by one-way ANOVA.(TIF)Click here for additional data file.S3 Figcis-acting RNA elements in ORF1 (A) and ORF2 (B) of Orthohepevirus A were not conserved in other Orthohepevirus species (Orthohepevirus B-D) or the Piscihepevirus genus. Alignment was performed by MEGA6 software. The conserved sequence is shaded in yellow. G113 and G7335 are highlighted by the arrow.(A-B) Sequences of the (TIF)Click here for additional data file.S4 FigWT, synonymous mutant  or GAD mutant replicon RNA of SAR55 (gt1), pSHEV3 (gt3) or TW6196E (gt4) were transfected into HepG2C3A cells. Cell culture medium was collected two days after transfection, and Gaussia luciferase activity was quantified. The numbering denotes the positions of the Kernow C1/p6 viral genome. Values are means plus SD (n = 4). Values are means plus SD (n = 4). ***, P < 0.001 by one-way ANOVA.(TIF)Click here for additional data file.S5 Figright panel. Values are means plus SD (n = 3). ***, P < 0.001, by one-way ANOVA.(A) Protein expression of ORF1 synonymous mutants. The WT and synonymous mutants of ORF1 cDNA were cloned into pLVX-IRES-zsGreen vector under the control of a CMV promoter and then transduced into HepG2C3A cells. Immunoblotting assay was performed to determine the expression level of ORF1. (B) A schematic representation of the HEV RNA pull-down procedure to examine the interaction of HEV RNA genome with ORF1 protein. (C) The ORF1 protein associated with HEV WT or mutant genomes was analyzed by immunoblotting assay, and the ORF1 abundance was quantified using ImageJ software. This assay was repeated three times, and the data was pooled as presented in (TIF)Click here for additional data file.S6 Fig(A) The 293T cells overexpressing ORF1 (GAD)-Flag were transfected with GAD or synonymous mutants of the negative-strand viral RNA genome (Kernow C1/p6). The cells were lysed and then incubated with Flag antibody to perform the immuno-precipitation assay, with lgG as the negative control. The immune-precipitated complex was subjected to RNA purification, and the ORF1-associated RNA were detected by RT-qPCR analysis. Enrichment of RNA binding to ORF1 is shown as fold change normalized to input. Immunoblotting analysis was performed to confirm the efficacy of ORF1 (GAD)-Flag immunoprecipitation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) Schematic illustration of the positions of the primers in the negative-strand RNA-specific RT-qPCR assay. For the RT primer (THU-2009), a specific tag sequence was added at the 5′ end for RT to generate cDNA derived from the negative viral RNA genome. The forward (Fwd.) primer (THU-0298) and reverse (Rev.) primer (THU-1240) were used in this study for the qPCR assay. (C) HepG2C3A cells were transfected with the indicated rHEV-Gluc RNA replicon. After 2 days, cells were washed, and intracellular total RNA was extracted and subjected to HEV negative-strand-specific RT-qPCR assay to measure the abundance of antigenome. The data are presented as the percentage of viral negative strand RNA relative to that of the WT. Values are means plus SD (n = 3). *, P < 0.05; **, P < 0.01; n.s., not significantly different by one-way ANOVA.(TIF)Click here for additional data file.S7 Figcis-acting RNA elements in ORF1 region 97nt-131nt. RNAalifold [http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAalifold.cgi) was utilized to predict the secondary structures for 97nt-131nt and suggested that G113 resides in a loop region. The cis-acting RNA element 102nt-131nt are highlighted in blue. (B) G113C and T116G, G113T and T116G double mutations did not rescue HEV replication. (C) RNA structure prediction of the secondary structures of cis-acting RNA elements 6nt-121nt. RNAstructure [http://rna.urmc.rochester.edu) was utilized to predict the secondary structures, indicating that G113 potentially base pairs with U23 in the 5’UTR. The cis-acting RNA element is shaded in blue. (D) G113C and T23G, G113T and T23G double mutations did not rescue HEV replication. Values are means plus SD (n = 3). n.s., not significantly different by Student’s t test.(A) RNA structure prediction of the secondary structures of Aalifold  (http://tructure  (http://(TIF)Click here for additional data file.