Sulfolobus solfataricus type III-D CRISPR complex generates cyclic tetra-adenylate (cA4), activating the ribonuclease Csx1, and showed that subsequent RNA cleavage and dissociation acts as an ‘off-switch’ for the cyclase activity. Subsequently, we identified the cellular ring nuclease Crn1, which slowly degrades cA4 to reset the system , and demonstrated that viruses can subvert type III CRISPR immunity by means of a potent anti-CRISPR ring nuclease variant AcrIII-1. Here, we present a comprehensive analysis of the dynamic interplay between these enzymes, governing cyclic nucleotide levels and infection outcomes in virus-host conflict.Cyclic nucleotide second messengers are increasingly implicated in prokaryotic anti-viral defence systems. Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) upon detecting foreign RNA, activating ancillary nucleases that can be toxic to cells, necessitating mechanisms to remove cOA in systems that operate via immunity rather than abortive infection. Previously, we demonstrated that the  Type IIportions . cOA secportions . To date systems . The rib systems , particu systems .Sulfolobus solfataricus synthesises predominantly cA4, which activates the CRISPR ancillary ribonuclease Csx1. We examined the first regulatory step in cOA synthesis in detail and demonstrated that target RNA cleavage and dissociation from the complex shut-off cOA synthesis (S. solfataricus encodes CRISPR-associated ring nuclease 1 (Crn1) family enzymes  of cA4 synthesis by the S. solfataricus type III effector as 0.04 ± 0.01 min−1 at 70°C . Using 4 second messenger binds to CARF family proteins to elicit an immune response. To understand the concentration of cA4 required to activate an antiviral response, we determined the dissociation constant of the major ancillary ribonuclease Csx1 for the cA4 activator. Using radioactive cA4, we titrated an increasing concentration of Csx1 protein and subjected the mixture to native gel electrophoresis (4 was bound by Csx1 with a dissociation constant (KD) of 130 ± 20 nM. Thus, even one viral target RNA detected by the type III CRISPR system should generate enough cA4 (6 µM) to fully activate the Csx1 ribonuclease for defence. We proceeded to estimate the binding affinity of a ribonuclease-deficient Csx1 variant for its RNA target, yielding an apparent dissociation constant of approximately 5 µM (kcat) for cA4-activated RNA cleavage by Csx1 as 0.44 ± 0.03 min−1 at 70°C , allowing the generation of the equivalent concentration of cA4 with the measured rate constant of 0.04 min−1 . As a result, Csx1 activity and therefore RNA degradation were strongly supressed  and simulated RNA cleavage  levels.Biochemical comparison of Crn1 and AcrIII-1 revealed that both enzymes bind cA4 by AcrIII-1 over a wide concentration range greatly limits RNA cleavage by deactivating defence enzymes. Therefore, the deployment of AcrIII-1 upon viral infection may not only promote viral propagation but also safeguard cellular integrity until viral release by lysis. Recent studies have uncovered that sequentially infecting phage evade CRISPR defences by exploiting the immunosuppression achieved by Acr enzymes from failed infections , the AcrIII-1 gene B116 is expressed early in the viral life cycle  and cyclic penta-adenylate (cA5) molecules. Whereas no signalling role has yet been ascribed to cA5, cA3 has been demonstrated to activate a family of DNases termed NucC which abort infection by degrading the host genome prior to completion of the phage replication cycle (Cyclic nucleotide-based defence systems are emerging as powerful cellular sentinels against parasitic elements in prokaryotes. Mirroring the role of cyclic GMP-AMP synthase (cGAS) in eukaryotic defence against viruses as part of the cGAS-STING pathway, bacterial cGAS enzymes have recently been discovered that abort infection by activating phospholipases through cGAMP signaling . Termed on cycle .4, eukaryotic poxviruses utilise Poxins to subvert host immunity by destroying cGAMP  made per RNA target  was investigated in a 20 μl reaction volume incubating A26 RNA target or A26 phosphorothioate RNA target (Sulfolobus solfataricus (Sso)Csm complex (~470 nM carrying A26 CRISPR RNA) in Csx1 buffer containing 20 mM MES pH 5.5, 100 mM K-glutamate, 1 mM DTT and 3 units SUPERase•In Inhibitor supplemented with 1 mM ATP, 5 nM α-32P-ATP and 2 mM MgCl2 at 70°C for 2 hr. All samples were deproteinised by phenol-chloroform extraction (Ambion) followed by chloroform (Sigma-Aldrich) extraction prior to separating the cOA products by thin-layer chromatography (TLC). TLC was carried out as previously described  diameter.Calculation determining the concentration of cA4 is made per 1 molecule of RNAAs ~1000 molecules of cAConcentration (M) = moles / Volume (L)4 generated using the SsoCsm was incubated with increasing concentrations of Csx1  in buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM MgCl2 supplemented with 2 µM Ultrapure Bovine Serum Albumin (Invitrogen) for 10 min at 25°C. A reaction volume equivalent of 20% (v/v) glycerol was then added prior to loading the samples on a 15% polyacrylamide, 1 X TBE gel. Electrophoresis was carried out at 28°C and 250 V. Gels were phosphor imaged overnight at −80°C. For investigating RNA binding, 50 nM 5’-end radiolabelled and gel purified A1 RNA was incubated with Csx1 variant H345N  in the presence or absence of 20 µM cA4 for 15 min at 40°C. To examine cA4 binding by Crn1,~10 nM radiolabelled SsoCsm cA4 was incubated with Sso2081  on ice for 15 min before gel electrophoresis as described above but at 300V and at 4°C. cA4 binding by AcrIII-1 was examined by incubating ~10 nM radiolabelled SsoCsm cA4 with SIRV1 gp49 H47A  for 10 min at 25°C before gel electrophoresis at 30°C as described above. For analysis densiometric signal corresponding to cA4 bound protein was quantified. The densiometric count corresponding to cA4 bound to 20 µM Csx1 dimer was used to represent 100% binding and densiometric counts from other lanes were normalised to this value within each replicate. Error of the 100% bound (20 µM Csx1 dimer) densiometric count was derived by calculating the area adjusted count for each replicate and then the standard deviation of their mean, reporting the standard deviation as a fraction of the mean set as 100% bound. The data were fitted to a quadratic equation with an adjust for nonspecific binding (Y = (ymax-ymin)*((x + [ligand] + Kd) - ((x+[ligand]+Kd)^2–4*x*[ligand])^0.5)/(2*[ligand]) +a*x+b) using GraphPad Prism 8.~20 nM radioactively-labelled cA4 were included. 10 μl reaction aliquots were quenched by adding to phenol-chloroform and vortexing and the different time-points at which reactions were quenched for each RNA concentration is indicated in data transparency. Deproteinised products were run on a 7 M urea, 20% acrylamide, 1 X TBE gel at 45°C as previously described  with radiolabelled (50 nM) and unlabelled RNA A1 to a final concentration of 1.25, 1.5, 2.0, 5.0, 10.0, or 20.0 μM RNA in Csx1 buffer at 70°C. Control reactions with no protein and with protein and RNA in the absence of cAescribed , and phohttps://kintekcorp.com/software). Experiments were modelled and simulated using kinetic and equilibrium paramters detemined experimentally as described in Modelling was carried out using the KinTek Explorer eight software package , which itarget_Csm_ATP = target_Csm + cA4 (irreversible)cA4 + Csx1 = cA4_Csx1cA4_Csx1 + RNA = cA4_Csx1_RNAcA4_Csx1_RNA = cA4_Csx1_cleavedRNA (irreversible)cA4_Csx1_cleavedRNA = cA4_Csx1 + cleavedRNAcA4 + Crn1 = cA4_Crn1cA4_Crn1 = A2 + Crn1 (irreversible)cA4 + Vrn = cA4_Vrn cA4_Vrn = A2 + Vrn (irreversible)Simulations were carried out varying target_Csm_ATP concentration  while Csx1, Crn1 (Sso2081) and AcrIII-1 concentrations were fixed at 1 µM dimer, or varied depending on the simulation, with total substrate RNA in the cell fixed at 1000 µM. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Gisela Storz as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Philip Kranzusch (Reviewer #1); Martin Lawrence (Reviewer #2); Rotem Sorek (Reviewer #3).Thank you for submitting your article "The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling" for consideration by The reviewers universally agree that this work is nearly ready for publication. No additional experiments are requested.Summary:4) produced by the type-III CRISPR system in Sulfolobus solfataricus, the immune proteins that are activated by cA4, and viral anti-CRISPR proteins that degrade cA4. Biochemical data is used to build a kinetic model, which helps explains how cyclases and ring nucleases govern cyclic nucleotide levels and determine infection outcomes in virus-host conflict.It is becoming increasingly clear that cyclic nucleotides play a critical role in the host response to a viral infection and that viruses have evolved mechanisms to intercept these signaling molecules. In this work, the authors focused on the synthesis of cyclic-oligo-adenylate . These rates should be incorporated into the mathematical model to understand how the relative rates of cA4 synthesis and cA4 degradation may influence effector function dynamics.1) Rates of cA4 synthesis wanes after ~10 min if no new activating RNA is delivered to the system. Please discuss this in the context of your model.2) The current model does not take into account previously observations from the lab demonstrating that cA Essential revisions:4 synthesis by the S. solfataricus Csm complex have been previously measured . These rates should be incorporated into the mathematical model to understand how the relative rates of cA4 synthesis and cA4 degradation may influence effector function dynamics.1) Rates of cA4 synthesis  from previously published data  and have incorporated this into the model. We have also adjusted the kinetic parameters of our model to the reaction temperature of 70 °C, which better represents infection kinetics at the growth temperature of S. solfataricus. We have also increased the cellular RNA concentration to 1000 µM, from 100 µM in the previous model, which we believe better represents cellular conditions and means that RNA is never fully depleted in the simulations. For comparison and transparency, we include a supplementary version of Figure 6 with simulations starting at fixed cA4 concentrations  carried with the current model as per our initial submission. See Author response images 1 and 2. Ultimately, the changes we have made, including incorporation of the rate of cA4 synthesis do not radically alter any of our previous conclusions.We agree that this would strengthen the model. We have determined the rate of cA4 synthesis wanes after ~10 min if no new activating RNA is delivered to the system. Please discuss this in the context of your model.2) The current model does not take into account previously observations from the lab demonstrating that cA4, dependent on the concentration of target RNA.This query relates to point 1, and we have addressed this point in the new model, which now incorporates the time dependent synthesis of cA


Human apurinic/apyrimidinic (AP) endonuclease APE1 is one of the participantsin the DNA base excision repair. The main biological function of APE1 is tohydrolyze the phosphodiester bond on the 5′-side of the AP sites. It hasbeen shown recently that APE1 acts as an endoribonuclease and can cleave mRNA,thereby controlling the level of some transcripts. The sequences of CA, UA, andUG dinucleotides are the cleavage sites in RNA. In the present work, weperformed a comparative analysis of the cleavage efficiency of model RNAsubstrates with short hairpin structures in which the loop size and thelocation of the pyrimidine–purine dinucleotide sequence were varied. Theeffect of various divalent metal ions and pH on the efficiency of theendoribonuclease reaction was analyzed. It was shown that site-specifichydrolysis of model RNA substrates depends on the spatial structure of thesubstrate. In addition, RNA cleavage occured in the absence of divalent metalions, which proves that hydrolysis of DNA- and RNA substrates occurs viadifferent catalytic mechanisms. Human apurinic/apyrimidinic endonuclease APE1 is one of the most exhaustivelystudied DNA damage repair enzymes , 3. HoweIt was shown earlier that APE1 causes RNA chain degradation in DNA–RNAduplexes  . Later, 2+ ion actsas a nucleophile.Therefore, human AP endonuclease is versatile in terms of the functions itplays in the cell. Structural data, kinetic studies and a mutation analysishave made it possible to identify the key stages of interaction between APE1and damaged DNA harboring the AP site , 16, 17,2+ ions in the binding of damaged DNA, itscleavage, and release of the reaction product has been vigorously discussed ina number of studies , APE1 preferentially cleaves the CA,UA, and UG dinucleotide sequences in single-stranded or weakly paired RNAregions. Furthermore, weak RNA cleavage at the UC, CU, AC, and AU sites wasalso observed [11]. It is fair to assume that, along with thepyrimidine–purine sequence, the substrate structure  is essential for thecatalytic complex formation. Indeed, the RNA cleavage observed insingle-stranded regions near the hairpin stems  may be an indirectindication that a substantial contribution is made by the secondary structureof the substrate. Therefore, we used a series of 14- to 17-nucleotide-longmodel RNA substrates, the short hairpin structures in which the stem was 6 bplong and the loop length ranged from 2 to 5 nucleotides; the positions of theCA or UA dinucleotide in the hairpin loop or stem were also varied. Meanwhile, the enzyme exhibited noactivity in the presence of Zn2+ ions. In addition, theyield of hydrolysis products dropped noticeably when pH of theEDTA-containing buffer was increased to 8.5, while pH reductionto 6.5 produced side products due to statistical hydrolysis.It was established for the HP6 RNA substrate that 3′-5′-exonucleaseand endoribonuclease reactions occur in the presence of the MgFig. 2C)characterizing theinteraction between APE1 and HP6 and came to the conclusion that the initialrates of the endoribo nuclease reaction in the presence of EDTA orCa2+ ions are similar, while the fluorescence intensity of FAM inthe presence of Mg2+ ions increases more rapidly (presumably due tothe appearance of the 3’-5’ endonuclease reaction). It isnoteworthy that the changes in the FAM fluorescence intensity in the presenceof Mg2+ ions can also be related to the formation of a catalyticexonuclease complex, which increases the distance between the BHQ1 quencher(residing at the 3’ end) and the 5’-end FAM label.We compared the kinetic curves,three buffer solutions (pH 7.5) containing 5 mM MgCl2, 5 mMCaCl2, or 1 mM EDTA were used to analyze various types of enzymeactivity in further experiments with different RNA substrates.According to these findings.Indeed, comparison of the sites ofcleavage of the RNA substrates of RNase A, which statistically cleaves RNA atall pyrimidine nucleotides, indicates that the UA dinucleotides within theduplex portion of a hairpin are not recognized by AP endonuclease as cleavagesites. Meanwhile, in the absence of divalent metal ions, APE1 does not catalyzethe 3′-5′-exonuclease reaction with any of the RNA substrate understudy .The interaction between APE1 and our model RNA substrates in the absence ofdivalent metal ions causes the accumulation of products of the endoribonucleasereaction for HP3–HP6 RNA substrates carrying the CA or UA dinucleotide inthe loop portion .Meanwhile, PAGE has demonstrated that both sites arecharacterized by similar cleavage efficiencies, thus proving that thisparameter is independent of the position occupied by the CA dinucleotide withinthe loop. Meanwhile, when APE1 interacts with HP4 carrying a single CAdinucleotide, cleavage efficiency reaches 16%. The HP5 hairpin carries the UAand CA dinucleotides in its loop; the total cleavage efficiency is 17%.However, cleavage at the UA dinucleotide is much less efficient than cleavageat the CA dinucleotide ,which is also consistent withthe low (8%) cleavage efficiency of HP3 carrying a single UA dinucleotide.However, when comparing the cleavage efficiencies of these substrates, oneneeds to take into account the substrate structure (different loop sizes anddifferent positions of the hydrolyzed bond within the loop) in addition to theCA/UA context of the hydrolyzed phosphodiester bond. Since the cleavage site inthe HP3 (UA) and HP4 (CA) hairpins has the same location with respect to thestem, it is fair to assume that the HP4 hairpin loop containing fivenucleotides is more readily adapted in the enzyme substrate-binding sitecompared to the HP3 hairpin with the 4-nucleotide loop. The HP5 hairpin, whoseloop is also five nucleotides long, differs from HP4 in the positions ofcleavage sites, which presumably impedes efficient formation of the catalyticcomplex for HP5.Interestingly, the HP6 hairpin harboring two CA cleavage sites becomes the mostefficient substrate, its cleavage efficiency being as high as 25%,which indicates that the double-stranded portion of RNA substrates plays acrucial role in the formation of the catalytic enzyme– substrate complex.Binding of RNA substrates to APE1 in the presence of EDTAFig. 3)allowed us to carry out experiments involvingfluorescent titration of the substrates by the enzyme and estimate thedissociation constant of the enzyme–substratecomplex. Figure 4Ashows the fluorescence intensity at 520 nm as a function of enzymeconcentration. For the HP1–HP6 substrates labeled with the FAM/BHQ1 pair,the FRET signal increased as the enzyme–substrate complex formed.Fluorescence quenching was observed in the case of linear substrate L harboringonly the 5’-FAM label. Such differing changes in the FAM fluorescenceintensity can be attributed to the fact that the enzyme can bind both to theloop and the 5’/3’-end of the hairpin structures. The presence ofproducts of endoribonuclease hydrolysis in the reaction mixtures indicates thatbinding occurred in the loop portion of the substrate, while the presence ofthe products of 3′-5′-exonuclease degradation proves that analternative complex with the 5’/3’-end region formed. Since theBHQ1 quencher resides at the 3’ end of the hairpins, the higherfluorescence intensity of FAM for the HP1–P6 substrates harboring theFAM/BHQ1 pair can be attributed to the fact that the distance between the FAMand BHQ1 labels increases when the enzyme–ubstrate complex with the5’/3’-end region is formed. In the case of substrate L, which doesnot harbor the BHQ1 quencher, the fluorescence intensity of FAM decreases as acomplex with an enzyme molecule is formed. The values of the dissociationconstant Kd were calculated(Table 2).The low cleavage efficiency of RNA substrates during a 1-h reaction, and therespective dissociation constants Kd were calculated(Table 2).However, the titration curve for substrate Lrecorded by MST did not allow us to determine the Kd valueusing this method because of the low signal-to-noise ratio. A comparison of thedissociation constants(Table 2)revealed rather good agreement between the values obtained using differentmethods. Hence, an analysis of the stability of the enzyme–substratecomplexes usingfluorescence titration and MST demonstrates that the highest dissociationconstants were observed for the complex that formed between APE1 and HP1 orHP2, which contain a short loop and carry no specific dinucleotide in theirloop. The complexes formed between the enzyme and the HP3–HP6 substrateshave similar dissociation constants lying within the range of 2.2–4.9μM.An additional microscale thermophoresis (MST) study of the binding between APE1and the substrates was carried out.Strong quenching of substrate fluorescence in the presence ofCo2+, Ni2+, and Zn2+ made it impossible todetermine the Kd value, whereas reduction of theKd value in the presence of Ca2+ may have been anindication that the enzyme–substrate complex had been stabilized.We also estimated the dissociation constants of the complex containing HP6 as asubstrate in the presence of divalent metal ions.It is interesting to note that in the case of HP4, Ca2+ions slightly reduced the APE1 activity, while exhibiting no effect when HP3and HP5 were used as substrates. These effects are presumably caused by thefact that Ca2+ ions have a small impact on the dissociation constantof the enzyme–substrate complex, which agrees with the fluorescencetitration data(Table 2).The interaction between APE1 and RNA substrates in the presence ofCa2+ ions were recorded.Interestingly, when a threefold excess of the substrate was chosen, itscleavage efficiency reached a plateau within 15 min . The low cleavage efficiency mayindicate that: (1) efficiency in the catalytic complex formation is low and (2)the enzyme becomes strongly bound to the reaction product. Indeed, the newportion of the enzyme added to the reaction mixture led to additionalaccumulation of the reaction products, which demonstrates that APE1 remainsbound to the RNA product after a single catalytic event.The kinetics of accumulation of the products of APE1 endoribonuclease activityon the HP6 RNA substrate in the absence of divalent metal ions and in thepresence of Ca2Hydrolysis of RNA substrates by APE1 in the presence ofMgCl2.The endoribonucleasereaction products did not form when HP1 and HP2 substrates with the shortestloop  that did not carry the specificpyrimidine–purine sequence were used. The products formed on theHP3–HP6 substrates due to the endoribonuclease activity corresponded tocleavage of the CA and UA dinucleotides within the loop. The3′-5′-exoribonuclease reaction was found to occur on all thehairpin RNA structures harboring 3′-BHQ1. In this reaction, 3′-BHQ1was removed and a typical increase in the mobility of the exoproduct in PAGEwas observed .Products of both the endoribonuclease and 3’-5’-exoribonucleasereactions accumulated when APE1 interacted with RNA substrates in the buffercontaining MgCl2+ ions demonstrated that cleavage of the 3′-BHQ1 is moreefficient than cleavage within the loop.Meanwhile, when the enzyme was returned to the reaction mixture, the rate ofproduct accumulation increased for the exonuclease reaction but not for theendonuclease one.This difference may be anindication that the hairpin harbors several enzymebinding sites. APendonuclease is tightly bound to the loop portion of the hairpin in order toslowly perform the endonuclease reaction. In this case, the additional enzymeintroduced into the reaction mixture results in additional binding only to thevacant opposite end carrying the FAM/BHQ1 dyes, which is accompanied by theexonuclease reaction. It can also be assumed that the catalytic complex thatforms between APE1 and the 5’/3’ end of HP6 in the presence ofMg2+ ions causes steric hindrance in the formation of the catalyticcomplex with the loop.An analysis of the accumulation kinetics of the products of the endo- andexonuclease reactions of RNA-substrate conversion in the presence ofMgIn this study, we have analyzed the interaction between human AP endonucleaseAPE1 and model RNA substrates with different structures. APE1 was shown toefficiently bind both to the linear RNA substrate and to the RNA substratesforming the hairpin. The endoribonucleolytic cleavage of the substrates tookplace in the loop fragments at the CA and UA sequences. The efficiency ofcleavage at the UA dinucleotide is lower than at the CA dinucleotide. However,when comparing the cleavage efficiencies of these substrates, one needs to takeinto account not only the CA/UA nucleotide context of the hydrolyzedphosphodiester bond, but also the substrate structure (including both the loopsize and the position of the bond being hydrolyzed within the loop). Since inthe HP3 (UA) and HP4 (CA) hairpins the cleavage site occupies the same positionwith respect to the hairpin stem, these findings allow one to assume that the5-nucleotide-long HP4 hairpin loop is more readily adapted in thesubstrate-binding site of the enzyme compared to the HP3 hairpin with the4-nucleotide-long loop. The HP5 (CA/UA) hairpin, whose loop also consists offive nucleotides, differs from HP4 in terms of cleavage site positions, whichpresumably impedes efficient catalytic complex formation in the case of HP5.Hence, a conclusion can be drawn that the formation of a catalyticenzyme–substrate complex depends both on the conformational strain of theloop in the hairpin-shaped RNA substrate and on the context and position of thephosphodiester bond to be hydrolyzed. It is worth mentioning that cleavageefficiency of linear substrate L was also low compared to that of theHP4–HP6 hairpin substrates. Given this fact, it is fair to assume thatthe structured duplex portion of the hairpin plays a crucial role in theformation of nonspecific contacts in the enzyme’s active sites, which areessential for catalytic complex formation.


RNA sequencing (RNA-seq) is a common tool for obtaining data related to gene expression  as well as the representative bulk methods .Regarding the first criticism, we previously showed the need for asymmetry and developed a robust normalization method (dubbed TbT) for manipulation in both symmetric and asymmetric scenarios (Kadota et al., Related to the second criticism, Vieth et al.  found thTaken together, there is no special reason to distinguish between scRNA-seq and bulk RNA-seq, especially in DE analysis. Despite the advances in experimental technology from bulk RNA-seq to scRNA-seq, universally applicable algorithms do exist.KK drafted the manuscript. KS supervised the critical discussion and refined the paper. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Disease-associated mutations in the FUS gene were sufficient to disrupt formation of the FUS-SFPQ complex, resulting in deregulated alternative splicing of MAPT pre-mRNA and a consequent increase in the 4-repeat (4R)-tau/3-repeat (3R)-tau (4R-T/3R-T) ratio, ultimately leading to neurodegeneration that recapitulated key aspects of FTLD , amyotrophic lateral sclerosis (ALS), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) have distinct clinical features from presenting symptoms through to management. However, accumulating evidence suggests some convergence in molecular and genetic pathology, which may argue against their taxonomy as completely distinct entities. Indeed these disorders are often referred to as the FTLD spectrum, for which fused in sarcoma (FUS), transactive response (TAR) DNA-binding protein 43 (TDP-43), and tau are pathological hallmarks  context. Notably, in their previous study, Ishigaki and co-workers demonstrated that both FUS-silenced and SFPQ-silenced mice similarly exhibited increased 4R-T/3R-T ratios along with FTLD-like behaviour, hippocampal neuronal loss and phosphorylated tau accumulation  protein aggregates. However, deregulation of RBPs such as TDP-43, SFPQ and FUS, and other aspects of RNA metabolism, are increasingly recognized as crucial players in these disorders. The mechanisms by which these defects in protein homeostasis and RNA metabolism conspire to cause neuronal degeneration are of key importance to resolve. Indeed, a common site of molecular convergence here is the ribonucleoprotein (RNP) complex, which is composed of both RNA and proteins (RBPs). RBPs implicated in FTLD spectrum disorders commonly contain prion-like low complexity domains (LCDs) and intrinsically disordered regions, which render them aggregation-prone. These RBPs possess the important physiological capacity to undergo liquid-liquid phase separation (LLPS), which refers to the condensation of molecules into liquid-like non-membrane delimited organelles (the aformentioned nuclear paraspeckles are an example of this phenomenon). LLPS likely occurs through tightly regulated prion-like polymerization driven by weak, transient molecular interactions between LCDs and other multivalent protein or RNA interaction domains. LLPS can be regulated by specific protein:protein, protein:RNA, and/or RNA:RNA interactions and is a molecular principle governing transient intracellular compartmentalization. LCD mutations can promote the fibrillization of molecular assemblies from  liquid-like droplets. Such perturbation in the kinetics of LLPS (i.e. hyperassembly and/or defective disassembly) may ultimately lead to some of the most recognized features of FTLD spectrum disorders including insoluble protein aggregates within the cytoplasm. Additionally, deregulated LLPS may lead to the sequestration of RBPs , in turn causing their functional deficiency and the misregulation of key splicing events, such as Specifically, future studies might aim to extend the important findings by Ishigaki and co-workers by addressing the potential relationship between spatial dissociation  of RBPs and deregulated LLPS. Another key consideration is the cell type specificity of such molecular events, particularly with respect to glia given their well recognized roles in neurodegeneration. Furthermore, it would be important to understand whether the molecular phenomena reported in this study relate in some way to the increasingly recognized prion-like spread of pathology between cells in these disorders (


Saccharomyces cerevisiae, as a potential model organism for the production of RNA nanoparticles on a large scale.RNA aptamers are becoming increasingly attractive due to their superior properties. This review discusses the early stages of aptamer research, the main developments in this area, and the latest technologies being developed. The review also highlights the advantages of RNA aptamers in comparison to antibodies, considering the great potential of RNA aptamers and their applications in the near future. In addition, it is shown how RNA aptamers can form endless 3-D structures, giving rise to various structural and functional possibilities. Special attention is paid to the Mango, Spinach and Broccoli fluorescent RNA aptamers, and the advantages of split RNA aptamers are discussed. The review focuses on the importance of creating a platform for the synthesis of RNA nanoparticles in vivo and examines yeast, namely  Aptamers are single-stranded DNA or RNA oligonucleotides, characterized by their various 3-D conformations, resulting in a distinctive ability to recognize and bind to numerous targets with high specificity. Such targets include metal ions, nucleic acids, proteins, polysaccharides, and other organic compounds, in addition to viruses, subcellular organelles, and cells ,5,6,7,8.The first nucleic acid-based aptamers were concurrently produced in both Gold’s and Szostak’s laboratories in 1990 using SELEX . This technology depends on a library of random single-stranded DNA or RNA molecules, the sequences of which are flanked by identical sequences at both 3′ and 5′ ends [SELEX is designed to enrich the oligonucleotides with high affinity to the target molecules by means of three main steps for each cycle: A) the oligonucleotides from the library bind to their specific targets, B) the unbound oligonucleotides are eliminated, and C) the bound oligonucleotides are amplified using PCR using the common 3′ and 5′ flanking sequences. After the enrichment step, the oligonucleotide libraries showing the desired results are selected and the sequences are synthesized in sufficient quantities in order to carry out functional tests .Methods of automated selection of aptamers have been developed in the interest of simultaneously determining aptamers for a number of different targets . In siliOther promising in silico tools are making the most of the advances in artificial intelligence and machine learning. A number of tools for RNA aptamer clustering, motif finding, and aptamer optimization are discussed by Hamada, 2018 . For RNANowadays, aptamers have high rates of demand in both fundamental and applied research. Aptamers with a reporter group are successful tools for the identification and localization of different molecules in cells, hence evaluating their roles in certain biological processes ,18.Aptamers can also help to identify specific sequences of nucleic acid-binding proteins and give rise to affinity reagents for protein purification ,20,21,22Another application of aptamers is in being the sensitive element of various biosensors for biomedical and environmental applications. Microorganisms can be detected using aptamer-based biosensors, including some aptamers designed to target HBV, HCV, SARS-CoV, Vaccinia, and Ebolavirus, reviewed by Zou et al. . AptamerAs potential therapeutic agents, DNA aptamers have been shown to effectively bind and inhibit thrombin, offering a promising anti-coagulant after optimizing some pharmacokinetic properties . AptamerDue to the high affinity and specificity of aptamers after many rounds of selection, in addition to their superior properties regarding shelf life, the ability to restore the 3-D structure after denaturation, low molecular size, low immunotoxicity, short time of development, and, more importantly, the wide range of targets, aptamers could replace antibodies at any point in the foreseeable future ,29. AccoIn the past few years, awareness of the importance of RNA has steadily increased amongst researchers, owing to the ever-growing list of various RNA functions (reviewed by Breaker and Joyce) . There iIn fact, RNA aptamers selectively bind to various targets and influence their functions . AptazymThe functional diversity of RNA molecules unveils the huge potential for the development of new RNA-based drugs. The main candidates of such molecules fall into the groups of RNA aptamers, miRNAs, and siRNAs. The latter two molecules regulate and suppress protein synthesis, therefore making great antineoplastic drug candidates ,50. RNA One of the major drawbacks in embracing RNA for therapeutic purposes is the short half-life, due to the prevalence of nucleases in blood plasma. This pharmacokinetic parameter determining the distribution and clearance is of crucial importance for any drug. In fact, oligonucleotides exist for no longer than a few tens of minutes in blood plasma, rendering such drug candidates disadvantageous. However, this problem has been solved by introducing some chemical modifications to the nucleotides to protect the RNA polymer from the hydrolyzing effect of nucleases and by improving the pharmacokinetic properties while maintaining the pharmacodynamic activity. Another solution to prolong the half-life is by using RNA nanoparticles ,52,53.O-methylpyrine, and 2′-O-methylpyrimidine. Aided by special RNA polymerases, e.g., T7 RNA polymerase, 2-O-methylated pyrimidine monomers can be catalyzed to synthesize RNA polymers [In the case of using chemically modified nucleotides, they can be added before or after SELEX. For the first option, nucleoside triphosphate can be used for aptamer synthesis after introducing one of the following modifications to the 2′ position of the ribose sugar: 2′ amino pyrimidine, 2′ fluoropyrimidine, 2′-polymers . For thepolymers ,55,56.As a matter of fact, the 3-D structure and the function of any aptamer are interconnected. Some RNA structural motifs include open and stacked three-way junctions , four-waLys is one of the well-studied RNA scaffold models, in addition to other tRNAs [To create an aptamer and support its structure, it should be embedded in the supramolecular scaffold of an RNA molecule. Human tRNAer tRNAs . Embeddier tRNAs .Vibrio proteolyticus (V5) as an aptamer-bearing scaffold has also been reported, in which the helical domain III and loop C have been replaced by an aptamer. As a result, a functional aptamer against vascular endothelial growth factor (VEGF) has successfully been produced in V. proteolyticus (V5) [Resistance to intracellular nucleases is considered one of the most important criteria in the process of selecting supramolecular scaffolds to include the aptamer. The cleavage of the RNA scaffold can lead to the dissociation of the aptamer and loss of its action on the target. This is the disadvantage of tRNA-based scaffolds . Employicus (V5) .More common scaffolds include phi9 3WJ, a stacked three-way junction (3WJ) motif from the bacteriophage phi29. This motif consists of three short RNA fragments (≤20 nucleotides); upon assembly, the overall structure shows high thermodynamic stability. It has been reported that this structure is stable in a solution containing 8 M of urea and it does not dissociate at lower concentrations .The branching structure of Phi29 3WJ is very useful for inserting different functional modules into each of the three helices. This scaffold facilitates the correct folding of other molecules merged in its structure. Thus, this scaffold is capable of carrying different molecules, including aptamers, miRNAs, ribozymes, and even ligands that bind to cell receptors—each can be placed on a separate branch of the scaffold. Thanks to the correct folding of each molecule, their functions are maintained, including cell-binding, cell penetration, suppression of gene expression, catalytic functions, and others ,67,68.Nevertheless, in mammalian cells, along with the full-sized aptamers, based on Phi29 3WJ scaffold, some shortened variants of the aptamers have been found. One of the likely reasons for this issue is that near RNA-polymerase III terminator, there is a UUUGUU sequence, causing premature termination of transcription. Filonov et al. designed an F30 scaffold based on Phi29 3WJ after mutating the sequence, and premature termination discontinued .The variety of RNA functions in living cells have led to advanced method development to detect and study the dynamics of RNA in vivo .For many years, the MS2-MCP method has been one of the most popular methods for RNA labeling, and it is based on the high-affinity binding of the bacteriophage coat protein MS2 (MCP) to the unique RNA hairpin sequence, the MS2 binding site (MBS). Therefore, cloning the MBS sequence into the RNA of interest and the simultaneous synthesis of MCP fused with the fluorescent protein GFP (green fluorescent protein) allows RNA localization in the cell ,71.However, background fluorescence from unbound MCP-GFP strongly affects the signal-to-noise ratio. In addition, it was found that the MS2 viral envelope proteins, associated with the MBS site in the 3’-untranslated region (UTR) of yeast mRNA, block the activity of Xrnp1 exonuclease and 5’-3’-degradation of mRNA. This leads to the accumulation of 3’ mRNA fragments that still bind to MCP-GFP, complicating in vivo full-length mRNA localization .An alternative method for in situ RNA imaging is the use of fluorogenic RNA aptamers ,74. SincIn 1999, Grate and Wilson proposed an RNA aptamer that binds to malachite green (MG) as a molecular biology tool . The welIn today’s RNA research, the most promising fluorescent RNA aptamers are the Mango, Spinach, and Broccoli.The Mango RNA aptamer has an exceptionally high affinity to TO1-biotin, a thiazole orange derivative fluorophore, and upon binding, the fluorescence of the fluorophore is increased by 1100 times. The high affinity facilitates in situ low-copy RNA imaging, allowing the use of low concentrations of the fluorophore and thereby reducing the fluorescence background noise level and the adverse effect of the dye on cells . The excThe analysis of the crystal structure of the Mango–TO1-biotin complex showed that the core of this aptamer, consisting of 23 nucleotides, is a three-level G-quadruplex . TO1-biotin is adjacent to one of the nearly planar T3 faces of the G-quadruplex, and each of the three heterocycles of the fluorophore carries out stacking interactions with the nitrogenous bases of the RNA loop  Figure .Caenorhabditis elegans gonads using fluorescence microscopy demonstrates the potential of this system for studying RNA in living cells. The incorporation of the aptamer into bacterial 6S rRNA has provided a useful tool not only to label the molecule but also to purify it using affinity chromatography on streptavidin agarose [Imaging of the Mango aptamer in  agarose .Three new variants of Mango aptamers, I, II, III, with increased affinity, increased fluorescence, and resistance to salts and formaldehyde, were obtained by selection in the presence of a TO1-biotin competitive inhibitor. The latter circumstance allows the use of Mango aptamers not only in living cells but also in solutions ex vivo. Mango aptamers I, II, III folded with the F30 framework were successfully used for labeling and subsequent imaging of human 5S ribosomal RNA .Increased levels of fluorescence can be achieved using RNA molecules with tandem repeats of the aptamer. It was shown that Mango II in triplex provides around 2.5 times higher fluorescence intensity than a single copy of the aptamer sequence. In this case, the localization of the target RNA —actin mRNA and NEAT1 long noncoding RNA—does not change .Jaffrey et al. have synthesized several derivatives of 4-hydroxybenzylidene imidazolinone (HBI), which acts as a fluorophore in GFP reporter assay. Further, using SELEX technology, several aptamers were discovered that bind to the obtained fluorophores. The strongest fluorescence was demonstrated by 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) in the presence of a 98-nucleotide aptamer called Spinach . SpinachChemical modifications of DFHBI, specifically adding a trifluoroethyl substituent to the methyl group (DFHBI-1T) or to the second carbon atom (DFHBI-2T), in the first case, result in an increase in the fluorescence intensity and, in the second, a shift in the excitation and emission maxima to the long-wavelength region of the spectrum. Thus, the fluorescence filters developed for YFP (yellow fluorescent protein) can be employed with DFHBI-2T .Despite the above-mentioned advantages, the Spinach aptamer is not devoid of disadvantages, the main of which are thermal instability and a tendency towards improper folding at 37 °C, leading to a decrease in the fluorescence intensity. Using mutagenesis, Spinach2 aptamer was designed, which is more stable than Spinach, and it demonstrates the correct folding at 37 °C regardless of the fused RNA, particularly the 5S and 7SK RNA .In addition, the use of tRNA as a scaffold increased the proportion of correctly folded aptamers of both types .Crystallographic analyses of Spinach aptamer structure showed that the RNA molecule has an elongated structure containing two helical domains separated by an internal loop. The loop folds into a G-quadruplex motif and it is flanked on both sides by antiparallel A-form duplexes. The G-quadruplex motif and the adjacent nucleotides form a partially formed fluorophore binding site. The intermolecular bonding between the fluorophore and the RNA aptamer is mediated by hydrogen bonds and π–π stacking interactions  Figure .Determining the molecular structure of Spinach and understanding the role of different regions in the sequence allowed the removal of parts of the sequence and the design of variants of the aptamer, e.g., Baby Spinach, which consists of 51 nucleotides only, with a fluorescence intensity comparable to that of the original variant .To enhance the Spinach fluorescence signal and detect low-copy RNAs, tandem repeats of the aptamer were used. It was shown that 64 repeats of the aptamer increased the fluorescence intensity by 17 times .Escherichia coli was observed [Spinach aptamer has been used to create the sensitive element of biosensors designed for specific metabolites in bacterial cells. For this purpose, RNA sequences responsible for binding to certain metabolites were inserted into one of the stems of the Spinach aptamer. This modification of the aptamer structure led to our understanding that the correct structure of Spinach and the ability to interact with DFHBI were determined by the presence of these metabolites, and the level of fluorescence depended on their concentration . Thus, tobserved ,94.On the basis of Spinach, similar biosensors were also developed for monitoring proteins, namely thrombin, streptavidin, and the envelope protein of the MS2 phage .The combination of SELEX and FACS (fluorescence-activated cell sorting) technologies has provided a powerful tool to develop a new version of the Spinach aptamer. This 49-nucleotide aptamer, named Broccoli, activates DFHBI or DFHBI-1T fluorescence, folds faster, shows high stability, and does not require a tRNA scaffold in vitro. The excitation and emission wavelengths of this aptamer are 472 and 507 nm, respectively . The BroThe secondary structure of Broccoli includes a hairpin-stem-loop and allows the production of aptamer dimers by replacing the terminal loop with a second aptamer molecule, leading to a 70% increase in fluorescence .An additional advantage of Broccoli, like Spinach, is the ability to image the aptamers in vitro; fluorescence can be observed in microcentrifuge tubes  or by elBoth Spinach and Broccoli aptamers fused to the tRNA backbone have been successfully expressed in bacterial and mammalian cells. The ability of the Broccoli aptamer to fold in vitro without the aid of a tRNA scaffold has been confirmed in vivo. The RNA of the aptamer was fused to the 3’ end of 5S RNA and the resulting plasmids were transfected into HEK293T cells. Using flow cytometry, 5S RNA–Broccoli was detected in the cells, and the brightness of the cells was higher compared to cells containing 5S RNA–tRNA–aptamer. This supports the idea that the tRNA backbone is often cleaved by cell nucleases and, thus, has a negative effect on the expression of RNA aptamers. It should be noted that Spinach2 folding requires a tRNA backbone, and no fluorescence was detected when using the 5S RNA–Spinach2 construct .Expression of the Broccoli–DFHBI-1T and Spinach–DFHBI aptamers in the 16S ribosomal RNA has allowed ribosomal imaging and provided a unique opportunity for studying translation in prokaryotes .Broccoli–DFHBI-1T and Spinach–DFHBI were inserted into the 5’-hairpin of one of the yeast H/CA small nucleolar RNAs (snoR30), which is involved in rRNA maturation. The yeast cells were transformed with plasmids containing these constructs under the control of the GAL promoter. The growth of the transformants did not significantly differ from the growth of the parent line; aptamers did not disrupt the localization and function of snoR30 and provided fluorescence in the nucleoli .The Spinach2–tRNA and Broccoli–F30 aptamers were used to study the regulation of RNA synthesis of the SINV virus, which can cause seasonal outbreaks of rash and arthritis in humans and encephalomyelitis in experimentally infected mice. Consequently, aptamers were inserted into the 3’UTR of viral RNA. The resulting recombinant viruses replicated well in nerve cells and BHK fibroblast cell culture. The fluorescence level correlated with the Broccoli–F30 copy number .On the basis of the Broccoli aptamer, a fluorometric RNA substrate was developed, the fluorescence of which was proportional to the activity of RNA-modifying enzymes. Thus, a variant of the aptamer with modified nucleotides, such as N6-methyladenosine (m6A), was synthesized. Such an aptamer is unable to function normally, or restore its function, and special demethylases are required to restore its function. This approach facilitates the search for not only enzymes that modify RNA but also for their inhibitors and factors that affect the levels of RNA methylation in living cells .New split RNA aptamers were developed to evaluate RNA co-transcriptional folding, RNA–RNA interaction dynamics, and RNA aptamer assembly in vivo. The split RNA aptamer consists of a pair of oligonucleotides that re-associate, when located in close proximity, and form the entire aptamer that can bind to the fluorophore and exhibit fluorogenic properties. A split Broccoli consisting of two strands of RNA—Broc and Coli—was developed, demonstrating high but incomplete complementarity. The dependence of the fluorescence of F30–Broccoli cleaved aptamers on temperature, the concentration of magnesium ions, and the presence of certain oligonucleotides allow these aptamers to be used as “molecular thermometers”, biosensors, and “molecular switches” ,102,103.Thus, aptamers can be used for biological imaging of nucleic acid and to study their dynamics in the cell and, therefore, studying the regulation of gene expression and metabolism. In addition, aptamers play important roles as biosensors for proteins and various other metabolites  ,82. RNA Hybrid RNA–DNA molecules represent another variant of aptamers. The therapeutic potential of the RNA–DNA aptamer has been demonstrated for the treatment of melanoma. When these structures enter the cells, si-RNA and DS-DNA are released. si-RNA suppresses the mutated BRAF gene in melanoma cells. DS-DNA, which contains the binding site for NF-kB, holds it in the cytoplasm and blocks the activation of the NF-kB pathway, which increases the death of melanoma cells treated with vemurafenib .A promising application of aptamers is targeted drug delivery, among which microRNAs and siRNAs are the most important. However, the bottleneck remains in effectively delivering the RNA to the target with minimal damage to healthy cells and tissues. These problems are partially solved thanks to nanoparticles ,109.The half-life of nanoparticles in the blood plasma, the target-oriented delivery, and the bioavailability of the active substances all depend on many parameters, particularly the composition, size, and shape of the nanoparticle and the method of ligand presentation. Various materials are used to obtain nanoparticles  ,111,112.Peptides, proteins, and oligonucleotides such as DNA and RNA have significant potential for the formation of supramolecular functional nanostructures. Due to their physicochemical properties, these molecules allow the creation of nanostructures of a certain conformation to be used in various fields of biology, biotechnology, and medicine  . In receThe DNA-based technology for producing nanoparticles called DNA origami  allows fFor assembly of DNA nanoparticles, circular single-stranded DNA of bacteriophage M13mp18 is usually used  .The application of DNA nanotechnology is especially promising in the field of nanomedicine and targeted drug delivery. To use DNA/RNA nanoparticles in medicine, it is necessary to study their efficacy, safety, and immunogenicity ,121.Research in recent years has demonstrated the potential of molecular engineering in the precise design of nucleic acid scaffold devices that are capable of performing complex tasks, such as targeting drug delivery and triggering a response in the cell or the entire body ,123. ForEach nanoparticle also contains three folic acid molecules to bind to numerous folate receptors found in various tumors. Nanoparticles accumulating in tumor cells reduced the expression of pathologically associated genes by around two times. In the course of studies on mice with a xenograft of the human adenocarcinoma cell line KB, it was found that the blood circulation time of the siRNA in association with nanoparticles increased by six times; nanoparticles accumulated almost only in tumor cells and did not cause any significant immune response . RecentlDespite the fact that the first nucleic acid-based nanoparticles were designed using DNA oligonucleotides, and these experiments served as the basis for the emergence of DNA origami technology, in recent years, RNA has become an increasingly important alternative in oligonucleotide nanotechnology ,127.Structural differences between DNA and RNA determine the differences in their properties. DNA is more chemically stable and has a higher melting point. Meanwhile, RNA is able to form more diverse 3-D structures than DNA .The accumulation of knowledge about RNA in combination with the methods of synthesis and the selection of RNA molecules with certain properties has given rise to a new field of science—RNA nanotechnology . RNA nanSingle-stranded oligonucleotides are the building blocks for creating complex and functional RNA nanostructures. The ability to devise nanoparticles with a specific structure and arrangement of functional groups opens up prospects for their use in biology and medicine ,126.The secondary or tertiary structure of RNA and RNA nanoparticles can be predicted using in silico analysis of the nucleotide sequence ,132. TheThe 3WJ motif of the “packing” RNA of bacteriophage phi29—Phi29 3WJ—is used as the central core for the production of RNA nanoparticles, as well as for aptamer folding  .The first RNA nanoparticles on the Phi29 3WJ platform were designed in 1998 . The cryThe branched structure of the 3WJ motif allows the facile integration of various functional modules into three helical sections . This “pStructural elements of tRNA can be used as a framework for obtaining three-dimensional multifaceted nanoparticles. Such nanoparticles provide the intracellular and extracellular localization of ligands ,139.The integration of aptamers with RNA nanoparticles allows the targeting of a number of molecules, particularly cellular receptors, and aids in the active penetration of nanoparticles into cells through receptor-mediated endocytosis, or the passive penetration through the plasma membrane ,141,142.Replacing RNA strands with either DNA or chemical analogs increases the enzymatic and thermodynamic stability of nanoparticles. It has been shown that nanoparticles of the same shape, size, and charge, but differing in chemical composition, show noticeable differences in their physicochemical properties, subcellular localization, and immunomodulatory effect. A composition of nanoparticles can be proposed that will provide targeted drug delivery, weaken unwanted inflammation, or enhance the required immune response. The design of nanoparticles with sequences that enhance or weaken the immune response allows them to be used as vaccine adjuvants in the first variant, as well as immunoquiescent drug delivery in the second one ,145.A series of publications devoted to the use of RNA nanoparticles demonstrates the positive experience of using nanoparticles with appropriate ligands for the treatment of cancer. The studies indicate that RNA nanoparticles are characterized by chemical and mechanical stability and can be assembled in vitro from modular blocks, which simplifies their quality control  ,146.The main approach used to develop nanoparticles is in vitro synthesis, primarily using reverse transcription methods. To date, a wide variety of self-assembling structures have been obtained in vitro, such as squares, mosaics, filaments, cubic frameworks, and polyhedra .However, the high cost of synthesizing large amounts of RNA nanoparticles using reverse transcription, as well as the need to study their properties and stability within living cells, encourages research on nanoparticle synthesis approaches in vivo .E. coli, which formed spatial structures necessary for carrying out chemical reactions [In vivo production of nucleic acid-based nanoparticles is a serious problem. This idea was originally expressed by Seeman in 1997 . Howevereactions .E. coli [In subsequent years, several studies investigated the synthesis of ssDNA in bacterial cells and their in vitro folding into specified nanostructures ,152. ResE. coli .The study of the opportunity to synthesize RNA nanoparticles in eukaryotic organisms is an important stage in the development of nanotechnology. On the one hand, this can contribute to improving the technology for producing RNA nanoparticles in vivo. On the other, it will allow us to approach the study of the properties of RNA nanoparticles and their functioning in eukaryotic cells.Saccharomyces cerevisiae, which is one of the most popular model organisms in molecular biology and genetics. This is due to the combination of simplicity of cultivation, with the possibility of performing complex genetic manipulations, as well as the conservation of many key processes in yeast and mammalian cells. One of the distinguished features of S. cerevisiae is the absence of an RNA interference (RNAi) system, which was lost during evolution.Among the possible eukaryotic organisms for RNA nanoparticle production, the most interesting is the baker’s yeast As a result, stable endogenous RNAs can be found in yeast cells, and this was demonstrated in the example of double-stranded RNA viruses of the genus Totivirus (L-A viruses) and single-stranded RNA viruses of the Narnavirus genus, belonging to the Narnaviridae family . Yeast iRNA has also been an interesting molecule in drug design, including the first FDA-approved RNAi therapy for hereditary transthyretin-mediated amyloidosis in 2018  and the For the synthesis of RNA aptamers or RNA nanoparticles, yeast strains with the T7 RNA polymerase structural gene integrated into the genome can be used ,158. TraRecent advances in the production of modified RNA aptamers and the improvement of their folding methods increase the efficiency and reliability of their production. Due to their unique properties—high affinity and selectivity of binding of the target molecule—aptamers are widely used in various fields of biological and medical sciences. They can be used to detect various target molecules and inhibit their activity and also to create sensitive and specific biosensors. Aptamers can be considered as promising candidates for the targeted delivery of drugs to certain cells and tissues .The creation of fluorogenic RNA aptamers has facilitated the localization of target molecules in the cell, monitoring the transcriptional activity of the cell, studying the regulation of transcription, evaluating the activity of regulatory proteins, and assessing the level of low molecular weight metabolites. Fluorogenic aptamers can be used to diagnose diseases, localize tumors, and identify pathologically relevant proteins .The development of RNA nanotechnology has paved the way for developing RNA nanoparticles with controlled structure, size, and favorable chemical and thermodynamic properties. RNA nanoparticles can be used as a means of targeted delivery of various drugs, including miRNAs, siRNAs . IntegraE. coli cells [For the widespread use of RNA nanoparticles, it is necessary to improve the methods of their preparation. Until now, RNA nanoparticles have mainly been produced in vitro, relying on chemical or enzymatic synthesis. This requires the development of special separation methods. In vivo synthesis of RNA nanoparticles is an important aspect of RNA nanotechnology, because it will facilitate not only the obtaining of the nanoparticles but also the study of their properties and stability inside the cells. Some progress has been made in this area using li cells . HoweverAdherence to good laboratory practice (GLP), good manufacturing practice (GMP), the development of standardized protocols for production and use of aptamers and nanoparticles, and the concerted efforts of scientists, industrial partners, clinicians, and other stakeholders are the prerequisites for the realization of RNA/DNA nanotechnology in clinical practice .


A new human coronavirus named SARS-CoV-2 was identified in several cases of acute respiratory syndrome in Wuhan, China in December 2019. On March 11 2020, WHO declared the SARS-CoV-2 infection to be a pandemic, based on the involvement of 169 nations. Specific drugs for SARS-CoV-2 are obviously not available. Currently, drugs originally developed for other viruses or parasites are currently in clinical trials based on empiric data. In the quest of an effective antiviral drug, the most specific target for an RNA virus is the RNA-dependent RNA-polymerase (RdRp) which shows significant differences between positive-sense and negative-sense RNA viruses. An accurate evaluation of RdRps from different viruses may guide the development of new drugs or the repositioning of already approved antiviral drugs as treatment of SARS-CoV-2. This can accelerate the containment of the SARS-CoV-2 pandemic and, hopefully, of future pandemics due to other emerging zoonotic RNA viruses. More2+) instead of magnesium (Mg2+) as cofactor [SARS-CoV-2 is a positive-sense RNA virus belonging to the Orthocoronavirinae  family and, in particular, to the genus beta (group 2) together with the other two new human coronaviruses SARS-CoV and MERS-CoV. Similarly to all RNA viruses, the viral genome replication and transcription processes of SARS-CoV-2 depend on an RNA-dependent RNA polymerase (RdRp), which is encoded by the RNA virus to catalyze the RNA synthesis from RNA templates. Consequently, RdRp is the key enzyme in the viral biological cycle of all RNA viruses, regardless the polarity of the viral RNA genome , 10. Altcofactor .The alignment of RdRp sequences from the three epidemic/pandemic coronaviruses, confirms the high homology and conservation along the sequence Fig. a. Such h2+) instead of magnesium (Mg2+) as cofactor.On the contrary, such homology is very poor when RdRp sequences from the three epidemic/pandemic coronaviruses are aligned with negative-sense RNA viruses, namely Ebola, Influenza, Rabies, Vesicular Stomatitis virus, Measle, LCMV, Respirovirus and Orthopneumovirus Fig. c. As preThe structure modeling confirms the differences between the RdRps from positive-sense (HCV and SARS-CoV-2) and negative-sense (i.e. Influenza) RNA viruses. Indeed, considering the Motif C β-strand structure, only the alignment of RdRp structures from the two positive-sense RNA viruses results in the superimposition of the two Motifs  , FavipirIn conclusion, as for all RNA viruses, the RdRp of the newly identified positive-sense human SARS-CoV-2 RNA virus represents the most optimal target for an antiviral drug. Linear amino acid sequence as well as molecule structure show the highest homology to RdRps of other positive-sense RNA viruses. Therefore, it is highly predictable that an antiviral developed for an RNA virus with a genome of the same polarity (i.e. Sofosbuvir for HCV) could have a higher inhibitory efficacy against the SARS-CoV-2, compared to those developed for negative-sense RNA viruses. Overall, the possibility of repositioning already available drugs will significantly accelerate the containment of the SARS-CoV-2 pandemic and, hopefully, of future pandemics due to other emerging zoonotic RNA viruses.


This affected the confidence that the authors could place in the results published in The authors therefore repeated some of these experiments with newly purchased antibodies; they also changed the RT-qPCR results. In the published manuscript, the authors investigated the expression of LYPD8 mRNA expression in tissues from stages I, II, and III whereas in the revised manuscript they have investigated stages II and IV. In addition, the authors have supplemented the manuscript with new transwell assays. The revised figures are presented on the next two pages –4. RepeaIV tissue.” (‘III’ has been changed to ‘IV’).i) The fourth sentence in the Abstract, on p. 2389, should read as follows: “The results revealed that the expression of LYPD8 was significantly reduced in the CRC tissue compared with that in precancerous tissue and normal tissue, particularly in stage Histological analysis” subsection on p. 2390, the last three sentences should be replaced with the following text: “The histological sections were then stained with the DAB Kit . All sections were observed under a bright-field microscope .”ii) In the Materials and methods section, “Cell culture” subsection in the right-hand column, the first four sentences should be revised to the following: “Four CRC cell lines  were used. ® CCL-228™, organism, human; tissue, colon; disease, colorectal adenocarcinoma), SW620 , HCT116  and RKO  cells were purchased from the American Type Culture Collection . The fourSW480  In the “Correlation of the expression of LYPD8 with STAT3/P65 phosphorylation and IL-6/TNF-α secretion in patients with CRC” subsection, in the second sentence, “immunofluorescence” should have been written as “immunohistochemistry”, and the fourth sentence should have read as follows: “The results of the western blotting showed that the levels of p-P65/P65 and p-STAT3/STAT3 gradually increased between stage II and IV  In the Results section, the following changes to the text are necessary: In the “I and IV .” Then, analysis . CompareConstruction and overexpression of LYPD8 in CRC cells”, the first sentence should have read as follows: “The plasmid DNA for overexpressing LYPD8 was constructed using the eukaryotic expression vector (pIRES2), as shown in RTO, SW480 HCT116 and SW620 cells were examined by RT-qPCR analysis.v) In the subsequent subsection, “Overexpression of LYPD8 inhibits CRC cell proliferation and migration” subsection, the penultimate sentence as it appears towards the foot of p. 2392 should now read as follows: “As shown in control, LYPD8 OE + IL-6 and LYPD8 OE + TNF-α groups.”vi) In the “II and IV CRC tissues.”vi) In the Discussion, the sentence starting on p. 2394, right-hand column, line 10 should read as follows: “By contrast, the expression of LYPD8 was significantly reduced in stage vii) Finally, some revisions were necessary to the descriptions in the figure legends for immunohistochemistry images revealing activated STAT3 and P65 in colonic cancer tissue and precancerous tissue. Scale bar, 100 µm. (B) Representative western blotting revealing the expression of p-P65, P65, p-STAT3 and STAT3 in stages II and IV colonic tumor tissues. GAPDH was used as a control. (C) Band intensities of western blotting for p-P65/P65 and p-STAT3/STAT3 in stage II and IV tissues were analyzed. The data are reported as the mean ± standard deviation of experiments (n=4). **P<0.05, phosphorylation levels of STAT3 in stage II tissues vs. in stage IV tissues.II and IV colonic tumor tissue and precancerous tissue. β-actin was used as a control. The data are reported as the mean ± standard deviation of experiments (n=6). **P<0.01, LYPD8 mRNA expression of normal tissue, precancerous tissue vs. colonic tumor tissue in stage II and IV tissues.LYPD8 OE + IL-6 and LYPD8 OE + TNF-α groups of SW480 cells. (B) SW480 cells were treated with different concentrations  of niclosamide and different concentrations  of JSH-23, respectively. (C) Numbers of migratory SW480 cells from the Control, LYPD8, LYPD8 OE + IL-6 and LYPD8 OE + TNF-α groups. (D) Transwell assay of SW480 cells from the (a) Control, (b) LYPD8 OE, (c) LYPD8 OE + IL-6 and (d) LYPD8 OE + TNF-α groups .Oncology Reports for allowing them the opportunity to publish this Corrigendum; furthermore, they apologize for any inconvenience caused to the readership of the Journal.Note that the replacement of the original figures and these revisions made to the text do not drastically alter the overall conclusions reported in the study. The authors are very grateful to the Editor of


Phytophthora spp. is an oomycetes pathogen which causes serious damage to a wide range of crops. Bud rot disease of coconut palm, caused by P. palmivora, causes huge economic losses since it cannot be detected at an early stage. Utilizing dual RNA-sequencing (RNA-seq), we have simultaneously investigated the gene expression patterns in both, the infecting oomycete  and infected host (coconut leaflets). Samples were collected at three time points viz., 12, 24 and 36 h, from both infected and uninfected (control) tissues and subjected to RNA-seq on an Illumina Hiseq™ 2500 sequencing platform. High quality reads obtained were subjected to mapping with corresponding reference genomes by using the HISAT2/ StringTie package. A total of 81,683 transcripts were generated against the coconut reference genome, while 9340 transcripts were generated against P. palmivora genome. Out of these, a total of 64,639 coconut transcripts and 9168 P. palmivora transcripts could be annotated using BLASTx. Gene ontology (GO) analysis, carried out using Blast2GO, resulted in 212,643 coconut and 30,736 P palmivora transcripts being functionally classified, with a single gene product described by numerous terms under the three classifications. The insights obtained could contribute to an understanding of pathogenesis of P. palmivora and inducible defense response of coconut leaves to P. palmivora. Specifications table•P. palmivora interaction studies and can be leveraged in deciphering the molecular mechanisms underlying response of coconut to P. palmivora attack.The dual RNA-seq dataset provides the first resources for coconut–•P. palmivora that possibly contribute to manipulation of host processes and promote infection.The data allows further analysis to identify candidate effector genes in •This data could be useful for selection of disease resistant varieties for coconut breeding programs and design of novel disease management startegies.•Phytophthora spp.It is a useful reference transcriptome to other researchers working in palm diseases caused by 1Phytophthora palmivora genome is given in P. palmivora in three Gene Ontology (GO) categories viz., biological processes, molecular functions and cellular components, respectively.Schematic outline of the experimental design and RNA-seq data analysis workflow is given in P. palmivora genome. A total of 64,639 coconut transcripts and 9168 P. palmivora transcripts could be annotated using BLASTx program. Gene Ontology (GO) analysis, performed to assign GO identity to the annotated transcripts, revealed that 212,643 coconut transcripts could be assigned with GO terms    . Among t = 6396) . We prov22.1in vitro inoculation assay, standardized earlier in our laboratory 4 mL−1) of a virulent isolate of P. palmivora (GenBank accession no. MK500842.1), isolated from bud rot infected coconut palm in our laboratory and maintained in carrot agar (CA) medium, was used for the inoculation experiments. P. palmivora was inoculated onto pin pricked coconut leaflets. Typical necrotic lesions, indicating infection, were first observed after 12 h post inoculation (hpi). Samples were taken at 12 hpi, 24 hpi and 36 hpi. Un-inoculated, but pin pricked leaflets, served as controls (hc). Three biological replicates were sampled at each time point and used for isolation of RNA.The Phytophthora-specific primers (ITS6 and ITS4) P. palmivora (GenBank accession no. GU111653).To confirm the presence of the pathogen at the infection area, the infected area was wiped with alcohol, DNA was extracted from the specific area using DNeasy Plant Mini Kit  and amplified using 2.2Ⓡ RNA Plant Kit (Macherey-Nagel). Total RNA was extracted from three independent samples per treatment. The quality and the purity of the extracted RNA were assessed by OD 260 nm/280 nm ratio and RNA integrity number was analyzed using an Agilent Technologies 2100 Bioanalyzer with the Agilent RNA chip with RIN (RNA Integrity Number) > 8.0. The isolated RNA samples, from each particular satge, were pooled together and used for RNA-seq library construction . After quality assessment, the constructed messenger RNA libraries were subjected to paired-end sequencing on an Illumina HiSeq 2500 platform, as per the procdure described in Rajesh et al. Total RNA was extracted from 150 mg of tissue at each specific time points [from the infection area in case of inoculated samples and pricked area in the case of control samples] using the NucleoSpin2.3Phytophthora palmivora  fasta files were subjected to quality check. Initially, the raw reads were checked for the ambiguous bases, Phred score >Q20, read length, nucleotide base content and other parameters using FastQC 2.4Phytophthora megakarya  for funding (Distributed Information sub-center).The authors would like to thank Indian Council of Agricultural Research (ICAR) and The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.K.P. Gangaraj: Data curation, Writing - original draft. M.K. Rajesh: Conceptualization, Methodology, Supervision, Writing - review & editing.


Plant viruses cause massive crop yield loss worldwide. Most plant viruses are RNA viruses, many of which contain a functional tRNA-like structure. RNase P has the enzymatic activity to catalyze the 5′ maturation of precursor tRNAs. It is also able to cleave tRNA-like structures. However, RNase P enzymes only accumulate in the nucleus, mitochondria, and chloroplasts rather than cytosol where virus replication takes place. Here, we report a biotechnology strategy based on the re-localization of plant protein-only RNase P to the cytosol (CytoRP) to target plant viruses tRNA-like structures and thus hamper virus replication. We demonstrate the cytosol localization of protein-only RNase P in Arabidopsis protoplasts. In addition, we provide in vitro evidences for CytoRP to cleave turnip yellow mosaic virus and oilseed rape mosaic virus. However, we observe varied in vivo results. The possible reasons have been discussed. Overall, the results provided here show the potential of using CytoRP for combating some plant viral diseases. New approaches to plant disease control are important for pathogens that are difficult to control by existing methods. Here, the authors report a potential strategy to combat plant viruses by cytosolic expressed protein-only RNase P and show its ability for in vitro cleavage of tRNA-like structures existing in many plant viruses. The development of biotechnologies for the control of viral diseases is thus required to sustain food production.One of the major challenges of the 21st century is to produce sufficient food resources for a rapidly growing humanity, while the availability of cultivated land is decreasing and the environment is changing. Currently, about 900 million people suffer from chronic malnutrition, and in this context, the increase of agricultural yields will be decisive. Plant viruses are among the most devastating pathogens, being responsible for significant damages on most agronomically important crops, estimated at tens of billions U.S. dollars annually6. Viruses containing TLS, including many widespread viruses such as cucumber mosaic virus (CMV), oilseed rape mosaic virus (ORMV), and its close relative tobacco mosaic virus (TMV) or turnip yellow mosaic virus (TYMV) (Supplementary Table More than 80% of plant viruses have an RNA genome and a significant number of them bear a tRNA-like structure (TLS) at the 3′ end of their genomic RNA, which is critical for viral infectivity and for virus replication7 and is thus essential to obtain functional tRNAs required for translation. Until recently, all characterized RNase P enzymes were ribonucleoproteins, containing an RNA holding catalytic activity7. However, another type of RNase P only composed of protein was discovered in eukaryotes8. Beyond tRNA maturation, these enzymes called PRORP also participate in mRNA maturation by cleaving TLS present at the 5′ or 3′ termini of some plant mitochondria mRNAs, as shown in Arabidopsis both in vitro and in vivo10.RNase P is an endonuclease activity responsible for one of the key steps of tRNA precursors maturation. It removes 5′ leader sequences of pre-tRNAs11, while PRORP enzymes accumulate only in compartments where endogenous gene expression takes place, i.e. the nucleus, mitochondria, and chloroplasts9. We thus develop an approach to allow RNase P enzymes to accumulate in the cytosol. Early work had already shown that bacterial RNA-based RNase P is able to cleave the TLS of plant viruses, in particular that of the TYMV12. However, the manipulation of RNase P ribozyme localization is hardly feasible, and we thus modify a plant PRORP enzyme to allow its accumulation in the cytosol  conserved in land plants nuclear PRORP enzymes  domain responsible for substrate-specific RNA binding, and a C-terminal Nedd4-BP1, YacP nuclease (NYN) domain that holds the endonuclease activityE. coli, affinity-purified , its catalytically inactive mutant (CM) as well as wild type PRORP2 (P2) were then used in activity assays to test their ability to cleave the TLS of in vitro transcripts corresponding to the 3′ end of TYMV genomic RNA. Results using both 5- and 3′-end radiolabelled transcripts showed that CytoRP is able to cleave the TYMV TLS in vitro, as cleavage products with sizes matching those expected to result from canonical RNase P activity were detected in lanes containing CytoRP  was expressed in oRP Fig. . As a po12. This result thus demonstrates that CytoRP recognition and binding of the 3D fold of the TYMV TLS is similar to that of PRORP2 recognition of a pre-tRNA13. Minor cleavages also occurred 1 or 2 nt upstream or downstream of the main cleavage site, thus suggesting a flexibility of the TYMV TLS 3D fold in vitro. In order to better characterize the cleavage of the TYMV TLS by CytoRP, kinetic analyses were also performed in single turnover conditions, revealing a Km of 222 × 10−6 μM and a Vmax of 2.4 × 10−7 nM/s, whereas wild type PRORP2 cleaved the TYMV TLS with a Km of 52 × 10−6 μM and a Vmax of 4.6 × 10−7 nM/s, thus showing that PRORP2 is a somewhat better catalyst in vitro than its mutant CytoRP  within TYMV TLS, the 5′ cleavage product of the TLS was then characterized by circular RT-PCR, cloning, and sequencing. This revealed that the majority of cleavage events had taken place in the TLS pseudoknot, 20 nt upstream of the RNA 3′ end. This indicates that CytoRP performed canonical RNase P activity and cleaved the TLS at the position predicted from the analysis of a three-dimensional model of the CytoRP/TLS complex Fig. , and simThen, in order to show that the CytoRP strategy could be applicable to different viruses, a transcript representing the 3′ end of ORMV genomic RNA composed of a TLS was used for RNase P activity assays. RNA cleavage was observed and precisely mapped by circular RT-PCR. This showed that most cleavages occurred in a predicted pseudoknot of the TLS, 43 nt upstream of the RNA 3′ end Fig. . AltogetCauliflower mosaic virus. In addition, cisgenic plant lines were also generated by genome editing through the deletion of 16 amino acids of PRORP2 starting at the 11th amino acid using CRISPR-Cas9 technology  reductions of both TYMV and ORMV RNA content as compared to wild type plants in some sets of experiments expressing a viral protein that may interfere with the viral life cycle due to its imbalanced and/or uncoordinated expression in the host cell or through RNA silencing19. Available approaches include expression in plants of artificial microRNAs20 or the expression of plantibodies, i.e. recombinant antibodies raised against viral proteins21. These approaches are sequence-specific or epitope-specific and thus result in narrow ranges of antiviral resistance.In all cases, what differentiates the CytoRP antiviral strategy from current antiviral approaches is that it should theoretically allow disease management of many viruses at once, i.e. of all viruses harboring a TLS in their genome  at 18 °C overnight with 1 mM IPTG for expression induction. Bacteria were lysed and pelleted 30 min at 20,000×g 4 °C. Cleared lysates were incubated with the Ni-NTA resin (Qiagen). Proteins retained were washed with buffers including 50 mM imidazole, 20 mM MOPS pH 7.8, 150 mM NaCl and 10% (v/v) glycerol, and 75 mM imidazole, 20 mM MOPS pH 7.8, 250 mM NaCl and 10% (v/v) glycerol. Protein elution was carried out with buffers containing 200 mM imidazole and 500 mM imidazole. Proteins were further purified by size-exclusion chromatography, with a Superdex 200 10/300 Increase column, using an automated Äkta pure system in buffer containing 20 mM MOPS pH 7.8, 150 mM NaCl, and 10% (v/v) glycerol.CytoRP corresponds to Arabidopsis PRORP2 amino acids 25–519 amplified with oligonucleotides CRP5′ and CRP3′. All oligonucleotides sequences used here and mentioned hereafter in the “Methods” section are available in Supplementary Table BamHI, T7 promoter sequence, and EcoRI restriction sites, respectively. The PCR product was cloned in pUC19. The clone used to express an Arabidopsis pre-tRNA Cys was used by Gobert et al. 9. Linearized plasmid DNA (200 ng) was transcribed in a 10 µL reaction comprising 7.5 mM rNTP, 5 U T7 RNA polymerase and buffer supplied with the enzyme  for 4 h at 37 °C. Plasmid DNA was then digested with 1 U of DNase I (Thermo Scientific) for 15 min at 37 °C and transcripts were cleaned by phenol chloroform (v/v) extractions. RNAs were dephosphorylated with 1 U FastAP alkaline phosphatase (Thermo Scientific) for 30 min at 37 °C and where then either 5′ radiolabelled with 32P-γATP and polynucleotide kinase, or 3′ radiolabeled with 32P-pCp and T4 RNA ligase (New England Biolabs).A cDNA fragment representing the 3′ end of TYMV genomic RNA was amplified with oligonucleotides TYMV5′ and TYMV3′, as well as the 3′ end of ORMV genomic RNA was amplified with oligonucleotides ORMV5′ and ORMV3′ containing the 2, 20 µg/mL BSA, and 2 mM DTT, at room temperature for 15 min. RNA molecules were separated on 12% polyacrylamide urea gels and visualized by autoradiography and/or by ethidium bromide staining under UV light. For kinetic analyses, cleavages were performed with 1 μM CytoRP or PRORP2, 20, 40, 80, and 120 pM of TYMV TLS RNA substrate for times varying from 5 min to 2 h, in triplicates for each condition.Cleavage reactions were performed in a 10 µL volume containing 100 ng proteins and 100 ng 5′ or 3′ radiolabelled transcript, in buffer containing 20 mM Tris–HCl pH 8, 30 mM KCl, 4.5 mM MgClTYMV and ORMV 5′ RNA fragments generated from RNase P cleavages were visualized by Ethidium bromide staining, purified from gel and incubated with 5 U of T4 RNA ligase (New England Biolabs) and 20 U RNase inhibitor  in a reaction volume of 25 µL. Circular RNA was used for cDNA synthesis with Super-script III (Invitrogen), with primers CRTTYMV5′ and CRTTYMV3′ for TYMV as well as CRTORMV5′ and CRTORMV3′ for ORMV.Agrobacterium tumefaciens GV3101. Plants were selected for hygromycin resistance, by genotyping with primers GtTg5′ and GtTg3′. Plants were also selected for CytoRP expression. The selected plants contain the three PRORP expressed from endogenous genes, as well as the additional CytoRP expressed from the transgene and localized in the cytosol. Six distinct Arabidopsis lines (4 with PRORP2 endogenous promoter and 2 with the 35S promoter), resulting from different insertions of the CytoRP gene randomly in the genome were selected for further use.A PCR-amplified fragment containing the CytoRP cDNA in frame with an HA tag, inserted after either the promoter sequence of Arabidopsis PRORP2 or the CaMV 35S promoter, was cloned in a binary vector and used to generate transgenic plants using Agrobacterium-mediated transformation, by floral dip with AAGAAGAAGA. It was cloned into pENchimera entry vector and transferred to pDECas923. Transgenic plants expressing Cas9 and the guide RNA were produced using Agrobacterium-mediated transformation. Deletion were selected by PCR in the T2 generation with primers GtCg5′ and GtCg3′. The selected plant was backcrossed three times with WT Col-0 in order to get rid of the T-DNA insertion and possible off-targets mutations.A guide RNA targeting the conserved lysines (underlined codons) of PRORP2 NLS was designed AAGACCCAACCytoRP cDNA was inserted into the pART7EYFP vector. Arabidopsis mesophyll protoplasts were isolated and transformed with pART7PRORPEYFP plasmids using polyethylene glycol-mediated transformation. EYFP fluorescence was observed by confocal laser scanning microscopy using a Zeiss LSM700 based on an Axiovert 200M microscope (Zeiss).A. thaliana plants were grown at 19–21 °C under a 16 h light/8 h dark photoperiod in a growth chamber, and plants were inoculated ~5–6 weeks after seed germination, before the occurrence of floral transition. The inoculum consisted of TYMV and ORMV viral RNA (4 µg per plant) diluted in 45 µL of water. Plants were mechanically inoculated by rubbing the upper surface of three rosette leaves using celite as an abrasive24. After inoculation, the plants were allowed to grow in the same conditions, harvested at 28 dpi for TYMV and 7 dpi for ORMV, frozen in liquid nitrogen and stored at –80 °C. A total of 17–20 plants was inoculated in two independent experiments.http://boxplot.tyerslab.com/).Total RNA extraction was performed using Nucleospin RNA kit (Macherey Nagel) and cDNA synthesis was performed with SuperScript III reverse transcriptase (ThermoFisher). The abundance of viral RNAs in plant samples was performed by quantitative RT PCR, mainly using a Roche LightCycler 480 with EF1α and 18S rRNA as reference genes. Data relative to CytoRP plants were then expressed as a percentage of the mean value of the data obtained with Col-0 plants that were inoculated simultaneously and analyzed by qPCR in the same run. Data are represented as box-plots using BoxPlotR  and molecular representations were prepared with PyMol26.Subcellular localization predictions were determined with SUBA4Further information on research design is available in the Supplementary Information FileReporting Summary


Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP target RNAs. Here, we develop an ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing method, easyCLIP. easyCLIP provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize RNA libraries during sequencing library preparation. Measurement of >200 independent cross-link experiments across >35 proteins identifies an RNA cross-link rate threshold that distinguishes RBPs from non-RBPs and defines target RNAs as those with a complex frequency unlikely for a random protein. We apply easyCLIP to the 33 most recurrent cancer mutations across 28 RBPs, finding increased RNA binding per RBP molecule for KHDRBS2 R168C, A1CF E34K and PCBP1 L100P/Q cancer mutations. Quantitating RBP-RNA interactions can thus nominate proteins as RBPs and define the impact of specific disease-associated RBP mutations on RNA association. It is important to develop quantitative criteria to identify RNA-binding proteins and target RNAs. Here, the authors present a method to quantify cross-linking and apply it to recurrent cancer mutations in RNA-binding proteins. Landmark proteomic efforts from multiple groups have identified many potential novel RBPs7; some, such as sequestosome-18, were subsequently verified and studied by cross-linking immunoprecipitation (CLIP), while the vast majority of which have not yet been evaluated by non-proteomic approaches9. Many important proteins studied in a different context have been categorized as also binding RNA, yet few or no experiments have been published on their functions in RNA binding. For example, proteins important to cancer, such as BRCA1, SMAD3-4, SPEN, CHD2, and JUN, have been categorized as RBPs, yet are not generally studied as such, raising the question as to whether they actually act in that role. Addressing this question for such proteins, and for additional potentially novel RBPs, has been hindered by the lack of a test that quantitates RNA interaction events per protein molecule to provide a global cutoff level of RNA binding to nominate a protein as an RBP.Approaches to quantify protein–RNA cross-links on a per-molecule basis are not widely available, leading to confusion both as to what constitutes an RNA-binding protein (RBP) and to the quantitative impact of disease-associated RBP mutations. For example, only roughly half of the proteins either in the RBP census10, are ultimately but indirectly determining if the absolute count of an RNA–protein complex in the cell is abnormally high. Defining RNA–protein interaction events per cell and per protein in absolute quantities, in contrast, may provide a framework for describing a global and widely reproducible view of RNA–protein interactions.Currently, there is no general method to estimate absolute RNA–protein interaction frequencies and a quantitative test is needed to assess whether any nonrandom interaction with an RNA exists. The frequencies of RNA–protein complexes, per-cell and per-interaction partner, would enable the fundamental characterization of RNA–protein interaction networks. Determining the targets of an RBP by conventional approaches, such as enrichment over negative control immunopurification or by clustering of cross-links12, indicating that there must be unknown mutations within or affecting RNA metabolism pathways that are collectively common. There are widespread RBP expression changes in tumors, and the alternative splicing of tumor cells is predicted to affect cancer hallmarks, with some tumor types reverting to a more undifferentiated splicing pattern13. Many cancer-associated genes are potentially RBPs and some RBPs contain recurrent missense mutations. More generally, recurrent mutations that are not exceptionally frequent are of unknown significance14.A number of RBPs are mutated in human cancers; however, the impact of such mutants on their association with RNA has not been quantitated. Most tumors have aberrant splicing without apparent mutational causeHere, we report a refinement of current CLIP protocols, termed easyCLIP. easyCLIP quantifies RNA cross-links per protein and provides visual confirmation of each step. easyCLIP enables the calculation of the distribution of cross-linking for the average protein and we propose a quantitative threshold for whether a protein is an RBP. Establishing a distribution for non-RBPs enables the definition of specific target RNAs for any RBP as those interactions with a frequency per protein are unlikely to occur with a randomly selected protein. easyCLIP is applied to the top 33 most frequent missense mutations across 28 RBPs, identifying quantitative changes in RNA binding in specific RBPs that are recurrently mutated in cancer. easyCLIP represents a method with built-in verifications that enables quantification of the number of RNA cross-links per protein in wild-type (WT) and disease-associated mutant RBPs.15 to identify 93 RBPs associated with cancer  were compared with the COSMIC  list of cancer-associated genescer Fig. , of whiccer Fig. . Proteincer Fig. . Notablency Fig. , which r 14 Fig. . The tis 14 Fig. . Of thos. 6 Fig. . Further19 of 3′ adapters (termed L3) and 5′ adapters (termed L5), each with a different fluorescent dye  in the manner in which eCLIP was benchmarked against iCLIP , using Rbfox2. easyCLIP was more efficient , target RNA numbers  cross-linked, hnRNP C immunopurified, and RNA digested, a series of bands were visible  all RNA and (b) minimal region RNA Fig. . “All RNked Fig. .Fig. 5RN7 cells), had total RNA cross-link values of 0.03–4%  in cross-link rate vs WT controls  and RNA biotypes. RBPs tended to have a significant reduction in the fraction of reads mapping to ribosomal RNA (rRNA), and an increase in reads mapping to mRNA, relative to non-RBPs Fig. . AssuminTarget RNAs were next identified as those bound per read at an unusually high rate. The success of this method is supported by the identification of the expected motif for all RBPs as the top motif Fig. , and tarP < 0.05, two-sided t test, Fig. R6C , the lncRNA PURPL (p53 upregulated regulator of p53 levels30) was one of the five most significant changes for both KHDRBS2 and A1CF  than WT PCBP1, and CDKN1A was the eighth most significantly downregulated RNA in PCBP1L100Q-expressing cells  and A375 (ATCC) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) media  with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C under 5% CO2. HCT116 cells (ATCC) were grown in McCoy’s 5A (modified) medium , supplemented with 10% FBS and 1% penicillin–streptomycin, and grown at 37 °C under 5% CO2. HepG2 cells (ATCC) were grown in Eagle’s minimum essential medium (ATCC 30-2003), supplemented with 10% FBS and 1% penicillin–streptomycin, and grown at 37 °C under 5% CO2.A260 ratio using an approximate ε = 368,050 M−1. Oligonucleotides were diluted to 10 nM in ligation buffer , 1 µL was blotted onto a nylon membrane, and fluorescence was measured in an Odyssey CLx machine (LI-COR). This was typically ~15,000 fluorescence units/fmol for full labelling.IRDye 680RD DBCO (0.5 mg)  was resuspended in 42.9 µL phosphate-buffered saline (PBS) for a concentration of 10 mM. The L5 linkers (Azide-DNA-RNA oligonucleotides) were ordered from IDT (Integrated DNA Technologies) and resuspended in PBS. Oligonucleotides were run through a Zymo RNA-clean-and-concentrator kit (purification was required for labelling), using ~7 µg oligonucleotide per column, and eluting at ~0.5 mg/mL (~40 µM) in water. Before binding to columns, we added ethanol to a final concentration of 67% instead of the 50% recommended by the manufacturer. During column purifications, washes were performed using an 85% ethanol in water solution made fresh each time, in place of the kit’s wash buffer. Five microliters of 10 mM dye (~50 nmol) was added to 10–150 µg purified oligonucleotide (~1–12 nmol) in PBS for a total volume of 200 µL and reacted for 2 h at 37 °C. Oligonucleotides were then run again through a Zymo clean-up kit and eluted in water. Concentrations were determined by 40, purified gluthathione S-transferase (GST)-tagged protein standards were run alongside the samples to be quantified. Purified GST-hnRNPC2 and purchased FBL  were diluted in protein dilution buffer  to 200 ng/µL. Twofold dilutions down from 20 to 100 ng/µL were made for a total of eight concentrations; this solution was then delivered as 14 µL aliquots to multiple striptube aliquots and frozen at −80 °C. When running gels, 10 µL from each concentration was combined with 10 µL loading buffer (3.6× NuPAGE loading buffer with 10% β-mercaptoethanol), heated at 75 °C for 15 min, and loaded on a 4–12% NuPAGE gel. Standards were therefore present at ~3–2000 ng per lane. Immunoblotting against the HA epitope was performed with 1:3000 αHA conjugated to Alexa Fluor 488 and incubating for 1 h at room temperature in PBS blocking buffer (LI-COR); images were taken in a GE Typhoon scanner . When small aliquots of immunopurification beads were loaded on a gel, BSA was first added to 0.2 mg/mL to prevent absorption. Primary antibodies were diluted 1:1000 and secondary antibodies diluted 1:3000 for immunoblots, including nonquantitative immunoblots, unless otherwise noted.Following the method of JanesFour replicates of 906–1600 µg of HCT116 lysate from cross-linked cells were added to ~20 µL Protein G Dynabeads  coupled with 25 µL (5 µg) anti-hnRNP C (4F4) antibody per replicate. Immunoprecipitation was carried out at 4 °C for ~1 h, followed by the standard easyCLIP protocol for cross-link rate determination. The RNAse digestion was performed with half of the samples treated with 0.1 U/µL RNAse ONE for 10 min, and the other half of the samples were treated with 0.05 U/µL RNAse ONE for ~5 min. The polynucleotide kinase (PNK) reaction was 14 min at 37 °C. The ligation was performed overnight (17 h) with 20 pmol L5 (barcode 23) and 2 µL high concentration of T4 RNA ligase (NEB). Samples were combined, and ~20 aliquots comprising 2.5% of the beads  in ~15 µL 1.6× NuPAGE buffer were frozen in dry ice and kept long term at −80 °C. Immunoblotting was performed with ~1:3000 αhnRNP C conjugated to AF790 , which is visible on the 800 nm channel in a LI-COR Odyssey scanner, in PBS blocking buffer (LI-COR) for ~1 h at room temperature.2. After cross-linking, 1 mL 4 °C lysis buffer (15 cm plates) or 0.5 mL lysis buffer (10 cm plates) was added to each plate, cells were harvested with a rubber spatula, and frozen in dry ice. CLIP lysis buffer was as in Zarnegar et al.23, except the concentration of Triton X-100 was 1%  in 10 or 15 cm plates in a Stratalinker at 0.3 J/cmCloning primers are included in Supplementary Data 7 cells) with a pLEX vector bearing a uORF to lower expression. Expression levels were low compared to endogenous protein , using a >50% confluent 10 cm or 15 cm plate with HEK293T cells per replicate. RBFOX2 was purified using antibodies to the endogenous protein  and 20 million HEK293T cells. PCBP1 was stably integrated outside its genomic locus in HCT116 cells (harvesting one ~70% confluent 15 cm plate per replicate). The others were transiently transfected into HEK293T cells growing in 10 cm plates (~2 × 107 cells) or 15 cm plates (~3 × 107 cells) with either a pLEX vector bearing a uORF to lower expression or a vector modified from pLEX to remove vector sequences to boost copy number, increase expression, or form viral particles. HA-STAU1 and randomly selected non-RBPs were expressed from a pLEX vector. Expression was 18–28 h before harvesting. WT and mutant proteins were always expressed, harvested, and processed together. Expression levels were low compared to endogenous protein , transferred to nitrocellulose, and visualized. The amount of RNA that was neither 5′ nor 3′ ligated was determined by the following reasoning. First, let P5 be the probability of a 5′ ligation, and P3 be the probability of a 3′ ligation. Let a = RNA with no ligation; b = RNA with a 3′ ligation only; c = RNA with a 5′ ligation only; and d = RNA with a 5′ and 3′ ligation. Let T = the total amount of RNA. It follows that:The ligation efficiency test with hnRNP C was performed in three replicates. hnRNP C was purified by incubating 600 µg of clarified HEK293T lysate with 4 µg anti-hnRNP C1/C2 antibody for 1.5 h at 4 °Ca × d = b × c. Since d, b, and c are determined by direct visualization of fluorescence, it follows that the RNA with no ligation (a) is also known.Rearranging terms shows that github.com/dfporter/easyCLIP. For the peak locations used for motif finding in Fig. Scripts used for CLIP analysis are available at σ2/µ was >2 for these samples, they were fit to a negative binomial, and they were fit to a Poisson if σ2/µ was <2. P values were calculated accordingly before finally adjusting all P values for each protein by the Benjamini–Hochberg method into FDR equivalents.Inputs to statistical analysis were either reads per million or reads per ten billion proteins, both treated the same. To speed up analysis, for the randomly selected non-RBPs constituting background, if a replicate had no reads it was assigned one-tenth the minimum positive count present in that dataset . The average count across replicates for each protein was determined, resulting in a sample of eight values taken from the null distribution . If Additional methods are described in Supplementary Data Further information on research design is available in the Supplementary InformationReporting SummarySupplementary Datas 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Description of Additional Supplementary Files