Nucleobase deamination, such as A-to-I editing, represents an important posttranscriptional modification of RNA. When deamination affects guanosines, a xanthosine (X) containing RNA is generated. However, the biological significance and chemical consequences on RNA are poorly understood. We present a comprehensive study on the preparation and biophysical properties of X-modified RNA. Thermodynamic analyses revealed that base pairing strength is reduced to a level similar to that observed for a G•U replacement. Applying NMR spectroscopy and X-ray crystallography, we demonstrate that X can form distinct wobble geometries with uridine depending on the sequence context. In contrast, X pairing with cytidine occurs either through wobble geometry involving protonated C or in Watson–Crick-like arrangement. This indicates that the different pairing modes are of comparable stability separated by low energetic barriers for switching. Furthermore, we demonstrate that the flexible pairing properties directly affect the recognition of X-modified RNA by reverse transcription enzymes. Primer extension assays and PCR-based sequencing analysis reveal that X is preferentially read as G or A and that the ratio depends on the type of reverse transcriptase. Taken together, our results elucidate important properties of X-modified RNA paving the way for future studies on its biological significance. Deamination of the nucleobases in DNA and RNA is a result of spontaneous hydrolysis, endogenous or environmental factors as well as deaminase enzymes ,2. As a 6A modification sites in RNA by exploiting the fact that nitrous acid deaminates all exocyclic amino groups present in nucleobases and thus significantly changes sequence information but leaves m6A intact  for column chromatography was purchased from Macherey-Nagel. The procedures for chemical synthesis of xanthosine phosphoramidite 9 and the characterization data are available in the Supporting Information  are reported relative to tetramethylsilane (TMS), referenced to the residual solvent signal . The following abbreviations were used to denote multiplicities: s = singulet, d = doublet, t = triplet, q = quadruplet, m = multiplet, b = broad. Signal assignments are based on 1H–1H-COSY, 1H–13C-HSQC and 1H–13C-HMBC experiments. High resolution mass spectra were recorded in positive ion mode on a Thermo Scientific Q Exactive Orbitrap, ionized via electrospray at 3.7 kV spray voltage.Reagents were purchased in the highest available quality from commercial suppliers  and used without further purification. All reactions were carried out under argon atmosphere, unless otherwise noted. Analytical thin-layer chromatography (TLC) was performed on Macherey-Nagel Polygram® SIL G/UV9 (>98% coupling yield). 2′-O-TOM and acetyl protected nucleoside phosphoramidite building blocks and 2′-O-Tbs 1000 Å CPG solid support (>15nt) were purchased from ChemGenes, Primer supportTM 5G (<15nt) was purchased from GE Healthcare. 15N-labeled nucleoside phosphoramidites were provided by INNotope (https://www.innotope.at). All oligonucleotides were synthesized on an ABI 392 nucleic acid synthesizer following standard methods: detritylation (90 sec) with dichloroacetic acid/1,2-dichloroethane (4/96); coupling (5.0 min) with phosphoramidites/acetonitrile  and benzylthiotetrazole/acetonitrile ; capping (2 × 25 sec) with Cap A/Cap B (1/1) for unmodified and xanthosine modified RNA, Cap A: 4-(dimethylamino)pyridine/acetonitrile (500 mM), Cap B: acetic anhydride/sym-collidine/acetonitrile (2/3/5); oxidation (60 sec) with iodine (20 mM) in tetrahydrofuran/pyridine/H2O (35/10/5). Solutions of phosphoramidites, tetrazole and Cap were dried over activated molecular sieves (3 Å) overnight.Standard phosphoramidite chemistry was applied for RNA strand elongation and the incorporation of xanthosine building block 2O/ethanol  and once with tetrahydrofuran. Combined supernatant and washings were evaporated to dryness and the residue was dissolved in a solution of tetrabutylammonium fluoride in tetrahydrofuran  and incubated for 14 h at 37°C for removal of 2′-O-silyl protecting groups. The reaction was quenched by addition of triethylammonium acetate/H2O . Tetrahydrofuran was removed under reduced pressure and the sample was desalted with size-exclusion column chromatography  eluting with H2O; collected fractions were evaporated and the RNA dissolved in H2O (1 ml). The crude RNA was purified by anion exchange chromatography  on a semipreparative Dionex DNAPac® PA-100 column (9 mm x 250 mm) at 80°C with a flow rate of 2 ml/min . Fractions containing RNA were evaporated an the residue redissolved in 0.1 M triethylammonium bicarbonate solution (10 to 20 ml), loaded on a C18 SepPak Plus® cartridge (Waters/Millipore), washed with H2O, and then eluted with acetonitrile/H2O (1/1). Crude and purified RNA were analyzed by anion exchange chromatography  on a Dionex DNAPac® PA-100 column (4 mm × 250 mm) at 80°C with a flow rate of 1 ml/min. For RNA up to 15 nucleotides in length, a gradient of 0–44% B in 30 min was applied; for longer RNA a gradient of 0–60% B was applied; eluent A: 20 mM NaClO4, 25 mM Tris·HCl, pH 8.0, 20% v/v acetonitrile; eluent B: 600 mM NaClO4, 25 mM Tris·HCl, pH 8.0, 20% v/v acetonitrile. HPLC traces were recorded at UV absorption by 260 nm. RNA quantification was performed on an Implen P300 Nanophotometer.For deprotection of unmodified and xanthosine modified RNA, the solid support was mixed with aqueous methylamine  and aqueous ammonia  for 15 to 25 minutes at 65°C. The supernatant was removed and the solid support was washed twice with H2H2EDTA) . Water was added to obtain a total volume of 30 μl. The sample injected onto a C18 XBridge column  at a flow rate of 0.1 ml/min and eluted using a gradient 0 to 100% B gradient at 30°C . RNA was detected by a Finnigan LCQ Advantage Max electrospray ionization mass spectrometer with 4.0 kV spray voltage in negative mode.RNA samples (∼200 pmol in ∼3 μl) were diluted with aqueous soulution of ethylenediaminetetraacetic acid disodium salt dihydrate (Na2HPO4 (pH 7.0) containing 150 mM NaCl) and transferred into UV permeable high precision cells made of quartz SUPRASIL® with a light path of 10 mm or 1 mm. UV melting profiles were recorded at 250 and 260 nm on a Varian Cary-100 spectrophotometer equipped with a multiple cell holder and a peltier temperature control device. Each RNA was measured at five different concentrations (between ∼1 and ∼100 μM) and with at least four ramps . Tm values were determined by calculating the first derivative, usually from data of the fourth ramp (cooling). Thermodynamic parameters were determined according to reference  and transferred into 5 mm NMR tubes. Sample concentrations varied between 0.1 and 2.0 mM and experiments were run at 298 K unless otherwise stated. All NMR experiments were conducted on a Bruker 600 MHz Avance II + NMR or a 700 MHz Avance Neo NM both equipped with a Prodigy TCI probe. Assignments of imino proton signals were achieved by a combination of 1H–1H jump and return NOESY and 1H–15N sofast HSQC experiments.RNA samples were lyophilized as triethylammonium or sodium salts, dissolved in 500 μl NMR buffer  (2H2EDTA (1 mM), pH 8.0. Both RNA samples were then heated to 55°C and cooled down to 10°C using a temperature-controlled device equipped with a Peltier element. For Xan2648-SRL, only one unique cubic-shaped crystal could be obtained. It grew after one month at 20°C using vapor diffusion method by mixing one volume of RNA sample with one volume of a crystallization buffer made of ammonium sulfate (2.5 M), magnesium acetate (10 mM), and 2-(N-morpholino)ethanesulfonic acid (MES) (50 mM), pH 5.6 . Prior data collection, the crystal was cryoprotected for about 5 min in a reservoir solution containing 15% of glycerol and 3.0 M of ammonium sulfate, flash-frozen in liquid ethane and then transferred into liquid nitrogen. The Xan-14-mer RNA was crystallized at 20°C by mixing one volume of RNA sample with one volume of a solution made with 25 mM MgSO4, 50 mM Tris-HCl pH 8.5, 1.8 M ammonium sulfate. Crystals were cryoprotected for about 5 min by adding ∼5 volumes of a solution made with 2.5 M ammonium sulfate and 20% glycerol, then flash frozen in liquid ethane. The Xan-12-mer RNA was dissolved in water at a concentration of 1 mM, heated at 80°C for 10 min and cooled at 20°C at a 1°C min–1 rate. Crystals were grown at 20°C by mixing 2 μl of RNA sample with 2 μl of a solution made with 10% (v/v) 2-methyl-2,4-pentanediol (MPD), 40 mM sodium cacodylate pH 7.0, 12 mM spermine, 80 mM NaCl, 30 mM MgCl2, against a reservoir made by 35% MPD. Crystals were frozen in liquid ethane without any further cryoprotection. X-ray diffraction data at atomic resolution for all three crystals was performed on the X06DA beamline at the SLS synchrotron, Villigen, Switzerland. Processing of the data was done with the XDS Package  ,21 and cop (SRL)  but cont Package  and struThe 37 nt RNA 5′-AUUCCUCXUCAUCCAUACAGACAGAACUAACGAUUCG  and 4 μl of Alexa Fluor 647 5′-end labelled DNA primer  were annealed for 5 min at 65°C, then incubated at 35°C for 5 min and cooled on ice for 1 min . Then, 8 μl of a ‘primer extension mix’ containing 4 μl of 5× SS IV RT buffer (provided by supplier), 1 μl of 0.1 M 1,4-dithiothreitol (DTT) solution, 1 μl of 5 mM dNTPs mixture (1.25 mM for each dNTP), 2 μl of dimethyl sulfoxide (DMSO), and 0.4 μl of SuperScript IV reverse transcriptase  were added and the reaction mixtures incubated at 55°C for 10 min ,29. The 2, 100 mM DTT, 5 mM ATP (pH 7.8 at 25°C)), poly(ethylene glycol) (50% PEG 4000 solution) (20 μl) and T4 DNA ligase  were added to obtain a total volume of 200 μl. The mixture was incubated for 5 h at 37°C. The reaction was stopped by a phenol/chloroform extraction (twice) and extracted three times with chloroform to remove remaining phenol from the reaction. Analysis of the reaction and purification was performed via anion exchange chromatography on analytic columns  was prepared by splint ligation using T4 DNA ligase (Fermentas). Both RNAs fragments  and the DNA splint oligonucleotide  were dissolved in water (140 μl), heated to 90°C for 2 min, and cooled to room temperature. Then, 10× ligation buffer  using either the GoScript Reverse Transcription System (Promega) or SuperScript IV (ThermoFisher) according to the manufacturer's instructions. The resulting cDNA was amplified by PCR using primers Xant_fw (5′-AAUGUAAAACGACGGCCAG) and Xant_rev (5′-CGAATCGTTAGTTCTGTC) and GoTaq polymerase (Promega). PCR products were purified from agarose gels, ligated to pGEM-T vector (Promega) and used to transform bacteria. Plasmid DNA was isolated from single colonies and subjected to Sanger sequencing.O-tert-butyldimethylsilyl (TBS) phosphoramidite. This building block was used in an early mutational study from 1993 to evaluate the activity of active site nucleosides in the hammerhead ribozyme  and the resulting derivative 2 was treated under Mitsunobu conditions to introduce the O6-(4-nitrophenyl)-2-ethyl (NPE) protecting group which gave compound 3. The selective cleavage of the DMT under acidic conditions provided the free NH2 functionality of compound 4 that was further transformed into the 2-oxo group of compound 5 via the diazonium intermediate using aqueous sodium nitrate in acetone and acetic acid. Then, alkylation of O2 using 1-(2-iodoethyl)-4-nitrobenzene (NPE-I) and silver carbonate proceeded smoothly with high selectivity to yield compound 6. Selective removal of the 3′,5′-O-di-tert-butyl silylether was accomplished with hydrogen fluoride (HF) in pyridine and gave the diol 7 in high yield, leaving the 2′-O-tert-butylsilyl group unaffected. Nucleoside 7 was then transformed into the dimethoxytritylated compound 8 using 4,4′-dimethoxytriphenylmethyl chloride in pyridine without any additional base to avoid migration of the 2′-O-tert-butylsilyl group. Finally, phosphitylation was executed with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite in the presence of 1-methylimidazole and sym-collidine. Starting from guanosine, our route provides phosphoramidite 9 with 21% overall yield in nine steps and with nine chromatographic purifications; in total, 2.1 g of 9 was obtained in the course of this study.Our novel synthetic route to obtain a powerful nucleobase protected xanthosine phosphoramidite started from guanosine by applying Beigelman protection of the 5′ and 3′ hydroxyl groups to regioselectively install the 2′-9 into oligoribonucleotides proceeds with excellent coupling yields using standard RNA solid-phase synthesis protocols. Importantly, deprotection of the NPE groups also proceeds under standard conditions. Figure 9 was applied in combination with standard N-acetylated 2′-O-[(triisopropylsilyl)oxy]methyl (TOM) phosphoramidites and the oligomers were assembled on controlled pore glass (CPG) supports . Of note, we had no indication for depurination at the RNA xanthosine lesions at physiological and lower pH values (down to pH 4.5). The latter were found problematic for the corresponding 2′-deoxyxanthosine lesions in DNA   to C(N3+-H) is conceivable  Figure , top; fue Figure , bottom.nthosine , and theTm) and the corresponding thermodynamic parameters .The sequence design of the RNAs investigated here is illustrated in Figure Ic/If and Ic/Ig). Also for C opposite of X, destabilization is significant and of comparable extent . Only when U is located opposite of X, destabilization is less .Table IIa) and at 59.7°C . Replacement of the purine-7 by xanthosine causes destabilization that is larger for C opposite of X  and smaller for U opposite of X .The same trend is observed for the palindromic set of RNAs. The native type II RNAs melt at 74.5°C . Substitution of G4 by X results in a X4–C11 pair accompanied by a drastic decrease of 31.7°C in Tm value (IIIb), while substitution of A3 by X results in a X3–U12 pair that gave a minor decrease of 5.3°C only (IIIc). This reflects the same trend as observed for bimolecular systems, showing that out of the four natural nucleosides, uridine is tolerated best for stable pairing to xanthosine.Next, we analyzed the impact of xanthosine in a monomolecular RNA system  melting at 71.1°C . In. Inw1U–XKaN3-H value of 5.2 was reported , IIb (U–A) and IIe (U–G) displayed little pH dependence in the same pH range  is involved in base pair formation with X , the imino proton of both, purine (N1-H) and pyrimidine (N3-H), are observable. This is comparable with the imino proton signature observed for the wobble U–G reference RNA .Hydrogen-bonded protons in Watson–Crick base-pairs are detected straightforward by NMR spectroscopy. The corresponding NMR resonances of the ‘imino protons’ directly reflect double helical segments within folded RNA. The chemical shifts of these signals are characteristic for A–U (> 14 ppm) and C–G base pairs (∼12–13 ppm), and the linewidths reflect proton exchange with the solvent. Obviously, imino protons are very sensitive to modifications, in particular, if the modification concerns the nucleobase. Comparative imino proton y Figure ,D, all rse pairs , or for se pairs ,48. Addiw1U–X and w2U–X conformations  and NH(2) of the C4–NH2 group . This is consistent with an C4–NH2 group that is not involved in H-bonding with one of its H atoms . However, at acidic conditions (pH 4.5), a significant upfield shift for the 15N3-H resonance is observed, and interestingly, the C4–NH2 resonances of the N3 protonated cytidine-4 are emerging at 9.3 ppm .Finally, we investigated if the pH dependence of base pairing strength is also reflected in the imino proton spectra of the X–C containing palindrome GGACCCGXUCC To summarize, our NMR study on short RNA palindromes indicates that in solution, xanthosine can form well defined base pairs. Both, X opposite of U and X opposite of C adopt a wobble geometry Figure , B whichIIc and IId that we used above for NMR spectroscopic investigations did not crystallize. We therefore utilized the 27 nt fragment of the E. coli 23S rRNA sarcin−ricin loop (SRL)  forming X-N1H•••O2-U and X-O6•••HN3-U hydrogen bonds similar to a typical wobble G–U pair  Figure  20,21),,21,IIc a) Figure  22,51),,51,IIc ar Figure –E. Charar Figure . Superimr Figure ,C. Of fur Figure , E. Finaw2U–X, Figure w2U–X geometry .Therefore, we switched to a different crystallization scaffold, namely 5′-CGCGAAUUACGC ,51, and y Figure , 6F enery Figure ,53. Thesw1C–X in Figure 2 of C5′. Of note, for both X–C pairs, the riboses are all in C3′-endo conformation.To maximize our chances to achieve a crystal structure also for the X–C base pair we tested a further RNA scaffold, namely 5′-GGUAUUGCGGUACC . Replace1H-NMR spectroscopic investigations on the 14 nt unmodified RNA scaffold indicates competition between bimolecular (duplex) and the monomolecular (hairpin) conformations . The equilibrium seems to be shifted towards the duplex if G10 and U5 are replaced by X10 and C5 . Furthermore, the 1H,1H-NOESY spectrum shows the typical correlations for G–U and X–U wobble pairs , however, the structural dynamics of the 14 nt RNA prohibited a more detailed NMR spectroscopic analysis of the nature of the two X–C base pairs in solution.With respect to the Watson–Crick shaped C5–X10′ pair, a tautomeric form of xanthosine must be involved, however, at the current state we are not able to distinguish between two interaction possibilities involving neutral nucleobases Figure , top verw1U–X and w1C–X wobble pairing, the X-ray structures show w2U–X wobble and Watson–Crick-type pairing modes (C–Xtaut or C+–Xtaut–) depending on the sequence context. We speculate that crystal packing influenced the conformations captured in the crystal. In particular, for the 12 and 14 nt RNAs, solution NMR spectroscopy indicated structural dynamics that we could not resolve further with standard NMR experiments applied. The alternative pairing modes might exchange on a fast time scale (below milliseconds) in solution. To verify this, more detaled NMR studies are planned in the future.Conclusively, our X-ray studies on short X–U and X–C containing RNAs revealed that xanthosine can form a significantly larger diversity of base pair conformations that were implied by the original NMR spectroscopic investigations. Beside Finally, to the best of our knowledge, we mention that only one crystallographic study on DNA with 2′-deoxyxanthosine is found in the literature that sheds light on the pairing mode with one of the standard nucleosides. This crystal structure concerns human DNA polymerase η (polη) with a single 2′-deoxyxanthosine containing DNA template. In the catalytic site of polη, the xanthine base forms three Watson–Crick-like hydrogen bonds with an incoming dCTP, indicating the O2-enol tautomer of xanthine involves in the base pairing .N1-methyladenosine (m1A) found that GoScript showed lowest mismatch and highest arrest rates, while Superscript IV had the highest mismatch and lowest arrest rates  is obtained. The cellular consequences of this transformation in RNA are scarcely known, and the chemical and biophysical consequences on RNA have been unclear. Our comprehensive study now sheds light on the properties of X-modified RNA. As thorough foundation, we developed the synthesis of a novel phosphoramidite building block allowing efficient access to X-modified RNAs by solid-phase synthesis. Thermodynamic analyses of such RNAs revealed that compared to the fully Watson–Crick paired counterparts, pairing strength is reduced to an extent that is similar to that observed for a G•U replacement. Applying NMR spectroscopy and X-ray crystallography, we demonstrate that X can form two distinct wobble geometries with uridine depending on the sequence context. In contrast, X pairing with cytidine occurs either through wobble geometry involving protonated C or in Watson–Crick shape-like mode. Strikingly, we identified the two distinct X–C pairing modes in the X-ray structure of a palindromic RNA duplex, breaking C2 symmetry. This indicates that the two pairing modes are of comparable stability separated by low energetic barriers. We furthermore demonstrate that the flexible pairing of X directly affects the reading of X-modified RNA by reverse transcription enzymes. Using primer extension assays and PCR-based sequencing analysis, we show that X is preferentally read as G or A and that the ratio depends on the type of reverse transcriptase. Taken together, our results elucidate important properties of X-modified RNA paving the way for future studies on the biological significance of xanthosine-containing RNA.Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data bank under accession numbers 7QSH, 7QUA and 7QTN.gkac477_Supplemental_FileClick here for additional data file.


Immunology and its philosophy are a primary source for thinking about biological individuality. Through its discriminatory function, the immune system is believed to delineate organism and environment within one generation, thus defining the physiological individual. Based on the paradigmatic instantiations of immune systems, immune interactions and, thus, the physiological individual are believed to last only for one generation. However, in recent years, transgenerationally persisting immune responses have been reported in several phyla, but the consequences for physiological individuality have not yet been explored. In this article, I will introduce an invertebrate immune system that is RNA-based and operates through a heritable silencing/licensing paradigm. I will discuss how such a perspective on immune systems can illuminate our conceptions of individuality. I will particularly introduce an account of immunological individuality that is not restricted to one generation. What is immunogenic, that is, triggers an immune system, and what is not, sits at the core of immunology and its philosophy C. elegans as a transgenerational immune system. I show that in the nematode C. elegans, small RNAs occupy an essential role in negotiating the boundaries between organism and environment through a silencing/licensing system and therefore serve as an excellent case to re-examine the immunological approach towards physiological individuality. Next, I will consider the impact of the silencing/licensing system on accounts of physiological individuality and ask how transgenerational immune systems might modify our perspective on the demarcation of physiological and evolutionary individuality. Finally, I will conclude and summarize how RNA-based systems illuminate our perspective of individuality in particular and the epistemic benefits of daring an invertebrate perspective on immunological individuality in general.Given the rise of data on transgenerational instantiations of adaptive(-like) immunity, I contend it is necessary to re-examine physiological individually. Is it necessarily confined to one generation? And how does transgenerational physiological individuality  – if it exists – relate to evolutionary individuality? In this article, I will introduce an invertebrate, RNA-based immune system that persists transgenerationally to discuss immunological individuality beyond jawed vertebrates. First, I will introduce small RNA inheritance in the nematode C. elegans. Second, I will introduce heritable small RNA responses to environmental stimuli. Third, I will introduce the capacity of small RNAs to protect germline messenger RNAs (mRNAs) from being degraded. Through its capacity of “silencing” certain transcripts and “licensing” others, I will argue that the heritable small RNA-based silencing/licensing system fulfills the discriminative characteristics of an (adaptive-like) immune system.This section will introduce an empirical case study to assess its fit with the immunological conception of physiological individuality. I will first explain some basics about small RNAs in the nematode Small RNAs are gene-regulatory, non-coding RNAs identified in almost all known species. Studies on small RNA-related effects in the 1980 and 1990 s culminated in identifying the phenomenon of “RNA interference” . viRNAs, in turn, guide effector molecules, such as argonautes, to viral RNAs, leading to the viral RNAs’ destruction. Thus, antiviral immunity is instantiated through sequence complementarity. Small RNAs are also involved in other responses to environmental stimuli, such as orchestrating gene regulatory changes after starvation or heat shock.In C. elegans was first described in the early 2000s, and evidence started accumulating in and after 2006  might be polyvalent (have more than one binding site).Does the silencing/licensing system instantiate a case of immune interactions? Immune reactions are often characterized by their intensity, instantiated through specificity, affinity, and avidity Pradeu, . This isThis gives the idea of the “RNA state” further depth. In a particular context, effector molecules are limited resources, and environmental fluctuations will slightly alter the silencing/licensing state. For instance, an excess of exogenously supplied small RNAs, such as viRNAs, alters endogenous small RNA dynamics since they compete for the same effectors  of a licensing profile, then what is left to ask is whether the overall response’s intensity decreases. It is important to note that experiments to investigate these hypotheses have not been conducted yet. It is nevertheless possible to come up with testable predictions regards the chronicity of antigen exposure in the small RNA-based system.While there are initial amplificatory cycles after second triggers .“ Settling this issue requires a separate paper. The silencing/licensing system is not functionally equivalent to jawed-vertebrate adaptive immunity, that is, for every entity and process of the jawed-vertebrate immune response, there does not exist a corresponding small RNA or small RNA effector. Also, key processes are different. For instance, while the jawed vertebrate immune response is “customized” (the immune receptor repertoire predates the first trigger), the small RNA-based immune response is “tailor-made” (complementary immune effectors are synthesized after exposure to the trigger) . What counts as the individual is thus quantitative, continuously renegotiated, and responsive to environmental fluctuations. What defines the small RNA-based immune system is the capacity to establish complementarity to every possible RNA through (1) genetically encoded small RNAs; (2) mismatch tolerance; (3) RNA-dependent RNA polymerase-dependent synthesis of complementary small RNAs (that could be of exogenous origin).The small RNA-based silencing/licensing system, defining the results of immune interactions as the expression or repression of a target, introduces a new perspective on immunological individuality. In One particular aspect of the silencing/licensing system is its heritability. Heritability, however, is usually not considered an aspect of the immunological individual but rather constitutes one of the minimal, necessary, but not sufficient criteria of evolutionary individuals. Given immunology’s focus on jawed vertebrates, immune interactions are generally believed not to be heritable in a stable way, even though some immune effectors are transferred through material overlaps from parent to offspring in jawed vertebrates . These transfers are, however, believed not to extend the intergenerational as there are no dedicated processes yet uncovered that could ensure the transgenerational persistence of these effectors. We thus have to ask how much our notion of physiological individuality is based on a particular notion of immunity that, in turn, is inspired to a large degree by a particular instantiation of an immune system.While the immunological account of the physiological individual necessarily has to apply to all individuated biota, it is strongly influenced by how the jawed vertebrate immune system negotiates the borders of the organism. If our account of the immunological/physiological individual, however, mainly rests on the fact that cell-based immunity or its effects are not heritable, we have to ask whether we should take the special case of the jawed vertebrate immune system as a determinant for all physiological individuals and, as a result, the relation of evolutionary and immunological individuality. With the jawed vertebrate immune system considered one of the major evolutionary transitions  small RNAs are candidates of evolutionary individuals  is an open debate. Let us, however, consider the minimal criteria for evolutionary individuality: First of all, small RNA pools are heritable. Also, it seems highly likely that a particular heritable small RNA pool contributes to differential fitness. Expressing and repressing specific RNAs in particular scenarios might contribute to the organism’s fit to the environment and alleviate or overcome specific selection pressures. In addition, the silencing/licensing state leaves room for variation since we do not expect a particular environmental change, such as viral presence, or starvation, to cause an identical response in each organism in a Darwinian population. While, e.g., viral infection causes the synthesis of antiviral small RNAs, we will expect stochastic fluctuations in small RNA states on the one hand, on the other hand, fluctuations based on previous exposures, previous ways of blending organism and environment in different individuals. Given that previous exposures influence the small RNA state and thus differential fitness, there will be a higher similarity between parent and offspring than between lines of ancestry. Thus descent with modification could be ensured.It is necessary to mention that when characterizing the persistence of environmentally induced immune interactions as inheritance phenomena, this characterization entails a Lamarckian interpretation Veigl,  of the porganisms-in-the-world and not organisms in the world , we have to understand the immunological individual as established through a particular expression/repression profile that persists. Also, of those entities that cannot be targeted through RNA-RNA interactions directly, such as proteins or cells, however, the transcripts that brought them into being interacted with the silencing/licensing system. Both within one generation as well as beyond one generation, the individual’s boundary is constantly renegotiated. Persistence and constant re-negotiation might, at first glance, lie in conflict. The small RNA perspective, however, shows how stability and change are both necessary and are mediated through the same process. To maintain what is licensed, the organism also needs environmental cues that stimulate amplificatory loops. Thus, environmental cues are needed to maintain the boundary of the individual. This also suggests a particular view regarding the relation between organism and environment. It suggests a commitment to the inseparability of organisms and environments , conceptualizing them as d Smith, . It suggBut I have not yet addressed what it means for the physiological individual that the immune reactions of the silencing/licensing system are heritable. This first necessitates the question of what is meant by a “generation” in the context of transgenerational immune responses. Generations of organisms are most of the time defined by reproduction events, also called “the bottleneck” view: As an individual reproduces, a new generation begins through progeneration. This definition is situated, however, in the evolutionary discourse about units of selection. But with regards to individuality conferred by the silencing/silencing system, there is an incongruency: a reproduction event, an event that makes discernable parent and offspring, does not mark a decisive event for small RNA-based individuality. Placatively speaking: If pools of small RNAs are inherited, and thus, the silencing/licensing state persists, the first encounter of a virus changes the small RNA state, the silencing/licensing equilibrium, and thus, the boundaries of the organism more than a reproduction event. In other words – an encounter with a virus will change what is expressed and what is repressed more than the start of a new generation within a stable environment.If we follow the immunological reading of physiological individuality, that is, an individual is defined by local biochemical interactions and negotiated by systemic immune interactions Pradeu, , we woulWe could start with addressing this puzzlement by asking why we have the intuition that the physiological individual is confined to a generation (a concept developed in an evolutionary/genetics context). Immunological individuality was formulated with cell-based immunity in mind. Even though the philosophy of immunology aims for generality, it is highly influenced by a particular way of thinking about immune systems. Insisting on the distinction between the evolutionary and the physiological  individual is caused by a jawed-vertebrate perspective on immune systems. Introducing the small RNA-based perspective opens several possibilities for consolidating individuality, evolutionary and physiological. Also, it helps to question why physiological individuality needs to be confined in the same way as evolutionary individuality (in terms of generations). With regards to these two concepts, the notion of the evolutionary individual has been quite dominant in 20th -century discourse, and the concept of the physiological individual, while aimed at separating a distinct phenomenon, might have, to a degree, been morphed after that of the evolutionary individual. In the next section, I will assess whether it is possible to separate both concepts regarding their generationality.In the previous section, I considered the possibility of the physiological individual being maintained as long as the systemic immune interactions show continuity. I argued that in some cases, one physiological individual might start and end at different points than one evolutionary individual. It might even last longer than one evolutionary individual. But where are the limits to physiological individuality? If, for instance, a small RNA state is transgenerationally heritable – would parent and offspring constitute one single organism, as long as the offspring maintains the small RNA state of the ancestors?This lies in conflict with several accounts of physiological individuality. Pradeu argues that the study of a physiological individual is the study of its life from conception to death Pradeu , p. 5. ASuch developmental processes involve “an ongoing, dynamic, construction and reconstruction of individuals by sub-organismic, non-organismic, and other-organismic factors.” Smith, , p. 6. AGiven this perspective, the essential question for the C. elegans example is whether the reconstruction .” , p. 2.Suárez and Stencel, on the other hand, support part-dependence:“a whole is a biological individual only if all the elements that are included in the whole fulfil a certain criterion in relation to a given element of the whole that one is prioritizing.” (ibd.)Thus, the ontological status of the holobiont differs depending on the perspective, even if the same concept of individuality is used. Suárez and Stencel explain this partly through a difference in scale between host and microbe: “the physiological necessities of the former are radically different from the physiological necessities of the latter” , p. 9. FBut part-dependence also has a second consequence: if we take the perspective of the host’s microbiota, there are many lineages of evolutionary individuals throughout the host’s lifetime. However, they constitute one physiological individual, the host and its microbiota, the functionally integrated holobiont. In this case, the holobiont’s physiological individuality lasts longer than several evolutionary individuals of microbiota. But what about heritability? There is currently a debate about whether the holobiont is also an evolutionary individual . As this condition does not hold empirically, and the small RNA state is responsive to environments, it will thus not remain the same for an extended amount of generations.But the heritability of a small RNA state only for one generation already presents problems. C. elegans has many offspring. Worms can lay up to 140 eggs per day (and live for around 16 days)  notion of evolutionary individuality on formulations of physiological individuality. Discussing the small RNA-based silencing/licensing system, I tried to show that physiological/immunological individuality is not necessarily confined to one generation and thus also provides a further potential instantiation where the physiological and the evolutionary meet.I hope to have shown that the silencing/licensing system provides an insightful perspective on how the organismal and the environmental relate. Furthermore, it provides a mechanistic basis for how the environment can become organismal and how the organism can become environment. Finally, through its focus on the quantitativeness , it instantiates a perspective on the boundaries of the organism as being eternally fluid and morphed by its environment. With that perspective in hand, I tried to show that while there are approaches towards physiological individuality – developmental, organismic, part-dependent – that dissuade looking at the physiological individual as persisting inter- or transgenerationally, if the small RNA state remains the same, from a particular perspective, which is eco-immunological and organism-in-the-world focused, it makes sense to doubt that physiological individuals necessarily have to begin at progeneration events.It is important to note that heritable small RNA-based responses to environmental stimuli and the silencing/licensing system, in particular, have been discussed in the literature exclusively in a space that is disciplinarily separated from immunology. They are considered genetic phenomena, are endowed with geneticist terminology, and are mobilized to discuss theories of inheritance and evolution in some cases. While RNA interference in a very general way, is registered as performing immune functions, both in the biological and the philosophical literature, its heritability is not part of these accounts. Heritability of immune effectors is generally not assumed to characterize immune responses. What about a particular phenomenon is included in discussions on immunology and what is neglected thus also seems partly to be caused by the dominant jawed-vertebrate perspective.While such “problems of the paradigm” might never be entirely evaded, there are remedies to keep them in check in the philosophy of immunology. One, demonstrated here, is to question membership. It is highly likely that phenomena that could be conceptualized as immune phenomena but do not resemble the respective realization in jawed vertebrates are overlooked or left out when studying instantiations of immune systems in non-jawed vertebrate species. But it is precisely these aspects of immune responses, those that do not resemble jawed-vertebrate instantiations, we need to foreground in order to assess our perspectives on immunology and immunological individuality. We thus need to broaden our focus and expect phenomena that could be fruitfully studied as immune phenomena to be hidden within different taxa and disciplinary contexts.C. elegans small RNA-based immune system to argue that our notion of physiological individuality confined to one generation is situated in a particular epistemic context. If we go beyond that particular context and admit other instantiations of immune systems to contribute to the discourses on immunological individuality in terms of how they differ from the established epistemic context, and not only in terms of what is similar, we arrive at diversified but more fine-grained accounts. Investigating other systems that negotiate  individuality will provide an even better understanding of where and how the physiological and the evolutionary meet. If the genetic and the immunological interact and coincide differently in different phyla, the same must be true for physiological end evolutionary individuals.For the specific case of this article, I unearthed the silencing/licensing system from the disciplinary context of genetics. I used the particularities of the In conclusion, the small RNA perspective shows that the immunological/physiological individual is not necessarily confined to one generation because silencing/licensing equilibria persist transgenerationally. How should this change our perspectives on individuality? Whether separating the physiological and evolutionary individual is empirically adequate is sensitive to the particularities of the phylum in focus. And whether the physiological individual can extend the evolutionary individual might be as well. It is vital to move away from generalist statements when we have only focused on particular instantiations of the processes we are interested in. As pointed out above, we should consider the physiological and the evolutionary more or less hybrid dependent on the respective species. Also, how both relate to each other temporarily  might be species-dependent. With this article, I aim to extend this perspective and argue that physiological individuality displays different durations depending on the species. Thus, transgenerational (adaptive(-like)) immune systems are a further instantiation of an overlap between physiological and evolutionary individuality. When it comes to individuality in organisms with small RNA-based immune systems, how the organism in one generation becomes countable (identifiable as one) is causally relevant to how the organism becomes countable in the next generation. The immune system is not only adaptive in the immunological sense but might also be adaptive in the genetic, evolutionary sense. The boundary between both is fluid.


Characterizing RNA structures and functions have mostly been focused on 2D, secondary and 3D, tertiary structures. Recent advances in experimental and computational techniques for probing or predicting RNA solvent accessibility make this 1D representation of tertiary structures an increasingly attractive feature to explore. Here, we provide a survey of these recent developments, which indicate the emergence of solvent accessibility as a simple 1D property, adding to secondary and tertiary structures for investigating complex structure–function relations of RNAs. It has been estimated that 98.8% of the human genome is not involved in the coding of proteins but instead is mostly transcribed into RNAs. That is, most RNAs are not protein-coding information carriers, but are transcribed with unknown functions. Some were found to be involved in the regulation of nearly all aspects of biological processes. Examples are protein synthesis, transcriptional regulation, RNA stability, chromosome replication and catalyzation of essential biological processes . MoreoveCurrently, RNA structure determination relies on X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and cryogenic electron microscopy (cryo-EM). However, due to intrinsic physio-chemical properties of RNAs, it is more challenging to determine RNA tertiary structures than protein structures . In factin vivo, genome-scale, structural interrogation has advanced our understanding of RNA–RNA and RNA–protein interactions as well as the overall RNA structure–function relationships [The lack of tertiary structural knowledge for most RNAs has led to the development of an array of chemical techniques for probing secondary and tertiary structural properties . The advionships , 15.Meanwhile, the cost and labour-intensive nature of the above experimental techniques have inspired the development of many computational modelling approaches , 17. Mosin vitro, and in vivo states [One important, simplified measure for RNA structural properties is solvent accessible surface area (SASA). Unlike RNA secondary structure, which is made of mostly local canonical base-pairing contacts, SASA is a direct tertiary-structure descriptor, indicating whether a base is exposed to or buried from the solvent. Those bases exposed to the solvent will more likely participate functional activities such as binding and catalysis. Indeed, SASA has been found useful for characterizing the conformational change due to the binding with other molecules , identifo states , 28 as wo states . Here, wo states , 30, 31.R, the radius R is given by the sum of the VdW’s radius of the atom and the selected radius of the solvent molecule, Solvent accessibility is defined as the accessibility of residues of a macromolecule to the solvent. SASA is calculated by ‘rolling’ a probe sphere  over the van der Waals surface (VdW) of a biomolecule, which is a surface created by spheres in atomic van der Waals radii around the atoms of a given molecule . The sur2+, K+), which cause the structural transition to playing different cellular roles [The adoption of particular RNA structures is required for their functional varieties. The specific structures are linked to the precise biological functions and cellular processes such as gene expression regulation , transcrar roles , 39. Simar roles . Therefoet al. [et al. [et al. [SASA is a 1D representation of the tertiary structure of a macromolecule such as RNA or protein. SASA values of proteins were first found to be significantly correlated with hydrophobicity, molecular weight, radius of gyration, intermolecular hydrogen bonding and transfer-free energy. For example, Chothia  establiset al.  calculat [et al.  successf [et al.  showed tet al. [et al. [In addition, SASA can be naturally applied to measure conformational transitions induced by binding interactions. Bustamante et al.  develope [et al.  compared2, respectively [Unlike proteins, RNAs are only made of four nucleobases with two different sizes: purine (adenine and guanine) and pyrimidine (cytosine and uracil) bases, which have a maximum accessible surface area of 300 and 260 Åectively . Weeks aectively . They reectively . Computaectively . That isectively .Thus, considering these vital relationships with structural and functional properties, SASA has been considered as a key parameter to study for macromolecules. For RNA SASA studies, both experimental and computational methods have been developed.in vitro applications, the recent methods are being developed based on either small-molecule modifications or crosslinking and proximity ligation [Due to the dynamic nature of RNA structures and the challenges in RNA structure determination, different enzymatic or chemical probing methods have been developed to interrogate RNA structures, some of which are at a single nucleotide resolution. They characterize RNA structures genome-wide across many species and conditions . These mligation , 14. MosThere is a wide range chemical probes, which can overcome the limitations of enzymatic probing because of their small sizes. They can be either base-specific or non-base specific, which are being used to reveal the Hoogsteen and/or Watson-Crick face of the bases, as well as the sugar-phosphate backbone in RNA structures. A list of common probes employed and their target sites are illustrated in in vitro or in vivo environment. This chemical probe yields a combined effect of solvent exposure and secondary structure profile [•) cleave the RNA backbone by reacting with the hydrogen atom at C4′ and/or C5′ ribose position(s) [One base-specific, chemical probe is dimethylsulfate (DMS) that methylates N1-A and N3-C at neutral pH. It is mainly used to recognize unpaired, accessible adenosine and cytosine nucleotides  profile . Other b profile , and die profile . Recentl profile . Non-basition(s) . Ethyl-nition(s) . A numbeition(s) .These types of probe reagents cause the strand scission or adduct formation at a particular nucleotide that can be deciphered by proper readout methods. Recently, instead of using denaturing polyacrylamide gel or capillary electrophoresis, HTS-based readout methods are widely used . To addr•) are short-lived, highly reactive, oxidative species generated upon probing experiments [• production is the reduction of H2O2 via the Fenton reaction [Hydroxyl radical footprinting is a unique method for mapping RNA solvent accessibility directly. Hydroxyl radicals  and 16S Escherichia coli rRNA (r = 0.56). HRF-Seq identified protein footprints on RNA  and used to examine the changes in RNA tertiary structures. This method made it feasible to study long and multiple RNA molecules in a single tube by using sequencing barcodes and random primers.After hydroxyl radical probing, the cleavage sites are identified by analyzing the sample on polyacrylamide gel or capillary electrophoresis techniques . The cleet al.  integratin vitro, hydroxyl radical probing in cells has been slow to take hold due to the experimental difficulties associated with employing synchrotron radiation.When it comes to determining the solvent accessibility of RNA, hydroxyl radical probing was likely to be the gold standard. It was particularly useful for measuring the accessible backbone of RNA, and commonly used to track RNA structural changes and RNA-protein interaction because of its fast rate constant. In spite of its widespread usages et al. [N,N-(dimethylamino) dimethylchlorosilane (DMAS-Cl) that selectively reacts at the N2 position of guanosine nucleobase (Bacillus subtilis mgtE aptamer domain) with a known high-resolution crystal structure. They obtained a strong correlation (r ≥ 0.82) between the DMAS-Cl reactivity and the guanosine N2 solvent accessibility, albeit with limited, base-specific coverage of a short RNA transcript. They also observed that DMAS-Cl was reactive to N2 guanosine in a structure-selective manner in solution, more reactive when RNA lacked tertiary structure (in the absence of Mg2+) than the extensive higher order tertiary conformation (in the presence of Mg2+). Kethoxal, a carbonyl electrophile, also attacks N2 of guanosine and forms cyclic adduct with N1 position of the same base [r ≤ 0.35) when kethoxal was employed, indicating that different chemicals may have different effects.Motivating from the hydroxyl radical solvent accessibility probing, Mortimer et al.  developecleobase . It formame base . Howeverin vivo monitoring of RNA structural properties and the influence of proteins on them. By integrating HTS, the first in vivo, genome-wide RNA structure probing method, Structure-seq, was developed based on DMS [et al. [in vivo compared to in vitro. The receiver operating characteristic (ROC) curve revealed a strong relationship between in vivo probing signals and crystal structure models with a 90% true positive rate (solvent accessible and unpaired bases). The application of DMS-seq on mammalian cells (human foreskin fibroblasts and K562 cells) also exhibited a similar result to yeast ribosomal RNA and found that mRNAs in vivo, in rapidly dividing cells, are less structured than in vitro. They also performed genome-wide structure probing at different temperatures  to evaluate thermal stability of mRNA. The results showed that most structures became unfolded at high temperature and RNAs with structures in vivo had high thermostability. At low Mg2+ concentration (1 mM), most RNA structures were also found unfolded in vitro, whilst at 2–6 mM they were similarly structured. The depletion of ATP on yeast revealed a significant increase in mRNA structures in vivo. In addition, the structural transition was substantially correlated (r = 0.54) to the interchanges between in vivo and in vitro samples upon ATP depletion. The reactivity readout of these techniques was based on RT-stop, whilst Zubradt et al. [In addition to probing secondary structural properties, DMS is also used for RNA solvent accessibility study . It is hd on DMS , 79. Latd on DMS . Rouskin [et al.  first det et al.  presenteet al. [r = 0.82). It worked with delicate sensitivity and distinguished compact structural changes in SAM-I RNA due to ligand (SAM) binding. In addition, LASER identified the key residues (A46 and A45), which are in direct contact with the ligand based on their solvent accessibility information. Probing in vivo 18S rRNA and U1 snRNP (complexed with proteins) revealed the ability of this method to reading RNA structures inside cells and detecting RNA-protein interactions in native environments. The use of NAz made the LASER method unique as other classical structure probing reagents (such as DMS) work by identifying single stranded regions.Light activated structural examination of RNA (LASER) is the most recent and advanced technique for RNA solvent accessibility study. It was firstly developed by Feng et al.  and lateet al. [in vitro and in vivo to compare against solvent accessibility calculated by using high-resolution crystal or cryo-EM structures. The results showed that LASER-Seq and LASER-MaP were able to detect solvent accessible nucleotides in both (in vitro and in vivo) environments. However, the authors preferred LASER-Map to LASER-Seq for further experiments. They compared NAz with 1 M7, BzCN used in SHAPE-MaP [E. coli and Saccharomyces cerevisiae ribosomal RNA, respectively, and demonstrated LASER-Map as a reasonable method for quantifying the solvent accessibility. In addition, this technique was used in detecting binding sites of small molecules or ligands on RNAs and conformational changes in complex RNA in an unbiased fashion. Upon ligand binding, the nucleotides at/close to the binding sites become less accessible or protected from the probing reagents. The secondary protection of nucleotides, far away from the ligand-binding sites, may indicate the massive conformational changes because of ligand binding. The application of LASER-MaP on intact E. coli ribosomes incubated with EF-G (elongation factor G), in the presence or absence of non-hydrolyzable GTP analogue (GDPNP), and proline-rich antimicrobial peptide onc112 detected the conformational changes and dynamics in both paired and unpaired regions.Zinshteyn et al.  expandedHAPE-MaP  and DMS HAPE-MaP  and founin vivo click LASER (icLASER), and it significantly enriched RT-stopped cDNAs. The mapping analysis revealed the enrichment had happened at A and G nucleotides as expected. This new probing technique allowed transcriptome-wide RNA solvent accessibility measurement and environment specific (in and outside of cells) RNA structures interrogation. Moreover, integrating icLASER (RNA solvent accessibility) with other reactivity-based measurements such as icSHAPE (RNA flexibility) has broadened its application to transcriptome-wide identification of RNA functional elements. Studying the IRE (iron response element) in UTR of mRNA, these methods determined the pattern of protein binding, flexibility and solvent accessibility of nucleotides based on the different chemical reactivities. The transcriptome-wide mapping of mRNA functional elements revealed the start codons are highly open and solvent accessible at the A and G of AUG, while the last two positions of stop codons were largely accessible. In addition, the solvent accessibility differences based on in vivo and in vitro accessibility profiles suggested that icLASER could be used for mapping protein-RNA interactions. The authors implemented Support Vector Machines (SVM) to generalize the icLASER and icSHAPE signals (in vivo) and compared with enhanced crosslinking and immunopurification (eCLIP) [More recently, a bi-functional LASER probe has been developed from the same research group to measure transcriptome-wide purine C-8 solvent accessibility . An alky (eCLIP)  datasets (eCLIP) .While predicting protein solvent accessibility has been around for three decades , 88, theDuring the inference mode, these models take sequence-derived information to predict RNA solvent accessibility. Inference mode is computationally less expensive as compared to the training mode. Next paragraphs discuss individual methods in more details in terms of the datasets used and their performance on the test sets.et al. [in vivo mRNA accessibility determined by the DMS probing technique, not to in vitro. It also showed a positive correlation (PCC > 0.8) between the projected SASA of the mutation site of single nucleotide variant (SNV) with the minor allele frequency in the 1000 Genomes Project, which indicated damaging effects of inaccessible RNA core structures. A study of 15 642 gene transcripts with RNAsnap revealed introns are more solvent-exposed than exons. The application of this tool to monitor temperature adaptation of rRNA, tRNA, and mRNA from 200 bacterial species showed that rRNA and tRNA were more exposed to solvent but remained structured in hyper-thermophilic bacteria [In 2017, Yang et al.  developebacteria .RNAsol was developed to improve over RNAsnap by using a relatively larger training set and a deep learning technique . This meRNAsnap2 was developed in 2020 . The acce-value of 10. To further evaluate these models, we prepared a benchmarking test set of 57 high-resolution (<3.5 Å) X-ray structures from PDB. This test set is non-redundant from training data of all three predictors according to CD-HIT-EST at lowest allowed sequence identity cut-off of 0.8 followed by BLASTN with 3) are only purine base specific. Thus, the development of a new method that can cover all nucleobases or integration with other probing techniques may be needed to overcome this limitation.The main experimental approaches for RNA solvent accessibility study include hydroxyl radical probing, LASER, and its improved versions . The integration of HTS technology, similar to other advanced RNA structure probing methods, has allowed transcriptome-wide solvent accessibility studies. However, the reactivity readout procedures are based on either the RT-stop at cleavage points or mutational profiling at the adduct formation sites  and 5. TComputationally, only three methods were developed for RNA solvent accessibility prediction. These methods obviously can be improved further given constantly improving deep learning techniques and increasing availability of training data. However, the major challenge for RNA solvent accessibility prediction remains the relatively small number of RNA structures that are available in the protein databank. There is a risk of over-training if not trained appropriately. Thus, transcriptome-wide experimental data may be used for initial training, following by transfer learning with SASA from known structures. Moreover, a newly developed automated tool for RNA sequence profile and correlated mutation analysis  can be ein vivo and in vitro. This method also investigated the RNA structural stabilities at different temperatures, Mg2+ concentrations, and in the presence or absence of ATP [Currently, applications of predicted or experimentally estimated RNA SASA values remain limited. The analysis of RNA SASA provides information on possible solvent-exposed interaction sites, disease-causing mutations, structured and unstructured areas based on the pattern of solvent accessibility. For example, RNAsnap  detectede of ATP . Using se of ATP , 82. Thee of ATP .ab initio protein structure prediction [In future, we expect that high-throughput experimental SASA probing data will be employed as a part of training data for initial learning to supplement the scarcity of RNA structural data for secondary and tertiary structure prediction, as exemplified by the use of approximate secondary structure database for transfer learning in SPOT-RNA . The proediction , templatediction  and natiediction .This work surveys current experimental techniques and computational methods for characterizing RNA solvent accessibility, an underexplored area of research.Solvent accessibility is a simple 1D measure for characterizing RNA 3D structure, complementary to 2d RNA secondary structure.The experimental techniques include hydroxyl radical, DMS and LASER probing and computational predictors are machine-learning-based RNAsnap, RNAsol and RNAsnap2.RNA solvent accessibility will be useful for improving structure prediction and discovering potential functional sites.


Short repeated sequences of 3−6 nucleotides are causing a growing number of over 50 microsatellite expansion disorders, which mainly present with neurodegenerative features. Although considered rare diseases in relation to the relatively low number of cases, these primarily adult-onset conditions, often debilitating and fatal in absence of a cure, collectively pose a large burden on healthcare systems in an ageing world population. The pathological mechanisms driving disease onset are complex implicating several non-exclusive mechanisms of neuronal injury linked to RNA and protein toxic gain- and loss- of functions. Adding to the complexity of pathogenesis, microsatellite repeat expansions are polymorphic and found in coding as well as in non-coding regions of genes. They form secondary and tertiary structures involving G-quadruplexes and atypical helices in repeated GC-rich sequences. Unwinding of these structures by RNA helicases plays multiple roles in the expression of genes including repeat-associated non-AUG (RAN) translation of polymeric-repeat proteins with aggregating and cytotoxic properties. Here, we will briefly review the pathogenic mechanisms mediated by microsatellite repeat expansions prior to focus on the RNA helicases eIF4A, DDX3X and DHX36 which act as modifiers of RAN translation in C9ORF72-linked amyotrophic lateral sclerosis/frontotemporal dementia (C9ORF72-ALS/FTD) and Fragile X-associated tremor/ataxia syndrome (FXTAS). We will further review the RNA helicases DDX5/17, DHX9, Dicer and UPF1 which play additional roles in the dysregulation of RNA metabolism in repeat expansion disorders. In addition, we will contrast these with the roles of other RNA helicases such as DDX19/20, senataxin and others which have been associated with neurodegeneration independently of microsatellite repeat expansions. Finally, we will discuss the challenges and potential opportunities that are associated with the targeting of RNA helicases for the development of future therapeutic approaches. C9ORF72)-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Several of these repeat expansions are bi-directionally transcribed including in Fragile X associated mental retardations, C9ORF72-ALS/FTD, HD, DM and several subtypes of spinocerebellar ataxia . For recent reviews, we refer to (Microsatellite expansions have been attributed to numerous neurodegenerative diseases which are subdivided into either polyglutamine (poly-Q) and non-polyglutamine disorders. Poly-Q expansions encode a CAG trinucleotide repeat expansions and have been reported in Huntington disease (HD), Huntington disease-like 2 (HDL2), 7 subtypes of spinocerebellar ataxia , Dentatorubral-pallidoluysian atrophy (DRPLA), Spinal-Bulbar Muscular Atrophy (SBMA) and some Schizophrenia/migraines. Non-poly-Q expansion disorders encompass expansions of various lengths, ranging from trinucleotide to hexanucleotide repeats. Trinucleotide repeats include: 1) CGG repeats in Fragile X associated mental retardations; 2) CTG repeats in myotonic dystrophy (DM) and SCA8; 3) GAA repeats in Friedreich’s ataxia; 4) pentanucleotide  or hexanucleotide (GGCCTG) repeat expansions in spinocerebellar ataxia (SCA) type 10, 31, 36 respectively, and bi-directionally transcribed CTG/ CAG repeats in SCA8; 5) hexanucleotide (GGGGCC) repeat expansions in chromosome 9 open reading frame 72  translation in all frames and from coding or non-coding regions of genes were also evidenced in multiple repeat expansion disorders (reviewed in ).FMR1), leading to the Fragile X syndrome (FXS), the most common inherited form of intellectual disability, due to haploinsufficiency and loss of the FMRP protein, which plays a role in repressing the translation of specific mRNAs for synaptic plasticity in dendrites. In contrast, the fragile X–associated tremor ataxia syndrome (FXTAS) is a late adult onset disease that involves intermediate expansions of 55–200 CGG repeats in the 5′-UTR of the FMR1 gene. Unlike FXS, it leads to neurodegeneration and elevated levels of CGG-expanded FMR1 transcripts in intranuclear neuronal and astrocyte inclusions  in the 5′-UTR region of the fragile X mental retardation 1 gene  and propansions , which apansions . For recpansions .HTT) cause HD, while >41 CAG repeats in the 3′terminal exon of Junctophilin 3 (JPH3) are involved in HDL2. In addition, >32-88 CAG repeats lead to autosomal-dominant cerebellar ataxias (ADCAs) when inserted in the exonic regions of the following genes: 1) ataxin  in SCA types 1-3,7; 2) calcium voltage-gated channel subunit alpha1A (CACNA1A) in SCA6; 3) protein phosphatase 2 regulatory subunit B beta (PPP2R2B) in SCA12; 4) TATA-binding protein (TBP) in SCA17; 5) atrophin 1 (ATN1) in DRPLA; 6) X-linked androgen receptor (AR) in SBMA, 7) potassium calcium-activated channel subfamily N member 3 (KCNN3) in some Schizophrenia/migraines. In poly-Q disorders, the CAG repeat expansions are found in polymorphic regions encoding poly-glutamine stretches that lead to the translation of proteins with extended poly-Q domains that promote misfolding/ aggregation, reduced interactions with binding protein partners and abnormal interactions with other factors (toxicity through protein loss- and gain-of-functions). Moreover, RNA-mediated toxicity was also reported to directly contribute to pathogenesis of poly-Q disorders potentially through abnormal interactions with the muscleblind protein in Drosophila  that are toxic and aggregate in a CAG repeat length dependent mechanism in autopsied brains from human HD patients  in exon 1 of the Huntingtin gene (osophila  and nuclosophila . In addipatients . For recpatients .ATXN8OS (ATXN8 opposite strand) gene  in the 3′-UTR of the nd) gene . The samclusions . Moreoven tissue  suggestin tissue  and protn tissue  toxic garefer to .DMPK) and of CCTG repeats  in intron 1 of the Zinc Finger Protein 9 gene (ZNF9) lead to myotonic dystrophy (DM1) and myotonic dystrophy type 2 (DM2) respectively. In DM1, these expansions are thought to predominantly cause RNA-mediated toxicity through loss-of-function of regulatory RNA-binding/splicing factors (including the muscleblind-like splicing regulator (MBNL) and CUG binding protein and ETR3-like factor (CELF) families of proteins) sequestered on hairpin structures formed by the CUG repeat transcripts that form characteristic intranuclear RNA foci. In addition, RAN translation of various repeat proteins were evidenced in DM1 and DM2 post-mortem brains as well as in vitro  in the non-coding 3′-UTR of the myotonic dystrophy protein kinase gene  and frontotemporal dementia (FTD)  in the first intron of the ia (FTD) . Bi-direintron-1 , and subintron-1  as one orefer to .RNA helicases are motor proteins that catalyse the ATP-dependent unwinding of RNA duplexes in a wide array of RNA metabolic processes, from pre-mRNA splicing, RNA nuclear export, ribosome biogenesis up to translation and mRNA decay/degradation, with a large number of RNA helicases functioning in multiple steps . They plRNA helicases are involved at multiple steps of translation, however most function at the initiation step of translation. Once exported to the cytoplasm the cap-binding complex (CBC) of capped mRNA is replaced by the cap binding protein eIF4E, which interacts with eIF4G and eIF4A to form the eIF4F complex. The RNA helicase eIF4A comprises two isoforms, eIF4A1  and eIF4A2 , which actively unwinds secondary structures within 5′-UTRs along with its cofactors eIF4B and eIF4H . Other ROther RNA helicases facilitate or repress translation through resolving specific secondary structures such as G4 quadruplexes (G4s). These include DHX9, DHX36, DDX5, DDX17 and MOV10L1 facilitate the translation of specific mRNAs which harbour G4s within their 5′-UTR . DHX9 R; while DOther RNA helicases are recruited to the 3′-UTRs and are involved in translation control and termination. DDX19B (DBP5) facilitates stop codon recognition and recruitment of eukaryotic Release Factor eRF3, the polypeptide chain release factor to promote terminating complexes but also plays additional roles with eukaryotic elongation factors eEF1/eEF2 in stabilizing ribosomal elongation complexes ; while DInterconnected with translation is miRNA gene silencing of translation and several RNA helicases function in both processes. miRNAs are short non-coding RNA sequences which form miRNA-induced silencing complexes (miRISCs) with Argonaut proteins and mediate post-transcriptional gene silencing . SilenciMAPT) and amyloid β precursor protein (APP) genes encoding Tau and amyloid-beta peptide  are highly conserved family of RNA helicases, functioning in the unwinding of RNA secondary structures, catalysation of RNA annealing and displacement of proteins from RNA. While eIF4A1 and eIF4A2 are involved in the control of translation, eIF4A3 is a subunit of the exon junction complex which stimulate mRNA nuclear export as well as translation but is also involved in inducing nonsense mediated mRNA decay (NMD) of transcripts with premature stop codons. They form large, multi-protein complexes to fulfil these tasks, interacting through their N and C-terminal domains. While research attention has predominately come from the field of cancer biology, helicases are now understood to also play an important role in neurodegenerative diseases. In both canonical and RAN translation, the RNA helicases eIF4A unwind and facilitate the scanning of mRNA for a start codon, with translation proceeding upon recognition of an AUG in the case of canonical translation and in some cases a near-cognate start codon (reviewed in ). An eve peptide . Interes peptide .C9ORF72-repeat expansions are particularly prone to forming highly stable G-quadruplex structures due to their G-rich nature  and dysgregates . Both DDgregates  resultingregates . Furthergregates .Another RNA helicase shown to modulate DM1 pathology is DDX6, which appears to regulate the homeostasis of nuclear foci in DM1, countering the effects of DDX5 and DDX17 . DepletiFUS gene recruit nuclear DDX17 into cytoplasmic stress granules disrupting its regulation of the DNA damage pathway  inflammatory response and neuronal death in neural cells and mice, implicating further pathological activation of the atypical DExD/H-box RNA helicase MDA5  CNG dsRNAs . The genase MDA5 . It is aneration . The preneration . FurtherThe RNA helicase UPF1 is an essential component of the mRNA surveillance pathway responsible for sensing and degrading aberrant mRNA transcripts, e.g., mRNAs harbouring premature stop codons (PTCs), termed the nonsense mediated mRNA decay (NMD) pathway. UPF1 specifically enables the processive unwinding NMD target mRNAs, remodelling the mRNP complexes present on the mRNAs and facilitating their degradation by the NMD machinery . UPF1 exC9ORF72-repeat driven neurodegeneration. The DPRs polyGR and polyPR have been shown to inhibit UPF1 mediated decay, including NMD  is involved in multiple processes, however its most documented role is as a core member of the essential multiprotein survival of motor neurons (SMN) complex, which is involved in snRNP assembly during splicing . SMN is SETX, ALS4), Spastic Paraplegia 11  and sigma non-opioid intracellular receptor 1 . ALS4, like the adult form of the disease, is characterised by limb weakness, muscle atrophy, and pyramidal features with associated degeneration of motor neurones in the brain and spinal cord  through a loss-of-function mechanism , senataxin  forms of neurodegeneration, including DDX6, DHX30 and IFIH1. DDX6 is an RNA helicase which has functions in enhancing decapping and repressing translation (reviewed in ). As disusing ID . All mutusing ID . DHX30 iusing ID . De novoisorders . The misisorders . DHX30 hociation . Additioociation . Finallyociation . A diagrDrosophila eIF4B and eIF4H1, two translation initiation co-factors stimulating the activity of eIF4A, lead to varying degrees of suppression of the rough eye phenotype in in vivo in both FXTAS and C9ORF72-ALS/FTD flies (in vivo in Drosophila. Further studies in mouse models of FXTAS and C9ORF72-ALS/FTD will be necessary prior to validating or infirming these eIF4A co-factors as potential therapeutic targets. The eIF4H gene is deleted in the Williams syndrome, a multisystem developmental disorder, and the specificity and level of depletion in therapeutic approaches is also likely to be critical. On the other hand, improving the UPF1 function through overexpression is generating a large interest in cell and Drosophila models of C9RF72-ALS/FTD (A large number of RNA helicases plays key functions in driving and regulating multiple steps of the mammalian expression of genes. Their roles in unwinding RNA structures is critical in both physiological and pathophysiological conditions. Expansions of short microsatellite sequences of usually 3 to 6 nucleotides are the hallmark of repeat expansions disorders, which mainly encompass neurodegenerative conditions in over 50 diseases . Repeat TD flies  while deTD flies . Whilst -ALS/FTD  as well -ALS/FTD . However


Furthermore, (TGGAA)n was isolated as the only repeat explaining the pathogenesis because other repeats were encountered in control Japanese. From the genomic point of view, the complex penta-nucleotide repeat lies in an intronic segment shared by two genes, BEAN1  and TK2 (thymidine kinase 2) transcribed in mutually opposite directions. While TK2 is ubiquitously expressed, BEAN1 is transcribed only in the brain. Thus, the complex repeat is bi-directionally transcribed exclusively in the brain, as two independent non-coding repeats. Furthermore, the complex repeat containing (UGGAA)n was found to form abnormal RNA structures, called RNA foci, in cerebellar Purkinje cell nuclei of SCA31 patients’ brains. Subsequent investigation by over-expressing (UGGAA)n in Drosophila revealed that the RNA containing (UGGAA)n exerts toxicity in a length- and expression level-dependent manner, whereas its toxicity could be dampened by (UGGAA)n-binding proteins, TDP-43, FUS and hnRNP A2/B1. It seems rational to formulate a treatment strategy through enhancing the role of RNA-binding proteins against (UGGAA)n-toxicity in SCA31.Spinocerebellar ataxia type 31 (SCA31) is one of the most common forms of autosomal-dominant cerebellar ataxia in Japan. SCA31 has a strong founder effect, which is consistent with the fact that this disease is basically absent in other ethnicities. After searching the entire founder region of a 2-megabase (Mb), we finally identified a 2.5 to 3.8 kb-long complex penta-nucleotide repeat containing (TGGAA) Clinical features of SCA31 were summarized as, late-onset (average around 60 years old), slowly progressive ataxia. Extra-cerebellar features such as pyramidal tract signs, neuropathy or gaze limitations were not detected. In accord with this, magnetic resonance imaging showed cerebellar atrophy without brainstem involvement . The presence of strong founder effect made us to collect many families as much as we could, in order to search for a rare recombination within the locus. Thanks to many neurologists all over the country sending us their DNA samples. Continuing such efforts, we finally reached to seize a 900-kb critical region limited by two recombination events, first one as the C-to-T change in PLEKHG4 and the other end at rs11640843 [We embarked a linkage analysis for these families, and three years later, we found their locus to a long arm of chromosome 16 . Subsequrophin-1 , 6, suggIn order to isolate the cause of SCA31, we had to check all the polymorphic markers that existed in the 900-kb critical region one by one. To do this, we took three different approaches: (1) Southern blot analysis for genetic rearrangement, (2) constructing BAC  tiling-path contig for the entire region followed by shot-gun sequencing for identifying smaller genetic changes, and (3) PCR-based Sanger sequencing of the entire 900-kb region. We embarked all the three approaches independently, and finally coincided with one genetic changes which all the affected members across different families harbored a 2.5 to 3.8 kb-long sequence not listed in public database . Therefon, (TAGAA)n, (TAAAA)n and (TAAAATAGAA)n [8–20 was seen. As we initially did not understand the implication of this change, we examined the relation between the length of this complex repeat and their age-of-onset for all available samples. Although the association was initially unclear, continuing this search by increasing new patients led us to find a weak inverse correlation between the two factors, supporting our discovery that the complex penta-nucleotide repeat containing (TGGAA)n, (TAGAA)n, (TAAAA)n and (TAAAATAGAA)n is indeed the mutation [n, and the other two chromosomes were complex repeats with (TAAAA)n, (TAGAA)n and (TAAAATAGAA)n. Thus, (TGGAA)n was never observed in controls. From these observations, we concluded that (TGGAA)n was the only repeat segregating with the phenotype, suggesting its importance in pathogenesis. The presence of TGGAA repeat in SCA31 patients was also confirmed independently by other researchers on different set of families [Cloning and sequencing of this aberrant sequence disclosed that this was a complex penta-nucleotide repeat containing (TGGAA)ATAGAA)n   and TK2 (thymidine kinase 2). As databases at that time suggested that two genes were transcribed in mutually opposite directions, and their genomic structure did not contain the complex penta-nucleotide repeat, we suspected that there could be additional exons downstream for both genes. In addition, it was suggested that BEAN1 drives brain-specific expression, while TK2 is expressed in all human tissues. We, therefore, underwent extensive 3′-RACE experiments for both genes on brain-extracted complimentary DNA (cDNA), and found that they indeed had multiple downstream exons that had not been deposited in databases. This implied that the 2.5 to 3.8 kb-long insertion is in an intronic region shared by the two genes, BEAN1 and TK2 [Public databases suggested that the complex penta-nucleotide repeat containing (TGGAA) and TK2 . Furthern was never found in the Caucasian SCA families (n = 320) in French and German cohorts nor in the 588 healthy control subjects [n, (TAGAA)n, and (TAAAATAGAA)n are present.As described, the SCA31 shows a strong founder effect. While SCA31 is a common ataxia in Japan, this disease has been reported from only a few countries such as Korea , Taiwan subjects . From thn regardless of the ethnicity. In Caucasians, different repeats such as (TACAA)n, (GAAAA)n, (TGAAA)n, and (TAACA)n are frequently seen n, (TAGAA)n, and (TAAAA)n. It seems possible that all the Caucasian repeats and the Japanese normal repeat (TAGAA)n, all arose through a single nucleotide transition. For example, (TACAA)n could arise from a A-to-C transition of the second A in the (TAAAA). If this view is correct, the (TGGAA)n could be regarded as the consequence of two transitions of A-to-G. If this is the case, some unknown genetic factor that predisposes to transitions may be present in SCA31 genome. Furthermore, such factors may also be present in other repeat expansion disorders.Apart from this notion, we also found the fact that nearly 5.5% of the tested Caucasian cohorts harbored penta-nucleotide repeat expansions different from Japanese repeats. This raised an intriguing question why the SCA31 penta-nucleotide repeat locus is highly unstable depending on the ethnic backgrounds. The basic repeat is (TAAAA)n is expressed in both directions: TK2 gene drives (UUCCA)n in virtually ubiquitous tissues, while BEAN1 gene is expressed exclusively in the brain. As patients with SCA31 do not show any clear manifestation outside the brain [BEAN1 gene expression fitted better than TK2 for explaining the pathogenesis. Therefore, we tested whether (UGGAA)n or (UAGAAUAAAA)n, the BEAN1-derived penta-nucleotide RNA repeats, in human cerebellum. In situ hybridization using a locked nucleic acid (LNA)-oligonucleotide (TTCCA)5 probe, Yusuke Niimi and our colleagues identified RNA foci within SCA31 Purkinje cells’ nuclei [n21. We also tested whether (UGGAA)n is toxic in cultured cells by creating transient and stable expression cell systems. We found that cell toxicity and formation of RNA foci were both consistently observed upon expression of (UGGAA)n than by expressing (UAGAAUAAAA)n. These observations led us to conclude that (UGGAA)n could be toxic in cells.As described, (TGGAA)he brain , 16, we ’ nuclei . SimilarDrosophila models expressing an expanded (UGGAA)n repeat RNA [n are toxic in Drosophila as well. In this model, the length of penta-nucleotide repeat had been contracted to 80–100 repeat (UGGAA)n compared to human SCA31 genome. Nevertheless, abundant RNA foci and remarkable eye degeneration were seen: some of the strongest lines were lethal, and some other lines with dramatic eye degenerations had a strong transgene expressions. On the other hand, lines with mild phenotypes had a low (UGGAA)n expression, in which the number of RNA foci was also small. The control repeat and a short UGGAA22 RNA, the latter happened to be generated by spontaneous contraction of the TGGAA repeats, had no significant effect.Collaborating with Professor Yoshitaka Nagai, Taro Ishiguro and our colleagues created peat RNA . We convn binding proteins that may mediate some sort of disease mechanisms. Therefore, Sato N. and our colleagues screened for potential (UGGAA)n-binding proteins by in vitro RNA pull-down assay using nuclear fraction of PC12 cells, and found that TDP-43 , FUS, and hnRNPs may bind to (UGGAA)n. After confirming that TDP-43 would bind to (UGGAA)n in western blot [n expressing flies with a line that expresses human TDP-43, and found that the phenotype and RNA foci brought by (UGGAA)n were both alleviated. Similar effects were also seen for other (UGGAA)n binding proteins, FUS and hnRNPA2/B1. When the (UGGAA)n expressing flies were crossed with the fly, in which the endogenous protein homologous to TDP-43 was deleted, the (UGGAA)n toxicity was further enhanced. Further details of our findings could be seen in reference [n, we considered these proteins act as RNA chaperone.Considering that one of the pathogeneses of noncoding repeat expansion disorders is mediated by RNA-binding proteins by binding to noncoding repeat responsible for human diseases , we thouern blot , we croseference . As thesn-toxicity and its counteracting effect by the (UGGAA)n-binding proteins as RNA chaperones are maintained in a well-balanced state when healthy, but once it tilts towards the RNA toxicity due to overexpression of (UGGAA)n, SCA31 emerges.From these and other related findings, we built a new idea that the RNA toxicity initiated by (UGGAA)Drosophila models.If our hypothesis is true for SCA31 human brains, overexpression of RNA binding proteins to an appropriate level may be rational, as we saw in n-containing RNA foci is low. Therefore, it seems that there are many other hidden factors behind SCA31 pathogenesis. Further studies are needed to discover mechanisms that (TGGAA)n in SCA31 patients’ genome leads to neurodegeneration.However, the number of (UGGAA)


Pleurotus tuoliensis. However, the molecular mechanism underlying primordium formation is largely unknown. This study investigated the transcriptional properties during primordium formation of P. tuoliensis by comparing transcriptome. Clean reads were assembled into 57,075 transcripts and 6874 unigenes. A total of 1397 differentially expressed genes were identified . GO and KEGG enrichment analysis showed that these DEGs were involved in “oxidoreductase activity”, “glycolysis/gluconeogenesis”, “MAPK signaling pathways”, and “ribosomes”. Our results support further understanding of the transcriptional changes and molecular processes underlying primordium formation and differentiation of P. tuoliensis.Primordium formation is extremely important for yield of  P. tuoliensis, also known as Bailinggu, is a precious, rare, and edible fungus with extremely high nutritive, medicinal, and industrial value [P. tuoliensis is rich in fungal polysaccharides, and modern pharmacological studies have shown that the fungal polysaccharides have a variety of biological functions, such as antioxidant activities, antitumor activities, and immune activity [P. tuoliensis is becoming more and more popular in China, Japan, and Korea, so it has become widely commercially cultivated [P. tuoliensis is usually distributed in Xinjiang province [P. tuoliensis, such as cottonseed hulls, sawdust, and corn stalks [al value ,2. It coal value . Additioactivity ,5,6. Witltivated . Wild P.province . Many agn stalks ,10.P. tuoliensis has been commercially cultivated on a large scale since 1997. By 2012, the output of P. tuoliensis had reached 300,000 tons. However, the yield is unstable and has gradually decreased in recent years [P. tuoliensis industry has been seriously restricted by the small number of cultivated varieties, single main cultivated variety, long cultivation period, and strain degradation [P. tuoliensis requires multiple environmental factors to initiate the primordium, such as humidity, special temperature and light conditions, and so on [P. tuoliensis, the formation of the primordium is generally induced by alternating day and night light [FBH1 positively regulated the primordium formation of P. ostreatus. The downregulated expression of FBH1 resulted in fewer primordia [Pofst3 played a negative role in the primordium formation. The primordium formation of Pofst3-overexpressing strains was inhibited [2 could inhibit tripe development and thus affected the primordium formation [nt years . The devradation . The fornd so on ,15. Tempnd so on . Cold stnd so on . Light ind so on ,19,20,21ht light . Many strimordia . Pofst3 nhibited . CaCl2 cormation . HoweverGanoderma lucidum [Cordyceps militaris [P. tuoliensis [hsp70, hsp90, and fes1 genes, which were involved in the early stages of fruiting body development in Flammulina filiformis [P. ostreatus [In recent years, high-throughput sequencing technologies have developed rapidly, and transcriptomics, metabolomics, and other omics have been widely applied to explore the growth and development of edible mushrooms ,25,26,27 lucidum , Cordyceilitaris , and P. oliensis  have expliformis . GC-MS astreatus . These sP. tuoliensis have been carried out to investigate mycelial maturity [P. tuoliensis. In this study, Illumina sequencing technology was used to perform a transcriptome analysis during four primordium formation stages. Twelve cDNA libraries were constructed and differentially expressed genes and pathways were identified. Our results provide new insights into the mechanism of primordium formation. Moreover, the sequences identified here can be used in future marker-assisted breeding programs to speed up the breeding progress of P. tuoliensis.Many studies of maturity , the resmaturity , and thematurity . HoweverP. tuoliensis (CCMSSC00489) was obtained from the Agricultural Culture Collection of China. The strain was activated on potato dextrose agar (PDA) medium at 25 °C for 7 days. We selected four different stage samples during the process of primordium formation. Pre-cultured mycelia were inoculated onto the substrates in the culture dishes. These culture dishes were then incubated at 25 °C for 7 days without light (Stage I). We randomly selected fifteen culture dishes of Stage I and placed the mycelia into three tubes on average and froze them in liquid nitrogen for RNA extraction. The rest of the culture dishes were cultivated at 4 °C for 3 days in the dark (Stage II). The mycelia of Stage II were also collected using the same method. In Stage III, the rest of the culture dishes were treated with alternate 12 h light and 12 h dark per day at 16 °C for 7 days to induce primordium formation. The method of collecting mycelia remained unchanged. The rest of the culture dishes were also cultivated under 12 h light and 12 h dark up to primordium formation (Stage IV). The primordia were taken into three corresponding tubes on average and frozen in liquid nitrogen. All samples were frozen at –80 °C.The ® spectrophotometer . RNA with a 260/280 nm ratio of 1.8–2.0 was chosen for analysis.The RNA was isolated separately from each sample using a plant genomic RNA kit . The quality of RNA was measured on agarose gels. RNA was checked for purity, concentration, and integrity using the NanoPhotometer® Ultra™ RNA Library Prep Kit for Illumina® . After that, paired-end sequences of the twelve libraries were performed on an Illumina HiSeq 2500 platform by Novogene Co., LT. .A total amount of 1.5 µg RNA per sample saved in each tube was used as the input material for the RNA sample preparations. Three cDNA libraries used as three biological replications were constructed for each stage and twelve sequence libraries were generated using NEBNextRaw data in FASTQ format were firstly processed through in-house perl scripts. Adapter sequences, sequences containing ploy-N, low-quality reads (<Q20), and sequences with lengths less than 50 bp were removed to obtain high-quality clean data by Cutadapt (version 1.15). In addition, the Q20, Q30, GC-content, and sequence duplication level of the clean data were calculated. Clean data were used for transcript and unigenes by Trinity software . The unip-value < 0.05, |log2 Fold Change ≥ |1) [Differential expression analysis was performed on each adjacent developmental stage of primordium formation. Analyses were based on three biological replicates per stage. RSEM was used to calculate transcript abundance by mapping clean reads to the unigene database . The DESge ≥ |1) .GOseq and KOBAS software  were useGAPDH) was used to as an internal reference gene [2O). Amplification conditions were 95 °C for 5 min, 95 °C for 15 s, 60 °C for 1 min, 72 °C for 30 s, three cycles. The relative gene expression levels were calculated using the 2−∆∆Ct method [qRT-PCR was used to validate the results and 10 DEGs were chosen randomly. The RNA was the same as that used for RNA-seq analysis. Primers were designed by Primer 5.0 software and are shown in nce gene . The qRTt method .P. tuoliensis includes four major stages (P. tuoliensis were as follows: mycelia were first grown at 25 °C in cultivation dishes for up to 7 days in the dark (Stage I), then cold stimulation of mycelia at 4 °C for 3 days without light was carried out (Stage II). Then, the mycelia were grown at 16 °C in light 12 h/day for 7 days (Stage III). The mycelia were then incubated at 16 °C in light 12 h/day for another 7 days (Stage IV). The mycelia turned into primordia in Stage IV. The environmental conditions required for the formation of the primordium had previously been studied, but the molecular mechanism remained unknown. To investigate the molecular basis of primordium formation, mycelia and primordia of four stages were collected for further analysis.The developmental process of primordium formation of r stages : myceliaTwelve cDNA libraries were constructed from the four stages, with three replicates per stage. A total of between 21,091,886 and 23,211,965 raw reads were obtained after Illumina sequencing. After quality control, 76.31 Gb of clean reads was used for further study . The Q20−100 had the largest ratio, which was 46.7%  category, 55.41% of unigenes were related to cellular anatomical entity, 36.19% to intracellular, and 26.84% to protein-containing complexes. Within the biological process (BP) category, the majority of unigenes were assigned to cellular process (69.81%), metabolic process (66.62%), biological regulation (28.49%), and regulation of biological process terms (26.20%). Moreover, unigenes related to binding (68.00%) and catalytic activity (58.43%) were enriched in the molecular function category .KEGG analysis showed that unigenes were represented in five pathways: “Cellular Processes” (332 unigenes), “Environmental Information Processing” (202 unigenes), “Genetic Information Processing” (515 unigenes), “Metabolism” (1033 unigenes), and “Organismal Systems” (307 unigenes). The number of unigenes related to carbohydrate metabolism was the highest .P. tuoliensis transcriptome. These unigenes were assigned to 25 categories. Among them, “General function prediction only”, “Posttranslational modification, protein turnover, chaperones”, “Signal transduction mechanisms”, and “Translation” were the most represented KOG pathways . Oxidoreductase activity (GO: 0016705) was enriched in most DEGs, indicating that oxidation–reduction processes may be highly active in primordium formation.To investigate the biological functions of DEGs involved in primordium formation, GO analysis was performed. Compared to Stage I, upregulated DEGs were enriched in 92 terms  and downTo better understand the functions and interactions of these DEGs, KEGG pathway enrichment was also conducted. In the Stage II:Stage I comparison, DEGs were enriched in 39 pathways . The topTo identify genes involved in primordium formation, we analyzed 26 DEGs, which were communally altered in all stages. The 26 DEGs were selected for heat map analysis . No geneTo verify the accuracy of transcriptome sequence data, 10 unigenes were randomly selected for quantitative RT-PCR (qRT-PCR), including ABC transporter (Cluster-198.6107), C2H2 Zinc finger (Cluster-198.616), CDC45-like protein (Cluster-198.5286), cellulase (Cluster-198.720), F-box-like (Cluster-198.215), Glycosyl hydrolase family 25 (Cluster-198.6122), PEX11 (Cluster-198.3467), TMPIT-like protein (Cluster-198.3145), transcription factor ZPC3 (Cluster-198.6036), and TRIAD3 (Cluster-198.1396). As shown in P. tuoliensis is longer than other Pleurotus species, especially the formation of the primordium, which lasts approximately 70–100 days [P. tuoliensis. A previous study was carried out to identify genes associated with the fruiting body. Fu et al. found that DEGs involved in morphogenesis, primary carbohydrate metabolism, cold stimulation, and blue-light response were related to Bailinggu mushroom formation [P. tuoliensis was performed by Du et al. Transcriptomic analysis revealed that nucleotide synthesis and energy metabolism were highly active during the physiological maturation of mycelia. NDPK was predicted to be essential for mycelia maturation [P. tuoliensis. The content of CA-asp may be an indicator for mycelial maturation [P. tuoliensis, including the mycelia, vegetative mycelia, one hyphal knot, and one primordium stages.The formation of the primordium is vitally important for the development of the fruiting body and the yield of edible fungi. The production cycle of 100 days . The lonormation . A compaturation . GC-MS-bturation . HoweverWhite collar 1 (WC-1) was upregulated in Stage III. Genes related to cell wall function were significantly upregulated in Stage IV, including CHS1 and CHS4. In Stage IV, we also found oxidoreductase activity enriched with most DEGs. We speculated that genes related to oxidation–reduction activities may also play a vital role in primordium formation.Primordium formation is a complex physiological process. The vegetative growth development occurred in Stage I and the rapid growth of mycelium required more nutrients and energy. Cold stimulation of mycelia occurred in Stage II. GO and KEGG pathway analysis found that these DEGs of Stage II were associated with gluconeogenesis and protein metabolism pathways. Light stimulation is necessary for primordium formation. Schizophyllum commune, the deletion of the transcription factors HOM1 and GAT1 resulted in the formation of more but smaller fruiting bodies with an unusual morphology compared to the wild type [Agaricus bisporus [Flammulina filiformis [Transcription factors that are essential for the development of fruiting bodies in fungi have been identified. In ild type ,42. The bisporus . Liu et liformis . In thisFlammulina filiformis, ribosomal protein encoding genes were hub genes in the young fruiting body. The results indicated that more protein is needed during reproductive growth [Coprinopsis cinerea and other fungi [Many KEGG pathways are associated with the development of fungi, such as carbon metabolism , oxidatie growth . The TCAe growth . Similarer fungi ,49. HoweLentinula edodes was performed by Yoo et al. This study systematically researched the expression patterns of light-induced browning-related genes, which were significantly associated with light sensing via photoreceptors such as FMN- and FAD-bindings [Pleurotus eryngii revealed DEGs were significantly associated with light sensing, signal transduction, cell wall degradation, and melanogenesis [Light is the key signaling element for every living cell . In fungbindings . A compaogenesis . In thisP. tuoliensis. The formation of the primordium is a very complex process, which involves functions related to, e.g., the cell wall, ribosomal proteins, light sensing, cold stimulation, oxidoreduction, and carbohydrate metabolism. In this work, 1397 DEGs were identified, including ribosomal protein encoding genes, white-collar 2 type of transcription factor genes, cytochrome encoding genes, MAPK signaling pathway genes, and other metabolism related genes. Among these DEGs, 26 DEGs were communally altered in all stages, which played an important role in many pathways, such as protein phosphorylation, transcription, translation, the oxidation–reduction process, and so on. These data provided a valuable resource for further studies on P. tuoliensis. Further studies should focus on the functional features of the 26 DEGs to enable better understanding of primordium formation and provide a foundation for future breeding.This study broadened our knowledge of the molecular mechanism of primordium formation by comparative transcriptome analysis of four stages of growth in