The SHAPE technique, chemical modification with DMS and RNA cleavage with Pb2+, were performed in order to characterize the RNA structure. The experimental results were used to improve the computer prediction of the structural models, whereas a phylogenetic analysis was performed to check universality of the newly identified structural elements for twenty CVB3 genomes and 11 other enteroviruses. Some of the RNA motifs turned out to be conserved among different enteroviruses. We also observed that the 3′-terminal region of the genome tends to dimerize in a magnesium concentration-dependent manner. RNA affinity chromatography was used to confirm RNA–protein interactions hypothesized by database searches, leading to the discovery of several interactions, which may be important for virus propagation.Here we present a set of new structural elements formed within the open reading frame of the virus, which are highly probable, evolutionarily conserved and may interact with host proteins. This work focused on the coding regions of the CVB3 genome , which, with the exception of the  Over the last decade the importance of the secondary structure of the RNA of viral genomes has become apparent, which has led to the mapping of the RNA of whole viral genomes, and transcriptomes isolated from simple organisms ,2,3,4,5.By using global analysis of viral genomes in search of secondary structures, it was possible to identify new structural patterns and motifs present in both the coding and non-coding regions ,8. NumerThe degree of RNA structure complexity in the protein coding regions can affect translation by changing the reading frame with a pseudoknot or temporarily stalling the ribosome, for example. Identification of thermodynamically stable and evolutionarily conserved RNA secondary structures within Epstein–Barr Virus (EBV) coding regions revealed the importance of their function in the viral life cycle. Analysis of the structure of the EBV genome led to the tentative identification of twenty-two structures, which include the START and STOP codons. RNA structure has been shown to influence the regulation of translation by modulating the accessibility of these codons. A high degree of RNA structure in the vicinity of the STOP codon can lead to “readthrough”, or continuation of translation past a stop codon. In contrast, manipulation of the START codon can result in a reduction in the efficiency of translation .Enterovirus of the Picornaviridae family showed only two potential secondary structure motifs located inside the open reading frame [cis-acting replication element) also called oriI . It folds into a stem-loop RNA structure within the 2C-ATPase coding site of poliovirus (PV), coxsackievirus B3, and other members of the genus and it plays a crucial role during the replication process [The GORS  occurrence prediction for the genus ng frame . One of  process ,14. The  process . Later,  process ,15. AnotIn contrast, a new extended structural element was found experimentally that had not previously been predicted by bioinformatic screening. In the 3′-terminal of the type C enteroviruses, which includes poliovirus, a complex RNA structure has been found that inhibits ribonuclease L: i-RNase L. This element is encoded in the 3CPro site of poliovirus and several types of coxsackievirus A, all members of the subgenus Enterovirus C ,17,18. TCVB3 and another ssRNA(+) virus, Hepatitis C virus (HCV) are sensitive to RNase L digestion, and they do not have the known i-RNase L structure within their genomes. It has been shown that fragments generated by this enzyme from the HCV genome are able to refold into hairpin structures, inducing an interferon response. Among them are so called “super-inductors” of interferon, which is probably a function of their secondary structure .Picornaviridae family, consists of positive-sense single-stranded RNA molecule, 7400 nucleotides (nt) long [The genome of coxsackievirus B3 (CVB3), a member of the nt) long . It contnt) long ,23,24. Hnt) long . The knoIn this study, the goal was to find very well-defined and evolutionarily conserved RNA structural elements in the CVB3 genome, particularly in its protein-coding region. We consider well-defined structural elements as those that remain unchanged in alternate predictions of the structure, and therefore have a high probability of occurrence in vivo. The selected regions of CVB3 genome were subjected to secondary structure probing, and to phylogenetic analysis. We focused on the V4-, V1-, 2C-, and 3D-coding regions. By analogy to other RNA viruses, we expected to find structurally well-defined and potentially essential RNA elements, especially in the highly conserved sequence encoding viral polymerase (3D). Additionally, we investigated the 3D-coding region for RNase L digestion sites, the homodimerization process, and RNA–protein interactions.2, ATP, β-mercaptoethanol) were from Sigma-Aldrich Corp  or BioShop Canada Inc. . Enzymes: Q5 DNA polymerase , Superscript III reverse transcriptase (Life Technologies), recombinant RNase L (in an active state) were purchased from Cusabio Technology LLC .The materials used in this study were from the following sources: Unlabeled DNA primers were from Genomed . Fluorescently 5′-labeled “reverse” primers were from Life Technologies  and all the chemicals  were used. The cDNAs were amplified by PCR with the primers shown in In the next step, the ca. 750-nt long RNAs were transcribed. Transcription reactions were performed using the TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. In case of RT products intended for capillary electrophoresis, 5′-end fluorescently labeled primers were purchased and added to the reverse transcription reaction and incubated at 55 °C for 10 min. The synthesized RNAs, were checked for size, integrity, and homogeneity on a denaturing agarose gel, and purified with the GeneJet RNA Cleanup and Concentration Micro Kit (Thermo Fisher Scientific). The quantity of RNA obtained was measured spectrophotometrically at λ = 260 nm.2+), and DMS methylation were performed on the basis of protocols published elsewhere [The in vitro SHAPE  reaction, RNA cleavage in the presence of lead ions , reverse transcription reactions were performed with 0.2 μg of the RNA and a suitable reverse DNA primer  . For pre2+-induced cleavages were taken as additional information by choosing one of the models generated by the computer program [Based on experimental data and theoretical thermodynamic calculations, the program generated models of analyzed secondary RNA structures. The resulting reactivity values of individual nucleotides from SHAPE reactions were analyzed in the ShapeFinder program, normalized, and then entered into the RNAstructure tool, together with the sequence of the analyzed RNA fragment . The sec program ,28,33,34The fragments F1, F1–2, and F10 are known to contain complex autonomous structures, which were omitted for better structure modeling of the remainder of the sequences. For F1 and F1–2, the excluded domain was a part of the IRES elements . In the The RNA or cDNA sequences of twenty CVB-3 isolates were aligned with sequences of analyzed structural elements in the Clustal Omega program ,36 usinghttps://www.ebi.ac.uk/Tools/msa/clustalo/.The Clustal Omega program can be found at http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAalifold.cgi.The RNAalifold program can be found at: 2, 50 µM ATP, and 7 mM β-mercaptoethanol. Four identical samples, each of 14 µL, were prepared, denatured for 5 min at 70 °C, and renatured by incubating for 10 min at 37 °C. Subsequently either 0, 0.5, 1, and 2 µL of 4.15 mM RNase L was added and filled with water to bring the volume to 16 µL. The mixture was incubated for 1 h at 37 °C and the RNA was subsequently purified with the GeneJET RNA Cleanup and Concentration Micro Kit .Before the procedure, 5 µg of the F10 RNA was combined with a cleavage buffer containing 25 mM Tris pH 7.4, 100 mM KCl, 10 mM MgCl2. The mixture was prepared in 10 µL samples, denatured for 5 min at 70 °C and renatured by incubating for 10 min at 37 °C. For dimer formation inhibition, either 5- or 50-fold molar excess of DNA oligomer “antydimer” (5′-ATCTA TAGGC CATGG GTACG ATGC GATCAC-3′) was added before renaturation. RNA was subsequently loaded onto a 1% agarose gel with standard non-denaturing 6 × loading dye and subjected to electrophoresis in 0.5 × TAE buffer containing 0.1 mM EDTA. As an RNA length marker, RNA Riboruler was used (Thermo Fisher Scientific).From 0.5 to 1 µg of the F10 RNA (for each reaction option) were combined with a cleavage buffer containing 10 or 25 mM Tris pH 7.4, 100 mM KCl or 40 mM NaCl, and 0.5 or 10 mM MgClHomo sapiens. The database can be found on: https://attract.cnic.es/index#.Potential interactions with proteins were proposed with the help of the ATtRACT database, which is a database of experimentally validated RNA binding proteins and associated motifs . We uplo7 MCF7 cells were used per extract for one RNA-affinity chromatography procedure. The cells were washed with PBS buffer and then they were collected by centrifugation at 1000 rpm. The pellet was resuspended in 5 pellet volumes of CE buffer  NP40, 1 mM DTT, and 1 mM PMSF, pH 7.6) and incubated on ice for 3 min. Then cell lysate was centrifuged at 1000–1500 rpm for 4 min and the cytoplasmic extract was collected.MCF-7 cells  were maintained in DMEM medium. All medium solutions were supplemented with 10% fetal bovine serum, non-essential amino acids , 100 U/mL of penicillin G, 0.1 mg/mL of streptomycin sulphate  and the cells were maintained at 37 °C in a 5% carbon dioxide atmosphere. Genotoxic stress was generated by addition of doxorubicin to a final concentration of 0.5 μg/mL. Approximately 4 × 10p/v) glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF). The RNA coupled to beads was incubated with 40% (v/v) cytoplasmic extract with addition of 1.5 mM MgCl2, 25 mM creatine-phosphate, and 5 mM ATP for 30 min at 37 °C with shaking at 400 rpm. Next, the beads were washed four times with 1 mL of buffer D containing 1.5 mM MgCl2 and twice with Milli-Q water. Subsequently, RNA coupled to the beads was incubated in 60 μL reaction mixture containing 10 mM Tris-HCl (pH 7.2), 1 mM MgCl2, 40 mM NaCl, and 5 μL of A/T1 ribonuclease mix  for 30 min at 37 °C, shaking at 1400 rpm for 10 sec every minute. The concentration of RNases in the A/T1 mix was 500 U/mL for RNase A and 20,000 U/mL for RNase T1. The resulting reaction mixture was centrifuged for 1 min at 4 °C and the supernatant was collected.RNase-assisted RNA chromatography was performed as described previously ,42. BrieHomo sapiens database. Subsequently, the Mascot results list including proteins represented by at least one peptide with a score above the threshold was analyzed by using MScan software available at proteom.ibb.waw.pl/mscan/index.html.The proteins obtained after RNase-assisted RNA chromatography were identified using MS/MS analysis . MS analDue to the extreme evolutionary pressure viruses are subjected to, a conservation of RNA structural motifs within their open reading frames is generally an indication that they play an important role in the life cycle of the virus. For example, they may be sites of interaction with host proteins, as observed with poliovirus and Coxsackie A viruses, where an RNA structural element has recently been demonstrated to bind to cellular ribonuclease L and inhibits its activity. Stable RNA secondary structures in the coding part may also affect the proper folding of viral proteins by slowing the ribosome, as proposed for HIV , or playCoxsackie B3 virus, as an enterovirus and representative of the picornavirus family, is an excellent model for research on the occurrence of structural motifs throughout the entire genome and for discovering their function. Moreover, in a broader sense, CVB3 is a representative of viruses with an RNA genome.To the best of our knowledge, with the exception of the CRE in the 2C region, the coding part of the CVB3 genome has not yet been structurally characterized. We decided to search the coding region of the viral genome for the presence of other RNA structural elements that are highly probable, evolutionarily conserved, and thus potentially important for the virus propagation.2+) [In order to investigate the structure of the entire genome of the CVB3 virus, especially the part encoding the viral polyprotein, nineteen RNA fragments corresponding to the entire viral genome were synthesized. We decided to experimentally study the secondary structure of several of those RNA fragments, each less than or equal to 800 nt in length . This le2+) ,33.These particular fragments of CVB3 genomic RNA were selected for their location in different regions of the genome, representing both conserved non-coding regions , coding for variable structural proteins (F1–2 and F4), and conserved non-structural proteins: viral protease 2C (F6–7), and 3D polymerase . The selection of the F8–9_ F10 fragments was partly dictated by the relatively high level of conservation of the sequences encoding this polymerase within related viruses. This offered the chance of finding potentially relevant motifs in more than one isolate of RNA virus. The 2C-coding region was chosen in order to test our experimental approach by confirming the presence of the CRE and to search for other structures in the close neighborhood of CRE. Region 3D was chosen because in analogous regions of related viruses, secondary structure elements have already been found, such as: iRNasel (group C enteroviruses), E-10 hairpin (bioinformatic predictions for enteroviruses), and the 3D-7000/WT-α and WT-β elements important for the replication of poliovirus. The V4 region was selected for its proximity to the 5′-UTR. In other viral genomes, such as HCV, stable structures have been found in this position. The V1 region, in contrast, was chosen as the least conserved of all protein coding regions in the CVB3 genome. We were interested to see if there is any difference in abundance of structurally well-defined RNA elements in comparison to the most conserved regions. Another objective was to find strictly CVB3-specific RNA structures in the V1 coding part.The structural characterization of selected CVB3 genome fragments was performed in vitro using the SHAPE technique. The obtained experimental data were used as constraints in the process of generating the eight RNA secondary structure models (SM1). The RNAstructure program proposed a set of alternative structural models for each fragment, which lie within 10% of the minimal energy. By comparative analysis of those alternative structure models, the probability of the formation of particular RNA motifs was calculated. Based on such comparisons and calculations, 21 structural motifs were identified within 8 RNA fragments . Their pHere we present a set of individual structures of newly identified, high probability RNA elements , and theStructure of fragments F1 and F1–2 were analyzed on the basis of the experimental results using the RNAstructure program in two ways. First, the full-length sequence of the fragment was analyzed, and then part of the sequences responsible for coding the I–V domains, mostly belonging to the IRES elements of the 5′-UTR region, were removed. This facilitated the calculation of the secondary structures of the 5′-terminal part of the 5′-UTR region, because the IRES region probably does not interact with the rest of the CVB3 genome, as it is believed to be autonomous. As expected, the presence of a previously described structure, an IRES domain VI-VII, was confirmed in our analysis.TP53 transcript, in that it occurs partly in a bulge of the longer stem, and the second and third letters of the codon remain paired [The F1 fragments with and without the IRES region were 801 and 221 nt in length, respectively. Close to the 3′-end of the fragment, the program proposed a hairpin-type structure with one single-nucleotide bulge and four several-nt bulges, ending with a 9 nt apical loop rich in adenosines named SL665-748 . In addin paired ,45,46,47It is also important to emphasize the existence of the SL665-748/652-752 motif in the structure projections for the two overlapping RNA molecules: F1 and F1–2, albeit with slight differences in the apical part and probability calculations. The length of the analyzed fragment F1–2 was 765 nt for the option with the IRES and 585 nt without the I–V domains. For the purpose of the analysis of the individual motifs, a structure with a minimal free energy ΔG = −202.3 (without the domains I–V IRES) was selected . In bothThe length of the F4 fragment subjected to analysis was 763 nt. Based on the structural models generated by the RNAstructure program, the one with the lowest energy of ΔG = −228.7 was selected . Within For the F6–7 fragment of the CVB3 virus genome, a secondary structural model was chosen that had the lowest energy level of ΔG = −281.7 . The lenThe proposal with the lowest free energy of ΔG = −266.7 was chosen as the secondary structure model for the 756 nt long F8–9 fragment . Three sThe structure of the 288 nt F9 molecule with the lowest energy, ΔG = −120.4, contains five interesting motifs, representing a highly probable RNA secondary structure  and one less probable one, SL6581-6597 . ClosestFor the F9–10 molecule, a 587 nt fragment was experimentally mapped. This fragment has 127 nucleotides shared with F9 and contains the overlapping F10 region, examined with the 9–10R primer. RNAstructure predicted the structure model F9–10, with a free energy of ΔG = −64.0, which contains four highly probable motifs. The region SL6581–6597 forms a short, simple hairpin also observed within the overlapping fragment F9 . It is p2+ ions were essential while choosing one of several alternative structural models generated by the computer program. In general, results from structural experiments, such as chemical modifications with DMS and RNA cleavage with lead ions, corresponded well to one of the models proposed by computer program RNAstructure and are displayed on the chosen structure  .Early research based on the prediction of RNA structure in silico and bioinformatic analysis found that, apart from the well-characterized non-translated regions, the genome of enteroviruses does not appear to contain conserved structural motifs , aside fFrom studies on the hepatitis C virus, which, like Coxsackievirus B, is susceptible to digestion with RNase L, it has been reported that a dozen or so RNA structural motifs are excised from the coding part of its genome during infection, and that at least some of them can serve the host by signaling the presence of an infection through the antivirus defense signal cascade . In turnWe decided to characterize which fragments of the CVB3 genome are cut out by human ribonuclease L from its terminal region. Observed RNase L digestion sites within the F10 RNA matched single-stranded regions of the structural model well, in accordance with specific activity of the enzyme, providing additional evidence of the accuracy of the model . Some ofWe also investigated the possibility of genome dimerization and RNase L digestion in the 3′-terminal region of the CVB3 genome. Both processes seem to involve the RNA element SL6981-6920. During electrophoresis of RNA products in the RNase L assay, we found that the F10 RNA fragment migrates in the form of two major bands. Moreover, the shorter band was of the expected length of the full-length F10 fragment (ca. 750 nt) and thus could not represent a digestion or degradation product. The upper band migrated close to 1500 nb of the RNA marker (and 1.8S rRNA), suggesting that it represents a double-length F10 fragment . This su+ and K+ ions gave the same results, so we could exclude G-quadruplex formation, which is dependent on the presence of potassium ions [2+ ions  we did a sequence search for each newly identified RNA motif to find potential protein binding sites , polyadenylate binding protein 1, and poly(rC) binding protein 2, to name but a few. Several proteins hnRNP K, A1, F, H1; KHSH, and PCBP1, which were found to bind to the 3′-terminal of the CVB3 genome, were also predicted by ATtRACT, and may be involved in viral replication processes . InteresTaken together, the RNA-centric affinity chromatography approach combined with MS analysis revealed several proteins, which were able to bind to the newly identified RNA structure motifs within the 3ʹ-terminal region of CVB3 RNA, that potentially might have functional implications for the CVB3 propagation .In this study we used both experimental and bioinformatic methods to study the secondary RNA structure and comparative phylogenetic studies to find new stable and conserved structural elements in the CVB3 genome, mainly in its protein coding part. Among twenty one of the RNA secondary structure motifs, we identified many with a potentially significant role for the propagation of the virus due to their high probability of occurring in the genomes of twenty different CVB3 virus isolates. Some of these RNA elements were found in other enteroviruses in similar forms as well. The RNA-centric affinity chromatography approach, combined with MS analysis, revealed several proteins that were able to bind to the newly identified RNA structure motifs within the 3ʹ-terminal region of CVB3 RNA, which potentially have functional implications for the propagation of CVB3. We also investigated the possibility of genome dimerization and RNase L digestion in the fragment corresponding to the 3′-terminal region of the CVB3 genome. Both processes can be important in the viral life cycle of CVB3 and other enteroviruses or in the antiviral response of the infected organism. The results presented in this study contribute to a better understanding of relationships between the structure and function of RNA elements found in the coding region of CVB3 and will serve as a starting point for functional studies of their importance in viral life cycle and host persistence.


NMT1 motif,” which forms a hairpin structure with an internal bulge that includes numerous highly conserved nucleotides. This motif associates with genes annotated to encode various dioxygenase enzymes, transporters, or proteins that have roles associated with thiamin or histidine metabolism. Biochemical evaluation of numerous ligand candidates revealed that NMT1 motif RNA constructs most tightly bind 8-azaxanthine, xanthine, and uric acid, whereas most other closely related compounds are strongly rejected. Genetic assays revealed that NMT1 motif RNAs function to turn off gene expression upon ligand binding, likely by regulating translation initiation. These results suggest that NMT1 motif RNAs function as aptamer domains for a riboswitch class that specifically responds to high concentrations of oxidized purines. Members of this “xanthine riboswitch” class appear to regulate genes predominantly related to purine transport and oxidation, thus avoiding the effects of overproduction of these common purine derivatives.Dozens of candidate orphan riboswitch classes have been discovered previously by using comparative sequence analysis algorithms to search bacterial genomic sequence databases. Each orphan is classified by the presence of distinct conserved nucleotide sequences and secondary structure features, and by its association with particular types of genes. One previously reported orphan riboswitch candidate is the “ Riboswitches that respond to metabolites or elemental ions are routinely used by many bacterial species to control the expression of genes coding for proteins that regulate metabolic or elemental ion homeostasis processes, or that make physiological changes in response to signaling molecules . Over 45In addition, many classes of “orphan” riboswitch candidates  have beeNMT1 motif is an orphan riboswitch candidate that was previously identified through comparative sequence analysis as a structured, noncoding RNA of unknown function  dependent dioxygenases, alpha-ketoglutarate dependent dioxygenases, nucleoside transporters, and adenosine and guanine deaminases. These bioinformatic analyses thus implicated the genes associated with NMT1 motifs in purine degradation, rather than thiamin metabolism.However, further bioinformatic analysis of the There are also various known riboswitch classes that sense purines and their biosynthetic intermediates, including guanine , adenineNMT1 motif RNAs selectively recognize oxidized purine nucleobases. Structure activity relationship (SAR) data reveal that natural ligands xanthine and uric acid are bound most tightly. In addition, genetic analyses demonstrate that representatives of these RNAs, now renamed xanthine riboswitches, function as genetic “OFF” switches to repress the translation of mRNAs whose gene products would otherwise generate oxidized purines. These findings reveal that some bacterial cells monitor the levels of oxidized purines, presumably to avoid accumulating unnecessary or even toxic levels of these purine degradation waste products.In the current study, we demonstrate that NMT1 motif RNAs  junction, wherein stem P2 forms a small internal bulge to divide the stem into two parts (P2a and P2b)  A. Key diSupplemental Fig. S1). This type of translation control mechanism is frequently used by members of many riboswitch classes  of the adjacent open reading frame (ORF) , to NMT1/THI5-like proteins (see above), or to ABC-type nitrate/sulfonate/bicarbonate transporters (tauA). Additional associated genes code for proteins similar to various dioxygenases , metallodependent amidohydrolases (hutI), amidohydrolases similar to 8-hydroxyguanine and guanine deaminases (PRK12393), nucleoside transporters (tsx), and in rare instances tRNA adenosine deaminases (tadA). Notably, these associated genes sometimes are found in series following an NMT1 motif RNA, indicating that some members of this candidate riboswitch class regulate an operon of genes related to the same metabolic process  of ∼3.7 µM , which differs from xanthine by oxidation of the C8 position of the purine ring .An RNA construct called 53 tauA A carryin binding . Initial binding B, which µM e.g., C based otauA ORF from S. plymuthica S13 called 68 tauA . Likewise, construct M3 . These results are consistent with our hypothesis that the highly conserved secondary structure and core of NMT1 motif RNAs forms a binding pocket for certain oxidized purines such as xanthine and uric acid.Similar losses in ligand binding function were observed for constructs M1 and M2 when exposed to uric acid , and additional active compounds . Most analogs tested, including the common natural pyrimidine nucleobases, are strongly rejected by the aptamers , indicating that the ligand-binding pocket is highly selective for specific oxidized purine compounds.To further examine the ligand binding characteristics of  53 tauA A or the uA . Binding is also precluded by the presence of a methyl group on N9 of uric acid, or the presence of a ribose moiety at this same position on hypoxanthine (as the compound inosine). These results highlight the importance of these purine ring positions as molecular recognition contact points, although the additional bulk presented by methyl or ribose group appendages could cause a steric clash with the aptamer. Alteration of the carbonyl oxygen atoms on C2 and C6 is also disruptive, suggesting these oxygens might be involved in forming hydrogen bonds, or possibly forming contacts with Mg2+ ions as is common with structured RNAs such as riboswitches . These findings again support our conclusion that NMT1 motif representatives function as high-affinity aptamers for xanthine and uric acid.In the process of validating NMT1 motif RNAs, we created a construct for gene expression studies in Escherichia coli as a surrogate organism by fusing the NMT1 motif representative from Hydrogenophaga intermedia strain S1 to a β-galactosidase reporter gene  resides downstream from the conserved portion of the NMT1 motif, suggesting that the expression platform might involve the regulation of ribosome binding to the mRNA. Furthermore, there is no evidence for the presence of an intrinsic terminator stem (strong stem followed by a run of U nucleotides) , which suggests that members of this riboswitch class do not directly use a transcription termination mechanism.The eotides)  in this tauA M1 construct, which is known to cause a loss of ligand binding activity (Two genetic reporter constructs were tested: a wild-type (WT) version and a mutant version (M4) that carries a single nucleotide change at the equivalent location to the 53 activity A. In liqactivity B.NMT1 motif are predicted to catalyze reactions on compounds that are earlier than xanthine and uric acid in the metabolic pathways for purine degradation. Therefore, the reporter assay results are consistent with the genetic contexts of NMT1 motif RNAs. An excess of oxidized purine degradation products might need to trigger the repression of genes involved in the transport of nucleosides by nucleoside transporters, the deamination of purines by deaminases and amidohydrolases, and the oxidation of purines by dioxygenases, or the cell risks the toxic effects of these molecules. For example, uric acid is famously disruptive to biological systems due to its insolubility, and can cause joint and kidney damage in humans  were discovered by using the comparative sequence analysis algorithms CMfinder  and most compounds used to conduct SAR analyses were obtained from Sigma-Aldrich. Exceptions include 3- and 9-methyluric acid , isoxanthopterine and 7-methyluric acid , and 8-hydroxyguanine (Carbosynth). [γ-32P] ATP (specific activity: 6000 Ci/mmol) was purchased from PerkinElmer. Genetic reporter constructs were designed  by the authors and subsequently purchased from GenScript VectorArt Gene Synthesis.Synthetic oligonucleotides (Synthetic DNA oligonucleotides templates with a T7 RNA polymerase (T7 RNAP) promoter on the 5′ terminus were transcribed, purified by 10% polyacrylamide gel electrophoresis (PAGE), and extracted, using protocols similar to those previously described .32P-labeled RNAs for in-line probing assays, 80 pmols of the RNA transcript was first dephosphorylated using rAPid alkaline phosphatase (Roche Life Sciences) following the manufacturer's protocol. Subsequently, 20 pmoles of the resulting RNA was 32P-radiolabeled at the 5′ terminus using T4 polynucleotide kinase in a 20 µL reaction mixture consisting of 25 mM CHES (pH 9.0 at 19°C), 5 mM MgCl2, 3 mM DTT, and 20 µCi [γ-32P]ATP. The reaction was incubated for 1 h at 37°C. The resulting radiolabeled RNA was purified by denaturing 10% PAGE and the desired RNA was extracted as noted above.To generate 5′ 32P-labeled RNA were incubated at 19°C for 36 h with the desired ligand in the presence of 100 mM Tris-HCl (pH 8.3 at 23°C), 100 mM KCl, and 20 mM MgCl2. Denaturing 10% PAGE was performed to resolve the reaction products. Binding curves and apparent KD values were estimated as previously described  gene by GenScript VectorArt Gene Synthesis. WT and M4 reporter constructs in plasmid pRS414 were transformed into E. coli strain BW25113 from the Keio Collection (600 of 0.1 and grown in both rich (LB) and minimal (M9) media in the presence of 50 µg mL−1 of X-gal . Changes in culture color were recorded after 11 h incubations.Genetic reporter constructs were designed with a d pRS414  upstreamllection  and grow-galactosidase activity measurements were performed as described elsewhere (Liquid β-galactosidase assays were performed to quantify the observations in media supplemented with X-gal. Reporter gene expression was quantified as described previously  with thelsewhere .Supplemental material is available for this article.


Xenopus lævis oocytes. Compared to dilute solution, the dsRNA A‐helix is more compact in cells. We recapitulate this compaction in a densely crowded protein solution. Atomic‐resolution molecular dynamics simulations of dsRNA semi‐quantitatively capture the compaction, and identify non‐specific electrostatic interactions between proteins and dsRNA as a possible driver of this effect.The structure and flexibility of RNA depends sensitively on the microenvironment. Using pulsed electron‐electron double‐resonance (PELDOR)/double electron‐electron resonance (DEER) spectroscopy combined with advanced labeling techniques, we show that the structure of double‐stranded RNA (dsRNA) changes upon internalization into  Xenopus lævis oocytes compared to the dilute solution. Atomic‐resolution molecular dynamics simulations in a dense protein solution semi‐quantitatively capture the compaction, pointing to the molecular interactions involved.A combination of advanced labeling techniques for nucleic acids and in‐cell pulsed electron‐electron double‐resonance spectroscopy reveals compaction of the dsRNA A‐helix inside  The biological function of RNA is tightly related to its structure and flexibility, which are both sensitive to the conditions and microenvironments present in the cell.The structure of RNAs inside the cell has been deduced primarily from chemical probing experiments, which have achieved single‐nucleotide resolution for base‐pairing interactions in whole‐cell measurements.3, 7 SPulsed electron‐electron double‐resonance (PELDOR)/ double electron‐electron resonance (DEER) spectroscopy,17, 18III chelates[gem‐diethyl substituents to sterically shield the spin label from reduction.[ImUm  and the stability of the shielded nitroxide inside cells verified .Early in‐cell PELDOR experiments on nucleic acids24, 25Xenopus lævis oocytes by microinjection (see Supporting Information). The in‐cell Q‐band PELDOR data .Xenopus lævis oocytes.[In an attempt to identify the origin of these in‐cell structural changes in RNA I, we incubated the labeled nucleic acid with a cytoplasmic extract, obtained by centrifugation of the oocytes (see Supporting Information). Remarkably, the PELDOR data for this sample .Realistic atomistic simulations of mixed‐component systems such as RNA and proteins pose a challenging problem, because the molecular interactions between RNA, proteins, water and ions all need to be balanced. We found that the commonly employed RNA force field OL368, 69Simulations of the dsRNA in dense lysozyme solution showed that the RNA retains its helical structure Figure  and decrImUm spin label, revealed a subtle compaction of the structure of RNA duplexes in oocytes. MD simulations of complex protein‐RNA mixtures at atomic resolution demonstrated how interactions with positively charged proteins slightly compact the RNA, and provide a possible structural explanation for the mean compaction demonstrated independently by the PELDOR measurements. It will be interesting to further investigate our findings for other cell types and states, and to correlate them to proteomic data on the abundance of basic and/or RNA‐binding proteins.In summary, in‐cell PELDOR experiments, enhanced by the highly performant EThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.


RNA modifications were discovered decades ago, and more than 150 different marks have been found decorating various RNA species, including coding and non-coding transcripts . A computational workflow dedicated to 2′-O-methylation marks was optimized, allowing a more accurate detection of these marks and a more precise quantification of their level variations . A novel tool (LITOPHONE) was developed that adopts an ensemble predictor relying on sequence features to predict m6A sites in long non-coding RNAs . A web server (PIANO) was implemented that relies on various genomic features, including sequence information, for the prediction and functional annotation of pseudouridine sites . Finally, a bioinformatic pipeline  was developed for the study of small non-coding RNAs derived from tRNAs (tRFs), allowing the study of tRFs biogenesis in Drosophila melanogaster .This Research Topic collects a number of contributions in this field. Few manuscripts focused on the development of novel methods for the prediction of RNA modifications. A Galaxy-based user friendly graphical workflow was developed that cover the preprocessing of omics data, the quantification of mismatch and arrest rates with single-nucleotide resolution, and the subsequent machine learning, modification calling and visualization . A second contribution compared m6A genome-wide maps generated in various studies based on eight different methods, discussing the agreement of the data and the challenges in their comparative analysis, revealing an expression bias in the detected genes .Two additional contributions discussed pitfalls in the analysis of specific marks. A first study discussed the impact of different bioinformatics steps on the detection of RNA editing events, describing key metrics for the quantification of their level of activity . A second study introduced MasterOfPores, a NextFlow workflow that facilitates the analysis of these data, allowing the prediction of RNA modifications and the estimation of polyA tail lengths .Two contributions were focused on the use of direct RNA sequencing through the Nanopore platform that enables long-reads sequencing of native transcripts. A perspective discussed how these data could allow quantifying the dynamics of modified RNAs at the level of individual isoforms . In the second study, bioinformatics analyses revealed a risk signature based on three m6A regulators, proposing candidate prognostic markers predictor of the clinicopathological features in hepatocellular carcinoma . In the third study, integrated bioinformatics analyses led to the identification of differentially expressed transcripts with aberrant methylation patterns in malignant pheochromocytoma .Finally, three different studies introduced bioinformatics workflows for studying the impact of RNA modifications in various tumor types. In the first study, a workflow based on consensus clustering and gene set enrichment analysis was presented that allowed the subsequent construction of a prognostic risk model suggesting the involvement of three m6A-related genes in liver cancer (www.epitran.eu) (Jantsch et al., Despite the rapid advance of the field, which allowed expanding the set of known marks, profiling their pattern, and disclosing their functional roles, a number of open questions remain (Frye et al., All authors contributed writing this Editorial and managing the corresponding Research Topic.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Genome-wide analysis of R-loop alterations in U2OS cells deficient of DDX5, XRN2, and PRMT5 identify >50,650 DRIP-seq peaks spanning ∼4.5% of the genomic sequence. R-loops near TSS generated intergenic antisense transcription. DDX5, XRN2, and PRMT5 have been shown to resolve DNA/RNA hybrids (R-loops) at RNA polymerase II transcription termination sites at few genomic loci. Herein, we perform genome-wide R-loop mapping using classical DNA/RNA immunoprecipitation and high-throughput sequencing (DRIP-seq) of loci regulated by DDX5, XRN2, and PRMT5. We observed hundreds to thousands of R-loop gains and losses at transcribed loci in DDX5-, XRN2-, and PRMT5-deficient U2OS cells. R-loop gains were characteristic of highly transcribed genes located at gene-rich regions, whereas R-loop losses were observed in low-density gene areas. DDX5, XRN2, and PRMT5 shared many R-loop gain loci at transcription termination sites, consistent with their coordinated role in RNA polymerase II transcription termination. DDX5-depleted cells had unique R-loop gain peaks near the transcription start site that did not overlap with those of siXRN2 and siPRMT5 cells, suggesting a role for DDX5 in transcription initiation independent of XRN2 and PRMT5. Moreover, we observed that the accumulated R-loops at certain loci in siDDX5, siXRN2, and siPRMT5 cells near the transcription start site of genes led to antisense intergenic transcription. Our findings define unique and shared roles of DDX5, XRN2, and PRMT5 in DNA/RNA hybrid regulation. R-loops are three-stranded structures consisting of a DNA/RNA hybrid and the displaced strand of single-stranded DNA. R-loops are typically, but not exclusively, formed co-transcriptionally where there is reannealing of the nascent transcript to its complementary DNA template with an estimated frequency of 5% depending on the locus and its sequence, transcription levels, and overall gene length . These RR-loops pose a major threat for the cell as the lack of their resolution can lead to DNA breaks causing genomic instability . R-loopsThe processing of the nascent RNAs into mRNAs and their export into the cytoplasm requires the coordinated effort of numerous factor and machineries such as splicing factors, RNA processing, and export machineries . If thesThe clearance and the prevention of unscheduled R-loops are tightly regulated by enzymes, including topoisomerases, helicases, and RNases. DNA topoisomerases such as Top1 and Top3B alter the topology of DNA by removing transcription-induced negative supercoiling to restrict access of the newly transcribed RNA transcripts to its complementary genomic DNA . In addiDExD/H RNA helicases are known to be implicated in various aspects of RNA metabolism, including regulation of R-loop accumulation . MultiplA variety of different families of helicases including senataxin (SETX) , RNA helLittle is known about how posttranslational modifications regulate R-loop metabolism. Accumulating evidence indicates that protein arginine methylation plays an important role in R-loop metabolism . ProteinSupplemental Data 1). Consensus peaks were obtained by taking the union of the peaks identified in each condition relative to its corresponding input. Each pair of biological replicates (denoted by replicates A and B) showed a high correlation in peak intensity . IntereS23 loci .Supplemental Data 1.Consensus R-loop peaks among the siLuciferase (siCTL), siDDX5, siXRN2, and siPRMT5 conditions.q-value < 0.1 and log-fold-change > 1) covering 5.33, 5.70, and 15.29 Mb in DDX5-, XRN2-, and PRMT5-deficient cells, respectively . In addition, 89, 48, and 145 R-loop peaks had reduced signals (termed loss peaks) spanning 0.43, 0.18, and 0.40 Mb . DDX5-deficient cells had 184 and 345 gain peaks that overlapped with the gain peaks of XRN2- and PRMT5-deficient cells, respectively , suggesting that these three proteins coordinately control cellular R-loop accumulation at specific genomic loci. Moreover, the XRN2-depleted cells had 480 overlapping gain peaks with PRMT5-knocked down cells (P < 4.9 × 10−324). A significant overlap of loss peaks in these three conditions was identified as well .Statistical analysis identified 762, 1,059, and 2,632 R-loop peaks with significant increase in intensity .To determine whether the increased R-loop accumulation in the DDX5-, XRN2-, and PRMT5-deficient cells was simply caused by an increase in RNA gene expression, we defined the transcriptomic changes of DDX5-, XRN2-, and PRMT5-depleted U2OS cells compared with siluciferase (siCTL) cells using RNA sequencing (RNA-seq). Unsupervised clustering analysis based on principal component analysis of sample correlations show high reproducibility among the replicates, with the first two PCs explaining 76% of the variance . The expSupplemental Data 2.RNA-seq gene expression in siLuciferase (siCTL), siDDX5-, siXRN2-, and siPRMT5-transfected U2OS cells.Supplemental Data 1). Importantly, the RNA expression of overlapping or nearest genes which showed increase in R-loop peak signals in DDX5-, XRN2-, and PRMT5-deficient cells did not significantly vary between control and knockdown cells . This was further quantified using Venn diagrams and, for example, of the siDDX5 697 genes containing at least one R-loop gain peak, only 10 and 15 overlapping or nearest genes were down-regulated and up-regulated for RNA expression in DDX5-deficient cells, respectively . The 2,632 R-loop gain peaks of siPRMT5 were closer to a neighboring gene compared to the unchanged R-loop bulk peaks  identified without a gain or loss in signal relative to siCTL in any of the samples revealed that more than half of the peaks (62.9%) were located in intronic regions, representing nascent transcripts, whereas 22.6% were located near defined noncoding and coding genes at the promoter-TSS, exons including 5′-UTR and 3′-UTR, and the TTS . FurtherP = 2.8 × 10−11 and P = 1.1 × 10−11, t test) or to the TTS , but further from TSS (P < 1.7 × 10−06). Importantly, the siDDX5 gain peaks were more likely to be located closer to the TSS (56%) than to the TTS (44%), whereas the siXRN2 and siPRMT5 gains behaved in an opposite manner  increased in DDX5-, XRN2-, and PRMT5-deficient cells, the R-loop gain peaks at the 5′-terminus (promoter-TSS and 5′-UTR) relative to the unchanged peaks  and TTS downstream of 2 kb . Genomic loci were identified with an increase in intergenic RNA-seq reads upstream of the TSS or downstream of the TTS of genes near to the R-loop gain peaks in siDDX5, siXRN2, and siPRMT5 cells using the threshold described in the Materials and Methods section . The unique list of loci for each siDDX5, siXRN2, and siPRMT5 cells  suggest that the proteins function independently of each other for this function. For DDX5, we selected eight loci for further reverse transcription (RT)-PCR confirmation of the increase of intergenic RNA. IGV tracks showed that EGR1, ACTG1, RHOB, and UBALD1 had increases of RNA-seq intergenic reads upstream of the TSS and downstream of the TTS, whereas RB1CC1, SOGA1, and STIL had increases of RNA-seq intergenic reads only upstream of the TSS in DDX5-deficient cells , consistent with GC-rich regions forming both G-quadruplexes and R-loops. DDX5 associates with the estrogen receptor α and is recruited to estrogen-dependent promoters in a cyclic fashion, suggesting DDX5 plays a role in transcription initiation , as these are likely occurring in a tissue-specific manner in breast cancer cell lines  than by DDX5 (762) or XRN2  deficiency. As PRMT5 posttranslationally modifies proteins, it is not surprising that PRMT5 has an elevated number of cellular R-loops. RNA helicases known to resolve R-loops containing arginine methylation sites (RGG/RG motifs) and potential substrates of PRMT5 include DHX9  and DDX2The presence of unscheduled R-loops is associated with increased DNA damage and genomic instability . Thus, tTaken together, our data identify a shared role for DDX5, XRN2, and PRMT5 in R-loop metabolism at the TTS region. In addition, we uncovered genomic loci where the loss of DDX5, XRN2, or PRMT5 leads to R-loop–associated antisense expression near the TSS regions. Thus, our data provide a valuable resource of the genome-wide R-loops modulated by DDX5, XRN2, and PRMT5 using classical DRIP-seq.2. siRNA oligonucleotides were transfected using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. All siRNAs were purchased from Dharmacon. siRNA sequences are as follows: siDDX5: 5′-ACA UAA AGC AAG UGA GCG AdTdT-3′; siXRN2, SMARTpool siGENOME human XRN2 siRNA (M-017622-01); siPRMT5, 5′-UGG CAC AAC UUC CGG ACU UUU-3′. The siRNA 5′-CGU ACG CGG AAU ACU UCG AdTdT-3′, targeting the firefly luciferase (GL2), was used as control. 20 nM siRNA was used for transfection.Human osteosarcoma cells U2OS cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium containing 10% vol/vol FBS at 37°C with 5% COBsrGI, EcoRI, HindIII, SspI, and XbaI) in the New England Biolabs CutSmart buffer with 2 mM Spermidine and 1× BSA. Digested DNAs were cleaned up by phenol–chloroform extraction using MaXtractTM High Density (200 × 2 ml) followed by treatment or not with RNase H (20 units/100 μg nucleic acids) overnight at 37°C in the New England Biolabs RNase H buffer. DNA/RNA hybrids from 4.4 μg digested nucleic acids, treated or not with RNase H, were immunoprecipitated using 10 μg of S9.6 antibody (ATCC) and 50 μl of protein A/G agarose beads (Sigma-Aldrich) at 4°C for 2 h or overnight in IP buffer . The beads were then washed four times with IP buffer for 10 min at room temperature, and the nucleic acids were eluted with elution buffer  at 55°C for 1 h. Immunoprecipitated DNA was then cleaned up by a phenol–chloroform extraction followed by ethanol precipitation at 20°C for 1 h. For each replicate and condition, three IP were combined and sent to IGM genome center of the University of Californa San Diego for library construction and sequencing. Data were generated at the UC San Diego IGM Genomics Center using an Illumina NovaSeq 6000.DRIP assays were performed as described . BrieflySingle-end reads of length 76 were first trimmed with fastq-mcf  to removTotal cellular RNA was isolated from siRNA-transfected U2OS cells using GenElute Mammalian total RNA Miniprep Kit (Sigma-Aldrich) according to the manufacturer’s instructions. For each experimental condition, three independent siRNA transfection experiments were carried out for the isolation of RNA. NEB rRNA-depleted (HMR) stranded library preparation was performed using Illumina’s platform following the manufacturer’s protocol (New England BioLabs). Purified libraries were subjected to sequencing on Illumina HiSeq4000 PE100 in pools of five per lane. Sequencing produced an average of 79.2 million reads per sample (range 71.1–87.7 million).2 fold change larger than one and FDR cutoff of 0.05, Wald test. Overlaps between differentially expressed genes and genes lying nearest gain or loss R-loop peaks were determined by HOMER. To detect cases of antisense transcription, the intergenic reads of each replicates were extracted with BEDTools v2.28.0  a positive log2 fold change of differential intergenic RNAseq expression relative to the control at the 2 kb adjacent to the TSS or TTS, (2) log2 fold change of differential gene expression relative to the control smaller than 2, (3) log2 fold change of R-loop peak signal relative to the control larger than one, and (4) FDR of R-loop peak signal relative to the control smaller than 0.1.Paired-end reads of length 100 were aligned to the human genome (hg19/GRCh37) with STAR v2.7.1a  and the  v2.28.0  using th v2.28.0 . DiffereGapdh housekeeping gene expression was used to normalize antisense expression. DRIP-qPCR analysis was performed as described previously For antisense RNA analysis, total cellular RNA was isolated as described above. cDNA was synthetized using Promega M-MLV reverse transcription kit. For determining which strand of RNA was transcribed, two different primers were used in the RT reaction. The sense primer hybrids with the antisense strand and the antisense primer anneals to the sense strand. A random primer was used as positive control and for the negative control, no primer was added in the RT reaction. Regular PCR reaction was performed using the cDNA transcribed with different primers, and the PCR product was revealed in agarose gel. Antisense expression was quantified by RT-qPCR using SYBR Green PCR Master Mix (Applied Biosystems) and cDNAs transcribed using random primers. The sequence of the primers for RT reaction and PCR to identify sense or antisense transcription are listed in


The objective of the present experiment was to construct self-draining beds to keep surface bedding materials clean and dry for beef cattle comfort in a temperate climate.In Experiment 1, a self-draining bed was covered with sand at depths of 10 cm (S-10a), 15 cm (S-15), and 20 cm (S-20) respectively. In Experiment 2, self-draining beds of different sizes were covered with 10 cm of sand (S-10b) and wood shavings (WS) at depths of 15 cm and 20 cm (WS-15 and WS-20). Fifteen cattle were engaged to evaluate the comfort of self-draining beds covered with different bedding materials.No cattle lay in the feed alley and cattle spent more time lying on S-10a than S-15 or S-20 in Experiment 1 (p<0.01). No difference in lying time was detected between S-15 and S-20 (p>0.05). In Experiment 2, no cattle selected the feed alley as the lying area. Cattle preferred WS-15 as the lying area and time spent lying on WS-20 was slightly higher than on S-10b (p<0.05). Feces weight was higher in the feed alley than in the different bedding areas in both Experiments 1 and 2 (p<0.01).Sand-bedding depth at 10 cm and WSs at 15 cm above the self-draining bed can provide for the lying comfort of beef cattle. Design of a special feed alley to hold most of the feces to keep bedding materials clean and dry is desirable for organic beef cattle in a loose barn. Production of beef cattle is a major agricultural industry and significant part of the rural economy in Yunnan Province, China. Great technological advancements in beef cattle production have been achieved during the past few decades, but profitability is still low in most indoor beef cattle operations. To improve this situation and satisfy the demand for higher quality beef, the Yunnan Government issued a policy for the organic beef cattle industry in 2018. Though a series of standards have been set, organic beef production faces great challenges regarding improvement in animal housing conditions.Animal-friendly flooring is a basic requirement for organic beef production, and floor type significantly affects production performance and animal welfare. Concrete is widely used as flooring material in dairy farms as this is affordable and convenient to manage. However, bulls kept on concrete floors display uncomfortable lying behavior and less lying time than bulls kept in a bedded lying area . Fully sStraw and other crop residues have been used as cushion ing materials to improve the lying comfort of cattle. However, the use of straw, saw dust and wood shavings (WS) as bedding materials on a solid floor for large numbers of cattle is impractical due to the production of large quantities of urine and the seasonal shortage of bedding materials ,5. To rePrevious research has predominantly focused on the effects of bedding materials on behavior of dairy cows while knowledge of similar effects on behavior of beef cattle is scarce due to economic constraints and short raising time. Keeping bedding material dry is challenging because there may be seasonal shortages of appropriate material that lead to untimely supplementation or replacement. Poor quality organic bedding may cause reproductive disorders in cattle due to the presence of bacteria and mycotoxins that can be transferred to the animals ,10.Inspired by a product made by Sponge city  designedA feed alley in front of the feed bunk was 3.5 m wide. A pen with a self-draining bed serving as the lying area was 12.5 m long and 12.0 m wide. The lowermost layer of the self-draining bed consisted of impervious concrete and had a 2% slope and the middle layer, 30 cm deep rubble stone. The top layer consisted of 20 cm deep cobblestone and 10 cm deep gravel. The self-draining bed was divided into three areas (12.5 m× 4.0 m) demarcated with round-timber and cattle had access to all three areas in Experiment 1. The three areas of the self-draining bed were covered with sand (2 mm screen) at depths of 10 cm (S-10a), 15 cm (S-15), and 20 cm (S-20), respectively . In Expe2) could meet the lying requirements for all animals. Cattle were fed the whole corn silage and concentrate diet and had free access to fresh drinking water.The study was conducted in Dehong State, China  in December 2018 and animal handling procedures were approved by the Animal Ethics Committee under the Yunnan Province Animal Welfare Act China (20071001). Fifteen healthy female Simmental cattle with similar body condition scores were engaged in the study, and each bedding area  to detect the behavioral preference at different sand bedding areas in Experiments 1. The same 15 cattle were transferred to another self-draining bed to detect the behavioral preference at different bedding areas covered with sand and wood shavings using continuous sampling for four consecutive days (96 hours). Cattle behavior included standing and lying down. Animals were considered to be lying down when their body trunk was in contact with the ground in the feed alley or on bedding materials regardless of posture. Cattle were considered standing when their body weight was supported with four legs. After completing the field trail, all cattle behavioral recordings were observed continuously by the same observers.A comfort index (CI) was used to evaluate the lying comfort of different bedding materials. CI is calculated by dividing the number of cattle lying on the bedding surface by the total number of cattle ,13. The Analyzes were performed using IBM SPSS 21.0 software . Behavioral parameters  were obtained from individual cattle. Standing and lying time, and weight of feces were analyzed using one-way analysis of variance following a Shapiro-Wilk analysis. The Kruskal-Wallis test was applied to analyze the effect of the self-draining bed covered with different bedding materials on behavioral frequencies .Cattle behavioral time allocation and frequencies in Experiment 1 are shown in Cattle behavioral time allocation and frequencies in Experiment 2 are shown in The cattle lying CI is summarized in Cattle lie down to ruminate and sleep for approximately 12 to 15 hours per day in a comfortable environment . Cattle Lying time plays an important role in increasing bouts of rumination and feed efficiency of beef cattle, both of which depend significantly on bedding base and materials. Lying preference is an effective indicator of the lying CI for cattle. Time spent lying on a thin layer of sand bedding (S-10a) was unexpectedly longer than for both S-15 and S-20 in Experiment 1, contrary to previous results showing that cows preferred more sand bedding in stalls  considerAfter transferring to another similar self-draining bed covered with sand bedding at 10 cm deep (S-10b) and WSs at 15 cm (WS-15) and 20 cm (WS-20) deep in Experiment 2, cattle were found to prefer WS-15 and WS-20 to S-10b. The difference in lying preference in Experiments 1 and 2 indicated that the lying CI of WSs is greater than that of sand in a temperate climate and this is consistent with previous studies . The difCI as a critical factor in production is widely used to eval uate the quality of the environment of dairy cows and similar criteria were used to evaluate the lying comfort of cattle and quality of bedding materials. Lying is a high priority behavioral requirement for cattle . CI was Cattle skin cleanliness is an important parameter for cattle health, thermoregulation, reproductive performance and meat hygiene ,27, but Self-draining beds covered with sand at a constant depth of 10 cm or WS at a depth of 15 cm can provide beef cattle with comfortable lying surfaces. In our experiments, no cattle selected the hard concrete alley floor as a lying area when provided with comfortable bedding materials. Designing a special feed alley to hold most of the feces to keep bedding materials clean and dry for organic beef cattle in a loose barn is desirable.


Both lung adenocarcinoma and coronavirus disease 2019 would cause pulmonary inflammation. Angiotensin-converting enzyme 2, the functional receptor of SARS-CoV-2, also plays a key role in lung adenocarcinoma. To study the risk of SARS-CoV-2 infection in lung adenocarcinoma patients, mRNA and microRNA profiles were obtained from The Cancer Genome Atlas and Gene Expression Omnibus followed by bioinformatics analysis. A network which regards angiotensin-converting enzyme 2 as the center was structured. In addition, via immunological analysis to explore the essential mechanism of SARS-CoV-2 susceptibility in lung adenocarcinoma. Compared with normal tissue, angiotensin-converting enzyme 2 was increased in lung adenocarcinoma patients. Furthermore, a total of 7 correlated differently expressed mRNAs  and 5 correlated differently expressed microRNAs  were screened. Interestingly, the most frequent toll-like receptor signaling pathway was enriched by mRNA (interlukin 6) and miRNA (miR-125b-5p) sets simultaneously. In conclusion, it was assumed that miR-125b-5p-ACE2-IL6 axis could alter the risk of SARS-CoV-2 infection in lung adenocarcinoma patients. In December 2019, there was an outbreak of coronavirus pneumonia (COVID-19) caused by SARS-CoV-2 in Wuhan, Hubei province in China. SARS-CoV-2 could encode spike (S) protein like SARS-CoV. In addition, a same cell entry receptor and manner were used to mediate infection. Unlike other type I fusion proteins, the S protein of coronaviruses is not cleaved in the virus-producing cell . The S1 Angiotensin-converting enzyme 2 (ACE2), a terminal carboxypeptidase, catalyzes the conversion of angiotensin II (Ang II) to angiotensin 1-7 (Ang 1-7). Ang II, the major effector molecule of the conventional renin-angiotensin system, is implicated in the pathogenesis of cardiovascular disorders like hypertension, atherosclerosis, and myocardial infarction. Whereas ACE2 and its product Ang 1-7 are thought to prevent the detrimental effects of angiotensin II . In respLung adenocarcinoma (LUAD), the most common primary lung cancer which falls under the umbrella of non-small cell lung cancer (NSCLC), has a strong association with smoking. LUAD usually evolves from the mucosal glands and represents about 40% of lung cancers. LUAD usually occurs in the lung periphery, and in many cases, may be found in scars or areas of chronic inflammation , 22. Thein vivo but also markedly decreases their vessel density .The potential target genes of differentially expressed miRNAs in LUAD were acquired from the widely used online databases TargetScanHuman 7.2  and DIANTotal RNA from tissues was isolated using Trizol extractions (Invitrogen). The RNA quantity was assessed by NanoDrop®ND-1000 spectrophotometer and RNA 6000 NanoChips with the Agilent 2100 Bioanalyzer . 100 ng of total RNA was amplified using the Ambion® WT Expression Kit . Small RNA libraries were prepared using 1μg of total RNA according to the TruSeq Small RNA Sample Preparation Guide . To generate count data, the raw sequences were compared to human mature miRNA sequences (from miRBase version 17) and non-coding RNA sequences (Rfam version 10) by MEGABLAST.Background deletion, quantile normalization, and probe assembly were performed. Different expression mRNAs between normal vs. LUAD samples were detected by the R package DESeq . P-valueResults are presented as mean values ± standard error of the mean (SEM). Unless mentioned otherwise, the statistical comparison between groups was performed by using t-test, a maximum of three comparisons were performed per panel, and robustness of statistical significance was verified after correction for multiple testing. Probability was considered to be significant at p < 0.05.Publicly available datasets were analyzed in this study. All data was available.Supplementary Figures


Model results show that the correlation between the occurrence of CCA and the development of true coral reefs increased with CCA diversity and cooler ocean temperatures while the diversification of herbivores had a transient negative effect. The evolution of novel herbivore groups compromised the interaction between CCA and true reef growth at least three times in the investigated time interval. These crises have been overcome by morphological adaptations of CCA.Crustose coralline red algae (CCA) play a key role in the consolidation of many modern tropical coral reefs. It is unclear, however, if their function as reef consolidators was equally pronounced in the geological past. Using a comprehensive database on ancient reefs, we show a strong correlation between the presence of CCA and the formation of true coral reefs throughout the last 150 Ma. We investigated if repeated breakdowns in the potential capacity of CCA to spur reef development were associated with sea level, ocean temperature, CO Mitigating the threats to modern coral reef ecosystems will thus benefit from a better understanding of the underlying causes in the rise and fall of ancient coral reefs2. Reefs, broadly defined as laterally confined limestone “structures built by the growth or metabolic activity of sessile benthic aquatic organisms”3 comprise a large array of constructional styles and biota, and grew in a variety of environments. The concept of ‘true reefs’ in ancient reefs derives from the constructional style and environment of recent tropical, shallow-water coral reefs. True reefs with a syndepositional relief and a rigid framework constructed by skeletal organisms are known since Cambrian times4 but occur alongside other reef types such as reef mounds, mud mounds and biostromes. There is a significant increase of both the relative and absolute abundance of true reefs over the Phanerozoic5. This trend may be caused by intrinsic  factors as there are few significant correlations with earth system parameters such as temperature, sea level, or oceans chemistry5. Although hypothesized to play a major role6, the relative importance of biotic interactions in reef evolution is still poorly known. The evolution of crustose coralline red algae  may underline such interactions in an exemplary fashion. CCA play a key role in the construction of many modern coral reefs10 in several regards: CCA are not only primary producers and contributors of calcareous sediment but also often act as consolidators and binders of coral reefs. The algae can form a distinct ridge providing a surf-resistant reef crest and bind loose sediment11. Even though there are examples of true reefs that grow successful without this CCA ridge under wave-exposure13, CCA are still considered as ‘the glue that holds coral reefs together’9 in many cases. Another key factor for the success of modern coral reefs are grazing organisms such as echinoids and parrot fish, because they remove fleshy algae from the reef surface15. The evolution of grazers also led to an increased feeding pressure on CCA, followed by adaptive strategies of the CCA16. It seems plausible that this development also impacted a potential capacity of CCA to facilitate coral reef formation in the geological past. However, this hypothesis has not been assessed quantitatively until now.Coral reefs support the biologically most diverse marine ecosystems and have done so over substantial parts of earth history, starting in the Late Triassic, when scleractinian corals became prolific reef builders17. However, a finer temporal and taxonomic resolution and the inclusion of environmental parameters may explain if and why a potential importance of CCA for reef development varies over time. Using data from the PaleoReefsDatabase (PARED), a comprehensive compendium of geological and paleontological data of Phanerozoic reef sites as described previously20, we evaluate the role of CCA in coral reef development at the level of geological stages from the Early Cretaceous to the Pleistocene while considering additional factors such as ocean temperature and chemistry as well as interactions with relevant groups of grazing organisms. We test three hypotheses: (1) The probability of true reef formation is correlated with the presence of CCA reef cementers; (2) the capacity of CCA to reinforce coral reefs is linked to various oceanographic and ecological parameters; (3) the diversification of grazing organisms led to transient crises in the capacity of CCA to support true reef formation. Additionally, we highlight alternative explanations for the correlation between CCA occurrences and the formation of true reefs. This includes the impact of herbivore radiation on corals rather than on CCA directly and ecological niches for CCA provided by increased reef growth.There are hints for a long-term positive interaction between corals and CCA over geological timescalesThe general hypothesis that the occurrence of true reefs is strongly correlated with CCA as secondary reef builders is supported by the linear regression model Fig. . Regress2 concentration, species diversity of CCA, and the origin and diversification patterns of the grazer clades echinoids and parrot fish are visualized in Fig. The temporal patterns of the investigated variables, proportions of true CCA-reefs, sea level, ocean temperature, COAll environmental variables except grazers are correlated with each other Table . The hig. Nevertheless, visualization of the GLM shows that species diversity of CCA is positively correlated with the presence of true CCA-reefs while higher ocean temperatures and the origination and diversification of grazers have negative effects  Foslie 1909, P. gardineri (Foslie) Foslie 1909, P. craspedium (Foslie) Foslie 1909, and Lithophyllum kotschyanum Unger 1858 in the Indo-Pacific. In the Atlantic, the main reef reinforcers are Porolithon onkodes (Heydrich) Foslie 1909 and Lithophyllum congestum (Foslie) Foslie 1900. All these species belong to the ‘cool’-water adapted Corallinales. Thus, the increasing capacity of CCA to stabilize coral reefs is in line with the general trend of decreasing ocean temperatures.CCA occur worldwide from the tropics5. On shorter time scales, sea level is expected to influence the formation of coral reefs, but probably not the CCA’s reef enforcing capacity. We conclude this because the environmental tolerances of CCA in terms of sea level fluctuation are much wider than those of reef corals. Most CCA species appear uniquely tolerant of aerial exposure10. Additionally, many CCA are very well adapted to changes in salinity and especially to low photon irradiances30. The environmental tolerances of reef corals are narrower32.A change in sea level does not impact the capacity of CCA to reinforce coral reefs, likely because sea level changes measured on the level of geological stages have no effect on reef formationAcropora Oken, 1815, is well adapted to cope with rapid sea-level changes. First observed as an important reef builder in the Oligocene33, Acropora has become a dominant reef builder from the Pleistocene until today, when sea-level fluctuations increased in rate and magnitude34. Indeed, there is a temporal overlap between the first decline in the fraction of CCA reefs—between the Turonian and the Campanian—and a maximum in sea level. Despite this, sea level is not selected as a relevant explanatory variable for the fraction of CCA reefs by the GLM because the relationship between the fraction of CCA reefs and sea level varies inconsistently throughout entire time series of the analysed 150 million years. While the decline in the fraction of CCA reefs may additionally be linked to an increase in temperature before and a significant drop in CCA diversity during the period with a low fraction of CCA reefs, data of the analysis are not suitable to conclusively identify the driver for this particular CCA crisis.Considering our assumption that there is a mutual relationship between the presence of CCA and the growth of true reefs, another reason might be that one of the most important genera in modern coral reefs, 2, was identified by the model as a minor driver, which may be explained by the fact that an increase of atmospheric pCO2 has only little to no impact on mean ocean surface pH on timescales exceeding 10,000 years35. A plausible reason is that slow rates of CO2 release lead to a different balance of carbonate chemistry changes and a smaller seawater CaCO3 saturation response. This is because the alkalinity released by rock weathering on land must ultimately be balanced by the preservation and burial of CaCO3 in marine sediments. The burial is controlled by the CaCO3 saturation state of the ocean and therefore, the saturation is ultimately regulated by weathering on long time scales, and not by atmospheric pCO2. The effect of weathering on atmospheric pCO2 is much weaker than the effect of weathering on ocean pH. The much stronger effect of weathering on ocean pH allows pH and CaCO3 saturation to be almost decoupled for slowly increasing atmospheric pCO235.For the entire time series, RCO37, we have to rely on morphological characters since molecular methods are not available for the identification of fossil CCA. Because CCA show a pronounced phenotypic plasticity depending on environmental factors, their taxonomic identification depends on morphological characters like conceptacles (i.e. spore chambers) and the arrangement of cells in different areas of the thallus, features often not adequately preserved in fossil CCA. This has led to a great number of fossil CCA taxa that have been described on the basis of only a few anatomical characters of doubtful taxonomic value38. The inclusion of such taxa precludes fully reliable diversity estimations. To circumvent such problems, we used rarefied species data reviewed by experts on fossil CCA taxonomy28.The quantification of CCA species diversity in the geological past is associated to a number of challenges. While for recent CCA the extensive use of molecular phylogenetic methods resolved the four orders  currently recognized in the subclass Corallinophycidae as monophyletic lineages39. As discussed in detail in the next section, the abundance of CCA in true reefs was transiently reduced four times since the Early Cretaceous. Except for the earliest crisis, this was likely caused by the origin and diversification of echinoids and parrot fish, prominent groups of bioeroding organisms that denude CCA. However, the CCA-coral reef system successfully recovered all times. We argue that this was supported by functional redundancy of CCA, because a diverse group of abundant species with a wider range of responses can help absorb disturbances39. This redundancy of responses to events among species within a functional group—the reef cementers—is an important component of resilience and the maintenance of ecosystem services. The amount of CCA biomass is critical in terms of the cementing capacity. Multi-species community models40 have shown that with consecutive native species’ extinctions at high diversity levels, species extinction usually only leads to a slight decrease in the total biomass of the native community. However, when starting from a lower initial diversity, a few consecutive species extinctions cause a relatively large biomass loss that ultimately leads to collapse. It should also be stressed that sometimes single species are responsible for the functioning of an ecosystem , even if the ecosystem features a generally high biodiversity. Therefore, such ecosystems will decline if this key species is removed41.Our results show that high CCA diversity is linked to a higher abundance of CCA in true coral reefs. This might seem to contrast with the fact that in modern reefs, the wave-pounded intertidal algal ridges are built predominantly by only a few species while the ones making up the majority of diversity have a cryptic, hidden mode of life protected from full or direct exposure to major physical environmental factors and therefore do not contribute significantly to reef stabilization. However, if several CCA species were contributing to the same ecosystem function, a higher species diversity may have buffered reef systems from losing all species associated with the key function of supporting reef development42 showed that functional diversity maintains ecosystem functioning. At heavily grazed sites, some species dominant in the ungrazed communities were lost or substantially reduced. In four out of five cases, the minor species that replaced these lost ones were their functional analogues. Accordingly, we suggest that formerly less dominant but functionally analogous grazing-tolerant species increased in abundance and contributed to the maintenance of ecosystem functions. CCA species removed or reduced in biomass by grazing pressure can be replaced in terms of their ecosystem service, i.e. reef cementation, by other CCA that are better adapted to grazing.Experiments with plants in rangelands43, this resilience possibly applies also to future ocean acidification.This implies that in recent coral reef environments, areas with high CCA diversity—potentially including species occupying cryptic habitats—are more resilient against disturbance. Because the skeletal mineralogies of CCA vary considerably among species20 might be that our study focuses on the number of true reefs, while the Paleocene-Eocene crisis is expressed by a change in cumulative metazoan reef volume. Except for the first one, all crises observed here occurred synchronous with pronounced evolutionary events in clades of grazing organisms. Cementing and binding is the main function of CCA in the facilitation of true coral reefs. The decline in CCA abundance during the Selandian–Thanetian corresponds with a marked increase in the rate of morphological evolution in echinoids  may have become major players45. Although reef-grazing fish have existed for nearly 400 Ma, specialized detritivores feeding on macroalgae have only been known since the Miocene46. This is also in line with the radiation of acroporid corals since the mid Miocene47, whose branched morphologies create interstitial niches for parrot fish but also for cryptic CCA species. The parrot fishes (Scarinae) first appeared in the Serravallian45, which may have caused the third crisis in CCA reef cementing capacity. The lineage diversification of Scarinae was most pronounced during the Zanclean-Piacenzian, which we deem responsible for the third crisis.The data reveal four crises in the abundance of CCA within true reefs, during the Cretaceous (Turonian–Campanian), the Paleocene (Selandian–Thanetian), the Miocene , and the Pliocene (Zanclean–Piacenzian). The reason that the timing of the Paleocene crisis differs from the known Paleocene–Eocene crisis16, which is critical for CCA with thin crust morphologies. The resulting decline of thin crust morphologies led to the occupation of niches by branching CCA16. The twig-like morphologies of branching CCA prevent echinoids from denuding CCA thallus and confine this process to the tips of the branches. CCA are able to transfer nutrients within their thallus16. Therefore, these superficial grazing wounds can be rapidly healed if sufficient nutrient reservoirs are present in other, ungrazed parts of the algae. Meristems and conceptacles engulfed in the thallus may be another adaptation pertinent to the relatively low impact of echinoid grazing, as this is a plausible strategy to protect the reproductive and growth structures of the CCA. The more intense grazing pressure exerted by the parrot fishes, which bite CCA to an average depth of 288 µm16 and are able to eat the tips of branched CCA48 may have resulted in a greater abundance of CCA with very thick crusts. Thick-crust CCA possess larger nutrient reservoirs making them capable to recover also from grazing exerted by parrot fishes. All these adaptations and their development are congruent with the origination and diversification of the grazer clades as already outlined in other studies50. Today and potentially already during the geological history, CCA did not only successfully adapt to various grazer clades but even required the grazing pressure to stay free of epiphytes49. Here we show for the first time that the process of grazer evolution may also have affected the potential capacity of the CCA to reinforce coral reefs for three times during the geological past.The abundance of CCA in true coral reefs recovered relatively fast after all crises probably due to morphological adaptations developed within the CCA. Experiments have shown that echinoids are able to graze tissues to depths averaging 88 µm2, the potential future impact of the ongoing global change on the capacity of CCA to reinforce coral reefs should become a focal point of reef research. Despite the implementation of numerous mesocosm and aquaria experiments53, long-term data in the magnitude of months on CCA responses to modified environmental parameters are still sparse. Also, the change from ambient to modified parameters  happens much faster than at natural rates.As it concerns some of the most important biodiversity hot spots on our planetpCO2 on CCA depends on the rate of change. While fast rates are critical, slow pCO2 increase may even result in increased net calcification at moderately elevated pCO2 levels54. However, this comes at the cost of structural integrity of the CCA skeleton which, in turn, makes the CCA likely more susceptible to bioerosion. Bioerosion by echinoids and parrot fishes is beneficial to CCA at the present state, as it removes fast growing fleshy algae and other epiphytes49, but nothing is known about the future of this interaction when the integrity of the CCA skeletons is altered. Additionally, it has been shown that elevated pCO2 levels accelerate sponge reef bioerosion57. Therefore, a combination of increased bioerosion rates affecting corals and CCA might lead to strongly deteriorated conditions for coral reef formation. As outlined above, a greater CCA diversity might also increase their resilience against ocean acidification because of the great variety in skeletal mineralogies.The impact of elevated 30, some change their skeletal chemistry in terms of dolomite concentration58, and others respond with strongly impaired germination success59 or declining skeletal densities60. Due to the positive influence of cooler temperatures on CCA’s abundance in true reefs detected in our study, elevated temperatures will likely have a negative outcome but also here, the rate of change might be similarly important as the magnitude.Regarding elevated temperatures, the outcome for CCA is unpredictable. Depending on the examined species, elevated temperatures affect CCA primary production in different ways: some species show no or negligible responseTo estimate the future of CCA’s potential to facilitate coral reef growth in the face of global change, we encourage long term experiments—preferably in near-natural mesocosm studies—including the main reef stabilizing CCA species.61, pre-Cretaceous occurrences are scarce. We downloaded data on all reefs occurring from the earliest Cretaceous  to the Late Pleistocene (0.01 Ma) and having corals as the main reef builders from the PaleoReefs Database  in May 2018. The 736 Cretaceous to Pleistocene coral reef sites were grouped in 33 geological stages  representing 145 million years of Earth history with information on reef diversity and environmental parameters.While CCA have existed at least since the Silurian19. We focus on coral reefs in the constructional style of true reefs, because we hypothesize that their rigidity and three-dimensionality depend on the CCA’s cementing capacity. The data in PARED also contain information on the secondary reef builders and we only asserted a significant functioning as reef cementers to the CCA when they were listed as secondary reef builders within the particular coral reef. For each geological stage, we consequently tabulated the absolute numbers of four reef types:All reefs all reefs in this study, regardless of their constructional style and if they have CCA as secondary reef builders.True reefsreefs with the constructional style of true reefs, but regardless if they have CCA as secondary reef builders or not.CCA-reefsreefs that have CCA as secondary reef builders, but regardless of their constructional style.True CCA-reefsreefs both features apply for: the constructional style of true reefs and CCA as secondary reef builders.The data in PARED contain information on the constructional style of each reef, distinguishing between true reefs, reef mounds, mud mounds or banks, and biostromes. True reefs are those where skeletal organisms in growth position form a dense, rigid framework. Reef mounds share abundant skeletal organisms with no evidence for a rigid skeletal framework. Mud mounds or banks predominately consist of carbonate mud, often of microbial origin. Biostromes consist of skeletal organisms but there is no syndepositional reliefp(reef type) is the calculated proportion of true reefs, CCA-reefs, and true CCA-reefs, respectively.From these data, we calculated the proportions (mean ± one standard error) of true reefs, CCA-reefs, and true CCA-reefs for each geological stage by dividing the number of reefs of each type by the number of all reefs Table . The sta2 concentration, species diversity of CCA, and the origin and diversification patterns of the grazer clades echinoids and parrot fish. Information on sea level represents the relative sea level based on ocean volume change as mean value per geological stage63. Temperature data derive from the oxygen isotope dataset published by Veizer and Prokoph64. CO2 concentration for focal time periods  is expressed relative to the current level . Data on CCA diversity represent rarefied species diversities per stage28.Our analyses aim to identify the environmental parameters, which are crucial for the capacity of CCA to facilitate the formation of true reefs. As potential explanatory variables, we explor sea level, ocean temperature, CO44 and represent rates of morphological evolution, which are measured as the mean number of character changes per lineage per million years. To quantify the diversification rates of herbivorous parrot fish, we used lineage-through-time (LTT) estimates in a dated phylogeny , 45 species of Scarus Forsskål, 1775 (scraping feeding mode), and two species of Hipposcarus Smith, 1956 (scraping feeding mode), all belonging to the family Labridae Cuvier, 1816. Testing the hypothesis that novel herbivore characteristics influence the capacity of CCA to facilitate the formation of true reefs, we distinguished background intervals from intervals in which distinct increases in character change or lineage origination occurred . All environmental parameters are compiled in Table Data on echinoids are from Hopkins and Smith66 and removed all stages containing missing values in any parameters  before analyses. We tested the general hypothesis that the occurrence of true reefs is strongly linked to CCA as secondary reef builders using a linear regression model between the proportions of true reefs and the proportions of CCA-reefs and tested the regression residuals for autocorrelation.We performed all statistical tests in R version 3.5.22, Mc Fadden’s pseudo-R2, maximum likelihood pseudo-R2), tested model residuals for autocorrelation and visualised the model predictions.Prior to further analysis, we tested all explanatory variables for collinearity. We then assessed the influence of the environmental parameters on the coral-coralline interaction by implementing multivariate analyses . As the dependent variables represent proportions , we implemented GLMs with a binomial error distribution (logit-link function). The binomial error distribution for percentage values has the advantage to account for the fact that a particular percentage value is more accurate if it is based on a larger number of observations (here the number of reefs). We used stepwise model selection based on a version of the Akaike information criterion , that has a correction for small sample sizes (AICc) to estimate the relevance of different environmental parameters. We quantified model performance using different goodness of fit measures (Nagelkerke’s pseudo-RSupplementary information.


Defects in the functions of RNA binding proteins (RBPs) are at the origin of many diseases; however, targeting RBPs with conventional drugs has proven difficult. PROTACs are a new class of drugs that mediate selective degradation of a target protein through a cell's ubiquitination machinery. PROTACs comprise a moiety that binds the selected protein, conjugated to a ligand of an E3 ligase. Herein, we introduce RNA‐PROTACs as a new concept in the targeting of RBPs. These chimeric structures employ small RNA mimics as targeting groups that dock the RNA‐binding site of the RBP, whereupon a conjugated E3‐recruiting peptide derived from the HIF‐1α protein directs the RBP for proteasomal degradation. We performed a proof‐of‐concept demonstration with the degradation of two RBPs—a stem cell factor LIN28 and a splicing factor RBFOX1—and showed their use in cancer cell lines. The RNA‐PROTAC approach opens the way to rapid, selective targeting of RBPs in a rational and general fashion. RNA‐PROTACs are new chimeric structures that mediate the proteasomal degradation of RNA‐binding proteins (RBP). They comprise an E3‐recruiting peptide conjugated to a short modified RNA, which is iso‐sequential with the native binding element to which the RBP binds in cells. RNA‐PROTACs offer a fast, rational and general approach to target an entire class of protein (RBPs) that has proven difficult until today to target pharmacologically. RNA binding proteins (RBPs) constitute a large fraction of a cell's proteome.Here we introduce a new concept in targeting RBPs using a novel class of chimeric structure, which we have termed RNA‐PROTACs. Conventional PROTACs (PROteolysis‐TArgeting Chimeras) comprise three components: a target‐binding ligand, a ubiquitin E3 ligase‐recruiting ligand and a connecting linker group.The targeting ligand of a conventional PROTAC is a drug‐like small molecule that binds selectively to the POI. Ligands have been reported for a variety of proteins, including enzymes and receptors.Intuitively, the RNA binding site of an RBP represents a viable target site for drugs, since it is functionally important and structural data for RBPs is often available. However, with the exception of a few natural products inhibiting splicing,  of an RBP for the first time as the targeting moiety for the PROTAC Figure . The oliRBE), a conserved sequence present in microRNAs, to which Lin28 binds  ribonucleotides and conjugated it to a E3‐recruiting peptide derived from the HIF‐1α protein. This RNA‐PROTAC binds selectively to Lin28 in vitro, and suppressed levels of Lin28A in two cancer cell lines in ubiquitin‐dependent fashion. Taken together, these findings pave the way to a new class of rationally designed inhibitors for RBPs, a protein family that until now has been difficult to address with conventional drugs.To target Lin28, we used 5′‐AGGAGAU‐3′  substituentsA short oligoribonucleotide corresponding to the consensus RBE was a conceptually natural choice for the targeting element of an RNA PROTAC. Short oligonucleotides have been investigated previously as antisense2 and G5 in 5′‐A1G2G3A4G5A6U7‐3′ .YI via the peptide N‐terminus  or a control sequence Pcon, in which norleucine was exchanged for the hydroxyproline  was immunoprecipitated using an anti‐Myc antibody. The isolated protein was then analyzed by western blotting using the anti‐HA and anti‐c‐Myc antibodies. Consistent with other previously described PROTAC reagents,LA[Hyp]YI (1ORN9P) using the same protocol as for the Lin28 RNA‐PROTACs. In parallel, we prepared two negative control reagents: 1ORN10P comprises a random oligonucleotide sequence, whereas conORN9P has the FOXRBE conjugated to the mutated peptide BP1RBE) aconORN9P and 1ORN10P did not affect levels of RBFOX1 protein, suggesting that 1ORN9P is selective for its target. In order to demonstrate further the selectivity of 1ORN9P for RBFOX1, the blot was probed with an antibody against heterogeneous nuclear ribonucleoproteins HNRNPC. HNRNPC contains an amino‐terminal sequence that is known to bind strongly uridine (U)‐rich sequences, and therefore provided a strict test of selectivity for the FOXRBE analogs.1ORN9P, 1ORN10P and conORN9P affected significantly the level of endogenous HNRNPC protein  cells were treated with the three reagents at increasing concentrations, under similar conditions for the Lin28 RNA‐PROTACs. Western blotting of protein isolated from treated cells showed an approximate 50 % reduction of RBFOX1 protein at 2 μM Figure . ORN9Pcon Figure .Conventional drugs can be classified into distinct structural types, such as small‐molecules, therapeutic proteins and oligonucleotides. Single‐stranded oligonucleotide drugs have cellular RNA as their common target, with which they hybridize according to Watson–Crick rules. Amongst them, the most prevalent groups are the antisense‐ and splice‐switching‐oligonucleotides that target mRNAs. However, new targeting mechanisms mediated by anti‐miRNA and decoy oligonucleotides are also under investigation  should be addressed to the authors.SupplementaryClick here for additional data file.