We have biochemically validated the predictedRNA thermometer in the 5′-UTR of the blyA gene.Our study suggests that RNA thermometers may play an underappreciatedyet critical role in the lytic life cycle of bacteriophages.RNA thermometers are noncoding RNA structures locatedin the 5′untranslated regions (UTRs) of genes that regulate gene expressionthrough temperature-dependent conformational changes. The fourU classof RNA thermometers contains a specific motif in which four consecutiveuracil nucleotides are predicted to base pair with the Shine-Dalgarno(SD) sequence in a stem. We employed a bioinformatic search to discovera fourU RNA thermometer in the 5′-UTR of the  In most characterizedexamples, they result in an increased level of gene expression inresponse to higher temperatures. This temperature-dependent upregulationoccurs as the RNA structure becomes denatured and reveals a previouslysequestered gene expression platform.2 FourU thermometersmake up a well-characterized class of RNA thermometers containinga specific motif in which the ribosome-binding site, the SD sequence,is predicted to base pair in a stem across from four consecutive uracilnucleotides.3 With an increase in temperature,base pairing within the fourU motif is disrupted, causing destabilizationof the stem and increased accessibility to the SD sequence, enablingtranslation of a downstream gene.3 SeveralfourU RNA thermometers have been found in the 5′-UTR of genesinvolved in the heat shock response or bacterial virulence. Theseinclude the ompA gene in Yersinia pseudotuberculosis in Bacillus subtilis. that is a non-fourU RNA thermometer.8RNA thermometers are mostly subtilis.B. subtil9 was usedto identify potential fourU RNA thermometers based on the predictedsecondary structure of the fourU agsA RNA thermometer3 (Methods in the Supporting Information). The results of our in silico approach revealed a potential RNA thermometer in the 5′-UTRof the blyA gene , which encodes an autolysin enzyme  that facilitates the hydrolysis of cell wallglycopeptides10 (B. subtilis phage SPβc2 (SPβ) andits host, the Gram-positive bacterium B. subtilis 168 (B. subtilis). Previously, only one RNA thermometerhas been described in a phage genome.22The homology among several previously characterizedfourU RNA thermometermotifs was exploited to search for novel fourU RNA thermometers. TheRNA motif search tool, RNArobo,ptides10 1A,B. ThiblyA, which plays a crucial role in thelytic activity ofthe phage in the host cell.10 SPβintegrates into the genome of its bacterial host and has been foundto be ubiquitous in B. subtilis 168.12 SPβ is a lysogenic phage with a temperature-dependent lyticlife cycle in B. subtilis 168.14 Previous studies have demonstrated thatthe level of expression of blyA in B. subtilis 168 increases at high temperatures, and its heat-induced expressionis necessary for the lysis of the host cell.10 Although it is known that heat stress induces the expression of blyA and the proliferation of the SPβ prophage in B. subtilis 168,10 the underlyingmolecular mechanism that drives this process has yet to be elucidated.The prophage SPβ contains the open readingframe for 15 was employed to predict the full-length structure of the RNA thermometersequence in the 5′-UTR of blyA  wascloned into a reporter plasmid containing an arabinose-inducible pBADpromoter upstream of a heat-stable β-galactosidase (bgaB) from Bacillus stearothermophilus..RNA thermometer function is dependent upon a conformational changein the RNA secondary structure. RNAfold of blyA 1C. The pmophilus. The 5UTREscherichia coli cells expressing the 5UTR_blyA–bgaB fusion. Cells containing 5UTR_blyA–bgaB fusions were incubated for 30 min at 25, 37, or 42 °C. Subsequently,β-galactosidase activity was measured for each temperature (Methods in the Supporting Information). Heatshock of cells expressing 5UTR_blyA-bgaB fusionsresulted in heat induction factors [activity in Miller units (M.U.)at 37 °C/25 or 42 °C/25 °C] of ∼4.0- and ∼5.2-foldat 37 and 42 °C, respectively , which is not expected to be thermally regulated,was tested and exhibited minimal heat induction of 0.94- and 0.95-foldat 37 and 42 °C, respectively . Cells were harvested under the same conditions of β-galactosidaseassays at 25 and 42 °C. Heat induction resulted in an approximately2.6-fold increase in transcript abundance at 42 °C, indicatingsome regulation at the transcriptional level  is considerably higher than that of the wild type (69.4± 0.4 °C)  2C. Altog19 At increased temperatures,the stem containing the RBS undergoes a zipper-like, unwinding meltingmechanism, allowing access to the RBS.20 To furtherinvestigate the conformational changes induced by heat, structuralprobing, using 2-methylnicotinic acid imidazolide (NAI) in DMSO, wasperformed on the blyA RNA thermometer sequence. Selective2′-hydroxyl acylation analyzed by primer extension (SHAPE)provides secondary structure information at a single-nucleotide resolution.The reactivity of each RNA base is correlated with the flexibilityof the 2′-OH, with single-stranded or flexible regions exhibitingincreased reactivity in opposition to regions engaged in base pairingor other interactions.21In vitro SHAPE analysis at 25 and 42 °C reveals a prominent temperature-dependentincrease in the reactivity of the fourU motif, indicating that thefourU motif is more flexible and accessible in response to an increasein temperature (The prevailing mechanism through which most RNAthermometers actrequires the destabilization of the RNA secondary structure at hightemperatures.perature 3. The moperature 2B. Interperature 2B. The UbgaB fusioncontaining only the P2 stem (mini-5UTR_blyA)  4A was teectively4B. Heat 22 The fieldof synthetic biology has also shown more interest in RNA thermometersfor their potential use in biotechnology and medicine. In this study,we discovered a fourU RNA thermometer located in the 5′-UTRof the blyA gene within the genomes of the SPβprophage and its host, B. subtilis 168. The 5′-UTRof the blyA gene was identified as an interestingcandidate because of its pivotal temperature-dependent role in theSPβ lytic life cycle and because it is widely integrated intothe extensively studied B. subtilis 168 and has beenfound in other B. subtilis strains.12 Furthermore, it has been reported that increasing the temperatureof the host to 50 °C for a brief period leads to the activationof SPβ through heat induction, causing the phage to replicateand the host cell to undergo lysis. It is postulated that heat inductionresults in the deactivation of a temperature-sensitive repressor.13 Our results revealed that the 5′-UTR of the blyA gene is an RNA thermometer, indicating an additional level of thermoregulationin SPβ. Our study suggests that similar RNA thermometers mayregulate bacteriophage lytic life cycles and may be more common thanpreviously anticipated.Although the fourU motif is a common structurefound in many RNAthermometers, all previously annotated fourU thermometers have beendiscovered in bacteria. Additionally, to date, only one other RNAthermometer has been found in a bacteriophage. However, this previouslydiscovered bacteriophage RNA thermometer inhibits gene expressionat increased temperatures.


Small RNAs (sRNAs) are bioactive molecules that can be detected in biofluids, reflecting physiological and pathological states. In plasma, sRNAs are found within extracellular vesicles (EVs) and in extravesicular compartments, offering potential sources of highly sensitive biomarkers. Deep sequencing strategies to profile sRNAs favor the detection of microRNAs (miRNAs), the best-known class of sRNAs. Phospho-RNA-seq, through the enzymatic treatment of sRNAs with T4 polynucleotide kinase (T4-PNK), has been recently developed to increase the detection of thousands of previously inaccessible RNAs. In this study, we investigated the value of phospho-RNA-seq on both the EVs and extravesicular plasma subfractions. Phospho-RNA-seq increased the proportion of sRNAs used for alignment and highlighted the diversity of the sRNA transcriptome. Unsupervised clustering analysis using sRNA counts matrices correctly classified the EVs and extravesicular samples only in the T4-PNK treated samples, indicating that phospho-RNA-seq stresses the features of sRNAs in each plasma subfraction. Furthermore, T4-PNK treatment emphasized specific miRNA variants differing in the 5′-end (5′-isomiRs) and certain types of tRNA fragments in each plasma fraction. Phospho-RNA-seq increased the number of tissue-specific messenger RNA (mRNA) fragments in the EVs compared with the extravesicular fraction, suggesting that phospho-RNA-seq favors the discovery of tissue-specific sRNAs in EVs. Overall, the present data emphasizes the value of phospho-RNA-seq in uncovering RNA-based biomarkers in EVs. Small RNAs (sRNA) are regulatory species involved in the targeted modulation of gene expression at different levels. The highly regulated expression of the different types of sRNAs is key for the control of cell physiology, and the identification of meaningful species is an active field of research.The expression patterns of sRNAs are highly dynamic, reflecting the cell physiological and pathological states. Thus, the detection of extracellular sRNAs in biofluids, such as blood, has opened new avenues in the search for disease and biological biomarkers ,2,3. In To date, the characterization of sRNA content within EVs has been largely focused on microRNAs (miRNAs), the best-known class of sRNAs. However, miRNAs represent a minor portion of the EV transcriptome. Several studies have revealed that other types of sRNAs, including fragments of messenger RNA (mRNAs), transfer RNA (tRNA), ribosomal RNA (rRNA), and Y RNA (yRNA), are highly abundant in EVs ,15,16,17In recent years, advances in sRNA sequencing strategies have allowed us to analyze and decipher the complex composition of the human transcriptome across a wide range of contexts ,25. StanTo analyze the effects of phospho-RNA-seq in different plasma compartments we obtained the total plasma of three different donors  and isolated EVs  and protein-enriched  fractions using size-exclusion chromatography (SEC) A. TetrasRNA was purified from each plasma and the corresponding EV- and protein-enriched SEC-fractions were treated in parallel with T4-PNK or a buffer (no-T4-PNK) . AfterwaExceRpt , SeqClusp < 0.0001, chi-squared test), respectively, thus increasing the pool for alignment and annotation  analysis confirmed that T4-PNK treatment favors the detection of additional types of sRNAs H. Out ofIn the data pre-processing step, ExceRpt filters out the sRNAs mapping onto rRNAs; however, these species are highly abundant in the plasma samples , and recTo obtain a more in-depth analysis of the rRNA fragments and tRFs, we additionally processed the sequencing data with the SeqCluster tool that detects units of sRNAs (clusters) using a heuristic iterative algorithm to deal with multi-mapped events ,38. SeqCsRNA biotype distribution  and the Overall, these data suggest that phospho-RNA-seq highlights the particularities of the sRNA profiles in different plasma compartments, offers complementary sources of information, and refines the possible identification of the RNA-based biomarkers.p < 0.05, Wilcoxon-matched paired test). Furthermore, specific miRNAs were exclusively captured with phospho-RNA-seq  which differ with respect to the reference, canonical, sequences . IsomiRsIn both plasma subfractions, 3′-isomiRs were highly abundant under standard library prep conditions E. RefereIn EVs, the sensitivity of the 5′- and 3′-isomirs to T4-PNK was more variable F. On theCurrent efforts that are being made to highlight the biomarker potential of circulating RNAs are focused on the identification of the sources (tissues) contributing to the plasma sRNA pool ,54. To bp < 0.05, Chi-squared test). The 55 EV-enriched mRNA-sRNAs with a TSS > 3 were enriched in 14 tissues/cells, with the brain, testis, muscle, and blood cells being the more represented sources  with at least five reads in each sample  and took sources A. Overalp < 0.05, chi-squared test)  B. In theChanges in functional states are accompanied with modifications in the RNA expression patterns, including small regulatory RNAs (sRNAs), which accommodate cell responses to environmental physiological or pathological stimuli. Since sRNAs can be detected in the peripheral biofluids, there is a growing interest in the study of these circulating species as highly sensitive biomarkers. Phospho-RNA-seq has been developed to highlight the diversity of the sRNA transcriptome, providing opportunities to uncover novel informative species. Here, we have applied phospho-RNA-seq to plasma EVs and extravesicular protein-enriched fractions and focused on the 18–42 sRNA fraction that concentrates many of the known functional species. Clustering analysis indicates that T4-PNK stressed the differences between the sRNA profiles in these two plasma compartments, providing a scenario to highlight the specific features of sRNAs.Standard RNA-seq methods rely on the 5′-P/3′-OH ends of RNAs, such as miRNAs and many tRFs, and RNAs showing alternative ends cannot be properly captured . IndepenPhospho-RNA-seq revealed a heightened detection of sRNAs mapping onto protein-coding genes (mRNAs) and rRNAs, which is consistent with previous findings . The incIn addition, T4-PNK treatment has the potential to reveal a subset of sRNAs with end configurations that are generated in vivo through regulated mechanisms unrelated to random degradation. RNA metabolism involves enzymatic cleavage that generates OH- or P- in the 5′- and 3′ -ends, or a cyclic phosphate that results from a phosphate bridge between the 2′- and 3′- positions of the pentose (cP) . MultiplBy conducting a paired comparison between the T4-PNK-treated and non-treated samples, our data suggest that specific characteristics of sRNAs can be highlighted under different biological conditions and disease states. For instance, in our study, the paired comparison of the T4-PNK-treated and non-treated samples suggests that miRNAs have differential 5′- and 3′-ends in the EVs and extravesicular fractions. Endogenously, diverse RBPs facilitate miRNA loading into EVs and other RBPs retain miRNAs in the cytoplasm, negatively regulating their sorting into EVs . The AgoThe different properties of sRNAs are also stressed by T4-PNK when evaluating tRFs, which have been pointed out as a novel class of biomarkers. Variable susceptibility of tRFs to phospho-RNA-seq has been shown across different tissues and cells . For insIn recent years, there have been substantial scientific attempts to comprehend the RNA composition of EVs since: (1) vesicles act as intercellular communication agents transferring biological information, including RNA between cells ,68; (2) n = 3) were provided by the Banc de Sang i Teixits . The blood, collected using citrate-based anticoagulant bags (ACD), was certified by the provider as testing negative for HBs Ag, Anti-HCV, Anti-HIV 1+2, syphilis serology, VHB, HCV, and HIV. Cell-free plasma was obtained through sequential centrifugations, as previously described [g and the cellular pellet was discarded. The obtained supernatant was then centrifuged at 2500× g for 15 min followed by a final centrifugation step at 16,000× g for 15 min.Whole blood samples  for overnight incubation at room temperature. EV-coated beads were then labeled at 4 °C with anti-CD9 (Clone VJ1/20), anti-CD63 (Clone TEA 3/18), and anti-CD81 (clone M38), which were all purchased from Immunostep , and FITC-conjugated secondary goat anti-mouse antibody  was used for detection. Samples were analyzed using flow cytometry in a BD SRFortessaSORP + HTS cytometer and FACS Diva Software .Two mL of cell-free plasma were used to isolate EVs using size exclusion chromatography (SEC), as previously described . BrieflyThe four fractions showing the highest mean fluorescence intensity (MFI) fold change were pooled and identified as the EV-enriched fraction. Additionally, the four fractions with the highest protein concentration according to the Nanodrop determinations were pooled as the protein-enriched fraction.TM Protein Assay kit, ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions.Total protein concentrations from the different pooled fractions were determined using the bicinchoninic acid assay  following the manufacturer’s instructions. EV samples were diluted at 1:40 with sterile and filtered PBS, and three videos of 60 s at 25.0 frames per second (FPS) at a camera level of 10 were recorded. The mean values for size (nm) and concentration (particles/mL) of the three replicates were considered as the final measurement.Additionally, EV-enriched fractions were examined using cryo-transmission electron microscopy (cryo-TEM), as previously described . BrieflyTM Protein Assay Kit  was considered.EV- and protein-enriched fractions were subjected to Western blotting for the detection of specific EV markers. Equal amounts of protein (25 μg) of each sample measured by the Micro BCA® Transfer Stack supports . Immunoblots were probed with the EV-markers anti-Alix antibody , anti-Flotilin1 , and anti-Syntenin . Anti-Calnexin  was used as a negative control. Membranes were incubated with the primary antibodies overnight at 4 °C under conditions of agitation. After washing them, membranes were incubated with the secondary antibody anti-rabbit HRP-conjugated IgG  or with the anti-mouse HRP-conjugated IgG . Immunoreactive bands were visualized using the Western blotting SuperSignalTM West Pico PLUS Chemiluminescent Substrate Reagent  and images were acquired using the ChemidocTM Imager .Samples were then loaded in NuPAGE 4–12% Bis-Tris polyacrylamide gels  and transferred to a nitrocellulose membrane using the iBlot2EV-enriched pools were further concentrated with the Amicon Ultra-2 100 kDa  following the manufacturer’s protocol before RNA isolation. Protein-enriched fractions were concentrated 10-fold by the speed vacuum at room temperature (RT) . RNAs from the total plasma, EV-, and protein-enriched fractions were isolated using the miRNeasy Serum/Plasma kit  according to the manufacturer’s protocol, and were further treated with T4-PNK  as previously described , and folhttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/ accessed on 17 December 2020). Adapter trimming was performed with Cutadapt [The quality of the sequenced fastq files was checked using FastQC software (v0.12.1)  package . To takep-values for multiple testing. A 5% statistical significance level was considered throughout all the analyses.The chi-squared test was used to evaluate the tissue-enriched proportions of the protein coding fragments. A Benjamini–Hochbergh correction was used to correct the In summary, our results confirm that the use of phospho-RNA-seq can provide a more comprehensive/extended view of the plasma sRNA transcriptome, stressing the difference between the plasma compartments and highlighting the novel species and potential RNA-based biomarkers. Our data highlight that the study of sRNAs in plasma EVs and extravesicular compartments after T4-PNK treatment can offer complementary information and may be useful in precision medicine settings to finely define the physiological and pathological states. The present findings suggest that alternative sRNA biotypes beyond miRNAs have potential in RNA-based biomarker discovery, and that EVs may offer a more informative source of the meaningful biomarkers.


Concurrent existence of lncRNA and circular RNA at both nucleus and cytosol within a cell at different proportions is well reported. Previous studies showed that circular RNAs are synthesized in nucleus followed by transportation across the nuclear membrane and the export is primarily defined by their length. lncRNAs primarily originated through inefficient splicing and seem to use NXF1 for cytoplasm export. However, it is not clear whether circularization of lncRNA happens only in nucleus or it also occurs in cytoplasm. Studies indicate that circular RNAs arise when the splicing apparatus undergoes a phenomenon of back splicing. Minor spliceosome (U12 type) mediated splicing occurs in cytoplasm and is responsible for the splicing of 0.5% of introns of human cells. Therefore, possibility of cRNA biogenesis mediated by minor spliceosome at cytoplasm cannot be ruled out. Secondly, information on genes transcribing both circular and lncRNAs along with total number of RBP binding sites for both of these RNA types is extractable from databases. This study showed how these apparently unconnected pieces of reports could be put together to build a model for exploring biogenesis of circular RNA.As a result of this study, a model was built under the premises that, sequences with special semantics were molecular precursors in biogenesis of circular RNA which occurred through catalytic role of some specific RBPs. The model outcome was further strengthened by fulfillment of three logical lemmas which were extracted and assimilated in this work using a novel data analytic approach, Integrated Cellular Geography. Result of the study was found to be in well agreement with proposed model. Furthermore this study also indicated that biogenesis of circular RNA was a post-transcriptional event.Overall, this study provides a novel systems biology based model under the paradigm of Integrated Cellular Geography which can assimilate independently performed experimental results and data published by global researchers on RNA biology to provide important information on biogenesis of circular RNAs considering lncRNAs as precursor molecule. This study also suggests the possible RBP-mediated circularization of RNA in the cytoplasm through back-splicing using minor spliceosome. A complete physicochemical mechanism of biogenesis of circular RNAs is still under scrutiny. In some study back-splicing supported by the presence of inverted repeats in flanking Introns are considered as root cause of their biogenesis , 2, whilInterestingly, both long non-coding RNAs (lncRNAs) and Circular RNAs with length greater than 200 bp are found to share many common properties , 9. BothRole of lncRNAs in post-transcriptional regulation is well reported . lncRNAsIn the present study the process of circularization of RNA was investigated by considering results of research works so far published under an introductory data processing framework, Integrated Data Geography (IDG) in general and Integrated Cellular Geography (ICG) in particular following well known Integrated Geography (IG) approach . Under tTowards resolving the first problem of finding precursor molecule, as for primary guess, first off, each of the exonic circular RNAs collected from 2 databases were BLASTed against the lncRNAs distributed within 3 databases. The objective was to see whether an lncRNA has the region inside their sequence from which their circular form may have originated in particular condition. As for supportive reports we find that circular RNA mostly originated from exonic regions. Other regions for their origin are also reported like, UTR and Intronic regions, lncRNA loci and antisense of known transcripts , 27. AlsNext part of this work was primarily devoted to identify biomolecular factors and the spatiotemporal conditions catalyzing circularization of these precursor molecules  producing Circular RNA. The RNA-binding protein FUS regulates circRNA biogenesis by binding the introns flanking the back-splicing junctions . WhereasBesides the major U2 spliceosome responsible for splicing of 99.5% introns, 0.5% of introns are spliced via minor spliceosome U12. Therefore, role of minor spliceosome for circulization of lncRNA within cytoplasm was also considered in this study which corroborates the phenomenon of cytoplasmic enrichment of CircRNAs.As stated in the introduction section, the objective of this work was to study molecular mechanism leading to formation of Circular RNA. To accomplice it, first possible precursor molecules were searched and thereafter, possible factors leading to conversion of these precursor molecules into Circular RNA were studied. Therefore, the results were listed in a manner so that each of the results lead to formation of a logical lemma  that served as a building block to construct the proposed ICG based model of biogenesis. A simple chemical representation of this model was shown in Fig. In this regard, methodologies as described in methodology section under subheading "Reasonable percentage of lncRNA (~ 5%) out of total hits obtained was found to have complete sequence similarity with circular RNA as shown in Table RBPs helping circularization from precursor lncRNA to circular RNA should first attach with lncRNA and remain attached till the process of circularization.For the above conjecture to be true, RBPs solely binding with lncRNA or circular RNA might not be considered for molecular agent helping circularization.RBPs common to lncRNA and circular RNA might be considered as agent for the circularization of RNA.For the conjectures 1–3 to be true, there must be existence of reasonable amount of common RBPs.The lemma related to identification of RBPs as possible molecular agent stood on the following conjectures:Furthermore, for validation of this lemma, the role of RBPs common to both lncRNA and circular RNA was explored. Towards this direction, methodology section under subheading “Also, for validating both Lemmas 1 and 2, steps as described in methodology section under subheading “Identification of QKI and FUS RBPs, as common to both Circular and lncRNAs", were used to extract information on number of genes producing both lncRNA and circular RNA transcripts whereas, both of these transcripts had binding sites for FUS or QKI as shown in Table At the onset of this study, the target was to zero in the RBPs common to both the lncRNA and circRNA types. Therefore, the statistical significance of the identified genes was not a matter of concern, where the result of search was utilized to prove the basis of this research, which is: RBPs common to both of these RNA types are responsible for circularization. In this study, the first objective was to find out the lncRNAs present in the POSTAR database having the binding sites for RBPs  corroborates the availability of the machinery required for the circularization of the lncRNA in the cytoplasm.Minor spliceosome and circularization of lncRNA.Partially spliced pre-mRNAs containing minor-class introns undergo nuclear export followed by splicing via U12 spliciosome in the cytoplasm –39. SpliAs stated earlier in introduction, methodology and initial part of result section, ICG approach considered to build a theorem through assimilation of lemmas supporting it. Following this approach we first built the proposition in the form of a model that there should be existence of a precursor molecule of circular RNA which upon the involvement of some molecular agents would yield exonic circular RNA as shown in Fig. Towards this direction, the lemmas Lemmas 1, 2 and 3) as a result of outcomes extracted in this work from various published experiments  formalism to study biogenesis of circular RNA". The concept of ICG was pressed in for such task as a generalization of naturally evolved solution instead of being forced upon into this study. The reason was a plethora of already published reports on experiments done on circular RNA and allied biomolecules which were although unconnected apparently in their present form, yet could be studied further in their assimilated form for confirming a target objective, i.e., a theorem. Although methodology for assimilation of published data, like Meta Analysis existed, it was found to be applicable only for data generated using same experimental protocol. The essence of ICG was actually that of Integrated Geography at microscopic, i.e., cellular scale which was further generalized in this work in a logical lemma based formalism as especially shown in the methodology section under subheading "Integrated Cellular Geography (ICG) formalism to study biogenesis of circular RNA" and the result section.Circular RNAs are known as biomarkers of quite a number of diseases including cancers. This work was motivated from this fact to partially contribute to the knowledge on events leading to biogenesis of Circular RNA assuming it to be central to unearth pathobiochemical mechanism of these diseases. In this regard, the problem was divided into two segments, first to search for molecular precursors being converted into circular form and identification of factors leading to its circularization to produce Circular RNA. Secondly, the objective was to extract mechanism of biogenesis of circular RNA from the spatio-molecular distribution of lncRNA, circular RNA and RBPs. As described in the result section, lemmas were extracted as supports for solving both of these problems. These lemmas were constructed using existing databases and results of published works as research materials and through judicious assimilation of those under ICG.To solve the first problem of identifying molecular precursor for Circular RNAs, lncRNAs were considered as primary guess material through such indication from the published research works , 26. SinIn continuation with first problem, identification of factors leading to circularization of RNA was tried to be resolved using methodology section under subheading "Regarding solution of the second problem, in this work it appeared to be interesting to extract spatio-molecular root of Circular RNA via its relationship with RBPs and earlier molecular form i.e., as indicated from this study, lncRNAs. That said, problem reduced to find out overall spatial distribution of lncRNAs, Circular RNAs and RBPs within nucleus and cytosol of cells. Regarding this, the results shown in Tables In the context of utilization of existing databases and apparently unconnected published reports, as stated in the beginning of this discussion and methodology section under subheading "Integrated Cellular Geography (ICG) formalism to study biogenesis of circular RNA", it appears to be quite justified to employ the formalism of integrated cellular geography (ICG) following the concept of general Integrated Geography (IG) . It grosThe interesting part of this work was to consider studying the role of RBPs in circularization of RNAs. In this regard, it appeared to be quite obvious that RBPs specific for Circular RNAs might not have any role for their biogenesis since they would bind with circular form only, which was not possible before circularization. The inference stemmed from the same reasoning led to the fact that RBPs common to both of these RNA types should be prime molecular factors that could be expected to help circularization of lncRNAs and thus would have to remain attached throughout transition of lncRNA to Circular RNA. This fact was further supported by the presence of such RBPs, QKI and FUS with their established role in circularization , 30. HowHowever, for RBPs specific for lncRNAs the chance of their having role in the transition from linear to circular form of RNA being less following the above-mentioned reasoning, discussion on them was avoided although possibility of their involvement might not be overruled.We have already discussed the features: sequence semantics, RBPs, and localization due to which lncRNAs have been considered as template molecules in our study. There are various types of circular RNAs, e.g., exonic, intronic etc., indicating possible existence of different precursor molecules responsible for their biogenesis. This work is actually intended to establish one among few more such relationships to see whether the present circular RNA in question is rooted from lncRNA. However, we can proceed with other RNAs molecules adopting the similar methodology. dsRNA has been indicated as interconvertible RNA with circular RNAs , and canOverall, this study showed a possible model of biogenesis of exonic circular RNAs from lncRNAs as precursor molecule on the foundation of judicious fusion of results and data of already performed experiments under the paradigm of integrated cellular geography approach as analytical basis. Sequence analysis of both RNA types revealed significant similarity making the foundation of a logical lemma based study related to possibility of biogenesis of Circular RNAs from lncRNAs. Additionally, information extracted in this work from distribution of these RNA types within cellular spaces, cytosol and nucleus, in relation to their binding with specific types of RBPs further helped in building of other two lemmas in support of spatio-molecular mechanism of biogenesis of exonic circular RNA. The theorem built from these four complimentarily supportive lemmas strongly supported model of biogenesis of Circular RNA as provided in this work. According to this model: Biogenesis of exonic circular RNA is mostly post-transcriptional event which starts with the binding of RBPs common to both lncRNAs and circular RNAs with precursor molecule lncRNAs with some fraction within nucleus and also some within cytosol with exceptions following spatio-molecular arrangements of lncRNA, circular RNA and all types of RBPs. Therefore, this model indicated that the biogenesis of exonic circular RNA was mostly a post-transcriptional event. The concept of ICG, as first time introduced in this work, appeared to have prospective of a very useful methodological ingredient of systems biology for holistic study of events of cellular system.Integrated Geography (IG) in its definition is the branch of geography that describes and explains the spatial aspects of interactions between human individuals or societies and their natural environment, called coupled human–environment systems . Since bserve as supports to prove theorem, represent complimentary form of necessary and sufficient proof of T,For a pool of logical lemma, We may consider the analysis of the system is compatible and doable under the broad paradigm of Integrated Data Geography (IDG) and specifically, for this work, Integrated Cellular Geography (ICG).In our work which dealt with biomechanical root of formation of circular RNA, the geographical space of our choice was a biological cell and its very important sub-cellular spaces (nucleus and cytosol) from where the referred biomolecule was generated. We analyzed the spread of this biomolecular entity outside and inside the sub-cellular space along with assimilating other reported pieces of information  to come up with necessary and sufficient conditions of root of formation of such molecule.Data type 1 (DT1): Apparently unconnected reports of published papers, and,Data type 2 (DT2): Formal data collected from databases.The objective of data collection for this work was primarily to study the effectiveness of the newly proposed Integrated Cellular Geography approach in predicting biogenesis of circular RNA. Under this approach, following two types of data were considered for their utilization:possible precursor molecules of circular RNA,molecular agents involved in circularization, and,cellular spatiotemporal condition supporting circularization.Therefore for the model constructed under this approach, the intention was to look into the convergence of DT1 and DT2 to extract information on:Considering the specific class of circular RNA that were exonic and were intuitively formed from lncRNAs, the sequences of circular RNAs were collected from circBase  and circAs mentioned in the introduction section, RNA Binding Proteins were reported to be involved in the circularization of some RNAs. To get a comprehensive view and extract relevant information, circInteractome , starBasFor this purpose, online database and web server were used by taking specific queries like, RBP-types or lncRNAs to search out their special location inside cellular spaces. UniProtKB  was usedDatasets for reference data of BLAST  tool: GEQuery-set: Queries obtained from circular RNA datasets, circBase and circRNADbSearch tool: BLASTN of BLAST+ 2.8.0 package.In this phase, the objective was to search out the region of homology of circular RNAs on lncRNAs to initially check if there was any indication for lncRNAs to serve as precursor molecules of circular RNAs. For this purpose, we have taken two circular RNA databases, circBase and circRNADb in our study. Also for lncRNAs we have taken datasets from three lncRNAs database like GENCODE, LNCipedia and NONCODE. Following dataset, query-set and tools were used for the mapping:Mapping process was given in Table The process was performed for all possible combinations of query and reference databases.As clarified in the introduction section, in this work, role of some RBPs was studied in relation to circularization of RNA. Therefore, it was important to get account of their spatial existence especially within nucleus and cytosol of a cell along with information on the types of RNAs they bound. Therefore, databases, circInteractome , starBasCytosol onlyNucleus onlyBoth nucleus and cytosol, and,Secreted from the cellSubsequently percentage existences of RBPs for each of the RNA-types along with RBPs unique to both Circular and lncRNAs were computed in:Step 1 First, POSTAR.csv file was downloaded and the list of lncRNAs was extracted by parsing of this file.Step 2 Each of the lncRNAs within the list was submitted as query to POSTAR to find out the RBPs having binding sites on it.Step 3 The RBPs extracted in step 2 were further processed to eliminate redundant instances of them.To accomplish this task, supplementary files extracted from databases, CircInteractome and starBase were utilized to extract RBPs and RNAs they bound to by writing Perl scripts to read these files. However, in case of POSTAR database following methodological steps were utilized to extract list of RBPs for lncRNAs:Furthermore, to get information on spatial location of RBPs, each one of them was inputted into UniProtKB  to get iEpithelial to mesenchymal transition (EMT) coincides with regulation of many circular RNAs,Circular RNA formation was regulated by QKI during EMT,Binding sites of QKI was found to be flanking circRNA-forming exons, and,Circularization of linear RNA could be induced by insertion of QKI binding site into them.Among many different types of functions mediated by QKI type of RBPs, in this work, its role for circularization was investigated from the report . This reSimilarly the role of another RBP, FUS in regulating circularization was documented in . TherefoStep 1 Specific RBP (either QKI or FUS) was submitted as query in POSTAR database.Step 2 The database returned the names of the genes and corresponding RBP-type binding sites on RNAs transcribed from these genes.Stage1: At this stage following steps were applied to get first hand information about RNAs for a specific RBP-type:Step 1 Out of the output gene pools extracted through stage 1, only those transcribing for lncRNAs were screened through writing Perl script.Step 2 Each of the screened genes was submitted as query in CircInteractome database and the outputs yielding at least 1 hit were chosen only.Step 3 Each of the outputs obtained from step 2 was further analyzed to check manually the RNA types transcribed by this gene , RBP types for these RNA types  and binding sites for these RBPs on these RNA types. Only those genes transcribing for Circular RNA with specific RBP-types (QKI or FUS) were retained for further analysis.Stage2: Since direct description of RNAs (whether mRNA or lncRNA) could not be extracted at stage 1, following steps were applied to get such information:Based on the total BLAST hits obtained only those hits were extracted (A) in which the circular RNAs sequences fully mapped in lncRNAs.http://lin-group.cn/server/iLoc-LncRNA/predictor.php') was used to know probable spatial location of these lncRNAs inside the cellular space.Using A, specific web-server, iLoc-LncRNA  in which the circular RNAs sequences were not at all mapped in lncRNAs.Finally cellular location of such lncRNAs was found using iLoc-LncRNA web server.Following steps were performed for this purpose:To reduce computational complexity in dealing with very large database where number of data is greater than 1000, sample datasets were used through random selection of data from the original database.Workflow depicting the ICG model for biogenesis of circular RNA was shown in Fig.


The oncogenic function of circ_63706 was characterized in vitro and in vivo. Further, circ_63706-depleted cells were subjected to RNA-seq and lipid profiling to identify its molecular function. Finally, we mapped the circ_63706 secondary structure using an advanced random forest classification model and modeled a 3D structure to identify its interacting miRNA partner molecules. Circ_63706 regulates independently of the host coding gene pericentrin (PCNT), and its expression is specific to the SHH subgroup. circ_63706-deleted cells implanted into mice produced smaller tumors, and mice lived longer than parental cell implants. At the molecular level, circ_63706-deleted cells elevated total ceramide and oxidized lipids and reduced total triglyceride. Our study implicates a novel oncogenic circular RNA in the SHH medulloblastoma subgroup and establishes its molecular function and potential as a future therapeutic target.Medulloblastoma (MB) develops through various genetic, epigenetic, and non-coding (nc) RNA-related mechanisms, but the roles played by ncRNAs, particularly circular RNAs (circRNAs), remain poorly defined. CircRNAs are increasingly recognized as stable non-coding RNA therapeutic targets in many cancers, but little is known about their function in MBs. To determine medulloblastoma subgroup-specific circRNAs, publicly available RNA sequencing (RNA-seq) data from 175 MB patients were interrogated to identify circRNAs that differentiate between MB subgroups. The online version contains supplementary material available at 10.1186/s40478-023-01521-0. TP53 wildtype, SHH-activated and TP53 mutant, and non-WNT/non-SHH .FASTQ files for RNA-seq data were collected from the European Genome-Phenome Archive  obtained from the St. Jude Hospital. We also isolated RNA samples from patient-derived xenografts (PDXs). The Wechsler-Reya lab established the DMB006, DMB012, RCMB28, RCMB32, RCMB38, RCMB40, RCMB45, and RCMB51 PDXs. The Olson lab at the Fred Hutchinson Cancer Research Center established MED211FH, MED511FH, and MED1712FH PDXs. The Milde lab at the German Cancer Research Center (DKFZ) established the BT-084 PDX, and the Cho lab at Oregon Health and Sciences University established the MB002 PDX. The Wechsler-Reya lab maintained all PDXs.circ_63706 (Thermo Fisher Scientific). Hybridization was performed according to the manufacturer’s instructions.RNA was visualized in formalin-fixed, paraffin-embedded tissue (FFPE) sections using the QuantiGene ViewRNA ISH Tissue Assay Kit . Tissue sections were rehydrated and incubated with proteinase K. Subsequently, we incubated the sections with ViewRNA probesets designed to target human circ_63706 were purchased from Integrated DNA Technologies . Cells were transfected with 20 nM siRNA targeting each gene or control non-targeting siRNA (negative control siRNA)  for 48 h using Lipofectamine RNAiMAX (Thermo Fisher Scientific). Knockdown efficiency was assessed using qRT-PCR. The following siRNAs sequences targeted circ_63706 (#1): CAGCTGGAGACCCTGAAGGAA and (#2): ACAGCTGGAGACCCTGAAGGA.siRNAs targeting circ_63706 knockdown into the cerebellums of NOD-SCID mice . Cerebellar coordinates were − 2 mm from lambda, + 1 mm laterally, and 1.5 mm deep. We evaluated tumor growth with weekly bioluminescence imaging using an in vivo spectral imaging system .All mouse studies were performed following the policies and regulations of the Animal Care and Use Committee of Johns Hopkins University, which approved the studies. We established intracranial medulloblastoma xenografts by injecting DAOY, ONS76, and DAOY cells with The circRNA detection pipeline is depicted in Fig. vs. the other three groups. 114 circRNAs in the WNT subgroup, 48 in the SHH group, 13 in G3, and 21 in G4 MBs were upregulated , we took a similar number (n = 15) of upregulated circRNAs from other groups for experimental validation and functional studies  machine learning algorithm provides efficient and high predictive accuracy for many data types, including clinical and molecular data. RF is particularly useful for genomic data analysis, which is typically of small sample size but high feature dimension. Differential expression (DE) analysis packages are not optimized for circular RNA analysis due to inherent complications with normalization. Therefore, to validate the top 58 circRNAs obtained from DE analysis, we built and applied an RF model to identify subgroup-specific circRNAs. The model used 460 circRNAs across 126 samples, selected using recursive feature elimination (RFE) with RF. The heatmap of loading coefficient of the top 28 circRNAs contributing to the classification model appears in Additional file ACTB was used as a control.The two combined analytic methods identified 65 subgroup-specific circRNAs  and one (circ_21305) in G4. Six circRNAs were validated in PDX samples, including all four SHH-specific circRNAs  and two G4-specific circRNAs (circ_21305 and circ_33068)  and circ_30598 had no coding potential.We predicted the protein-coding potential of these six circRNAs using the RNAsamba tool , which ucirc_30598, circ_63706, circ_64014, circ_66962, circ_21305, and circ_33068), only circ_63706 showed statistically significantly higher expression in the SHH subgroup by qRT-PCR in PDX samples compared with the other three groups. The cell line results are shown in Additional file circ_63706 and further confirmed its expression by RNA-FISH in formalin-fixed paraffin-embedded tissue samples from patients with MBs. Out of 20 medulloblastoma samples, circ_63706 was highly expressed in six SHH samples but not in any G3 or G4 MBs .Of the six SHH subgroup-specific circRNAs  in these SHH MB cell lines and PDXs , , 340 genes were upregulated and 395 genes were downregulated in cultured DAOY cells treated with si-circ-63706   and glycerolipids (DG and TG) were mainly oxidized , the breakdown product of TGs, were slightly elevated, whereas total TGs (n = 258) were significantly reduced in circ_63706 KO cells, suggesting impaired glycerolipid metabolism that may interfere with cellular proliferation  itself and its different subclasses such as CerNS (nitroso-ceramide), CerP (phosphorylated-ceramide), and CerG (glycosylated-ceramide)  Fig. . Lipid ools Fig. A, suggessurvival . Using tcirc_63706 knockdown significantly enriched for active lipid ontologies including sphingosine, sphingomyelin, and ceramide, while circ_63706 overexpression significantly reduced the active lipid component. Unsaturated fatty acids, specifically fatty acids with 2, 3, and 6 double bonds, were elevated in circ_63706 knockdown cells. Interestingly, saturated lipids were elevated in circ_63706-overexpressing cells  and their biochemistry are increasingly recognized as important in cancer and therapeutic development. We found that . Therefore, we mapped the secondary structure of the circ_63706 sequence using MXfold2, which uses deep learning to integrate thermodynamic information to accurately predict secondary structures of newly discovered ncRNAs [circ_63706 with FARFAR2, which uses an RNA fragment assembly method to model RNA structures.Characterizing the 3D structures of circRNAs is crucial for understanding their cellular functions such as microRNA sponging and interactions with RNA-binding proteins and other RNAs. The experimental determination of RNA structure is challenging, but circRNA structure determination is especially difficult due to significant overlap between circRNA and linear RNA sequences . There id ncRNAs . This incirc_63706 showed that the double-stranded pairings were stable, while the overall tertiary structure remained flexible. Figure circ_63706 structure has two major dsRNA helix stems with 29 and 19 base pairs, respectively, as well as several other shorter segments ranging from 2 to 9 base pairs. Similarly, the structural features also include three 3-way junctions, one 4-way junction, six hairpin loops, and 12 internal loops. To explore the binding regions of the miRNA, we used the database of 87 mature miRNA human sequences from miRBase paired with the circ_63706 sequence using IntaRNA. Six top-ranked circRNA-miRNA duplexes, along with their binding energies compared with those within the circRNA structure, are shown in Additional file A 500-ns molecular dynamics simulation of the modeled circ_63706  was a potential SHH subgroup-enriched molecule, confirmed by qPCR and RNA-FISH of clinical tissue samples.To identify medulloblastoma subgroup-specific circRNA biomarkers, we subjected publicly available RNA-seq data to the CIRI2 circular RNA detection pipeline. By applying machine learning and statistical methods, we identified group-specific circRNAs. Among these identified circular RNAs, TP53-mutant SHH tumors, are less aggressive than G3 and G4 tumors. Biomarkers for these two groups include immunohistochemical stains for YAP1, nuclear β-catenin, monosomy 6 in WNT tumors, and identifying activating pathway mutations through sequencing [WNT and SHH MBs generally contain mutations activating those pathways and, aside from rare quencing . SHH MBsSNCAIP, a gene associated with Parkinson’s disease, exquisitely restricted to group IV alpha. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1 arising through chromothripsis, were limited to G3 tumors. Numerous targetable SCNAs, including recurrent events targeting TGF-beta signaling in G3 and NF-kappaB signaling G4, are attractive candidate molecular markers. Circular RNAs can act as tumor suppressors or oncogenes, but little is known about their role in MBs. In a recent study, Lv et al. [circ-SKA3) and circ-DTL, promoted the malignant phenotype of medulloblastoma when upregulated. Significantly higher expression of circ-SKA3 has also been reported in medulloblastoma compared with normal tissues [A recent study identified somatic copy number aberrations (SCNAs) in 1,087 unique MBs . The mosv et al.  selected tissues .circ_63706; hsa-PCNT_0003, CircAtlas 2.0) in medulloblastoma patient tissues. The availability of specific classes of lipid is critical for successful cancer cell proliferation. TGs are critical to cell membrane homeostasis, lipid droplet formation, and signaling through lipid rafts for oncogenesis [circ_63706 is important for modulating TG and DG levels in SHH cells  acting as a sponge promotes drug resistance in hepatocellular carcinoma [hsa-miR-22-5p as another potential circ_63706-binding miRNA, which is reported as a suppressor of breast cancer metastasis [Our molecular dynamics simulation of the modeled osarcoma  and breaosarcoma . Similare cancer  and in se cancer . Interesl cancer . Also, sarcinoma . We detetastasis .Overall, this study provides several important insights into novel molecular signatures of medulloblastoma subgroups for diagnostic and therapeutic purposes.Additional file 1: Supplementary File.Additional file 2: Supplementary Methods.Additional file 3: Supplementary Figures.Additional file 4: Supplementary Tables.


The ways in which humans can support good welfare for animals in their care is an ongoing subject of debate: some place emphasis on the animals’ physical health; others, on animals’ ability to live “natural lives”; and others on animals’ affective states or psychological wellbeing. Recently, there has been an increase in interest in how an animal’s ability to exercise control over its environment could impact their welfare. In this article, we take the stance that the relevant aspects of the first two concepts of animal welfare  are largely addressed when an animal’s psychological wellbeing is prioritized. Through that lens, we review the current state of the literature regarding the psychology of control, and the intersection between choice, control, and welfare. We clarify terms to support future work, and propose future directions that could lead to a better understanding of the psychological benefits of choice and control and ultimately inform animal care decisions. There is some philosophical controversy about how welfare should be defined, and care for an animal can differ dramatically depending upon which definition of welfare one adopts .unnatural behaviors are rewarding to animals and support good health and positive experiences r tasks; ].A third perspective, and perhaps the dominant perspective now, focuses on affective states, or “feelings”, of animals as central for determining animal welfare e.g., , 11, 14], 1411, 12.Dawkins , 19 prop2.1.choice as the act of choosing or selecting from more than one alternative  and competence  are considered innate psychological needs for humans  challenge are necessary for an animal to experience re plans , 27. Agere plans , 28. Furre plans , and there plans . But indre plans , 25, 29.re plans .choice to animals.In interim summary, to promote positive welfare in animals, we must strive to increase the perception of agency. Špinka  and Špin2.2.Leotti et al.  argued tper se, but the ability to start or stop aversive or rewarding stimuli. For example, Seligman and Maier  and even when the assigned order was yoked to match animals’ established preferences. Interestingly, the monkeys who demonstrated improved performance in the Beran et al.  that all animals would reap benefits from being functional agents in their environment, the psychological impact of choice may differ from species to species. Most primates evolved to live in groups with complex social dynamics, consume varied diets, and inhabit heterogenous environments. These traits could make primates especially sensitive to the availability of choice in their environment; or, it is possible that we are better prepared to consider the value of choice for primates due to a bias in our science and publication history , 51. Cermeasured . More remeasured .3.3.active use of the chosen alternative. Only recently  have scy e.g., , 41, 43], 43activy [e.g., , yet they [e.g., . Incorpo4.In this article, we have reviewed the literature on welfare, choice, and control, and discussed the ways in which these topics intersect. The majority of research has investigated the role of control on psychological wellbeing via stimulation management. However, we also know that making choices is a means by which to exercise control, and that studies on choice behavior have indicated that making choices can have a direct positive impact on welfare. Yet gaps in the literature remain, including how non-primates are affected by choice opportunities, the impact of the duration of choice or number of options available, and the benefit of unselected alternatives. We are optimistic that providing choices to captive animals will continue to prove an excellent strategy to improve their welfare while also enabling us to learn about their preferences, choice behavior, and cognition more generally.ME and KC developed the concept of the manuscript and reviewed and revised the article. ME wrote the first draft. All authors contributed to the article and approved the submitted version.This work was supported by Lincoln Park Zoo.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Methylation of carbon-5 of cytosines (m5C) is a conserved post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. It can be further modified to yield 5-hydroxymethylcytidine (hm5C), 5-formylcytidine (f5C), 2´-O-methyl-5-hydroxymethylcytidine (hm5Cm) and 2´-O-methyl-5-formylcytidine (f5Cm). How m5C, and specially its derivates, contribute to biology mechanistically is poorly understood. We recently showed that m5C is required forCaenorhabditis elegans development and fertility under heat stress. m5C has been shown to participate in mRNA transport and maintain mRNA stability through its recognition by the reader proteins ALYREF and YBX1, respectively. Hence, identifying readers for RNA modifications can enhance our understanding in the biological roles of these modifications.Methods: To contribute to the understanding of how m5C and its oxidative derivatives mediate their functions, we developed RNA baits bearing modified cytosines in diverse structural contexts to pulldown potential readers inC. elegans. Potential readers were identified using mass spectrometry. The interaction of two of the putative readers with m5C was validated using immunoblotting.Results: Our mass spectrometry analyses revealed unique binding proteins for each of the modifications.In silico analysis for phenotype enrichments suggested that hm5Cm unique readers are enriched in proteins involved in RNA processing, while readers for m5C, hm5C and f5C are involved in germline processes. We validated our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m5Cin vitro. Finally, sequence alignment analysis showed that several of the putative m5C readers contain the conserved RNA recognition motif (RRM), including ALY-1 and ALY-2.Conclusions: The dataset presented here serves as an important scientific resource that will support the discovery of new functions of m5C and its derivatives. Furthermore, we demonstrate that ALY-1 and ALY-2 bind to m5C inC. elegans. Nucleoside chemical modifications are a common feature in RNA molecules. More than 160 post-transcriptional modifications have been reported since the discovery of pseudouridine in 1957 . However5C) is now known to be a conserved and widely distributed feature in biological systems. m5C is catalysed by tRNA aspartic acid MTase 1 (DNMT2) and RNA methyltransferases from the Nop2/Sun domain family (NSUN1-7 in humans), and has been shown to be involved in tRNA stability, ribosome fidelity and translation efficiency , forming 5-hydroxymethylcytidine (hm5C) and 5-formylcytidine (f5C) (5Cm) and 2´-O-methyl-5-formylcytidine (f5Cm)  readers added new layers of complexity to this pathway, revealing context- and stimuli-dependent functions previously not appreciated (5C. The Aly/REF export factor (ALYREF) was identified in an RNA pulldown experiment and shown to interact with NSUN2-modified CG-rich regions and regions immediately downstream of translation initiation sites. NSUN2-mediated mRNA methylation is interpreted by binding of ALYREF to modulate nuclear-cytoplasm shuttling of transcripts.  seeded withEscherichia coli and transferred to fresh plates regularly to avoid starvation  and used as food source . Bristol N2 was used as the wild type strain.rvation . The str5Cm), 5-hydroxymethylcytidine (hm5C) and 5-formylcytidine (f5C) phosphoramidite monomers was performed as described inThe synthesis of 2´-O-methyl-5-hydroxymethylcytidine  were synthesised in collaboration with ATDBio, Southampton, UK. Purity and integrity of all modified RNA oligonucleotides were confirmed by LC-MS analysis.RNA oligonucleotides  and the animals were resuspended in 4 ml of lysis buffer before the addition of a protease inhibitor cocktail . The animals were pelleted by centrifugation at 2,000 rpm for 2 min (Eppendorf 5810) and the supernatant was removed. Samples were frozen in liquid nitrogen drop-wise, using a Pasteur pipette. Frozen droplets were grinded to powder in metallic capsules for 25 sec in a mixer (Retsch MM 400 Mixer Mill). The powder was stored at -80°C until required. The powder was defrosted at 4°C and the lysate was sonicated for 10 cycles of 20 sec, with breaks of 20 sec in between, in a Bioruptor Pico (Diagenode). The sample was centrifuged in an Eppendorf 5430R microcentrifuge at maximum speed for 15 min at 4°C and the protein concentration of the supernatant was determined using a Bio-Rad Bradford protein assay (Bio-Rad 5000006) according to the manufacturer’s instructions.Nematodes were grown until gravid adults in 140 mm NGM plates seeded with concentrated HB101RNA pulldown protocol was adapted fromSamples were processed and analysed as described inXcalibur Software version 2.8-280502/2.8.1.2806 (RRID:SCR_014593) in a data dependent mode selecting the 15 most intense ions for HCD-MS/MS. Raw MS data was processed usingMaxQuant (version 1.3.0.5) (RRID:SCR_014485) (RP-LC was performed using a Dionex RSLC nano HPLC (Thermo Fisher Scientific). Peptides were injected onto a 75 μm x 2 cm PepMap-C18 pre-column and resolved on a 75 μm x 50 cm RP- C18 EASY-Spray temperature controlled integrated column-emitter (Thermo Fisher Scientific). The mobile phases were: 2% ACN incorporating 0.1% FA (Solvent A) and 80% ACN incorporating 0.1% FA (Solvent B). A gradient from 5% to 35% solvent B was used over 4 h with a constant flow of 200 nL/min. The spray was initiated by applying 2.5 kV to the EASY-Spray emitter and the data were acquired on a Q-Exactive Orbitrap (Thermo Fisher Scientific) under the control of014485) .-9  was then added to all median values to calculate fold changes.Putative readers were ranked based on the median of the intensity-based absolute quantification (iBAQ) values (oC in a heat block (Eppendorf 5355). Boiled samples were then spun at 13,000 rpm in a microcentrifuge (Eppendorf 5430R). Pulldown eluates (100%) and input controls (5%) were loaded alongside a protein ladder  in a 4-12% Bis-Tris gel and ran in NuPAGE MOPS SDS running buffer (Invitrogen) at 200V for 50 min in a XCell SureLock electrophoresis system (Thermo Fisher Scientific) connected to a Bio-Rad PowerPac Universal Power Supply (Bio-Rad 1645070).Samples were prepared and validation pulldowns were performed as described in “Protein extraction” and “RNA pulldown”, respectively. Proteins bound to RNA-baits were eluted in 50 µL of sample buffer  and boiled for 5 min at 95Proteins resolved in SDS-PAGE were transferred onto a nitrocellulose membrane  using NuPAGE Transfer Buffer (Invitrogen), for 2 h, at 250 mA, 4°C in a Mini Trans-Blot Cell (Bio-Rad). Membranes were blocked in 3% non-fat dry milk in TBS-T buffer for 30 min at room temperature. Membranes were incubated with primary antibody  at 1:1000 dilutions in 3% milk-TBS-T overnight rotating at 4°C. Following three washes in TBST-T for 10 min each, membranes were incubated with goat polyclonal anti-mouse IgG conjugated to HRP  diluted in milk/TBS-T for 1–2 hours at room temperature. Following three washes in TBS-T, bands were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore) according to manufacturer’s instructions. Medical X-ray films  were exposed to luminescent membrane for the time required and the films were developed on a Compact X4 automatic X-ray film processor (Xograph Imaging Systems Ltd).bona-fide modification readers, we used modified RNA baits with biologically relevant characteristics. Considering that m5C is deposited in tRNAs in a structure-dependent manner, we designed baits bearing modifications in three structural contexts: single-strand, double-strand, and loop -biotin , or ALY-2 (OP217), tagged at the C-terminus with TY1::eGFP::3xFLAG, to confirm these findings  domain, which is a known RNA-binding domain , highlighting these as potentialbona fide m5C readers.In summary, we have identified putative readers for m5C oxidative derivatives hm5C, hm5Cm and f5C. Stable isotope labelling experiments suggest that hm5Cm is a stable modification  is an abundant and conserved post-transcriptional modification found on a variety of RNA molecules. Thus far, much effort has been placed on identifying m5C modification sites and unraveling the biological significance of these modifications through “writer” loss-of-function experiments. More is currently known about the “writers” and “erasers” of the m5C modifications than the “readers”, which could introduce specificity and unique biological effects through combinatorial modification plus target context.RNA modifications have gained much interest in the recent years as they become associated with important biological functions. 5-methylcytosine  median across three (3) replicates could be wildly misleading if there is a high degree of variability among the replicates and if pairing of the data exists. Use of a median essentially reduces the analysis to a single replicate comparison between the experimental conditions. One way to bolster the confidence in the data would be to provide a principal component analysis  of the proteomics data to demonstrate data consistency between control and m5C (and derivatives) experimental conditions.    Additional mass-spec data analysis should be presented to increase the scientific readers’ confidence in the identified lists of putative m Figure 1 legend and manuscript text: Please define what you mean by “Proteins uniquely enriched in pulldowns”. Does that mean 1. proteins that were enriched 1.5 or 2-fold over control in each experiment, 2. proteins that were unique to modification type but not enriched over control, or 3. proteins that were 1.5 or 2-fold enriched over control and unique to m5C pulldown vs. other modifications? The reader assumption is that it’s likely the latter, however, Figure 1B highlights “uniquely enriched proteins” that appear to fail the 1.5 or 2-fold enrichment threshold. Please address this discrepancy. In addition, use of 1.5- or 2-fold lines on Figure 1B plots, as well as color-coding fold enrichment vs. uniqueness in the plots would further improve the clarity of the data analysis. Labeling highly changed/high confidence candidate readers on the plots  would also provide context of these proteins’ enrichment for the reader. As shown, Figure 1B provides limited and unclear information.5C RNA modification and its derivatives hm5C, hm5Cm, and f5Cm. A discussion addressing the justifications for this expectation is important to set the stage for the reader to understand the appropriateness of the “uniquely binding” criteria of the co-precipitating proteins. A protein appears to be disqualified from being considered a candidate “reader” if it is not unique to any given modification derivative. Is there a precedent of this assumption and could this assumption perhaps erroneously eliminate “readers” capable of reading m5C and some of its derivatives? Further discussion on this is needed to ensure the manuscript audience can evaluate the validity of the approach used to qualify any given putative interactor as a potential “reader”.  The expectation inherent to the described experimental approach is that the “reader” proteins are uniquely and specifically able to discriminate between m5C within the oligos. Some discussion about how the baits were designed and potential limitations would be helpful to understand the significance of the findings. Given that many m5C modification occurs on tRNAs, were the baits designed to mimic the tRNA structure ? Is there evidence that RNAs contain these specific modifications at multiple sites? If so, is it known whether modification “readers” can differentiate between molecules containing multiple modifications and single modifications? If single-modified baits were used, how might that impact the identified readers? It seems plausible that some “readers” might differentiate between single-stranded, double-stranded and loop modifications. An expanded discussion of some of these points would help clarify the significance and limitations of this study, necessary to identify potential avenues for further analysis. In a similar vein of thought, the manuscript does not address the justifications for using the specific oligos and the three modification sites within each oligonucleotide. Thus, the manuscript would greatly benefit from a discussion of what previous data drove the design of the RNA oligonucleotides used in this study, as well as the justification for using the three (3) specific locations for met al., 2017: “residues in ALYREF required for m5C binding previously identified by Yanget al. are highlighted in red”. However, these residues do not appear to be defined in that study. According to Yanget al., 2017, the same residues are described as being “the relatively conserved amino acids used for constructing mutants” . The authors should also clarify the relevance of the Yanget al., 2019 citation, as it appears to have been referenced in error.   2. In Figure 3A, the residues required for RNA binding are highlighted, with the authors referring to Yang In Figure 3A and 3D, numbering of amino acid positions would help orient manuscript readers to positions of functional domains and understand the take-home message. In Figure 3A, the highlighted residues appear to be different from the RNP1 and RNP2 motifs highlighted in Figure 3D. What is the basis for RNP1 and RNP2 being highlighted in Figure 3D as RNA binding domains? This should be clarified in both the manuscript text and figure legend. Further, these reviewers are not convinced that the provided alignment shows a “high degree of conservation”, as shown in Figure 3D. Usage of protein isoforms identical for the shown region can skew the Muscle analysis and more information is needed for why the authors deem the highlighted regions (RNP1 and 2) in Figure 3D as “important for nucleic acid binding”. Finally, the language should be softened to propose the highlighted “RNP” regions as potentially important, with qualifications that the highlighted regions have not been demonstrated to bind nucleic acids. Justifications for highlighting the proposed regions should still be provided.5C readers. ALY-1/2 pulldowns/Western findings would be bolstered by providing all replicates in Supplemental data. In addition, language within the manuscript should be softened to avoid stating that pulldowns and Western unambiguously confirm ALY-1 and ALY-2 as mAdditional comments:C. elegans gene names. This would also allow the readers to independently assess the nature of the putative interacting proteins beyond the provided GO term analysis. These reviewers appreciate the list of putative interactive proteins provided as a resource in The Supplemental Table 1. However, the practical usability of that resource would be dramatically improved by including the  Figure 3C: The legend should state that only the RRM domains of the proteins are shown. This will prevent manuscript readership from misinterpreting the shown predicted structures as structurally conserved across the entire protein length. Based on GO-term analysis, the authors claim that hm5C and f5C modifications show “exclusively germline related phenotypes”. However, one of the phenotypes for hm5C was sluggish, which seems unlikely to result from a germline defect. Please state in the Methods section how much protein was used per each pulldown experiment going into mass spectrometry. As written, it appears that only 2mg of protein has been used, an inadequate amount for most mass spectrometry experiments. Alternatively, please reference the literature that supports that 2mg of protein is sufficient for RP-LC mass spectrometry.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?Not applicableAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:RNA biology, C. elegans, RNA pulldown techniquesWe confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. 5C is a conserved RNA modification that has been detected in tRNAs, rRNAs, and mRNAs from other systems that has roles such as promoting tRNA stability and translational fidelity. InC. elegans, m5C is non-essential but nonetheless prevalent in rRNAs and tRNAs, as shown by the same research group in previous work . mRNA modification appears to be less common. Loss of m5C fromC. elegans leads to developmental and fertility defects as well as a decrease in translational efficiency. Here, Navarro et al seek to identify readers of the m5C modification inC. elegans. Only two m5C readers have been identified to date in any organism, so identification of more candidate proteins is an important question. To this end, Navarroet al, synthesize RNA baits modified with m5C and derivatives, incubate the baits with RNA lysate, and following pulldown, perform mass spec for interacting proteins. A significant number of candidate readers were identified, including orthologs of ALYREF, a previously known m5C reader, which were then further validated by m5C RNA pulldown and western blot. Further analysis will be needed to parse the false positives from the true m5C readers and to determine the biological function of each protein.m5C candidate readers were found using one or all three of these structures. And were unmodified baits also used in all three structural contexts?RNA baits were designed in three structural contexts, single-strand, double-strand and loop – but it is not discussed in the results whether the identified m5C variants. Is it possible that interesting candidates would have been eliminated at this step? At a minimum, the full list of mass spec interactors, including those found binding multiple m5C variants should be provided.The text indicates that candidates were limited to those that bound only one of the modified RNA baits, but it is not discussed whether the known readers are unable to bind the m5C modification.Figure 3A and text “Protein alignments show that some of these residues are conserved in ALY-1 and ALY-2” – Of the residues highlighted in the red box, several are weakly conserved or not conserved at all. It is not discussed whether the loss of these critical residues will likely affect the ability of these proteins to bind the mIn the last sentence of the results, the word “bona fide” should be removed since these interactions have not yet been validated. Comments –Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:C. elegans RNA silencingI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Besides mapping chemical modifications in RNA, the vast majority of epitranscriptomics studies is currently reporting on associations between the function of a gene  and a multitude of observable phenotypes. These approaches are usually very simplistic and classically involve gene knockdowns or even knockouts, followed by the description of phenotypic changes, many of which seem to be rather pleiotropic resulting in little insight which of the RNA substrates of a particular writer, reader or eraser protein has contributed to the observed changes. Interestingly, even though mapping studies have provided positional clues about the occurrence of particular modifications, there seems to be little incentive in following up on assigning functional impact of specifically modified nucleotides in specific RNAs on a particular trait and/or phenotype.th to 10th mRNA molecule should account for physiologically meaningful impact on the biology of a cell. It is accepted that RNA modifications are “written” onto RNA sequences by a staggering number of dedicated enzymes. While non-coding RNAs are often stoichiometrically modified with a great number of different RNA modifications  , mRNAs, while being modified with a few different RNA modifications, carry only sub-stoichiometric levels of a particular modification at specific nucleotides. It follows that modified nucleotides in mRNA are not only rare within a transcript  but also stoichiometrically low . This is an important and seemingly overlooked conundrum in the field, since it begs the question how a few nucleotides within thousands of nucleotides, and those at every 5 A solution for this presents the concept of reader proteins. This concept has been put forward by epitranscriptomic influencers borrowing heavily from the notion of epigenetic readers. If there are only a few RNA modifications within a sea of transcripts, then proteins binding preferentially to this modified nucleotides (readers) might make the difference. Is this concept correct? Do experimentally validated data support the assumption that a few RNA modifications will make a difference through interpretation of particular proteins? In the last years, especially the m6A-focused field has reported on the identification of various m6A readers. A few attempts also exist in the public record that identified readers of other RNA modifications, including m5C. Notably, much of the current identification and characterization of these RNA modification readers relied on in vitro pull-down experiments using either defined (synthetic) RNA baits (rather than those identified by epitranscriptomic mapping strategies) combined with mass spectrometry or RNA-IPs (and derivatives of the method) to identify RNA substrates of specific reader proteins. From these reports one can deduce that all readers of specific RNA modifications also bind to unmodified RNA sequences. This begs the question if readers bind with lower Kd to modified RNAs than to unmodified RNAs? Curiously, very little has been published reporting on actual interaction parameters for specific readers and their substrates, which is common when studying enzymes or protein-substrate interactions. Instead, fixed end-point assays with readouts based on RNA sequencing or western blotting are currently substituting for deriving quantitative and kinetic parameters from the identified interactions, which would be needed to better understand the biology of putative readers of RNA modifications. This manuscript by Navarro et al., 2022 is therefore welcome for attempting to identify reader proteins for m5C as potential mediators of RNAs modified with this chemical modification. Assuming that C. elegans contains readers for the m5C modification and its oxidized derivatives, the authors use an in vitro RNA pull-down approach with differently modified RNA baits to identify proteins differentially binding to m5C, hm5C, hm5m and f5C. Setting rather tight cut-offs for enrichment of controls, they report on numbers and GO annotations of the identified proteins. Since they found the orthologues of a previously identified human m5C reader protein (ALYREF) in their data, they repeated the pull-down experiment with m5C-modified oligos and identified both worm orthologues of ALYREF to bind preferentially to m5C-modified oligos.Wellcome, without editorial bias including papers reporting single findings, replications studies, and null results or negative findings. This reviewer will therefore only assess the validity of the article content, rather than the novelty or interest levels. This reviewer accepts that Wellcome Open Research publishes articles reporting any original research that has been funded (or co-funded) byComments:, which reported on only 188 positions in the worm transcriptome as putative m5C sites. Their final conclusion about m5C in C. elegans: “In summary, we found no evidence of a widespread distribution of m5C in coding transcripts. To investigate whether the presence of common characteristics could support a subset of the aforementioned 188 positions as bona fide methylated sites, we performed gene ontology, motif search, genomic localisation and secondary structure analyses on these transcripts and sites; however, no significant shared features were found. While our data do not completely rule out the existence of m5C methylation in mRNAs, it demonstrates that this mark cannot be detected in high stoichiometry in C. elegans, as observed in tRNAs and rRNAs.” These published results hints towards the fact, that putative readers of m5C in worms will read this modification likely in tRNA and rRNA but not in mRNA. This reviewer suggests to place a reminder of their findings into the introduction of the manuscript, since the current version will be read by many scientists, eager to put meaning into exceptions, as proof that C. elegans contains m5C in mRNAs which affect its adaptability to high temperatures.This manuscript cannot be disconnected from Navarro et al., 2020The RNA bait is depicted as a secondary structure in Figure 1a. Please specify in the M&M, how you achieved formation of such structures before performing pull-down experiments with protein extracts.PAGE 4: “To increase the chances to identify bona-fide modification readers…” Since synthetic and not naturally occurring modified cytosine-containing RNA oligos were used, this reviewer would not call the “to-be-identified readers” bona fide .Figure 1b: To this reviewer, mustard is not the color of choice to highlight enriched proteins. Please make this readable. Please, indicate what the vertical and horizontal lines of proteins should signify. Based on the Figure legend, this reviewer would expect them to be all in mustard since they are clearly enriched of unmodified oligo. However, these identities are marked by all colors.et al. identified amino acid residues on ALYREF required for binding to m5C . Protein alignments show that some of these residues are conserved in ALY-1 and ALY-2 .” Yang et al., reported on exactly one (1) amino acid (K171) that seemed to be required for binding of ALYREF to m5C-modified RNA oligos. Please, correct this sentence. Figure 3a shows that exactly this lysine (human K171) is a glutamine (Q) in both worm aly-1 and -2. Even though a few other residues in the shown sequence are conserved, none really matters since lysine within the sequence of AMKQ in human ALYREF is the only residue to be important as published by Yang et al., 2019. Spoiler alert for readers of this review: in S4G of Yang et al., 2019, exactly at the level of ALYREF (with the K171A mutation), the binding to the 5mC oligo is obscured by a blind spot (air bubble?), which makes it questionable if that the signal is really reduced.PAGE 6: “YangFigure 3a: “*” represents identical amino acids while “:” and “.” represent amino acids with strongly and weakly similar properties, respectively.” Please indicate, which protein substitution/similarity index was used to support this statement.5C-modified RNA oligos and relative quantification (right). Mean ± SD, quantification performed using ImageJ, n = 2 independent biological replicates.“ Provided raw blots athttps://data.mendeley.com/datasets/k6z3c5xftr/3 do not show replicates of pull-down followed by aly-2-FLAG identification. Please provide, or state that the data come from a single experiment. A comment for interested readers: quantification of overexposed inputs is notoriously difficult and likely not possible. Hence, either a titration curve on input protein or lower exposure would convince this reviewer of the stated enrichment factors using density measurements.Figure 3b: “Anti-FLAG western blotting (left) showing enrichment of ALY-1 and ALY-2 following pulldown using m).” same overstatement as before. Yang et al., 2017 only reported on K171, which lies at the border of the RRM and is not conserved in any of the proteins shown in Figure 3. Please, rewrite this statement to avoid that AI-based manuscript writing picks up on it and propagates this inconsistency.“Amino acid residues shown to be important for m5C binding in ALYREF also reside in the RNA recognition motif (RMM) domain, which is a known RNA-binding domain (Yang et al., 2017Given that aly orthologues of ALYREF were “only” 1.9x enriched to bind to m5C over C-oligo, what about the other binders listed in Supp Table 1? Provided that m5C is likely present in tRNA and rRNAs in C. elegans, how many of the identified proteins in the experiments bind to tRNAs or rRNA?Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?Not applicableAre all the source data underlying the results available to ensure full reproducibility?NoIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:RNA biologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.