Previously published sequencing data from 573 metastasis samples were also analyzed. Through transcriptome profiling and sample classification based on a set of predefined MetA‐C‐differentiating genes, we found that most metastases were heterogeneous for the MetA‐C subtypes. Overall, MetA was the most common subtype, while MetB was significantly enriched in castration‐resistant samples and in liver metastases, and consistently associated with poor prognosis. By gene set enrichment analysis, the phenotype of MetA was described by high androgen response, protein secretion and adipogenesis, MetB by high cell cycle activity and DNA repair, and MetC by epithelial‐to‐mesenchymal transition and inflammation. The MetB subtype demonstrated single nucleotide variants of RB transcriptional corepressor 1 (RB1) and loss of 21 genes at chromosome 13, including RB1, but provided independent prognostic value to those genetic aberrations. In conclusion, a distinct set of gene transcripts can be used to classify prostate cancer metastases into the subtypes MetA‐C. The MetA‐C subtypes show diverse biology, organ tropism, and prognosis. The MetA‐C classification may be used independently, or in combination with genetic markers, primarily to identify MetB patients in need of complementary therapy to conventional androgen receptor‐targeting treatments.To improve treatment of metastatic prostate cancer, the biology of metastases needs to be understood. We recently described three subtypes of prostate cancer bone metastases (MetA‐C), based on differential gene expression. The aim of this study was to verify the clinical relevance of these subtypes and to explore their biology and relations to genetic drivers. Freshly‐frozen metastasis samples were obtained as hormone‐naive , based on their transcriptomic profiles. This study verifies the clinical relevance of the MetA‐C subtype classification and demonstrates its association with patient prognosis and tumor biology in several separate cohorts. The MetA‐C classification may be used to identify MetB patients in need of complementary therapy to conventional androgen receptor‐targeting therapies. ADTandrogen deprivation therapyARandrogen receptorARSIandrogen receptor signaling inhibitorsCNVcopy number variantEMTepithelial‐to‐mesenchymal transitionGSEAgene set enrichment analysismCRPCmetastatic castration‐esistant prostate cancerMetmetastasisNEPCneuroendocrine prostate cancerNESnormalized enrichment scoresOPLS‐DAorthogonal projections to latent structures discriminant analysisPARPpoly‐ADP ribose polymerasePCprostate cancerPCAprincipal components analysisPSAprostate specific antigenRMArobust multi‐array averageSNVsingle nucleotide variant1Prostate cancer (PC) is the most common malignancy in men and a major cause of cancer mortality worldwide. Patients with lethal PC develop bone metastatic disease that is primarily treated with androgen deprivation therapy (ADT). In most cases, ADT reduces metastasis growth, but eventually metastatic castration‐resistant PC (mCRPC) develops. Several treatment strategies for mCRPC exist of which the majority aim at inhibiting androgen receptor (AR) signaling, while others include chemotherapy, immunotherapy, bone‐targeting therapies, and poly‐ADP ribose polymerase (PARP) inhibitors . PatientRB1 alteration as a marker for poor prognosis ,,7], the U tests. Genes showing significant differences (P < 0.05) in both tests were selected for modeling using orthogonal partial least squares discriminant analysis (OPLS‐DA) based on corresponding RNA counts.For analysis of copy number variation (CNV), all coding genes were retained and called as having a gain if the copy number was ≥ 4 and loss if the copy number was ≤ 1. Genes were kept in downstream analyses if they had an RNA count ≠ 0 in > 97% of the samples and had a CNV in ≥ 3% of the samples. Remaining genes were tested for difference in MetA, B, or C fraction levels and RNA levels between samples harboring an CNV and samples with no CNV in the specific gene using Mann–Whitney 33.1Rs = −0.68, P = 1.9E‐11 and Rs = −0.58, P = 1.8E‐8, respectively, n = 103), while the MetB and MetC showed no clear relationship . Replicate samples obtained from the same metastasis showed high concurrence in the estimated fractions of MetA‐C , and that of MetA significantly lower , in CRPC compared with hormone‐naive cases  and at metastasis sampling , while the fraction of MetB showed an inverse relation to serum PSA levels    or chemotherapy Table . However3.3n = 99) and the Abida (n = 332) cohorts and evaluated in relation to available clinical data [n = 266) or capture (n = 208) libraries. All three cohorts gave results that were in good agreement with results from the Clariom D cohort, as specified below. In addition, they provided the possibility of evaluating metastasis content of the MetA‐C subtypes in relation to metastatic site.The content of MetA‐C was estimated also in external metastasis samples profiled by RNA sequencing within the Quigley .3.4To explore biological differences between the MetA‐C subtypes, GSEA was performed, based on the dominating subtype of each sample. Enrichment of hallmark gene sets was analyzed for each subtype in comparison with the other two in all cohorts examined Table , and halRs = 0.76, P = 6.2E‐15), while it was inversely correlated to the proliferation  and NEPC scores . In contrast, the MetB and MetC estimates showed inverse correlations with the metastasis AR activity scores  , while increasing MetC content was associated with decreasing proliferation score  and increasing NEPC score   Table .3.5The estimated MetA‐C content of the metastasis samples of the validation cohorts was evaluated in relation to reported ETS gene fusions , 7, gainRB1  showed significantly higher MetA content than the negative cases, while no clear association between the MetA‐C content and ETS gene fusion status was observed in the Quigley cohort Fig. . The MetRB1 Fig. , while tRB1 Fig. . The MetRB1 Fig. . OverallRB1 Fig. . DiverseRB1 (deleterious SNVs and/or CNV loss) were observed in 63% and 18‐31% of the metastases with a dominant MetB subtype in the Quigley and Abida data, respectively. As previously reported, RB1 aberrations were associated with poor patient prognosis after mCRPC diagnosis  and afty cohort ), but im4By analysis of 676 metastasis tissue samples originating from four patient cohorts, this study verifies the clinical relevance of classifying PC metastases into the transcriptomic subtypes MetA‐C . By estiMost metastases were estimated to be heterogeneous for MetA‐C subtypes, with MetA being most frequently observed. If this heterogeneity represents a mixture of tumor epithelial cell clones with different characteristics, as have been previously demonstrated for primary PC , 16, or The MetA‐C frequencies observed here were comparable to those originally reported . The conRB1 and P53 aberrations [The MetA‐C subtypes were originally identified in bone metastases . Here, trrations , we wereThe GSEA confirmed the biological characteristics of MetA‐C originally described , with MeThe MetA‐C metastasis subtypes show similarities with, but are not identical to , the traRB1 aberrations, would probably benefit from early chemotherapy and treatment with drugs currently being suggested for NEPC, such as cell cycle inhibitors and epigenic modulators [Recently, we identified a DNA methylation signature associated with AR activity in PC bone metastasis samples . High prdulators , 28. Givdulators , and the5PC metastases can be differentiated into clinically relevant subtypes, based on distinct transcriptomic profiles. The MetA‐C subtypes show different biology, organ tropism, and prognosis after AR targeting therapies Fig. . PhenotyET, AB, and PW have a pending patent application . The other authors declare no conflict of interest.ET, LK, JN, and PW conceived and designed the study. LK, JS, HJ, ML, SC, and CTK acquired the data. ET, LK, JS, EF, and PW analyzed and interpreted the data. PW wrote the manuscript draft that all authors critically revised. CTK, JED, AW, SC, AJ, KW, and AB contributed with technical and materiel support. AB and PW supervised the study.https://publons.com/publon/10.1002/1878‐0261.13158.The peer review history for this article is available at Fig S1. Associations between the metastasis subtypes A‐C (MetA‐C) and ETS gene fusion status.Click here for additional data file.Fig S2. Associations between the metastasis subtypes A‐C (MetA‐C) and deleterious single nucleotide variants (SNVs).Click here for additional data file.Table S1. Patient and sample characteristics.Click here for additional data file.Table S2. Gene transcripts used for classification of the metastasis subtypes A‐C (MetA‐C).Click here for additional data file.Table S3. Estimated fractions of the metastasis subtypes A‐C (MetA‐C) in replicate samples collected from the same metastatic site at one time‐point.Click here for additional data file.Table S4. Estimated fractions of the metastasis subtypes A‐C (MetA‐C) in samples collected from the same patient at two different time‐points.Click here for additional data file.Table S5. Estimated fractions of the metastasis subtypes subtypes A‐C (MetA‐C) in relation to tumor and patient characteristics of the Abida polyA, Abida capture, and Quigley metastasis cohorts.Click here for additional data file.Table S6. Hallmarks being consistently enriched in the metastasis subtypes A‐C (MetA‐C), respectively, in the Clariom D, Abida capture, Abida polyA, and Quigley metastasis cohorts.Click here for additional data file.Table S7. Copy number variation (CNV) gains and losses of coding regions in relation to corresponding transcript levels and estimated fractions of metastasis subtypes A‐C (MetA‐C) in samples of the Quigley, Abida polyA and Abida capture cohorts.Click here for additional data file.


Approximately 93% of the human genome is translated into RNAs, of which only 2% code for proteins and the rest 98% are noncoding RNAs. Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs of > 200 nucleotides length that are emerging as novel players in the field of cancer diagnostics or prognostics. Recently, lncRNAs are known to be associated with oral squamous cell carcinomas (OSCC). The demonstration of stable lncRNA has been a challenge in formalin-fixed paraffin-embedded tissues (FFPE). The survivability and expression level of lncRNA in FFPE tissues compared with fresh tissues is not well documented in the literature. Hence, we designed the current pilot study with the main aim to optimize modified TRI  reagent RNA isolation protocol to identify the lncRNA expression in archived FFPE tissues of OSCC in comparison to the standard RNA isolation kit method. The findings of our study demonstrated that the RNA quantity and quality were comparatively better with the optimized TRI reagent modified protocol than the standard RNA isolation kit method. Furthermore, ct (cycle threshold) values after reverse-transcription and qRT-PCR  were comparable and almost equal in both the methods for normal mucosa (control) and OSCC samples.  Specifications tableAn optimized protocol for total RNA Isolation from Archived Formalin-fixed Paraffin-embedded Tissues to Identify the Long non-coding RNA (lncRNAs) in oral squamous Cell CarcinomasThe long non-coding RNAs (lncRNAs) are a class of RNAs >200 nucleotides length. These lncRNAs are emerging as novel players in the field of cancer diagnostics or prognostics as they are involved in oncogenic and tumor-suppressive regulatory functions The ability to predictably retrieve sufficient RNA for cDNA template generation and subsequent quantitative polymerase chain reaction (qPCR) facilitates differential gene transcriptional analysis With this background, we designed the current pilot study with the main aim to optimize the modified TRI reagent RNA isolation protocol to identify few important lncRNA expression in archived FFPE tissues of OSCC. Normal mucosa was used as control. In addition, we also aimed to compare commercially available column-based RNA isolation kit viz. NucleoSpin, Total RNA FFPE, Germany with modified TRI reagent RNA isolation protocol to check for the quality, and its usefulness in lncRNA expression analysis.•Take FFPE tissues sections of 6-8 µ thickness using soft tissue microtome.•Clean the blades with xylene after each sample to remove paraffin residues.•Transfer 4 to 5 cut paraffin sections into 1.5 mL Eppendorf tubes.Note: Slightly rolled up sections can be better handled and this could be obtained by decreasing the temperature of the paraffin blocks by placing ice on the cutting surface or putting paraffin blocks in a freezer before cutting.•Add 1 ml of xylene, vortex, and then incubate in the water bath at 56 ˚C for 10 min.•Centrifuge the sample at 14,000 rpm for 10 min at room temperature and discard the supernatant•Repeat the above procedure three times till the tissue sections are completely deparaffinized.•Centrifuge again at 14,000 rpm for 2 min at room temperature and discard the supernatant completely. Avoid the residues of xylene.•Add 1ml of absolute ethanol, vortex, and centrifuge at 14,000 rpm for 10 min at room temperature and discard the supernatant.•Again, centrifuge the tubes at 14,000 rpm for 5 min at room temperature to remove the residues of ethanol.•To wash the pellet with Phosphate Buffer-Saline (PBS), add 500 µl of PBS, vortex and then centrifuge the tubes at 14,000 rpm for 5 min at room temperature.•Dry the pellet for 5 minutes at 37 ˚C in thermomixer or air dry till completely evaporation of traces of PBS.Note: To remove the paraffin and unmask hidden or latent epitopes in preparation for downstream application. The procedure of Xylene and absolute alcohol can be repeated to ensure the complete removal of paraffin from tissue.•Add proteinase K digestion buffer containing390 µl lysis buffer and 10 µl of proteinase K (500 µg/ml) and vortex it.•Incubate the tube in the water bath at 56 ˚C for 60 min, after incubation immediately transfer the tube into the ice.•Add 1ml TRIZOL reagent, vortex for 2–5 s, and incubate for 1-2 min at room temperature•Add 0.2 ml of chloroform, vortex, and incubate at room temperature for 5 min•Centrifuge at 13,000 rpm for 15 min at 4 °C and collect the supernatant.Note: To digest proteins and remove contamination from nucleic acid preparations. Buffer was added in nucleic acid preparations for the inactivation of nucleases that could degrade RNA during isolation and purification applications. Carefully Removing the aqueous phase (supernatant) is a very crucial step and avoids contamination with the interphase and organic phase.•Add 0.6 ml of isopropanol and incubate at -20 °C overnight•Following day, centrifuge at 13000 rpm at 4 °C for 15 mins•Discard the supernatant and add 500 µl of 75% chilled ethanol•Centrifuge at 10,000 rpm for 15 min at 4 °C•Discard supernatant completely.Note: Isopropanol precipitation is based on the principle of salting out, in the presence of salts that renders nucleic acid preferentially to become insoluble and the precipitate is collected by centrifugation. The process also purifies the RNA leaving out alcohol soluble salts, organic solvents, and detergents. The addition of glycogen was not done in the present protocol as it may cause contamination. Maintaining temperature during incubation and optimal centrifugal force is important to avoid degradation of RNA.•Dry the pellet in thermomixer at 37 °C for 5min•The RNA was eluted in 30–50 µL of nuclease-free waterNote: Ethanol should completely evaporate or else it prevents the RNA solubilization into nuclease-free water.If the pellet dries out too much, the RNA crystallizes and is very difficult to resolubilize.Main modifications made in the present optimized protocol compared to TRI reagent baseline protocol [RNA isolation was also carried out from the study samples using a column-based kit  following the manufacturer's protocol.The concentration of RNA was estimated at 260:280 absorbance using a Bio-Spectrophotometer  in nanograms. Then the RNA was stored in aliquots of the required quantity at -20 °C in Eppendorf tubes sealed with parafilm.After the RNA quantification, the integrity of RNA was verified using 200 ng of RNA in 1% formaldehyde agarose gel electrophoresis in 1X MOPS buffer at 100 V for 30 min and stained with ethidium bromide (ETBR) to visualize the RNA bands. This helps to know the quality of the extracted RNA and also to determine the presence of any contaminants like DNA or Protein.st strand cDNA synthesis kit  as per the manufacturer's instructions. DNA sample was diluted with nuclease-free water (1:10) and stored at -20 °C until further use.All RNA extracts were prepared at 1µg/µl per sample and transcribed into cDNA using Prime script 1https://bioinfo.ut.ee/primer3-0.4.0/) and were checked for specificity using basic local alignment search tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi).The ct values of study cases were compared in TRI reagent modified protocol and kit method. Agarose gel electrophoresis was carried out to determine the molecular weight of the qRT-PCR end product.qRT-PCR (Rotor Gene Q MDx) was performed with TB green Mix  in a total volume of 20 µl. Primer sequence for lncRNAs and endogenous control gene (GAPDH) used to normalize the expression level were custom-designed . PrimersThe flow chart of the work carried out is represented in The total RNA concentrations using TRI reagent modified protocol was better than the kit method . The A26The cyclic threshold values  of study cases in TRI reagent modified protocol and kit method were found to be comparable and the mean difference between them was < 1 and 5. MThe present study results demonstrated that RNA quantity and quality was comparatively better with TRI reagent modified protocol than the kit method. The ct values after reverse-transcription and qRT-PCR were comparable and almost equal in both the methods for normal mucosa (control) and OSCC samples.Furthermore, the TRI reagent modified RNA isolation approach was cost-effective, costing roughly 1/3 of the cost of the kit method, and is particularly advantageous when there are a large number of samples. Additionally, while the column-based kit method took less time to isolate RNA, it required more steps, which could lead to human error.The TRI optimized protocol for RNA isolation effectively demonstrated lncRNA expression in oral tissues without demanding any additional training or extra time and also saves money. Hence, we recommend to use TRI optimized method for RNA isolation in lncRNA expression studies using FFPE tissues.0.2M Phosphate Buffer Saline (PBS), pH 7.0(a)Dissolve 27.8 g of monobasic sodium phosphate in 1 L deionized water – solution A(b)Dissolve 53.65 g of dibasic sodium phosphate in 1 L deionized water – solution BMix 19.5 ml of solution A, 30.5 ml of solution B, 1 g of sodium chloride and adjust the volume to 100 ml with deionized water.Proteinase K Digestion Buffer (FFPE Tissue Dissolver) Tris HCl – 20 mM (pH 8.0)2 – 1 mM CaCl SDS – 0.5 % Proteinase K – 500 µg/ml.2X RNA Loading Dye Formamide Ultrapure- 95% EDTA (pH 8.0) – 5 mM Bromophenol Blue – 0.025% Ethidium Bromide – 10 µg/ml.6X DNA Loading Dye Tris Base – 10 mM (pH 8.0) EDTA – 50 mM (pH 8.0) Glycerol – 30% Bromophenol Blue – 0.025%10X MOPS Buffer MOPS – 200 mM (pH 7.0) EDTA – 10 mM Sodium Acetate – 50 mM Note: Adjusted the pH with KOHFormaldehyde Agarose Gel Mix Agarose – 1 gm 10x MOPS buffer - 10 ml Distilled Water – 72 mlNote: Melt agarose in the oven, then let it to cool at 55 °C, then add 18 ml of Formaldehyde(37%).1X Gel MOPS running Buffer 10 X MOPS buffer – 100 ml 37% Formaldehyde – 20 ml Distilled water – 880 ml Total volume – 1000 ml Note: Make up a fresh buffer for each gelThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Dear Editor,Drosophila RNA-editing enzyme adenosine deaminase acting on RNA (ADAR) carrying E488Q mutation (HyperADARcd) outperforms other RNA editing enzymes and its RBP fusion allows proximity editing of dynamically associated RNAs in phase-separated condensates, e.g., during plant immune response in this study. This method will foster our understanding of the role of the interplay between RBPs and their RNA companions in cellular reprogramming through phase separation in any organism.RNA binding proteins (RBPs) often phase separate RNAs into condensates to reprogram transcription and translation. Here, we find that catalytic domain of the Arabidopsis genome1. However, many of them are annotated beyond nucleic acid-related functions , leaving the immediate question: how to prove them as authentic RBPs? Certainly, the most straightforward way is to identify their RNA targets.The interaction between RBPs and RNAs secures the correct flow of the genetic information and confers spatiotemporal reprogramming of cellular responses. This importance is highlighted by the presence of a large percentage of RBP candidate genes (> 6%) in the Arabidopsis RBPs have been reported, most likely due to their dependence on high-quality antibodies, low UV-cross linking efficiency, and particularly complex procedures4. This situation becomes even severer when RBPs are sensitive to the microenvironment and are prone to cluster or aggregate into condensates via phase separation either due to genetic mutation, developmental and environmental stimuli, or in vitro incompatible assay conditions6. Given the intrinsic multivalent interactions between RBP and RNA, RNA and RNA, and RBP and RBP, such a situation will be commonly encountered for most RBPs, for example, Arabidopsis AtUBP1c in this study.Although as the standard methods, ultraviolet crosslinking and immunoprecipitation-derived methods  underwent pronounced phase separation indicated by enhanced turbidity in the presence of the crowding agent that enhances intermolecular interaction, but failed to do so at a high salt concentration that disrupts such interaction  further demonstrated that these condensates became hardened and less dynamic with bare detection in the dilute phase upon activation of effector-triggered immunity  and without (−ETI) ETI induction while non-treatment leaf served as a control   and this increase was due to more bound mRNA targets  plus higher RNVs/RNA   were well expressed (RPKM ≥ 1) and maintained at a steady RNA level  in both conditions. These target genes were enriched for GO terms related to normal metabolism, catabolism, and transport  upon ETI induction (HyperADARcd-AtUBP1c/ 1) Fig. . Most ofort Fig. . This inort Fig. , S4d, e.ort Fig. .Arabidopsis7 and its Nicotiana plumbaginifolia homolog. RBPs usually selectively bind to RNA targets through recognition of a continuous binding sequence. To illustrate the spatial relationship between RNVs and RBP binding sites, we proposed a one-dimensional (1D) editing mode to describe RNVs by linear proximity editing and a three-dimensional (3D) editing mode that shows structurally spatial proximity editing , in crops. Compared to the non-transgenic parent and transgene-negative progeny, the transgene-positive plants showed obviously increased A>G RNV number (Fig. In an attempt to find alternates, we in parallel compared other four editing enzymes with HyperADARcd upon ETI induction, including adenosine deaminase TadA and cytidine deaminases rAPOBEC1, PmCDA1, and AtCDA1, but found that none of their AtUBP1c fusions could effectively edit the target genes Fig. . Therefober Fig. . It is wber Fig. .10. HyperADARcd is still under development and requires extensive improvements, such as using controllable systems (e.g., β-estradiol; Supplementary Fig. Global profiling of the RBP binding spectrum is an essential part of the human Encyclopedia of DNA Elements project, and great progress has been made in studies of both methodology and mechanism. Our whole toolkit and case studies here will assist to fill these gaps and pave the way for understanding the interplay between RBPs and RNA targets involved in cellular reprogramming of plant development and stress responsesSupplementary figures and textsSupplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5Supplementary Table S6Supplementary Table S7


The evolutionary origin of the genome remains elusive. Here, I hypothesize that its first iteration, the protogenome, was a multi-ribozyme RNA. It evolved, likely within liposomes (the protocells) forming in dry-wet cycling environments, through the random fusion of ribozymes by a ligase and was amplified by a polymerase. The protogenome thereby linked, in one molecule, the information required to seed the protometabolism  in newly forming protocells. If this combination of autocatalytic sets was evolutionarily advantageous, the protogenome would have amplified in a population of multiplying protocells. It likely was a quasispecies with redundant information, e.g., multiple copies of one ribozyme. As such, new functionalities could evolve, including a genetic code. Once one or more components of the protometabolism were templated by the protogenome , and/or addiction modules evolved, the protometabolism became dependent on the protogenome. Along with increasing fidelity of the RNA polymerase, the protogenome could grow, e.g., by incorporating additional ribozyme domains. Finally, the protogenome could have evolved into a DNA genome with increased stability and storage capacity. I will provide suggestions for experiments to test some aspects of this hypothesis, such as evaluating the ability of ribozyme RNA polymerases to generate random ligation products and testing the catalytic activity of linked ribozyme domains. The origin of life, i.e., the transition from abiotic molecules to replicating entities subject to Darwinian evolution, remains elusive. There are two main hypotheses that can be summarized as ‘RNA-first’ (the RNA world hypothesis), or, more broadly, ‘replicator-first’, and ‘metabolism-first’. The former (described in detail below) suggests that the ancestor of the genome arose spontaneously as a self-replicating oligo- or polymer of RNA, and that the metabolism emerged as a consequence of the evolving RNA. The latter hypothesis posits that a metabolism, i.e., a set of chemical reactions and their respective catalysts, within a primordial cell (the protocell) existed and evolved in complexity without any form of genome. Compelling arguments for both scenarios have been made in recent decades theoretically as well as by mathematical modeling and experimentation. Both hypotheses can arrive at some entity with the basic characteristics of life, i.e., the ability to multiply and to undergo Darwinian evolution. However, today’s cells contain two components, a replicating genome and a metabolism. A convincing scenario explaining both the evolution of the metabolism and the genome has not been formulated to the best of my knowledge.In the hypothesis presented here, I suggest that a protometabolism of catalytic RNAs (ribozymes) evolved first and gave rise to the first iteration of the genome, the protogenome. I will start by summarizing three key concepts underlying my hypothesis: the RNA world hypothesis , the conThe RNA world hypothesis suggests that there was a stage during Earth’s evolutionary history in which self-replicating RNA molecules proliferated . From th2O) were likely formed from precursors such as water (H2O), methane (CH4), ammonia (NH3), and hydrogen (H2). The subsequent chemical reactions that yielded more complex organic molecules such as nucleobases, amino acids and lipid precursors must have required regions that allowed for a concentration of the reactants, creating the ‘warm little pond’ envisioned by Charles Darwin. Such regions may have been hydrothermal fields undergoing wet–dry cycles as proposed by Damer and Deamer (described below) [4, NH3, H2 and water vapor, when energy was provided by electric discharge to simulate lightnings. Even though early Earth’s atmosphere, according to more recent data, might have looked different to the one simulated in the Miller–Urey experiment [Life on Earth likely evolved about four billion years ago . The redd below)  or hydrod below) . The abid below) . This experiment , these eperiment ,13,14,15periment  for a reperiment . An addiperiment .The next step in the RNA world scenario is the formation of random RNA oligo- or polymers. Oligomers can form in solution ,19 and oThe naturally occurring ribozymes in today’s world appear to have limited functionalities, mainly catalyzing peptide bond formation in ribosomes, as well as cleavage and ligation of phosphodiester bonds of RNAs ,26,27,28Given this rich catalytic repertoire of ribozymes, the RNA world scenario seems plausible and even likely to have existed in the form of ribozyme-catalyzed metabolisms . HoweverEscherichia coli [The concept of autocatalytic sets  provides an explanation on how a metabolism can emerge and evolve without the need of a dedicated information storage device such as the genome. The idea was pioneered by Stuart Kauffman ,3. In anhia coli , providiMathematical and computational studies on autocatalytic sets, formally described as reflexively autocatalytic and food-generated (RAF) sets (with food being a set of building blocks such as organic molecules on early Earth) have shown that autocatalytic sets have a high probability of emerging even with moderately active catalysts . MoreoveIn summary, autocatalytic sets have likely spontaneously emerged from prebiotic, organic precursors present on early Earth, were self-sufficient, replicating, and capable of evolution. RNA oligo- or polymers, whose precursors were likely present on early Earth , are a lToday’s cellular organisms are physically contained by a membrane mainly composed of a bilayer of phospholipids (bacteria and eukaryotes) or ether lipids (archaea). It is thus likely that the precursors of cells, the protocells, were also surrounded by a lipid bilayer, i.e., a liposomal container . ArtificAn answer to this question has been provided by Damer and Deamer . In areaNow we have all the components that are required to form a protocell with an RNA-based protometabolism, as laid out above, consisting of RNA oligo- or polymers that form an autocatalytic set.The abiotic synthesis of precursors for RNA and lipid molecules in chemically plausible conditions on early Earth has been experimentally verified see ;Autocatalytic sets have a high probability of forming spontaneously, as per the mathematical RAF theory, and RNA (ribozyme)-based autocatalytic sets have been experimentally generated see ;Liposomes can form spontaneously in dry–wet cycles and can grow, divide, and evolve see . Such drAltogether, the dry–wet cycles appear to be a plausible scenario for the first protocells to evolve. Yet, to resemble present-day cells, these protocells require a genome that contains or encodes the information for the protometabolism.First, let me point out why a ribozyme-based protometabolism is more likely to evolve increasing complexity than one composed solely of peptides. RNAs are more likely to evolve into more complex molecules  because they can be complementary to each other and are less chemically diverse than peptides. This allows for replication of RNA species  and sequence-specific cleaving and joining. Thus, it appears more plausible that, initially, ribozymes evolved, while peptide-based catalysts likely remained relatively simple, restricted to short oligomers. Moreover, before the evolution of a genetic code, the sequence-specific assembly of long peptides would have been limited in length due to the chemical diversity of amino acids, as their joining would have required highly specific catalysts . Of note, these additional amino acids were of limited relevance for the evolution of protein biosynthesis whose code likely became ‘frozen’ in its present form, as originally proposed by Francis Crick ,69—I wilOf note, ribozymes can be relatively small. Thus, simple representatives have likely emerged from the random oligomerization of ribonucleotides in dry–wet cycles. For example, the smallest ribozyme identified to date consists of only five nucleotides with a three-nucleotide active center, able to catalyze aminoacylation of another RNA , and minAn early protocell might have contained autocatalytic sets composed of such small ribozymes. As the autocatalytic sets evolved, more complex ribozymes might have emerged. A ribozyme-based protometabolism likely contained the ribozymes required for RNA assembly and copying, i.e., RNA ligases and polymerases. The polymerase might have evolved from a ligase, similar to in vitro evolution experiments that have successfully generated polymerases from ligases .B1’, since RzB1 contains start sites for polymerization . The reThere are three prerequisites for the above scenario to be plausible. First, random ligation products of ribozymes must have a probability to occur. Second, the joined ribozymes must retain their catalytic activity when part of a longer RNA molecule. Third, there must have been an RNA polymerase with sufficient fidelity to copy such a long RNA without running into an error catastrophe.Thermococcus kodakarensis, respectively (both are protein enzymes) [Regarding the first point, template-independent ligation of single-stranded DNA and RNA molecules has been demonstrated to be catalyzed by T4 DNA ligase and an RNA ligase from the archaeon enzymes) ,74. RNA enzymes) , yet, toenzymes) , it is cAvsunviroidae viroids [For the second prerequisite there is evidence from naturally existing RNAs as well as from experiments that ribozymes can remain catalytically active when they are part of longer RNA molecules. For example, active hammerhead ribozyme domains are embedded in the larger circular ssRNA genomes of  viroids , virus s viroids , and hep viroids , as well viroids , Penelop viroids , and ret viroids . Moreove viroids ,83,84, R viroids ,86, up t viroids , and up  viroids . TherefoWith respect to the third point, there are indeed length restrictions to the initial protogenome that are imposed by the fidelity of the RNA polymerase. The error threshold defines the maximum length of an RNA molecule beyond which deleterious mutations will accumulate, as proposed by Manfred Eigen . For ribIf a protocell ‘escaped’ the dry–wet cycle scenario and became a free-living entity in the primordial ocean, or in areas such as tidal pools with higher concentrations of the organic molecule food sources, the evolutionary dynamics would have changed. The ability for liposome division must have had already evolved, e.g., via a phospholipid synthesis pathway (see above). The protogenome would have been an advantage for transmitting the metabolic information to the daughter cell during protocell division.Yet, for the protogenome to become stably inherited during protocell division, the protometabolism had to become dependent on the protogenome. One way might have been that a genetic code evolved in the protogenome that enabled the generation of a peptide catalyst which provided an evolutionary advantage to the protocell. The protogenome, as it would have existed as a quasispecies due to the error-prone replication by an RNA polymerase enzyme, and likely contained redundant information  could have provided a ‘playing field’ for such a genetic novelty to evolve. Of note, small peptides as short as four amino acids have been shown to be efficient catalysts ,92,93. SAnother dependency mechanism might have been the evolution of addiction modules or toxin–antitoxin systems, in which the antitoxin RNA or templated peptide might have been present in the protogenome and neutralized a toxin in the protometabolism. There is evidence that a certain class of such toxins, Small Alarmone Synthetases, was already present in the last universal common ancestor (LUCA) as they are found in both bacteria and archaea ,102.In order to resemble present-day cellular genomes, the protogenome must have increased in size. As the RNAs of the protometabolism increased in complexity and size, entities resembling present-day transposable or virus-like elements might have emerged, acting cooperatively within the protocell, as proposed by Villarreal and Witzany . They coIt has been proposed previously that complex ribozymes such as the RNA polymerase might have emerged by the random ligation of smaller hairpin-loop structures, which was supported by in silico studies . A similThe herein-described hypothesis is supported by computer simulation studies which have confirmed that compartments such as protocells can be favorable for linkage of smaller RNAs to longer strands to occur evolutionarily ,121 and Some of the aspects of this hypothesis could be tested experimentally. For example, are any of the in vitro generated RNA ligase ribozymes  able to


The coronavirus disease 2019 (COVID-19) pandemic has greatly disrupted the normal treatment of patients with liver cancer and increased their risk of death. The weight of therapeutic safety was significantly amplified for decision-making to minimize the risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Herein, the safety and effectiveness of carbon ion radiotherapy (CIRT) for unresectable liver cancer (ULC) were evaluated, and Chinese experiences were shared to solve the predicament of ULC treatment caused by SARS-CoV-2. Worldwide studies were collected to evaluate CIRT for ULC as the world has become a community due to the COVID-19 pandemic. We not only searched five international databases including the Cochrane Library, Web of Science, PubMed, Embase, and Scopus but also performed supplementary retrieval with other sources. Chinese experiences of fighting against COVID-19 were introduced based on the advancements of CIRT in China and a prospective clinical trial of CIRT for treating ULC. A total of 19 studies involving 813 patients with ULC were included in the systematic review. The qualitative synthetic evaluation showed that compared with transarterial chemoembolization (TACE), CIRT could achieve superior overall survival, local control, and relative hepatic protection. The systematic results indicated that non-invasive CIRT could significantly minimize harms to patients with ULC and concurrently obtain superior anti-cancer effectiveness. According to the Chinese experience, CIRT allows telemedicine within the hospital (TMIH) to keep a sufficient person-to-person physical distance in the whole process of treatment for ULC, which is significant for cutting off the transmission route of SARS-CoV-2. Additionally, CIRT could maximize the utilization rate of hospitalization and outpatient care (UHO). Collectively, CIRT for ULC patients not only allows TMIH and the maximized UHO but also has the compatible advantages of safety and effectiveness. Therefore, CIRT should be identified as the optimal strategy for treating appropriate ULC when we need to minimize the risk of SARS-CoV-2 infection and to improve the capacity of medical service in the context of the unprecedented COVID-19 crisis. The coronavirus disease 2019 (COVID-19) pandemic, an infectious disease caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) . COVID-1The weight of therapeutic safety is enlarged due to SARS-CoV-2 –12. TherA pre-retrieval procedure was implemented to ensure that the best results of literature retrieval could be obtained, which started on March 11, 2020. A preliminary and rapid systematic review was conducted before this study to ascertain how to design this study scientifically and accurately.Studies were included if they matched the following criteria based on the pilot study of a systematic review. (1) Participants: patients were diagnosed with liver cancer by histopathology and imageological examination, ineligible or infeasible for resection; regardless of primary liver cancer or metastatic liver cancer. (2) Intervention and comparison: there were few studies with a control group for the assessment of CIRT in treating patients with liver cancer on the basis of pre-retrieval. Therefore, a study should be included if CIRT was evaluated with effectiveness and/or adverse effects in treating liver cancer, whether there was a comparison group or not. (3) Outcomes: the outcomes of evaluation included overall survival (OS), local control, short-term effects, adverse effects, and complications. (4) The study type was unrestricted due to the development stage of CIRT. All study types of clinical research were included to evaluate CIRT for liver cancer on the basis of the pre-retrieval and preliminary systematic review. Publications were excluded if they had inappropriate research designs including cellular or animal experiments, letters, editorials, commentaries, protocols, reviews, systematic reviews, or meta-analyses.The pre-retrieval was performed on March 11, 2020 and the comprehensive retrieval was started on April 15, 2020, following the pilot systematic review. The retrieval was updated every month during the research process in order to acquire the latest data of reports. The final retrieval time was May 31, 2021.We searched five international databases including the Cochrane Library, Web of Science, PubMed, Embase, and Scopus from the database inception to May 31, 2021. We also searched other supplementary resources, such as Google Scholar, Medical Matrix, reference lists of relevant reviews and included papers, COVID-19 Open Research Dataset Challenge (CORD-19), COVID-19 Research Database (WHO), and the WHO International Clinical Trials Registry Platform. The search terms contained the target disease group and intervention groups, such as liver neoplasms, CIRT, SARS-CoV-2, and COVID-19. No restrictions were set for the study language, publication date, and publication status. All relevant clinical trials were collected to evaluate CIRT for patients with liver cancer.All records were imported into the EndNote software of the X9 version  for further management and screening. Studies were selected according to the inclusion and exclusion criteria. The articles were reviewed by the researchers independently in two stages for the study screening: the first stage was an evaluation of the titles and abstracts, followed by a full-text review as the second stage. The researchers discussed the discrepancies and re-evaluated the articles until a consensus was reached.Data were extracted from each included article using standardized forms. The subset of interventions that satisfied the inclusion criteria was kept in the analysis after having discarded the groups that did not satisfy the inclusion criteria when the trials have multiple groups. The list of the collected data included: (1) the basic characteristics of the included studies; (2) the outcomes from the research results. All data were extracted from the text, tables, or figures of the included papers. CIRT was assessed using the method of qualitative synthetic analyses due to its development stage. The data of CIRT were synthesized in both tabular and narrative formats according to the qualitative analysis method of the systematic review.via the included studies combined with the experience of Chinese citizens. No ethical approval or patient consent was required for the systematic review as the data originated from previously published studies online. The clinical trial of the first Chinese carbon ion therapy system (CITS) in Wuwei, China was conducted in accordance with the Good Clinical Practice Guidelines and the Declaration of Helsinki, and this trial was approved by the ethics committee of the research institute. All patients provided written informed consent.The experiences of fighting against SARS-CoV-2 from different countries are necessary due to the unprecedented crisis worldwide. We explored the optimal strategy for treating ULC in the context of the COVID-19 crisis From our systematized search, a total of 1,065 records were imported into the EndNote software for further identification, including 1,049 records identified through traditional database searching and 16 records identified through additional sources. A total of 575 reduplicative records were removed because of the repeats included by the different databases. After the elimination of duplicates, 490 records were screened for eligibility by their titles and abstracts at the first stage and by full-text screening at the second stage. A total of 19 eligible studies –54 were All of the included studies –54 were A total of 19 studies –54 were p < 0.05), 3-year local control rate , and 3-year progression-free survival rate , respectively. Compared with TACE, CIRT was associated with a significant reduction regarding the number of patients whose liver function progressed to a worse Child-Pugh class within 3 months from the initiation of treatment (p < 0.01). There were two studies , Chinese Academy of Sciences in 1993, successfully completed the treatment of 46 cancerous cases as a clinical trial and was officially registered as a medical device of Class 3 in China on September 29, 2019. The CITS in Wuwei is the first Chinese CIRT equipment with the serial number 20193050713 and the type specification HIMM-01-GS-WW-1, and several CITSs in other areas of China are now under construction. A total of 47 cancer cases were enrolled into the clinical trial for the medical device registration of CITS in Wuwei. One patient withdrew from the trial after enrolment, and 46 subjects completed the trial finally. There were a total of seven patients with ULC in the trial, including six cases with primary hepatocellular carcinoma and one case with hepatic metastasis from rectal cancer. All of these patients were advanced and intractable cancer cases. No severe radiation morbidities and treatment-related deaths of CIRT were observed during the treatment and follow-up. The data acquired from the clinical trial in Wuwei, China demonstrated that the Chinese CITS encouraged safety and anti-cancer effectiveness in treating liver cancer .According to the experience of Chinese citizens, compared with other locoregional treatment (LRT) , CIRT allows TMIH with controllable risk of SARS-CoV-2 infection in the whole process of treatment for ULC. Compared with photon (or proton) radiotherapy modalities, CIRT could achieve the optimal utilization rate of hospitalization and outpatient care (UHO). Therefore, non-invasive CIRT is identified as the optimal treatment strategy for appropriate patients with ULC concerning the need to cut off the transmission route of SARS-CoV-2 and to improve the capacity of healthcare service in the context of the unprecedented COVID-19 crisis. Based on Chinese foundations, ultramodern projects of CIRT have been planning and preparing to bring its superiorities into full play. A schematic diagram for the development planning of the CIRT center is exhibited in Globally, COVID-19 has caused unprecedented social turmoil, triggering a comprehensive transformation of global healthcare systems , 55–59. Aitken et al.  suggesteThe qualitative synthetic analyses results of CIRT for liver cancer demonstrated encouragingly high rates of OS and local control and low rates of hepatotoxicity. One of the most limiting factors of the use of radiotherapy for liver cancer is the significantly poor radiation tolerance of the normal liver tissues, especially when the liver function is impaired by some chronic liver disease , 51. CIRThe statistics showed that liver cancer was the third leading cause of cancer death worldwide in 2020, with about 906,000 new cases and 830,000 deaths annually . In addiThe IMP of China has started to develop CITS independently since 1993, and now, we have many original innovations not only in the equipment and clinical technology but also in the supporting theoretical basis, such as the relative biological effectiveness (RBE) modeling for CIRT . Why wasIn the summer of 2021, the SARS-CoV-2 Delta Variant surge has caused a new wave of the epidemic peak in America and other countries , 72, 73.The present study has a few limitations. With the exception of a PSM study  for CIRTIn order to optimize the treatment strategy of patients with ULC due to the COVID-19 crisis worldwide, multi-angle methods were implemented to evaluate the non-invasive CIRT for treating ULC, from which we concluded that CIRT could obtain favorable anti-cancer effectiveness and concurrently, minimize toxicities to patients for preserving patients in a relatively good systemic and immune condition. In addition, CIRT has the ability of TMIH with the controllable risk of SARS-CoV-2 infection exposure, as well as the optimal utilization rate of both hospitalization and outpatient care concurrently. Therefore, we have definitively judged CIRT as the optimal treatment strategy for appropriate patients with ULC when we need to minimize the risk of SARS-CoV-2 infection and improve the capacity of medical service in the COVID-19 crisis. We believe that CIRT will be greatly helpful to reduce the risk of SARS-CoV-2 infection and cancer malignant death concurrently during the COVID-19 pandemic. We also firmly believe that the trajectory of this unprecedented pandemic caused by SARS-CoV-2 will become better and better worldwide with international cooperation and mutual assistance, innovation, and sharing.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Ethics Committee of Gansu Provincial Cancer Hospital and the Ethics Committee of Gansu Wuwei Tumor Hospital. The patients/participants provided their written informed consent to participate in this study.ZL and QL developed the study conception and design. ZL, QL, and XW supervised the whole study process and coordinated all the work. ZL, QL, XW, SL, and WC collected the data and prepared the figures and tables. ZL, QL, XJ, and XGL contributed to selecting the analytical tools and methods. ZL, QL, ZD, XXL, XZ, PL, HZ, QZ, HL, and RL analyzed, interpreted, reviewed, and cross-checked the data. ZL and QL wrote and revised the manuscript. All authors critically reviewed and approved the final manuscript.This work was jointly supported by the China Postdoctoral Science Foundation (Grant No. 2019M663860), the National Natural Science Foundation of China (Grant No. 11875299), the Key Deployment Project of the Chinese Academy of Sciences (Grant No. KFZD-SW-222), and the West Light Foundation of Chinese Academy of Sciences (Grant No. 29Y86205).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.


Our study provides insight into the assembly pathway when the still-connected nascent 40S and 60S subunits are beginning to separate.Synthesis of ribosomes begins in the nucleolus with formation of the 90S pre-ribosome, during which the pre-40S and pre-60S pathways diverge by pre-rRNA cleavage. However, it remains unclear how, after this uncoupling, the earliest pre-60S subunit continues to develop. Here, we reveal a large-subunit intermediate at the beginning of its construction when still linked to the 90S, the precursor to the 40S subunit. This primordial pre-60S is characterized by the SPOUT domain methyltransferase Upa1-Upa2, large α-solenoid scaffolds, Mak5, one of several RNA helicases, and two small nucleolar RNA (snoRNAs), C/D box snR190 and H/ACA box snR37. The emerging pre-60S does not efficiently disconnect from the 90S pre-ribosome in a dominant   •Affinity purification of primordial pre-60S particle via split-tag method•Primordial pre-60S contains specific snoRNAs and assembly factors•mak5 helicase mutantArrest of 90S-pre-60S pre-ribosome in •90S-pre-60S pre-ribosome exhibits a bipartite structure Ismail et al. report the isolation of a primordial pre-60S particle, composed of a specific set of assembly factors and snoRNAs. This assembly intermediate is visualized by cryo-EM while emerging from the 90S pre-ribosome, revealing a 90S-pre-60S bipartite structure. Saccharomyces cerevisiae, in which ribosomal RNA (rRNA) is first transcribed in the nucleolus from ribosomal DNA repeats by RNA polymerase I to produce a polycistronic transcript. This precursor, called the 35S pre-rRNA (47S pre-rRNA in human), contains both external (5′ ETS and 3′ ETS) and internal (ITS1 and ITS2) transcribed spacer sequences, which flank the mature 18S rRNA (part of the 40S), and the 5.8S and 25S rRNAs (parts of the 60S subunit). 5S rRNA is transcribed separately by RNA polymerase III and, together with Rpl5 (uL18) and Rpl11 (uL5), this nascent 5S ribonucleoprotein (RNP) is incorporated into the early pre-60S subunit in a subsequent step, the details of which remain unclear. Initially, ribosome assembly proceeds through a number of endonucleolytic RNA cleavages, followed by RNA modification and folding reactions of the nascent pre-rRNA and recruitment of ribosomal proteins. All these steps are facilitated by a myriad of ribosome assembly factors, which sequentially associate with the evolving pre-ribosomal particles and drive the whole process from the nucleolus into the cytoplasm , a first set of early ribosome assembly factors associate with the emerging nascent pre-rRNA to gradually form the 90S pre-ribosome (SSU processome), which is the first stable pre-ribosomal assembly intermediate that can be biochemically isolated  and stru emerges , which c emerges .2, Rrp5 is thought to detach from the 90S pre-ribosome but remain bound to the nascent pre-60S along with Noc1 and Noc2, which together form the Rrp5-Noc1-Noc2 module , which revealed the initial folding of rRNA domains and the associated assembly factors together with the ribosomal proteins -guided RNP complexes, of which there are two classes, the C/D box and H/ACA box snoRNAs, which form specific snoRNPs characterized by their core factors (O-ribose methylation and contain the methyltransferase Nop1 (fibrillarin in human) and the core factors Nop58, Nop56, and Snu13. H/ACA snoRNPs are responsible for pseudouridylation, for which Cbf5 (dyskerin in human) acts as a pseudouridine synthase along with the core factors Nhp2, Gar1, and Nop10  that encodes a core factor of the C/D box snoRNAs  to visualize the long-sought particle formed between the nascent small (90S) and large subunit (pre-60S), giving insight into how the pre-40S and pre-60S assembly pathways begin to diverge.To shine further light on the very early 60S assembly pathway and find out whether specific snoRNPs play a role in this process, we performed biochemical and genetic analyses, with a first focus on a specific mutant allele of  snoRNAs . Based oNOP1 , which encodes an essential subunit of the U3 snoRNP that is also a core factor of the many other C/D box snoRNAs  tag on the Nsa3 did not exaggerate the growth defect  carrying the mutated Nop1 Ala245>Val. Hence, we searched for snoRNP-containing pre-60S particles that precede the classical and structurally well-characterized nucleolar pre-60S particles, which are all devoid of snoRNPs , which specifically no longer revealed these two snoRNA bands  and 90S (Noc4-Dhr1) particles A. Based NA bands . To demoNA bands C.Figure To identify additional snoRNAs of lower abundance in the early pre-60S particles, we performed small RNA Illumina sequencing of the total RNA extracted from the different preparations, that is, from the Urb2-Noc1 and Urb2-Nop58 eluates, from a Nop58 affinity purification to detect the entire pool of C/D box snoRNAs, from Kre33 as bait for 90S, and from Arx1 as bait for nuclear and cytoplasmic pre-60S particles . Analysinop1 mutant allele, nop1-5  were markedly diminished in the Urb2-Noc1 particles . NotablyConsidering all these findings, the nascent 60S subunit that precedes the previously described nucleolar pre-60S particle is characterized by, among other features, the presence of two large α-solenoid scaffold complexes (Urb1 and Rrp5 modules), a cluster of little-studied RNA helicases, and two prominent snoRNPs (snR190 and snR37).O-ribose methylation in the ribosomal RNA , might contribute to the synthetically enhanced phenotype observed between snR190 and snR37.To investigate the roles of snR190 and snR37 that are so prominent in the earliest pre-60S particles, we conducted genetic and biochemical studies. It is known that snR190 does not guide 2′-omal RNA , and henomal RNA ; howeveromal RNA A  growth defect at all temperatures tested , reminiscent of what has been found for Nsa3 particles isolated from the nop1-4 strain. However, a specific set of other pre-60S factors, including Dbp10, Spb1, and Nug1, were decreased if Nsa3 was purified from the snoR190Δ rsa3Δ double mutant  and other proteins from this family, including Emg1, that function as homodimers .Since Upa1 and Upa2 were found to be specific factors of the Urb2-Noc1 particle with a link to snR37, we further analyzed these uncharacterized paralogs. Both belong to a group of methyltransferases that carry the SPOUT domain, with representatives in all three domains of life. SPOUT-class proteins can bind  protein . The yea protein . Hence,  protein . Apparen protein A or semi protein . Accordipre-rRNA . When weTo gain a first insight into their structure, we analyzed the Upa1-enriched pre-60S particles by negative-stain EM and 2D classification. According to the results, Upa1 particles are approximately 32 nm long and 23 nm wide, and are typically U shaped in appearance C. In a nmak5 D333A under the control of the GAL promoter in the yeast strains expressing different baits for isolation of the various pre-60S particles , medium-sized particles (∼42 nm), and very large bipartite particles ; the latter far exceed the size of either pre-60S or 90S particles C. Accord pre-60S C. Thus, 1. EMDB: 11359; To gain deeper insight into the structure of these different particles, we performed cryo-EM of the Urb2-Noc1-Mak5-333 preparation, from which we obtained cryo-EM maps for the major intermediates, albeit only at lower resolution, but sufficient for a prudent assignment . The smamak5 helicase mutant, we could arrest the early pre-60S maturation pathway at a stage at which the newly forming large subunit is still connected to the enormous precursor of the 40S, the 90S pre-ribosome. This welcome side effect allowed us to reveal for the first time the overall shape of the largest assembly intermediate in the eukaryotic ribosome biogenesis pathway: the bipartite 90S-pre-60S particle.The results of this study give us a molecular insight into the early phase of the pathway eukaryotes use to assemble the large ribosomal subunit. This revealed a primordial pre-60S particle that is dominated by two scaffold modules, Rrp5-Noc1-Noc2 and Urb1-Urb2-Nop10-Dbp6-Rsa3, and further distinguished by two prominent snoRNPs, snR190 and snR37; several poorly characterized RNA helicases, including Mak5; and the SPOUT-domain-containing methyltransferase pair Upa1-Upa2. The huge and α-solenoid-dominated Urb1-and Rrp5-containing modules  and the The two principal snoRNAs in the primordial pre-60S, snR190 and snR37, which are C/D box and H/ACA snoRNAs, respectively, are not shown to be essential when individually deleted, but the snR190 snR37 double disruption induced a synergistic growth defect at higher temperatures. Notably, the uncharacterized protein YCR016W, which exhibits a unique WKF domain , like its human ortholog C7orf50, which has been shown to bind to RNA , was synBecause snR190 does not guide a known base modification, it might instead play a structural role in the primordial pre-60S, as U3 snoRNP does in the 90S pre-ribosome. Specifically, snR190 provides a linkage to the Urb1 module, with direct contact to Urb1, demonstrable by CRAC analysis  and, as nop1-4 allele, which is expected to impair all C/D box snoRNPs, generated a strong defect in the early pre-60S assembly pathway, suggesting that the C/D box snoRNAs work synergistically together in the primordial pre-60S. However, deletion of snR37 clearly reduced H/ACA core factors , which is consistent with the finding that H/ACA snoRNAs are less prominent than C/D box snoRNAs in the primordial pre-60S.The pre-60S particles that were isolated via the Urb2-dependent split-tag approaches were significantly enriched in several snoRNAs and a cluster of RNA helicases. Three of these helicases, Dbp7, Dbp9, and Mak5, were reduced if the Urb2-Noc1 particle was isolated from the snR190-disruption strain, which might point to direct functional link in the primordial pre-60S. Indeed, recent studies suggested that Dbp7 is involved in the release of snR190 from pre-60S particles . By cont4RG1), or the SPOUT-domain-containing methyltransferase Emg1, which is present as a homodimer on 90S particles  might stabilize these highly dynamic 90S-pre-60S assembly intermediates, consistent with earlier findings that depletion of the Drs1 helicase caused an enrichment of 90S factors on Nop7- or Nog2-isolated pre-60S particles .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Ed Hurt  followed by U14 and TRP marker (snR190Δ-U14-native terminator-TRP). The W303 yeast strain was then transformed with this construct to replace genomic snR190-U14 by homologous recombination. The transformed strain was confirmed by colony PCR and sequencing.To construct an anscript . We used600 nm value of 2.0–3.0. Cells were lysed in purification buffer  glycerol, 0.1% (v/v) IGEPAL CA-630, and 1 mM dithiothreitol) supplemented with protease inhibitor cocktail (SIGMAFAST) and RiboLock RNase inhibitor. Lysis was performed mechanically either in a bead beater (Fritsch) using glass beads or in a cryogenic mill (Retsch MM400), and lysates were pre-cleared by centrifugation at 4°C for 10 min at 5000 rpm then for 20 min at 17,000 rpm. For the first affinity step, the supernatants were incubated with pre-equilibrated IgG Sepharose 6 Fast Flow affinity resin  for at least 2.5 h at 4°C, which was then washed with purification buffer. The resin was collected and bound proteins were eluted from it by cleaving with TEV protease at 16°C for 1.5 h. For the second affinity step, TEV eluates were incubated with pre-equilibrated Anti-Flag M2 Affinity Gel (Sigma–Aldrich) for 1.5 h at 4°C, which was then washed with purification buffer. Then, bound proteins were eluted from the anti-Flag beads with Flag peptide . Flag eluates were analyzed by on 4–12% polyacrylamide gels  with colloidal Coomassie staining .Yeast strains expressing single-bait proteins tagged with Flag-TEV-protA (FTpA) or strains expressing split tags—one protein with calmodulin-TEV-protA (TAP) and another with Flag—were cultured at 30°C and harvested at an ODUrb2–TAP Noc1-Flag YCplac111-GAL-mak5 D333A strain, the cells were cultured at 30°C in SDC-Leu (glucose) medium to early logarithmic phase then shifted to YPG  medium and grown for 8 h to induce mak5 D333A. Then, affinity purification of Urb2-TAP Noc1-Flag particles was performed as described above with final elution from the anti-flag beads using Flag peptide  in the following buffer 100 mM NaCl, 5 mM MgCl2, 50 mM Tris-HCl, pH 7.5 and 5% (v/v) glycerol for negative-stain EM analysis. For cryo-EM analysis, the final elution was done using Flag peptide  in the following buffer 100 mM NaCl, 5 mM MgCl2, 50 mM Tris-HCl, pH 7.5 and 0.05% Nikkol.For the + nylon membrane  and UV-crosslinked. The following oligos (5′-labeled with 32P) were used for detecting snR190, snR37, and U3, respectively: CGTCATGGTCGAATCGG, AAGCTCCTCATCACTCACAC, and GGTTATGGGACTCATCA.RNA was extracted from the Flag eluates of affinity-purified pre-ribosomes using phenol–chloroform extraction followed by precipitation with ethanol. For detection of snoRNAs, extracted RNA samples were resolved on 8% polyacrylamide/8 M urea gels. For RNA staining, the gels were incubated for 30 min with SYBR Green II RNA gel stain (Sigma–Aldrich) using a 1:5000 dilution in 1× TBE. For northern blot analysis, the RNA was transferred to a Hybond-N2, 50 mM Tris-HCl, pH 7.5) and centrifuged at 27,000 rpm for 16 h at 4°C. After centrifugation, fractions were collected and an equal volume from each was used for RNA extraction and northern blotting (in case of Urb2-Nop58). The remainder of each fraction was precipitated with 10% TCA, resuspended in SDS sample buffer, and analyzed on 4–12% polyacrylamide gels  with colloidal Coomassie staining.The Flag eluates of affinity purified pre-ribosomes were loaded on a 10–40% (w/v) linear sucrose gradient , where the proteins were identified by 1D nLC–ESI-MS/MS. Raw data was analyzed using MaxQuant software .2 and 0.05% Nikkol. Three microliters of the sample were pipetted onto Quantifoil R 2/1 holey carbon grids, which were glow-discharged for 45 s using the PELCO easiGlow system. The grids were blotted for 5 s using a blot force of 10 at 100% humidity using a Vitrobot Mark IV (Thermo Fisher Scientific) operated at 4°C, and immediately plunge-frozen in liquid ethane cooled with liquid nitrogen. Grids were mounted onto auto-grids and imaged using a Thermo Scientific Glacios cryo-transmission electron microscope equipped with a Falcon IIIEC detector and operated at an acceleration voltage of 200 kV. One thousand and one movies were recorded in nanoprobe mode with parallel illumination at a nominal magnification of 45,000 and a spot size of 4 in linear mode. The calibrated object pixel size was 3.17 Å. Micrographs were acquired using dose fractionation to record 119 frames per exposure with a dose rate of 1 e per Å2 per frame.Negative-stain EM of isolated pre-ribosomal particles was performed essentially as described by . For cryParticles were picked with CrYOLO  and imagFor particle picking, particles from 14 micrographs were manually picked with the CrYOLO box manager and used to refine the general model. A total number of 310,374 particles were picked, extracted using a box size of 180 × 180 pixels, and subjected to 2D classification in CryoSPARC. Good 2D classes were selected and the corresponding 69,486 particles were used for subsequent ab initio reconstruction with three classes. The resulting volumes were used as references for heterogeneous refinement with all picked particles. Homogeneous refinement was performed for the resulting pre-60S and 90S classes following re-extraction (box size 200 × 200) and 3D classification in RELION 3.1.2. Three-dimensional classification was performed on the pre-60S class, with image alignment and an angular sampling interval of 7.5°; no alignment was performed for the 90S class. The good pre-60S class and the 90S class were refined and local filtered with CryoSPARC. Furthermore, the 90S particles were re-extracted with a box size of 450 × 450 pixels and 3D refinement was performed with a mask of 1200 Å, which led to the reconstituted 90S connected to a large and flexible extra density. The particles were iteratively re-extracted, centered, and refined, allowing large shifts, and the final homogeneous refinement and local filtering was performed with CryoSPARC. ChimeraX was used to display cryo-EM structures and models .RNA was extracted from the selected particles  using a mirVana miRNA isolation kit, following the manufacturer’s protocol for enriching for small RNAs. After extraction, RNA was quantified using a Qubit RNA HS assay kit and used as input for library preparation with the NEBNext Small RNA Library Prep Set for Illumina, following the manufacturer’s instructions. Libraries were loaded on a MiniSeq and sequenced in single-end mode with reads of 150 nts length. To generate the final Log2 fold-change values, raw reads were quality checked and processed using in-house scripts. Raw data is available at ENA .See


To retrospectively analyze the incidence of hippocampal metastasis and the associated high-risk factors in patients with brain metastases and evaluate the safety of hippocampal avoidance whole-brain radiation therapy (HA-WBRT).We retrospectively analyzed the data of patients with brain metastases diagnosed by contrast-enhanced cranial Magnetic resonance imaging (MRI) at the First Hospital of Lanzhou University from 2017 to 2020. The boundaries of the hippocampus, hippocampus + 5 mm area, hippocampus + 10 mm area, and hippocampus + 20 mm area were delineated, and the distances from the brain metastases to the hippocampus were measured. Univariate and multivariate logistic regressions were adopted to analyze the high-risk factors of hippocampal metastasis.A total of 3,375 brain metastases in 411 patients were included in the analysis. The metastasis rates in the hippocampus and surrounding areas of the entire group were as follows: 7.3% (30/411) in the hippocampus, 16.5% (68/411) in the hippocampus + 5 mm area, 23.8% (98/411) in the hippocampus + 10 mm area, and 36.5% (150/411) in the hippocampus + 20 mm area. Univariate logistic regression showed that the pathological type, the number of metastases, the maximum diameter of metastases, and the volume of brain metastases were all correlated with hippocampal metastasis. Multivariate logistic regression showed that the pathological type, the number of metastases, and the total volume of metastases were correlated with hippocampal metastasis.The pathological type, the number of metastases, and the total volume of metastases are the high-risk factors associated with hippocampal metastasis. Small cell lung cancer (SCLC) has a significantly higher rate of hippocampal metastasis than other tumor types. The greater the number and total volume of metastases, the more likely the hippocampal metastasis. For patients with SCLC or a greater number and total volume of brain metastases, the implementation of HA-WBRT may bring a higher risk of tumor recurrence. As a regular site of malignant tumor metastasis, the brain metastasis rate in tumor patients is over 20% . Lung caThis study included 411 patients with brain metastases admitted to our hospital from 2017 to 2020, following the inclusion and exclusion criteria below. Patients pathologically diagnosed as malignant tumors and diagnosed as brain metastasis by contrast-enhanced MRI were included. Patients a) with two or more malignant tumors, b) incomplete medical records, or c) other craniocerebral diseases were excluded.The patients' head MRI data were acquired through the hospital's PACS system. The number and distribution of brain metastases were observed from the T1-weighted contrast-enhanced images. The brain is divided into the frontal lobe, parietal lobe, temporal lobe, occipital lobe, cerebellum, insular lobe, and brainstem according to the anatomical boundaries. All brain metastases were delineated after importing the contrast-enhanced MRI data into the Varian Eclipse Treatment Planning System. The anatomical boundaries of the hippocampus were delineated according to the standard established in RTOG 0933. Then, the boundaries were pushed outwards in every direction to get the boundaries of the hippocampus + 5 mm area, hippocampus + 10 mm area, and hippocampus + 20 mm area, respectively. Finally, the shortest distances between the boundaries of brain metastases and that of the hippocampus were measured. The location of brain metastases and the hippocampus is shown in The incidence of brain metastases in the hippocampus and surrounding areas was calculated. The differences in hippocampal metastasis rates among different tumor types were analyzed. Statistical methods were employed to determine the relationship between hippocampal metastasis and various clinical features so that the high-risk factors for hippocampal metastasis can be screened out.Logistic regression was conducted in SPSS 21 to analyze the correlation between hippocampal metastasis and the various clinical factors, including age, gender, pathological type, number of brain metastases, the maximum diameter of metastases, and total volume of metastases. The optimal cutoffs for the number of brain metastases, the maximum diameter of metastases, and the total volume of metastases were obtained by calculating the area under the receiver operating characteristic (AUROC) curve. The difference was considered statistically significant with P < 0.05.The protocol of this retrospective study was approved by the ethics committee of the First Hospital of Lanzhou University (LDYYLL-2021-346).The 411 eligible patients included in the analysis consist of 249 males and 162 females. The ages of the patients were between 18 and 86, and the median age was 62. The primary cancers of the patients included 341 (83.0%) lung cancers, 30 (7.3%) breast cancers, and 40 (9.7%) other cancers. As shown in According to the results of a study by Sun (2019) on 116 Chinese patients with brain metastases from multiple cancers, the metastasis rate in the hippocampus was 1.7%, and the metastasis rate in the hippocampus + 5 mm area was 11.2% . Based oIn addition, subgroup analysis of this study found that the metastasis rate in the hippocampus and surrounding areas of 105 patients with brain metastasis from SCLC were significantly higher than other tumor types such as non-small cell lung cancer (NSCLC) and breast cancer (BC). The hippocampal metastasis rate of SCLC patients 18.1%) was over twice that of the entire group of patients (7.3%). SCLC patients accounted for over 25% 105/411) of the entire group, which may be the reason for the high hippocampal metastasis rate in this study. Univariate and multivariate logistic regressions also found that the pathological type was correlated with the risk of hippocampal metastasis. The risk of SCLC metastasis in the hippocampus was 6 times that of NSCLC. Many previous studies also found that SCLC patients seemed to have a higher rate of hippocampal metastasis. Harth reported that the hippocampal metastasis rate of SCLC patients could reach 18.2%, and the metastasis rate in the hippocampal + 5 mm area was 27.2%, but only 11 SCLC patients were included in that study .1% was o. Chen re of the eIn the research on factors of hippocampal metastasis, many have reported the number of brain metastases as an influencing factor , 15, 17.As an analysis based on a large sample of patients with brain metastasis from multiple tumors, a higher proportion of patients with multiple brain metastases were included in this study, which may better reflect the risk of hippocampal metastasis in patients with brain metastases. This study has certain reference values for studying the characteristics of brain metastasis and the probability of hippocampal metastasis in the Chinese population. However, as a retrospective analysis, this study has certain limitations, and bias is inevitable in the selection and collection of cases. For example, this study included 40 patients with primary cancers located outside the lung and the breast. However, those lesions covered 8 histological types, i.e., kidney cancer, pancreatic cancer, colorectal cancer, esophageal cancer, gastric cancer, nasopharyngeal cancer, ovarian cancer, and endometrial cancer, and the brain metastasis rate in the hippocampus + 5 mm area of this subgroup was as high as 17.5%, which may have impacted the results.In conclusion, the risk of hippocampal metastasis is related to the pathological type, the number of brain metastases, and the total volume of metastases. Patients with SCLC have a significantly higher hippocampal metastasis rate than those with other tumors. When the number of brain metastases was no less than 10, the chance of metastasis in the hippocampus + 5 mm and hippocampus + 10 mm regions increased more than 4-fold; when the total volume of metastases was above 7400 mm³, the risk of hippocampal metastasis increased more than 6-fold. Therefore, for patients with SCLC or a greater number and total volume of brain metastases, the implementation of HA-WBRT may bring a higher risk of tumor recurrence, thus warrants caution.1005443068@qq.com.The data analyzed in this study is subject to the following licenses/restrictions: Any request for data and material may be sent to the first author or corresponding author. Requests to access these datasets should be directed to PX, The protocol of this retrospective study was approved by the local ethics committee of the First Hospital of Lanzhou University (LDYYLL-2021-346).PX: Data curation, Manuscript writing. HQ: Research designing, Manuscript revising. HH, WX, HZ, NL, XC, and YM: Date collection. All authors contributed to the article and approved the submitted version.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.


Bone is the most common site of metastatic breast cancer, and it is a leading cause of breast cancer-related death. This study aimed to explore bone metastasis-related long non-coding RNAs (lncRNAs) in breast cancer.cis nearby-targeted DEmRNAs of DElncRNAs were obtained. Quantitative real-time polymerase chain reactions (qRT-PCR) was used to detect the expression levels of selected DEmRNAs and DElncRNAs. LOC641518-lymphoid enhancer-binding factor 1 (LEF1) pair was selected to verify its role in migration and invasion capability of breast cancer cells by wounding healing assay and transwell invasion assay.Four mRNA datasets and two lncRNA datasets of bone metastasis, lung metastasis and liver metastasis of breast cancer were downloaded from Gene Expression Omnibus (GEO) database. The differentially expressed mRNAs (DEmRNAs) and lncRNAs (DElncRNAs) in group of bone metastasis vs lung metastasis and bone metastasis vs liver metastasis, as well as the overlap of the two groups, were identified. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and protein–protein interaction (PPI) network construction of DEmRNAs were conducted. The A total of 237 DEmRNAs were obtained in bone metastasis compared with both lung metastasis and liver metastasis. A total of three DElncRNAs in bone metastasis compared with both lung metastasis and liver metastasis were obtained. A total of seven DElncRNA-nearby-targeted DEmRNA pairs and 15 DElncRNA-nearby-targeted DEmRNA pairs in group of bone metastasis vs lung metastasis and bone metastasis vs liver metastasis, were detected, respectively. Four cis LncRNA-mRNA interaction pairs were identified, which are LOC641518-LEF1, FLJ35024-Very Low Density Lipoprotein Receptor (VLDLR), LOC285972-Retinoic Acid Receptor Responder 2 (RARRES2) and LOC254896-TNF receptor superfamily member 10c (TNFRSF10C). qRT-PCR using clinical samples from our hospital confirms the bioinformatics prediction. siRNA knocking down LOC641518 down-regulates LEF1 mRNA expression, and reduces the migration and invasion capability of breast cancer cells.We concluded that four LncRNA-mRNA pairs, including LOC641518-LEF1, may play a central role in breast cancer bone metastasis.The online version contains supplementary material available at 10.1186/s12935-021-02068-7. Globally, breast cancer is the most common malignancy in women . Bone isOver the past few decades, a great deal of information about lncRNAs has been generated, which facilitated the studies on diverse cancer etiology, including breast cancer . Increascis nearby-targeted DEmRNAs of DElncRNAs were obtained as well. Finally, we established in vitro cell based assay to investigate and confirm the role of novel LncRNA-mRNA axis in tumor metastasis. This study aimed to provide additional knowledge on the molecular mechanisms of breast cancer bone metastasis, especially the role of pro-bone metastasis LncRNAs.In this present study, a comprehensive integrated analysis of lncRNAs and mRNAs expression profiles of bone metastasis, lung metastasis and liver metastasis of breast cancer downloaded from GEO database were performed. The differentially expressed mRNAs (DEmRNAs) and lncRNAs (DElncRNAs) were acquired. The To acquire the mRNA and lncRNA expression profiles of bone metastasis, lung metastasis and liver metastasis of breast cancer tissues, datasets in GEO database with the following criteria were retrieved: datasets should be whole-genome mRNA/lncRNA expression profile by array; these data were derived from bone metastasis, lung metastasis and liver metastasis of breast cancer tissues; datasets were normalized or original. After screening, four mRNA datasets and two lncRNA datasets were enrolled in this study and shown in Table P-values were calculated using a significance threshold of p-value < 0.05. With R package “pheatmap”, hierarchical clustering analysis of DEmRNAs and DElncRNAs were performed.MetaMA  and limmhttp://genecodis.cnb.csic.es/analysis) was applied to perform GO and KEGG pathway enrichment analysis of DEmRNAs. The threshold was set as false discover rate (FDR) < 0.05.GeneCodis3 (http://www.uniprot.org/database/DB-0184), and PPI network was constructed with Cytoscape software .Top 50 up- and down-regulated DEmRNAs were searched with the BioGrid  according to the manufacturer’s protocols. One microgram RNA was applied to synthesize cDNA by Fast Quant Reverse Transcriptase (TIANGEN Bio). Then real-time PCR was performed in an ABI 7300 Real-time PCR system with SYBR R Green PCR MasterMix (TIANGEN). All reactions were carried out in triplicate. GAPDH was used for the internal reference. Relative gene expression was analyzed by fold change method. And all the primer sequence was shown in Additional file 2. Commercial RNA interfering sets targeting LOC641518 were purchased from Ribobio technology , and scramble siRNA was also transfected into breast cancer cells to regard it as control. Exogenous human full-length LEF1 plasmid vectors were purchased from Sino biological Inc. ; and empty vector was also transfected as negative control. Lipofectamine 3000 (Invitrogen) was used for all transfection studies by following the manufacturer’s protocol. Transfection efficiency was examined using qRT-PCR. Briefly, total RNA was extracted from cells using Trizol  and further purified by RNAeasy mini kit plus DNase I treatment . The relative mRNA level for each gene was quantified by real-time RT-PCR with SYBR Green , using GAPDH as a control.Human breast cancer cell line MCF-7 and MDA-MB-231, and non-carcinoma human breast epithelial cell line MCF-10A were purchased from ATCC  and were cultured in RPMI 1640 medium  supplemented with 10% fetal bovine serum (Invitrogen) and incubated at 37 °C and 5% CO4 cells were inoculated in 6-cm tissue culture dishes, cultured overnight, scratch was performed when cell growth fusion reached to 90%. Migration images were captured 24 and 48 h after scratching, the migration rate (width) of cells were calculated.The migration ability of cells was measured by wound-healing assay. Briefly, 2 × 105/mL were added in the upper chamber with (8 μm pore), 600 μL medium containing 10% serum was added in the lower chamber. After cultured for 6 h, medium in the chamber were removed and unmigrated cells were swabbed. Cells was fixed by 4% polyformaldehyde for 10 min, stained with crystal violet for another 10 min. The filter membrane was photographed under the inverted microscope (200×) after sealed with the neutral gum. Cells were counted by Image Pro Plus Version 6, three wells in each group and five vision fields of each well were randomly selected to calculate the average number of cells.Corning™ BioCoat™ Matrigel™ Invasion Chamber with Corning™ Matrigel Matrix  was used for cell invasion assay. One hundred microliters of cells with concentration of 5 × 10Compared with lung metastatic sites, a total of 1280 DEmRNAs (645 up- and 635 down-regulated mRNAs) were detected in tumor tissues from bone metastatic sites of breast cancer. Compared with liver metastatic sites, a total of 1482 DEmRNAs (851 up- and 631 down-regulated mRNAs) were detected in tumor tissues from bone metastatic sites of breast cancer. Hierarchical clustering analysis of top 100 up- and down-regulated DEmRNAs was exhibited in Fig. GO analysis indicated that DEmRNAs in group of bone metastasis vs lung metastasis were significantly enriched in negative regulation of cell proliferation , cytoplasm  and protein binding  , Pathways in cancer (FDR = 1.22E−05) and ECM-receptor interaction (FDR = 1.73E−05) , FGB (degree = 8) and FAM189A2 (degree = 7) were three hub proteins of the PPI network  were detected in tumor tissues from bone metastatic sites of breast cancer  and 91 DElncRNAs (bone metastasis vs liver metastasis) have been identified. By correlation study between these DElncRNAs and DEmRNAs, combined with cis-regulated the expression of LEF1 in group of bone metastasis vs lung metastasis, which might indicate that LOC641518 involved in breast cancer bone metastasis through regulating LEF1.By knocking down the LOC641518, we observed reduced migration and invasion ability which was consistent with lower mRNA levels of LEF1, and over-expression of LOC641518 reversed its effect. LEF1, a member of the T cell Factor (TCF)/LEF1 family of high-mobility group transcription factors, is a downstream component of the Wnt signaling pathway . LEF1 anLOC285972-RARRES2, is another interaction pair identified in the current study. RARRES2, also known as chemerin, was considered as a chemoattractant and an adipokine . The corVery low density lipoprotein receptor (VLDLR) has been considered as a multiple function receptor due to binding numerous ligands, causing endocytosis and regulating cellular signaling , 28. PreTNFRSF10C copy number variation has been reported that is associated with metastatic colorectal cancer , and itsOne of limitation is that we only verify the role of LOC641518-LEF1 interaction pair in migration and invasion capability of breast cancer cells, and the remaining interaction pairs also deserve further investigation. The other limitation is that there is no in vivo data to be provided in the current study, further, the experimental bone metastasis model should be established to investigate their role in mice. These findings may make contribution to understand the process of bone metastasis through lncRNA regulation, and further be helpful to develop new strategies that effectively predict occurrence of bone metastasis, also provide novel targets for drug design in the future clinical practice.In conclusion, four lncRNA-mRNA interaction pairs were identified to be closely associated with breast cancer bone metastasis in this analysis, and Loc641518-LEF1 axis was confirmed by in vitro study. All these results may provide the novel and additional therapeutic strategies for breast bone metastatic patients.Additional file 1: Table S1. Primer sequenceAdditional file 2: Table S2. Differential expressed mRNAs detected in metastatic tumor tissues of breast cancer between bone and lung metastatic sites (bone vs lung).Additional file 3: Table S3. Differential expressed mRNAs detected in metastatic tumor tissues of breast cancer between bone and liver metastatic sites (bone vs liver).Additional file 4: Table S4. A: Top 10 Gene oncology list enriched by differential expressed mRNAs detected in metastatic tumor tissues of breast cancer between bone and lung metastatic sites; B: Top 10 Gene oncology list enriched by differential expressed mRNAs detected in metastatic tumor tissues of breast cancer between bone and liver metastatic sites; C: Top 10 overlapped gene oncology between GO list (bone metastasis vs lung metastasis) and GO list (bone metastasis vs liver metastasis).Additional file 5: Table S5. Differential expressed LncRNAs detected in metastatic tumor tissues of breast cancer between bone and lung metastatic sites (bone vs lung).Additional file 6: Table S6. Differential expressed LncRNAs detected in metastatic tumor tissues of breast cancer between bone and lung metastatic sites (bone vs lung).


A recent publication by Kabinger et al. reported the unique molecular mechanisms of molnupiravir on inducing RNA mutations by RdRp during replication of SARS-CoV-2.3 To date, some NAs which can evade the ExoN proofreading function have shown potent anti-coronavirus activity in preclinical studies and have entered clinical trials for COVID-19 treatment, among which molnupiravir was just approved by Australia on August 9, 2021, for treating COVID-19.The outbreak of the COVID-19 pandemic has led researchers and clinicians to repurpose existing antiviral NAs to fight SARS-CoV-2 infection. Since RdRp plays a pivotal role in virus replication and transcription, broad-spectrum NA inhibitors have been used to target RdRp, which can recognize and incorporate the active form of NA inhibitors into the growing RNA strand, thereby terminating RNA chain elongation. However, the 3′–5′ exonuclease (ExoN) of SARS-CoV-2 may limit the antiviral effects of NA inhibitors incorporated into nascent RNA.d-N4-hydroxycytidine (NHC), the first mutagenic NA that was demonstrated to circumvent ExoN proofreading and has been identified as a potent inhibitor of murine hepatitis virus (MHV), respiratory syncytial virus (RSV), influenza B virus, influenza A virus and Middle East respiratory syndrome coronavirus (MERS-CoV). Although molnupiravir can inhibit SARS-CoV-2 replication in human lung tissue,1 the underlying mechanisms have not yet been clarified. Here we discuss the findings of a recent systematic biochemical analysis that proposed a two-step mechanism for molnupiravir-induced mutagenesis of coronavirus RNA.2Molnupiravir is an isopropyl ester prodrug of β-1 Nevertheless, the viral RdRp incorporated M into the RNA strand less efficiently than the correct nucleotides C or U. After incorporating NHC, RdRp was able to extend the growing RNA strand by 4 or 11 nucleotides until the end. Thus, molnupiravir shows a completely different mechanism from chain-terminating NAs such as remdesivir.4RNA elongation assays were first conducted using recombinant SARS-CoV-2 RdRp and four synthetic RNA template–product duplexes. The NHC triphosphate (MTP) incorporation by SARS-CoV-2 RdRp was quantified using a fluorescent label at the 5′ end of the RNA product strand. The results indicated that NHC monophosphate (M) can be readily incorporated into RdRp instead of cytidines cytosine (C) or uracil (U), and can base-pair with purines guanine (G) or adenine (A), which is consistent with the fact observed in MERS-CoV previously. This is similar to how favipiravir induces SARS-CoV-2 RNA mutagenesis.N4-hydroxy-N4-benzoylcytidine 3′-(2-cyanoethyl) diisopropyl phosphoramidite as the substrate. The purity of the resulting RNA oligonucleotide was assessed by denaturing high-performance liquid chromatography and high-resolution electrospray ionization mass spectrometry. The templating properties of NHC were investigated by annealing the M-containing RNA oligo with a fluorescently labeled product RNA. The M residue at position +1 effectively directed the incorporation of G or A into nascent RNA, forming M-GTP or M-ATP base pairs in the RdRp active site. Thermal melting experiments showed that the stability of RNA duplexes containing M–G or M–A base pairs was slightly lower than that of C–G-containing duplexes, which could explain how they can escape the proofreading mechanism. These results suggest that when RdRp uses M-containing RNA as a template, the enzyme can subsequently incorporate a correct or incorrect nucleotide into the growing RNA strand, leading to mutagenesis. This process is previously known as error catastrophe.1To clarify the role of M-containing RNA in the next step of RNA synthesis, RNA incorporating the M nucleotide at the templating position +1 was prepared by successive solid-phase synthesis using 2 Specifically, M-containing RdRp–RNA complexes with G or A at the 3′ end of the strand were prepared to form RdRp–RNA structures bearing an M–G or M–A base-pair at position −1. The respective structures were solved to overall resolutions of 3.3 or 3.2 Å, while the active site was resolved to a resolution of ~2.9 Å, allowing the characterization of a post-translocation state with a free nucleoside triphosphate (NTP)-binding site at position +1. The conformations of protein and nucleic acid did not differ significantly between the two structures or from the corresponding conformations in the structures of RdRp complexed to wild-type or remdesivir-containing RNA. Modeling of M–A or M–G base pairs into the electron density at the −1 position suggested that the amino tautomer of NHC can mimic C to hydrogen-bond with G, while the imino tautomer can mimic U to hydrogen-bond with A.To elucidate the mechanism of NHC-induced RNA mutagenesis, mutagenic RdRp–RNA complexes were prepared and analyzed by cryo-electron microscopy.4 Molnupiravir mimics the cytidines C and U, whereas favipiravir mimics the purines G and A to form non-canonical base pairs.3 Favipiravir has also been found to act against coronaviruses via other mechanisms, including non-obligate chain termination and slowing of RNA synthesis.3 By building on the known three-dimensional structure of SARS-CoV-2 RdRp,5 mechanistic studies of these drugs should contribute significantly to the discovery of promising drug candidates for SARS-CoV-2.Taken together, these experiments suggest a two-step mechanism in which molnupiravir induces SARS-CoV-2 RNA mutagenesis by forming stable M–G and M–A base pairs in the RdRp active site. So far, remdesivir, favipiravir, and molnupiravir are only approved NA drugs for the treatment of COVID-19, and all three can evade the ExoN proofreading function. Specifically, the incorporation of remdesivir allows the addition of three nucleotides and stalls RdRp through competitive inhibition, while favipiravir and molnupiravir participate RNA synthesis, inducing mutagenesis.5 Therefore, future studies should assess the potential long-term genotoxic side effects of molnupiravir. Future work should also explore the clinical efficacy of molnupiravir against COVID-19, especially in light of new SARS-CoV-2 variants, even though it has been proven effective against several variants in vivo.In summary, mechanism revealed in the currently discussed study by Höbartner and Cramer’s group may help explain and optimize drugs against the current and future coronavirus pandemics. At the same time, drugs that rely on inducing RNA mutations may exert genotoxic effects: molnupiravir-induced mutagenesis has been reported to occur in mammalian cells, indicating the possibility that molnupiravir and its similar drugs may fail due to mutagenic effects.