X-ray crystallography has been used to observe the synthesis of RNA in the absence of enzymes with atomic resolution. Related research article Zhang W, Walton T, Li L, Szostak JW. 2018. Crystallographic observation of nonenzymatic RNA primer extension. eLife7:e36422. doi: 10.7554/eLife.36422How life on Earth began remains one of the greatest scientific mysteries of our time. According to the phylogenetic record, all forms of life, both extant and extinct, are known to store genetic information in DNA and use proteins as enzymes to catalyze metabolic reactions . HoweverIt is well established that RNA can store information and catalyze reactions, so it is possible to envision how modern life could have evolved from RNA-based life. However, it is less clear how the RNA world came into being. Researchers studying this problem have focused on the transition from chemistry to biology, which can be broken down into a number of individual steps, each with its own set of testable hypotheses . ChallenRecent research advances include the prebiotic synthesis of nucleotides, which are the building blocks of RNA , and theNon-enzymatic chemical synthesis of RNA offers a possible bridge between prebiotic chemistry  and the RNA world . For ovePreliminary structural insights into the functional role of the dinucleotide intermediate were initially obtained by X-ray crystallography using crystals of an RNA primer-template duplex bound to a structural analog of the predicted intermediate . HoweverZhang et al. first made crystals of the RNA primer-template complex with a non-reactive monomer. Activated monomers were then introduced into the crystal by a process of soaking, and as the activated monomers replaced the non-reactive monomers, primer extension began. The researchers used liquid nitrogen to freeze the samples at different times and X-ray crystallography to determine the structures with atomic resolution. This process provided a sequence of events that included all the steps of non-enzymatic RNA synthesis: the activated nucleotides bind to the template, pairs of nucleotides form the intermediate and, finally, a bond is formed between the RNA primer and the intermediate to extend the primer by one nucleotide – all without the involvement of any enzymes.The observations made by Zhang et al. are nothing short of amazing and will likely influence the field of non-enzymatic RNA synthesis for years to come. In addition to revealing the reaction mechanism, the structures also explain why the primer reacts more readily with the activated intermediate than a template-bound monomer. Namely, pre-organizing the intermediate on the template reduces the distance between the primer and the adjacent nucleotide, which enables the primer to react more rapidly with the dinucleotide intermediate than it could with an individual monomer. While further questions persist, these findings serve as a monumental achievement and a stepping stone towards understanding the origin of the RNA world and the evolution of life on Earth.


We propose herein a simple, rapid, efficient, and cost effective method to clone and sequence single, yet unknown, small ncRNA. This technique that we called “Pocket-sized RNA-Seq” or psRNA-seq is based on  Small non-coding RNAs (ncRNAs), so named because of their size arbitrarily set below 200 nt, form a heterogeneous family of molecules holding important regulatory functions that have fundamentally transformed our understanding of gene regulatory networks ,2. PromiIn recent years, great efforts have been devoted towards the systematic identification of novel miRNAs and their target mRNAs, as well as their origin and biogenesis.The biogenesis of miRNAs is a multi-step process; miRNA genes are transcribed by RNA polymerase II, giving rise to long primary miRNAs (pri-miRNAs) that are cleaved in the nucleus by the enzymes Drosha/DGRC8 of the microprocessor complex to produce a 70/100 nucleotide-long precursor (pre-miRNA) and then transported into the cytoplasm by Exportin 5 . InMost miRNAs are encoded by independent transcription units , locatedTo date, the only ways of studying mature miRNAs are (1) with high-throughput methods, which allows the sequencing of any RNAs detected in a cell; or (2) small-scale approaches, such as stem-loop RT-qPCR that all require to know the exact sequence of the mature miRNA.®, Taqman-based arrays or high-throughput sequencing. However, because deep sequencing-based expression analysis produces huge amounts of sequence data, there are substantial bioinformatic challenges to appropriately analyze and handle such sequence information. Another important consideration is the difficultly to identify miRNAs that are expressed at low levels, at highly specific stages of development, or in rare cell types. It is indeed well established that low read counts yielded by low expressing sequences show poor consistency between technical replicates, and exclusion of sequences with read counts less than a low threshold in each sample is a common strategy used to eliminate sequencing errors [Current methods of large-scale profiling of miRNAs include miRNA microarrays, bead-based capture methods, NanoString Technologiesg errors . In pring errors ,18,19.In essence, despite rapid advancement in sequencing technologies and because of the general excitement surrounding miRNA discoveries and their potential therapeutic applications in numerous diseases, additional tools are required to enable researchers to accurately identify and study novel miRNAs when sensitivity on low-abundant miRNAs is an issue or bioinformatical analysis of substantial data represents a challenge. For these reasons, we propose herein a method to systematically identify, clone, and sequence unique unknown small ncRNAs, using miRNA as a proof of principle, from a genomic region of interest, that we named Pocket-sized small RNA-Seq (psRNA-seq) as a wink to the tiny sequencing reads it requires.Detailed experimental protocol is given in As a proof of principle for the efficiency of psRNAS-seq method in identifying miRNAs produced by a genomic region of interest, we used three 60–90 nt long sequences surrounding the precursor of hsa-miR-21 and proceeded as described in the Material and Methods section. Out of the 75 clones that were sequenced (25 per each three baits), 47 showed a hundred percent match with the mature hsa-miR-21 sequence  as referThe presence of hsa-miR-21 in the sample material was also verified by stem-loop RT-qPCR , which ci.e., a miRNA highly expressed in cancer cells and not at all or at very low levels in normal cells. We carried out the psRNA-seq method using sample RNAs isolated from cells of the breast cancer line MCF-7 or from human primary myoblasts and indeed verified that mature hsa-miR-21 was only captured from cancer MCF-7 cells We originally selected the well-characterized hsa-miR-21 as a proof of principle for the efficiency of psRNA-seq since hsa-miR-21 is a well-known oncomiR In addition, we decided to use our method to identify a miRNA of mirtron origin, hsa-miR-1237 , processa priori to identify, clone and sequence even weakly expressed new miRNAs, although it implies to delimit a genomic region of interest already implicated in normal development or disease for instance. We list below the main clear benefits offered by psRNA-seq over high-throughput approaches:The reason for developing yet another technical approach to uncover small RNAs stemmed from the realization that if alternative pathways in the production of small regulatory RNAs exist, then the analysis of RNA-seq data is complicated by the lack of predictive tools and basic data about their characteristics. In addition, several reports suggest that the large majority of mature miRNAs are expressed at very low levels in eukaryotic cells ,26,27,28(1)psRNA-seq is a highly sensitive technique that can detect even low amounts of mature miRNAs (or any other short RNA), as exemplified by the amplification of the weakly expressed has-miR-21 star and hsa-miR-1237, that would not pass low-thresholding or elimination of isolated reads applied to eliminate high-throughput sequencing errors ,17,18,19(2)psRNA-seq does not require knowing the biogenesis pathway of the small RNA of interest. Therefore, not only miRNA, but also mirtrons, simtrons, snoRNAs, snRNAs, and others small ncRNAs can potentially be detected and cloned using psRNA-seq provided the genomic location, even approximate, of the sequence containing the small RNA is known. It is indeed simple to refine the position using overlapping sequences as we have done with hsa-miR-21. We used 60 to 102 nt long baits to minimize secondary structures that may form, although we have experience of RNA pull down assays using much longer bait RNAs, which efficiency has to be tested individually ,30.(3)psRNA-seq can be performed on rare samples like adult stem cells or patient biopsies since it does not require large amounts of material. While microarrays and RNA deep sequencing needs are in the range of several micrograms of sample RNA, we used as less as 10 ng of fractionated RNA, an amount that can be further reduced. Indeed, from all the RNA samples that we prepared, we used 1/100th by PCR reactions and obtained thousands of colonies after bacterial transformation, clearly suggesting that 0.1 ng of fractionated RNA should be enough to obtain similar results .(4)psRNA-seq is a simple, rapid, and inexpensive method to set up. It does not require special skills in molecular biology or bioinformatics. Indeed, it does not require long and tricky procedures to prepare libraries to be sequenced by RNA-seq technologies or challenging bioinformatical analysis of large datasets.(5)psRNA-seq is highly flexible. It is not specific to miRNAs  and can also be used to capture plenty of other ncRNAs such as snRNAs, snoRNAs and many other still-unknown small RNAs. The method is also easily transposable to other species, with some restrictions (see below).We should mention some limitations to the technique. First, psRNA-seq relies on the initial knowledge of the genomic sequence; therefore, it cannot be used for discovering small RNAs in species with unannotated genomes. Second, psRNA-seq was designed as a qualitative method to capture RNA with unknown sequences and thus, does not allow a quantitative analysis. However, once captured and sequenced following psRNA-seq, mature miRNAs can be quantified using classical stem-loop RT-qPCR or other techniques ,31,32 anIn support of the use of psRNA-seq, this method recently allowed us to identify and clone a novel mirtron produced by a human gene in specific developmental conditions  and from another pathogenic locus is disease conditions .Detailed experimental protocol is given in in vitro transcribed RNA from the genomic region of interest that will be used as bait. We chose to use MessageMuter™ shRNA production kit  to produce RNAs complementary to the human precursor of hsa-miR-21, or the irrelevant luciferase gene as a negative control, according to the manufacturer’s instructions and as previously described [The first step requires the production of escribed . We chosescribed ,35,36. TRNA samples that will be hybridized to the bait were isolated from MCF-7 breast cancer cells or primary myoblasts with TRI reagent (Sigma) as previously described ,37. To rAfter phenol/chloroform extraction, 400 pmol of RNA bait were denature at 95 °C for 2 min and placed on ice for an additional 2 min. RNA was then annealed to 500 pmol of a specific biotinylated oligonucleotide (sequences available at the end of this subsection) in 50 mM KCl, 1 U/mL RNaseOUT (Life Technologies) for 1h at RT. Streptavidin magnetic beads , prewashed twice in 0.1 M NaOH, 0.05 M NaCl and once in 0.1 M NaCl, were incubated with the RNA/oligonucleotide complex in binding/washing buffer  for 30 min at RT. Pre-incubated beads were washed twice in binding/washing buffer and added to 100 ng of small purified RNAs (<200 nt), as described above, for an additional 1 h at RT. “Small RNA/RNA bait/magnetic beads” complexes were washed twice with RNA binding buffer  adjusted to 300 mM KCl and once with RNA binding buffer.After phenol/chloroform extraction, 1 U of poly (A) Polymerase (Life Technologies) was then used to add a poly(A) tail to the recovered RNAs following the manufacturer’s instructions, along which, 1 h incubation generally adds between 50 and 200 A at the 3′-ends. Polyadenylated RNAs were then purified by phenol-chloroform extraction.We then performed full RACE-PCR using the GenRacer kit (Life Technologies) as previously published ,15 with http://mirnadesigntool.astridresearch.com). All reactions, including no-template controls and RT minus controls, were run in duplicate. Normalization was performed relative to the signal of RNU6-1 amplification. Stem-loop primers for reverse transcription of miR-10a and miR-21 were 5′-GTT GGC TCT GGT GCA GGG TCC GAG GTA TTC GCA CCA GAG CCA ACC ACA AA-3′ and 5′-GTT GGC TCT GGT GCA GGG TCC GAG GTA TTC GCA CCA GAG CCA ACT CAA CA-3′, respectively. For qPCR, forward primers were 5′-TGT TTT TTT TTA CCC TGT AGA TCC GAA-3′ and 5′-GTT TGG TAG CTT ATC AGA CTG A-3′, for miR-10a and miR-21, respectively, while reverse primer was universal primer (5′-GTG CAG GGT CCG AGG T-3′). U6 forward and reverse primers were 5′-CTC GCT TCG GCA GCA CA-3′ and 5′-AAC GCT TCA CGA ATT TGC GT-3′, respectively.Stem-loop RT-qPCR was performed as previously described  using stet al. (2008), and deposited to the Short Read Archive section of GEO at NCBI under accession number GSE12946 [Genome-wide RNA-Seq data sets used here were generated by Chris Burge’s lab at the Massachusetts Institute of Technology  and by Roderic Guigó’s lab at the Center for Genomic Regulation  published by Wang GSE12946 , and by GSE12946 ,39,40. DGSE12946 ,37,39,41


Paracentrotus lividus  is a keystone herbivore in the Mediterranean Sea due to its ability to transform macroalgal-dominated communities into barren areas characterized by increased cover of bare substrates and encrusting coralline algae, reduced biodiversity and altered ecosystem functions. P. lividus is also an excellent animal model for toxicology, physiology and biology investigations having been used for more than a century as a model for embryological studies with synchronously developing embryos which are easy to manipulate and analyze for morphological aberrations. Despite its importance for the scientific community, the complete genome is still not fully annotated. To date, only a few molecular tools are available and a few Next Generation Sequencing (NGS) studies have been performed. Here we aimed at setting-up an RNA extraction method to obtain high quality and sufficient quantity of RNA for NGS from P. lividus embryos at the pluteus stage. We compared five different RNA extraction protocols from four different pools of plutei : TRIzol®, and four widely-used Silica Membrane kits, GenElute™ Mammalian Total RNA Miniprep Kit, RNAqueous® Micro Kit, RNeasy® Micro Kit and Aurum™ Total RNA Mini Kit. The quantity of RNA isolated was evaluated using NanoDrop. The quality, considering the purity, was measured as A260/A280 and A260/230 ratios. The integrity was measured by RNA Integrity Number (RIN). Our results demonstrated that the most efficient procedures were GenElute, RNeasy and Aurum, producing a sufficient quantity of RNA for NGS. The Bioanalyzer profiles and RIN values revealed that the most efficient methods guaranteeing for RNA integrity were RNeasy and Aurum combined with an initial preservation in RNAlater. This research represents the first attempt to standardize a method for high-quality RNA extraction from sea urchin embryos at the pluteus stage, providing a new resource for this established model marine organism.The sea urchin  NGS technologies have found broad applications in functional genomic research, including gene expression profiling, genome annotation, small ncRNA discovery and profiling, determination of DNA sequences associated with epigenetic modifications of histones and DNA, and profiling DNA methylations –6.“Omic” approaches are powerful instruments not only for genomic studies but also for many other research fields. For example starting from 2007, ecologists used this approach to address several important ecological questions, introducing the new discipline of “ecological genomics” and/or “molecular ecology” –10. The Paracentrotus lividus is considered a suitable organism to study the ecotoxicological responses to xenobiotics and the physiological reactions to physical stressors [Among the echinoderms, the sea urchin tressors –29. It htressors ,31.P. lividus represents a well-established model organism, to date the complete sequence of its genome is still not available [P. lividus. However, high-throughput NGS of genomes and transcriptomes requires high-quality, clean, and concentrated DNA/RNA.The species represents a useful test organism for several reasons: it is an important component of benthic communities in the Mediterranean Sea and Atlantic Ocean; extraction and maintenance of gametes are easy; the embryos grow rapidly and synchronously (pluteus stage is reached 48 hours post-fertilization) and embryos are transparent and suitable for microscope detection of sub-lethal effects of pollutants on development; they have a sufficiently long reproductive season (from October to May). Although vailable . Thanks P. lividus to define the best method for obtaining high-quality RNA for NGS applications. We fertilized four pools of eggs and performed RNA extractions from embryos at the pluteus stage (at 48 hour post-fertilization) preserved in RNAlater®, using five different protocols: a guanidinium-thiocyanate-phenol-chloroform (GTPC) extraction protocol with TRIzol®, and four widely-used Silica Membrane kits, namely GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), RNAqueous® Micro Kit (Ambion from Life Technologies), RNeasy® Micro Kit (Qiagen) andAurum™ Total RNA Mini Kit (Biorad). The quantity and quality of isolated RNA was evaluated taking into account the purity, measured as A260/A280 and A260/230 ratios, and the integrity, measured as RNA Integrity Number (RIN). The results yielded a first protocol for high-quality RNA extraction from sea urchin plutei, increasing the resources for such a well-established model organism.This study compares five RNA extraction protocols using embryos of the sea urchin P. lividus (Lamarck) were collected from a site in the Bay of Naples that is not privately-owned or protected in any way, according to Italian legislation . Field studies did not include endangered or protected species. All experimental procedures on animals were in compliance with the guidelines of the European Union (Directive 609/86).Adult sea urchins were collected during the breeding season by scuba-diving in the Gulf of Naples, transported in thermic box to the laboratory within 1 hour after collection and maintained in tanks with circulating sea water until testing. Sea urchins were injected with 2 M KCl through the peribuccal membrane to obtain the emission of gametes. Eggs were washed with filtered sea water (FSW) and kept in FSW until use. Concentrated spermatozoans were collected, dried and kept undiluted at +4°C until use.later®, an RNA Stabilization Reagent , and then frozen in liquid nitrogen and kept at -80°C.Different quantities of eggs  from five females were fertilized, using sperm-to-egg ratios of 100:1, and embryos were then collected at the pluteus stage (48 hpf) by centrifugation at 1800 rcf for 10 min in a swing out rotor at 4°C. Immediately after harvesting, the embryos were placed in at least 10 volumes of the RNAFive different methods of RNA extraction were compared.® RNA extraction methodi) TRIzolTotal RNA was extracted using TRIzol  according to the manufacturer’s instructions, homogenizing with TissueLyser  with 3 mm sterile aluminium beads at 20.1 Hertz (Hz) for 5 minutes. Extractions with chloroform/isoamyl alcohol (24:1) were performed, followed then by RNA precipitation by adding glycogen and isopropyl alcohol. Total RNA was suspended in 0.1% v/v diethylpyrocarbonate (DEPC)-treated water. Contaminating DNA was degraded by treating each sample with a DNase RNase-free kit  according to the manufacturer’s instructions. The samples were then stored at -80°C.ii) GenElute™ Mammalian Total RNA Miniprep KitEmbryo lysis was performed in 500 μl Lysis Solution/2-ME mixture and then RNA was extracted following the manufacturer's protocol. An additional step with DNase RNase-free kit  was used to remove contaminating DNA. Finally, RNA was eluted with 50 μl Elution Solution provided by the manufacturer. The samples were then stored at -80°C.® Micro Kitiii) RNeasyEmbryos were lysed with 350 μl Buffer RLT/2-ME (10 μl β-mercaptoethanol for each ml of Buffer RLT) and homogenized with TissueLyser  using 3 mm sterile aluminium beads at 20.1 Hz for 5 minutes. RNA was extracted following the manufacturer's protocol. DNA contaminations were avoided using the RNase-Free DNase Set, provided by the kit. RNA was eluted with 14 μL RNase-free water. The samples were then stored at -80°C.® Micro Kitiv) RNAqueousPellets were suspended in 500 μl Lysis Solution by vortexing vigorously. Elution was performed in two steps, adding 10 μl Elution Solution each time, and preheating at 75°C. Finally a DNase treatment was performed adding 1/10 volume 10X DNase I Buffer and 1 μL of DNase I to the sample. DNase was finally blocked using a DNase Inactivation Reagent . The samples were then stored at -80°C.™ Total RNA Mini Kitv) AurumSamples were disrupted with 350 μl Lysis Solution , pipetting up and down several times to lyse cells thoroughly. After treatment with 80 μl of diluted DNase I (75 μl DNase Dilution Solution and 5 μl DNase I), RNA was eluted with 40 μl of Elution Solution. The samples were then stored at -80°C.The amount of total RNA extracted with the five methods was estimated by measuring the absorbance at 260 nm and the purity at 260/280 and 260/230 nm ratios, by a Nanodrop  were fertilized and embryonic development was followed until the pluteus stage, which is reached at about 48 hpf. The results of different RNA extractions are summarized in Increasing amounts of sea urchin P. lividus embryos  and 18S (47s) with very high average RIN values ranging from 7.9 for GenElute, 8.7 for RNAqueous, increasing to 9.3 for RNeasy and reaching a maximum value of 9.7 with the Aurum kit. Such high RIN values were considered suitable for NGS technologies.Total RNA extracted with TRIzol showed a single peak corresponding to 18S at 42 seconds (s), absence of a 28S peak, a high amount of small size RNA occurring between 25s and 42 s and RNA degradation with a very low RIN value (RIN = 3.90). The integrity of In samples exhibiting A260/280 and A26030 ratios lower than 1.8 or over 2.0 , we obseP. lividus embryos at the pluteus stage. Since the sea urchin P. lividus is one of the most common sea urchins in the Mediterranean Sea, as well as in the Northeast Atlantic from Ireland to the coasts of Morocco, the Canary Islands and the Azores [P. lividus is considered a keystone herbivore, able to transform communities dominated by macroalgae into barren areas thereby reducing biodiversity and altering ecosystem functions [In the present work we focused our attention on optimizing RNA extraction protocols from sea urchin e Azores , settingunctions ,36. The unctions .P. lividus. To our knowledge, our work represents the first attempt to standardize a method for RNA extraction from sea urchin embryos at the pluteus stage. Our aim was to obtain high-quality RNA in sufficient quantities for NGS technologies. Firstly, we extracted RNA from different numbers of sea urchin P. lividus embryos at the pluteus stage, applying five different protocols. Our findings revealed that all extraction procedures from 500 and 1000 embryos did not yield sufficient amounts of total RNA as requested for NGS approaches . Sufficient RNA quantity was obtained from 2500 and 5000 embryos. The most efficient procedures to produce such quantities were obtained with GenElute, RNeasy and Aurum, producing more than 10 and 20 μg of RNA, using 2500 and 5000 embryos, respectively.Despite the well-recognized ecological significance of the sea urchin in marine environment, to date there are few molecular tools available for this species. For this reason, NGS approaches are increasingly being applied to this species, in order to increase the molecular data on Having established the number of embryos necessary to obtain the required quantity of RNA, we assessed RNA quality by measuring different features: A260/280 and A260/230 ratios and estimation of RIN values ,37–39. Ilater ensured greater stability for our samples since this a nontoxic storage reagent able to permeate cells and/or tissues and to stabilize and protect cellular RNA. This step minimizes the need for immediate processing of the samples, without jeopardizing the quality or quantity of RNA obtained after subsequent RNA isolation. RNA extraction from fresh cell/tissues has been used in many cases with success [later is a reliable preservative for RNA in a wide array of tissues improving the yield of total RNA [Interestingly our findings demonstrated no significant correlations between A260/280 and A260/230 ratios and RNA integrity. A possible explanation for this may be due to the different stability of the RNAs. In fact, ribosomal RNA is more stable than mRNA , 40–43.  success ,43, but otal RNA ,39.P. lividus plutei. A similar methodological analysis was performed by Pérez-Portela and Riesgo [P. lividus, but tissues from another sea urchin, Arbacia lixula. In fact, their study aimed to optimize a preservation protocol for the isolation of high-quality RNA from three different A. lixula tissues: gonad, oesophagus and coelomocytes. Extractions of total RNA were performed with a modified TRIzol protocol for all tissues, applying four preservation treatments. The results showed high values of RNA quantity and quality for all tissues, indicating non-significant differences among samples. Insufficient RNA amount and great variability in RNA integrity were found in coelomocytes in RNAlater. The most efficient system to stabilize RNA was the TRIzol method that produced high RNA quality and quantity. These data are comparable with our results (see above), because no correlation was found between RNA integrity and absorbance ratios, as in our experiments. Furthermore, the evaluation of RIN values by Agilent Bioanalyzer chips was the best approach to evaluate the RNA integrity (as in our case).This study fills a gap considering that no comparative studies for RNA extractions are available in the literature for sea urchin d Riesgo , althougThe extraction of high-quality RNA represents a fundamental step, considering that transcriptomic information and NGS approaches represent significant tools to acquire information regarding several biological processes. Several attempts, mainly for non-well-established model organisms, were recently made to establish methods for high-quality RNA extractions, because NGS represents the only way to address specific ecological and evolutionary questions ,44.®, Aurum Total RNA Mini Kit and Qiagen RNeasy Micro Kit, in combination with preservation reagents TRIzol® or RNAlater®, to obtain high-quality and large quantities of RNA from the copepod Calanus helgolandicus. Their results confirmed that the preservation of copepods in RNAlater® and the extraction with Qiagen RNeasy Micro Kit were the optimal isolation method for high-quality and quantity of RNA for NGS studies of C. helgolandicus.Asai et al.  comparedP. lividus.In conclusion, our results provide for the first time a standard protocol for isolating high-quality RNA from sea urchin plutei for high-throughput NGS studies thereby increasing the resources available for a well-established model organism such as S1 FigThe boxes extend from the 25th to the 75th percentile, and the line in the middle is the median. The error bars extend down to the lowest value and up to the highest. Dashed line at the RIN value of 8 is reported, because higher values than 8 are considered suitable for NGS analysis.(PPT)Click here for additional data file.S1 TableWe concern as statistically significant the p-values lower than 0.05.(DOC)Click here for additional data file.


Self-rated health (SRH) and socioeconomic position (SEP) as important determinants of health differences are associated with health and economic changes in society.The objectives of this paper were (1) to describe trends in SRH and (2) to analyze associations between SRH and SEP among adults in Estonia in 1996–2014.n = 18757) of postal cross-sectional surveys conducted every second year in Estonia during 1990–2014. SRH was measured using five-point scale and was dichotomized to good and less-than-good. Standardized prevalence of SRH was calculated for each study year. Poisson regression with likelihood ratio test was performed for testing trends of SRH over study years. Age, nationality, marital status, education, work status and income were used to determine SEP. Logistic regression analysis was used to assess association between SRH and SEP.The study was based on a 25–64-year-old subsample (The prevalence of dichotomized good self-rated health increased significantly over the whole study period with slight decrease in 2008–2010. Until 2002, good SRH was slightly more prevalent among men, but after that, among women. Good SRH was significantly associated with younger age, higher education and income and also with employment status among both, men and women. Good SRH was more prevalent among Estonian women and less prevalent among single men.There was a definite increase of good SRH over two decades in Estonia following economic downturn between 2008 and 2010. Good SRH was associated with higher SEP over the study period. Further research is required to study the possible reasons behind increase of good SRH, and it’s association with SEP among adults in Estonia. Health as a basic human right  is assocAt the same time life expectancy at birth increased steadily from 1996  to 2014   can be used as an option which is recommended by WHO . SRH is Sociodemographic and socioeconomic factors can predict how a person might rate their health , 18. FacThe objectives of this paper were (1) to describe trends in SRH and (2) to analyze associations between SRH and SEP among adults in Estonia in 1996–2014.The study was based on data drawn from the cross-sectional postal survey of Health Behaviour among Estonian Adult Population conducted among 16–64-year-old adults in every second year. The survey started in Estonia in 1990 as part of the Finbalt Health Monitor project. The surveys were approved by the Tallinn Medical Research Ethics Committee. A random sample, stratified by age, gender and place of residence, of the Estonian population aged 16–64 was taken from the Population Registry. The methodology of survey of Health Behaviour among Estonian Adult Population is described in more detail elsewhere , 26.In 1996–2002 the initial sample consisted of 2000 adults, in 2004–2014 the sample size was 5000 adults (Table  The subsample consisted of 18757 adults including 7660 men (40.8%) and 11097 women 59.2%) in 1996–2014  and less-than-good (average/rather bad/bad) SRH.To describe SEP, the variables age, nationality, marital status, education, employment status and income were used and categorized as described below.Age was measured in full years and analyzed in four age-groups: 25–34, 35–44, 45–54, 55–64. Nationality referred to self-determined national identity and data was categorized into two groups: Estonians and non-Estonians. Marital status was categorized into married/cohabiting; single, divorced/widowed. Education was based on the highest completed education levels and designated as follows: basic; secondary; higher education. Economic activity was measured using employment data and categorized to four groups: currently employed; unemployed; retired and not working; other . Income was determined by average monthly income per family member. Data was categorized into four groups based on quartiles that were calculated separately for each year and designated as follows: I (the lowest); II; III; IV (the highest).The data was analyzed separately for men and women. Age-standardized prevalences of SRH for each study year were calculated, using the European standard population . Poissonn = 142) were excluded from the analysis. A total of 18757 questionnaires (7660 men and 11097 women) were used in the analysis. Questionnaires with missing information concerning SEP were excluded from the logistic regression analysis. A total of 7211 questionnaires for men and 10465 for women were used in the model.Questionnaires which lacked information about SRH . Among men, good health increased from 2.7% in 1996 to 20.1% in 2014  in 1996–2014   Fig. . In 1996Compared to the first study year, good health was significantly higher in 2006–2014 among men and in 2004–2014 among women Table . SRH wasThe study analyzed trends in SRH, and associations between SRH and SEP among 25–64-year-old adults in Estonia over the period of 1996–2014.The main findings from the study were, first, that prevalence of good self-rated health increased over the whole study period with only a slight decrease between 2008 and 2010. Second, until 2002, good SRH was slightly more prevalent among men. After that, women rated their health as good more often. Third, SRH was associated with higher SEP from 1996 to 2014 throughout the whole study period..The survey of Health Behaviour among Estonian Adult Population presents a great, and also the only opportunity to analyze self-rated health over more than two decades in Estonia. It is considered as a strength of the study, that the survey design and methodology have remained largely the same across the study periodHowever, this study has several limitations that need to be considered. The survey was conducted as postal questionnaire and used self-reported data. Possible lower participation of adults with lower SRH or SEP has to be considered –30. AnotBased on five-point-scale, the most prevalent response of SRH was ‘average’ in every study year. Similar findings have been reported based on previous studies in Estonia , 11. At The age standardized prevalence of dichotomized good SRH increased significantly over the study period. In 2014, the age-standardized prevalence of good SRH was 47.7% among men and 52.7% among women. Until 2002 the prevalence of good SRH was higher among men, after that, women rated their health as good more often. Earlier international studies have shown good SRH to be more prevalent among men in countries like Greece, Italy, Latvia, Armenia, Russia and elsewhere , 13, 21.. Concerning economic recession, it has been suggested that for high-income countries it is unlikely that the recession will have any major overall negative health effects (e.g. on life expectancy or causes of death) [The results of the present study showed that compared to 2008 the prevalence of good SRH was lower in 2010. This finding is similar to results published earlier –40. Whenf death) . In low-f death) , 43. Thef death) . DespiteAssociation between good SRH and SEP in this study were stable over the period of almost two decades in 1996–2014 being similar to the results published earlier , 23, 44.Younger age groups had higher odds to assess their health as good. These findings were in accordance with the results from previous studies , 20, 28.Compared to the married or cohabited men, the odds of having good SRH were lower among single men. Previous studies have shown the opposite results – the odds of having average or lower SRH were lower among single men . HoweverCompared to adults with basic and secondary education, the odds of having good SRH were more than three times higher among men and women with higher education. Higher education has been related to good SRH in studies in Estonia , 10, 11 This paper provided, on one hand, a new information concering a definite increase of good SRH over two decades following economic downturn in Estonia. Men assessed their health as good more often until 2004 but since then the prevalence of good SRH was higher among women. On the other hand, the association between good SRH and higher SEP was persistent over the whole study period. This study provides evidence-based information that could support identifying risk groups to decrease health inequalities in Estonia. A more in-depth analysis is required to determine the possible reasons behind increase of good self-rated health among adults in Estonia.


The organization of molecules into cells is believed to have been critical for the emergence of living systems. Early protocells likely consisted of RNA functioning inside vesicles made of simple lipids. However, little is known about how encapsulation would affect the activity and folding of RNA. Here we find that confinement of the malachite green RNA aptamer inside fatty acid vesicles increases binding affinity and locally stabilizes the bound conformation of the RNA. The vesicle effectively ‘chaperones’ the aptamer, consistent with an excluded volume mechanism due to confinement. Protocellular organization thereby leads to a direct benefit for the RNA. Coupled with previously described mechanisms by which encapsulated RNA aids membrane growth, this effect illustrates how the membrane and RNA might cooperate for mutual benefit. Encapsulation could thus increase RNA fitness and the likelihood that functional sequences would emerge during the origin of life. So far little is known about how encapsulation affects the activity and folding of RNA, which is of interest for understanding the origin of cellular life. Here the authors show that encapsulation of functional RNA in vesicles increases RNA activity and improves RNA folding through a biophysical confinement effect. Encapsulation into primitive cells (protocells) is believed to be critical for the early evolution of life, since this spatial organization prevents dilution of interacting components and prevents parasites from sabotaging the evolution of ribozymes4 with enhanced replication7. Indeed, compartmentalization has become an important strategy for experimental evolution of catalytic nucleic acids8 and replicases9. Moreover, creating a cellular system with emergent properties requires cooperative interactions between the genome and membrane11. Some mechanisms have been described in which membrane vesicles derive a ‘benefit’ from encapsulating RNA. In particular, RNA-containing vesicles acquire a growth advantage over empty vesicles12, the binding of RNA bases can stabilize vesicles against salts13, ribozyme-catalyzed peptide synthesis might have helped protocells to divide14, and ribozyme-catalyzed lipid synthesis would stabilize vesicle membranes15. However, little is known about the converse interaction, i.e., whether the RNA might derive a benefit from the surrounding membrane vesicle. Vesicle growth can regulate encapsulated ribozymes by diluting inhibitory sequences16, and dry–wet cycling with lipids may aid the synthesis of sugar-phosphate backbones for nucleic acids18, but mechanisms by which encapsulation itself could improve RNA function have not yet been described. Studying how encapsulation affects functional RNA  is important for understanding the cooperative interactions that are fundamental to the early evolution of simple cells.The ability of RNA to act as both an informational and catalytic molecule suggests that a simple living metabolism may require only RNA, which is of high interest for the origin of life as well as for the construction of minimal synthetic cells19. Therefore, there is a need to understand how encapsulation inside simple cells affects RNA function as well as folding, since proper folding is generally required for catalytic or binding activity20. Previous studies have focused on the effect of model crowding agents on RNA folding and activity, indicating that these agents can stabilize the folded state22 as well as increase the rate of ribozyme reactions24. For example, polyethylene glycol (PEG) and Ficoll stabilize the folded state of the Azoarcus group I intron, resulting in increased activity27 which can be attributed to the excluded volume effect. Similarly, the HDV-like ribozyme adopted a more native structure and exhibited greater self-cleavage activity in the presence of PEG 8000 and dextran28. However, it should be noted that chemical interactions between a nucleic acid and a specific crowding agent can complicate the effect observed from crowding agents29.While most studies of RNA have been conducted in dilute aqueous solutions, it is well-known that an encapsulated cellular environment differs in important ways from such solutions. For example, the cellular environment may be characterized by depletion effects, membrane surface interactions, and increased effective concentrations, among other effects31. An increase in the number and activity of functional sequences could uncover feasible evolutionary pathways for evolution, effectively improving the ability of natural selection to optimize function in sequence space33.We hypothesized that confinement inside vesicles could improve RNA aptamer activity. Physical mechanisms that lead to increased activity are of special interest for the prebiotic RNA world, because they may raise the likelihood of emergence of functional RNA. Moreover, early studies suggest that the fitness landscape of functional RNAs consists primarily of evolutionarily isolated peaks with distinct structural motifs linked by generally unfavorable paths of mutation36. Unlike phospholipids, fatty acids have been synthesized under plausible prebiotic scenarios38. An earlier study found a modest reduction in self-cleaving activity of a hammerhead ribozyme encapsulated in vesicles assembled from myristoleic acid  and a cognate glycerol monoester39. However, the mechanism of this effect was not investigated. In particular, it is not known whether the reduction was caused by mere exposure to fatty acid vs. by the confinement of encapsulation. In the present study, in order to isolate the effect of confinement from effects of exposure , we compare all encapsulation experiments against a control in which aptamer is exposed to vesicles but not encapsulated  RNA aptamer inside model protocells and characterize binding affinity and local RNA folding. While modern cell membranes are primarily composed of phospholipids, such membranes are not appropriate for protocells due to their impermeability and slow molecular dynamics, which require protein transporters and flipases to allow internal metabolism and membrane growth. On the other hand, vesicles made from fatty acids are relatively permeable and exhibit faster dynamics, allowing passive diffusion of small molecules  for RNA replication as well as sustainable membrane growth and division cyclescid MA; C4:1 and a40. To study mechanisms that are not simply caused by changes in effective concentration, we encapsulated the RNA aptamer for MG, which is permeable and thus equilibrates across the membrane. Here we show that the affinity and local structural interactions of the MG aptamer are improved by confinement inside vesicles. We discuss the mechanistic interpretation of these results, which suggest an excluded volume effect due to confinement, as well as the evolutionary implications for early life.In principle, reactions involving macromolecular interactions may be subject to changes in effective concentration brought about by encapsulation, and indeed compartmentalization can accelerate ribozyme reactions involving macromolecular interactions through this mechanism42. Vesicle size after extrusion was confirmed by dynamic light scattering (DLS). The hydrodynamic diameter of the vesicles was ~60 nm, consistent with a previous study41, and was stable after overnight incubation. Separation of vesicles encapsulating RNA and free RNA was achieved by size exclusion chromatography (SEC) with Sepharose 4B resin34  in a 2:1 ratio, and MA with myristoleyl alcohol (MAOH) in a 10:1 ratio41), given a surface area per MA molecule of ~68 × 10−20 m2 (ref. 43), the total bilayer surface area in solution is ~410 m2 L−1. Since a single vesicle of diameter 60 nm has a surface area of ~10−14 m2, we estimate a concentration of ~4.1 × 1016 vesicles L−1. The concentration of encapsulated RNA (after dispersion into bulk solution) was found to be on the order of ~58 nM by RT-qPCR, or 3.5 × 1016 molecules L−1, so on average we estimate ~0.85 RNA molecules per vesicle. Given the volume of a single vesicle (~10−22 m3 ≈ 10−19 L), the bulk-equivalent concentration of RNA in the encapsulated volume would be ~14 μM. This value is roughly consistent with the expectation based on random enclosure of RNA during vesicle formation (see Methods). The discrepancy may be due to estimation inaccuracies  or from losses caused by charge–charge repulsion between the RNA and MA surface.To determine the quantity MG RNA aptamer encapsulated, we measured the amount of RNA encapsulated using quantitative reverse transcription PCR (RT-qPCR) and compared it to the value expected from random encapsulation. In a solution containing 2 mM MA in vesicles  of 4 mM44. To determine the dissociation constant (KD) of the MG aptamer in buffer  lacking fatty acid, we measured the fluorescence of the aptamer-MG complex at varying concentrations of MG. The binding curve indicates a KD of 2.5 ± 0.4 μM , indicating that the presence of lipid reduced the affinity of the aptamer  Fig. ; in thismer Fig. , so smalons Fig. .Fig. 2MGKD value of the encapsulated aptamer. Samples containing vesicles were incubated for several hours to allow MG to equilibrate across the membrane  compared to aptamer exposed to empty vesicles as control ) or POPC  vesicles. DLS confirmed the formation of both types of vesicles in the same buffer conditions. We first measured the KD of the aptamer exposed to empty DOPG and POPC vesicles, which gave a KD of 20 ± 1.7 and 48 ± 8.6 μM  of 4.8 ± 1.1 °C. Encapsulation inside MA vesicles increased the Tt to 20 ± 0.5 °C  in the presence of empty MA vesicles. As expected, the Tt was not significantly altered at high concentrations , a single transition was observed with a transition temperature  by circular dichroism (CD) spectroscopy. The Tm of the aptamer in aqueous buffer, without vesicles, monitored by CD, was found to be 71 ± 2 °C  was used to monitor the conformational transition upon binding to MG. A pronounced leftward shift was observed in IA30/IA31 at varying concentrations of MG when the RNA was encapsulated (midpoint at 2.7 ± 0.5 μM) vs. exposed to empty MA vesicles  reactivity of the MG aptamer at different concentrations of the MG dye. The ratio of band intensity of A30 to A31 was found to increase monotonically with increasing MG concentration, consistent with the known participation of these residues in the MG-binding pocket Fig. 45. ThereKD of the MG aptamer in the presence of standard crowding agents. Different crowding agents caused qualitatively different effects. Dextran 40,000 (polymer of glucose) decreased the KD ~2-fold compared to an equivalent amount of glucose .The MG aptamer is known to be relatively insensitive to Mgles Fig. . Also, a2+ Figs.  and 6c. 2+ as a cofactor49, and in icy conditions, a polymerase ribozyme can accurately synthesize an RNA sequence longer than itself50. While RNAs would have been exposed to lipids in the prebiotic milieu, compartmentalization inside a protocell was critical for several reasons, including preventing the evolution of genetic parasites. To understand the effect of vesicle encapsulation on RNA activity and folding, crowded conditions can be used as an analog of confinement, but crowding agents show variable effects on RNA depending on the specific crowding agent29. Alternatively, RNA could be studied inside living cells51, but such an environment is much more complicated than a primitive protocell52. In the model system studied here, we encapsulated the MG aptamer inside vesicles made from prebiotically plausible fatty acids to mimic protocells.The earliest cells likely existed in an environment that was fairly complex and at least included lipids and various ions. The physico-chemical environment is known to be an important factor in RNA function. For example, in anaerobic conditions RNA can catalyze electron transfer reactions using FeKD and decreased the thermal stability of the MG-aptamer complex, suggesting that mere exposure to fatty acid causes some denaturation of the RNA. Interaction between RNA and lipids is believed to be mediated by both electrostatic interactions with the headgroup and hydrophobic interactions to nucleobases53, and may be enhanced by an ordered bilayer54. Electrostatic effects appear to dominate the interaction between fatty acids and minerals55. Consistent with the importance of headgroup charge, we found that the zwitterionic lipid POPC decreased aptamer activity more (KD = 48 ± 8.6 μM) than the negatively charged lipids MA (KD = 6.8 ± 1.4 μM) and DOPG (KD = 20 ± 1.7 μM). Anionic detergents  have been previously observed to completely inhibit a self-cleaving ribozyme, while non-ionic and zwitterionic detergents substantially enhanced activity56. Conversely, the cationic detergent cetyltrimethylammonium bromide (CTAB) has been shown to promote self-cleaving ribozyme reactions58, likely by increasing the rate of association and dissociation, potentially by 3–4 orders of magnitude59. Since the prebiotic milieu of protocells would have contained lipids, the comparison between RNA exposed to empty vesicles vs. RNA encapsulated inside vesicles probes how an RNA molecule that became encapsulated in a protocell would differ from an otherwise identical molecule that did not.To properly interpret experiments encapsulating RNA in model protocells, it is necessary to first determine what effect simple exposure to the lipid has on RNA folding and function Fig. . We init60. However, the activity of the ribozyme was not tested when exposed to vesicles without encapsulation, so it is unknown whether the observed effect was due to greater denaturation or to a smaller encapsulation effect compared to the aptamer studied here. It is intriguing to speculate that encapsulation might inhibit a self-cleavage reaction that produces two product molecules but promote an association between two molecules. Further studies are needed to understand how different aptamers or reactions would be affected.We found that the affinity of the MG aptamer increases when encapsulated inside vesicles Fig. . A simil40. However, this mechanism does not apply here, because the small-molecule ligand equilibrates across the membrane within few hours , well within experimental error. Thus an increased concentration of MG inside the vesicles is unlikely to explain the effect on affinity that was observed. Second, RNA–RNA interactions might be stabilizing and be enhanced inside a minority of the vesicle population that encapsulates multiple RNAs. Chance encapsulation of two RNAs in a vesicle of volume 10−19 L would correspond to ~30 μM RNA. However, the Tt of the MG aptamer was not increased when measured at this higher concentration. A related possibility would be that membrane-bound MG dye is more favorable for binding by the RNA compared to aqueous MG. However, the observation that confinement in POPC vesicles  induced an increase in MG aptamer affinity compared to an equivalent amount of monomer glucose molecules. However, as seen in other studies19, the chemical nature of the crowding agent is important, and indeed we observed qualitatively different effects using Dextran, PEG, and Ficoll. Several studies suggest that PEG in particular interacts nonspecifically with nucleic acids and proteins, leading to nontrivial effects64. These results indicate that the excluded volume effect is not necessarily the primary driver of the effect of crowding agents on the MG aptamer, and chemical interactions with cosolutes would be an important factor affecting aptamer affinity. As seen by the range of effects we observe in the presence of different crowding agents and lipids  of different states. In essence, the confinement inside a vesicle disfavors extended structures and shifts the equilibrium toward compacted structures. Although this effect is typically examined using crowding agentsTt monitored by fluorescence when RNA was exposed to MA vesicles, indicating decreased stability of interactions relevant to ligand–RNA binding . A substantial decrease in the transition temperature was also observed in the presence of 18% glucose (34 vs. 18 °C), indicating that this cosolute is also destabilizing. However, both encapsulation and the crowding effect of 18% dextran substantially increased the Tt  than for the global structure. To probe the structural transition in more detail, we assessed local nucleotide flexibility in the RNA using the SHAPE assay, which measures the relative nucleophilicity of the ribose 2′-OH groups68. The MG aptamer is known to change conformation during complex formation (‘induced fit’)69. SHAPE assays confirm that a structural transition to the bound conformation occurs at lower [MG] when the aptamer is encapsulated vs. exposed to empty vesicles. The results from SHAPE are consistent with a confinement effect, and demonstrate the utility of SHAPE for encapsulated reactions. These results are consistent with prior work, in which small RNAs were probed by NMR spectroscopy and their structures appeared to be stabilized in reverse micelles70, likely due to restricted local motion. In our work, we show that confinement (in vesicles) also leads to functional improvement for the MG aptamer. Previous studies of a small DNA duplex indicated that binding of ethidium bromine was decreased by DNA condensation in reverse micelles71. Thus while nonspecific intercalation in DNA may be inhibited by confinement, the specific binding interaction between an RNA aptamer and its target can be enhanced.If the excluded volume effect is based on a change in equilibrium between folded and unfolded structures, this change should be found when assaying the conformation of the RNA. We found a large decrease in the Tt Figs.  and 6b, 72, in which a polymer is modeled as existing in either the folded conformation or as a freely jointed chain (unfolded). The effect of confinement on the ΔΔG between the folded and unfolded states can be predicted from this model. Using parameters for ssDNA for the unfolded state , and the inner diameter of the vesicle (52 nm for MA vesicles), the ΔΔG is predicted to be −0.4kT for encapsulation inside MA vesicles (where k is the Boltzmann constant and T is absolute temperature). The three-fold increase in affinity observed in our experiments translates to ΔΔG = −1.1kT stabilization. Given the simplifying assumptions made by the model , the experimental observation is in rough agreement with the theoretical prediction. In principle, varying the confinement size and the RNA size might allow further probing of this effect, although the quantitative variation of this effect within the regime of experimentally tractable vesicle sizes and aptamers of typical size is predicted to be small , and thus raise the probability that an active sequence would emerge. Encapsulation favors compact structures relative to extended structures, an effect that would be more important for poorly folded RNAs. To the extent that fitness depends on proper folding, encapsulation would also ‘flatten’ the fitness landscape, permitting greater sequence diversity. The importance of chaperone-like activity has been emphasized in the early co-evolution of ribosomal RNA and proteinN-methylisatoic anhydride (NMIA) (Molecular Probes), dimethyl sulfoxide (DMSO) (Life Technologies), Mag-Fura-2, tetrapotassium salt  and GlycoBlue (Invitrogen) were used as received. The MG RNA aptamer (5′-GGAUCCCGACUGGCGAGAGCCAGGUAACGAAUGGAUCC-3′) and other oligonucleotides were obtained by chemical synthesis and HPLC-purified by IDT. The MG aptamer construct for SHAPE study was prepared by in vitro transcription. Other chemicals not mentioned above were purchased from Fisher Scientific.Myristoleic acid , monomyristolein (GMM), and Δ9 myristoleyl alcohol  were purchased from NuChek Prep . Phospholipids POPC and DOPG were purchased from Avanti Polar Lipids . Bicine (Alfa Aesar), Sepharose 4B (Sigma-Aldrich), malachite green (MG) chloride (Sigma-Aldrich), Dextran 40,000 , PEG 8000 (Promega), Ficoll PM 70 (Sigma-Aldrich), 41. Briefly, 3.7 μL of neat MA was mixed with one equivalent of 1 M KOH (15 μL) in water to produce a clear solution of 800 mM MA (micellar). To this solution 120 μL of 1 M bicine and 60 μL of 10× salt mix  and water were added to obtain a visibly turbid solution of 25 mM MA in 0.2 M bicine, 10 mM HEPES, 1 mM Mg-Citrate, 100 mM KCl, pH 8.5. KOH was used instead of NaOH as vesicles were noted to be less prone to aggregation in the presence of chelated Mg2+ ions74. After extrusion through 100 nm polycarbonate membrane filters (Whatman) using a Mini-Extruder , the vesicle solutions were tumbled overnight for equilibration before any subsequent experiments. For mixed composition lipid vesicles, the neat oils were mixed by pipetting before dispersion in buffer solutions39. The molar ratio of GMM and MAOH to MA was 1:2 and 1:10, respectively.Previous methodology was adopted in order to prepare vesicles from MA using the pH-drop methodThe absorbance at 600 nm was monitored with serial dilution using the cuvette-based application of the IMPLEN P300 nanophotometer. Two milliliter of a 4.3 mM vesicle suspension was prepared and the solution was serially diluted with 100–600 μL of buffer solution containing 10 mM HEPES, 1 mM Mg-Citrate, 100 mM KCl, 0.2 M bicine, pH 8.5. The point of inflection of the curve was used to estimate the CAC of the vesicles . The DOPG suspensions were stirred at 45 °C for 2 h before freeze–thawing five times with dry ice and a water bath at 45 °C. For POPC, the vesicles were freeze–thawed repetitively (10 times) with dry ice and a 25 °C water bath. Vesicles were extruded through 100 nm pores as described above, and equilibrated overnight with end-over-end tumbling.DLS measurements of the extruded vesicle solutions were performed using the Zetasizer Nano ZSP  at room temperature. Photons were collected at 173° scattering angle and the scattering intensity data were processed using the instrumental software to determine the hydrodynamic size of the vesicles.2+ (1 mM) instead of MgCl2 reduces the destabilizing effect of Mg2+ on fatty acid membranes75. Vesicles were extruded (see above) and purified from unencapsulated (free) RNAs using a Sepharose 4B size exclusion column34 with a mobile phase consisting of 10 mM HEPES, 1 mM Mg-Citrate, 100 mM KCl, 0.2 mM bicine, pH 8.5, 4 mM MA. The presence of 4 mM MA ensures membrane integrity of the vesicles during column purification and dilution, as the CAC of MA is ~4 mM41. For the purification of mixed composition vesicles, the corresponding lipid concentration used during purification was 1 mM for MA:GMM vesicles12 and 2.8 mM for MA:MAOH vesicles  than the lipid concentrations used here.The RNA aptamer solution  was heated to 90 °C for 3 min and annealed by cooling on ice for 15 min. The annealed RNA was mixed with buffer so as to obtain a final concentration of ∼33–58 μM RNA in 0.6–2.5 mM Tris, 10 mM HEPES, 1 mM Mg-Citrate, 100 mM KCl, 0.2 M bicine, pH 8.5, after addition of MA. This solution was added to the MA micellar stock (see above) to prepare vesicles encapsulating RNA. The use of citrate-chelated MgKD measurements, 20 μL of this solution was added to 20 μL of solution containing 4 mM MA, 10 mM HEPES, 1 mM Mg-Citrate, 100 mM KCl, 0.2 M bicine, pH 8.5, and varying concentrations of MG, and treated as described below. For MA:GMM and MA:MAOH vesicles, the same procedure was followed except that the buffer included the appropriate concentration (≥CAC) of the corresponding lipid solution. For phospholipid vesicles, no additional lipid was added during dilution steps due to the low CAC of these lipids.A 50 μL solution was prepared containing 4 μM RNA aptamer, 6.6 mM Tris, 10 mM HEPES, 1 mM Mg-Citrate, 100 mM KCl, 0.2 M bicine, pH 8.5. This solution was added to 100–150 μL of preformed MA vesicles  and 200–250 μL of buffer  to obtain a solution containing 0.5 μM RNA (non-encapsulated) and 8.8–11 mM MA. For λ = 660 nm) of a corresponding control sample containing the dye in the presence of the same concentration of vesicles (without aptamer) was subtracted from the fluorescence (λ = 660 nm) of vesicles with aptamer-bound dye (encapsulated or non-encapsulated). This background correction was not necessary for POPC, which did not bind MG detectably  was plotted for binding curve analysis and fitted in Origin Pro 9 by the Hill equation77F = (Fmax x Cn)/(KDn + Cn), where C is the concentration of ligand, n is the Hill coefficient, and KD is the dissociation constant. Since the interaction is known to be 1:1, curve fitting to the Hill equation was implemented with n = 1. The reported KD is the average of at least three independent experiments. During KD measurement, the average concentration of RNA was estimated to be ∼58 nM  or 0.25 μM (for RNA outside the vesicles). The concentrations of RNA were well below the KD, which allowed the use of Hill equation instead of the analytical quadratic solution78. To confirm the use of the Hill equation, KDs calculated by fitting to the quadratic solution . KD measurements in the presence of crowding agents were performed in analogous fashion without vesicles.MG was dissolved in 4 mM MA solution containing 10 mM HEPES, 1 mM Mg-Citrate, 100 mM KCl, 0.2 mM bicine, pH 8.5, to obtain a stock solution of 1–80 μM MG. Purified vesicles were incubated with various known concentrations of MG. The mixture was incubated at room temperature overnight, which allowed equilibration of the dye across the membrane. The steady-state fluorescence of the RNA aptamers at multiple concentrations of dye ligand was measured using the TECAN M200 Pro plate reader. The MG dye was excited at 617 nm and the emission monitored at 660 nm. Fluorescence intensity was normalized to a minimum of 0 and a maximum of 1. In cases including negatively charged membranes, to account for fluorescence signal arising from MG bound to the membrane /(1 + exp((Tt − T)/s)), where F refers to fluorescence (λ = 660 nm) and Fmin and Fmax are the minimum and maximum fluorescence, respectively, T is temperature, Tt is the transition temperature, and s is a fitting parameter. Tt values presented here are average of three independent experiments. Fatty acid vesicles are known to be thermostable across this temperature regime79. For samples containing vesicles, the background fluorescence (λ = 660 nm) of a control sample containing dye in the presence of vesicles (without aptamer) was subtracted from the fluorescence of vesicles with aptamer-bound dye at each temperature.The fluorescence of the aptamers was measured at different temperatures using a Fluoromax 4C (Horiba) with a Peltier-controlled temperature attachment . The fluorescence of the aptamer–dye complex was recorded in 3 °C increments from 1 to 70 °C, with a 10 min incubation at each interval. Fluorescence intensity spectra  were recorded and the baseline was subtracted from raw fluorescence values. Melting curves were fitted in Origin Pro 9 software using the Boltzmann sigmoidal equation θ = θmin + (θmax − θmin)/(1 + exp((Tm − T)/s)), where θ refers to the ellipticity at 264 nm and θmin and θmax are the minimum and maximum θ, respectively, T is temperature, Tm is the melting temperature, and s is a fitting parameter.Temperature-dependent CD spectra were acquired using a JASCO J-1500 spectrophotometer  equipped with a Peltier-controlled cell holder . A sample containing 3.8 μM RNA, 12 μM MG, 1.7 mM Tris, 10 mM HEPES, 1 mM Mg-Citrate, 100 mM KCl, 0.2 M bicine, pH 8.5 was prepared. The CD signal of the aptamer was monitored at 264 nm in 5 °C increments from 1 to 91 °C, with a 10 min incubation at each interval. The spectra were recorded using a 1 mm path length cuvette. Melting curves were fitted in Origin Pro 9 software using the Boltzmann sigmoidal equation 68 . The ssDNA was obtained from IDT and amplified through 30 cycles of PCR using KAPA Taq ReadyMix PCR kit (Kapa Biosystems) in Bio-Rad C1000 thermal cycler . PCR was carried out in 50 μL reactions , with initial melting at 95 °C for 3 min, followed by 30 cycles of 95 °C for 30 s, 57 °C for 30 s, 72 °C for 1 min, with an additional final extension at 72 °C for 1 min. The dsDNA transcription template was purified by 8% native-PAGE using gel extraction with the G-Biosciences electroelution kit and quantified by spectrophotometry (yielding 50–250 ng μL−1 in 1× TAE buffer). RNA was transcribed in 30 μL reactions  using the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB) with a 16 h incubation time at 37 °C. Transcripts were 8% native-PAGE purified, extracted by electroelution into 1× TAE, quantified with spectrophotometry, and pooled for a stock solution. RNA for all treatments was annealed simultaneously using the method described above. Encapsulation and purification of vesicles containing the MG aptamer SHAPE construct were achieved using the methods described above. Varying concentrations of MG dye were added, keeping the same RNA concentration in each MG concentration series. One hundred and thirty millimolar NMIA stock was prepared in anhydrous DMSO and diluted to a final reaction concentration of 12 mM in the samples containing RNA (0.2 μM for RNA outside vesicles and ∼50 nM for RNA inside vesicles). After a 10 h incubation of the RNA sample with MG at room temperature, NMIA was added as the acetylation agent for the SHAPE reaction. Negative control samples received the same volume of anhydrous DMSO. All samples were incubated at 10 °C overnight to allow diffusion and reaction of NMIA . Vesicle rupturing was achieved with 0.6% Triton X-100 and a 30 min, room temperature incubation. NMIA-modified RNA was recovered by ethanol precipitation with 0.3 M sodium acetate and 15 μg of GlycoBlue, followed by a wash with ice-cold 95% ethanol. Pellets were dried and resuspended in 0.5× TE buffer. SuperScript IV reverse transcriptase (Invitrogen) was used for cDNA synthesis and for transcript sequencing reactions, which used a 2:1 ratio of dNTPs to ddNTPs. Both the reverse transcription reactions and the sequencing reactions used a 5′ fluorescently tagged (Rhodamine Green) RT primer to label products for visualization on a gel (RT Primer sequence: /5RhoG-XN/GAA CCG GAC CGA AGC CCG). Reverse transcription reactions were conducted as follows: 10 μL of NMIA-reacted RNA (or 5 μL of transcript for sequencing reactions), 2 μL of 2 μM reverse primer, and 1 μL 10 μM dNTP mix were mixed and incubated at 65 °C for 5 min and annealed on ice for 5 min. Four microliters Superscript IV buffer mix, 1 μL 0.1 M DTT, and 1 μL Superscript IV reverse transcriptase were added and the reaction was incubated at 55 °C for 10 min, and then deactivated at 80 °C for 10 min. RNA was removed by base hydrolysis by addition of 1 μL of 4 M NaOH followed by neutralization by 4 μL of 1 M Tris-HCl. Samples were analyzed by 12% urea-PAGE run on an Apogee S2 sequencing gel apparatus at 1200 V for 6 h. The gel was imaged using an Amersham Typhoon 5 or Typhoon FLA9500 from GE, and band quantitation was performed using GE ImageQuant software. Band intensities were adjusted for gel background intensity, and ratiometric measurements were made to account for variations in loading and/or reverse transcription efficiency. The gel band intensity ratios were baseline-subtracted and then normalized to 1 (by dividing the maximum of the intensity ratios) for fitting to the Boltzmann equation. KD values presented here are the average of three independent experiments. Experiments were performed in triplicate.The malachite green aptamer was adapted for the SHAPE assay by adding a T7 promoter, 5′ and 3′ linker regions, and a 3′ reverse transcription (RT) primer binding siteThe amount of encapsulated RNA present in a sample of vesicles was determined by RT-qPCR. Although primers could not be designed for the MG aptamer itself due to its small size, the MG aptamer SHAPE construct was amenable to RT-qPCR. The MG construct used for SHAPE was encapsulated in vesicles as described above, and the purified vesicles were ruptured with 0.6% Triton X-100 incubated for 30 min at room temperature. RNA stocks were serially diluted two-fold in 4 mM MA containing 10 mM HEPES, 1 mM Mg-Citrate, 100 mM KCl, 0.2 mM bicine, pH 8.5, and 0.6% Triton X-100 to create a standard curve ranging from 0.08 to 0.002 μM . Additional data  are available from the corresponding author on reasonable request.Supplementary InformationPeer Review File


The RNA of desert truffles and yeast were blotted and hybridized with probes complementary to LSU extremes. RACE of LSU rRNA was carried out to determine the LSU rRNA breakage point. LSU rRNA of desert truffles presents a post-transcriptional cleavage of five nucleotides that generates a hidden gap located in domain D7. LSU splits into two molecules of 1.6 and 1.8 kb. Similar to other organisms, a UAAU tract, downstream of the breakage point, was identified. Phylogenetic comparison suggests that during fungi evolution mutations were introduced in the hypervariable D7 domain, resulting in a sequence that is specifically post-transcriptionally cleaved in some desert truffles.Despite of the integrity of their RNA, some desert truffles present a non-canonical profile of rRNA where 3.3 kb is absent, 1.8 kb is clear and a band of 1.6 kb is observed. A similar rRNA profile was identified in organisms belonging to different life kingdoms, with the exception of the Kingdom Fungi, as a result of a split LSU rRNA called  Terfezia (Tul. & Tul.) Tul. & Tul., Picoa Vittad. 1831 and Tirmania Chatin 1892. Most of the species of Terfezia, Picoa, and Tirmania establish mycorrhizal symbiosis with plants of the family Cistaceae, mainly with perennial or annual species of the genus Helianthemum , which produce hypogeous and edible fruit bodies. Their distribution is limited to arid and semi-arid regions, mainly in the Mediterranean Basin and the Middle East . The mosanthemum . This syanthemum . In addiltivated , 2017. Dltivated .Our most recent studies on desert truffles used molecular biology techniques, which often require the isolation of intact RNA , 2013. AT. claveryi RNA is analyzed in a bioanalyzer after thermal denaturation, the non-canonical band profile is displayed, where LSU rRNA is almost missing and a typical SSU rRNA (1.8 kb) and a peak similar to bacterial 16S rRNA (1.6 kb) are clearly observed. We are perhaps faced with a unique event throughout the evolutionary history of the Kingdom Fungi. Our hypothesis is that the non-canonical rRNA band profile observed in some desert truffles is due to a specific post-transcriptional processing of LSU called “hidden gap,” even though it had not been observed in fungi before. To explain this pattern, the structure of T. claveryi LSU rRNA was studied using molecular techniques, with the main objective of uncovering the existence and mapping the position of a hidden gap in the LSU rRNA in fungi.The LSU rRNA hidden break has not been reported in fungi yet. However, when T. claveryi, Terfezia arenaria (Moris) Trappe and Picoa lefebvrei (Pat.) Maire, mycelium of Tirmania nivea (Desf.) Trappe or yeast using spin columns  according to the manufacturer’s instructions. In the case of T. claveryi, the CTAB protocol . Samples were denatured at 65°C for 10 min, placed on ice, and then loaded. Electrophoresis was carried out at ∼50 V for 6 h in 1X MOPS containing 1,5% formaldehyde as running buffer. Samples were blotted to positively charged Nylon membranes (Roche) in SSC 10X overnight. The membranes were hybridized with probes  overnight. Stringency washings were performed with 0.1X SSC- 0.1% (w/v) SDS, at 68°C for 30 min. Target RNA was detected according to the manufacturer’s protocol  using CSPD as chemiluminescent substrate.RNA was electrophoresed in denaturing 1.2% (w/v) agarose gels prepared in double-distilled HT. claveryi 5′ RT specific primer. After digestion of residual RNA with 1 U of RNase H (Invitrogen), cDNA was purified with a silica column  and poly(A) tailed with terminal transferase (Roche). Three rounds of PCR were performed, the first with primers 5′ RACE oligo dT adaptor, LR9 and 5′ RACE outer adaptor, the second with primers 5′ RACE adaptor and 26S 5′ RACEnest2 and the third with primers 5′ RACE adaptor and 26S 5′ RACEnest3. For 3′ RACE, 2 μg of DNase-treated RNA  were polyadenylated with Poly(A) polymerase (Ambion) and reverse transcribed with an oligo-(dT)-adapter primer. First round and nested PCRs were performed with the primers shown in Table 1 and Supplementary Figure RACE experiments were carried out as described by 1) for T. boudieri , T. claveryi , Tuber melanosporum , Pyronema confluens  and Morchella conica , and Saccharomyces cerevisae (NR_132209.1) from NCBI. Moreover, we sequenced and deposited at NCBI the region encompassing the hidde gap of T. arenaria (MH160402), Terfezia extremadurensis (MH160403), Terfezia fanfani (MH160404), T. nivea (MH160405). All the sequences were aligned using the program ClustalO . In addition, a peak similar of 1.6 kb was barely but clearly observed in T. claveryi, T. arenaria, and T. nivea . In T. arenaria the LSU peak seems smaller than in other species but this is the result of a partial denaturation during RNA extraction . On the other hand, the peak belonging to a fragment of approximately 1.8 kb, typical of the 18S rRNA, was still observed and the intensity of the 18S band had increased, as indicated by a greater FU (fluorescence unit) value in comparison with the FU value of the 18S rRNA peak for each non-heat-denatured sample . The presence of a second anomalous peak with an approximate size of 1.6 kb and intensity similar to the 18S peak was observed only in T. nivea and Terfezia species RNA . Low molecular weight RNA fractions, including a peak with an approximate size of 190 bp corresponding to 5.8S, were observed in both heat-denatured or non-heat-denatured conditions with similar intensity. Similar band profiles were observed in several ascocarps and with different RNA extraction protocols for all the species. For T. claveryi, the band profiles were also confirmed in RNA extracted from mycelium.The integrity of total RNA samples from traction . The thiT. claveryi LSU rRNA, respectively. They were used in Northern blot experiments with total RNA from T. claveryi, T. arenaria and yeast. In the case of yeast total RNA, both 26S_5′ and 26S_3′ probes detected the same RNA species of approximately 3.3 kb . However, in the case of T. claveryi and T. arenaria RNA, the 26S_5′ probe detected an RNA species of approximately 1.6 kb and the 26S_3′ probe detected another of approximately 1.8 kb . A very faint 3.3 kb band was also detected in T. claveryi RNA, suggesting that very low amounts of the uncut species were still present. When the blot hybridized with the 3′ probe was reprobed with the 5′ probe, no change in the 26S band of yeast rRNA was observed, but in both Terfezia species a distinct signal of about 1.6 kb was observed below the pre-existing 1.8 kb band . These results allowed us to conclude that: (i) the hidden gap maps within 1.6 kb from the 5′extreme and 1.8 kb from the 3′extreme of LSU, (ii) the 1.8 kb band, co-migrates with uncleaved 18S RNA and corresponds to the 3′ portion of LSU rRNA, (iii) the faint 3.3 kb band observed in T. claveryi corresponds to an uncut version of LSU rRNA and (iv) the absence of 28S rRNA is not due to a non-specific degradation.Preliminary attempts to amplify different portions of the LSU rRNA by RT-PCR always failed when primers where designed encompassing a region of about 1 kb asymmetrically positioned within the 3′ half of the LSU rRNA molecule. On the basis of this observation and to confirm the presence of the hidden gap, two probes were designed, named 26S_5′ and 26S_3′, complementary to the 5′terminal and 3′terminal regions of T. claveryi 28S rRNA breakage point, primers to perform 5′ RACE and 3′ RACE were designed. Two cleavage sites, and between them a gap of five nucleotides, were identified after aligning with the T. claveryi LSU rDNA sequence . Different rDNA sequences of Terfezia spp. and T. nivea, which are known to present non-canonical rRNA profiles, were aligned with other Pezizomycetes fungi. All the tested Terfezia and Tirmania rDNA sequences are conserved in the five nucleotide gap, nine nucleotides upstream the gap and 10 nucleotides downstream the gap. In fact, these 24 nucleotides  seem to be a hypervariable region D7 within the LSU. This region D7 has been identified for being the closest downstream variable region to the conserved primer LR7 in the alignment. Four nucleotides downstream of the 26Sβ_5′extreme can be identified as a UAAU tract .To determine the site and sequence of the The LSU gene is organized into several highly conserved nuclei interrupted by 12 divergent domains, also called “expansion segments”  or “D doT. claveryi allowed also us to identify a very faint band, suggesting that very low amounts of the immature species were still present . In this respect, the Northern blot of  present  or that  present , which cT. claveryi LSU rRNA results in two molecules of 1.6 and 1.8 kb, corresponding to the 5′ and 3′ ends, which has not been previously observed in fungi even when it seems to be widespread in others kingdom, including bacteria, protozoa, worms, several arthropods, fish and rodents , T. extremaduriensis, T. fanfani, T. boudieri (data not shown)] and even to the closest genus Tirmania , but have not been reported in other Pezizomycetes such as Picoa lefbvrei , Tuber melanosporum, Morchella conica, or Pyronema confluens. During Pezizomycetes evolution, the introduction of sequences susceptible to cleaving to form a hidden gap must have appeared relatively late in their evolution, since, as far as we know, few genera share the hidden gap within the Pezizomycetes order.To date, the species where the hidden gap has been observed belong to Sciara coprophila rDNA into Xenopus laevis oocytes confirmed that during pre-28S rRNA fragmentation, specific processing machinery is not required. Instead, oocytes may provide the required accessory factors, suggesting that the gap processing mechanism is served by an evolutionarily conserved apparatus (T. claveryi and T. boudieri present the UAAU tract downstream of the 26Sβ rRNA.The molecular mechanism behind the introduction of this hidden break remains unclear. Microinjecting pparatus . Alternapparatus . Nishimupparatus . The clepparatus . As wellin vitro within tetraploid Cyprinid species (Arabidopsis thaliana mutant, which exhibit defective chloroplast 23S rRNA fragmentation, have a drastically reduced level of ribulose-1,5-bisphosphate carboxylase/oxygenase and of other chloroplast-encoded photosynthetic proteins (Terfezia species, which are also adapted to extreme environments, display high resistance to drought and high temperatures (Terfezia 28S rRNA influences translation efficiency and what impact this can have on its biological cycle is still unknown and deserves further research.Regarding the possible biological role of this post-transcriptional maturation, several interpretations have been proposed. One theory proposes a compensatory protein translation rate of hidden gap ribosomes since they show reduced efficiency for protein synthesis  species . The sec species . Additioproteins . The thiproteins . Terfezieratures . WhetherAN-R, AC, and AM conceived and designed research. AN-R and AC completed the experiments and analyzed the data. AN-R wrote the manuscript. AC and AM reviewed the manuscript. AM provided financial support.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. The handling Editor declared a shared affiliation, though no other collaboration, with one of the authors AC.


Cancer patients are likely to undergo osteoporosis as consequence of hormone manipulation and/or chemotherapy. Little is known about possible increased risk of fracture in this population. The aim of this study was to describe the biomechanical effect of a metastatic lesion in an osteoporotic lumbar spine model.A finite element model of two spinal motion segments (L3-L5) was extracted from a previously developed L3-Sacrum model and used to analyze the effect of metastasis size and bone mineral density (BMD) on Vertebral bulge (VB) and Vertebral height (VH). VB and VH represent respectively radial and axial displacement and they have been correlated to burst fracture. A total of 6 scenarios were evaluated combining three metastasis sizes  and two BMD conditions .15% metastasis increased VB and VH by 178% and 248%, respectively in normal BMD model; while VB and VH increased by 134% and 174% in osteoporotic model. 30% metastasis increased VB and VH by 88% and 109%, respectively, when compared to 15% metastasis in normal BMD model; while VB and VH increased by 59% and 74% in osteoporotic model.A metastasis in the osteoporotic lumbar spine always leads to a higher risk of vertebral fracture. This risk increases with the size of the metastasis. Unexpectedly, an increment in metastasis size in the normal BMD spine produces a greater impact on vertebral stability compared to the osteoporotic spine. The spine is the most common site for skeletal metastasis, with one third of all cancer patients developing metastases of the spine . BecauseMany patients with metastases of the spine are likely to decrease their bone mineral density (BMD), leading to osteopenia or osteoporosis, as a consequence of hormone manipulation and/or chemotherapy , increasBiomechanical studies investigating the risk of fracture in metastatic spines lack realistic models and are not ideal for parametric analyses . BecauseThe aim of this study was to evaluate the biomechanical effects of a metastatic lesion in an osteoporotic model of the lumbar spine.A finite element model of two spinal motion segments (L3-L5) was extracted from a previously developed and validated three-dimensional, nonlinear, ligamentous L3-Sacrum model , 26 Fig. DevelopThe finite element mesh was generated using HyperMesh 10.0 .The model included the vertebral bodies consisting of a cortical shell, cancellous centrum, endplates and posterior elements. Intervertebral discs consisted of a nucleus pulposus, annulus ground substances, and annulus fibrosus (seven layers). All major ligaments were represented: . The ligaments were modeled with 3-D two node truss elements and assigned nonlinear hypoelastic material properties, which allow axial stiffness to be a function of axial strain. At initially low strains the ligaments exhibit low stiffness, but as the strains increase the ligament stiffness increases. The cross-sectional areas of each ligament were obtained from literature. All elements representing the ligaments were aligned in the anatomical fiber direction and unstressed at the unloaded state .The metastasis was represented as a lytic bone lesion. Material properties for all structures are summarized in Table Nodes at the lower surface of the L5 vertebra, corresponding to the endplate, were encastred and constrained from all three axes of rotation and translations. A reference node was placed 10 mm above the L3 vertebra and surface nodes from the vertebra and the reference node were selected to form rigid bodies and create a kinematic coupling. This constrain limited the motion of aforementioned nodes to the motion applied to the reference node . An axiaA parametric analysis was performed varying metastasis size and BMD. Metastatic lesion was varied to represent 15% and 30% of the vertebral body volume by selecting elements at the central core of the L4 vertebral body. Fig. . OsteopoVertebral fracture risk was analyzed based on vertebral bulge (VB) and vertebral height (VH) outcomes . VertebrThe distance between two standard nodes at the mid-height of L4 on the sagittal plane was measured . In normal BMD scenarios, 15% metastasis increased VB by 178% (0.139 mm) and VH by 248% (0.522 mm); while a 30% metastasis increased VB by 424% (0.262 mm) and VH by 626% (1.09 mm). A comparison of metastatic lesion size showed a 30% metastasis to increase VB by 88% (0.139 mm vs 0.262 mm) and VH by 109% (0.522 vs 1.09) when compared to a 15% metastasis. In osteoporotic scenarios, a 15% metastasis increased VB by 134% (0.288 mm) and VH by 174% (1.145 mm); while a 30% metastasis increased VB by 272% (0.458 mm) and VH by 479% (1.996 mm). Comparing the two metastatic scenarios (15% and 30%), VB increased by 59% (0.288 vs 0.458) and VH by 74% (1.145 vs 1.996). When same metastasis size scenarios were compared, the osteoporotic spine had larger VB and VH values compared to normal BMD scenario. Results are summarized in Table The purpose of this study was to describe the biomechanical effect of a metastatic lesion in an osteoporotic lumbar spine model in order to better understand the risk of vertebral fractures in this population. A finite element model of two spinal motion segments (L3-L5) was used to analyze the effect of metastasis size and osteoporosis on VB and VH. Results from the study showed osteoporosis can represent a risk of fracture regardless of metastasis size compared to patients with normal BMD. Furthermore, an increase in metastasis size has a greater impact on the risk of fracture in patients with normal BMD compared to osteoporotic patients.A previous study by Taneichi et al.  identifiTo the best of our knowledge, this is the first three-dimensional, anatomical model of two spinal motion segments (L3-L5) that investigates a metastatic lesion in an osteoporotic spine. A previous study by Whyne et al., investigated the effects of tumor size, material properties and compressive loading rate on vertebral strength, using a two-dimensional, symmetric finite element model of the L1 vertebral body without posterior elements . Two addThis study has limitations. The L3-L5 finite element model was extracted from a previously developed and validated three-dimensional, nonlinear, ligamentous L3-Sacrum model , 26. PriA metastasis in the osteoporotic lumbar spine always leads to a higher risk of vertebral fracture. This risk increases with the size of the metastasis. Unexpectedly, an increment in metastasis size in the normal BMD spine produces a greater impact on vertebral stability compared to the osteoporotic spine.


Fighting external pathogens requires an ever-changing immune system that relies on tight regulation of gene expression. Transcriptional control is the first step to build efficient responses while preventing immunodeficiencies and autoimmunity. Post-transcriptional regulation of RNA editing, location, stability, and translation are the other key steps for final gene expression, and they are all controlled by RNA-binding proteins (RBPs). Nowadays we have a deep understanding of how transcription factors control the immune system but recent evidences suggest that post-transcriptional regulation by RBPs is equally important for both development and activation of immune responses. Here, we review current knowledge about how post-transcriptional control by RBPs shapes our immune system and discuss the perspective of RBPs being the key players of a hidden immune cell epitranscriptome. Immune cell development and function are not only regulated by gene networks controlled by transcription factors but also by post-transcriptional regulatory mechanisms, controlled by RNA-binding proteins (RBPs) and non-coding RNAs, that are essential for immune cell lineage commitment, maintenance, and modulation of immune responses , 2. HereTnf, Il1, Ifng, and Csf2 (Pioneering studies discovered the presence of nucleotide regulatory sequences in the 3′ untranslated region (3′UTRs) of dozens of messenger (m) RNAs encoding cytokines, such as and Csf2 , that weand Csf2 –9. With and Csf2 , 11. RNANeST, Xist, Air, and Hotair, modulate transcription by binding to proteins in histone-modifying complexes and targeting them to selected genes including Ifng  –15. In ming Ifng –19. Otheolerance , 21. Cirolerance . cRNAs aolerance , 24. AltRecent progress in RNA biology has brought us to the realization that chemical modification of the RNA  can exert key roles in cell maintenance, development, and differentiation in the immune system. Methylation, hydroxylation, and uridinylation of ribonucleotides were discovered over 50 years ago . But, itRNA-binding proteins control all aspects of mRNA biology Figure A. In theIn the cytoplasm, RBPs control the dynamic location and assembly of mRNAs into polyribosomes for protein synthesis. Recognition of the 5′-CAP and RNA regulatory motifs in the 5′UTR  by eukaryotic initiation factors enables formation of the 43S pre-initiation complex, ribosome assembly, and translation. mTOR-dependent regulation of the 4E-BP/eIF4E axis is essential for lymphocyte growth, proliferation, activation, and differentiation –56. OtheRc3h1 and Rc3h2). Stable and actively translated mRNAs are bound by the RBP PABP, which also interacts with eIF4E to assemble the 43S pre-initiation complex and the ribosome. Recognition of constitutive decay AU- and GU-rich elements by mRNA decay activators such as Roquin1/2, ZFP36, KSRP, and CUGBP/CELF recruit polyA ribonucleases  Figure B. NMD isThe importance of RBP in miRNA biogenesis and function has been reviewed extensively –102. BriRNA location and storage are important mechanisms for timely protein synthesis in immune cells. Cytoplasmic RNA granules, including processing (P-) bodies and SGs, are assembled in both T and B cells upon activation with mitogens , 115. P-in vivo and in vitro  present in the Bcl2 3’UTR. Gene targeting deletion of these Bcl2 AREs diminishes Bcl2 mRNA stability and protein levels in primary B cells, decreasing life- span of transitional (T) and FO B cells  is required for BCL2, A1, and BCL-XL expression and maintenance of follicular (FO) B cells . hnRNPC  B cells . Stringe B cells .The role of RBPs such as ZFP36, Roquin, Regnase-1, hnRNPC and ELAVL1 in myeloid cell activation has been studied extensively. Mice lacking the expression of ZFP36, Roquin, Regnase-1, and AUF1 share pro-inflammatory syndromes arising from a failure to limit cytokine production. Timely expression of cytokines and growth factors is regulated by RBPs that bind to RNA regulatory elements present in their 3′UTR. Constitutive decay elements (CDEs), AU-rich elements (AREs), and miRNA recognition elements control cytokine mRNA stability and decay. CDE and ARE show very low sequence complexity and can be bound by multiple RBPs sometimes mediating opposing functional outcomes. Widespread 3′UTR shortening and removal of decay elements may be a general mechanism used by T cells and macrophages to increase the expression of cytokines upon activation , 44, altCD274 mRNA decay, and promotes tumor-immune evasion   . ELAVL1 nd Ccl2) . These rrophages , 173. Si protein . This co protein , 175. In protein . Regulat protein –179. Cel protein , 152. ThRNA-binding proteins coordinate global changes in mRNA expression and splicing upon TCR- and CD28-mediated T-cell activation –183 Fig. Intron Malt1 exon 7 inclusion acting as a negative feedback regulator  is expressed upon B-cell activation and is necessary for affinity maturation in GCs. Affinity maturation is the process by which SHM of the Ig locus enables the expansion of the antibody repertoire. AID is an RNA/DNA binding protein that binds to G-quadruplex structures formed upon active transcription of the Ig locus    to reguleticulum , 218. Byque mice .Post-transcriptional control by RBPs is an essential extra layer of gene regulation that is fundamental for the development, homeostasis, and function of the immune system. The involvement of RBP at all stages of the biology of RNA can be conceptualized by considering RBPs as writers, editors, readers, and erasers of the transcriptome; an analogy that has been used previously in the context of epigenetic regulation at the level of DNA and histones. RBP writers  affect transcription and RNA processing. RBP editors  modify the sequence content of the transcriptome. RBP readers bind RNA and define subcellular location , and translation . Finally, RBP erasers  induce RNA decay. Acting in concert with transcription factors, epigenetic regulators, and signal transduction networks, they comprise a global regulatory network that we are just starting to appreciate.6A modification of the RNA might differ between different RNA species and during T-cell activation (A reductionist approach to study the role of RBPs at a single gene level is likely to be insufficient to uncover the broad implications of post-transcriptional RNA control in immune cell development, differentiation, and function. An integrative analysis of the protein:RNA interactome, transcriptome, and translatome is required to understand the global mechanisms of RBP-mediated control. Gaining knowledge of how individual RBPs target single RNA molecules and their function is a first step to further define these global cellular mechanisms. Collection of the protein:RNA interactome from hundreds of RBPs is extremely useful in order to infer possible interactions of different RBPs for single or global gene expression. Capturing the dynamic changes of cellular RBP-content in concert with the quantitative and qualitative changes in the transcriptome is a key to understand immune cell development and activation. For example, it has been reported that mtivation , 220. Cetivation . HoweverMDD-M, manuscript conception, literature review, and writing. MT, conception and writing.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.