The early detection of metastasis based on biomarkers in plasma may improve cancer prognosis and guide treatment. The aim of this work was to characterize alterations in metabolites of the arginine pathway, energy metabolism, and structural and signalling lipids in plasma in the early and late stages of murine breast cancer metastasis.Mice were orthotopically inoculated with 4T1 metastatic breast cancer cells, and plasma was analysed along the pulmonary metastasis progression using LC-MS/MS-based targeted metabolomics and lipidomics.Based on primary tumour growth and pulmonary metastases, 1–2 weeks after 4T1 cancer cell inoculation was defined as an early metastatic stage, and 3–4 weeks after 4T1 cancer cell inoculation was defined as a late metastatic stage. Early metastasis was featured in plasma by a shift of L-arginine metabolism towards arginase (increased ornithine/arginine ratio) and polyamine synthesis (increased putrescine). Late metastasis was reflected in plasma by further progression of changes in the arginine pathway with an additional increase in asymmetric dimethylarginine plasma concentration, as well as by a profound energy metabolism reprogramming towards glycolysis, an accelerated pentose phosphate pathway and a concomitant decrease in tricarboxylic cycle rate (“Warburg effect”). The late but not the early phase of metastasis was also characterized by a different lipid profile pattern in plasma, including a decrease in total phosphatidylcholines, a decrease in diester-bound phospholipid fraction and an increase in lysophospholipids associated with an increase in total sphingomyelins.The early phase of metastasis in murine 4T1 metastatic breast cancer was associated with plasma metabolome changes characteristic of arginase activation and polyamine synthesis. The late metastasis was reflected in plasma not only by the alterations in arginine pathways but also by a shift towards glycolysis and the pentose pathway, remodelling of structural lipids and activation of lipid signalling, all of which coincided with metastasis progression.The online version of this article (10.1186/s13058-018-1075-y) contains supplementary material, which is available to authorized users. Although significant progress has been made in cancer research during recent decades, breast cancer is still the most frequently diagnosed tumour in women worldwide. The survival rate of primary breast cancer is now close to 100%, but it decreases to only 25% once distant metastasis has occurred , and sevSeveral recent publications have demonstrated that targeted metabolomics can prove useful not only for diagnosis and monitoring response to treatment but also for finding a relationship between clinicopathological characteristics of breast cancer and various metabolites in blood, urine and tissue specimens . DepletiArginine is a dibasic, semi-essential amino acid involved in many biosynthetic pathways that can significantly influence carcinogenesis and tumour biology. It is metabolized mainly by arginase (ARG) to urea and ornithine, which can be further transformed into polyamines, and by nitric oxide synthase (NOS) into nitric oxide (NO) and citrulline . PolyamiMetabolism reprogramming towards aerobic glycolysis—the so-called ‘Warburg Effect’ —represenFinally, changes in lipid metabolism were shown to be closely associated with cancer progression. Alterations in lipid composition and abundance have been established as a hallmark of cancer aggressiveness  that mayGiven that changes in arginine metabolism –21, enerWe identified that the first systemically detectable metabolic changes in plasma were linked with a shift in arginine metabolism toward ARG, associated with early changes in immune response as it coincided with infiltration of granulocytes and extramedullary haematopoiesis. Later changes in arginine metabolism also involved an increase in plasma asymmetric dimethylarginine (ADMA) concentration, suggesting endothelial dysfunction. In turn, the late phase of metastasis was associated with plasma changes suggesting energy metabolism reprogramming, modulation of structural lipid content, and increased release of pro-inflammatory lipid mediators, all of which coincided with tumour growth and metastasis formation.4 viable mouse mammary adenocarcinoma 4T1 cells  cultured according to a previously described protocol [Seven- to eight-week-old BALB/c female mice  were orthotopically inoculated into the right mammary fat pad with 1 × 10protocol . HealthyAll experimental procedures involving animals were conducted according to the Guidelines for Animal Care and Treatment of the European Communities and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All procedures were approved by the Local Ethical Committee on Animal Experiments at the Jagiellonian University (17/2016).g at 4 °C for 12 min, with plasma then kept for further analysis. Tumour size was calculated by measuring the tumour length (L) and width (W) using an electronic calliper and expressed as tumour volume V = 0.52 × L × W2. In order to determine pulmonary metastasis, representative lung tissues from four mice were kept in buffered formalin solution, and the macroscopic lesions were counted in the lungs, as previously described [Blood count was measured by using an ABC Vet analyser , and remaining blood was centrifuged at 3000 × escribed . For exaIn order to determine the concentration of selected metabolites from studied pathways, three LC-MS/MS targeted metabolomics-based methods were applied. The arginine metabolites were evaluated on the basis of LC-MS/MS measurements using the AbsoluteIDQ p180 Kit  and a developed LC-MS/MS targeted-metabolomic method, which was also used for energy metabolite determination. The lipid profile was evaluated using a flow injection analysis (FIA)-MS/MS approach from the AbsoluteIDQ p180 Kit. The concentration of sphingosine-1-phosphate (S1P) was measured by using an LC-MS/MS-based method, described below.Energy metabolites , amino acids and other intracellular metabolites measured in plasma) were measured by employing an LC-MS/MS technique on a QTRAP 5500 mass spectrometer  coupled to a UFLC Nexera chromatograph  using an Acquity UPLC BEH C18 column, 1.7 μm, 3.0 × 100 mm  as an analytical column. Samples were analysed twice in positive and negative ionization multiple reaction monitoring mode. The detailed methodology is included in Additional file Amino acids, biogenic amines and lipids in plasma were measured using the commercially available AbsoluteIDQ p180 kit, which allows for simultaneous quantification of 21 amino acids, 14 biogenic amines, 40 acylcarnitines, 90 glycerophospholipids, 15 sphingolipids and sum of hexoses. The sample preparation and measurement were carried out according to the kit manufacturer’s instructions. Briefly, 10 μl of standards, quality controls and plasma samples were pipetted onto filter paper within each well plate of the 96-well plate provided by the kit manufacturer. The samples were dried under nitrogen, followed by application of phenylisothiocyanate , then they were dried again, and metabolites were extracted from the filter paper using 5 mM ammonium acetate in methanol solution and diluted. Samples were measured twice by LC-MS/MS (amino acid and biogenic amines) and FIA-MS/MS (lipids and hexoses) using the QTRAP 5500 mass spectrometer coupled to a UFLC Nexera chromatograph. Quantification of metabolite concentration and quality assessment were performed with MetIQ software .The concentration of S1P in plasma was determined by using an LC-MS/MS technique with a TSQ Quantum mass spectrometer system  coupled to a UHPLC UltiMate 3000 . Chromatographic separation was achieved with a Hypersil GOLD analytical column . The detailed methodology is included in Additional file P values are provided in the figure legends. For multivariate analysis, data of all experiments were merged into a matrix table. Partial least squares discriminant analysis (PLS-DA) and heat map clustering of the data were performed using web-based software  [Statistical analysis was performed using STATISTICA 12 software . All values are expressed as mean ± SEM. The assessment of normality and homogeneity of variances was performed using the Shapiro-Wilk and Levene tests, respectively. To assess the statistical significance, one-way analysis of variance (ANOVA), Tukey’s posthoc test and a non-parametric Kruskal-Wallis ANOVA were performed. yst 4.0) .3, respectively, which represented c.a. 1.18% of body weight, whereas 4 weeks after 4T1 cell inoculation, the mean tumour weight and volume were 1.85 g ± 0.65 g and 1285 ± 415 mm3, respectively, which represented 10% of total body weight  and reached 50.6 ± 15.89 × 103/μl and 189.9 ± 73.7 × 103/μl 3 and 4 weeks, respectively, after cancer cell inoculation. Cancer-associated leucocytosis was linked with a relative increase in number of granulocytes and monocytes and a decreased number of lymphocytes, which was pronounced 3–4 weeks after cancer induction  and included a decrease in total phosphatidylcholine level  with a simultaneous increase in the concentration of lysophosphatidylocholines (LysoPC), the changes being compatible with the increase in phospholipase A2 activity Fig. a, b. TheThe decrease in PC fraction was related to a concomitant increase in sphingomyelin (SM) fraction in the plasma phospholipid profile, also visible to some extent in the early phase of metastasis. The SM content was elevated by c.a. 15% and 30% at 2 and 4 weeks, respectively, after cancer cell inoculation   Fig. b. Hierar.In the present work, taking advantage of targeted LC-MS-based metabolomics and lipidomics focused on arginine metabolism, energy metabolism and structural and signalling lipids, we demonstrated that in 4T1 metastatic breast cancer in mice, the early phase of metastasis was mirrored in plasma by changes in arginine metabolism characteristics for ARG activation and polyamine synthesis. The late metastasis, apart from alterations in arginine pathways, was characterized by a shift of energy metabolism towards glycolysis and PPP, remodelling of structural lipids and activation of lipid signalling Fig. . AltogetThe salient finding of this study was the early detection of a significant change in arginine metabolism that was detected in plasma even before the first breast cancer metastasis to the lung and involved acceleration of arginine catabolic rate as well as an increased polyamine synthesis. Acceleration of ARG activity seemed to coincide with the increased production of leukocytes Table , which wThese changes were followed by alterations of ADMA synthesis regulating NOS-dependent function in the late phase of metastasis Fig. f. The foNω-hydroxy-nor-l-arginine reduced the inhibitory effect of MDSCs on T-cell proliferation and reduced the number and size of pulmonary metastasis [Altogether, we claim that induction of ARG activity detected in plasma in the early phase of metastasis formation in 4T1 metastatic breast cancer in mice contributes to metastasis progression. Indeed, it was shown that the ARG inhibitor tastasis . Moreovetastasis . Novel cSurprisingly, in contrast to changes in arginine metabolism that occurred in an early phase of metastasis, changes in energy metabolism Fig.  were obssee review [In the present work, we also identified a number of changes in lipid profile that all occurred in the late phases of cancer and metastasis Fig.  and invoe review ). In ture review , 58. Thee review . Additioe review  and therIn the present work, on the basis of multivariate statistical analysis, we confirmed that the plasma profile of arginine metabolism, energy metabolism and lipids was clearly distinct for 4T1 breast cancer-bearing mice and control mice, as well as for the late metastatic group (3 and 4 weeks after cancer cell inoculation) and the early metastatic and control groups (1–2 weeks after cancer cell inoculation), taken together. The early metastatic group was also to some extent differentiated from the control group on the basis of changes in arginine metabolism and some lipids. Our multivariate statistical analysis was performed based on ten animals per experimental groups. Obviously, this approach was sufficient for the detection of meaningful changes in targeted analysis of selected metabolites, but it was not sufficient to achieve high statistical power in multivariate statistical analysis.In the present work, we demonstrated that progression of 4T1 metastatic breast cancer in mice was linked to distinct metabolomic changes in plasma in the early and late phases of metastasis. The early phase of metastasis was featured by an increase in ARG activity and polyamine synthesis that can be associated with ARG-dependent immunosuppression and polyamine-dependent cellular proliferation, respectively. The late phase of metastasis was reflected in plasma by alterations in arginine pathways involving not only ARG activity and polyamine synthesis but also increased ADMA synthesis that can be linked with an impairment of endothelial function. However, the late phase of metastasis but not the early phase was featured in plasma by reprogramming of energy metabolism toward glycolysis and the pentose phosphate pathway at the expanse of the TCA, as well as by remodelling of structural and signalling lipids, all of which coincided with metastasis progression. Altogether, the ARG pathway constitutes an early plasma biomarker of cancer and metastasis and an important target for therapy, whereas metabolism reprogramming towards glycolysis and changes in signalling and structural lipids, if detected in plasma, constitute biomarkers for advanced cancer and the late phase of metastasis.Additional file 1:Table S1. Gradient elution program applied for UPLC-MS analysis for positive ionisation mode. Table S2. Gradient elution program applied for UPLC-MS analysis for negative ionisation mode. Table S3. LC-MS/MS parameters in positive and negative ESI mode for determination of metabolites in plasma. Table S4. LC-MS/MS parameters for determination of S1P in plasma. (DOCX 24 kb)


Stylonychia, somatic macronuclei differentiate from germline micronuclei during sexual reproduction, accompanied by developmental sequence reduction. Concomitantly, over 95% of micronuclear sequences adopt a heterochromatin structure characterized by the histone variant H3.4 and H3K27me3. RNAi-related genes and histone variants dominate the list of developmentally expressed genes. Simultaneously, 27nt-ncRNAs that match sequences retained in new macronuclei are synthesized and bound by PIWI1. Recently, we proposed a mechanistic model for ‘RNA-induced DNA replication interference’ (RIRI): during polytene chromosome formation PIWI1/27nt-RNA-complexes target macronucleus-destined sequences (MDS) by base-pairing and temporarily cause locally stalled replication. At polytene chromosomal segments with ongoing replication, H3.4K27me3-nucleosomes become selectively deposited, thus dictating the prospective heterochromatin structure of these areas. Consequently, these micronucleus-specific sequences become degraded, whereas 27nt-RNA-covered sites remain protected. However, the biogenesis of the 27nt-RNAs remains unclear. It was proposed earlier that in stichotrichous ciliates 27nt-RNA precursors could derive from telomere-primed bidirectional transcription of nanochromosomes and subsequent Dicer-like (DCL) activity. As a minimalistic explanation, we propose here that the 27nt-RNA precursor could rather be mRNA or pre-mRNA and that the transition of coding RNA from parental macronuclei to non-coding RNAs, which act in premature developing macronuclei, could involve RNA-dependent RNA polymerase (RDRP) activity creating dsRNA intermediates prior to a DCL-dependent pathway. Interestingly, by such mechanism the partition of a parental somatic genome and possibly also the specific nanochromosome copy numbers could be vertically transmitted to the differentiating nuclei of the offspring.In the ciliate  Arabidopsis [Tetrahymena (class: Oligohymenophorea) [Stylonychia or Oxytricha, since this key mechanism seems to differ fundamentally from Tetrahymena or Paramecium—two genera for which much more data is available to date.Many small non-coding RNAs (ncRNAs) inhabit control function for cellular processes including the regulation of gene expression and chromatin structure. The RNA interference (RNAi)-dependent formation of heterochromatin might be best understood in fission yeast , Arabidobidopsis  and the ophorea) ,4, whereophorea) ,6. MacroTetrahymena, non-coding ‘scnRNA’ guide the programmed elimination of germline-restricted DNA sequences, which become targeted by sequence homology. Their precursors are bi-directionally transcribed from the micronuclear chromosomes encompassing germline-restricted and macronucleus-destined sequences. Then, Dicer-like activity by Dcl1p is required for the production of ~29nt-RNAs, which become bound by the PIWI-homolog Twi1p followed by slicing, i.e. the passenger strand removal, and 3’-end 2’-O-methylation via the methyltransferase Hen1p. Subsequently, a genome comparison/selection process (‘scanning’) is undergone in the degrading parental macronucleus, leading to the degradation of ~29nt-RNAs matching to homologous macronucleus sequences. The resulting scnRNAs target micronucleus-specific sequences not required in the mature macronucleus. Subsequently, H3K9me3 and H3K27me3 become enriched at these sequences. This process involves the activity of the histone lysine methyltransferase (KMT) EZL1. Prior to their elimination, these sequences become transformed into heterochromatin through binding of the chromodomain protein PDD1p at both H3K9me3 and H3K27me3 [Paramecium. Here, ~25nt-RNAs result from precursors, which derive from whole micronuclear genome transcription. In the parental macronucleus, they undergo a genome comparison against macronuclear non-coding transcripts, which are constitutively produced in vegetative cells [In H3K27me3 . A similOxytricha trifallax and Stylonychia lemnae comprises of so-called nanochromosomes harbouring mostly one or few genes flanked by discrete telomeric repeats. In a mature macronucleus, each nanochromosome occurs in a specific copy number [Oxytricha it was proposed that these 27nt-RNAs protect specific sequences from being degraded. However, no deeper mechanistic insight was provided [Oxytricha 27nt-RNAs demonstrating that they are not modified by 2’-O-methylation at their 3’-end, in contrast to Tetrahymena scnRNAs [Stylonychia, a telomeric G-quadruplex resolving RecQ-like helicase activity (in association with telomerase) was exclusively observed in the replication band of vegetative macronuclei, but not in the parental macronulcear fragments during sexual reproduction [Stylonychia justifies an alternative hypothesis how the biogenesis of 27nt-RNAs could occur. Our thoughts will be exemplified in the following paragraphs.Interestingly, in ciliates belonging to the class Spirotrichea an apparently inverse role for developmental small non-coding RNAs was observed. Here, 27nt-RNAs are synthesized from the parental macronucleus. The macronulceus genome in y number ,9. In boy number ,11,12,13provided . A valua scnRNAs . Outgoin scnRNAs ,16. To doduction . TherefoStylonychia has a long history as a model for macronuclear differentiation. Here, developmental chromatin reorganization eventually leads to the formation of >16,000 different gene-sized linear nanochromosomes in the mature macronucleus [Stylonychia histone post-translational modifications (PTM) have been extensively studied [Stylonychia the deposition of multiple histone variant-containing nucleosomes into chromatin of different nuclear types and their association with specific sequence-classes play a superior role in developmental chromatin reorganization with Stylonychia being among the list of eukaryotes encoding very high numbers of histone variants [Stylonychia is highly regulated, and we found evidence that the expression of some H3 variants might be influenced by PIWI1-dependent RNAi [onucleus , wherebyonucleus . Apart fonucleus . IESs caonucleus . Prior tonucleus . In thisonucleus , prior tonucleus ,6. In St studied ,23,24. F studied . Macronu studied  and the  studied ,24. Furt studied ,9,26,27.ent RNAi . Accordient RNAi . This imIt is obvious that in spirotrichous ciliates 27nt-RNAs are developmental key regulators targeting specific DNA sequences and influencing local chromatin structures. In search of the vanguard master regulatory process that leads to the de novo mobilization of these non-coding 27nt-RNAs during macronuclear development, we followed the rationale of minimalism. Thereafter, a basic biological process with known molecular actors could provide a more plausible hypothesis for a 27nt-RNA biogenesis mechanism than a theoretical mechanism that would require the taxon-specific evolution of multiple unrecognized molecular factors. In this context, we extract earlier and novel relevant observations, which enable us to shape the hypothesis that the substrate for the biogenesis of developmental non-coding RNAs could be the pool of coding mRNAs that become transcribed in the parental (old) macronucleus. Stylonychia, deep sequencing revealed two different fractions of small RNAs, which are 21–22 nt and 27 nt in size, respectively [Oxytricha [Stylonychia an occurrence of 27nt-RNAs over baseline is not observable during vegetative growth. 27nt-RNAs emerge at the onset of conjugation between mating cells. Shortly afterwards a massive, temporally restricted 27nt-RNA enrichment takes place, which is reminiscent of a selective 27nt-RNA amplification activity, possibly by a putative RDRP-activity. Interestingly, three nanochromosomes containing RDRP candidate genes were identified in Styloynchia [Stylonychia the substrate for the biogenesis of 27nt-RNAs (and 21–22nt-RNAs) could be mRNA [Stylonychia 27nt-RNAs between nucleotides 3–24 there are almost equal ratios of A/U or G/C, respectively (22ACU-3’) . IntereStylonychia, upon its first cellular appearance, the argonaute homolog PIWI1 that binds 27nt-RNAs and is involved in MDS protection accumulates in exactly those parental macronuclear fragments [Stylonychia  export and translation of mRNAs. It seems extremely coherent that in ragments ,24, whiclonychia A,B. Geneon sites ,30. In tStylonychia genes is represented in the mapped RNA reads 10 and 20 hrs post conjugation ([http://stylo.ciliate.org/index.php/home/downloads). Conveniently, while ‘vegetative growth’ transcripts are synthesized in the non-reproductive cycle of Stylonychia it was observed through actinomycin D treatment experiments that RNA synthesis continues and is essential for macronuclear development for approx. 6hrs after the beginning of conjugation before it abruptly stops. Then cells become insensitive to actinomycin D treatment [Oxytricha [Stylonychia [However, we have to consider that MDS protection through 27nt-RNAs can be efficient only, if all MDSs can be targeted. This would require the catholic presence of transcripts from all nanochromosomes upon initiation of 27nt-RNA biogenesis, regardless of whether they are required for vegetative growth or whether they become differentially expressed during development. How this holistic accumulation of usually differentially regulated transcripts could be achieved is unknown, but transcriptome data do indeed support this idea, since the vast majority of annotated ugation  and Dicer-like activity (27nt-RNA processing prior to PIWI1 loading). If this proposed mechanism exists it could provide an elegant explanation of how protein-coding information (mRNA) from parental somatic nuclei (thus their transcription profiles determining the phenotype) can be converted into non-coding information that is used to transmit the functional partitioning of the parental genome to the offspring.Taken together, in our review we summarize evidence that the small RNA species occurring in


Arachis hypogea) in Haiti was determined in the effort to build epidemiological knowledge of the virus.Tomato chlorotic spot virus (TCSV) is emerging as a significant constraint to vegetable and legume crops in the Americas. The complete genome sequence of a TCSV isolate naturally infecting peanut ( Arachis hypogea) in Haiti was determined in the effort to build epidemiological knowledge of the virus.Tomato chlorotic spot virus (TCSV) is emerging as a significant constraint to vegetable and legume crops in the Americas. The complete genome sequence of a TCSV isolate naturally infecting peanut ( Tospovirus, family Peribunyaviridae  with the default parameters in CLC Genomics Workbench 8.0 . The quality-filtered reads were assembled de novo into contigs using the default parameters in CLC Genomics Workbench 8.0. To validate the sequence data from HTS data, individual genomic segments were amplified with segment-specific primers, cloned, sequenced, and assembled as previously described . The cDNA library preparation was carried out with the TruSeq Stranded Total Ribo-Zero rRNA kit (plant)  and subjected to HTS on an Illumina HiSeq 2500 platform at the Huntsman Cancer Institute . After trimming adapters, raw reads were quality filtered to remove reads with a read length below 125 bp and mapped to the peanut genome (GenBank accession numbers escribed .NC_035482 to NC_035484). The L-RNA sequence is 8,873 nucleotides (nt) long and encodes the putative RNA-dependent RNA polymerase (RdRp) in the viral complementary (vc) strand with a predicted molecular weight of 331 kDa. The M-RNA sequence is 4,847 nt long and encodes a 33.94-kDa nonstructural movement protein (NSm) on the viral (v) strand and a 127.68-kDa glycoprotein precursor (GN/GC) on the vc strand. The S-RNA sequence is 3,311 nt long and encodes the 52.5-kDa nonstructural silencing suppressor (NSs) on the v strand and the 28.5-kDa nucleocapsid protein (N) on the vc strand. The length of the intergenic region in the M-RNA and S-RNA sequences is 346 nt and 886 nt, respectively. The eight conserved nucleotides and complementary sequences present at the 5′ (AGAGCAAU) and 3′ (AUUGCUCU) termini of the L-RNA, M-RNA, and S-RNA sequences are identical to corresponding sequences in other tospoviruses. The complete genome segment sequences of TCSV from peanut will provide leads for inferring the evolutionary history of the virus and will lead to a better understanding of the extrinsic and intrinsic barriers that influence genome segment exchanges between TCSV and other tospoviruses , MH427862 (M-RNA), and MH427863 (S-RNA). Raw high-throughput sequencing reads were deposited in the Sequence Read Archive under accession number SRX5643916.The TCSV genome segment sequences in this announcement are publicly available in GenBank under the following accession numbers:


The molecular mechanisms underlying bipolar disorder (BPD) have remained largely unknown. Postmortem brain tissue studies comparing BPD patients with healthy controls have produced a heterogeneous array of potentially implicated protein-coding RNAs. We hypothesized that dysregulation of not only coding, but multiple classes of RNA  RNA, circular (circ) RNA, and/or alternative splicing) underlie the pathogenesis of BPD. Using non-polyadenylated libraries we performed RNA sequencing in postmortem human medial frontal gyrus tissue from BPD patients and healthy controls. Twenty genes, some of which not previously implicated in BPD, were differentially expressed (DE). PCR validation and replication confirmed the implication of these DE genes. Functional in silico analyses identified enrichment of angiogenesis, vascular system development and histone H3-K4 demethylation. In addition, ten lncRNA transcripts were differentially expressed. Furthermore, an overall increased number of alternative splicing events in BPD was detected, as well as an increase in the number of genes carrying alternative splicing events. Finally, a large reservoir of circRNAs populating brain tissue not affected by BPD is described, while in BPD altered levels of two circular transcripts, cNEBL and cEPHA3, are reported. cEPHA3, hitherto unlinked to BPD, is implicated in developmental processes in the central nervous system. Although we did not perform replication analyses of non-coding RNA findings, our findings hint that RNA dysregulation in BPD is not limited to coding regions, opening avenues for future pharmacological investigations and biomarker research. Bipolar disorder (BPD) is characterized by one or more manic episodes alternating with euthymia or depressive episodes. Bipolar spectrum disorders carry a lifetime prevalence of up to 2% and are estimated to be the seventeenth leading cause of disability worldwide . They ende novo variation contributing to BPD. The best powered GWAS has highlighted 30 loci for BPD and has provided insight into genes and pathways involved in the disease [To date, the biological mechanisms underlying BPD remain largely elusive, although research hints that both genetic and environmental factors contribute. For example, twin-based heritability estimates vary between 70 and 90% . Recent http://resource.psychencode.org/), with intriguing results for a range of psychiatric disorders (https://science.sciencemag.org/content/362/6420) [In one of the first comprehensive gene expression analyses in BPD, peripheral blood cells for microarray-based transcriptome analysis were used to identify changes in levels of transcripts involved in G-protein signalling . More re62/6420) . RNA seq62/6420) ,16,17,1862/6420) , approxi62/6420) ,23,24,2562/6420) , other s62/6420) , resting62/6420) , and alt62/6420) . Thus, bSequencing experiments of non-polyadenylated transcriptomes have led to the discovery of new RNA classes, such as circular RNAs (circRNAs), a category of lncRNAs produced by back-splicing reactions that covalently link the 3′ end of an exon to the 5′ end of an upstream exon ,30,31. cTo comprehensively probe the implication of numerous RNA classes in BPD, we performed the first multi-class RNA sequencing experiment in the frontal gyrus of BPD patients, regardless of polyadenylation status. We thus compared the relative abundances of both protein-coding and non-coding RNAs in BPD with healthy controls. We find several differentially expressed genes (DEGs) and long lncRNA transcripts.www.brainbank.nl). All experimental protocols were approved by the NBB where this project was assigned id number 909. All material has been collected from donors for or from whom written informed consent was obtained by the NBB. Procedures, information—and consent forms of the NBB have been approved by the Medical Ethics Committee of the VU Medical Centre at 30 April 2009 . The informed consent includes permission for a brain autopsy and the use of the material and clinical information for research purposes. A first cohort that consisted of four cases and four controls—matched on gender, age, post-mortem interval (<9 h), and pH (6.0–7.0)—was used for RNA sequencing experiments. A second cohort also consisting of four cases and four controls and matched on age, post-mortem interval (<9 h), and pH (= 6.0–7.0) was used for replication experiments. Extensive phenotype information available from the NBB was used to verify diagnosis and healthy control status. An overview of detailed phenotypic information (including medication) is presented in the All methods were carried out in accordance with relevant guidelines and regulations. Postmortem material from all BPD and healthy control subjects used in this study was obtained from the Netherlands Brain Bank (NBB), Netherlands Institute for Neuroscience, Amsterdam, the Netherlands  according to the manufacturer’s instructions and then DNase-treated to eliminate genomic DNA contamination. Finally, RNA integrity (RIN) was verified on an Agilent 2100 bioanalyzer  and only samples with RIN ≥ 5.0 were used for RNA sequencing. Detailed information about postmortem tissue conditions is provided in the A total of 1 ug of RNA per sample in a 5-μL volume was rRNA depleted using the Ribo-Zero Magnetic Kit  before library preparation. Stranded, paired-end sequencing libraries were prepared by the ServiceXS sequencing facility  and sequenced on the Illumina HiSeq 2500 platform . The reads  were de-multiplexed and converted to FASTQ format using CASAVA software from Illumina by the ServiceXS sequencing core facility. FASTQ files were mapped to the hg19/GCh37 reference human genome  with TopHat2 (version 2.0.13) , using twww.r-project.org) using the Bioconductor package edgeR version 3.12.1 [Count data for genes and lncRNA transcripts were analyzed in R  we compared the log2-fold change values of all genes in our dataset with two independent brain tissue RNAseq datasets [Furthermore, to test the concordance between our DEG results and previous RNAseq findings in BPD, using Pearson correlation analyses in R  in our independent replication cohort  we were unable to identify any significantly correlated modules. Gene ontology (GO) term enrichment analysis, however, showed significant enrichments of biological processes related to angiogenesis and vascular system development . In our −50; p-values ≤ 6.5 × 10−6; −13; The total number of AS events was higher in BPD subjects than in controls  overlapped with the DEGs. Three additional circRNA transcripts encompass this locus , while 1511 circRNAs map outside the genomic region of known genes . When apis locus . At the is locus .cNEBL) and one additional circRNA at FDR < 0.1  and healthy controls. By applying the bioinformatics pipeline that we developed to total RNA libraries we first signal several differentially expressed genes and then validate and replicate our findings by PCR. We then demonstrate that alternative splicing events are increased in BPD and that expression levels of several lncRNAs differ between cases and controls. Finally, we describe a reservoir of circular RNAs in human medial frontal cortex and altered levels of circular RNA transcripts in BPD.STAB1 in that study too (at p = 0.0013 and FDR-corrected p = 0.039 in that study). The other 35 DEGs highlighted by us were not found to be associated with BPD in that study [neurobiology of BPD remains to be unraveled. An alternative explanation for this pathway finding relates to medication use in BPD as lithium and valproic acid promote angiogenesis [Functional annotation analysis of the differentially expressed genes (DEGs) showed significant enrichments of biological processes related to angiogenesis and vascular system development. The largest BPD GWAS did not find evidence of angiogenesis or vascular system development being implicated in BPD , which mat study , which mat study . The resat study ,57. Baseogenesis ,59, evenogenesis . A potenUsing gene-set enrichment analysis (GSEA) we identified enrichment in histone H3-K4 demethylation: the expression levels of genes associated with this GO term were upregulated in BPD brains compared to controls. Numerous studies indicate that histone modifications have important roles in BPD , althougNEBL and EPHA3 loci, respectively. Particularly EPHA3 may be of relevance to BPD as it is implicated in developmental events, mostly in the central nervous system (CNS). The Eph receptor belongs to the ephrin receptor subfamily of the protein-tyrosine kinase family. Eph receptors and ephrins (their ligands) regulate key CNS processes, such as neurotransmitter release, postsynaptic glutamate receptor conductance, synaptic glutamate reuptake, and dendritic spine morphogenesis [CircRNAs are increasingly being discovered as a functionally important kind of ubiquitous, endogenous non-coding RNAs ,34. Receogenesis . They arogenesis  and anxiogenesis . Memory ogenesis ,66,67: Eogenesis . In addiogenesis . Althougogenesis . If futuPrevious RNAseq studies in BPD have focused on other brain regions, in particular hippocampus and dorsal striatum tissue where immune response pathways were found to be implicated ,17. A diSeveral limitations should be borne in mind when interpreting the results of the current study. First, the modest sample size may have impeded power to detect any significant modules in our WGCNA. Second, having chosen a non-polyadenylated library as opposed to a small RNA library by consequence precluded us from examining miRNAs, molecules of particular relevance to schizophrenia. Third, having focused on coding and non-coding RNA in rather unique and scant sections of postmortem tissue prevented us from designing analyses at the protein level, a limitation that future studies may be able to overcome. Fourth, we chose to target a single brain area which comes with the disadvantage of lack of generalizability of our findings to other brain regions. And finally, while our circRNA findings in both healthy and affected brain tissues are intriguing, this relatively unexplored domain of RNA molecules currently does not allow cross-validation in other datasets. These results consequently will have to await confirmation in other cohorts. Such cohorts will need to be better powered than the current study as power for replication in our modestly sized cohort is limited in particular for non-coding RNAs. Moreover, future studies may disentangle the specificity of our findings to BPD by incorporating other psychiatric disorders.In conclusion, our study hints that RNA dysregulation in bipolar disorder is not limited to coding regions.


However, in RNAcompete, most synthetic RNAs are unstructured, which makes machine learning methods not effectively extract RBP-binding structural preferences. Furthermore, RNA structure may be variable or multi-modal according to both theoretical and experimental evidence. In this work, we propose ThermoNet, a thermodynamic prediction model by integrating a new sequence-embedding convolutional neural network model over a thermodynamic ensemble of RNA secondary structures. First, the sequence-embedding convolutional neural network generalizes the existing k-mer based methods by jointly learning convolutional filters and k-mer embeddings to represent RNA sequence contexts. Second, the thermodynamic average of deep-learning predictions is able to explore structural variability and improves the prediction, especially for the structured RNAs. Extensive experiments demonstrate that our method significantly outperforms existing approaches, including RCK, DeepBind and several other recent state-of-the-art methods for predictions on both in vitro and in vivo data. The implementation of ThermoNet is available at https://github.com/suyufeng/ThermoNet.Predicting RNA-binding protein (RBP) specificity is important for understanding gene expression regulation and RNA-mediated enzymatic processes. It is widely believed that RBP binding specificity is determined by both the sequence and structural contexts of RNAs. Existing approaches, including traditional machine learning algorithms and more recently, deep learning models, have been extensively applied to integrate RNA sequence and its predicted or experimental RNA structural probabilities for improving the accuracy of RBP binding prediction. Such models were trained mostly on the large-scale  in vitro and in vivo datasets demonstrate the superior performance of our method compared to several state-of-the-art approaches. This study suggests the great potential of our method as a practical tool for identifying novel protein-RNA interactions and binding sites of RBPs.RNA-binding proteins (RBPs) play a key role in modulating various cellular processes, including transcription, alternative splicing, and translational regulation. Identifying protein-RNA interactions and the binding preferences of RBPs are critical to unraveling the mechanism of post-transcriptional gene regulation. In the current study, we present a computational approach that integrates both structure and sequence contexts for protein-RNA binding prediction. We propose to incorporate the structure information using a thermodynamic ensemble of secondary structures, which effectively identifies RBP-binding structural preferences, especially for structured RNAs. Our model is further empowered by a deep neural network that combines the sequence and structure information to achieve improved protein-RNA binding prediction. Extensive experiments on both  RNA-binding proteins (RBPs) modulate the processing of cellular RNAs, including their production, transportation, splicing, stability, translation, and degradation , 2. TherIn vitro and in vivo methods have been developed for determining RBP binding specificities. RNAcompete is a high-throughput in vitro assay that quantifies the relative affinity of a specific RBP to a pre-defined set of more than 240,000 short RNAs. In a recent work, more than 200 human RBPs are analyzed by RNAcompete, generating the first large-scale dataset of protein-RNA interaction measurements , , , and , respectively. The secondary structure of the RNA is a string of the same length ℓ over the alphabet {P, H, I, M, E}, representing the five types of structural contexts, namely, paired (P), hairpin loop (H), inner-loop (I), multi-loop (M) or external region (E). Existing approaches, including DeepBind , Click here for additional data file.


L. monocytogenes infection. Here, we identify specific sRNAs, secreted by the bacterium, with the capacity to induce type I IFN. Further analysis of the most potent sRNA, rli32, links the ability to induce RIG-I-dependent induction of the type I IFN response to the intracellular growth properties of the bacterium. Our findings emphasize the significance of released RNA for Listeria infection and shed light on a compartmental strategy used by an intracellular pathogen to modulate host responses to its advantage.Interferons are potent and broadly acting cytokines that stimulate cellular responses to nucleic acids of unusual structures or locations. While protective when induced following viral infections, the induction of interferons is detrimental to the host during  Listeria monocytogenes induces high levels of type I interferons  to create a growth-permissive microenvironment during infection. We previously demonstrated that RNAs secreted by L. monocytogenes (comprising the secRNome) are potent inducers of IFN-β. We determined the composition and diversity of the members of the secRNome and found that they are uniquely enriched for noncoding small RNAs (sRNAs). Testing of individual sRNAs for their ability to induce IFN revealed several sRNAs with this property. We examined ril32, an intracellularly expressed sRNA that is highly conserved for the species L. monocytogenes and that was the most potent inducer of IFN-β expression of all the sRNAs tested in this study, in more detail. The rli32-induced IFN-β response is RIG-I (retinoic acid inducible gene I) dependent, and cells primed with rli32 inhibit influenza virus replication. We determined the rli32 motif required for IFN induction. rli32 overproduction promotes intracellular bacterial growth, and a mutant lacking rli32 is restricted for intracellular growth in macrophages. rli32-overproducing bacteria are resistant to H2O2 and exhibit both increased catalase activity and changes in the cell envelope. Comparative transcriptome sequencing (RNA-Seq) analysis indicated that ril32 regulates expression of the lhrC locus, previously shown to be involved in cell envelope stress. Inhibition of IFN-β signaling by ruxolitinib reduced rli32-dependent intracellular bacterial growth, indicating a link between induction of the interferon system and bacterial physiology. rli32 is, to the best of our knowledge, the first secreted individual bacterial sRNA known to trigger the induction of the type I IFN response.Cellular sensing of bacterial RNA is increasingly recognized as a determinant of host-pathogen interactions. The intracellular pathogen  Mammalian cells detect invading pathogens by recognizing “non-self” bacterial structures (pathogen-associated molecular patterns [PAMPs]) through pattern recognition receptors (PRRs). While recognition of many PAMPs such as peptidoglycan, lipopolysaccharide, and flagellin does not necessarily indicate an active infection, bacterial mRNA is a viability-associated PAMP (vita-PAMP) present only in viable bacteria .Listeria monocytogenes (Mycobacterium tuberculosis (Francisella tularensis (Type I interferons (IFNs), which comprise several IFN-α subtypes, the single IFN-β subtype, and several other subtypes , are critical components of the antiviral defense of mammalian cells , 3. The ytogenes , 7, Mycorculosis , 9, and larensis .L. monocytogenes is a ubiquitously occurring facultative intracellular Gram-positive bacterium. It is the causative agent of listeriosis, a disease of low incidence but of considerable mortality  (L. monocytogenes RNA (comprising the secRNome), which triggers a strong IFN-β response  . This isresponse . Secreteytogenes .Listeria infection. For example, mice lacking type I IFN receptors (−/−Ifnar mice) show greater resistance to Listeria infections than wild-type mice  on antigen-presenting cells (APCs)  , increass (APCs) , enhances (APCs) , reduce s (APCs) , therebyl spread .L. monocytogenes, present in two extracellular subcompartments, were identified. We have operationally divided the secRNome into the supernatant and the membrane vesicle (MV) fractions. The cargo of bacterial membrane vesicles (MVs), which are released during all phases of bacterial growth and comprise peptidoglycan, lipopolysaccharides, proteins, toxins, and nucleic acids, was previously shown to exploit different mechanisms to enter the host cells , are enriched in the secreted fraction. Screening of individual sRNAs led to detection and identification of several sRNAs that specifically induce type I IFN. In-depth characterization of one of these sRNAs, viz., rli32, revealed features for RIG-I (retinoic acid inducible gene I) activation and for the functional activation of the immune response in tissue culture cell lines as well as in a mouse model. Furthermore, overexpression and deletion of rli32 affected both IFN-β production and intracellular bacterial growth.L. monocytogenes supernatant fluids is composed of “naked” RNA (sec-RNA) and intravesicular RNAs that are shed to the external medium by membrane vesicles (MV-RNA) . This was particularly true for the MV-RNA fraction .Transfection of the three RNA fractions into bone marrow-derived macrophages (BMDM) demonstrated that sec-RNA and MV-RNA induced higher IFN-β levels than cytosolic RNA  . TreatmeL. monocytogenes genome. The sequencing data showed that sec-RNA and MV-RNA comprised all forms of RNA, including tRNA, rRNA, mRNA, and sRNA. Interestingly, the sequencing data revealed a high proportion of noncoding small RNAs (sRNA) in sec-RNA (12.9%) compared to cytosolic RNA (2.4%) and MV-RNA (0.7%). Furthermore, some individual sRNAs demonstrated even higher enrichment levels in sec-RNA and MV-RNA than in cytosolic RNA  on sec-RNA, MV-RNA, and cytosolic RNA. Panel C of The relatively high abundance of sRNA reads in sec-RNA compared to cytosolic RNA prompted us to investigate the contribution of sRNA to the IFN-β induction by sec-RNA. We selected 20 sRNA candidates  and individually transfected (50 ng) in BMDM and HEK293 cells. The results demonstrated that rli32, rli99, and rli100 triggered the strongest IFN-β response in BMDM and HEK293 cells whereas rli48, whose activity may be cell type dependent, showed high levels of IFN-β induction only in BMDM  and is . Moreovytogenes .in silico using RNAfold WebServer .Listeria RNA  are involved in sensing of eria RNA , we invempletely . Howevermpletely . TreatmeFollowing binding of ligand to RIG-I, a conformational change is induced that renders the protein resistant to limited trypsin digestion –36. We e–As RIG-I is an essential receptor for sensing many viruses, including influenza virus , 38, we −/− and MDA5−/− deficient HEK293 cells and the respective supernatants were used for virus titer assay. The virus titer was unaffected in HEK293 cells deficient for RIG-I, whereas pretreatment of MDA5−/− deficient HEK293 cells using rli32 led to a strong decrease in virus titer , and strain Lm-Δrli32 (L. monocytogenes Δrli32 deletion mutant). Neither overexpression nor deletion of rli32 led to overall changes in the total amount of secreted RNA. Transfection of sec-RNA, MV-RNA, and cytosolic RNA isolated from the Lm-rli32 strain induced a strong IFN-β response in macrophages . The results showed higher numbers of transcripts of rli32 in all three RNA fractions than in the wild type . This dIn contrast to the results seen with rli32 overexpression, the absence of rli32 strongly reduced the capacity of the sec-RNA, MV-RNA, and cytosolic RNA fractions to induce IFN-β . HoweverMVs isolated from the supernatant of the rli32-overproducing strain added to macrophages were highly active in inducing IFN-β response and were more active than MVs isolated from either the wild type or the corresponding isogenic Δrli32 deletion mutant .L. monocytogenes, the ability of the Lm-Δrli32 and Lm-rli32 strains to grow intracellularly in BMDM macrophages was examined at 4, 8, and 24 h postinfection (p.i.). Intracellular growth of the Lm-rli32 strain was already enhanced at 8 h p.i., and significant restriction of intracellular growth was detected for the deletion mutant at 24 h p.i. . The inFig. S9A ).L. monocytogenes and to systematically elucidate changes in the RNA composition in sec-RNA, MV-RNA, and cytosolic RNA in the wild-type (Lm), Lm-rli32, and Lm-Δrli32 strains. Remarkably, Lm-rli32 and Lm-Δrli32 showed only minor differences in the sRNA composition of the sec-RNA and MV-RNA fractions compared to wild-type L. monocytogenes at a fold change (fc) cutoff value of ≥2. The only enriched sRNA species in the sec-RNA fraction of Lm-rli32 was rli32 (+5.5 fc)  . sRNAs s+5.5 fc) . Transfe+5.5 fc) . The amoof IFN-β . In the of IFN-β .Major differences were detected in the cytosolic mRNA fraction . For ex10.1128/mBio.01223-19.2TABLE S2Table S2, PDF file, 0.3 MB.Differentially expressed mRNAs in sec-RNA, MV-RNA, and cytosolic RNA in the overexpression strain (Lm-rli32) and deletion mutant (Lm-Δrli32) compared to the wild type (Lm). Download Copyright © 2019 Frantz et al.2019Frantz et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the We found that 17 sRNAs were upregulated and 6 sRNAs were significantly downregulated . In the L. monocytogenes cell envelope, induces expression of the sRNAs LhrC1–5  in the cytosolic fraction of Lm-rli32 . As sRNA LhrC1–5 –41. The 4 in the  LhrC1–5 .2O2), paraquat (PQ), and ethanol on the growth of the Lm-rli32 and Lm-Δrli32 strains. Additionally, we examined the impact of low pH values (pH 2.5 and 5) on the growth of the three strains. We found that Lm-rli32 was more growth resistant to a high (0.15%) H2O2 concentration than both Lm-Δrli32 and the parental strain, whose growth was completely inhibited  are key components of the macrophage response to invading pathogens, we tested the impact of different oxidative stress-inducing agents such as hydrogen peroxide , an inhibitor of IFN-signaling . In theig. S12A ). These Sensing of pathogen-derived nucleic acids from bacteria growing in the cytosol of infected host cells has emerged as a major mechanism for innate immune activation during infection. It was previously reported that an augmented type I IFN response and NLRP3 inflammasome-dependent activation are triggered by bacterial RNA for Gram-negative bacteria . HoweverPseudomonas aeruginosa and other bacteria such as Porphyromonas gingivalis and Treponema denticola also contain such sRNAs that have immune suppressive activities , there was unique enrichment of noncoding nonstructural sRNAs. Three of the sRNAs detected in the secRNAome of L. monocytogenes  have been previously characterized. Isogenic mutants that lack rli50 or rli60 are significantly attenuated for intracellular survival and proliferation in macrophages  or any of the various sRNAs described here, that target multiple cellular receptors, viz., STING and RIG-I, for inducing a type I interferon response implies an intrinsic need for live bacteria to create an environment that allows sufficient growth as a beachhead for further dissemination during infection. Further work will be required to address the role(s) of the other sRNAs detected. At least one species, rli48, exhibits cell-type specificity in the induction of the IFN response, and additional studies are required to address whether common and overlapping sRNAs are used, depending on type of cell infected. The diversity of sRNAs in a single bacterium inducing type I interferon signaling is intriguing. Thus, while c-di-AMP may be seen as a common vita-PAMP produced by many bacteria, the various sRNAs described here could be classified as variable vita-PAMPs that provide diversity that shapes both bacterial and cell type specificity.In summary, we provide direct evidence that secreted individual bacterial sRNAs have the capacity to induce type I IFN. However, the mechanisms for RNA secretion and sorting in the supernatants and MVs remain poorly understood and require further study. We describe rli32, which, to the best of our knowledge, is the first secreted individual bacterial sRNA that links RIG-I-dependent induction of the type I IFN response to intracellular growth of the bacterium. The diversity of RNAs deriving from a single bacterial species that target RIG-I-dependent type I interferon induction was unexpected, and the approach employed here can be used to identify vita-RNA species in other bacteria that contribute to immune sensing and evasion during infection.L. monocytogenes strain EGD-e (GenBank accession no. NC_003210)  (600] = 1.0). Escherichia coli DH10β cells (Invitrogen) were grown in Luria-Bertani (LB) broth containing 300 μg/ml erythromycin to select for the plasmid pERL3 (L. monocytogenes strains harboring pERL3 were grown in the presence of 5 μg/ml erythromycin. Primers used for the construction of rli32 overexpression vector and the rli32 deletion mutant are listed in _003210)  was usedoth (MM)  and incuid pERL3 . L. mono10.1128/mBio.01223-19.3TABLE S3Table S3, PDF file, 0.1 MB.Primers used in this study. Download Copyright © 2019 Frantz et al.2019Frantz et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the L. monocytogenes to generate the chromosomal deletion mutants as described previously  and the 3′ flanking region (with primers rli32-3-for and rli32-4-rev) of rli32. The primer sequences used to generate the isogenic mutants are presented in eviously .hly) promoter and hly terminator of L. monocytogenes. The primers used to generate the flanking regions are shown in the supplemental material  using an miRNeasy minikit (Qiagen). To isolate bacterial cytosolic RNA, additional steps were performed to lyse the bacterial cells before the addition of QIAzol . Details−/− cells were kindly provided by Veit Hornung (6 cells) or with IVT-RNA (50 ng/106 cells), complexed with Lipofectamine 2000 according to the manufacturer’s protocol (Thermo Fisher Scientific). HEK293 cells were transfected with sec-RNA, MV-RNA, and cytosolic RNA (100 ng/105 cells). Transfection of IVT-RNA was performed by using 50 ng RNA. The transfection efficiency was determined by qRT-PCR.BMDM were isolated from bone marrow of C57BL/6 mouse strains and used after 6 days of cultivation in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS) (Biochrom GmbH), 100 μg/ml streptomycin and100 U/ml penicillin (Thermo Fisher Scientific), and 30% supernatant from the L929 cell line. Cells of the HEK293 cell line (human embryonic kidney cells) were maintained in DMEM (Dulbecco’s modified Eagle medium) (Thermo Fisher Scientific) supplemented with 10% FCS. HEK293 RIG-I and MDA5 Hornung . BMDM ce2. For virus titration, MDCK-II cells (Madin-Darby canine kidney cells) were maintained in DMEM supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FCS. All cells were incubated at 37°C in the presence of 5% CO2.For RIG-I activation assay, A549 cells (lung carcinoma cells from human) were maintained in DMEM supplemented with 10% FCS, 50 U/ml penicillin, and 50 μg/ml streptomycin at 37°C and 5% COL. monocytogenes as described previously  of 10 bacteria per eukaryotic cell. Infection was carried out for 45 min in macrophages or for 1 h in HEK293 cells, followed by the addition of fresh medium containing 50 μg/ml of gentamicin to kill extracellular bacteria. Intracellular growth assays were performed at different time points . At each step, the plates were washed extensively with phosphate-buffered saline. For IFN inhibition, the macrophages were treated 30 min prior to infection with 1 μM or 10 μM ruxolitinib   or were PRJEB21884.Sequences originating from this study have been deposited in the European Nucleotide Archive. All the sequencing data are available under project identifier


Reef corals are currently undergoing climatically driven poleward range expansions, with some evidence for equatorial range retractions. Predicting their response to future climate scenarios is critical to their conservation, but ecological models are based only on short-term observations. The fossil record provides the only empirical evidence for the long-term response of organisms under perturbed climate states. The palaeontological record from the Last Interglacial , a time of global warming, suggests that reef corals experienced poleward range shifts and an equatorial decline relative to their modern distribution. However, this record is spatio-temporally biased, and existing methods cannot account for data absence. Here, we use ecological niche modelling to estimate reef corals' realized niche and LIG distribution, based on modern and fossil occurrences. We then make inferences about modelled habitability under two future climate change scenarios (RCP4.5 and RCP8.5). Reef coral ranges during the LIG were comparable to the present, with no prominent equatorial decrease in habitability. Reef corals are likely to experience poleward range expansion and large equatorial declines under RCP4.5 and RCP8.5. However, this range expansion is probably optimistic in the face of anthropogenic climate change. Incorporation of fossil data in niche models improves forecasts of biodiversity responses under global climatic change. Rising Several studies have used ecological niche modelling (ENM) to evaluate the environmental controls on coral reefs and predict global distribution under future climate change scenarios –17. Howeca 125 000 years ago. Several authors have provided approximations on global SSTs for the LIG, with estimates ranging from no significant difference to as much as 2°C warmer than today [et al. [et al. [Apparent range shifts have been observed in the Quaternary (2.58–0 Ma) fossil record of reef corals ,13,24,25an today –28. Kies [et al. ), they cHere, we use ENM to: (i) evaluate the extent to which the LIG equatorial decline of reef corals is the result of fossil bias or a genuine loss in habitability; (ii) quantify the variation in geographical range of reef corals during the LIG and under future climate scenarios, with implications for understanding the evolution of the reef coral latitudinal biodiversity gradient; and (iii) demonstrate the value of using fossil reef coral data to improve biodiversity risk assessment through consideration of the realized niche under varied climate states.2.2.1.When implementing ENM, most studies have focused on analyses of individual species ,30–33. Hwww.iobis.org) using the search criteria ‘Scleractinia’, yielding 589 817 occurrences. Fossil coral occurrence data were accessed on 19 July 2017 through the Paleobiology Database . The PBDB returned 8021 occurrences for Pleistocene Scleractinia. These datasets were then filtered to exclude data not identified to species level, as well as further quality control checks (e.g. synonymizations). We based our analyses on zooxanthellate corals, herein referred to as reef corals, which host unicellular organisms known as zooxanthellae in their tissue, forming a photosymbiotic relationship [Modern coral occurrences were downloaded on 5 July 2017 from the Ocean Biogeographic Information System  with 166 unique coordinates .Figure 2.2.Climate variables for the modern , LIG and two future (2100 AD) climate scenario simulations were derived from HadCM3M2.1N , a versiet al. [Environmental variables known to be ecologically limiting for modern corals  ,34,35, aet al. ,15 demon2.3.2.3.1.D [As previously noted, short-term observations of distribution dynamics can be inherently biased by a taxon not occupying its full geographical or environmental space. However, an additional fundamental question is whether environmental niches are conserved over evolutionary time scales . To evalD , with vaD  for deta2.3.2.To map the potential distribution of reef corals in the modern, LIG and under future climate change scenarios (RCP4.5 and RCP8.5), we implemented three ENM methods: BIOCLIM , randomF2.3.3.In order to measure distribution dynamics, we first generated 24 binary maps from the ensemble models using two different training datasets  and three different binary thresholds for the four climate scenarios . We selected three thresholds for binary conversion: the 5th-, 10th- and 20th-percentiles of the lowest tail of habitat suitability values  associated with the occurrence data for each model to provide insight into habitability dynamics . As short-term observations are intrinsically biased and perhaps incomplete in both environmental and geographical space, we combined binary predictions generated from both LIG and modern training datasets. This was done for each time-slice/climate scenario to establish a multi-temporal approach, estimating the full geographical localities reef corals may occupy. We then assessed whether this approach improved the models' capability to predict the occurrence of both LIG and modern localities, which would have implications for forecasting under future climate scenarios. The latitudinal range and habitability of reef corals for past, present and future climatic scenarios were computed as a function of the sum of suitable cells, within 5° latitudinal bins, for each model class’ projections .2.4.Model performance was assessed using the area under the curve (AUC) statistic , derived3.3.1.D) fall within the null distribution of the environment available to reef corals (p > 0.05), and thus we cannot reject the null hypothesis of niche equivalency. However, the observed overlap value for the niche similarity tests is greater than 95% (p < 0.05) of the simulated values, indicating modern reef corals occupy environments similar to those of the LIG.Niche margin analysis  indicates considerable niche stability (91%) between LIG and modern reef corals, with minimal expansion (8%) and unfilling (5%). Observed niche overlap values for niche equivalency tests , the MaxEnt models performed best for both modern-trained  and LIG-trained  models, across individual projections. Conferring to AUC evaluation criteria , all mod3.2.2.The results from binary habitability maps , the comThreshold selection for binary conversion has important consequences on predicted habitability for future climate scenarios. When comparing predictions from modern-trained ENMs and different binary thresholds, LIG and modern habitability predictions reduce uniformly between thresholds, whereas RCP4.5 and RCP8.5 do not . When ap3.2.3.The LIG binary maps (hindcasted and trained) indicate habitat suitability at equatorial latitudes figures  and 3 foUnder RCP4.5 and RCP8.5, the models predict a considerable decrease in habitability  within the tropics with a 5% (5LPT), 20% (10LPT) and 59% (20LPT) loss in habitable cells for RCP8.5 relative to the modern (SM3), notably in the centre of the hyper-diverse Coral Triangle (Indo-Pacific Ocean) . By cont4.The LIG has occasionally been considered as an analogue for the impacts of global warming on species' distribution ,60,61. AUnder climate scenarios RCP4.5 and RCP8.5, our results indicate that high latitude habitability should increase (i.e. range expansion), while equatorial latitudes will see a large decrease in favourable habitats (i.e. range retraction) compared to the modern. Most notably, this includes the centre of the Coral Triangle, in the Indo-Pacific Ocean, the ‘heart’ of modern-day coral biodiversity, with 605 reef coral species, representing over three-quarters of global reef coral diversity . In addi4.1.Attempts to reconstruct latitudinal biodiversity gradients in geological time have historically been achieved using direct reading of occurrence records , potenti‘Modern-type’ latitudinal biodiversity gradients, with tropical peaks in diversity, have been suggested to form under cold icehouse climate regimes, whereas flattened gradients or temperate peaks are restricted to warmer greenhouse regimes . Additio4.2.Variable contribution analyses computed within biomod2  clearly 2 emissions. However, this is conditional on reef corals being able to acclimatize to heightened temperatures or shift distributions to suitable temperature localities in the first place. Integrating palaeontological data into ENM promotes two clear advantages to aid model projections: (i) the opportunity to test models with empirical evidence across temporal windows and climate regimes and (ii) the ability to combine palaeontological and neontological data to consider the full ecological limits of a particular taxon, rather than just the occupied niche in its current biogeographic pattern.To avoid environmentally driven extinction, taxa need to be able to adapt to changing environments, either through distributional shifts (i.e. habitat tracking) or by altering their abiotic preferences ,30,74. TAlthough the LIG fossil record is biased, the inclusion of fossil reef coral data provides additional insight into the niche hypervolume and potential distribution of reef corals under future climate change. As with previous studies , predict5.Our results suggest that LIG reef corals might have expanded their poleward ranges during the LIG, but that they did not experience an equatorial decline. Instead, the apparent equatorial decline appears to reflect an aspect of bias (i.e. absence of reported fossil collections/occurrences) that cannot currently be ameliorated by existing subsampling approaches. Our findings are in agreement with the results from multiple modelling and palaeoclimatic studies, which suggest that annual mean SST and seasonal ranges at equatorial latitudes in the LIG were comparable to today ,28,70. U


Y gastric bypass (RYGB) for severe obesity.Patients' perceptions of health change after bariatric surgery are complex. The aim of this study was to explore whether self‐rated health (SRH), a widely used tool in public health research, might be relevant as an outcome measure after Roux‐en‐This was a single‐centre retrospective study of a local quality registry. SRH score was registered at baseline and 5 years after RYGB. SRH, one of the 36 items in the quality‐of‐life Short Form 36 (SF‐36®) questionnaire, is the answer to this single question: ‘In general, would you say your health is excellent (1), very good (2), good (3), fair (4) or poor (5)?’ Change in SRH was analysed in relation to change in weight, co‐morbidities and quality of life after 5 years.Of a total of 359 patients who underwent RYGB between September 2006 and February 2011, 233 (64·9 per cent) reported on SRH before and 5 years after surgery. Of these, 180 (77·3 per cent) were women, and the mean(s.d.) age was 40(9) years. Some 154 patients (66·1 per cent) reported an improvement in SRH, 60 (25·8 per cent) had no change, and SRH decreased in 19 patients (8·2 per cent). SRH in improvers was related to better scores in all SF‐36® domains, whereas SRH in non‐improvers was related to unchanged or worsened scores in all SF‐36® domains except physical function.Two‐thirds of patients reported improved SRH 5 years after RYGB for severe obesity. In view of its simplicity, SRH may be an easy‐to‐use outcome measure in bariatric surgery. Y gastric bypass for severe obesity reported improved SRH after 5 years.A single question on self‐rated health (SRH) could provide an easy‐to‐use outcome measure in bariatric surgery. Two‐thirds of patients who had Roux‐en‐Self‐rated health ‐ One question to replace a questionnaire? The patient's experience of improvement in general health is the ultimate goal for all medical treatment. The perception of health has several aspects, and the WHO defines health as ‘a state of complete physical, mental and social well‐being and not merely the absence of disease or infirmity’Generic as well as disease‐specific tools have been used to evaluate change in quality of life (QoL) after bariatric surgerySelf‐rated health (SRH) is a person's subjective evaluation of their general health, expressed as the answer to the question: ‘In general, would you say your health is excellent, very good, good, fair, or poor?’People suffering from severe obesity report lower SRH than the non‐obese, even in the absence of chronic diseaseY gastric bypass (RYGB) for severe obesity. The secondary aim was to explore the relationship between change in SRH to weight loss, co‐morbidity and change in QoL.The present study explored whether SRH, a patient‐reported, simple and robust instrument from public health research, is applicable as an outcome measure in bariatric surgery. The primary aim of the study was to evaluate change in SRH from before to 5 years after Roux‐en‐2 or a BMI above 35 kg/m2 with obesity‐related co‐morbidity in an adult population. The SRH response was collected as part of the Short Form 36  questionnaire about 1 month before the operation, at the end of a preoperative education dayThis study is a retrospective analysis of patients who had RYGB at Aalesund Hospital, a public, non‐academic, secondary referral centre covering a population of 260 000 in Norway. The indication for RYGB was a BMI above 40 kg/mSRH is the first question of the SF‐36®, and the version used in this study was the Norwegian translation of the question and alternative answers: ‘In general, would you say your health is (1) excellent, (2) very good, (3) good, (4) fair or (5) poor?’.Data for all patients who had RYGB at Aalesund Hospital between September 2006 and February 2011 were collected prospectively in a local quality registry, and data from routine visits at 6 weeks and 6, 12, 18, 24, 36, 48 and 60 months after surgery were updated to January 2018. Participation in postoperative support groups, adverse events, plastic surgery and new symptoms related to the bariatric procedure were also registered.The difference between baseline SRH scores and scores at 5 years was calculated, and the change in SRH was categorized as improvement, no change, or a decrease.Weight development from baseline through 5 years was reported by standard measures: percentage excess weight loss (%EWL), percentage excess BMI loss (%EBMIL), percentage total weight loss (%TWL) and change in BMIThe study was approved by the Regional Ethics Committee (REK 2016/331) and by the local Data Protection Officer.2 test was performed for comparison of categorical variables, and independent and paired t tests were performed for comparison of continuous variables. Multiple logistic regression analysis was used to explore whether baseline variables could predict changes in SRH. P < 0·050 was considered statistically significant for all analyses. All analyses were performed using IBM SPSS® version 23 .Categorical variables are given as proportions. All but one of the continuous variables  were normally distributed and are given as mean(s.d.) values. SRH acts as a categorical as well as a continuous variable. Pearson's χA total of 359 patients underwent laparoscopic RYGB as a primary bariatric procedure between September 2006 and February 2011. At baseline, 339 patients completed the SF‐36® questionnaire. After the operation, 322 patients (89·7 per cent) attended the 5‐year follow‐up visit, of whom 242 completed an identical questionnaire. There were complete baseline and postoperative SF‐36® data, as well as clinical information on weight, co‐morbidity, complications and blood test results, for 233 patients, representing 64·9 per cent of patients undergoing RYGB at this hospital in the study period.2. Nadir BMI was 27·7(4) kg/m2, and BMI at 5 years was 30·9(5) kg/m2. Details of co‐morbidity at baseline are shown in Table Of the 233 patients who formed the study cohort, 180 were women (77·3 per cent) and 53 were men (22·7 per cent). All participants were Norwegian/Caucasian by ethnicity. At baseline, their mean(s.d.) age was 40(9) years and BMI was 43·4(5) kg/mFig. Figs Mean(s.d.) preoperative SRH was 3·6(0·8), corresponding to a level between ‘good’ and ‘fair’. No patient reported excellent health at baseline, but 17 (7·3 per cent) reported very good SRH, 89 38·2 per cent) good, 98 (42·1 per cent) fair and 29 (12·4 per cent) poor SRH  had a better SRH score at 5 years, 60 (25·8 per cent) had no change, and 19 (8·2 per cent) had a decrease in SRH score (Table P = 0·044), and %EBMIL was 71·6(24·0) and 65·0(24·5) per cent respectively (P = 0·049). There was no significant difference in %TWL (29·2(9·6) per cent for improvers and 27·2(10·3) per cent for non‐improvers; P = 0·132), or change in BMI (12·7(4·9) versus 12·0(5·0) kg/m2 respectively; P = 0·295)  kg/m2 for non‐improvers (P = 0·057). Even though there was no significant difference in BMI at 5 years, the improvers had significantly lower weight regain from nadir to 5 years than non‐improvers: 8·7(7·0) versus 12·0(8·2) kg respectively (P = 0·002), equivalent to a difference in BMI of 1·2 kg/m2. Measured as weight regain in percentage of maximum weight loss, from their nadir weight improvers had a weight regain of 20·0(18·4) per cent and non‐improvers 26·6(18·0) per cent (P = 0·010)  BMI was 30·5(4·9) kg/m2. In total, 179 patients (76·8 per cent) had a BMI of 35 kg/m2 or less at 5 years. There was no significant relationship between BMI below or above 35 kg/m2 and change in SRH (P = 0·377).One of the success criteria for bariatric surgery is the achievement of a postoperative BMI of less than 35 kg/mP = 0·215). In multiple logistic regression analysis, none of the baseline variables predicted change in SRH (data not shown).Another criterion of success is %EWL of 50 per cent or more, which occurred in 124 (80·5 per cent) of improvers and 58 (73 per cent) of non‐improvers at 5 years (P = 0·065), bodily pain (P = 0·149) or role emotional (P = 0·137). At 5 years, the opposite relationship was found, as sum‐scores for improvers were significantly better (P < 0·050) than those for non‐improvers for all domains. For improvers, sum‐scores at 5 years were better than baseline scores for all eight SF‐36® domains (P < 0·005). Non‐improvers had better scores for physical function (P < 0·001) and worse scores in mental health (P = 0·017) at 5 years compared with the baseline, but no change in the other domains. Details on the relationship between changes in SRH and the eight domains in SF‐36® are given in Tables Fig. At baseline, improvers had worse sum‐scores than non‐improvers for all SF‐36® domains. However, this difference was not significant for physical function  before surgery. Of the 60 with no change in SRH, ten (17 per cent) had T2DM at baseline and six of these patients did not require medication at 5 years. Of the 154 patients with improved SRH, 28 (18·2 per cent) had T2DM at baseline and 21 did not require medication at 5 years (Table Abdominal surgery for suspected internal herniation was more common among the improvers, but there was no difference between improvers and non‐improvers for cholecystectomy, abdominal excess skin removal or births Table .Before RYGB, patients with severe obesity in the present cohort reported SRH far below that in the general populationQoL scores in SRH improvers were worse than those of non‐improvers at baseline, but they were better at 5 years. Although improved SRH was related to better scores in all SF‐36® domains, non‐improvement was related to unchanged or worsened scores in all domains except physical function. None of the baseline characteristics predicted in which patients perceived health would improve. In a clinical context, these findings may indicate that patients with severe obesity who perceive their health as poor have more to gain from bariatric surgery than patients who perceive their health as good. Moreover, in the long run SRH can be interpreted as the result of the patients' continuous negotiation between the positive and negative effects of the RYGB procedure on all aspects of life.2 or less, or %EWL above 50 per cent at 5 years, was similar for improvers and non‐improvers. However, non‐improvers regained 3·3 kg more than improvers from nadir to 5 years after RYGB, a significant difference. Whether this ‘marginal’ weight regain reduced SRH, or whether other health issues led to increased weight among non‐improvers, could not be explored further from the available data.It terms of the relationship of SRH with weight loss, the study found that the difference between SRH improvers and non‐improvers depended partly on the formula used: %EWL and %EBMIL were better for improvers than for non‐improvers, but %TWL and change in BMI were not different; and the proportion of patients attaining a BMI of 35 kg/mConsidering long‐term outcomes, a meta‐analysisThe strengths of this study are the close follow‐up and complete registration for many variables from baseline to 5 years after the RYGB, and that patients reported on SRH when they had long‐term experience of the positive and negative effects of the surgery on their general health status. Among the limitations of the study are the small sample size, and that the SF‐36® questionnaire was not given to all patients who attended the 5‐year follow‐up visit. In addition, the study did not consider socioeconomic factors or life events that may have affected SRH at baseline or during follow‐up after the bariatric procedure.SRH, expressed by the answer to one single question, seems relevant and valid as an outcome measure for bariatric surgery, and in this observational study RYGB for severe obesity resulted in improved SRH in two‐thirds of the patients. Focusing not only on weight, but also on health in general, might reduce the stigma experienced by people with severe obesity considering or undergoing bariatric surgery. The increased knowledge on what to expect from bariatric surgery will be useful for patient education, their choice of treatment, and their view of life after treatment for severe obesity. In clinical use, SRH might replace more comprehensive QoL tools, and SRH scores can be used to identify patients in need of closer follow‐up after surgery.


This mark can either prevent or stimulate the formation of RNA-protein complexes, thereby influencing mRNA-related mechanisms and cellular processes. This review focuses on proteins containing a YTH domain (for YT521-B Homology), a small building block, that selectively detects the m6A nucleotide embedded within a consensus motif. Thereby, it contributes to the recruitment of various effectors involved in the control of mRNA fates through adjacent regions present in the different YTH-containing proteins.The control of gene expression is a multi-layered process occurring at the level of DNA, RNA, and proteins. With the emergence of highly sensitive techniques, new aspects of RNA regulation have been uncovered leading to the emerging field of epitranscriptomics dealing with RNA modifications. Among those post-transcriptional modifications, N6-methyladenosine (m The regulation of gene expression plays a central role during development and upon cell response to stress exposure. Hence, living organisms have developed highly complex mechanisms at different steps of gene expression to tune various cellular pathways. Many of those events occur at the post-transcriptional level through the formation of protein-RNA complexes that will influence various aspects of messenger RNA (mRNA) maturation such as alternative splicing, editing, export, and polyadenylation. Therefore, RNA binding domains (RBDs) are key actors in these regulatory mechanisms through their recognition of specific RNA sequences or structures. The most common RBDs are the RNA recognition motif (RRM) , the hnRNP K Homology domains (KH), Piwi Argonaute and Zwille domains (PAZ), and double stranded RNA-binding domains (dsRBD) ,2,3,4. W6A (N6-methyladenosine), m1A (N1-methyladenosine), pseudouridine (Ψ), m5C (5-methylcytosine), and ac4C (N4-acetylcytosine) within mRNAs has shed light onto an additional layer of regulation now known as epitranscriptomics [6A. This modification was initially identified in mRNAs four decades ago [6A does not alter base pairing [6A sites has been a main obstacle for studying its biological significance. This field was reignited in 2011, following the discovery of the m6A demethylase FTO (fat mass and obesity-associated protein), a protein involved in human obesity, as a so-called ’eraser‘ enzyme that removes m6As present on mRNAs [6A at individual-nucleotide resolution using cross-linking and immuno-precipitation with m6A-specific antibodies. High-throughput sequencing of the immuno-precipitated RNA fragments revealed the presence of more than 10,000 m6A sites in human cells, affecting more than 25% of the transcriptome. Their detailed mapping showed an enrichment of m6A near the stop codon and in the 3′ untranslated region (3′ UTR) of the target mRNAs. The main ‘writer’ methyltransferase is a multi-protein complex composed of at least METTL3, METTL14, WTAP, and KIAA1429, which is responsible for m6A deposition on the consensus motif DRA*CH . d ALKBH5 ,25. Furtunctions ,27,28. T6A reader protein family is human YT521-B (hereafter termed YTHDC1), which was initially identified as a factor interacting with Tra2β, SC35, SF2, hnRNP G, and SAM68 splicing factors [Saccharomyces cerevisiae and Schizosaccharomyces pombe yeasts) through plants  to higher eukaryotes  [6A modification at the heart of the DRA*CH consensus sequence [The foremost member of this m factors ,30. DespHDF 1–3) ,32. AlthHDF 1–3) . YTH-consequence ,26.6A containing RNA complexes have revealed that this aromatic cage is responsible for the specific recognition of the m6A mark by YTH domains [6A containing RNA, the aromatic cage specifically accommodates m6A via the side chains from W377, W428, and L439 amino acids [6A methyl group forms methyl-π interaction with W428 side chain while the purine base is sandwiched between the W377 and L439 side chains (6A methyl group with W428 most probably explains the higher affinity (20 to 50-fold difference) of YTH domains for an m6A containing RNA oligonucleotide compared to the same unmodified oligonucleotide [6A since mutation of any of these three residues to alanine disrupts binding [6A recognition by forming specific hydrogen bonds with nitrogen atoms from the adenine base and then participating together with the aromatic cage to the selective recognition of m6A  or Am6AC (30%) motifs [6A is a G compared to an A [6ACU RNA fragment [6A forms a hydrogen bond with the main chain nitrogen group from V382  exhibit similar affinities for RNAs containing any of the four nucleotides at the position immediately upstream m6A [Zygosaccharomyces rouxii Mrb1/Pho92 fungal orthologue compared to YTHDC1 [The YTH domain is made of 150 to 200 residues and adopts an α/β fold, with four to five α-helices surrounding a curved six stranded β-sheet as revealed by the NMR structure of YTHDC1 YTH domain determined by the RIKEN Structural Genomics and Proteomics Initiative in 2007 (PDB code: 2YUD). At the center of the β-sheet lies a cavity delineated by conserved hydrophobic residues including three tryptophan amino acid side chains  forming a so-called aromatic cage A. Severa domains ,36,37,38e chains A. This i binding ,37,38. Pn of m6A A. Residuo YTHDC1 ,38. So f6A nucleotide at the center of a consensus signature motif matching that of the major m6A methyltransferase machinery identified to date.In summary, YTH-domain containing proteins interact with single-stranded RNAs and selectively identify the presence of a modified m6A-containing RNA fates, i.e., splicing, mRNA nucleocytoplasmic export, translation and mRNA decay [Most RNA binding modules are embedded within larger proteins and are surrounded by either structured domains or low complexity regions, all with various functions ,40,41. TNA decay ,45,46,47A. thaliana, which roles are being clarified. As this topic has been nicely discussed by Bhat et al. in this special issue [As mentioned above, bioinformatics analyses have identified 13 YTH-containing proteins in al issue , these pDrosophila melanogaster [6A as either METTL3 silencing or a YTHDC1 double mutant (two Trp residues from the aromatic cage of the YTH domain are substituted by Ala) strongly reduce binding of SRSF3 to RNAs in cellulo [6A-containing pre-mRNAs. This interaction between YTHDC1 and SRSF3 is not only involved in alternative splicing but also in the polyadenylation process of the pre-mRNAs through their association with the pre-mRNA 3′ end processing factors CPSF6 [6A control from the nucleus to the cytoplasm (YTHDC1 (previously known as YT521B) is the founding member of YTH family of proteins. In this protein, the YTH domain is the only region predicted to be folded and is surrounded by regions rich in charged residues  cofactor from methionine and ATP . The MAThis mRNA , a modifhis mRNA . Depletihis mRNA  and to rA 3′ UTR . Whether6A methylation in the alternative splicing of Sex lethal (Sxl), which encodes a master regulator of sex determination and dosage compensation [Sxl is expressed in males and females, the presence of an additional internal exon in males introduces a premature stop codon that results in both the production of a truncated and non-functional Sxl protein and the rapid elimination of the transcript most probably by the nonsense-mediated mRNA decay pathway [6A mRNA methyltransferase holoenzyme or the YTHDC1 protein in the female but not the male fly results in retention of the male-specific exon concomitant with the decrease of the female-specific isoform, clearly indicating a female-specific splicing defect linked to altered m6A deposition and recognition.In fruit-fly, the YTHDC1 orthologue is also involved together with mensation . Althoug pathway ,55. Inac6A sites and the preferential binding of YTHDC1 to m6A marks is necessary for XIST-mediated transcriptional silencing. The depletion of m6A ‘writer’ causes inhibition of XIST function and this defect can be restored by artificially tethering YTHDC1 to XIST in cells lacking m6A methylation machinery. A comprehensive understanding of the mode of action of YTHDC1 on XIST will necessitate further studies.Beyond its role in splicing, human YTHDC1 also helps in transcriptional repression of X chromosome genes by X-inactive specific transcript (XIST), a long non-coding RNA that plays a critical role in inactivation of one X chromosome in female cells . XIST is6A-rich mRNAs through its YTH domain [Compared to the other YTH domain-containing proteins, where the YTH domain is embedded within low complexity regions, members of the YTHDC2 family are multi-domain proteins . Apart fH domain ,62.Initially, YTHDC2 was shown to associate with hepatitis C virus protein NS5B to facilitate viral DNA replication  and to p6A-enriched mRNAs to address them to degradation by the XRN1 exonuclease. However, this model could be restricted to a subclass of mRNAs as various studies have observed different effects on mRNA translation and stability upon inactivation of YTHDC2 gene. Indeed, YTHDC2–MEIOC complex could stabilize meiosis-specific transcripts [In human cells, YTHDC2 interacts in an RNA-independent manner with the meiosis-specific MEIOC protein as well as with the 5′→3′ exonuclease XRN1 ,62,65,67nscripts ,62,65.6A-containing mRNAs to the ribosome but also decreasing mRNA stability by recruiting XRN1 to those mRNAs  in which YTHDF2 co-localizes with DCP1a and DDX6 proteins known to be involved in mRNA decapping [S. cerevisiae, also interacts with Pop2, another component of the CCR4-NOT complex [S. cerevisiae Pho92 are emphasized by (1) the ability of human YTHDF2 gene (but not YTHDC1) to complement for the deletion of PHO92 gene in S. cerevisiae and (2) the role of Pho92 as an enhancer of mRNA decay [6A-containing mRNA decay and that this role has been conserved throughout evolution. Interestingly, upon heat shock, YTHDF2 relocalizes to the nucleus and this is accompanied by a specific increase of m6A in the 5′ UTR of stress-inducible mRNAs and an increased ribosome occupancy in their coding region [6A sites located within mRNA 5′ UTRs [YTHDF2 was the first protein to be functionally characterized, especially regarding the repertoire of mRNAs that it binds to, as well as its mode of action. Photo-activable ribonucleoside crosslinking and immunoprecipitation (PAR-CLIP) combined with RNA-immunoprecipitation coupled to sequencing (RIP-Seq) experiments showed that human YTHDF2 selectively targets more than 3000 different transcripts and binds predominantly to their 3′ UTR and around the stop codon . Furtherted mRNA . This ace effect . This roecapping . YTHDF2 ecapping . This inecapping ). Intere complex . The simNA decay . Altogetg region . This co 5′ UTRs .6A/A ratio, indicating that this protein is unlikely to be involved in m6A-containing mRNA decay [6A-dependent manner. This mechanism is likely to occur through the recognition of m6A sites by YTHDF1 within the 3′ UTR of mRNAs on the one hand, as well as with the 40S subunit and components of the eIF3 complex bound in the vicinity of the start codon on the other  or enhance degradation of mRNAs (YTHDF2), respectively ,72. Inde6A marks are not restricted to cellular RNAs but are also found in viral mRNAs, where they are recognized by the YTHDF1–3 proteins [6A and YTHDFs on the regulation of viral infection leading to the description of various mechanisms. For instance, the recognition of m6A marks on viral mRNAs by YTHDFs has been shown to block reverse transcription of Zika virus genome [6A marks on the infection of human cells by various viruses.Interestingly, mproteins ,76,77,78s genome ). Finall6A ‘writers’ homologous to human METTL3 and METTL14 proteins are absent in S. pombe fission yeast [S. pombe Mmi1 (for Meiotic mRNA interception 1), a YTH-domain containing protein, is present in this organism. Mmi1 has the same organization as YTHDFs with a low-complexity N-terminal region rich in Prolines and charged residues followed by a C-terminal YTH  but not in other YTH containing proteins. Mmi1 YTH domain is incapable of binding a GGm6AC containing RNA, and N6-methylation at any position of the DSR motif weakens binding to the RNA [6A binding site in other YTH domain proteins shows that two Trp residues are conserved while the position corresponding to Trp, Leu, or Tyr in canonical YTH domains is occupied by a His in Mmi1. However, two main differences that could explain the Mmi1 YTH domain inability to bind m6A containing RNAs have been observed. First, the amino acid corresponding to the Asn, Asp, or His residues forming a hydrogen bond with N1 atom of the adenosine ring is substituted by Ala in Mmi1. Second, while the surface surrounding the m6A binding pocket is positively charged in canonical YTH domains, the corresponding region in Mmi1 is negatively charged, which is not favorable for RNA binding [6A modification and interacts with RNA in a completely different manner.Mmi1 is an RNA binding protein that specifically recognizes a consensus U(U/C/G)AAAC motif, present in multiple copies within larger regions called Determinant of Selective Removal (DSR). This motif radically differs from the m domains . Mmi1 YT domains . Althoug the RNA . Similar binding . Hence, S. cerevisiae Pho92, namely the regulation of the stability of its RNA targets [Mmi1 is a key cell fate regulator that promotes degradation of meiosis-specific transcripts in mitotic cells and hence seems to fulfill a similar function as human YTHDF2 and  targets ,68,81,85 targets ). The EM targets ,88,89,90 targets ,92). In in vivo as well as in the stimulation of deadenylation activity in vitro [S. pombe.Recent works have also reported a tight association between Mmi1 and the CCR4-NOT complex, resulting in its recruitment to meiosis specific transcripts in vitro ,86,93,94in vitro . This rein vitro ). Mei2 min vitro . Further6A. As a consequence, the best characterized m6A ‘readers’ are YTH-containing proteins and studies in human cells have shown that the five human YTH proteins are affecting mRNA fates such as splicing, export, translation, and decay by recruiting various protein partners. However, there is increasing evidence that proteins lacking YTH domains can also selectively recognize m6A marks. This is indeed the case for instance of the eIF3 initiation factor [The technological progresses made during the last decade have allowed the identification of RNA modifications present at low abundance and on RNA species with limited stability such as mRNAs and long noncoding RNAs in eukaryotic cells. This research has created the basis for the characterization of proteins and multi-protein complexes involved in the deposition, elimination, and detection of these modifications. So far, much of the studies have focused on the most abundant modification, namely mn factor , the FMRn factor ,96 or thn factor . Undoubtn factor , paving