Gag precursor undergoes a cascade of proteolytic cleavages, and whilst the structural rearrangements of the viral proteins are well understood, the concomitant maturation of the genomic RNA (gRNA) structure is unexplored, despite evidence that it is required for infectivity. To get insight into this process, we systematically analysed the interactions between Pr55Gag or its maturation products  and the 5ʹ gRNA region and their structural consequences, in vitro. We show that Pr55Gag and its maturation products mostly bind at different RNA sites and with different contributions of their two zinc knuckle domains. Importantly, these proteins have different transient and permanent effects on the RNA structure, the late NCp9 and NCp7 inducing dramatic structural rearrangements. Altogether, our results reveal the distinct contributions of the different Pr55Gag maturation products on the gRNA structural maturation.Maturation of the HIV-1 viral particles shortly after budding is required for infectivity. During this process, the Pr55 Gag and Pr160GagPol precursors triggers morphological rearrangements of the immature Gag shell and lead to formation of the cone-shaped capsid characteristic of the lentivirus family [Retroviruses, including human immunodeficiency virus type 1 (HIV-1), bud from infected cells as immature non-infectious particles [s family . At the s family ,6. This s family ,7,8. Inds family  and matus family  virions,s family , revealiGag is driven by ordered, sequential cleavages at five positions by the viral protease enzyme [Gag is necessary not only to form the mature matrix and capsid [Gag and its NC-containing maturation products with gRNA are expected to govern its structural rearrangements [Proteolytic processing of Pr55e enzyme ,3,7,8  of HIV-1 gRNA. We used RNA chemical probing to compare the protein footprints and their transient effects of the RNA structure, the importance of the zinc fingers in the binding of these proteins to gRNA, and the permanent refolding they impose to the gRNA structure. Indeed, while several Gag [Gag and its consecutive maturation products, which would provide insight into the gRNA structural maturation process, is available.However, the relationship between virion protein maturation and structural maturation of the gRNA is poorly understood, despite the fact that these two phenomena are intricately linked, as gRNA increases the kinetics of Pr55protease ,25. The protease . As an aeral Gag  and NCp7eral Gag ,34,35 foGag and its NC-containing maturation products with the 5ʹ-UTR of HIV-1 gRNA because this region is replete with sites that play key roles in HIV-1 replication and whose functions are directly linked to their structure  at the 3ʹend of tRNALys3, and other regions complementary to the tRNALys3 anticodon loop [Gag binding [We focused on the interaction of Pr55tructure ). Of spetructure ). The PBdon loop  and the don loop ,40. The  binding  and is c binding . This re binding . It cont binding ,47: SL1  binding ,48, wher binding . In addi binding ,50.Gag and its maturation products NCp15, NCp9 and NCp7 to bind and rearrange the 5ʹ-end region of the HIV-1 genome, we performed detailed RNA structure and protein binding analysis using high-throughput selective 2ʹ-hydroxyl acylation analysed by primer extension (hSHAPE). SHAPE experiments interrogate RNA structural dynamics using electrophilic anhydride that preferentially acylate the 2ʹ-hydroxyl (2ʹ-OH) ribose groups of single stranded RNA [in vitro transcribed RNA corresponding to the first 600 nts of HIV-1 gRNA under well-defined experimental conditions. In our analysis, we focused on the untranslated region (nucleotides 1–335), as it contains functional sites that play key roles in HIV-1 replication  and is Gag and its maturation products on the structure of the 1–600 gRNA, we analysed four conditions (NoProt condition), 2) gRNA 1–600 in complex with Pr55Gag, NCp15, NCp9, or NCp7 (Complex condition), 3) gRNA 1–600:protein complexes treated with aldrithiol-2 (AT-2), a compound that is able to eject the Zn2+ ions from the two zinc fingers located in the NC domain [AT-2 condition), and 4) gRNA 1–600:protein complexes treated with proteinase K (ProtK condition). Since we wanted to mimic conditions prevailing in viral particles, and viral particles are known to contain between 1,000 and 5,000 of Pr55Gag or its maturation products [Complex condition with SHAPE reactivity values of the NoProt condition allowed us to identify the protein binding sites on gRNA 1–600, as well as the effects of protein binding on the RNA structure. The reverse footprints of the two zinc-fingers located in the NC domain of full-length Pr55Gag and its maturation products : 1) gRNC domain   were idComplex, AT-2, and ProtK conditions , but since they did not show any significant differences amongst conditions, all NoProt datasets were pooled together (Supplementary Dataset 1).Separate controls without protein were performed for each of the nditions , left paSupplementary Figure 1), and if the correlation of a data set with the other ones was < 0.70 it was discarded. We averaged data from 3 to 6 experiments for Complex, AT-2, and ProtK conditions (Supplementary Dataset 1). For each protein, the averaged SHAPE values were then used to perform pairwise comparison between the different conditions.In order to minimize the errors on the SHAPE reactivity values, several data sets were obtained for each condition. Pair-wise comparisons performed between all data sets obtained under the same condition usually showed very good correlation (NoProt condition) . NoProt SHAPE reactivities (Supplementary dataset 1) and the deduced secondary structure model  correlated very well with previous studies [As a reference, we first analysed the structure of the 1–600 gRNA in the absence of protein (ndition) , left pa studies ,27, indiGag, NCp15, NCp9, and NCp7 complexes with gRNA 1–600 all show SHAPE reactivity profiles that differ from the NoProt profile, as well as between themselves (Complex and NoProt profiles (Pr55emselves ). Schemaprofiles ) indicatGag and its maturation products, they each bind to gRNA and affect its structure in different manners. NCp7 distinguishes itself as having the most pronounced destabilizing effect on the RNA structure, while on the contrary NCp15 barely destabilized the RNA structure at all, and both Pr55Gag and NCp9 had intermediate behaviours . This is due to the fact that these two proteins protect longer stretches of nts than Pr55Gag and NCp7, and these stretches contain ~40% of uridines  or destabilized (red) regions are common to several proteins, but only one-third (10/28) of protected regions and one amongst 16 destabilized regions are found for all four proteins ). This ihaviours ). The ovuridines .Table 1.Gag and its maturation products, we plotted the differences observed between the SHAPE profiles under the Complex and NoProt conditions on the RNA secondary structure model that best fits the SHAPE reactivity of the NoProt condition . While ondition ). This r binding  and is u binding ,43,45,57ing site ,58. An aing site , but onlGag, but not by NCp15 or NCp9 , and th or NCp9 ).Lys,3 and has been proposed to play a key role in the initiation of reverse transcription [Lys,3 [Outside of Psi, we observed that NCp7, but none of the other proteins included in this study, strongly destabilized the 3ʹ strand of the basal stem of the PBS domain (nts 217–223) ). The 5ʹcription ,40. Addin [Lys,3 . Altogetn [Lys,3 .Gag and its maturation products were found to bind gRNA 1–600 and to affect its structure in a protein-specific manner, we next asked whether the two conserved zinc fingers (2+ ions from the CCHC-type zinc fingers [Complex condition to those obtained in the AT-2 condition (Since Pr55 fingers ) also pl fingers , and comondition .Figure Gag:gRNA 1–600 complex had minimal effect on SHAPE reactivities, suggesting that once the complex is formed, the zinc fingers are not required to maintain the interaction with RNA . On thereatment ). Indeed compare  and 3(d)i.e. nts 241–242, 246–247, 271–273) addition of AT-2 to the Pr55Gag:RNA complex (Gag AT-2Pr55 condition) had little or no effect on the SHAPE reactivity of the complex, while treatment of the NCp15:RNA complex with AT-2 (NCp15 AT-2 condition) increased the SHAPE reactivity to levels similar to those observed in the absence of protein (NoProt condition)  were less clear-cut, distinct trends could be observed. AT-2 usually had limited effect on the SHAPE reactivity of the NCp9:RNA complexes , while AT-2 increased the reactivity of the NCp7:RNA complexes at most positions, without always reaching the same reactivity level as in the NoProt condition  ). Althouondition ). Similaondition ). In theondition ), sugges2 domain ), as als2 domain ).FigureGag or its maturation products resulted in numerous protein-specific protections as well as destabilization of gRNA 1–600 (i.e. if they required the protein to be present in order to be observed) or if they were permanent (i.e. if they would persist after removing the protein). Permanent effects on the RNA structure are the hallmark of an RNA chaperone activity, which has been documented for Pr55Gag and its maturation products [Complex, ProtK and NoProt conditions . Indeed, the ProtK SHAPE profiles were far more similar to the NoProt profile than to the Complex profiles, indicating that most of the effects of the proteins on the gRNA structure were transient. Nevertheless, while the differences between the ProtK and NoProt profiles were quite limited in the case of Pr55Gag and NCp15, they were more significant with NCp9 and NCp7 .FigureProtK data to analyse the effects of Pr55Gag and its maturation products on the gRNA secondary structure. In contrast to the Complex data sets, where SHAPE reactivities are modulated by protein binding, SHAPE reactivites in ProtK data sets can be used as constraints in RNA folding algorithms to obtain information on gRNA secondary structure. The first (i.e. the most stable) structure predicted when incorporating the ProtK SHAPE reactivities as constraints are shown for each protein in NoProt and Pr55GagProtK SHAPE   indicates that the sequences forming the CU:GA interaction are highly conserved amongst HIV-1 isolates. Likewise, the two lower stems of the U5:gag interaction are also highly conserved, indicating that the structural rearrangements we observed with the NL4.3 isolate are also possible in the other HIV-1 isolates, suggesting they are important for function.We next took advantage of the tK SHAPE ), it is g domain  and the g domain  folds ing domain . In the 440–446) ), ratherUG codon ). This nUG codon ). Similaabilized ). SignifSupplementary Figure 5).Importantly, the structural changes observed with the different proteins do not reflect differences in the dimerization status of gRNA 1–600. Indeed, gRNA 1–600 was 81 ± 2% dimeric in the absence of protein, and the dimer fraction further increased to 89–95% after incubation with the proteins, without any significant difference between proteins (Gag and its NC-containing maturation products regarding i) the protein binding sites and their transient structural effects, ii) the importance of the two NC zinc fingers in the stability of the complexes, and iii) the permanent effects of these proteins on the HIV-1 gRNA structure. Even though a few previous studies used structural approaches to identify Gag or/and NCp7 [in vitro [Gag, NCp15, NCp9, NCp7 binding sites has been reported. Furthermore, several of these studies were performed using fusion proteins [Gag. Similarly, AT-2 has only been used to analyse the contribution of the zinc fingers of mature NCp7 [Gag and its maturation products on the 5ʹ region of HIV-1 gRNA has never been described, even though others have analysed the in vitro RNA chaperone activity of these proteins using model systems [The structural rearrangements of the HIV-1 gRNA during maturation of the viral particles remain unexplored, despite evidence that they are required for infectivity ,19,23,24and NCp7 ,32,34,35in vitro ,41 or diin vitro ,35, no sproteins  or Gag∆pproteins  as a surure NCp7 , and, to systems ,15,38,64in vitro NCp7 binding analysis identifies the same binding sites as a similar analysis performed on mature viral particles, indicating that our in vitro analysis is relevant to the situation prevailing in viral particles [Gag, in agreement with experiments conducted on small model systems [For the part of this study that can be compared to published works, our results fit well with previous data. Of particular interest is the finding that our articles . Similar systems ,15,65.Gag, NCp15, NCp9, and NCp7 to the 5ʹ region of HIV-1 gRNA: some of the binding sites of these proteins are different and their transient effect on the RNA structure . Indeed, the zinc fingers have opposite contributions in maintaining the Pr55Gag:gRNA and NCp15:gRNA complexes once they are formed, but these two proteins both have minimal chaperone activity and minimal to low transient RNA destabilization ability . Remarkably, the effect of AT-2 on Pr55Gag (or NCp15) is the same at all RNA binding sites  . The primary role of the charge density in the RNA chaperone activity of Pr55Gag and its maturation products is in keeping with the observation that many proteins with disordered positively charged regions, including synthetic polypeptides, display RNA chaperone activity [Figure 6, central part).The RNA chaperone activity of Pr55activity . Likewisactivity  . This oaffinity ,73. The otect it . Indeed,otect it ,75. In f of gRNA . AnalysiS domain . In partGag and its maturation products suggest that the gRNA structure is progressively remodelled inside maturating viral particles. A key region involved in these rearrangements seems to be the CU:GA interaction, with the two interacting strands being progressively shifted as Pr55Gag is replaced by its successive maturation products, eventually causing a dramatic change in the overall gRNA secondary structure . While SL1, SL2 and SL3 all exist in more or less extended forms  and 6, outer part), the ability of NCp7 to transiently unwind RNA secondary structures , which is much more pronounced than that of NCp9, is likely crucial for efficient reverse transcription, explaining why infectivity is only acquired after complete maturation of the viral particles.The different RNA chaperone activity of Pr55ed forms , extensied forms ). Importg domain  and the g domain  folds ing domain . It thus process . It woulure ones , whereasGag with an appended C-terminal His6-tag was performed as described by McKinstry et al [Expression, purification and characterization of NL4.3 Pr55ry et al . Recombiry et al .in vitro transcription from plasmid pDR4607 [PvuII restriction enzyme. Transcription was performed using a MEGAscript T7 Transcription kit (Thermo Fisher Scientific) following the manufacturer’s instructions and purified by exclusion chromatography on a TSKgel G4000SW column as previously described [gRNA 1–600 corresponding to the first 600 nts of the wild type NL4.3 HIV-1 gRNA was synthesized by  pDR4607 , after lescribed . RNA int2) supplemented with 40 U RNasin® (Promega) and 1 µg total yeast tRNA. In parallel, 240 pmoles of protein  were incubated on ice for 15 min in the 1X Folding Buffer supplemented with 0.01% Triton X-100; 0.01 M DTT and 4 µg BSA in a final volume of 100 µl.In order to form RNA-protein complexes, RNA was folded in a buffer favouring dimerization. Briefly, four pmoles of gRNA 1–600 were denatured in water at 90°C for 2 min then placed on ice for 2 min. The RNA was then incubated at 37°C for 30 min in a 20 µl final volume of 1X Folding Buffer  complexes were formed by mixing 20 µl of RNA mixture with 100 µl of protein solution or 100 µl 1X Folding buffer as a control without protein and incubated at 37°C for 30 min then at 0°C for 15 min. The RNP samples were divided in two equal fractions that were treated with a SHAPE reagent (+) or with an equal volume of DMSO as a negative control (-) (see below).NoProt condition) or of protein-RNA complex (Complex condition) was treated with 12 µl of 10 mM NMIA (Sigma-Aldrich) in anhydrous DMSO (Sigma-Aldrich) (+) or with 12 µl anhydrous DMSO . After 50 min at room temperature, 128 µl Milli-Q water were added and material was precipitated with 3 volumes of ethanol, 1/10 volume of 3 M sodium acetate pH 5.0, 1 µl of glycoBlue (ThermoFisher) for 30 min in a dry ice/ethanol bath and collected by centrifugation at 20,800 g for 30 min at 4°C. The pellets were washed twice with 70% ethanol, dried and resuspended in 15 µl water and 4 µl 5X Folding Buffer. One µl of proteinase K (Roche) was added and the digestion was performed at 37°C for 30 min before adding 79 µl water. The modified RNA was purified by phenol-chloroform extraction  and ethanol precipitated as described above. RNA pellets were resuspended in 6 µl Milli-Q water.A 60 µl solution of refolded gRNA 1–600 (AT-2 condition).The RNP complexes (60 µl) were treated with 2 µl of 30 mM 2,2ʹ-dithiodipyridine  (Sigma-Aldrich) in DMSO for 1 h at 37°C. Then, RNA was modified by NMIA using the same protocol as above (ProtK condition). Milli-Q water was added up to 200 µl and the RNA was phenol-chloroform extracted and ethanol precipitated as described above.After formation of the RNP complexes as described above, including incubations for 30 min at 37°C and for 15 min at 0°C, the proteins were eliminated by proteinase K treatment prior to RNA modification. The 60 µl mixture was supplemented with 1 µl proteinase K and incubated at 37°C for 30 min. Then, RNA was modified with 12 µl of 10 mM NMIA in anhydrous DMSO or 12 µl DMSO (negative control) and incubated for 50 min at room temperature (2) and 10 min incubation at room temperature, reverse transcription was performed by adding 2 µl AMV RT Buffer 5x, 6 µl dNTPs 2.5 mM (Invitrogen), 2 U of AMV RT (Life Sciences) and water to 20 µl. Elongation was ensured by incubation 20 min at 42°C followed by 30 min at 50°C. The enzyme was inactivated at 60°C for 10 min. Simultaneously, a sequencing reaction was performed with 2 pmoles of unmodified RNA and 2 µl of a 2 mM AS primer 1 or AS primer 2 labelled with Ned . Reverse transcription was performed as for the SHAPE (+) and (-) elongation reactions except for the reaction mix added which was composed of 6 µl G10 , 2 µl ddGTP at 100 µM, 2 µl AMV RT Buffer 5x and 2 U of AMV RT (Life Sciences).RNA samples (6 µl) treated with NMIA (+) or DMSO (-) were mixed with 2 µl of a 1 mM AS primer 1 (5ʹ- AGC TCC CTG CTT GCC CAT ACT A-3ʹ: complementary to nts 436–457 of gRNA 1–600) or AS primer 2 (5ʹ- CTT CTG ATC CTG TCT GAA GG-3ʹ: complementary to nts 536–555 of gRNA 1–600) labelled with Vic . The mixture was heated at 90°C for 2 min and placed on ice for 2 min. After the addition of 2 µl AMV RT Buffer 5x . For each experiment, the modified (+) and unmodified (-) samples were pooled each with a ddG sequencing reaction before ethanol precipitation. The cDNAs were resuspended in 10 µl HiDi Formamide (Applied Biosystem), denatured at 90°C for 5 min, then placed on ice for 5 min and finally centrifuged for 5 min at 6,000 g. The primer extension products were loaded on an ABI 3130XL Genetic Analyser (Applied Biosystem) and the electropherograms were analysed with the QuShape software .i.e. irrelevant differences) when SHAPE reactivities are high in both conditions and may miss biologically relevant differences  when the SHAPE reactivities are low in both conditions. On the other side, using a relative difference threshold can generate false positive when SHAPE reactivities are low in both conditions, and the difference between reactivity values is not significant. We therefore considered differences between conditions biologically relevant only if the absolute SHAPE reactivities differed by ≥ 0.20 and if the relative difference differed by ≥ 40%.For pairwise comparisons between the different conditions, we first used deltaSHAPE  to identGag and its maturation products on the gRNA 1–600 structure ‘NoProt’ or ‘ProtK’ SHAPE reactivity values were used as constraints to fold the RNA secondary structure with the RNAstructure software version 6.0. No other constraints than the SHAPE reactivities were applied to the fold. Based on the RNAstructure data, the structure of naked RNA and deproteinated RNAs were drawn using the Structure Editor graphical tool, a module of the RNAstructure software.In order to compare the RNA chaperone effect of Pr55Click here for additional data file.


Infertility has been reported as one of the most common reproductive impairments, affecting nearly one in six couples worldwide. A large proportion of infertility cases are diagnosed as idiopathic, signifying a deficit in information surrounding the pathology of infertility and necessity of medical intervention such as assisted reproductive therapy. Small noncoding RNAs (sncRNAs) are well-established regulators of mammalian reproduction. Advanced technologies have revealed the dynamic expression and diverse functions of sncRNAs during mammalian germ cell development. Mounting evidence indicates sncRNAs in sperm, especially microRNAs (miRNAs) and transfer RNA (tRNA)-derived small RNAs (tsRNAs), are sensitive to environmental changes and mediate the inheritance of paternally acquired metabolic and mental traits. Here, we review the critical roles of sncRNAs in mammalian germ cell development. Furthermore, we highlight the functions of sperm-borne sncRNAs in epigenetic inheritance. We also discuss evidence supporting sncRNAs as promising biomarkers for fertility and embryo quality in addition to the present limitations of using sncRNAs for infertility diagnosis and treatment. C. elegans [Noncoding RNAs (ncRNAs) are transcripts that are not designed to be translated. Since their discovery in  elegans ,2, ncRNA elegans , as well elegans ,5,6,7. N elegans ,9 and ri elegans , were id elegans .Gametes are the only cell type that transmit information across generations of sexually reproducing organisms. Studies have shown that miRNAs, endo-siRNAs, and piRNAs are critical regulators of germ cell development, as depletion of enzymes essential for their biogenesis leads to infertility ,13. sncROverall, sncRNAs demonstrate increasingly clear importance in mammalian reproduction. In this review, we discuss the regulation and significance of five classes of sncRNAs, miRNA, piRNA, endo-siRNA, tsRNA, and rsRNA, in the context of germ cell development and disease predisposition. We focus on these sncRNA subtypes as carriers of paternal epigenetic information across generations, as well as the discovery of novel sncRNA species such as tsRNA by NGS technologies. The potential utilization of sncRNAs as biomarkers for infertility diagnostics and the improvement of assisted reproduction will also be discussed in this review. Reproduction is a complex process involving not only germ cells but also gonadal somatic cells and multiple organs. The roles of sncRNAs in other reproductive diseases, such as reproductive cancer ,33,34, oWe searched databases of PubMed, Scopus, and Web of Science for studies relevant to the role of sncRNAs in mammalian reproduction and infertility, with a focus on mammalian germ cell and epigenetic inheritance. The search was conducted before the end of September 2021. The following text words were used in the searching syntax: “sncRNA” or “small non-coding RNA” or “ncRNA” or “non-coding RNA” or “small RNA” or “miRNA” or “microRNA” or “piRNA” or “piwi-interacting RNA” or “endo-siRNA” or “tsRNA” or “tRNA-derived small RNA” or “tRF” or “tRNA-derived fragment” or “rsRNA” or “rRNA-derived small RNA” or “rRF” or “rRNA-derived fragment” or “sperm-borne RNA” or “extracellular RNA”, in combination with “mammalian” and “germ cell” or “primordial germ cell” or “PGC” or “oogenesis” or “oocyte” or “spermatogenesis” or “spermiogenesis” or “spermatogonia” or “spermatocyte” or “spermatid” or “epididymis” or “epididymosome” or “extracellular vesicles” or “early embryo” or “zygote” or “embryo culture” or “inheritance”. For example, the searching strategy in Scopus was listed as the following: TITLE-ABS-KEY  AND ALL  AND TITLE-ABS-KEY . All studies associated with the discovery, regulation, and function of sncRNAs in mammalian germ cell and epigenetic inheritance were then manually selected based on their publication year and the number of their citing articles, as we aim to include the most recent studies with high impact and novelty. The references and cited articles of included studies were also manually searched. The quality of these studies was assessed manually as the included studies, depending on their hypotheses and purposes, varied largely in methods and analyses.6-methyladenosine (m6a) writer, METTL3, to mRNA via a base-pairing mechanism and, in turn, regulate m6a abundance [miRNAs, endo-siRNAs, and piRNAs are the most well-studied classes of sncRNAs. They are mainly distinguished by their biogenesis mechanisms and interacting partners. In animals, the canonical pathway of miRNA processing begins with the transcription of primary miRNA (pri-miRNA) by RNA polymerase II. Processed by the Drosha-DGCR8 complex, the hairpin-shaped pri-miRNAs are trimmed to ~65 nt precursor miRNAs (pre-miRNAs) in the nucleus . Transpobundance .Endo-siRNAs, which are slightly shorter than miRNAs (~21 nt), were ubiquitously found in higher eukaryotic models ,45,46. TpiRNAs, as their name indicates, are associated with PIWI proteins, a germline-specific and highly conserved group of the AGO family. These small RNAs are around 24–32 nt in length. The 5’ end of piRNAs maintain a strong preference for uridine ,54 while5C) modification of tRNAs increases the stability of some tRNAs, making them less likely to be cleaved into tsRNAs [5C on tRNA to 5-hydroxymethylcytosine (hm5C) on tRNA, displaying an opposite effect to NSUN2 on regulating the tsRNA profile [A portion of miRNA and piRNA subtypes are suggested to be derived from tRNA- and/or rRNA-encoding regions, supporting the concept that tsRNAs and rsRNAs are functional sncRNAs ,78,79,80o tsRNAs ,85. TET2 profile . As a re profile ,87,88. r profile . rsRNA p profile ,91. The  profile ,93. As w profile ,90,94. t profile ,95,96,97TDRD1 [Agfg1 and Tbpl1, is implied to be critical for acrosome formation during spermiogenesis [pi6) induces defective sperm acrosome function in male mice, manifesting as impaired capacitation and defects in egg fertilization. These sub-fertile male mice also produce embryos with reduced viability in utero [During spermatogenesis in mouse testes, piRNA is the dominant species of the small RNA population. piRNA-related factors often locate to a germline-specific, electron-dense structure known as nuage . MutatioTDRD1 ,105; (2)TDRD1 ; and (3)ogenesis . Later sin utero . Similarin utero .Drosophila and zebrafish where the piRNA pathway is essential for both spermatogenesis and oogenesis [Mili mutant females, does lead to derepression of some retrotransposons in mouse oocytes, suggesting an uncoupling between retrotransposon expression and fertility [Unlike in ogenesis ,109, mutertility ,20,110. ertility , is neglertility . Notablyertility ,112. A rertility . Similarertility . Althougertility ,113.Pten transcripts which are suppressors of proliferation [6a, has been proposed to regulate the recognition and processing of pri-miRNA [6a writers and readers results in spermatogenic defects [6a in human mature spermatozoa may be indicative of spermatogenic impairments [6a modification occurs on miRNAs and other sncRNAs during spermatogenesis is largely unknown [Sry was shown to contain 16 sites for miR-138, acting as its sponge [miRNAs are also necessary for mammalian primordial germ cell (PGC) development and different stages of spermatogenesis. Depletion of miRNA processing components  in mouse germ cells leads to massive loss of miRNAs and male infertility ,117,118.feration . During feration ,121. miRferation . Althougri-miRNA ,124. Mut defects ,127,128. unknown ,127,128. unknown ,130 as w unknown . circRNAs sponge . Furthers sponge ,134,135.Dgcr8 mutant female mice, which display defects in the canonical miRNA pathway while retaining a fully functional endo-siRNA pathway, exhibit no meiotic defect and are fertile. Although DGCR8-depleted oocytes show profound loss of miRNAs such as DICER1-depleted oocytes, they are relatively similar to wild-type (WT) oocytes at transcriptomic level [Dicer1 and Ago2 mutant but not Dgcr8 mutant oocytes, increases levels of both transposon and protein-coding transcripts that are complementary to those endo-siRNAs [O), with higher cleavage activity compared to somatic DICER (DICERs). Expression of this mouse- and rat-specific DICERO is driven by an oocyte-specific promoter derived from retrotransposition [O -deficient oocytes, suggesting endo-siRNAs and AGO2 may predominantly mediate translational regulation. Further proteomics analysis confirmed this translation regulatory function of the endo-siRNA-AGO2 complex in mouse oocytes [While miRNAs are required for both male and female PGC development ,118 and ic level ,137. On position . Using a oocytes .Dgcr8 mutant male mice with a defected miRNA pathway but an intact endo-siRNA pathway are infertile, albeit with slightly less severe phenotypic outcome as compared to Dicer1 mutant mice [Numerous endo-siRNAs are also detected in mouse spermatogenic cells . Using aant mice . dsRNAs,ant mice . Over 80ant mice , whereasant mice .Taken together, recent studies employing advanced technologies have revealed the dynamics and complexities of sncRNA pathways during mammalian germline development and further emphasized their extensive influence on reproduction . DiffereAs the sperm leaves the testis and transits through the epididymis, the process of sperm maturation occurs, during which spermatozoa acquire full fertility capacity. The epididymis is a long, highly coiled tubule linking the testis and vas deferens. Conventionally, mammalian epididymides are comprised of three main regions , and each region can be subdivided into a number of morphologically and transcriptionally distinct segments ,145. Epi5C and N2-methylguanosine (m2G) have been detected in sperm tsRNAs [Although regulatory mechanisms of tsRNA and rsRNA in testicular sperm or epididymis remain largely unknown, several proteins may be involved in tsRNA biogenesis, including RNA modifiers DNMT2 and NSUN2 ,85. Givem tsRNAs ,84. A rem tsRNAs . RNA modm tsRNAs . In addim tsRNAs . It is tm tsRNAs .Immediately following fertilization, the early embryo remains transcriptionally quiescent. Transcriptional activity is first detected in mid to late one-cell mouse zygote and at 4- to 8-cell stage in human zygote. This resumption of transcription from the zygotic genome is termed maternal to zygotic transition (MZT). Embryonic development before MZT is therefore dependent on maternally stored transcripts and sperm-borne RNAs. In recent years, increasing evidence in mouse and rat models has established the existence of epigenetic inheritance in mammals, where the impact of parental exposure to environmental changes is transmitted to the offspring via epigenetic information in the germline . Sperm-bDicer1 and Drosha conditional knockout (cKO) mice can fertilize WT eggs but display moderate reduction in developmental potential from the pronuclei stage and abnormal gene expression during preimplantation development. This diminished developmental potential is enhanced by injecting WT sperm-borne small RNAs or total RNA, suggesting that paternal miRNAs and endo-siRNAs may contribute to the maternal transcript degradation and MZT [Gln-TTG (termed Gln-TTG) in IVF oocytes by injecting the antisense strand led to aberrant first cleavage. The depletion of Gln-TTGs in porcine IVF oocytes also resulted in downregulation of a LINE1 family retrotransposon, L1M3b [In general, evidence in multiple species and with various methods indicates that sperm-borne sncRNAs are most likely to be carried into oocytes during fertilization and can impact early development. miRNA- and endo-siRNA-deficient spermatozoa generated by germline-specific  and MZT . Using a and MZT . In bovi and MZT . Further and MZT . In porcn, L1M3b .It is worth noting that “readouts” of the impact of sperm-borne sncRNAs on early development, such as fertilization rate and blastocyst rate, are largely affected by the type of experimental procedures used in studies, including the intracytoplasmic sperm injection (ICSI) procedure and methods used to “neutralize” sperm-borne sncRNAs. Consequently, discrepancies have been reported between studies. For example, the sperm-borne miRNA miR-34c-5p was initially reported as essential for the first cleavage in mouse embryos  but was The above studies focus on the existence and functional mechanisms of sperm-borne sncRNAs in early embryos. In the meantime, other studies begin to investigate sperm-borne sncRNAs with a slightly different perspective, i.e., whether they can act as carriers of paternally acquired information that leads to phenotypic changes in offspring. The first pieces of evidence that outline the role of sncRNAs as hereditary epigenetic modifiers are presented in paramutation mouse models, where injection of miRNAs, which may not be sperm-borne naturally, into fertilized eggs was used as a signal to induce the hereditary transfer of the paramutated state ,201,202.Since 2015, a growing number of studies have demonstrated inheritance patterns of diet-induced metabolic disorders via sperm-borne sncRNAs. Grandjean et al. injected testis and sperm RNA of mice kept on a Western-like diet  into a normal zygote, both of which resulted in progenies developing WD-induced metabolic phenotype. Injection of a high amount of one specific miRNA that is increased in the testis of WD-fed mice into normal zygote similarly induced metabolic abnormalities in the offspring which were subsequently transmitted to the next generation . Strikin5C and m2G). When injected with 30–40 nt small RNAs from the sperm of HFD-mice, normal zygotes develop into adults with HFD-induced metabolic disorders. Moreover, RNA modifications may also be informative as injection of synthetic small RNAs did not reproduce the phenotypes in offspring [Chen et al. observed that in sperm of male mice kept on a high-fat diet (HFD), a subset of 30–40 nt 5′-tRNA halves exhibit changed expression profiles as well as RNA modifications , which they report induced altered liver gene expression in F1 offspring . Alteredididymis ,30. A reididymis ,204.Similar to the aforementioned observations in mice, the sperm sncRNA profile in humans is sensitive to changes in metabolic health  as well Psychological condition, including early traumatic experience, stress, and depression, is another transgenerationally influential factor that has been extensively linked to alteration in sperm sncRNA profiles. Using mice experiencing unpredictable maternal separation combined with unpredictable maternal stress (MSUS), Gapp et al. have investigated the transgenerational effects of early trauma. Abnormal behavioral and metabolic traits induced by early traumatic experience, such as depressive-like behavior and insulin hypersensitivity, can be transmitted to the third generation. Microinjection of total RNA from sperm of MSUS-exposed mouse (F1 MSUS) into normal zygote generated offspring that recapitulate these traits. Several miRNAs have been shown to be upregulated in F1 MSUS sperm, while some piRNAs were downregulated. However, the miRNA signatures observed in F1 MSUS sperm disappeared in F2 and F3, suggesting that additional mechanisms are involved to maintain and transmit the traits after F2 . InteresIn a mouse model of paternal chronic stress prior to conception, the offspring often exhibited a reduced hypothalamic–pituitary–adrenal (HPA) stress axis response and transcriptional changes in the hypothalamus cells . ExpressA more recent study has suggested that sperm miRNA, and potentially rsRNAs, also mediates depression susceptibility of offspring in an intergenerational but not transgenerational manner. F1, but not F2, offspring of the male depression-like model developed depressive-like behaviors when exposed to slight chronic variable stress lasting for 2 weeks. Both the depressive F0 and F1 showed aberrant neuronal gene activation, enhanced HPA axis activity, and impaired synaptic transmission. Altered expression profiles of miRNAs as well as rsRNAs were detected in sperm of the F0 depression-like model. Again, microinjection of the small RNAs (<200 nt) from those sperm into normal oocytes reproduced the depressive-like phenotype in offspring. Remarkably, this injection of small RNAs induced dysregulation of several genes with known neurological functions detected in early embryos. These dysregulated genes were also predicted to be direct targets of differentially expressed miRNAs in sperm of the F0 depression-like model, conveying the possible existence of an altered transcriptional cascade induced by sperm-borne sncRNAs .Although the majority of studies have focused on uncovering inheritance patterns of adverse phenotypes via sperm-borne sncRNAs, a few studies have also shown that paternally acquired cognitive benefit may also be transmitted to offspring. A mouse model of voluntary wheel running was used to investigate the transgenerational effect of paternal exercise. The male, but not female, offspring of the runners exhibited anxiolytic behaviors. Surprisingly, only a few miRNAs and tsRNAs showed alteration in the sperm of runners . In humasncRNAs in biological fluid hold great potential for use as novel biomarkers in various diseases, such as ovarian cancer ,207, andGiven the abundance of spermatozoa and existence of noninvasive retrieval methods, sperm analysis is standard during fertility evaluation. It is widely agreed upon that the miRNA profile of mature spermatozoa can be used as biomarkers for impaired spermatogenesis ,210. DifSeminal plasma, which is partly derived from epididymal fluid, generates sncRNA profiles that implicate seminal plasma quality in and male fertility. miRNA profiles in seminal plasma are correlated with specific spermatogenic defects and, more broadly, in male infertility ,218,219.Components of follicular fluid, including sncRNAs, are likely critical determinants of oocyte quality . MultiplThe major challenge of using sncRNA profiles from the aforementioned biological fluids for clinical diagnosis is that sncRNA profiling can be extensively affected by technical differences in materials  ,233, samAcross the past few decades, infertility has become a prevailing impairment to human health, affecting nearly one in six couples worldwide ,142. A sIn this review, we have discussed the relevance of sncRNAs in the pathology of infertility. As biomedical technology continuously advances, novel species of sncRNAs, as well as novel functions and regulation of known sncRNAs, have been uncovered during mammalian germ cell development. Those novel findings imply the existence of a highly complex and dynamic network of sncRNAs in the mammalian germline, where the functions and regulations of sncRNAs correlate to RNA modifications , RNA binThe roles of paternal sncRNAs in epigenetic inheritance have been relatively well-established, yet far less is known about how the environmental exposures are translated into the sperm RNA code, as well as how this sncRNA-encoded signal is maintained and amplified into the phenotypes observed in adults. A few studies have suggested the involvement of ROS and oxidative stress in sncRNA regulation in sperm, but detailed mechanisms remain to be explored ,205,240.Finally, we have highlighted the possibility of using sncRNAs in biological fluid as biomarkers for diagnosis of fertility as well as improvement of ART. Although the idea of sncRNAs as biomarkers for fertility and embryo quality is inspiring and promising, extensive work remains to be undertaken in validating their clinical utilization, standardizing clinical procedures, and improving the accuracy and sensitivity of their detection. For example, as RNA modifications have been shown to abort NGS, removal of RNA modifications must be employed when NGS is used as the detection method. Additionally, as bioinformatics analysis is vital to NGS data interpretation, a reliable pipeline is required for procedural standardization . Databas


Dishonesty in communication has important economic implications. The standing literature has shown that lying is less pervasive than predicted by standard economic theory. We explore whether biology can help to explain this behavior. In a sample of men, we study whether masculine traits are related to (dis)honesty in a sender-receiver game. We study three masculine physical traits: the second-to-fourth digit ratio, facial morphometric masculinity and the facial width-to-height ratio. These biomarkers display significant associations with lying and deception in the game. We also explore the extent to which these effects operate through social preferences or through beliefs about the behavior of receivers. Truthful communication is a pillar of human interactions. Economic exchanges rely on language being trustworthy. Many buyers consult financial advisors before acquiring stocks or probe sellers on the quality of their goods. Honest communication is also crucial in policy making. Central banks make pronouncements which influence the actions of investors and stock traders. Regulatory bodies consult private entities before setting new standards. A third area where communication is crucial is organizations. Division managers, for example, report local market conditions to the their superiors who then use this information to devise their plans for the firm.But better-informed agents often have incentives to misrepresent what they know in order to alter the decision-making process in their favor. Central bankers have an incentive to manipulate economic expectations Stein, . PrivateThe experimental literature on strategic information transmission has shown that individuals engage in truthful communication above standard game-theoretical predictions  and during puberty . The third one has been associated to antisocial and dominance behavior . We discuss these markers in detail and the debates about their relevance in the following section.We study how lying and deception by senders in this game correlate with a set of masculine physical traits. Two of the biomarkers we consider are related to testosterone exposure at two developmental periods, in utero and with more masculine facial features are more likely to send truthful messages. However, we also find that the latter engage more often in deception through truth-telling. In contrast, individuals exposed to higher prenatal levels of testosterone seem to display a stronger lie aversion as they are more likely to engage in costly truth-telling, i.e., send a truthful message when it is expected to be trusted.Our results suggest the existence of a significant relationship between the masculine physical traits we study and (dis)honesty in strategic communication. We find that individuals exposed to higher levels of testosterone Finally, we explore whether these associations between lying and the masculine physical features we consider operate mostly through social preferences  or through beliefs about the behavior of receivers. Results suggest that preferences are the main drivers of these effects.The present paper contributes to the rapidly expanding literature on the influence of biometric traits and sexual hormones on economic behavior. Studies in this area have shown that differences in circulating and basal levels of sexual hormones influence risk preferences  is the ratio between the length of the index and the ring fingers. The available evidence suggests that the 2D:4D ratio is related to the ratio of amniotic testosterone/estrogen concentrations +p(B∣θ) = 1.Our experimental design is based on Sanchez-Pages and Vorsatz . First, m and must choose a mixed strategy over his available actions, A and B. The action taken s∈{A, B} is relevant for both players as it determines in conjunction with the table selected θ the payoffs they receive. The payoff structure is of divergent interests  is the probability with which the receiver believes that the message m is truthful and action s = m will indeed earn him the highest payoff. Note that μ(A∣m)+μ(B∣m) = 1. Denote the mixed strategy of the receiver as q(A∣m)+q(B∣m) = 1. As it is customary in the literature, we will interpret that a receiver trusted (or followed) the sender's message if he took the action that maximized his payoff if the message was truthful, i.e., when s = m.The receiver holds a belief profile p(A∣A) = p(A∣B) = p∈, meaning that they lie with 50% probability. This renders messages completely uninformative, so receivers' posterior beliefs remain identical to the prior, i.e., Under these preferences, the standard game-theoretical prediction is that the set of sequential equilibria of the game are all "babbling": Senders send each message with the same probability regardless of the table chosen, i.e., content of the message, deception relates to the outcome the message is trying to induce . Therefore, a sender can be deceptive in our experiment either by lying when he expects the receiver to trust his message with more than 50% chance or by telling the truth when he expects the receiver to follow his message with less than 50% chanceLet us now introduce some behavioral considerations. Suppose that the sender expects the receiver to trust his message with more (less) than 50% probability. In that case the sender should tell a lie (the truth) under standard preferences (and independently of his risk attitude). It is at this point where we should make a crucial distinction between lying and deception: whereas lying is related to the e Sobel, . Obviousstrong truth-teller. A sender who tells a lie when he expects the receiver to distrust his message with more than a 50% chance is not maximizing his expected payoff either and in addition he is lying. Given that such sender is paying a monetary cost and probably a psychic (lying) cost also to make the receiver obtain a higher payoff, we refer to this sender as an altruistic liarOn the other hand, a sender who tells the truth when he expects the receiver to trust his message with more than a 50% chance is not maximizing his expected payoff. We will say that this sender is a The study was conducted with undergraduate students at the Universidad Autónoma de Madrid (UAM), Spain, in early 2016. It was approved by the UAM Research Ethics Committee (reference CEI 62-1086). Subjects were recruited from the subject pool of the Madrid Laboratory for Experimental Economics (MADLEE) and with posters and flyers distributed within the Faculty of Sciences where the experimental sessions took place. The invitations and promotional materials mentioned that participants would be taken images of their faces and their hands and that these images could not be linked to any personal information. No mention to the all-male nature of the experiment was made during the recruitment process or the sessions.A total of 168 males participated in 10 sessions composed by 12–24 subjects each. This sample size was meant to detect the associations between economic behavior and masculine physical traits identified in previous worksSubjects were called one by one to the lab and took sit at individual tables. Instructions were then read aloud see the . During control treatment, where they played the sender-receiver game described above. After that, they played a punishment treatment, a version of the Punishment Game in Sanchez-Pages and Vorsatz  choice as receiver; (2) choice as sender; (3) elicitation of beliefs about expected truth and trust rates. In the punishment treatment there were two additional rounds: (4) punishment choices and (5) elicitation of beliefs about punishment rates.At the end of each session, participants were called one by one to an adjacent room where morphometric measurements were taken in private by one experimenter and two research assistants. After this, one treatment was selected for payment. Roles were randomly assigned within each anonymously matched pair of participants and payoffs were determined according to their decisions. Subjects were paid their earnings in cash in addition to a 5€ show-up fee for this experiment. The exchange rate between points in the experiment and money was 100 points=1€. Average earnings were 7.82€.p = 0.099, n = 167) and between fMM and 2D:4D is -0.128 . No significant correlation exists between fWHR and 2D:4D. These weak correlations are in line with previous studies . They were also significantly different from each other . Trust rates were 63.1% in the control treatment and 58.9% in the punishment one. Both rates were significantly higher than 50%  and similar to those in Sanchez-Pages and Vorsatz .The percentages of untruthful messages in the control (37.5%) and punishment (25%) treatments were well below the theoretical prediction of 50% , suggesting that the possibility of punishment induced senders to switch from selfish lying to costly truth-telling. The threat of punishment was indeed very real: The punishment rate after history {lie,trust} was substantial, 27.38%.We next use regression analysis to study the association between lying and deception on the one hand and the three masculine physical traits we consider on the other. In Beliefs. Column (1) studies the association between masculine physical traits and participants' beliefs about trust rates in their session. All three markers display a significant coefficient, although in varying degrees of significance and directions. An increase in fMM (fWHR) by a standard deviation increases the expected trust rate by 3.5 (2.3) percentage points (pp). However, higher exposure to prenatal androgens as measured by 2D:4D, decreases the expected trust rate by 3.2 pp.Result 1: Higher levels of fMM are associated with senders expecting more receivers to follow their message. Higher exposure to prenatal testosterone and higher fWHR are associated with the opposite.Note that the coefficients of interest in column (1) are relatively small and on those for fMM and fWHR are weakly significant. This already suggests that the association between beliefs and our masculine traits is not strong. We will come back to this issue below.Lying. In the rest of columns of in utero engaged less in lying. The estimates for the other traits are non-negligible but less significant.The specification in column (2) estimates the total effect of the three masculinity markers we study on lying. Again, the traits display sizeable effects at different degrees of significance. 2D:4D is associated to a decrease in the probability of lying by 6.1 pp. This regression thus suggests that individuals exposed to more testosterone Column (3) includes subjects' expected trust rates as a control. The coefficient for fWHR increases in significance and absolute value whereas the one for 2D:4D decreases. An increase of one standard deviation in fWHR now leads to a reduction in lying by 6.6 pp, and an increase in the latter to a decrease by 5.4 pp.Result 2. Higher fWHR and higher exposure to prenatal testosterone are associated to less lying.The positive coefficient for beliefs implies that participants who believed that a higher fraction of receivers would trust messages were more likely to lie.Preferences vs. beliefs. The regression in column (3) is also important because it allows us to explore the extent to which the association between our masculine traits and lying behavior operates through beliefs about the behavior of others, through preferences, or both  see . These rDeception. At this point, the distinction between lying and deception becomes important. A higher likelihood of sending a truthful message does not necessarily indicates stronger prosociality. If the receiver is expected to distrust the sender, telling the truth becomes a form of sophisticated deception  are negatively correlated with lying, but the picture changes when we bring into consideration the expected consequences of messages. This is consistent with recent research on the role of testosterone in social interactions. This hormone makes the seek of social status and dominance a salient motivation, but this goal translates into aggression and competitiveness in some contexts and into prosocial behavior in others  and the events the individual experiences (including abiotic factors and their interaction with other living beings). From that point of view, the conjecture that prenatal hormone levels may influence human behavior is especially interesting . Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


RNA motifs may promote interactions with exosomes (EXO-motifs) and lipid rafts (RAFT-motifs) that are enriched in exosomal membranes. These interactions can promote selective RNA loading into exosomes. We quantified the affinity between RNA aptamers containing various EXO- and RAFT-motifs and membrane lipid rafts in a liposome model of exosomes by determining the dissociation constants. Analysis of the secondary structure of RNA molecules provided data about the possible location of EXO- and RAFT-motifs within the RNA structure. The affinity of RNAs containing RAFT-motifs  and some EXO-motifs  to rafted liposomes is higher in comparison to aptamers without these motifs, suggesting direct RNA-exosome interaction. We have confirmed these results through the determination of the dissociation constant values of exosome-RNA aptamer complexes. RNAs containing EXO-motifs GGAG or UGAG have substantially lower affinity to lipid rafts, suggesting indirect RNA-exosome interaction via RNA binding proteins. Bioinformatics analysis revealed RNA aptamers containing both raft- and miRNA-binding motifs and involvement of raft-binding motifs UCCCU and CUCCC. A strategy is proposed for using functional RNA aptamers (fRNAa) containing both RAFT-motif and a therapeutic motif  to selectively introduce RNAs into exosomes for fRNAa delivery to target cells for personalized therapy. Exosomes are small 30–100 nm) membrane nanovesicles originating from endosomes, that are released from the parent cell into the extracellular environment 00 nm mem,3, dendrSince the first description of microvesicles produced by platelets in human blood and the discovery of their procoagulant properties  the roleExosomes are formed as intraluminal vesicles (ILVs) by the inward budding of the multivesicular body (MVB) and are secreted into the extracellular space by fusion of MVB with the cell membrane . The memMembrane rafts are heterogeneous, dynamic, cholesterol and sphingolipid-enriched nanodomains (10–200 nm in size) within membranes, that compartmentalize cellular processes  and may Although the exact mechanism of RNA loading into exosomes is still being explored, it appears that exosomal RNAs are not loaded randomly. Studies on RNAs transported by exosomes show that exosomal RNA content may differ from that of the parent cell ,32,33,34The factors involved in the active regulation of the exosomal RNA sorting process are being actively explored. Research identified molecules such as neural sphingomyelinase 2 (nSMase2) , AGO2 prRNA motifs are short sequences in the RNA related to a particular property, such as binding to certain molecules and sorting into exosomes. RNA motifs that are significantly over-represented in exosomes can be designated as EXO-motifs . They arApplication of exosomes as an RNA-delivery system is based on isolation of exosomes from extracellular fluids with subsequent loading of exosomes in vitro with appropriate RNA. The methodology proposed in this study is based on the loading of cells with appropriate RNA  and allowing the cell to load fRNAa into ILVs with subsequent release of fRNAa within exosomes into the extracellular fluid. However, no clear relationship between the affinity of RAFT-motifs  to membranes and their location within the secondary or tertiary RNA structure was demonstrated, therefore, RAFT-motifs seem to function as sequence motifs rather than structural motifs.Since the selective binding of RNAs to the membrane of MVB may be regulated by the activity of a lipid raft region in the MVB membrane and the presence of the RNA motifs, in this study we investigate the significance of these factors  in the RNA-membrane relationship. We analyze the interactions of certain RNA oligonucleotides containing EXO-motifs and/or RAFT-motifs with both ordered and disordered regions of membranes using model membranes. The aim is to determine which of these RNAs binds best to membranes and what is the role of RNA motifs in that process. We also explore the importance of the location of certain EXO- or RAFT-motifs within the secondary structure RNA molecules for their interaction with the raft membrane.D) was calculated, showing the degree of the RNA aptamers’ affinity to the lipid bilayers.The experimental system was designed to characterize the interaction of individual RNA aptamers containing different RNA motifs with both ordered and disordered membranes. We used liposomes made of DOPC, sphingomyelin, and cholesterol (molar ratio 6:3:1) or solely DOPC liposomes as models of ordered and disordered membranes, respectively. The fluorescent methods used were focused on the FRET technique applied to RNA aptamer-to-membrane energy transfer. The decrease in the fluorescence in the donor channel (YOYO-RNA) at a constant concentration of the donor and increasing concentration of the acceptor (LissRh-PE-Liposomes) was measured. Based on fluorescent measurements the dissociation constant  was searched for EXO-motifs   and RAFTFor the measurements of the RNA-membrane interactions, ten RNA aptamers containing RNA motifs in various sequence locations were chosen . SimilarThe RNAs’ secondary structures were predicted using the m-fold program  and the To confirm the importance of lipid rafts in RNA-membrane interactions, fluorescence measurements were applied based on the FRET mechanism. For each selected RNA aptamer, the changes in donor emission intensity were observed during titration with disordered (DOPC) liposomal membranes or with liposomal membranes containing ordered domains . max is the maximal YOYO-1 fluorescence obtained before liposome titration. ΔF is the difference between Fmax and subsequent fluorescence measurements. The ratio of ΔF/Fmax increases during titration of both types of liposomes, showing the gradual saturation of RNA with lipid molecules.As shown on the emission spectra A for RNAD). The calculated KD values were higher for the interaction of RNA aptamers with DOPC liposomes in comparison with RAFT liposomes. For aptamer 54, the KD was 168.4 µM for the interaction with DOPC and 66.2 µM for interaction with RAFT liposomes  and RAFT liposomes (right) is presented in iposomes C. Similaembranes .\D values range is presented in D) value was over two times lower for RNA-RAFT interactions than the median KD value for RNA-non-RAFT liposomes. The comparison of median RNA-DOPC KD value (232 µM) and median RNA-RAFT KD value (113 µM) indicates that the presence of membrane rafts promotes the binding of RNA aptamers to the liposome membranes. A significant difference was also confirmed by the Mann–Whitney U test .The boxplot of the KD values were compared , was obtained for RNA 54 which contains three CCCU motifs (two of which are located entirely in loops). Another EXO-motif, UCCU, also occurs three times in this RNA and within the loop domain. Besides CCCU, three other RAFT-motifs are present in this aptamer: UCCC, CUCC, and UUGU. The two last motifs are present twice in this RNA. These RAFT-motifs share their location between the stem and loop domain. It should be noted that three groups of motifs overlap: UCCU with UUGU, UCCU with CUCC, as well as CUCC, UCCC, and CCCU. The second highest affinity (KD = 61.1 µM) was obtained for the aptamer 90, which has an EXO-motif GGAG and a RAFT-motif UUGU.To determine the importance of RNA motifs (both EXO and RAFT) for RNA affinity to membranes, the Kcompared ; Table 2D value is observed for RNA 78 with just one EXO-motif (UGAG) and no RAFT-motifs. In contrast, the lowest affinity for lipid rafts is measured for RNA 78 (KD = 225.2 µM) which, besides EXO-motif UGAG, has no other motifs. The UGAG motif is located partially in the loop and stem sections. The other eight aptamers have KD values in the range of 61 to 213 µM. Besides RNA 54, four other aptamers  have a KD value below 100 µM, and RNA 64, 24, 93, and 10, as well as RNA 78, have KD values above 100 µM, wherein a significant increase in the KD value is observed between RNA 64 (KD = 112.2 µM) and RNA 24 (KD = 187.5 µM).The highest KD value varies among aptamers with a different number of CCCU motifs. The average KD value for aptamers with a single CCCU motif  is equal to 139 µM (±29) and decreases to 121 µM (±27) for aptamers with two CCCU motifs . These findings, in conjunction with the KD value of aptamer 54 with three CCCU motifs (KD = 61 µM), may indicate a relationship between the number of CCCU motifs and RNA-membrane affinity, although the strong affinity of aptamer 90 (no CCCU motif) suggests that other raft-binding motifs, such as UUGU, also enhance the RNA affinity to membrane rafts.The KTo this point, we have documented the importance of both lipid rafts and RAFT-motifs for RNA-membrane interactions. We now wish to show that several raft-RNA aptamers  contain Among analyzed raft-RNA aptamers, there are a few special cases: RNA aptamers containing two different miRNA-binding motifs for two different miRNAs , RNA aptamers containing the same miRNA-binding motifs for two different miRNAs , two different RNA aptamers containing two different miRNA-binding motifs for one miRNA . Interestingly, 5-nts raft-RNA motifs UCCCU and CUCCC  appear to be the most frequent motif within miRNA-targeting raft-RNA aptamers. Three raft-RNA aptamers  contain a miRNA-binding motif and all have a strong affinity to rafts . SchematD) was calculated for the RNA-exosome complex. D = 0.0039 µM, whereas the least strong aptamer was RNA 10 (KD = 0.0109 µM). The results of the remaining RNA aptamers are attached in the We have also determined the raft-RNA aptamers’ activity in biological systems using exosomes. After purification and labeling , exosomeThe differences in affinity of eight analyzed RNA aptamers to exosomes, seem to resemble the variations of these same RNA aptamers’ relationship found in the liposome-RNA assay . In bothIn our study, the affinities of lipid membranes to RNA oligonucleotides containing both EXO- and RAFT-motifs were characterized to understand the role of RNA sequence and membrane lipid rafts on the aptamer-membrane interactions. Using the FRET approach, the affinity of RNA aptamers to lipid membranes was measured, while analyses of the secondary structure of the RNA provided data about the location of the RNA motifs within the RNA molecule.D) values were approximately two times lower for the RNA-RAFT interactions than for the RNA interaction with non-RAFT liposomes  emission intensity were measured after incubation with ordered RAFT (DOPC:SM:CHL) and disordered non-RAFT (DOPC) membranes. All RNA aptamers showed a higher affinity for RAFT liposomes compared to non-RAFT liposomes. The dissociation constant  to the liquid-ordered bilayer in comparison to the liquid-disordered (DOPC) bilayer, and the localization elements were suggested to function as novel lipid raft-binding RNA motifs. The presence of localization elements within the loop of an RNA hairpin structure enhanced RNA affinity for lipid rafts.Similarly, stronger RNA affinity to raft membrane was obtained in the study on the interactions of the  oocytes . The Vegβ-maximally ordered phase), there is a rather small amount of RNAs with the affinity for fluid membranes (Lα-liquid disordered phase). These studies emphasize that the state of the membrane order can regulate the RNA-membrane interactions and RNAs can moderately interact with the raft phase.The structure-dependent RNA binding was demonstrated previously for membranes in the liquid-disordered, liquid-ordered phase, and gel phase . AlthougLipid rafts were reported to be involved in the vesicle formation process itself  and it wD) values of aptamer-membrane affinity were compared. RNA aptamers containing both the EXO- and RAFT-motif CCCU bound stronger to lipid rafts compared to the control RNA aptamer without the RAFT-motif. RNA aptamer 54 with three CCCU motifs (two of them in loops) exhibited the highest affinity for the raft bilayer, suggesting that the number and location of RNA motifs may be meaningful in RNA binding to the membrane.Since the presence of specific RNA motifs in the sequence of RNA is postulated to regulate the binding of RNAs to the lipid raft regions of the membrane, we searched the original RNA pool for EXO- motifs   and RAFTVegT RNA structure enhances its binding to lipid raft domains  where: ΔFmax is the calculated maximal fluorescence change, x is the DOPC or RAFT lipid concentration, and KD is the equilibrium dissociation constant for RNA-liposome complex. Liposomes without fluorescence probes were used to correct for light scattering.Based on fluorescent measurements, the Kequation ,52,56: Δg, and the supernatant was subjected to 18 h centrifugation at 126,000× g. The obtained pellet was washed with PBS and dissolved by gentle agitation, before re-pelleting it with ultracentrifugation at 126,000× g for 70 min, and next re-suspending in 120 μL PBS. This suspension was incubated with a membrane fluorescence probe CTB-555 , followed by incubation with fDiI , and further purified using a Sephacryl S-1000 gel filtration column, followed by a 100-nm pore size ultrafiltration. The gel filtration column was eluted with RNA buffer. The gel filtration step removes both small lipoprotein aggregates and non-bound CTB-555 and fDiI.Exosomes were isolated from calf bovine serum (CBS) as described ,86. BrieD values were calculated in a similar way to the RNA aptamer-rafted liposome complex.Each analyzed RNA after folding (2.5 µM in RNA buffer) was added to SYTO-61 solution in RNA buffer . The exosome suspension was titrated with SYTO-61-RNA aptamer solution. Membrane-to-RNA aptamer FRET was monitored at room temperature as a decrease in donor (CTB-555 and fDiI) emission intensity (λex = 550 nm and λem = 570 nm) under increasing acceptor (RNA-bound SYTO 61) concentration. The K


E. coli, however, lack of 6S RNA (ΔssrS) was demonstrated to be unfavorable but not essential for cell survival under various growth conditions. In the present study, we revealed, for the first time, a lethal phenotype of the ΔssrS strain in the presence of high concentrations of H2O2. This phenotype was rescued by complementation of the ssrS gene on a plasmid. We performed comparative qRT-PCR analyses on an enlarged set of mRNAs of genes associated with the oxidative stress response, allowing us to identify four genes known to be involved in this pathway  that had decreased mRNA levels in the ΔssrS strain. Finally, we performed comparative proteomic analyses of the wild-type and ΔssrS strains, confirming that ΔssrS bacteria have reduced levels of the proteins AhpC and Tpx involved in H2O2 reduction. Our findings substantiate the crucial role of the riboregulator 6S RNA for bacterial coping with extreme stresses.6S RNA, a small non-coding RNA present in almost all bacteria, inhibits transcription via direct binding to RNA polymerase holoenzymes. The mechanism of 6S RNA action was investigated to a large extent in  Staphylococcus aureus, SgrS RNA from Escherichia coli and SR1 RNA from Bacillus subtilis [Regulation of transcription is a central mechanism in prokaryotes for adjusting gene expression to changes, in particular, under unfavorable environmental conditions ,2,3. In subtilis . To datesubtilis ,13. The subtilis .E. coli and B. subtilis. Historically, the first documented phenotype of an E. coli 6S RNA knockout (ΔssrS) strain was reduced survival compared to the parental strain under conditions of extended cultivation (20 days) and in competitive cultivation experiments [E. coli 6S RNA was shown to regulate the expression of various transcription and translation factors, transporters and enzymes involved in the metabolism of purines and degradation of amino acids. Notably, a number of stress-related genes were also shown to be dysregulated in ΔssrS cells, especially in the stationary phase, in particular, asr (acid shock protein), dps (DNA protection protein), cspA and yfiA (cold shock proteins), sra and smpB (ribosome stabilizing proteins), as well as uspG and uspF  [E. coli 6S RNA knockout also caused derepression of the relA gene encoding ppGpp synthase I in the early stationary phase, resulting in decreased rRNA expression and enhanced expression of genes involved in amino acid biosynthesis [pspABCDE and pspG operons involved in alkaline stress response. Upon 6S RNA knockout, these proteins are overexpressed and improve stationary cell survival under high pH conditions [6S RNA is an abundant bacterial ncRNA that directly binds to RNA polymerase (RNAP) holoenzymes, particularly in the stationary phase of cell growth ,16. The eriments . E. coliroteins) . The E. ynthesis . A prononditions .B. subtilis, although this bacterium is quite different in terms of natural habitats, physiology and metabolism. B. subtilis and other Firmicutes express two different 6S RNAs (6S-1 and 6S-2) [B. subtilis culture density in the late stationary phase (24 h cultivation) and was advantageous under extremely alkaline conditions (pH 9.8) [B. subtilis 6S-1 RNA  in the strain 168 caused a delay in the outgrowth of cell cultures from the stationary phase and resulted in earlier sporulation [B. subtilis PY79 derivative strains with knockouts of genes encoding its two 6S RNA paralogs, 6S-1 and 6S-2 RNA, also revealed changes in expression levels of many proteins involved in metabolisms of purines, amino acids and carbohydrates, including a number of stress response regulators, such as guaB, cysK, tpx (superoxide-inducible proteins), yvyD and yjlD (glucose starvation-inducible proteins), rplJ , greA, yraA, ahpC, sodA and nadE  [B. subtilis 6S-2 RNA, but not 6S-1 RNA, was demonstrated to regulate biofilm formation, swarming activity and sporulation in the undomesticated wild-type strain NCIB 3610 [B. subtilis PY79 background (see above), deletion of both 6S RNA genes in strain NCIB 3610 retarded outgrowth under alkaline stress (pH 9.5) relative to the parental and single knockout derivative strains [Similar and overlapping phenotypes were observed for the Gram-positive nd 6S-2) . Deletio(pH 9.8) . Additiorulation ,26. Protroteins) . RecentlCIB 3610 , phenotyCIB 3610 . Notewor strains .E. coli and B. subtilis. For example, lack of 6S RNA was reported to lead to slower growth of Rhodobacter sphaeroides under high salt stress (250 mM NaCl) [Synechocystis sp. PCC 6803 cells from nitrogen starvation [Salmonella enterica serovar Typhimurium under acidic stress (pH 3.0) [L. pneumophila during infection of the protist Acanthamoeba castellanii or mammalian THP-1 cells [Borrelia burgdorferi in a mouse infection model [Evidence for a key role of 6S RNA in the regulation of bacterial stress responses has also been growing owing to studies in other bacteria beyond mM NaCl) , to delaarvation  and to r(pH 3.0) . Moreove(pH 3.0) . Similar-1 cells , and foron model .In summary, 6S RNA functions seem to be quite diverse across different bacterial phyla, possibly species-specific in some cases. Yet, an emerging commonality of 6S RNAs is their importance for bacterial physiology, especially under adverse environmental conditions. Surprisingly, 6S RNA knockouts have so far not been reported to be lethal for their bacterial host, despite the RNA’s function as a global regulator of transcription. Seemingly, 6S RNAs are intertwined with additional, partly redundant mechanisms of gene expression control that can compensate for the loss of 6S RNA to some extent. E. coli and report the first-ever lethal phenotype of a 6S RNA-deficient strain at high concentrations of hydrogen peroxide. Notably, E. coli 6S RNA knockouts have been studied since 1985 [E. coli stress response that is critical for cell survival.In the present work, we observed the involvement of 6S RNA in the oxidative stress response in nce 1985 , but no E. coli MG1655 ΔssrS strain where we replaced the 6S RNA gene (ssrS) with a kanamycin resistance cassette [E. coli 6S RNA gene deletion on cell growth and survival under oxidative stress conditions. Upon inoculation of fresh medium containing 5 mM H2O2 with stationary phase cells (overnight culture), we observed an extended delay in outgrowth for the ΔssrS strain relative to the parental MG1655  strain (We previously constructed an cassette . In the ) strain .2O2 reduction. The phenotype was reproducibly observed for growth in liquid culture flasks  outgrowth delays for both, the WT and ΔssrS strain, with increasing variations between replicates (data not shown), thus excluding the calculation of meaningful average growth curves.This relative outgrowth delay reached 1 to 2 h  or even e flasks a and 96-e flasks b despiter ~ 1 h; . The pre2O2 treatment by plating of serial culture dilutions. To further accentuate the difference between the two strains, we increased the concentration of H2O2 in the culture medium to 50 mM and withdrew culture aliquots just after 30 min of incubation. In these experiments, ΔssrS bacteria showed at least 10-fold lower viability than the parental WT strain  resulting in complementation strains abbreviated as WT+S, ΔssrS+S, WT+0 and ΔssrS+0, respectively. Expression of 6S RNA in strains WT, WT+0, WT+S and ΔssrS+S, as well as the absence of 6S RNA in strains ΔssrS and ΔssrS+0, was confirmed by Northern blot analysis  and transition (8 h) phase were somewhat lower in the WT and WT+0 than in the WT+S and ΔssrS+S strains. All strains showed essentially uniform growth behavior in standard rich (LB) medium  medium . Compara strains . The samer small . We alsoondition . The Δss2O2 concentration and then inoculated with an overnight culture grown in standard LB medium. To further characterize the discovered phenotype of 6S RNA-deficient cells, we tested several oxidative stress regimens. The phenotype was exacerbated when 20 mM H2O2 (f.c.) was added to exponentially growing (OD600 ~ 0.5) E. coli cell cultures in flasks, resulting in the lethality of ΔssrS and ΔssrS+0 bacteria , the growth delay of the knockout strains correspondingly increased  in the absence of H2O2, and then diluted this culture 1:5 in media containing different amounts of H2O2; the resulting suspensions were transferred to 96-well plates for growth monitoring. In this type of experiment 20 mM H2O2 was very toxic to all of the strains, but 10 mM H2O2 was only lethal for ΔssrS and ΔssrS+0 bacteria , however, no difference between the WT and ΔssrS strain was observed  or even downregulation of mRNA levels, e.g., oxyR, that is in line with previous observations [ssrS versus WT strain under H2O2 stress (soxS (activator of superoxide response), ahpC , sodA (superoxide dismutase) and tpx (thiol peroxidase) mRNAs were ~2–3-fold decreased in the absence of 6S RNA. The strongest activation upon Hobserved . Moderatrvations . However2 stress b. NamelyB. subtilis [ssrS knockout strain by 2D-PAGE , the sigma factor specific for stationary phase transcription and stress response [E. coli 6S RNA regulates the expression of hundreds of genes, also including several stress response proteins, translation and transcription factors as well as enzymes involved in various metabolic processes [NcRNAs are widely activated in response to different stresses and/or adaptation to unfavorable conditions, where they regulate the expression of single or multiple targets. For example, DsrA, ArcZ, and RprA ncRNAs are known to activate translation of RpoS . Through complementation using a plasmid-borne ssrS gene we were able to fully rescue the lethal phenotype, thus proving that it is a direct consequence of the lack of 6S RNA expression. This is the first reported lethal phenotype of 6S RNA-deficient bacteria in general. So far, phenotypes of 6S RNA knockouts were primarily observed under extreme alkaline or acidic conditions: E. coli [B. subtilis [Salmonella [R. sphaeroides [B. subtilis [E. coli [B. subtilis [E. coli and related Enterobacterales, including pathogenic species [Here, we discovered a lethal phenotype of  E. coli , B. subtsubtilis ,27, Salmlmonella ; salt staeroides , B. subt[E. coli , B. subtsubtilis ,25,29; osubtilis ,32. Nota species . There a species . Moreove species .E. coli against damage by oxidative stress is well-studied, including the key regulators OxyR and SoxRS, the alternative sigma factor RpoS and many other proteins that are conserved among Proteobacteria [2O2, which triggers the formation of an intramolecular disulfide bond [katG , dps (involved in DNA damage repair & iron storage), the ahpCF operon  and oxyS (see below) [E. coli MG1655 cells [2O2-degrading enzymes, among them the major catalases KatG and KatE as well as alkyl hydroperoxide reductase AhpCF. Recent studies discovered some novel protein players as well, for example, the transcriptional regulator LsrR that directly inhibits ahpCF and katG promoters [The general defense of bacteria . OxyR iside bond . Genes te below) ,55,56,57romoters . ompF mRNA as a target, also affects the regulatory network involving the leucine responsive protein Lrp [2O2-inducible (via OxyR) ncRNA that represses expression of transcription factors, such as NusG, FhlA and RpoS, which entails pleiotropic alterations in the expression of many other genes [E. coli was also shown to cause lethality after growth for 20 min at 5 mM H2O2 [2O2 stress but is not transcribed under normal conditions [2O2 treatment, as shown in the present study  in the ΔssrS strain has its origin in reduced expression of SoxS. Moreover, ahpC is under control of OxyR whose mRNA levels were not significantly affected by the lack of 6S RNA, in line with previous observations that oxyR expression is not induced upon H2O2 stress [2O2 induction of ahpC expression in the ΔssrS strain . Interestingly, the dysregulated proteins identified so far in ΔssrS bacteria included AphA (acid phosphatase) and DsbA (thiol:disulfide interchange protein) whose mRNA levels were also found to be altered in microarray analyses of ΔssrS bacteria in standard growth conditions. Levels of aphA mRNA were reported to be upregulated (~1.7-fold) and those for dsbA mRNA to be downregulated (~1.6-fold) in ΔssrS cells [ssrS cells, while DsbA levels decreased  of the Δbacteria . This co to H2O2 b, despitrS cells . These decreased . Despiteibiotics .B. subtilis strains that lack either 6S-1 or 6S-2 RNA, while SodA was only upregulated in the 6S-2 knockout strain [B. subtilis strain, ∆6S-1&2 RNA double knockout cells also showed a prolonged lag phase under mild oxidative stress conditions [Notably, we previously observed that AhpC and Tpx were upregulated in the proteomes of t strain . In the nditions . These fE. coli strains used in this study are listed in ssrS complementation and corresponding control strains is detailed in w/v) agar. Further E. coli control strains (three pairs of WT/ΔssrS strains) are listed in All plasmids and E. coli strains were grown in the absence of antibiotic in liquid cultures (100 mL of LB medium) in Erlenmeyer flasks (500 mL volume) covered with a metal cap. In general, freshly autoclaved LB medium was inoculated with a stationary phase culture  to a starting OD600 of 0.05 or 0.1, followed by growth at 37 °C under stirring (200 rpm) in a waterbath shaker. Culture aliquots were withdrawn at indicated time points and diluted 1:10 in fresh LB medium for measurement of optical density at 600 nm. To plot growth curves, at least three biological replicates of the same growth experiment were conducted. For oxidative stress experiments, appropriate amounts of 1 M H2O2 prepared from an aqueous 30% (w/w) H2O2 stock  and standardized by KMnO4 (Sigma-Aldrich) titration were added to LB media prior to inoculation. In another set of experiments (indicated in the text), freshly autoclaved media were first inoculated with a stationary phase culture to a starting OD600 of 0.1. After incubation for ~2 h and achieving an OD600 of ~0.5, appropriate amounts of H2O2 (f.c. 10–20 mM) were added to the cell culture, followed by further cultivation with monitoring of optical density.For growth curve monitoring, 600 of 0.05 or 0.1 in LB medium without antibiotic but beforehand supplemented with H2O2 (concentrations indicated in the corresponding graphs), and then loaded with a multichannel pipette in 100-µL aliquots into the wells of a 96-well plate . In a set of initial experiments  in an automated TECAN Safire 2 Platereader  using flat bottom, transparent 96-well microtiter plates with plastic cover . Appropriate amounts of stationary phase culture  were diluted to an ODeriments , plates 2O2. From such aliquots, we made a number of serial 10-fold dilutions in fresh LB medium and plated 5-μL drops on solid agar plates without antibiotic. The number of surviving colonies was evaluated by visual inspection. To ensure that the decrease of optical density corresponds to enhanced cell mortality, we withdrew 100 μL aliquots of cell culture grown in flasks at several time points after the addition of HE. coli strains to oxidative reagents we performed classical inhibition zone assays as described previously [600 of ~0.5, then diluted to an OD600 of 0.1 with fresh LB medium, followed by adding a 100-μL aliquot to 6 mL of warm (42 °C) top agar (0.8% w/v); the mixture was subsequently poured on top of a solid agar (1.5% w/v) plate. A filter paper disk (d = 5 mm) was soaked with a H2O2 solution of defined concentration and positioned in the center of the rigidified top agar. The diameter of the inhibition zone was measured with a ruler after incubation for 24 h at 37 °C.For additional estimation of the sensitivity of eviously . In briessrS+S and WT+S E. coli strains were estimated by Northern blot analysis  following the protocols described in [Expression levels of 6S RNA in the WT and complementation Δribed in ,67. For E. coli by TRIzol reagent  were treated with DNase I  followed by reverse transcription using the OT-1 Kit with MMLV Reverse Transcriptase  according to the manufacturer’s protocol (reaction volume: 20 µL). Thereafter, the volume was increased to 40 μL with ddH2O and 2 μL of such diluted cDNA samples were mixed with 4 μL 5× qPCR mix-HS SYBR  and 0.4 μM (f.c.) of each reverse and forward primer (t) by the 2−∆∆Ct method using the amount of 16S rRNA for normalization. At least three biological replicates were measured for each sample and used to calculate mean 2−∆∆Ct values. Total RNA samples (0.5 μg) isolated from d primer  in a finE. coli MG1655 WT and ΔssrS knockout cells were grown in parallel in 100 mL LB medium starting from an OD600 of 0.1 . 50 mL aliquots were withdrawn during exponential growth at OD600 ~ 1; the rest of the cell culture was withdrawn in stationary phase after further incubation for up to 12 h. After centrifugation at 4000 rpm , cell pellets were snap-frozen in liquid nitrogen and stored at −80 °C. All procedures of sample preparation, labeling and 2D protein gel electrophoresis were carried out exactly as was described for the comparative proteome analysis of 6S RNA-deficient B. subtilis strains [v/v) ethanol, 10% (v/v) acetic acid) and silver staining of gels, protein spots of interest were excised, washed and subjected to tryptic digestion and identification of resulting peptides by MALDI-TOF mass spectrometry analysis. Proteins were identified using the Mascot software release version 2.4.2 (Matrix Science) in conjunction with the NCBIprot database.For this experiment,  strains ; for det


C9orf72 repeat expansion mutation as causative for chromosome 9-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in 2011, a multitude of cellular pathways have been implicated. However, evidence has also been accumulating for a key mechanism of cellular compartmentalization—phase separation. Liquid-liquid phase separation (LLPS) is fundamental for the formation of membraneless organelles including stress granules, the nucleolus, Cajal bodies, nuclear speckles and the central channel of the nuclear pore. Evidence has now accumulated showing that the formation and function of these membraneless organelles is impaired by both the toxic arginine rich dipeptide repeat proteins (DPRs), translated from the C9orf72 repeat RNA transcript, and the repeat RNA itself. Both the arginine rich DPRs and repeat RNA themselves undergo phase separation and disrupt the physiological phase separation of proteins involved in the formation of these liquid-like organelles. Hence abnormal phase separation may explain a number of pathological cellular phenomena associated with C9orf72-ALS/FTD. In this review article, we will discuss the principles of phase separation, phase separation of the DPRs and repeat RNA themselves and how they perturb LLPS associated with membraneless organelles and the functional consequences of this. We will then discuss how phase separation may impact the major pathological feature of C9orf72-ALS/FTD, TDP-43 proteinopathy, and how LLPS may be targeted therapeutically in disease.Since the discovery of the  C9orf72 mutation is an expansion of a GGGGCC (G4C2) repeat in intron 1 of the gene. In unaffected individuals the G4C2 is repeated 2 to 23 times, whereas in those with the mutation, the sequence is expanded to contain hundreds to thousands of repeats  and C4G2 (antisense) repeat RNA. These RNA species both form small RNA aggregates called RNA foci in patient brain  is a process in which a homogenous liquid solution consisting of RNA and/or protein separates into two different phases; with one of these separated phases containing an increased concentration of RNA and protein—the dense phase, and the other phase—known as the dilute phase—now depleted of them  are an important class of LCD sequence found within numerous RNA binding proteins that undergo LLPS, including those associated with neurodegenerative diseases such as FUS, hnRNPA1, and FMRP  in vitro similarly to RGG/RG domains , indicating both electrostatic interactions and base-pairing interactions  within its intrinsically disordered region 149 mice using postnatal adenovirus expression  similar condensation was observed, suggesting that repeat number is important, but these can be in cis or trans. It also required the presence of cellular RNA, suggesting that G-quadruplex G4C2 RNA enhances intermolecular RNA interactions and thereby promotes the nucleation behavior of RNA that causes protein condensation. Indeed, transfection of G4C2 RNA in cells leads to the formation of stress granules  was shown to accumulate in the central channel of the nuclear pore in isolated nuclei from Xenopus oocytes  binding protein PABPC  interaction. Poorly dynamic stress granules induced by expression of the arginine rich DPRs can sequester both nucleoporins and the transport receptors and thereby disrupt nucleocytoplasmic transport  causing solid compartments to form within HP1α droplets and their bursting, and resulting in a significantly reduced number of droplets in vitro. This was recapitulated by reduced HP1α protein levels in the mouse model with functionally disrupted H3 histone post-translational modifications and upregulation of repetitive RNA elements, known to localize to heterochromatin, and double-stranded RNA which form from these which can initiate interferon signaling and cell death in neurons; these changes could also be induced by HP1α knockdown in cells. Interestingly, these changes occurred concurrently with irregularities in the nuclear lamina, which can also cause heterochromatin dysregulation  but increased cytoplasmic accumulation, increased biochemical insolubility or phosphorylation have been observed in Drosophila, murine and cellular models expressing poly-GR and poly-GA  only occurred with a disrupted nuclear localization signal which localizes TDP-43 to the cytoplasm but not wildtype TDP-43 which remains in the nucleus  to stress granules and decreased persistent TDP-43 puncta in cells treated with cellular stressors, and also ameliorated toxicity from overexpression of a mutant TDP-43 in primary neurons  and enhance toxicity, therefore modulating this process via reducing ataxin-2 levels may provide a novel therapeutic target in C9orf72-ALS/FTD (Drosophila or spinal cord cultures, respectively (McGurk et al., C9orf72-FTD/ALS.We have described the evidence for the ability of the -ALS/FTD , panel 6-ALS/FTD  and thus-ALS/FTD . Knockdovia influencing FUS phase separation and reduce FUS toxicity in vivo (Wheeler et al., Drosophila models of poly-PR toxicity, with PRMT1 also colocalizing with poly-PR upon co-transfection in cell lines (Boeynaems et al., Drosophila studies. These opposing roles do not however align with their effect on LLPS, as both symmetric and asymmetric demethylation of poly-GR reduce propensity to undergo LLPS. Interestingly, a rapid demethylation of the stress granule protein G3BP1 is critical for stress granule assembly (Tsai et al., in vitro (Hofweber et al., Targeting DPRs or TDP-43 directly to prevent their aberrant phase separation and aggregation may also be effective. It has been recently shown that anti-cancer drugs partition into droplets formed by their molecular targets and change the properties of the condensate (Klein et al., Drosophila models of mutant FUS and hnRNP A2, respectively (Guo et al., in vitro LLPS and fibril formation of TDP-43 and could also disassemble preformed fibrils (Guo et al., via its classic NLS recognized by the importin-α/β complex, whilst FUS and the hnRNPs contain a PY-NLS which is bound and transported by transportin-1 (Mihevc et al., C9orf72-ALS/FTD where TDP-43 is the major pathology, the arginine rich DPRs bind to importin-α and β, reducing their solubility and lead to their precipitation and condensation; this results in defective nuclear import of TDP-43, and thus likely contributes to the dominant TDP-43 pathology (Hutten et al., in vitro and its reduced solubility in cells, and inhibit RNA-driven LLPS of poly-GR. Therefore, therapies boosting levels of importin-α and β could ameliorate both DPR and TDP-43 pathologies and prevent other pathogenic interactions, which may be beneficial for the wide range of cellular abnormalities observed in C9orf72-ALS/FTD (It has also been proposed that chaperones exist whose normal function is to modulate LLPS within a cellular environment that is particularly concentrated, unstable and oversaturated in order to prevent abnormal fibrillization (Elbaum-Garfinkle, -ALS/FTD , panel 6-ALS/FTD , and thuC9orf72 G4C2 repeat RNA and DPRs directly to reduce their levels and prevent pathological intra and intermolecular associations. An approach that has gained much traction and is being pursued in clinical trials is using ASOs to the C9orf72 repeat which can reduce the formation of repeat RNA and DPRs in C9orf72 repeat-expressing neurons and mice and patient-derived neurons with an associated reduction in glutamate-induced toxicity in cultured neurons and behavioral and cognitive phenotypes in mice (Donnelly et al., 4C2 G-quadruplexes can reduce the production of DPRs and rescue toxicity (Zhang et al., 4C2 repeat RNA and thus the translation of DPRs and their associated toxicity in model systems (Liu et al., 4C2 repeat RNA and its depletion increases production of repeat RNA and DPRs (Mori et al., Drosophila models and the former also in C9orf72 patient cells (Goodman et al., C9orf72 mutation mouse model (Zu et al., C9orf72-FTD/ALS patient brain (Mori et al., A complementary strategy to all of the above strategies is to target the C9orf72-ALS/FTD. Gain-of-function mechanisms associated with the G4C2 repeat expansion in C9orf72–G4C2 repeat RNA and the arginine rich DPRs poly-GR and poly-PR—undergo phase separation themselves and perturb the phase separation of LCD containing proteins, resulting in abnormal membraneless organelle formation and dissolution, impairing their physiological functions and leading to neurodegeneration. Further pathological phase separation induced by the arginine rich DPRs is strongly associated with TDP-43 dysfunction and aggregation, the major pathological hallmark of C9orf72-ALS/FTD correlating with neuronal cell death. The targeting of abnormally phase separated condensates using small molecules or gene therapy provides a novel strategy for future therapeutics, although a greater understanding is needed of phase separation in order to design targets which are both beneficial and precise.There is now substantial and clear evidence that a disruption in the phase separation behavior of proteins and RNA involved in the formation of liquid-like membraneless organelles explains much of the major pathological phenomena associated with DS and SM conceived the review. DS, RS and SM wrote sections of the manuscript. MR and DS designed and produced figures. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.