In an era of coral reef degradation, our knowledge of ecological patterns in reefs is biased towards large conspicuous organisms. The majority of biodiversity, however, inhabits small cryptic spaces within the framework of the reef. To assess this biodiverse community, which we term the ‘reef cryptobiome’, we deployed 87 autonomous reef monitoring structures (ARMS), on 22 reefs across 16 degrees latitude of the Red Sea. Combining ARMS with metabarcoding of the mitochondrial cytochrome oxidase I gene, we reveal a rich community, including the identification of 14 metazoan phyla within 10 416 operational taxonomic units (OTUs). While mobile and sessile subsets were similarly structured along the basin, the main environmental driver was different . Distribution patterns of OTUs showed that only 1.5% were present in all reefs, while over half were present in a single reef. On both local and regional scales, the majority of OTUs were rare. The high heterogeneity in community patterns of the reef cryptobiome has implications for reef conservation. Understanding the biodiversity patterns of this critical component of reef functioning will enable a sound knowledge of how coral reefs will respond to future anthropogenic impacts. Coral rThe high reef biodiversity is not only reflected in the spectrum of phyla inhabiting coral reefs  but alsoThe main limiting factors hindering advances in this research area are related to (i) the relatively small sizes of cryptic fauna, (ii) their preferential residence in hidden spaces, and (iii) the lack of standardized approaches to accurately assess this biological component. To investigate the global diversity patterns of reef cryptic communities, a joint effort by the Coral Reef Ecosystem Division (CRED), the Census of Marine Life (CoML), and Census of Coral Reef Ecosystems (CReefs) developed autonomous reef monitoring structures (ARMS). ARMS are long-term artificial substrate units made of a tiered structure of nine stacked polyvinyl chloride (PVC) plates, including a variety of niches . They mimic the structural complexity of a coral reef and attract organisms that are usually hidden in the reef matrix . ARMS enable both the analysis of sessile (species encrusting onto the plates) and mobile organisms (motile specimens colonizing the various niches between the plates) living in these habitats. These artificial structures represent a standardized and non-destructive monitoring tool, which, in combination with the increasingly available and cost-efficient high-throughput sequencing facilities, allows a comparable and fast assessment of cryptic diversity. ARMS have previously been used to describe the eukaryotic cryptic fauna in a variety of locations, but so far these studies have been limited in scale.° of latitude of the Red Sea coast. We characterized the diversity of the reef cryptobiome by targeting a region of the mitochondrial cytochrome oxidase I (COI) gene from the eDNA present in the ARMS. Rather than presenting a simple characterization of the composition of these biological assemblages, we provide insights into the intrinsic patterns of rarity and abundance of the reef cryptobiome. Additionally, the analysis of two different biological traits (sessile and mobile) allowed us to investigate the consistency in the structural and biogeographic patterns of different subsets within the reef diversity.Here, we focus on the distribution patterns across a broad spatial scale of what we have named the ‘reef cryptobiome’, which comprises reef organisms inhabiting hidden spaces within the reef matrix. Distribution patterns are often driven by habitat specificity and geographical distribution  with oth2.https://oceancolor.gsfc.nasa.gov/ downloaded 14 February 2017). Monthly averages, at 4 km resolution, were obtained from the MODIS A satellite system for chlorophyll a, sea surface temperature (SST), particulate organic matter (POC), and photosynthetic active radiation (PAR). Spatial resolution required that some reefs were assigned to the same grid point.Satellite data were obtained from the NASA Oceancolor website . The deployment, retrieval, and laboratory processing of the units was undertaken as in Leray & Knowlton   and sequences with the data being processed from the raw OTU table in the R  package phyloseq  (electroeosphere ). The deeosphere   and locally abundant (greater than 1% in a reef) as defined by Logares et al. . Rare ane method . Visuali[ggplot2 , ggdendrggdendro , and denndextend  packagesThe core community was characterized as OTUs accounting for on average 0.1% relative sequence abundance and 90% of reefs being occupied. The satellite community was determined by an average sequence abundance of less than 0.001% and being only present in a single reef (adapted from ).3.(a)In the present study, we identified 10 416 OTUs; the mobile and sessile traits had 8903 and 4450, respectively. A total of 2937 OTUs were present in both traits. On average, the mobile trait  was almost twice as diverse as the sessile trait . At the reef level (triplicate ARMS combined), we observed an average of 1471 OTUs in the reef cryptobiome . Along the Red Sea basin, the majority of the OTUs (53%) were only present in one reef with less than 2% of the OTUs (162) being present in all the reefs . A similar pattern was observed for the sessile and mobile traits.Arthropoda  and Annelida  dominated the reef cryptobiome community. Mollusca with 3% of the OTUs also showed a relevant contribution to the γ diversity . Chordata were prevalent in the reef cryptobiome, particularly in terms of the relative proportion of sequences (14%) . Other gWith regard to the number of OTUs, the sessile community was dominated by Arthropoda (34%), with these OTUs contributing to 12% of the sequences. Chordata contributed the most to the number of sequences (14%), with Bryozoa accounting for over 10% . Arthropoda also had the highest contribution in terms of the number of OTUs in the mobile community (38%). Together with Annelida, they also dominated the community with regard to the proportion of sequences  .(b)p < 0.001) with a Mantel R statistic of 0.55. The assessment of the local contribution to β diversity, based on Jaccard, indicated that the reefs at the extremes of the basin, especially those in the south, showed above average contributions to β diversity . This pattern was consistently observed independent of the dataset analysed . There was a significant difference in the number of OTUs observed in a region for both the pooled ARMS  and mobile sets  with the southern region having comparatively fewer OTUs. POC contributed the most to explaining the patterns of variance in observed OTUs for the pooled ARMS and the mobile trait and had a negative relationship to OTU number. For the sessile trait, while POC contributed substantially in explaining the observed number of OTUs, SST showed a higher relative contribution .The investigation of the similarity patterns of reef communities (Jaccard) showed a significant negative relationship with linear distance . Comparative analysis of the community patterns between the sessile and mobile traits showed a significant positive correlation . The southern region was characterized by five indicator OTUs, all positively correlated to SST and POC. Arthropoda, Annelida, and Chordata had representative indicators in all regions with Echinodermata represented with one OTU in the northern region.(c)b,c). Hierarchical clustering of the abundant OTUs showed a separation of the most southerly reefs (F18 and F24) with the remaining southern reefs more similar to the central reefs. The northern reefs in the main clustered together (figure 2a).The reef cryptobiome was characterized by an unbalanced distribution of abundant and rare OTUs. For the pooled dataset, locally abundant OTUs, on average, only accounted for approximately 2% of the diversity at the scale of the reef , and werfigure 2R = 0.69, p < 0.001). However, on average, pairwise comparisons based on Jaccard showed that the abundant subset (average similarity = 0.89) had a significantly higher similarity  than the rare subset of OTUs (average similarity = 0.28).On average, 78% of the OTUs of the pooled ARMS were classified in the category ‘locally rare’ . Similarp < 0.001). The core species (i.e. average 0.1% relative sequence abundance and 90% of reefs being occupied) accounted for less than 2% of the γ diversity (128 OTUs), whereas the satellite subset was represented by 52% (5471 OTUs)  . Similar71 OTUs) . For the71 OTUs) .Figure 4.In an era where the composition and structure of marine biological communities are changing at increasing rates as a result of anthropogenic activities and climate change, emphasis needs to be placed on understanding patterns across large spatial scales. This fact becomes especially important for groups of organisms that are relatively poorly described and where species could be driven to extinction before their role in the ecosystem is understood . By taki(a)In the current study, we revealed a total of 20 phyla, of which 14 were metazoans. While multi-phyla studies of the cryptofauna are limited , thus maThe diversity of the reef cryptobiome has likely been underestimated due to a combination of the small size of the organisms as well as the prevalence of nocturnal lifestyles , which hRegional differences in the community composition revealed a lower reef cryptobiome diversity in the southern region compared with the central and north. This trend has been previously observed in the Red Sea in plankton communities , where n(b)Marine communities are in general dominated by a few taxa with most of the diversity resulting from rare species that may change in space and time ,36.This trend has been observed from bacterioplankton  to soft-On a larger spatial scale , there is a generally acknowledged phenomenon that those species with a higher average abundance are, in general, found more frequently within the community ,56. This(c)Marine communities are spatially structured based on (i) dispersal limitation, mortality, and ecological interactions and (ii) environmental variation, with the resulting distribution patterns being affected by species functional traits ,59. EvenFurther differences in the traits were observed in their diversity with distinctly fewer OTUs being present in the sessile community. The lower diversity in the sessile community may be due to the limited space and niches available for sessile organisms in the ARMS  and the artificial nature of ARMS that may also restrict settlement of some species. The higher diversity for the mobile trait may in part be due to transient occupation of the ARMS unit  from the surrounding mature reef but could also be due to the creation of multiple niches by the sessile community allowing the higher diversity of the mobile trait.(d)et al.[https://mooreabiocode.org/) but needs to be expanded to other geographical areas as it is essential to catalogue species and understand their niche preferences before they are driven from existence [Despite the clear advantages that eDNA metabarcoding approaches show to reveal a great proportion of the biodiversity, molecular approaches are dependent on reference databases for taxonomic assignments, which are currently limited and biased towards common taxa in well-studied regions . The BOLet al. could imet al.. Indeed,xistence . The usexistence –72. Thisxistence , where txistence . Howeverxistence ,75. Whilxistence , the res2 concentrations).Moving forward, many interesting questions about the reef cryptobiome remain unanswered, such as: (i) the roles of its elements in ecosystem functioning, (ii) what are the unique and common ecological traits of rare-to-abundant taxa, (iii) how its abundance-occurrence patterns relate to the structure of larger benthic and fish communities, (iv) how does the reef cryptobiome change in time, and (v) what are the main drivers of community composition. To achieve these, further investigations could include the targeting of specific functional genes (e.g. nitrogen fixation genes) using qPCR methodologies for the assessment of functional abilities in the coral reef benthic environment. Functional diversity has been proposed as the way forward in regard to the assessment of natural and anthropogenic impacts in the marine environment . Until t5.The approach followed here confirms the reef cryptobiome as a phylogenetically rich component of the reef system. We identified 14 out of the 30 metazoan phyla  that havWe argue that ARMS combined with metabarcoding processing provides a standardized and non-destructive approach to describing the reef cryptobiome and should be promoted as a way to investigate patterns across variable spatial  and temporal  scales. Ultimately, we believe that incorporating the results from the reef cryptobiome into marine monitoring programmes alongside macrobenthos and fish which are typically studied will provide a better understanding of the reef community and enable more informed management decisions to be taken.


Recent studies have revealed thousands of A-to-I RNA editing events in primates, but the origination and general functions of these events are not well addressed.Here, we perform a comparative editome study in human and rhesus macaque and uncover a substantial proportion of macaque A-to-I editing sites that are genomically polymorphic in some animals or encoded as non-editable nucleotides in human. The occurrence of these recent gain and loss of RNA editing through DNA point mutation is significantly more prevalent than that expected for the nearby regions. Ancestral state analyses further demonstrate that an increase in recent gain of editing events contribute to the over-representation, with G-to-A mutation site as a favorable location for the origination of robust A-to-I editing events. Population genetics analyses of the focal editing sites further reveal that a portion of these young editing events are evolutionarily significant, indicating general functional relevance for at least a fraction of these sites.Overall, we report a list of A-to-I editing events that recently originated through G-to-A mutations in primates, representing a valuable resource to investigate the features and evolutionary significance of A-to-I editing events at the population and species levels. The unique subset of primate editome also illuminates the general functions of RNA editing by connecting it to particular gene regulatory processes, based on the characterized outcome of a gene regulatory level in different individuals or primate species with or without these editing events.The online version of this article (10.1186/s13059-019-1638-y) contains supplementary material, which is available to authorized users. Alus in primates, which are the preferred substrates of ADARs, A-to-I editing is the most common type of RNA editing in primates [RNA editing is a post-transcriptional mechanism that introduces differences between RNA and its corresponding DNA sequence . One typprimates . The recprimates –6, with primates .Alu elements [Most A-to-I RNA editing sites in primates are contributed by the expansion of primate-specific elements , 7. Of telements , 5, 8, 9elements –12. Whilelements , 13, 14,Recently, a group of A-to-I RNA editing sites has been reported in candidate gene studies to be genomically encoded as non-editable nucleotide in other closely related species , 16, repAlthough cases of the birth or death process of RNA editing through DNA point mutation have been reported, the generality of this phenomenon on a genome-wide scale, the models underpinning the phenomenon, and the applications of these events in evolutionary and functional interrogation of RNA editing regulation remain largely unresolved. In particular, this phenomenon on the population level, in which the A-to-I RNA editing sites detected in some individuals are genomically encoded as non-editable nucleotides in other individuals, would complement these analyses. However, this type of polymorphic editing sites is intentionally omitted primarily due to the potential false positives of these events and their consequent removal by the editing-calling computational pipelines , 18–20.Alu repeat elements. For these Alu-associated candidates, 2,638,838 sites (or 96.47%) were associated with A-to-I transitions, which were used in the following analyses  of the same macaque animal Fig. a. To thiAlu-associated A-to-I RNA editing sites detected in the macaque animal with correspondence to the human orthologous sites, 281,578 (12.67%) are genomically encoded as G in human on the basis of the reference genome, with 255,056 (11.47%) sites (hgG) being fixed in the human population on the basis of the 1000 Genomes Project [P value < 2.2 × 10− 16, Fig. Alu with the same trinucleotide context, the editing sites still showed a significantly higher level of overlap with A/G divergent sites between human and rhesus macaque  the DNA coverage of these A-to-I editing sites is not lower than other editing sites, ruling out the possibility that these sites were wrongly assigned as homozygotes due to biased representation under lower DNA sequencing coverage . These findings thus suggest that sites with A/G mutation might be the hotspot for the recent gain or loss of robust A-to-I editing events  or polyAG sites 4.19%) located on 9% locatents Fig. ”. For moP value < 2.2 × 10− 16, Fig. As hgG and polyAG editing sites showed opposite patterns for the allocation of the two models, we next sought to address possible biases introduced in the definition of these sites. In contrast to hgG sites, the definition of polyAG editing sites per se could introduce biased allocation of the two models. To this end, because the homozygous adenines in the macaque animal was the prerequisite for A-to-I editing identification, A is less likely to be the derived allele, as it is relatively difficult to identify derived, homozygous adenines in the macaque animal for A-to-I editing identification in such an occasion. The ancestral allele of these sites is thus more likely to be A, and more polyAG editing sites are thus presumably attributed to an “editing loss model”. To control for the biases, we thus introduced matched controls for the two types of sites “”. NotablAlu repeats. Notably, it seems the pattern of over-representations is largely contributed to by the sites with ancestral G  should be expected. Second, even when the “opposite C-ancestral G” pairing sites are not enriched during the primate evolution, a site with “opposite C-ancestral G” pairing may represent a favorable location for the origination of robust A-to-I editing event, leading to its detection in our analysis. To distinguish the two mechanisms, we introduced negative controls of hgG sites (regardless of the editing status) as a background, such as the Alu-associated, hgG sites with ancestral G , as well as Alu-associated, hgG sites with ancestral A . The fraction of opposite C is higher for hgG-ancG adenosines (56%) than hgG-ancA adenosines (28%), suggesting that the ancestral dsRNA is generally tighter than the current one. Notably, as a higher fraction of opposite C was found for hgG-ancG editing sites (75%) in comparison to the control group of all hgG-ancG adenosines (56%), it is plausible that the over-representation of “opposite C-ancestral G” pairing sites could be attributed to a bigger pool for these sites relaxing the dsRNA structures during the primate evolution, as well as the scenario that a site with “opposite C-ancestral G” pairing is a favorable location for the origination of robust A-to-I editing event.Notably, two possible mechanisms may underpin the over-representation of “opposite C-ancestral G” pairing sites: First, it is possible that the diversity between The model proposed here lends support to the features of the over-representation detected. In addition, the list of hgG and polyAG editing sites also represents a valuable resource to investigate the features and functions of A-to-I RNA editing events at the population and species levels.P value = 9.33 × 10− 10, Fig. P value < 1 × 10− 4, “ frequency than the control sites . Especially, to clarify the sequential order between the introduction of editing and the accumulation of mutations, we selected a subset of sites detected as editing in both rhesus macaque and out-group species . Overall, although we tried to control for these potential confounding factors, it is plausible that some other confounding factors may still perplex the explanations of the signals; it is thus cautious to use the signals in nearby regions as evidence to support the evolutionary significance of these sites, in contrast to the direct evidence of the focal editing sites.The identification and investigation of these newly originated A-to-I editing events also provided a basis for clarifying the evolutionary relevance of RNA editing regulation in primates. Through the population genetics analyses of the focal editing sites, we found that at least a fraction of these young editing events are evolutionarily significant. Besides this approach, the detection of signals for the valley of decreased polymorphism level flanking the editing sites would presumably be additional evidence for the evolutionary significance of the RNA editing events , 27. AccThe newly originated A-to-I editing events are evolutionarily significant in general, indicating that at least a fraction of these sites should already acquire functions during the primate evolution. In order for these RNA editing events to have a significant impact, the editing must occur at sites that could be linked to cellular functions. However, the majority of this editome discussed in this study are located on intronic 60.91%) or intergenic (38.56%) regions, and only a small portion of the identified sites is linked to regions with well-recognized biological functions, such as protein coding (2035 sites or 0.08%). As the majority of this editome are not connected to well-recognized functional entities, the genome-wide selection signals revealed by population genetics analyses are not readily explicable. Therefore, finding new general functions for the pervasive RNA editing sites located in non-coding regions remains a critical issue in the field % or inte, 12.ADAR1-knockdown experiments further linked intronic A-to-I editing to regulations of circRNA biogenesis. We also found that the RNA editing regulation in intergenic regions could crosstalk with piRNA biogenesis and contribute substantially to the diversification of the piRNA repertoire in primates [Recently, several studies have implicated a portion of RNA editing sites in non-coding regions in other regulatory processes, such as alternative splicing and microRNA regulation , 14, 28.primates , 14, 29.primates –33. ThesV/V). Deep sequencing was then performed on Illumina Hiseq X Ten sequencing systems with 151-bp paired-end reads mode.Total RNAs were extracted from frozen tissues following Trizol RNA isolation procedure. The quality of the input RNA was controlled using Agilent 2100. Total RNA samples were then applied to strand-specific, rRNA-depleted RNA-seq, or strand-specific, poly(A)-positive RNA-seq following the pipeline as previously described [The recent birth and death events of RNA editing were identified by first defining A-to-I RNA editing sites detected in some macaque animals while genomically encoded as G in other animals  or in other species (species-specific editing sites). Using a stringent computational pipeline previously reported by us , 10, we To identify the recent birth and death events of RNA editing within the macaque population, the polymorphism map of 31 macaque animals from the RhesusBase PopGateway  was usedAlu elements, probes covering the editing sites might not reach the desired specificity. We thus designed the probes corresponding to the nearby non-repetitive regions at a distance of about 100 bp to the focal editing sites. Three micrograms genomic DNA was then isolated from whole blood sample of each macaque animal, using the QIAamp DNA Blood Mini Kit . The genomic DNA was then sheared to fragments with a peak at 150–200 bp for the library construction following Agilent SureSelect XT Target Enrichment System’s instructions. Deep sequencing was performed on Illumina Miseq sequencing system, with 151 bp paired-end reads mode. After reads mapping to macaque genome (rheMac2), single nucleotide polymorphisms were then identified according to the standard GATK pipeline with HaplotypeCaller.For 54 randomly selected polyAG sites  pipeline . The proFor the newly originated polymorphic editing sites in rhesus macaque with ancestral state of G, the derived allele was defined by the EPO pipeline from eight primate species , in which the ancestral state for macaque and baboon was used to polarize the polymorphism site. In this pipeline, the potential mutation bias of CpG regions was considered in steps to simulate local sequence-dependent fluctuations in substitution and indel rates . A site On the basis of the polymorphism data from the population of 31 macaque animals, we also estimated the polymorphism levels (π) for the nearby regions and remote regions of A-to-I RNA editing sites. The distribution of nucleotide diversity nearby these editing sites was also calculated as a genomic background with 10,000 times of bootstrap to deduce the confidence intervals.Alu elements, the nearest inverted Alu pairs were located. The genomic distance between two inverted Alus should be less than 5000 nt and on the same transcript. The RNA secondary structure was then predicted by MFOLD (v3.6) [For RNA editing sites within D (v3.6) .Additional file 1:Table S1. Statistics of the deep sequencing data used in this study. (XLSX 12 kb)Additional file 2:Table S2. Genomic annotation of the A-to-I RNA editing sites in Alu elements. Table S5. Proportion of transcribed, Alu-associated hgG or polyAG sites with different ancestral status detected as editing. Figure S1. Accurate editome identification in rhesus macaque. Figure S2. Over-representation of A/G divergent and polymorphic sites at RNA editing sites. Figure S3. Proportions of human-macaque sequence differences and macaque polymorphic sites. Figure S4. Distribution of editing levels for various editing type in different tissues. Figure S5. Site frequency spectrum for derived A allele for polyAG editing sites with ancestral G. Figure S6. The polymorphism levels of newly originated RNA editing events compared with remote regions. Figure S7. Quantification of mutations accumulated after the origination of editing events. (DOCX 4790 kb)Additional file 3:Table S3. A-to-I editing events located on polymorphic sites in the macaque population. (XLSX 1879 kb)Additional file 4:Table S4. 54 randomly selected polyAG sites of targeted DNA sequencing. (XLSX 13 kb)


Xenopus. 7SL RNA in RBCs is not associated with the canonical proteins of the SRP. Instead, it coimmunoprecipitates from a lysate of RBCs with a number of membrane-binding proteins. Human and mouse RBCs also contain a previously undescribed 68 nt RNA, sRN7SL, derived from the “S domain” of 7SL RNA. We discuss the possibility that 7SL RNA is selectively protected from nucleases by association with the RBC membrane. Because 7SL is not associated with the canonical proteins of the SRP, it could represent a nonfunctional remnant of the protein synthetic machinery. Alternatively, it could play a new, as yet undefined role in RBC metabolism.We report that 7SL, the RNA component of the signal recognition particle (SRP), is an abundant noncoding RNA (ncRNA) in mature red blood cells (RBCs) of human, mouse, and the frog  In an earlier study we reported the existence of more than 9000 different circular intronic sequences (lariats) in the cytoplasm of the amphibian oocyte . RBCs exhibit a high level of autofluorescence throughout the visible spectrum, requiring the use of a chromogenic detection system (Fast Red dye). We found that RBCs of all three species are remarkably resistant to the hybridization procedure, when prefixed with ethanol and/or paraformaldehyde. X. tropicalis prefixed with ethanol and 4% paraformaldehyde. Unknown to us before we began these experiments, a small number of RBCs lyse during the spreading procedure. Because Xenopus RBCs are nucleated, these lysed cells can still be recognized by the residual DAPI staining of their extruded nuclei . We detected a band at ∼300 nt, confirming that full-length 7SL RNA is present in RBCs . However, this annotation was linked to a predicted piRNA (28 nt), piR-45120 locus DQ577008, identified by Intriguingly, the sRN7SL sequence overlaps a 111-nt 7SL S-domain fragment, namely 7SLrem (for 7SL remnant), found in virus-like particles . This prXenopus cell cultures and RBCs with a full-length 7SL RNA probe. We detected full-length 7SL RNA in all samples, but the 68-nt band was present only in the human and mouse RBC samples  might be associated with 7SL RNA in RBCs, we used a strategy based on “capture hybridization analysis of RNA targets” (“CHART”) method . This meO-methylated nucleotides and a desthiobiotin-conjugated 3′ end nucleotide, similar to oligos used in the CHART method. As a negative control, we used a desthiobiotin-conjugated oligo that is antisense to a Xenopus-specific RNA. Pulled-down RNA was run on an acrylamide gel and stained with SYBR Gold. 7SL RNA was detected in the pull-down with the “linker targeting” oligo but not in the control. Moreover, 7SL RNA was the major RNA in the pull-down . Four of the five most abundant proteins in the pull-down were spectrin α and β chains, band 3 and protein 4.1, consistent with the interpretation of the protein gel in Supplemental Fig. S1), raising the possibility that 7SL might be associated with SART3.We analyzed the proteins in the pull-down by mass spectrometry and focused on proteins enriched by at least twofold  and centrifuged at 1000g for 30 min at room temperature. RBCs accumulated near the bottom of the tube and were collected free of white blood cells. Before use, they were washed three times in 1× PBS.Blood samples were centrifuged at 300A drop of whole blood or red blood cells was spread quickly on a microscope slide and dried overnight or longer. In some cases the smear was fixed for up to 1 h in 100% ethanol and/or 4% paraformaldehyde.RNA was extracted with TRIzol reagent (Ambion) and purified with the Direct-zol RNA MiniPrep kit (Zymo Research). DNase I treatment was performed on the Direct-zol column. RNA concentration was determined with a Nanodrop apparatus (Thermo Fisher Scientific) and its integrity (size distribution) with a Bioanalyzer 2100 (Agilent).bases 99–138: CGATCGGGTGTCCGCACTAAGTTCGGCATCAATATGGTG,bases 165–199: GTTGCCTAAGGAGGGGTGAACCGGCCCAGGTCGGA, andbases 200–242: AACGGAGCAGGTCAAAACTCCCGTGCTGATCAGTAGTGGGATC.The probe consisted of three “ZZ” oligonucleotide pairs from Advanced Cell Diagnostics, which are complementary to the following three regions of human 7SL:The “ZZ” oligonucleotide pairs are attached to a proprietary amplification target, which in turn is detected by a proprietary set of oligonucleotides. The latter are finally detected by Fast Red dye. Each red “dot” in the final preparation presumably represents a single RNA molecule., Roche) and CDP-Star chemiluminescent substrate (Roche).Up to 1 µg of RNA per sample was separated on an 8% polyacrylamide/8 M urea gel and transferred onto a nylon membrane . RNA was probed with dsDNA labeled with digoxigenin (DIG)-dUTP in hybridization buffer (Roche) and detected using an anti-DIG antibody conjugated with alkaline phosphatase , one protease inhibitor cocktail tablet  and 1% Tween-20. Twenty microliters of cleared lysate was incubated 1 h at 37°C with 5 units of RNase H and 1 mM antisense oligo prior to RNA purification.Cells were homogenized in 500 µL RNase H buffer: 50 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgClRNA was run on an 8% polyacrylamide–8 M urea gel. RNAs ranging from 200–400 nt and 50–100 nt were cut from the gel and extracted overnight with 300 mM NaOAc, 1mM EDTA, 0.25% SDS at room temperature. Oligos were ligated to the 5′ and 3′ end according to the NEBNext Multiplex Small RNA Library Prep protocol. The RNA was converted to cDNA with an oligo that targeted the ligated sequence at the 3′ end. It was then amplified either with NEBNext oligos to make a library or with internal oligos to sequence the end of 7SL RNA specifically. All products were analyzed by TA cloning.2, 1 mM DTT, 0.2 unit of RNasin [Promega], one protease inhibitor cocktail tablet , and 1% Triton X-100) and incubated 1 h at 37°C with 5 µL of 10 µM biotinylated antisense oligonucleotide purchased from Integrated DNA Technologies. The sequence of the antisense oligonucleotide to mouse 7SL was /5BioTinTEG/iSp18/mAmUmCmGmGmCmAmUmAmGmCmGmCmAmCmUmAmCmAmGmCmCmCmAmGmAmAmCmUmCmCmUmGmGmAmCmUmCmAmAmG/iSp18/3BioTEG/. The sequence of the antisense oligonucleotides to Xenopus tropicalis faf2 intron 2 was mGmAmUmUmUmGmAmCmCmAmCmAmCmAmCmAmCmAmCmAmGmUmU/iSp18/3BioTEG/ and mGmUmAmUmUmUmGmUmAmUmGmCmUmCmAmGmAmCmCmUmGmC/iSp18/3BioTEG/.One hundred microliter of packed mouse red blood cells were homogenized in lysis buffer  was added, and the mix was further incubated at 37°C for 1 h. Beads were washed in 1 mL of lysis buffer five times for 10 min. RNA was purified from beads that were directly added to TRIzol (10%), run on an 8% acrylamide/8 M urea gel, and analyzed by SYBR Gold and Northern blotting. The pulled-down complexes were eluted with 50 µL of nanopure water by heating gradually (0.5°C/sec) to 70°C based on . Five miMass spectrometry was carried out at the Johns Hopkins Medical School Mass Spectrometry and Proteomics Facility. Pulled-down complexes were digested with trypsin, desalted, and analyzed by liquid chromatography tandem-mass spectrometry on a Q Exactive Plus instrument (Thermo Fisher Scientific). The output was searched against refseq2015_mus_musculus database with PD1.4-Mascot.2, 1 mM DTT, 0.2 U RNasin [Promega], one protease inhibitor cocktail tablet , and 1% Triton-X100). After centrifugation to clear the lysate, hemoglobin was depleted using 1 volume of NI-NTA agarose beads (Qiagen) for 1 volume of lysate . Sequencing was performed on an Illumina HiSeq 2000 sequencer with 100 bp single-end reads. Reads were aligned with TopHat (v2.0.7) to the mouse genome (v10) or the human genome (v19). 7SL and RNase MRP reads were quantified using Bedtools (v2.15.0).Supplemental material is available for this article.


Salmonella enterica serovar Typhimurium (S. Typhimurium) cultured under different conditions, which mimic host–pathogen interactions. S. Typhimurium was grown to exponential and stationary growth phases in minimal growth control medium , as well as in acidic and phosphate-depleted PCN, comparable to the macrophage environment and inducing therefore the expression of Salmonella pathogenicity island 2 (SPI-2) genes. Moreover, Salmonella pathogenicity island 1 (SPI-1), which is required for virulence during the intestinal phase of infection, was induced by culturing S. Typhimurium to the stationary phase in Lysogeny Broth (LB). For each condition, we identified OMV-associated RNAs that are enriched in the extracellular environment relative to the intracellular space. All RNA classes could be observed, but a vast majority of rRNA was exported in all conditions in variable proportions with a notable decrease in LB SPI-1 inducing media. Several mRNAs and ncRNAs were specifically enriched in/on OMVs dependent on the growth conditions. Important to note is that some RNAs showed identical read coverage profiles intracellularly and extracellularly, whereas distinct coverage patterns were observed for other transcripts, suggesting a specific processing or degradation. Moreover, PCR experiments confirmed that distinct RNAs were present in or on OMVs as full-length transcripts , whereas others seemed to be rather present in a processed or degraded form. Finally, we show by a digestion protection assay that OMVs are able to prevent enzymatic degradation of given full-length transcripts . In summary, we show that OMV-associated RNA is clearly different in distinct culture conditions and that at least a fraction of the extracellular RNA is associated as a full-length transcripts with OMVs, indicating that some RNAs are protected by OMVs and thereby leaving open the possibility that those might be functionally active.Bacterial outer membrane vesicles (OMVs), as well as OMV-associated small RNAs, have been demonstrated to play a role in host–pathogen interactions. The presence of larger RNA transcripts in OMVs has been less studied and their potential role in host–pathogen interactions remains largely unknown. Here we analyze RNA from OMVs secreted by  Salmonella . OMVs are 50–250 nm spherical particles derived from the outer membrane of Gram-negative bacteria, containing a wide range of molecules. Their release is a phenomenon observed constitutively in many Gram-negative bacteria, including lmonella . OMV forlmonella . Environlmonella . TherefoOuter membrane vesicles’ physiological roles are broad, ranging from functions such as nutrient acquisition, sharing of resistance, stress response or biofilm reinforcement, to more offensive roles like toxin concentration, delivery of virulence factors or modulation of host immune defenses . Their rListeria spp. are key components for developing immunity against the bacterial infection , on the chromosome and on the pSLT virulence plasmid  within Lysogeny Broth (LB)  . Similarlability . Salmoneogresses .Salmonella Typhimurium OMVs through OMV isolation and RNA sequencing, in different culture conditions representative of some of the environments encountered by the bacteria during the infection process.In this study, we aimed to characterize the RNA cargo of Salmonella enterica subsp. enterica serovar Typhimurium LT2 (henceforth referred to as S. Typhimurium) preculture triplicates were grown overnight in control (PCN), SPI-2 inducing (SPI-2ind) or SPI-1 inducing (SPI-1ind) conditions at 37°C and 160 Rotations Per Minute (RPM). Control conditions consisted of PCN buffer . SPI-2ind conditions  consisted of PCN buffer with lower pH  and low inorganic phosphate content (0.4 mM K2HPO4)  consisted of LB supplemented to 10 g.l-1 NaCl  grown u Miller, . 1.5 l cg, 10 min, 4°C) and the pellet was stored at -80°C in order to be able to extract intracellular RNA at a later time point. The intracellular pellet was conserved without any RNA preservation agent. 109 cells were used for extraction of intracellular RNAs. The whole 1,5 l supernatant was filtered using a 0.22 μm filter before being concentrated by ultrafiltration using tangential flow (Quixstand Benchtop System with 100-kDa hollow-fiber membrane). The collected OMVs-enriched liquid was then ultracentrifuged using a precooled Beckman ultracentrifuge with a 90Ti rotor during 3 h at 150,000 g and 4°C to obtain crude OMVs (before density gradient separation). After resuspension of the crude OMVs in 500 μl of buffer , pure OMVs were isolated by iodixanol gradient  using a precooled Beckman ultracentrifuge with a swinging bucket rotor (SW40 Ti) during 16 h at 100,000 g and 4°C. Continuous gradients were built with a bottom-up approach, loading vesicles in the 2 ml bottom fraction containing 45% w/v iodixanol, which was overlaid successively by 2 ml of 40, 35, 30, 25, and 20% w/v iodixanol fractions. After overnight (16 h) ultracentrifugation, 12 fractions of 1 ml were collected from the top low-density fractions to the bottom high-density fractions. The OMV-containing fractions were identified by subjecting 12.5 μl of each of the 12 isolated iodixanol fractions to SDS-PAGE followed by Western-Blot with an anti-OmpA IgG (Biorbyt) . Once identified, the remaining 987,5 μl of each OMV-containing fraction (positive for OmpA) was pooled and subjected to Trichloroacetic Acid precipitation  followed by centrifugation during 30 min at 17136 g and 4°C. The pellet was resuspended in 450 μl ice-cold 80% acetone before a further centrifugation step for 30 min at 17136 g and 4°C. After acetone evaporation, the pellet was resuspended in 100 μl of buffer . Vesicles were treated with freshly prepared lysozyme , even though this step might be superfluous as lysozyme digest sugars, but we aimed treating intracellular and extracellular samples as similarly as possible in order to be able to compare the different samples later on. RNA extraction was then performed with the All-in-One Purification Kit (Norgen), allowing the separation of the RNA within a small RNA (<200 nt) and a large RNA (>200 nt) fraction. The “large RNA fraction” (>200 nt) has been used for RNA-Seq, even though our data clearly indicates that all small RNAs (except tRNAs) can be resolved by RNA-Seq within the “large RNA fraction” . OMV-associated RNA was further concentrated using a Zymo Research RNA clean up and concentrator kit (including On-column DNaseI treatment) up to a volume of 6 μl . The extraction of intracellular RNA was also performed with the All-in-One Purification Kit (Norgen), followed by a DNaseI treatment. 1 μl of RNA was quality-checked with an Agilent 2100 Bioanalyzer. Detected RNAs were longer than 25 nucleotides in length .Outer membrane vesicles extraction was performed as described previously . Brieflyg at 4°C. The pellet was finally resuspended in 100 μl of the same buffer. A sample of the crude OMV preparation was also observed by TEM.The presence of OMVs after density gradient separation was confirmed by Transmission Electron Microscopy (TEM). For this purpose, the SPI-1ind condition has been used. OMV purification was performed as stated before but each of the 12 obtained iodixanol fractions obtained after gradient ultracentrifugation was fixed with 4% w/v paraformaldehyde during 10 min. OMVs were then rinsed with 8 ml of buffer  and pelleted using a precooled Beckman ultracentrifuge with a 90Ti rotor during 3 h at 150,000 A 5 μl drop of each sample was deposited on a glow-discharged carbon-coated Electron Microscopy (EM) copper grid . After 5 min of application, the excess of sample was blotted out using a Whatman filter paper and then the grid was washed with water three times to remove any buffer salts, which would react with uranyl acetate used for negative staining. To perform negative staining, 5 μl of uranyl acetate solution (2%) was applied on the grids. After 30 s of incubation, the excess of uranyl acetate was blotted out and then the grids were kept in a dry, dark, dust-free environment until observation with the electron microscope.Supplementary Figure S2).The EM grids were mounted on to a room temperature-equilibrated holder and subsequently introduced into a FEI Tecnai 20 transmission electron microscope (FEI Eindhoven Holland). Images (2048 pixels × 2048 pixels) were acquired using a US1000 camera (Gatan) at 50000× . OMVs were found in gradient fractions where OmpA was detected by Western-blot, demonstrating that the OmpA screening is a suitable method for identifying OMV-enriched iodixanol gradient fractions  and pelleted using a precooled Beckman ultracentrifuge with a 90Ti rotor during 3 h at 150 000 g at 4°C. The pellet was finally resuspended in 500 μl of the same buffer. A portion of the culture was plated on LB agar plates to determine CFU.ml-1 (Colony Forming Units). Quantification was not performed on samples subjected to density gradient separation, as it is not possible to obtain the dilution factor of this technique, which is needed to obtain OMVs/CFU in the original culture.In order to have an estimate of the amount of OMVs secreted by 3). The measurements were carried out in a 8 Well Lab-Tek II cover glass system. The sample fluorescence was collected through the same objective and directed by a dichroic mirror  to the lateral output of the microscope. After rejection of residual IR light by a short pass filter , the emitted photons were focused by a 200 mm achromatic lens on the 64 μm core of a multimode optical fiber coupled to an avalanche photodiode . The detected signal was correlated online by an ALV-5000E correlator  to generate autocorrelation curves G(τ). The analysis of G(τ) can provide information about the underlying mechanisms responsible for the intensity fluctuations such as number and diffusion of the particles. For an ideal case of freely diffusing monodisperse fluorescent particles undergoing a triplet-like blinking process in a Gaussian excitation volume, the correlation function, G(τ), calculated from the fluorescence fluctuations can be fitted according to:Quantification of OMVs was achieved by Fluorescence Correlation Spectroscopy (FCS). OMVs were stained with 100 nM of Nile Red lipophilic fluorescent dye (Sigma-Aldrich). Measurements were performed on a two-photon platform, as previously described . Brieflyd is the apparent diffusion time (a parameter that is inversely related to the diffusion constant of the molecule), N is the mean number of molecules within the excitation volume, S is the ratio between the axial and lateral radii of the sample volume (S ∼ 5), ft is the mean fraction of fluorophores in their dark state and τt is the triplet state lifetime. Using carboxytetramethylrhodamine (TMR) in water as a reference (TMRD = 421 μm2.s-1), the diffusion coefficient, expD, of the labeled OMVs was calculated by:where τd (TMR) and τd (sample) are the measured correlation times for TMR and the sample, respectively. Typical data recording times were 10 min. Quantification results gave us an estimate of 8.51 OMVs/CFU in SPI-1ind condition .where τPRJNA438234, available at https://www.ncbi.nlm.nih.gov/bioproject/438234.RNA sequencing was performed for Low OD and High OD samples in two distinct sequencing facilities, with minor changes indicated below. 1 μg of intracellular RNA was subjected to rRNA depletion using the Ribo-Zero Magnetic Kit (Gram-negative Bacteria) , to obtain a sufficient sequencing depth. For better insight in the composition of OMV-associated RNAs, the rRNA depletion step was omitted for OMV libraries. RNA-Sequencing libraries were prepared using the TruSeq Stranded mRNA LT kit  or the KAPA stranded RNA kit  without mRNA pull down steps according to the manufacturer’s protocol. The insert size and the quality of libraries were checked with an Agilent 2100 Bioanalyzer and quantified using the Qubit dsDNA HS assay kit. Libraries were diluted to 4 nM each, pooled, denatured and sequenced 2 × 80 (Low OD) or 2 × 75 (High OD) cycles on a NextSeq500 (Illumina) according to the manufacturer’s instructions. For each experimental condition, three biological replicates were sequenced. The raw sequence libraries are deposited in the BioProject database under the experiment accession number trim-fastq.pl script from PoPoolation package with the following parameters – quality-threshold 20 – fastq-type sanger – min-length 35  were downloaded from NCBI (genome accession number AE006468.2 and plasmid accession number AE006471.2). Samtools were used to merge the bam files  to avoid potential biases in the comparisons between intracellular and OMV-associated RNAs as they were experimentally removed from intracellular fractions only. Principal component analyses (PCA) were obtained with the plotPCA function from DESeq2. Analysis of variance (ANOVA) was performed with subsequent post hoc analysis to test if significant differences in relative amounts of exported RNA classes between the different culture conditions exist, using Tukey’s range test from the multcomp package . To produce the scatterplots in Figure 7, sum normalization was performed using the decostand function from the vegan package  over 2, and a corresponding FDR-adjusted p-value under 0.05 were considered as enriched.Exported RNAs associated to OMVs were filtered for a normalized count over 1, averaged from biological triplicates. Enriched RNAs associated to OMVs were determined using DESeq2 , calculaIntegrative Genomics Viewer software (v2.4.8) using default parameters  on independent replicates from high OD conditions.To confirm results obtained by RNA sequencing and to show that RNAs of interest are present in or on OMVs in full-length, PCR validation was performed for distinct RNAs ; cDNA libraries were prepared from 1 μl of OMV-associated RNA or 100 ng of intracellular RNA with the SuperScript III First-Strand Synthesis System (Invitrogen) using random hexamer primers according to the manufacturer instructions and without further cDNA fragmentation step.New Supplementary Dataset C). 0.5 μl of cDNA library was used for each 20 μl PCR reaction as template, together with 1 μM of primers. Intracellular libraries were used as positive control, and cDNA synthesis reactions were performed on OMV-associated RNA without adding reverse transcriptase (RT) as a negative control to check for the presence of residual genomic DNA. Three different Touch-Down PCR programs were used, depending on the target length and differed only by their extension time . The 3 first cycles used an annealing temperature of 72°C during 7 s, which was lowered by 3°C steps every 3 cycles until reaching 57°C for the last 20 cycles. This approach was used to ensure a good selectivity without the need of further optimization. The size of PCR products was checked by running 10 μl of PCR reactions on an agarose gel prepared with 3% w/v TopVision Agarose (Thermo Scientific) in Tris-Borate-EDTA buffer  for small size products or on a 1% w/v agarose gel in Tris-Acetate-EDTA buffer  for large size products, using the MassRuler DNA Ladder Mix (Thermo Scientific) as a size guide. In the case of 3% gels, the agarose solution was autoclaved to ensure an optimal dissolution before pouring the gel. Electrophoresis was run at 130 V during 200 min (3% gels) or 100 V during 100 min (1% gel) and DNA was stained with ethidium bromide. Pictures were acquired with a Canon EOS 1200D camera coupled to a BioDocAnalyzer (Biometra).Polymerase Chain Reaction was performed using Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo Scientific). A set of primers was designed to amplify the totality of the targeted Open Reading Frame (from start to stop codon), or the totality of the selected ncRNAs . The primers used are listed in Supplementary Dataset C. 0.75 μl of cDNA was used as a template for each 10 μl reaction, which was carried out with 100 nM of primers. Cycling conditions were as follow: initial denaturation 5 min at 95°C, 45 cycles of 15 s at 95°C, 30 s at the adapted annealing temperature (see Supplementary Dataset C), 30 s at 72°C, and a melting curve consisting of 5 s at 95°C and 1 min at 55°C. A first gradient run was performed on intracellular cDNA synthesis to select the optimal annealing temperature and to ensure that no coproducts were formed by analyzing the melting curves. The data was treated and analyzed with the LightCycler480 software to determine the Ct. Results with Ct equal or above 40 were considered as negative. Only technical duplicates with less than 5% variation were considered as reliable. For two given technical duplicates, if the difference between the average of +RT Ct and the average of -RT Ct is equal or superior to 3, the difference is considered to be significant .In addition to the PCR, qPCR was used to confirm for some RNAs their higher signal in RT-positives (+RT) libraries compared to RT-negative (-RT) controls, using absolute comparison between -1 and 37°C during 20 min. The fourth was digested by ProteinaseK  at 0.05 μg.μl-1 and 37°C during 30 min, followed by proteinase inactivation  and RNAseA digestion. The protocol was then followed until the cDNA libraries from the OMV-associated RNAs were obtained. End-point PCR was then performed with the same primers and conditions used previously for SsrS, CsrC, 10Sa, and rnpB genes. Resulting samples were deposited on a 3% w/v agarose gel in TBE buffer, subjected to electrophoresis and stained with Ethidium Bromide. The efficiency of RNase digestion was confirmed using an intracellular RNA extract (10 ng) subjected to the same protocol . The efficiency of proteinase digestion was confirmed on an intracellular protein extract (5 μg), which had been digested under the same conditions .To analyze if some nucleoprotein aggregates were co-purified with our OMV preparations, we performed a digestion protection assay . OMV isoS. Typhimurium cultured in five distinct conditions, with three biological replicates per condition, were purified and the associated RNAs were sequenced, together with their respective intracellular counterparts.Outer membrane vesicles originating from S. Typhimurium in order to study their RNA composition. A significant number of transcripts was found in OMVs under all conditions . Reads were mapped to the S. Typhimurium LT2 genome and pSLT plasmid and annotated using NCBI, and sRNA supplemental annotations from . As our annotated genome comprised 4964 genes, it means that on average, around 73% of the genes were expressed and identified in/on OMVs, even if it does not imply that these were native transcripts.RNA sequencing (RNA-Seq) was performed on the RNA isolated from OMVs secreted by ons from . Basic s samples . These cin vitro culture conditions chosen to mimic different life cycle steps of S. Typhimurium during host–pathogen interaction effectively induced the expression of the desired Salmonella pathogenicity island (SPI), we looked at the intracellular expression of the different SPI genes. As expected, SPI-1 expression was increased in SPI-1ind high OD conditions , in agreement with previous findings . Residual expression was also present in stationary phase for the same medium .To confirm that the specific findings . The actFigures 2A,B). The results confirm distinct clustering of the intracellular samples and, to a lesser extent, the OMVs triplicates. However, a general impression emerges from this data: most of the variance in these profiles is between the intracellular and OMV samples, which clearly points to a selective packaging of the exported RNAs. A difference is also observed in the high OD samples between SPI-1ind OMVs versus SPI-2ind OMVs triplicates. It is notable that these are conditions that mimic different infection steps . Moreover, when only RNAs (rRNA included) associated with OMVs within SPI-1ind and SPI-2ind conditions were used for a PCA, we could observe that 79% of the variance was explained by the growth conditions . When only intracellular RNAs from these same conditions were compared, 81% of the variance was explained . Thus, the OMV-RNA complement reflects the adaptation of S. Typhimurium to its environment.In order to acquire a broad view of the RNA expression profiles in the different samples, we performed Principal Component Analyses (PCA) on low and high OD samples . One can observe that for all but the OMVs originating from SPI-1ind, the vast majority of exported RNAs were rRNAs, as it has already been observed in other species  .As some exported RNAs appeared to be complete transcripts according to their coverage profiles, we designed a set of primers to validate their integrity experimentally by PCR amplification. The full coding-sequence of 20 mRNAs and the totality of 10 ncRNAs sequences were targeted and amplifications were performed on high OD condition samples (sseB and STM0972 in the case of SPI-2ind High OD). STM0277 was also detected in SPI-2ind High OD but not in SPI-1ind, whereas a greater number of reads was detected in OMVs from the later by sequencing. Interestingly, we observed a tendency toward the amplification of the smaller sized RNAs compared to the larger ones . This trend might be due to a higher degradation predisposition of larger RNAs compared to small RNAs, which are usually more structured and therefore more stable. Especially, protein-associated ncRNAs such as ffs (SRP RNA binding to Ffh and routing nascent proteins to the membrane), rnpB , SsrS (6S RNA that binds and regulates the σ70 transcription factor) or 10Sa (tmRNA associated to SmpB) were remarkably conserved in OMVs, in complete agreement with the RNA sequencing results . Fragments originating from tmRNA, SRP-RNA or 6S RNA were already reported to be present in the extracellular complement of E. coli, but completeness was not assessed . As neither RNase nor proteinase followed by RNase treatment prevented RT-PCR on SsrS, CsrC, 10Sa, and rnpB, we assume that these ncRNAs were packaged inside the OMVs instead of being passively enriched during vesicles purification. However, this association might also occur when these are bound to their partner proteins explaining their stability. Note that in the same digestion conditions, intracellular RNA or protein extract was almost completely digested and only rnpB gave a low signal by RT-PCR after RNase treatment .Hence, one may consider that the presence of these RNAs in our samples might be due to a “contamination” with nucleoprotein complexes occurring during the purification process of the vesicles. This issue has already been identified in the Extracellular Vesicle field, resulting in artificial enrichment of the OMV-associated cargo with protein-associated RNAs . TherefoSupplementary Figure S9) despite the DNAseI digestion step before cDNA synthesis. Therefore, we performed qPCR on +RT and -RT samples for several genes and confirmed that the amount of cDNA was greater in +RT than in -RT for ffs, CsrC, rnpB, sseB, and STM0972 (sopD2) (Table 1). For sseB and STM0972, both replicates were positive contrary to the full-length PCR, indicating that the transcripts were present but not in their native form or the end-point PCR was not sensitive enough to detect them on a gel.Some -RT controls showed a remaining DNA contamination .In contrast to the genes highlighted above, no full-length transcripts associated to OMVs could be detected for the following genes: Figure 7). In this context, it is important to note that we did not have the possibility to distinguish between full-length or processed RNAs which remain functional and degradation products for each individual transcript enriched in/on OMVs as described and explained in section “Native and Processed and/or Degraded RNA Co-exist Among OMV-Associated RNAs.” As, however, this remained true for all the different culture conditions, we performed pairwise comparisons between intracellular and OMV-associated RNAs among our five culture conditions, in order to obtain a global overview of RNA sequences which were differentially expressed among the different culture media. The RNAs that were enriched in/on OMVs compared to their intracellular counterpart were mainly coding sequences and to a lesser extent, ncRNAs . On the contrary, tRNAs seemed more internalized for the studied transcript libraries . Note that rRNAs are not displayed here as the intracellular samples had been experimentally depleted of the latter and thus intracellular versus OMV RNA sample comparisons became impossible. The pattern was clearly dependent on the condition, with a wider spread in high OD conditions and especially in SPI-1ind . Interestingly, the translation rescuing RNA tmRNA (10Sa), which was experimentally proven to be packaged as full-length transcript  was always highly represented in both fractions. As 10Sa binds other proteins for its activity, and among them elongation factors or ribosomal proteins, its OMV association could be favored by the presence of the elongation factor EF-G in the vesicles  (OMV/intracellular) ≥ 2, a p-value < 0.05 (Welch’s t-test), and an averaged number of reads per condition ≥ 5 in order to ensure a stringent selection of “enriched RNAs.” Henceforth, all RNAs that fulfill the aforementioned criteria are referred to as enriched RNAs, whereas we refer to exported RNAs when a given RNA is associated with OMVs, but not necessarily enriched compared to its intracellular counterpart. In each media condition, a significant number of transcripts was indeed selectively exported . In total, we found 1151 enriched transcripts in Control Low OD condition, 763 in SPI-2ind Low OD conditions, 881 in Control High OD condition, 643 in SPI-2ind High OD condition and 1196 in SPI-1ind condition (detailed lists are available in Supplementary Dataset D). A smaller core set of 193 RNAs was enriched in all conditions tested, with the notable absence of ncRNAs in this set, these being mainly found in condition-specific groups. However, these group sizes were very low compared to the totality of the RNA cargo, which originated from 4670 genes in total, from which 2217 were found in all the conditions tested . This difference may underline the dual nature of OMV-RNA association, reflecting the intracellular state of the transcriptome through global packaging, but also containing specific sets of read fragments mapping to distinct RNAs that depend or not on the current environmental state.In order to define more precisely the OMV-enriched transcripts in comparison to their intracellular counterpart, a differential expression analysis with the  package  was perfFigure 5, Table 1 and Supplementary Dataset C) and as most of the effectors were covered over the whole length of their gene , we looked at the more than 40 effectors known to be secreted by Salmonella during invasion and infection . However, even if almost all effectors’ mRNAs were found associated with OMVs in every condition, the OMV export route appeared to be more important when the corresponding T3SS (Type 3 Secretion System) was absent . This was confirmed by the analysis of enriched effectors RNAs, where a distinct pattern did exist : SPI-1 translocated effectors seemed enriched in/on vesicles produced in SPI-2ind conditions , whereas SPI-2 translocated ones were enriched in/on OMVs originating from non-SPI-2ind conditions, i.e., Control and SPI-1ind . Interestingly, we also identified prophage-encoded effectors. For instance, GogB is a secreted effector originating from Gifsy-1 prophage that can be translocated through both the SPI-1 and SPI-2 T3SS, but which is mainly expressed under SPI-2 control by ssrB (p < 0.001), emphasizing its multiple roles in pathogenicity as this protein might be involved in regulating immune processes from an early stage of the host–pathogen interaction. One can also note the stable presence of transcripts of T3SS-independent effectors such as ydgH (or sssB) and STM0082 (or srfN)  . No enriFigure 10). In this case, an increased intracellular expression caused a greater export by vesicles, as it was clearly visible in Control conditions at high OD and SPI-1ind. The in vitro expression level of SPI-6 is generally low, as reported earlier . Interestingly, the same was observed concerning the enrichment in OMVs, showing a correlated expression and enrichment in vesicles for most of the SPI-6 transcribed RNAs . The coverage data of the mapped reads was relatively constant between intracellular and OMV-associated fractions .Interestingly, a number of SPI-6 mRNAs were found to be exported and enriched in/on OMVs ( earlier . HoweverS. Typhimurium encodes for a Type 6 Secretion System (T6SS) that is involved in eukaryotic cell infection  and its cognate immunity-protein mRNA tai4 (STM0278) were only moderately present in the intracellular fraction  under the conditions studied, but their mRNAs were enriched in/on OMVs purified in SPI-1ind and Control conditions at high OD . Another pair of toxin/antitoxin encoded by the SPI-6 is rhs (STM0291 and STM0292)/rhsI (STM0293). Rhs has a putative nuclease activity and was expressed in the intracellular case together with RhsI . Both of their mRNAs were found associated with OMVs but only rhs was slightly enriched in several conditions .SPI-6 in nfection . Howevernfection . InteresFigure 5 and Supplementary Dataset C) and as we have seen in Figure 8 that a smaller proportion of these latter was also enriched in/on OMVs in a condition-dependent manner, we analyzed in more detail these sRNAs . The majority of them was found in the SPI-2ind and SPI-1ind cases, thus linked to virulence-like conditions. An enrichment in/on OMVs was mainly observed in high OD conditions; with only a few transcripts enriched at low OD. If these were exported in their native form through OMVs, they might be involved in pathway regulation in surrounding cells up-taking OMVs.As we could experimentally validate the presence of several full-length sRNAs . Among them, one can note the presence of IsrC, triggering the degradation of the mgrA mRNA, involved in Salmonella survival in macrophages (p < 0.001). No trace of it was found in/on OMVs from SPI-2ind conditions . IsrM is known to be expressed in gut tract conditions in vitro  were among the most exported sRNAs through OMVs, and the most abundant intracellular sRNAs . These sRNAs control the translational regulator CsrA that regulates SPI-1 genes and the expression of flagella components, playing a central role in virulence , was also highly expressed inside the cells and associated with OMVs. It is necessary for acidic-environment adaptation and epithelial cell invasion . These contaminating structures are present in crude OMV preparations . We cannot rule out the co-purification of nucleoprotein aggregates, however, these should be resistant to RNase A and the combination of RNase A and proteinase K. As the understanding of microbial membrane vesicles advances, we expect the validation of purity markers will provide the appropriate tools for even more stringent quality control.This study provides a first global picture of the RNA cargo (>25 nt) associated with  factors . RNA wasThe global quantity of OMVs and associated RNAs was not studied in this work. From western-blots, we could see differences in OmpA quantities between conditions. These do not automatically reflect differences in OMV secretion but can also originate from a change in the composition of the outer membrane. However, several studies already demonstrated the variation of membrane vesicles secretion induced by different cultivation conditions . The gloIn accordance with several other studies, the majority of OMV-exported large RNAs were ribosomal RNAs . It is iE. coli where they were among the most exported OMV-RNAs , indicating that most of the phage-encoded RNA identified in our RNA-Seq data is originating from prophages. Nevertheless, we cannot exclude that the purification method used to purify OMVs did result in a co-purification of some active Salmonella’s phages, as gradient purification is sometimes also used for phage isolation  which has been shown to be crucial for resistance and adaptation to acidic environment encountered during invasion , the strong presence of others is mysterious. Interestingly, most of them are linked to virulence and resistance of the cell . Anotherinvasion . As we dBorrelia burgdorferi cultures, an enrichment of plasmid-encoded RNAs over chromosome-encoded transcripts has been recently shown .Finally, in OMVs isolated from ly shown . We valiSalmonella-derived OMVs and emphasizes the impact of the bacteria-encountered environmental conditions on the RNA content of the vesicles. Much more work is needed to fully understand the underlying mechanisms and physiological processes that lead to this export, which results in the release of many virulence-associated RNAs and could have important consequences in host–pathogen interplay during infection. Whether this adaptable RNA export is a mechanism for targeted transfer or a way for cells to eliminate undesirable RNAs remains to be clarified. However, the confirmation of the export of full-length transcripts opens the door to numerous possible functional implications in cell-to-cell communication.In summary, this work provides a first insight into the differential association of RNAs (>25 nt) with JF and PW conceived the original idea. JH, JF, and AM designed the experiments. JH, JF, AM, AH-B, JG, RH, AE, and DG performed the experiments. AH-B, PM, AM, and JF analyzed the data. AM and JF wrote the manuscript. All authors analyzed results, commented on and edited the manuscript.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.


Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.”The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts,  The origin of life on Earth has recently regained attention following the discovery of exoplanets within possible habitable zones . The astPelagibacter ubique with about 1,400 genes , 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10132  before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs . They lalication .tRNA-amino acid complexes can find RNAs as “mRNAs.” Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: “The ribosome is a ribozyme” , supplemin vitro  as today, and allow for DNA polymerization . The samPelagibacter ubique to about 98% in the human genome.It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the “Encyclopedia of DNA Elements” (ENCODE) project. ncDNA amounts to more than 98% of the human DNA genome . Higher trans-activating response element (TAR), the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as well as during retroviral replication, proving a requirement of RNA , resembling these early RNAs . The TLSRibozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses . ViroidsFrom infected potatoes, Diener isolated the Potato spindle tuber viroid (PSTVd) whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural information in the form of hairpin-loops . They caPospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid (ASBVd). Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle.The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 . The genPospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost.Most viroids are often enzymatically active ribozymes – yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA (CarSV RNA) cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers , 2014. WThe circular RNA (circRNA) is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a “sponge” absorbing small RNAs. Micro RNAs (miRNAs) are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules . Their sThere is an exceptional viroid that gained coding information and entered the human liver . The virIn plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation – but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets . ViroidsIn summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma . Even anin vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as “Spiegelman’s Monster.” Coding RNA became replicating ncRNA  as a stable composition , 2017. DThe reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . AnalogoSalmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life  that act as promoters (Protein Kinase Inhibitor B (PKIB) gene we determined about 70% retrovirus-related sequences , disabling the receptor functionally. The allele frequency of the mutant CCR5-Δ32 mutant is about 10% in the European population, making these people resistant to HIV infections . This geViruses have been proven to be drivers of evolution , includiEndogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years . If the de novo infections by “superinfection exclusion”  in “real time” and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus (GALV). Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus (JSRV), whereby the endogenized viruses protect with a viral gene product, such as Env, against clusion” . The conclusion” .Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cell-surface receptors . A compaIntegration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus (SIV) in some monkey species may be explained by endogenization , 2018. IOne may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . How lonenv or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome , Mycobacterium leprae, M. tuberculosis, and M. mycoides , replicating (pol) or envelope (env) genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells (src (for sarcoma) oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome transposons in germline cells can result in diseases such as male infertility – presumably by “error catastrophe,” caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis .Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species.Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin’s concept of evolution.Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication (in vitro by Spiegelman’s Monster and Eigen’s follow-up experiments, explaining the great surviving potential of simple ncRNA.There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA . But genlication , 2013. TViruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled “Viruses: More Friends Than Foes” . The sce25. Thus, pure statistics make some people believe that there is extraterrestrial life.The early RNA world gained interest 20–30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the “pansperm hypothesis” and think that retroviruses are of extraterrestric origin . The recThe extraterrestric life is mimicked in laboratories on Earth with many assumptions – perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence.Both authors wrote the manuscript and approved the final 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.


Xist, the master regulator of the X chromosome inactivation in mammals, is a 17 kb lncRNA that acts in cis to silence the majority of genes along the chromosome from which it is transcribed. The two key processes required for Xist RNA function, localisation in cis and recruitment of silencing factors, are genetically separable, at least in part. Recent studies have identified Xist RNA sequences and associated RNA-binding proteins (RBPs) that are important for these processes. Notably, several of the key Xist RNA elements correspond to local tandem repeats. In this review, I use examples to illustrate different modes whereby tandem repeat amplification has been exploited to allow orthodox RBPs to confer new functions for Xist-mediated chromosome inactivation. I further discuss the potential generality of tandem repeat expansion in the evolution of functional long non-coding RNAs (lncRNAs). Xist mediates X chromosome inactivation (X inactivation), the process that, in mammals, equalises levels of X-linked gene expression in XX female relative to XY male cells [Xist RNA is expressed from the inactive chromosome-elect at the onset of X inactivation during early embryogenesis and then localises to a subnuclear domain corresponding to the X chromosome nuclear territory [Xist RNA induces chromosome inactivation by recruiting factors that modify underlying chromatin and repress gene activity [The long non-coding RNA (lncRNA) le cells ,2,3,4,5.erritory . Localisactivity . The repXist transgenes suggested that the localisation of Xist RNA and Xist-mediated silencing are separable processes [Xist RNA has come from recent studies identifying RNA binding proteins (RBPs) that interact with Xist RNA, and that function principally in Xist RNA localisation (hnRNPU and Ciz1) [Xist-mediated gene silencing  [A deletion analysis using rocesses . Furthernd Ciz1) ,10,11, o hnRNPK) ,15,16,17Xist RBPs and the elements to which they bind have provided important advances in our understanding of Xist RNA localisation and Xist-mediated silencing. Several observations have led to the idea that Xist RNA is anchored to the Xi territory through the interaction with the nuclear matrix: Thus, Xist RNA domains are retained in cells following nuclear matrix extraction, a procedure which yields nuclei from which the bulk of chromatin and soluble proteins are removed [Xist localization factors hnRNPU and Ciz1, both of which have been characterised as components of the insoluble nuclear matrix, leads to the delocalisation of Xist RNA to sites throughout the nucleoplasm [Xist RNA colocalises with hnRNPU and Ciz1 within channel networks that pervade interphase chromosome territories, including on Xi [Xist RNA molecules function to nucleate chromosome silencing through the recruitment/enrichment of Xist silencing factors  [Xist RNPs induces gene silencing across an entire chromosome, comprising some 150 Mb of DNA and around 1000 genes. One factor that may contribute is signal amplification through local tandemly repeated RBP binding sites within single Xist RNA molecules. Specifically, the sequence analysis of Xist RNA in mouse, human and other species has revealed that a significant proportion of the primary RNA sequence is comprised of blocks of local tandemly repeated elements, designated repeats A–F  . An impoeats A–F . Several species ,22, and ilencing ,13,15,17Xist A-repeat, located at the 5′ end of Xist RNA, comprises of 7–8 copies of a 24 nt GC-rich core sequence separated by uridine tracts of variable length [Xist RNA largely ablates Xist-mediated silencing, but does not grossly affect Xist RNA localisation [Xist RNA transgene spanning the first 4 kb of sequence, deletion of the A-repeat does lead to loss of Xist RNA localisation [Xist RNA. Given the essential role of the A-repeats, there has been considerable interest in defining the RBPs that interact with this element. To this end recent proteomic and genetic screening strategies have identified that the RBP Spen is recruited to the A-repeat and functions as a critical factor in Xist-mediated silencing [Xist-mediated silencing has also been shown to bind to the A-repeat region [Xist RNA to facilitate the silencing function [Xist silencing is not well defined but is thought to involve the m6A reader protein, YTHDC1 [The lisation , attestiilencing ,13,14,15ilencing . A closet region . Rbm15 ifunction . The pre, YTHDC1 .Xist RNA molecule, thereby enhancing the silencing activity. Spen is a large protein, some 3600 amino acids, which includes four closely spaced RNA binding (RRM) domains at the N-terminus, and a Spen Paralogue and Orthologue C-terminal (SPOC) domain located at the C-terminus [Xist RNA molecule has the potential to bind 3–4 Spen molecules, a moderate enhancement. It is interesting to note that the complementation of the A-repeat deletion can be achieved using a synthetic X7.5 A-repeat consensus sequence, but not with an X5.5 A-repeat [An interesting speculation is that the evolutionary expansion of A-repeat monomers has increased the number of Spen molecules that can concurrently bind to a single terminus ,25,26,27terminus . This coA-repeat . WhetherA-repeat , competeXist RNA and with NCoR-HDAC3 or the m6A MTase complex, respectively, is also potentially important in relation to signal amplification. Thus, relatively weak and, hence, dynamic interactions could contribute to signal amplification by creating a high local concentration of effector proteins in interchromatin spaces  [Xist RNPs, with the increased number of bound RBP molecules contributing to the strength of the interaction with the nuclear matrix. Evolution of strong interactions with the nuclear matrix would first serve to limit Xist RNP localisation in cis, and second, would be important in terms of modulating the range or distance of spread of Xist RNPs.The accumulated evidence that local tandem repeats in ression) . With thXist lncRNA may have a previously evolved interaction with a known transcriptional repressor. An example of this would be the RBP Spen, for which interaction with NCoR-HDAC3 is predicted to be evolutionarily conserved [Xist lncRNA may acquire an interaction with a known repressor complex as a neofunctionality. This could be the case for hnRNPK which has roles in diverse aspects of RNA biogenesis [Xist RNA [Xist RNA may have been exploited to enhance a relatively weak interaction with the Pcgf3/5 Polycomb protein to a level sufficient to initiate the Polycomb cascade.Using a similar argument, the amplification of binding sites for RBPs that generate a local concentration of silencing factors could be selected for on the basis of improved efficiency/completeness of gene dosage compensation. One possibility is that an RBP that binds to a single site in the prototypic onserved . Alternaogenesis , of whicXist RNA . Thus, tXist RNPs on the nuclear matrix is likely to be important in enabling the local concentration of silencing factors to reach a critical threshold. Accordingly, Spen/hnRNPK binding sites in other messenger RNAs (mRNAs) or lncRNAs may not lead to significant levels of chromatin modification in cis, i.e., in cases where the RNA is not anchored to the nuclear matrix for a significant time. Here it is interesting to note that hnRNPK binding to C-rich motifs has been implicated in retention of nuclear RNAs, including those arising from the Alu family of dispersed repeats [In the context of the above models, the long-term retention of  repeats .Xist RNA leads to the question of how general this model may be in the evolution of functional lncRNA. There are indeed examples that illustrate that this mechanism may be more widely utilised. Thus, the lncRNA RNA on the Silent X (Rsx), which evolved as the master regulator of X inactivation in marsupial mammals entirely independently of Xist, is also characterised by the presence of large blocks of local tandem repeated sequences [Xist RNA is limited, and at present nothing is known about their functional importance or bound RBPs. However, close parallels in the X inactivation process in eutherian and metatherian mammals, notably chromosome-wide hypoacetylation of histones and recruitment of the Polycomb system, imply that commonalities could extend to key Xist silencing factors such as Spen and hnRNPK. There may also be overlaps in terms of the factors and mechanisms regulating Rsx RNA localisation, as, like Xist RNA, Rsx RNA localises strictly in cis on the X chromosome from which it is transcribed [Our growing appreciation of the importance of local tandem repeat expansion in functionalisation of equences . The seqnscribed .Whilst the focus of this review up to this point has been on local repeat sequences that are tandemly arranged, it should be noted that many of the arguments put forward apply equally well to local repeats that are dispersed within a given locus, referred to simply as local repeats. An example of this has emerged from recent studies on the lncRNA Firre/FIRRE ,50. ThusRecent studies on the lncRNA Neat1, which is required to nucleate the formation of paraspeckles, a phase-separated membrane-less organelle , also poWhilst the amplification of RBP binding sites likely represents a key mechanism for the functionalisation of lncRNAs, it should be noted that the amplification of binding sites for other RNAs may also be an important evolutionary mechanism. Indeed, certain circular RNAs have evolved as molecular sponges, presenting multiple binding sites for specific miRNAs .Xist RNA provides an instructive example of how lncRNAs may become functionalised through natural selection. Conversely, the analysis of local tandem repeat expansions, or for that matter dispersed local repeats, in lncRNA, together with the identification of RBPs that bind to them, could provide a useful approach towards understanding the function of specific lncRNAs. An interesting starting point would be to analyse lncRNAs associated with imprinted gene clusters as, at least in some cases, these likely function analogously to Xist to induce chromatin repression over contiguous genomic regions [The expansion of local tandem repeats during the evolution of  regions ,57. Simi regions , pericen regions , and oth regions , are int regions .