Little is known about long-term changes in coral reef fish communities. Here we present a new technique that leverages fish otoliths in reef sediments to reconstruct coral reef fish communities. We found over 5,400 otoliths in 169 modern and mid-Holocene bulk samples from Caribbean Panama and Dominican Republic mid-Holocene and modern reefs, demonstrating otoliths are abundant in reef sediments. With a specially-built reference collection, we were able to assign over 4,400 otoliths to one of 56 taxa (35 families) though mostly at genus and family level. Many otoliths were from juvenile fishes for which identification is challenging. Richness (by rarefaction) of otolith assemblages was slightly higher in modern than mid-Holocene reefs, but further analyses are required to elucidate the underlying causes. We compared the living fish communities, sampled using icthyocide, with the sediment otolith assemblages on four reefs finding the otolith assemblages faithfully capture the general composition of the living fish communities. Radiocarbon dating performed directly on the otoliths suggests that relatively little mixing of sediment layers particularly on actively accreting branching coral reefs. All otolith assemblages were strongly dominated by small, fast-turnover fish taxa and juvenile individuals, and our exploration on taxonomy, functional ecology and taphonomy lead us to the conclusion that intense predation is likely the most important process for otolith accumulation in reef sediments. We conclude that otolith assemblages in modern and fossil reef sediments can provide a powerful tool to explore ecological changes in reef fish communities over time and space. Coral reefs are changing because of local and global human impacts –3. The fCoral reef fishes generally have a poor fossil record , but in 14C) dating of otoliths. Finally, we use these findings to explore the potential applications of otolith assemblages in fossil and modern sediments to establish baselines and monitor reef fish communities.To assist in the identification of sediment otoliths, we build an otolith reference collection from Neotropical coastal fishes . We then provide guidelines for extracting and identifying otoliths from reef sediments, and apply this approach to sub-Recent (modern) and mid-Holocene reef sediments from Bocas del Toro and the Dominican Republic. We test the fidelity between otolith sediment assemblages and the living fish communities on reefs and explore taphonomic controls  on otolith assemblages in reefs, including the amount of time-averaging  through pre- and post-bomb radiocarbon  where both mid-Holocene and sub-Recent reefs were available for study . In Bocasemblage  that accsemblage  28]. We. WeIn sisemblage  and extrin situ and in-life position corals and the matrix is neither consolidated nor indurated  and the Ministerio de Medio Ambiente y Recursos Naturales, República Dominicana (Permit number VAPB-02374), respectively.https://otolithspisciumpanama.jimdo.com/) [The bulk samples of sub-Recent and mid-Holocene reef matrix were dried and weighed, then wet-sieved over 2 mm, 500 μm, 250 μm, and 106 μm-mesh screens. Sediment in the 2 mm and 500 μm fractions was carefully scanned under a dissecting microscope and all otoliths picked from the carbonate sands. Each isolated otolith was identified using regional literature –38, estado.com/)  and our The abundance of otoliths in each taxon in each sample was counted. Other fish remains such as spines, teeth, and vertebrae can be found sporadically from the residues as well, but were much rarer in the > 500 μm fraction. These were kept but are not further discussed here. Average otolith density in each sample was computed as otolith count/dry sediment weight (kg). Richness and diversity estimates were calculated per sample and sample-based rarefaction and extrapolation with 95% confidence intervals calculated using EstimateS software . FurtherTo explore how faithful otolith sediment assemblages represent living fish communities we conducted small rotenone surveys on four reefs in Bocas del Toro and compared the survey results to our otolith assemblages from the sub-Recent bulk samples on the same reefs . These s2 of the reef at each station [Rotenone is the most effective tool for reef fish diversity surveys in small areas –44 and i station ,43. All  station ,46, theiSediment otolith assemblages and rotenone survey samples were compared based upon their taxonomic composition and abundance. Rotenone survey samples with less than 10 individuals of fishes were first excluded. Samples from the same site were then grouped together to increase their sample size in subsequent analyses. Higher taxonomic ranks were adopted and aligned  to make sediment otolith assemblages and rotenone assemblages comparable. In addition, fish otoliths that were measured to be less than 500 μm in the rotenone samples were excluded because the sediments were sieved at that mesh size. All anchovies (Engraulidae), herrings (Clupeidae), codlets (Bregmacerotidae), and silversides (Atherinidae and Atherinopsidae) were excluded from the sediment assemblages because the rotenone survey technique fails to capture mobile, epipelagic fishes . SimilarUranium-Thorium (U-Th) dates of coral skeleton show that coral pieces in branching reef framework undergo limited post-burial vertical mixing , presuma14C that can be used in determining time averaging from recent sediments. In this case, both the rise of atmospheric 14C and the post-peak decline can be used as a chronometer when properly analysed [Standard radiocarbon dating can be applied to small otolith masses but are subject to marine reservoir age , which can reduce temporal resolution. However, dates after 1950—the period of atmospheric testing of nuclear bombs—provides a sharp increase of environmental analysed .14C levels. Radiocarbon measurements were reported by NOSAMS as Fraction Modern—the measured deviation of the 14C/12C ratio from Modern. Modern is defined as 95% of the 14C concentration of the National Bureau of Standards Oxalic Acid I standard (SRM 4990B) normalised to δ13CVPDB (–19‰) in 1950 AD [13C measured concurrently during AMS analysis and are reported here as F14C [We, therefore, used bomb pulse dating to explore the ages of the small otoliths in modern seafloor assemblages. From two bulk samples, we randomly selected 20 Gobiidae otoliths, within mass ranges 0.56–0.89 mg . Otolithdard SRM 90B normaComparative systematics is critical for the correct identification of isolated otoliths . RegionaThe reference collection includes over 1500 otolith specimens belonging to 173 species of 52 families . Severalsensu Nelson [A list of otolith taxa recovered from fossil and sub-Recent sediment assemblages is presented in u Nelson  that we Anchoa and Anchovia [Our engraulid otoliths show variations in the posterior rim, otolith length-height ratio, and the length of rostrum suggesting that more than one species or genus are involved. Indeed, ca 13 species occur in the Bocas region (western Panama) . ReferenAnchovia . We, theJenkinsia are trapezoid in shape, with a deep and triangular ostium and a shallow, indefinite cauda. They have a robust rostrum but lack a pronounced antirostrum, resembling much to those of Etrumeus. The combination of these features make them readily distinguishable among clupeid otoliths, but very difficult to discriminate at the species level. To our knowledge, images of Jenkinsia otoliths have never before been published.The otoliths of Porichthys share many superficial similarities with those of gobiids; their otoliths are high-bodied with a centrally-located sulcus and a collicular crest on the crista inferior of the cauda. The diagnostic features for Porichthys otoliths compared to those of Gobiidae are a much wider and longer sulcus, a marked constriction at the collum of crista superior, and a wider ventral rim relative to the dorsal rim. The varied shapes in the dorsal rim of our Porichthys specimens, each from the mid-Holocene and sub-Recent sample of Bocas, is here considered ontogenetic differences [Porichthys otoliths from the Dominican Republic [Porichthys plectrodon based on recent comparative material .The otoliths of ferences . They arRepublic . We idenBathygobius . Average density of otoliths was marginally higher  in sub-Recent than mid-Holocene sediments in Bocas del Toro  differed substantially from other sites  were captured on four reefs  in BocasBoth cluster and nMDS analyses revealed that the composition of the standing living community at the shallowest reefs was distinct from the deeper reefs . In clus14C values were well above mean pre-bomb levels (mean F14C = 0.942 ± 0.015) covering an F14C range of 1.05–1.14 and could be assigned a calibrated date from either the rise or decline periods . One value was near the peak of the coral records and could be assigned a median year of ~1976 and may have had an uncertainty of ± 6 years at most from the observed range of peak values (~1970–1982). One pre-bomb value lying below mean pre-bomb levels (F14C = 0.9144 ± 0.0024) and aged using 14C dating, revealed a median date of 1550 AD after correction for regional reservoir age. Potential dates of formation were 1490–1640 AD (2 sigmas). Thus, 19 out of 20 otoliths could be assigned ages of between ~1966 and 2014, while one otolith was dated to 1550 AD.We used radiocarbon dating directly on 20 otoliths to explore the amount of time encapsulated by a single bulk sample sediment otolith assemblage from a modern, actively accreting, reef. Nineteen of the otoliths were of a modern origin (> 1950) . We foun periods . Dates fOur findings reveal that otoliths can be abundant and well-preserved in unconsolidated carbonate-rich reef sediments. We propose that these records can be used to extend monitoring of reef fish communities to complement ongoing research into the spatial and temporal variation in the biodiversity and ecological functioning of coral reefs. This work augments ongoing research on other skeletal elements preserved in reef matrices including molluscan shells , sponge High-level taxonomic identification of otoliths often relies on features that appear only in adult individuals . The higRarefaction curves and extrapolation suggest that sampling effort was, on the whole, fairly good, although mid-Holocene sites from the Dominican Republic are currently under-sampled . Given tOtoliths representing a wide spectrum of reef fishes were recovered from our sediment otolith assemblages, including 35 identified families, of which we were able to resolve 56 taxa Figs , Table 3The otolith assemblages were dominated by small fishes, with gobies (Gobiidae), anchovies (Engraulidae), and cardinalfishes (Apogonidae) always in the top taxa . Larger,per capita energy flow in reefs [The dominance of small cryptobenthic and epipelagic fishes in our sediment assemblages is in sharp contrast to the findings of visual surveys that are frequently dominated by large diurnal fishes. For example, Dominici Arosemena and Wolff  and Seemin reefs ,74 explain reefs –27. Thusin reefs .Our comparison between surveys in rotenone stations and the otolith assemblages in sediments from the same sites, reveals a high concordance between the two approaches . Both reAnchovies, herrings, and silversides are appropriately represented in the otolith sediment assemblages but are unsurprisingly absent in the rotenone surveys because these fast epipelagic fish swim around living reefs ,77, avoiThe otolith assemblages recovered from sub-Recent and mid-Holocene Dominican Republic and Caribbean Panama bulk-sampled reef sediments were, on the whole, well-preserved. The proportion of eroded and unidentifiable specimens was fairly consistent across samples and sites . When ot14C dating on the otoliths themselves. Nineteen of the 20 otoliths we analysed are estimated to have dated from between ~1966 to 2014  mortality of a fish, from disease or old-age, and the subsequent decay of its skeleton would lead to the release of the otoliths and their inclusion into the reef matrix. This process may be important in fishes or ecosystems that experience low rates of predation. However, predation on reef fishes is well documented to be extremely intense [There are two main routes by which otoliths may become incorporated into reef sediments. First,  intense , especia intense  where pr intense .Intense predation on reef fishes is supported by the fact that the majority of otoliths we observe in reef sediments are from juveniles. It is therefore likely that the overwhelming mode of incorporation of otoliths into reef sediments is through digestion followed either by regurgitation or defecation by predators. Three main groups of predators could be responsible; predatory fishes (including sharks), marine mammals, and seabirds. Epipelagic fishes, such as anchovies and silversides, likely experience intense predation from all three , whereasPredation by highly mobile predatory fishes, marine mammals and seabirds could, therefore, bring otoliths from other habitats onto reefs where they could become buried in the sediments. Dolphins  and reefWe describe a novel approach to reconstruct coral reef fish assemblages using otoliths accumulating in modern and fossil coral reef sediments. We present a reference collection which we use to identify otoliths found in reef sediments, showing that sediment otolith assemblages are on the whole representative of the native benthic and pelagic fish communities living on reefs. The relatively rare occurrences of some non-reef taxa support the general conclusion that predation is the most probable route by which otoliths accumulate in reef sediments. Preservation of otoliths in the Caribbean reef sediments we studied was generally good and the attainable taxonomic resolution was limited by the majority of otoliths originating from juvenile fishes rather than preservation state. Radiocarbon analysis conducted directly on representatively-small otoliths found in reef sediments in actively accreting branching coral framework show that time averaging is not excessive. In this habitat, the majority of otoliths in sea-floor sediments were modern in age, although this is highly unlikely to be the case in habitats where bioturbation is high.We conclude that modern and fossil otolith assemblages in reef sediments can provide a powerful approach to reconstruct reef fish communities, establish pre-human baselines and explore the spatial and temporal dynamics of reef fish communities. These condensations of fish remains can provide a complementary approach to classic fish surveys which tend to provide “snapshots” of fish communities. Otolith assemblages, on the other hand, are accumulations over time that average-out temporal ecological fluctuations. Otoliths in the sediments have the benefit over other survey approaches by including fishes that are often missed in surveys, such as nocturnal and cryptobenthic fishes. The majority of reef sediment otolith assemblages are dominated by the cryptobenthic components, a result which is likely driven by the fact that such fishes are highly abundant, grow fast and have relatively short reproductive life histories.A major constraint of this approach is the limited taxonomic resolution that is possible. Our improving reference collection will help go some way to advance this resolution. Nevertheless, it is likely that there will always be a substantially large proportion of reef sediment otoliths that cannot be identified to species because diversity is high and a large proportion of the otoliths are juveniles, making identification not only challenging but in many cases unresolvable. Nonetheless, in many cases, otoliths can be confidentially assigned to basic functional guilds on reefs, and this could represent an extremely powerful approach to explore the questions about trophic structure, energy flow and food web structure on reefs over space and time.S1 FigFacies, distribution of principal habitats (A) and stratigraphy (B) of mid-Holocene excavation sites in Bocas del Toro, western Panama. Description of trenches is ordered roughly from the youngest to the oldest in (B).(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigImages are inner views and scale bars = 1 mm unless otherwise indicated.(TIF)Click here for additional data file.S4 FigImages are inner views and scale bars = 1 mm unless otherwise indicated.(TIF)Click here for additional data file.S5 FigImages are inner views and scale bars = 1 mm unless otherwise indicated.(TIF)Click here for additional data file.S6 FigImages are inner views and scale bars = 1 mm unless otherwise indicated.(TIF)Click here for additional data file.S7 FigSamples from the same site are grouped. Box plot with lower (25th percentile), median and upper (75th percentile) boundaries, whiskers of 10th and 90th percentiles, and outliers (solid circle) outside of 10th and 90th percentiles are presented.(TIF)Click here for additional data file.S1 Table14C reference record (14C rise (~1958–1970) and decline periods (post-1982). Two specimens fell outside these periods and were either pre-bomb (earlier than ~1958) or during the peak period (~1970–1982). Uncertainty for formation dates is approximately ± 2 years for the rise period and ± 5 years for the decline period based on 95% prediction intervals.Measurements were correlated in time by alignment to a regional coral e record . There w(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableSD = standard deviation.(XLSX)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.


Recently, it was found that bacterial and eukaryotic transcripts are capped with cellular cofactors installed by their respective RNA polymerases (RNAPs) during transcription initiation. We now show that mitochondrial RNAP efficiently caps transcripts with ADP – containing cofactors. However, a functional role of universal RNAP – catalysed capping is not yet clear. In addition, a number of currently uncharacterised moieties were found attached to E.coli cellular RNA which could potentially also serve as 5’ RNA caps [+ modification (NADylation) in the cell varies greatly for different RNA species. The RNA species that are most heavily NADylated in vivo [+  ,2. NAD+  in vivo  are listan cells ,5.Figur+ as an initiating nucleotide (given its ADP moiety and free 3’ hydroxyl group) [Km for NAD+ in transcription initiation was much lower than the in vivo concentration of NAD+  was shown previously to use NADl group) . Studiesl group) ,8. These of NAD+ B. Furthein vitro . Eukaryo by RNAP .+, suggesting that the possible abundance of these caps is also lower [Other ADP-containing cofactors were shown to be efficiently incorporated at the 5’ end of RNA by RNAP, such as FAD and 3′-dephosphocoenzyme A (but not NADP and NADPH) ,8. The eso lower .+ in E. coli cells grown on rich media  remains somewhat controversial. Bird et al. showed that the identity of the base at position -1 (-1A vs -1C) affects the efficiency of capping [+ [+ incorporation, suggesting that observed stabilisation of short capped RNAs is due to interactions between the NAD+ cap and the RNAP rifampicin-binding pocket [+, the incorporation of UDP-containing cell wall precursors was not affected by the amino acid substitutions in the rifampicin-binding pocket.We showed that initiation with NADin vitro . Whether capping . Howeverpping [+ . Indeed,pping [+   had no effect on incorporation of NAD+, NADH, FAD, UDP-Glucose or UDP-GlcNAc. In contrast, the mature cell wall precursor UDP-MurNAc-pentapeptide, was incorporated by the σ 70Δ3.2 mutant of RNAP much more efficiently, suggesting that region 3.2 of σ70 may serve to prevent incorporation of advanced cell wall precursors [Cofactors bound at +1 position may potentially interact with the 3.2 region of initiation factor σe centre . Howeverecursors   and AMP [+/NADH cap to produces 5′-monophosphorylated RNA [Saccharomyces cerevisiae and Nudt19 in Oryza sativa, which both showed decapping activity in vitro [in vitro, including DP-CoA [The discovery of NudC (NUDIX nicotinamide pyrophosphohydrolase) as an enzyme that removes the NAD and AMP . Recentlated RNA . In eukain vitro . The actg DP-CoA , consistg DP-CoA .E. coli  . A numbeE. coli  suggests+ were detected in human cells [+ (up to 70%), where it is used for redox reactions and for signalling [+, NADH, FAD and DP-CoA on the light strand promoter  in vitro  RNA  RNA A – antitE.coli, the proportion of NAD+ capped RNAI found in stationary phase compared to exponential phase was two-fold higher [+/NADH balance plays key role in cellular redox homeostasis, capping could connect transcription directly to the cell's redox state. Given the affinity for NAD+ is roughly the same as for NADH (+ or NADH. The functioning of such signalling of course would depend on a mechanism recognising NADylated from NADHylated RNAs. UDP-Glucose and UDP-GlcNAc are the initial substrates for the cascade of reactions leading to the synthesis of cell wall components. It would be tempting to speculate that expression of some cell wall synthesising enzymes could be controlled directly by the pool of UDP-GlcNAc via capping of +1U transcripts. In general, being rare, RNA modification with cell wall precursors might provide a better regulatory potential, compared to ubiquitous capping with ADP analogs.The extent of capping could be responsive to the changes in cellular metabolism. For example, in d higher . Similard higher . Since tfor NADH B, changeCapping might potentially influence translation initiation on a leaderless mRNA.e.g. to the vicinity of the membrane in the case of UDP-GlcNAc capped RNA.Another potential cellular role of capping could be the targeting of a specific RNA species, via its cofactor cap, to a protein with affinity for the cognate cofactor, or to a specific subcellular location, Intriguingly, we showed that a number of rifampicin resistant RNAPs, including the most widespread clinical isolates, are deficient in capping . This de. More information is needed to put this type of RNA 5’ modification into the category of functional capping, rather than a side reaction of RNAPs. Currently it is hard to envisage a “classic” regulation by the stochastic process of capping. Nevertheless, this “unavoidable” side reaction has to be either used to some advantage or, alternatively, fought against. Both scenarios would have wide ranging cellular consequences with multiple regulatory mechanisms involved. It seems that RNAP has a limited ability to control capping process, apart from alternative sigma factors exchange. It is more feasible to regulate amounts of capped RNA post-transcriptionally, by the linked processes of decapping, alternative folding and RNA chaperons binding. Being a stochastic process, capping might generate variability in a clonal population, which can be exploited at a population level to benefit the organism in adaptation to rapid change in growth conditions. At present, an exact roles of various non-canonical caps in bacteria, eukaryotes and mitochondria are still to be established, as well as full repertoire of enzymes that process non-canonically capped RNAs are to be characterised.To conclude, despite recent progress, the understanding of non-canonical RNA capping by RNAPs is still patchy


Up to date, several chemically modified long non-coding transcripts have been identified and associated with different pathologies, including cancers. This review presents the current level of knowledge on the most studied cancer-related lncRNAs, such as the metastasis associated lung adenocarcinoma transcript 1 (MALAT1), the Hox transcript antisense intergenic RNA (HOTAIR), or the X-inactive specific transcript (XIST), as well as more recently discovered forms, and their potential roles in different types of cancer. Understanding how these RNA modifications occur, and the correlation between lncRNA changes in structure and function, may open up new therapeutic possibilities in cancer.In contrast to the amazing exponential growth in knowledge related to long non-coding RNAs (lncRNAs) involved in cell homeostasis or dysregulated pathological states, little is known so far about the links between the chemical modifications occurring in lncRNAs and their function. Generally, ncRNAs are post-transcriptional regulators of gene expression, but RNA modifications occurring in lncRNAs generate an additional layer of gene expression control. Chemical modifications that have been reported in correlation with lncRNAs include m Long non-coding RNAs (lncRNAs) have recently been recategorized from ‘junk’ non-coding material to master regulators of transcriptional, post-transcriptional, and translational levels of expression. LncRNAs are a category of non-coding RNAs with distinctive features and exhibit tissue specificity. These molecules display lengths between 200 nt and 100 kilobases (kb), and originate from the non-protein coding regions of the genome. LncRNAs resemble mRNA-like transcripts, since they possess a CAP structure, polyadenylated tail, and are transcribed by RNA polymerase II (RNA Pol II). However, these transcripts lack conserved open reading frames [To date, studies have shown that lncRNAs are involved in up- and down-regulation of gene expression at both transcriptional and post-transcriptional levels in all fundamental cellular processes—proliferation, differentiation, development, immunity, altered metabolism, and signaling, including in cancer states ,3. AlthoCancer is a dysregulated state, characterized by chaotic signaling and abnormal patterns of expression resulting from multiple genetic and epigenetic alterations, which have been frequently associated with ultra-conserved, non-coding sequences of the genome . These aRecent research and evolution of detection methods has led to the identification of several dynamic chemical modifications in RNAs that have been shown to modulate their activity, and therefore can be considered ‘switches’ ,9. NeverThe aim of this review is to explore the cancer-related lncRNA landscape, and to highlight to readers the interplay between the dynamic chemical modifications that may occur within lncRNAs’ structure, and the effect they may have on their function. Overall, these epitranscriptomic changes lead to modified lncRNAs that generate an additional layer of post-transcriptional control, having a great impact on human health and disease.6-methyladenosine (m6A), N1-methyladenosine (m1A), 5-methylcytosine (m5C), pseudouridine (Ψ), and adenosine to inosine transition (A-to-I) [5C, m6A, Ψ, CAP, splicing, editing, and U-tail. Next, the most common modifications will be further described in detail, showing their specific writers, erasers, and readers.Post-transcriptional modifications that occur in RNA molecules started being explored recently, giving rise to a new field of research called epitranscriptomics. Equivalent to epigenetics, which analyzes post-transcriptional molecular events occurring in DNA, epitranscriptomics investigates modifications resulting from all RNA processing events, such as RNA splicing, RNA editing, or methylation . Among t(A-to-I) ,13. Acco(A-to-I) , describ6-methyladenosine (m6A) is the most abundant and the first internal modification of messenger RNA [6A sites, adding up to between 0.1–0.4% modified sites in mRNA [6A sites are frequent around stop codons, in 3’-untranslated region (UTR), and in long exons [6A is mostly found: DRACH sequence  [6A modification was discovered in 2011, when the first RNA demethylase for m6A was identified [6A dynamics are modulated by the action of a few regulators organized in ‘writers’ (methyltransferases), ‘readers’ (binding proteins), and ‘erasers’ (demethylases) [Nnger RNA . This RNnger RNA ,17. One  in mRNA ,19,20. Tng exons ,22), and= A/C/U) . The reventified . The m6Ahylases) .6A modification is catalyzed by a complex of RNA methyltransferases (‘writers’) which includes one heterodimer, the methyltransferase-like 3 (METTL3)/METTL14, and other cofactors [The mofactors ,27. The ofactors ,28,29.6A writer complex is Wilms’ tumor 1-associating protein (WTAP), which binds both METTL3 and METTL14 and ensures their recruitment to nuclear speckles [6A methyltransferase associated (VIRMA) is also part of the complex and influences methylation around stop codons and in UTR. Depletion of KIAA1429 leads to a lower number of m6A sites in RNA [Another component of the mspeckles . WTAP alspeckles ,31. KIAAs in RNA ,33.6A sites [Among other adaptor proteins that interact with WTAP are RNA-binding motif protein 15 (RBM15) and its paralogue RBM15B ,32,34. TMETTL16 is a U6 small nuclear RNA (snRNA) methyltransferase with independent action from the METTL3 complex. METTL16 is responsible for methylation within the UACAGAGAA motif in U6 snRNA. METTL16 is also involved in S-adenosyl methionine (SAM) homeostasis control, by methylating inside an intron of methionine adenosyltransferase 2A (MATA2A) . Another6A binding proteins, also known as ‘readers’, are either direct readers or indirect readers. This distribution indicates the RNA-binding method [6A sites. The indirect readers are able to bind to RNA as a result of an RNA secondary structure relaxation induced by m6A modification [The mg method ,37. The fication ,39.6A, and affects their stability by inducing their degradation [6A-containing RNA [6A-containing lncRNAs [The proteins that belong to the YT521-B homology domain family (YTHDF) are direct readers with cytoplasmic (YTHDF1-3 and YTHDC2) or nuclear  distribution. While YTHDF1 regulates RNA translation , YTHDF2 radation . YTHDF3 ning RNA . YTHDFC1ning RNA  and also lncRNAs ,34.6A, which opposes poly-U hairpin RNA [Heterogeneous nuclear ribonucleoprotein A2/B1 (HRPNPA2B1) is another direct reader involved in miRNA processing . Heterogrpin RNA .6A modification can be reversed through the action of two m6A demethylases (‘erasers’) that belong to the Fe(II)/α-KG-dependent dioxygenase AlkB family: the mass- and obesity-associated protein (FTO) and α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) [6A eraser discovered [6 2’-O-dimethyladenosine (m6A m) [6A to N6-hydroxymethyladenosine (hm6A), and further to N6-formyladenosine (f6A) [6A levels in certain mRNA transcripts that further promote leukemogenesis [The m(ALKBH5) . Althougscovered , followi (m6A m) . Howeverne (f6A) . Demethyne (f6A)  and cap-ne (f6A) . Overexpogenesis .alkbh5 in mice affects mouse fertility and spermatogenesis, and in humans affects nascent mRNA synthesis and the rate of splicing [Knockdown of splicing . Hypoxiasplicing . Anothersplicing .6A, another important RNA modification is N1-methyladenosine (m1A) [1A is charged positively, which can affect the structure of RNA and the protein–RNA interactions. The role of m1A modification has not been completely elucidated, but it is suggested that it promotes protein production and a more efficient translation process [Although not as abundant as mne (m1A) . Initialne (m1A) ,53,54. I1A are tRNA m1A methyltransferases (MTase), and they are different for each specific m1A location [1A in rRNA [1A is catalyzed by tRNA methyltransferase 10 C, mitochondrial RNase P subunit (TRMT10C), and tRNA methyltransferase 61B (TRMT61B) [1A modification. MALAT-1, a lncRNA which is highly expressed in many types of cancer, can also suffer m1A modification in A8398 position [The writers for mlocation ,56,57. H in rRNA , and in TRMT61B) ,60. Usuaposition .1A sites are mostly the same as those for m6A modification: YTHDF1, YTHDF2, YTHDF3, and YTHDC1 [The readers for md YTHDC1 .1A modification is its reversibility, and there are 2 erasers identified that ensure m1A demethylation, ALKBH3 and ALKBH1 [An important aspect of md ALKBH1 ,64. Highd ALKBH1 ,66.5C) in mRNA, tRNA, and viral RNA, it has only recently been thoroughly characterized in RNA, as it was considered to be primarily a modification of DNA [5C is positioned about 100 nucleotides downstream of the translation initiation site in human and murine mRNAs, and it can also be found in the UTRs [Although early studies demonstrated the existence of 5-methylcytosine  family, with seven members, and DNA methyltransferase-2 (DNMT2), previously thought to methylate DNA. The substrate(s) for each member of NSUN family, as well as DNMT2, is mentioned in There are two groups of mNsun genes lead to different diseases, for example, alteration of Nsun7 in mice causes sperm motility problems, with various grades of infertility, while mutations in Nsun2 are associated with autosomal-recessive intellectual disability [Nsun2 by hypomethylation is present in human cancers, and is associated with metastatic progression in human breast cancer [DNMT2 have also been observed in human malignant cells [Mutations in sability ,81. Overt cancer ,83. Altent cells . More thnt cells .5C. This m5C binding protein promotes selective mRNA export from the nucleus, suggesting a potential role for m5C in RNA transport. The full functions of m5C remain to be discovered [A recent study suggested the Aly/REF export factor (ALYREF) as a reader for mscovered .5C to cytosine, m5C can be ‘erased’ and turned into 5-hydroxymethylcytosine (hm5C) by the ten-eleven family demethylases (TET) that also direct DNA demethylation [While there is no known protein that can fully determine the regression of mhylation ,88.1 and enables it to form additional hydrogen bonds, leading to a more rigid sugar–phosphate backbone and enhanced folding. Although ψ binds to adenosine in the same manner as uridine, its interaction with the other bases is stronger [Also called ‘the fifth nucleotide’ due to its high abundance, 5-ribosyluracil or pseudouridine (ψ) was discovered in 1951 and is the most predominant RNA modification, found in all types of RNA from mRNA to ncRNAs, such as rRNA, tRNA, snRNA, snoRNA, and lncRNAs ,90. It istronger ,92.According to recent studies, pseudouridylation can also affect mRNA’s coding potential. In yeast, the presence of ψ in stop codons suppressed translation termination by guiding the incorporation of new amino acids ,94. The The enzymes that catalyze the conversion of uridine to ψ, the so called ‘writers’, are pseudouridine synthases (PUSs). Their action can be conditioned or not by the presence of RNA, resulting into two categories: RNA-dependent and RNA-independent. The RNA-dependent PUSs associate with cofactors and the H/ACA box of snoRNAs, forming a complex that interacts with the RNA target in a site-specific manner based on sequence complementarity, e.g., H/ACA ribonucleoprotein complex subunit 4 (dyskerin) . This meUnfortunately, specific ‘readers’ and ‘erasers’ remain unknown. The absence of an eraser protein could be explained by the fact that the C–C bond formed between the base and the ribose in ψ is much more inert than the C–N bond in uridine, possibly making pseudouridylation irreversible .To date, the major technology used for the identification of dynamic RNA modifications relies on sequencing. However, there are some important downfalls of this technology. Next, we will present major sequencing variants developed for detection of each chemical modification, and their pro and cons. Last but not least, computational tools that identify modification sites from sequencing data are still under development.6A can be achieved with m6A-seq or methylated RNA immunoprecipitation sequencing (MeRIP-seq), two separate methods based on m6A-specific methylated immunoprecipitation, followed by NGS. After isolation, poly(A)-enriched RNA is fragmented to 100–150 nt and immunoprecipitated with m6A-specific antibodies. Libraries are constructed from the fragments that contain m6A, taking as controls non-immunoprecipitated samples, and subjected to high-throughput-sequencing. Although easily manageable, these methods have two significant disadvantages: slightly low resolution (100–200 nt) and antibody specificity , making identification of specific m6A sites at single nucleotide level difficult [A transcriptome-wide profile of mifficult ,102.6A sequencing (PA-m6A-seq) was developed in order to overcome some of the difficulties presented by m6A-seq and MeRIP-seq [6A at single-base resolution due to the incorporation of photoactivatable ribonucleoside 4-thiouridine (4-SU) in RNA. After m6A immunoprecipitation, the antibody is crosslinked to 4-SU under 365 nm UV light and the crosslinked RNA is fragmented by RNase T1 to about 30 nt, after which sequencing commences. During reverse transcription-polymerase chain reaction (RT-PCR), at the site of crosslinking, 4-SU determines a U/T to C transition, resulting in better signal-to-noise ratio of methylation detection. However, not all m6A modifications have a nearby site for 4-SU incorporation, and some m6A sites may be missed [Photo-crosslinking-assisted meRIP-seq . This ste missed .6A detection, it can also be used for identification of m5C and ψ [The methylation status of a single modified nucleotide can be quantified with site-specific cleavage and radioactive-labelling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET) . This me5C and ψ . In SCAR5C and ψ .1A-seq and m1A-ID-seq) have been used to carefully map m1A modification in the eukaryotic transcriptome [6A detection methods (m1A-seq is an adapted version of MeRIP-seq), as they also rely on the combination of immunoprecipitation and high-throughput-sequencing. Both of them have succeeded in generating high resolution maps, due to different strategies. While in m1A-seq an m1A-to-m6A rearrangement occurs under alkaline conditions, turning RT-interfering m1A to RT-silent m6A, in m1A-ID-seq an RNA/DNA demethylase reverts the modified base to regular A after immunoprecipitation. Peaks of m1A are detected by comparing the NGS reads of the demethylase treated and untreated fragments [Two methods  detection at single nucleotide resolution through bisulfite conversion, also called bisulfite sequencing (bsRNA-seq), (b) 5-azacytidine-mediated RNA immunoprecipitation (Aza-IP), and c) methylation individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) [For the detection of widespread m(miCLIP) .5C remains unchanged. This difference is detected by Sanger sequencing or by next-generation sequencing (NGS) after library preparation [5C in those regions. The same goes for other modifications of cytosine other than m5C, which will most likely be resistant to the treatment as well and misidentified as m5C sites [5C-specific antibody or a control antibody prior to sodium bisulfite treatment , followed by library construction and NGS [Bisulfite treatment determines the conversion of unmodified cytidine to uridine (read as thymidine during sequencing), while mparation ,106. How5C sites . These d and NGS .5C-RMTs. The RNA targets are isolated and identified by affinity purification of the tagged RMTs, partial RNase digestion, and RNA sequencing, with the resulting fragments being used for the construction of a cDNA library and finally NGS. During sequencing, modified cytidine residues are read as guanidine instead of normal cytidine. This facilitates the clear identification of the modified nucleotides. Direct targets of DNMT2 and NSUN2 could be identified using this technique [The second method, Aza-IP, relies on random incorporation of the anti-cancer drug 5-azacytosine into the nascent RNA of cells that overexpress affinity-tagged RNA methyltransferases (RMTs), followed by immunoprecipitation of those tagged enzymes and NGS. The incorporated 5-azacytidine acts as a suicide substrate because of the covalent bond that is formed between it and mechnique ,108.5C mapping with NGS commenced. During library preparation, reverse transcription terminated at the cross-link site of the cytosine with the modified protein, resulting in a high cytosine appearance in position +1 in cDNA libraries (the first nucleotide to be read), allowing for m5C detection at single nucleotide resolution [6A residues [The third method, called miCLIP, is a derivative of individual-nucleotide-resolution cross-linking and immunoprecipitation (iCLIP) method, and it relies on the use of an overexpressed affinity-tagged mutant RMT. This approach has been used before to asses NSUN2’s specific sites of methylation . In thissolution ,111. Usisolution . The miCresidues .As an isomer of uridine, mapping ψ in a site-specific quantitative manner can be rather challenging. Nevertheless, there are some methods we can employ in order to detect ψ, some more successful than others. One method consists of labelling ψ with N-cyclohexyl-N9-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulphonate (CMCT), followed by alkaline hydrolysis which removes adducts formed with U or G, leaving behind only CMCT-ψ. This CMCT-ψ terminates reverse transcription one nucleotide downstream of the pseudouridylated positions, resulting in short cDNA fragments that are used for library preparation. NGS is performed for cDNA libraries constructed with or without CMCT treatment, and ψ is mapped by calculating the differences between the stop rates of the two samples ,114,115.Increased detection sensitivity was achieved with the adaptation of this approach developed by Li et al. . They naThere are some other strategies employed apart from these sequencing-based methods, for example SCARLET, a good validating method for base modifications and determination of the stoichiometry ,104. AnoFrom the methodological point of view we are just starting to comprehend the need to increase the resolution and sensitivity of these methods. High-throughput sequencing techniques generate huge amounts of data that need further validation with new approaches. Combinations of third-generation sequencing, new chromatography methods, and new mass spectrometry approaches will improve studies in the field of epitranscriptomics.There are several lncRNAs involved in different types of cancers, which simultaneously acquire one or more dynamic modifications within their structures . Among tThe X-inactive specific transcript (XIST) is a lncRNA (17–20 kb) involved in the permanent inactivation of one of the two X chromosomes, an early developmental process in mammalian females. XIST is expressed from a specific region termed the X inactivation center (XIC), and only from the future inactive X chromosome. Once transcribed, XIST is not translated, instead accumulating in the nucleus where it coats the inactive X chromosome in cis. This transcriptional silencing mechanism provides dosage equivalence between males and females ,121,122.5C within repeat 8 of this region, using a protocol they developed based on bisulfite treatment and PCR amplification of poly(A)-enriched RNA. The analysis revealed five methylated cytosines in positions 701–703, 711, and 712 for 19–24% of the amplicons, while simultaneous methylation of all five residues was found in 19% of the sequences [In order for X inactivation to take place, a region composed of 8.5 repeats (R1–R8.5) with 26 nt per full repeat from the 5’-end of XIST, called the repeat A-region, associates with the polycomb-repressive complex 2 (PRC2), a chromatin-associated protein complex . Amort eequences . Howeverequences .Another chemical modification, specifically a ψ residue in U11249, was discovered by Li et al., but the impact of this structural change on the function of XIST is currently unknown .6A residues, and this methylation promotes XIST-mediated transcriptional repression [6A in adjacent consensus motifs relies on the interaction between m6A-methylation complex and two RNA-binding proteins, RBM15 and its paralogue RBM15B, that bind the complex to XIST. The methylated sites are recognized by YTH domain containing 1 (YTHDC1) protein and gene silencing occurs, although it is not clear how. Knockdown of RBM15, RBM15B, or methyltransferase METTL3 inhibits X-mediated gene silencing [It has been reported that, in human cells, XIST is methylated with no less than 78 mpression . The forilencing .X-inactive specific transcript is one of the best studied lncRNAs, and as such it has been searched for and found in many different human neoplasias. Its expression can either be upregulated or downregulated, acting as an oncogene or as a suppressor in multiple types of cancer. Overexpression of XIST is associated with advanced tumor stage, lymph node or distant metastasis, and overall poor prognosis in human cancers .In breast cancer, XIST acts as a tumor suppressor by positively regulating the expression of non-X-chromosome gene PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1), which in turn catalyzes dephosphorylation of protein kinase B (AKT) . In non-The complex roles of XIST in human carcinoma have been recently reviewed by Yang et al. .One of the most studied lncRNAs, MALAT1 is involved in several dysregulations found in cancer and is responsible for coordination of alternative splicing.This transcript results from a precursor containing a tRNA-like small ncRNA, known as MALAT1-associated small cytoplasmic RNA (mascRNA) after cleavage by RNAse P . It is aLittle is known so far about this abundant lncRNA’s molecular mechanism of action. MALAT1 has been shown to act either as a sponge or circular endogenous RNA (ceRNA) for miR-195  or as a Functionally, MALAT1 is involved in several cellular processes, such as alternative splicing and transcriptional regulation . Being lMALAT1 originates from a gene on chromosome 11 that is transcribed by RNA Pol II. In turn, MALAT1 controls transcription of several genes. By interacting with a member of PRC2 complex- Enhancer of zeste homolog 2 (EZH2), MALAT1 is actively involved in the methylation of histone H3 in lysine 27 (H3K27) and inhibition of tumor suppressor genes, therefore contributing to loss of proliferation control, cell migration, invasiveness, and pro-metastatic programs ,143,144.6A modification in position 2515/2577 of the triple helix at the 3’end in MALAT1 is catalyzed by the nuclear METTL16. This interaction with the m6A writer has been proven both in vitro and in vivo [6A methylation is a reversible modification [6A is able to modulate accessibility and interaction with heterogeneous nuclear ribonucleoprotein G (HPRNPG) [Several chemical modifications are evidenced in MALAT1. An m in vivo ,139, and in vivo ,20. Thisfication . For ins(HPRNPG)  or with 1A8398, previously described by Gutschner et al. [5C possible methylation sites [Additionally, another methylation occurs in MALAT1 in mr et al. . This chr et al. . By meanon sites . In addion sites ,95,96. TA recent study from Amodio et al.  places MBy regulating gene expression and coordinating splicing, MALAT1 is practically involved in cell cycle and proliferation dysregulation, as well as cell migration and metastasis in several types of cancer ,154,155.Considering its implications in cell proliferation, and generally in cancer progression, MALAT1 is a potential therapeutic target for cancer treatment ,157,158.HOX antisense intergenic RNA (HOTAIR) lncRNA is 2.2 kb long and results from the antisense transcription of the HOXC gene cluster . HOTAIR In the vicinity of the LSD1-binding site of HOTAIR, a methylated C was identified at position 1683 in two different cell types in HEK293 and NT2 cell lines. Methylation of C1683 was found to be invariable, regardless of the different levels of HOTAIR expression and the different types of cancer cells, such as Hs578T and BT-20 breast cancer and HOC7 ovarian cancer cell lines. Since methylated C1683 is found in the vicinity of the LSD1-binding site, it has been suggested that it affects the HOTAIR interaction with LSD1 complex .6A, the studies conducted so far did not identify an m6A site consistent in all cell types investigated. In HOTAIR from HEK293T cells, a single m6A peak region near the 5’-end region was identified, but in HepG2 cells and human brain tissue, there was no m6A signal detected in HOTAIR [Concerning other types of RNA modifications, in particular mn HOTAIR ,102.HOX antisense intergenic RNA is a lncRNA with numerous roles in cancer development. Altered expression of HOTAIR is found in many types of cancer, and promotes metastasis and tumor invasiveness through epigenetic gene silencing ,170,171.High levels of HOTAIR correlated with metastasis and poor prognosis have been found in lung cancer , hepatocEvidence supporting HOTAIR’s role in mediating drug resistance has emerged for many types of cancer investigated. Elevated HOTAIR expression was found in samples from drug-resistant patients with NSCLC . SimilarApart from the three main lncRNAs discussed in detail so far, other lncRNAs with roles in cancer development are under extended investigation to determine their role and whether or not they also carry RNA modifications . A correSome studies have carefully mapped ψ sites in lncRNA . These lSmall nucleolar RNA host gene (SNHG) 1 and 7 each have one modified uridine residue, SNHG1 in position 1766 and SNHG7 in position 292 ,96. WhilDICER1 Antisense RNA 1 (DICER1-AS1), another lncRNA with a ψ site (U463), is upregulated in osteosarcoma cells, promoting their proliferation, invasion, and autophagy via miR-30b/ATG5 .5C [Telomerase RNA component (TERC) has two modified uridines in its structure as well as three m5C ,117. Bae5C .5C modifications in antisense non-coding RNA in the INK4 locus (ANRIL), growth arrest-specific transcript 5 (GAS5), nuclear paraspeckle assembly transcript 1 (NEAT1), Pvt1 oncogene non-protein coding (PVT1), ribonuclease P RNA component H1 (RPPH1), SNHG12, and TERC [Squires et al. discovered several mand TERC . At the 5C positions [ANRIL has two sites for cytosine methylation  and is iositions , and it ositions . Also imositions .5C positions in its structure [SNHG12 is involved in both gastric and colorectal cancer, with two currently known mtructure ,195,208.5C sites have been found for NEAT1 [5C positions have been detected in its structure [NEAT1 can either be upregulated or downregulated, with aberrant overexpression in pulmonary, oesophageal, colorectal, and hepatocellular carcinoma, and low expression in acute promyelocytic leukaemia (APL), both associated with poor prognosis . Severalor NEAT1 . For PVTtructure . High letructure .Despite the current remarkable evolution of the epitranscriptomics and lncRNA fields of research, little is known about the functional roles of lncRNAs in cancer, or their complete mechanisms of action. In order to advance personalized cancer therapies or pharmacological studies targeting lncRNAs or the signaling pathways they govern, it would be crucial to elucidate the interplay between the dynamics of chemical modifications occurring in lncRNAs, the consequences upon lncRNAs or their targets’ function, and modulation of cancer features in response to these changes. Consequently, a correlation between chemical modifications in lncRNAs and possible cancer implications is further presented.6A interferes with pluripotency and cellular differentiation, both of which are associated with cancer progression. Modification of m6A is described as a potential target for treatment of human cancers, since it has implications in metabolism, stem cell self-renewal, and metastasis. As a result, modulation of metabolism in tumors would be reflected in the regulation of m6A. [m of m6A. .5C can be found in multiple lncRNAs which also play a role in malignant processes. These lncRNAs can be either upregulated  or downregulated  in order to promote cell proliferation and migration, and prevent apoptosis of cancerous cells [mus cells ,206,209.us cells ,209.The connection between ψ and cancer was first reported in studies conducted on urinary metabolites from patients suffering from this disease ,213. BeiMutations in PUSs lead to impaired activity of said enzymes, which has been recognized as a potential trigger for cancer in both inherited and sporadic tumors. In breast carcinomas, for example, low dyskerin expression, correlated with reduced rRNA pseudouridylation, promotes neoplastic transformations by suppressing translation of mRNA molecules that code for tumor suppressors. The reason behind this is the localization of the pseudouridylation sites for dyskerin in the ribosome; they are located in specific domains important for tRNA and mRNA binding ,217. In 6A. For instance, in breast cancer some erasers and writers seem to be related to hypoxic condition (ALKBH5), and respectively to a shorter survival of patients as a consequence of a lower expression (METTL14). In hepatocellular carcinoma tissue, a significant low expression of METTL14 has been found and correlated with metastasis inhibition [Deregulations have also been found at the level of readers, erasers, and writers of already described chemical modifications, particularly for mhibition .6A writers known to have a potential role in tumor course are METTL14 and METTL3, for which the experimental knocking-down expression in cellular models results in tumor cell growth. Moreover, the protein eraser ALKBH5 seems to display a high expression in glioblastoma and sustains tumor cells growth via fork head box M1, thus ALKBH5 deregulation is linked to a poor outcome [6A writers–readers–erasers expression in order to prevent tumorigenesis activation and progression [In glioblastoma tumor, the key m outcome . It has gression .6A modifications are now deeply explored for altering m6A chemical profiles in a drug development context. Meclofenamic acid is such a potential drug example as it is a very selective inhibitor for a certain eraser (FTO), but, although promising, such drugs are not specific for multiple cancers, because of cancer heterogeneity [These myriads of ‘switch’ deregulations contribute to setting a therapeutic stage as inhibitors of mogeneity ,225.6A and several pseudouridine modifications, has been reported as a master regulator of metastasis and a potential therapeutic target. MALAT1 has been found to control proliferation, migration, and apoptosis in many different human cancers, and its overexpression has also been correlated with drug resistance. Specific cytosine methylation was found in HOTAIR, which promotes metastasis in several cancer types, but no pseudouridylation was yet reported for this lncRNA. XIST includes all three types of chemical modifications—m6A, m5C, and ψ— and can act either as an oncogene or as a suppressor in multiple types of cancer. Overexpression of XIST is associated with advanced tumor stage and overall poor prognosis in human cancers. The molecular mechanisms triggered by chemical modifications in lncRNAs that lead to changes in their function are not yet fully understood, and need further molecular characterization to complete another ‘piece of the puzzle’ in the interplay between lncRNAs and cancer.This review integrates lncRNAs and their function into the cancer development landscape. Once considered ‘junk’ non-coding material, lncRNAs prove to be key regulators of gene expression and molecular events during transcription, and modulators of signaling pathways and post-transcriptional regulation guided by miRNAs. Therefore, lncRNAs have progressed rapidly to a privileged position of control—a second layer of post-transcriptional regulation. Chemical modifications in lncRNAs generally induce a dysregulated state, and convert altered lncRNAs to transcripts potentially critical for cancer progression. In this context, MALAT1, which carries an m


Small RNAs and their associated RNA interference (RNAi) pathways underpin diverse mechanisms of gene regulation and genome defense across all three kingdoms of life and are integral to virus–host interactions. In plants, fungi and many animals, an ancestral RNAi pathway exists as a host defense mechanism whereby viral double‐stranded RNA is processed to small RNAs that enable recognition and degradation of the virus. While this antiviral RNAi pathway is not generally thought to be present in mammals, other RNAi mechanisms can influence infection through both viral‐ and host‐derived small RNAs. Furthermore, a burgeoning body of data suggests that small RNAs in mammals can function in a non‐cell autonomous manner to play various roles in cell‐to‐cell communication and disease through their transport in extracellular vesicles. While vesicular small RNAs have not been proposed as an antiviral defense pathway per se, there is increasing evidence that the export of host‐ or viral‐derived RNAs from infected cells can influence various aspects of the infection process. This review discusses the current knowledge of extracellular RNA functions in viral infection and the technical challenges surrounding this field of research.Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAsRNA in Disease and Development > RNA in DiseaseRegulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of ActionThis article is categorized under: Small RNAs usually operate by guiding an Argonaute (AGO) protein to another nucleic acid target through Watson–Crick base pair complementarity  miRNAs regulate nearly every signaling pathway inside the cells and they work in concert with transcription factors to control initiation, maintenance, and resolution of cell response to injury and stress   polymerase of mammalian poxviruses polyadenylates host miRNAs resulting in their degradation . At the same time, emerging data suggest viral infections also impact the secretion of miRNAs from infected cells, and this can include both viral‐ and host‐derived miRNAs. In this review, we consider the extracellular realm when thinking about the role of miRNAs and other small RNAs in viral infection.3It has long been known that various RNA species exist outside of cells in different body fluids, and can be released from cells and internalized by other cells under physiological conditions  are 100–1,000 nm vesicles released by budding from the plasma membrane, while exosomes are small vesicles (~100 nm) of endosomal origin which are released by fusion of multivesicular endosomes (MVEs) with the plasma membrane  cells and this miRNA trafficking has been proposed to facilitate infection through the regulation of immune responses . The authors showed proof‐of‐concept with reporter assays that EVs transfer viral miRNAs from infected cells to other cells and demonstrated the immune‐stimulatory gene 5.1.2Small RNAs derived from the HIV genome have been reported both in intra‐ and extra‐cellular contexts , which is found in EVs derived from human placental trophoblasts ‐infected patients. Transfer of this miRNA to primary alveolar cells leads to the downregulation of the antiviral factor MX1, thereby potentiating IAV infection  , transfer RNA (tRNA) YRNAs, 7S RNA, and Vault RNAs are generally more abundant in EVs than individual miRNAs and their levels in EVs are enriched compared to intracellularly  are either depleted during library preparation or discarded in small RNA‐seq analysis (Kopylova, Noé, & Touzet, Further work on the functions of rRNA and tRNA fragments inside the cells will help create a framework for why and how specific sequences are exported and whether this could be used by viruses or the host defense system. Assuming the export of certain small RNA species from a cell has an important functional role, it is expected that this process would be controlled, and perhaps targeted, by the virus. There is still little understanding on small RNA export and uptake mechanisms but this is an area of active investigation.8Extracellular RNA is found outside of cells either protected in EVs or bound to proteins including AGO or high density lipoproteins (Fritz et al., 9Just as there is a variety of export vehicles for extracellular RNA that includes EVs, RBPs and lipoproteins, it is expected that there are different routes to cross the plasma membrane Figure . HoweverAfter target cell binding, internalization of EV cargo can be achieved by direct fusion with the plasma membrane (Del Conde, Shrimpton, Thiagarajan, & López, 10To date the above example is one of only a few reports providing information on the subcellular localization of EVs following internalization in recipient cells. Advances in single molecular imaging technologies and EV labeling strategies will continue to be important to advance the field, as well as studies under physiologically relevant conditions. Novel mouse transgenic approaches have recently provided compelling evidence for transfer of RNA between via EVs in vivo. The Cre‐loxP transgenic mouse system was used by Stefan Momma's group to show direct transfer of EVs containing Cre mRNA from the blood to the brain in response to inflammation (Ridder et al., 11A key hurdle in the field is purifying sufficient quantities and homogenous populations of EVs for functional studies. Many strategies are now focused on this aspect, which is reviewed further in Konoshenko et al.  In partiAnother major issue in EV purifications is contaminants from the culture media in the case of in vitro studies (Tosar et al., 12In summary, the field of extracellular RNA offers many new exciting possibilities for how cells communicate with one another, and how viruses have evolved to exploit or modulate these mechanisms during their lifecycles. However, there are many technical challenges and unknowns that come with the youth of this field Box . SequencHowever, many technical challenges exist that need to be addressed to move the field forward. In particular, the ability to study RNA export, including selectivity mechanisms, requires isolation of pure populations of extracellular RNA. We are still learning what those populations are and they include both vesicular and nonvesicular complexes. In the case of vesicular RNAs, we know the EVs themselves are heterogenous: cells produce multiple EV subsets whose cargo and functions can differ (Kowal et al., The authors have declared no conflicts of interest for this article.RNA in extracellular vesicles


Tetrastichus brontispae (TbRV-1) was determined to be 12,239 nucleotides in length with five non-overlapping, linearly arranged coding sequences (CDS), potentially encoding nucleoproteins, hypothetical proteins, matrix proteins, glycoproteins, and RNA-dependent RNA polymerases. Sequence analysis indicated that the RNA-dependent RNA polymerase of TbRV-1 shares a 65% nucleotide and 67% amino acid sequence identity with Hubei dimarhabdovirus 2, suggesting that TbRV-1 is a member of the dimarhabdovirus supergroup. This corresponded to the result of the phylogenetic analysis. The affiliation of TbRV-1 with members of the family Rhabdoviridae was further validated by similar transcription termination motifs (GGAACUUUUUUU) to the Drosophila sigmavirus. The prevalence of TbRV-1 in all tissues suggested that the virus was constitutive of, and not specific to, any wasp tissue. To our knowledge, this is the first report on the complete genome sequence of a dimarhabdovirus in parasitoids.The complete sequence of a novel RNA virus isolated from  Vesiculovirus, Cytorhabdovirus, Nucleorhabdovirus, Sigmavirus, Ephemerovirus and the superfamily dimarhabdovirus [Rhabdoviridae, a family of viruses in the Mononegavirales order, are negative-sense (–) single-stranded RNA viruses. Most of them infect plants and animals  and are categorized into arthropod-vectored viruses of plants or vertebrates, and vertebrate- and arthropod-specific viruses on the basis of the host range . Based obdovirus , where tbdovirus .2, such as the effect of sigmavirus on Drosophila [Among the arthropod-specific viruses, one group of predominantly insect-associated viruses is a sister group to the large clade of plant viruses, while the other group is the sigma virus clade, and are derived from the vector-borne dimarhabdoviruses . Dimarhaosophila ,8. Up toPteromalus puparum negative-strand RNA virus 1 (PpNSRV-1) reduced female offspring numbers, while it increased the longevity of the wasp P. puparum [Diachasminorpha longicaudata rhabdovirus (DlRhV) was introduced into the host Anastrepha suspense upon parasitism, and no indispensable role of DlRhV in successful parasitism was reported [Parasitoid wasps as vital biocontrol agents against pests are widely used in agro-ecosystems. In most cases, the higher efficiency of the parasitism ratio of Icheneumonidae and Braconidae wasps results in the presentation of viruses or virus-like particles detected in parasitoid wasps, such as the polydnavirus ,10,11,12 puparum . For thereported ,15. In aTetrastichus brontispae, a type of endoparasitoid wasp, is an efficient bio-control agent against several invasive leaf beetle species, such as Octodonta nipae and Brontispa longissima [T. brontispae to escape host encapsulation in O. nipae pupae [T. brontispae were detected by next generation sequencing (NGS).ngissima ,17,18,19ae pupae . The rolae pupae ,22. HencT. brontispae, named T. brontispae RNA virus 1 (TbRV-1) was reported. The TbRV-1 was somewhat similar to Hubei dimarhabdovirus, a novel virus discovered in Odonata by transcriptome and assigned to the dimarhabdovirus superfamily by bioinformatics [In this paper, the detection of virus types was performed by metatranscriptional analysis, and the complete genome of a novel dimarhabdovirus from ormatics .This stuT. brontispae, were reared on Octodonta nipae or Brontispa longissima (Coleoptera: Chrysomelidae) on the campus of Fujian Agriculture and Forestry University for nearly four years, after being collected from the Chinese Academy of Tropical Agricultural Sciences in 2012. The wasps, T. brontispae was sequenced as described by Tang et al. (2014) [The transcriptome of . (2014) . The totTM 2500 platform using the single-end/paired-end technology in a single run at Genedenovo .The complementary DNA (cDNA) library was constructed with NEBNext® Ultra RNA Library Prep Kit . Poly (A) mRNA was isolated using oligo-dT beads . All mRNA was broken into short fragments (200 nt) by adding a fragmentation buffer. First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by the synthesis of the second-strand cDNA using RNase H and DNA polymerase I. The cDNA fragments were purified using a QIAquick PCR extraction kit. These purified fragments were then washed with EB buffer for the addition of end reparation poly (A), and then they were ligated to sequencing adapters. Following agarose gel electrophoresis and the extraction of cDNA from gels, the cDNA fragments (200 ± 25 bp) were purified and enriched by PCR to construct the final cDNA library. The cDNA library was qualified and quantified with an Agilent 2100 Bioanalyzer and an ABI StepOnePlus Real-time PCR system, respectively, and then sequenced for 125 bp on the Illumina HiSeq E-value cut-off of 1 × 10−5 against the NCBI non-redundant (NR) database were chosen for further analysis because of the scarce reports regarding the Rhabdoviridae in Hymenoptera. For the reads derived from RNA-Seq, the clean data was obtained by filtering out unknown nucleotides larger than 5% and low quality reads and used for assembly with Trinity . Those aTM RACE PCR kit . The specific primers for amplifying the whole genome of the putative rhabdovirus were designed according to the obtained sequences -phosphoprotein (P)-matrix protein (M)-glycoprotein (G)-RNA-dependent RNA polymerase (L) . Based oequences .Escherichia coli T1. The final sequences were assembled using the DNAMAN 7 program . The full length of the coding sequence was further validated after sequencing the products of the routine PCR acquired with a fastfly pfu enzyme (TransGen Biotech).The PCR products were sequenced by Invitrogen  after ligation into the T1 vector  and transformation into https://www.ncbi.nlm.nih.gov/orffinder/). ORFs were analyzed on InterPro Scan [The complete genome of TbRV-1 was aligned to the known viruses by Blastx and Blastn to identify its closest relatives. The putative open reading frames (ORFs) were predicted by the ORF finder  were enriched from the total RNA of T. brontispae by polyacrylamide gel electrophoresis. After the ligation of 3’ and 5’ adapters, the ligation products were reverse-transcribed by PCR amplification, where the PCR products were enriched to generate a cDNA library and sequenced using IlluminaHiseqTM 2500 by Gene Denovo .The small RNA library was constructed as follows. Seventy individuals of Q-value ≤ 20) or unknown nucleotides. The clean reads were de novo assembled by Velvet with a k-mer of 17 [E value of 10−5.Before further analysis, the clean tags were retrieved by trimming the adaptors after removing low quality reads containing more than one low quality base . Then, the RNA was visualized by 1% agarose gel electrophoresis.The virus pellet was isolated following the procedure described by Suzuki and Tanake (2005) , with soTM RACE cDNA amplification kit . A pair of primers (MS and MA), amplifying 1541 bp, was used for PCR amplification. The PCR was run with fastfly pfu enzyme with the following conditions: 30 s at 94 °C, 30 s at 49.8 °C, and 45 s at 72 °C for 35 cycles. The PCR reaction, with distilled water as the template, was used as a negative control. The PCR results were visualized by agarose gel electrophoresis.To detect if there was TbRV-1 in the mixture, the RNA extracted above was reverse-transcribed to cDNA using the SMARTerRdRP, which codes the protein required for transcription, plays a role in genome replication, and is the most conserved sequence domain among RNA viruses [The main parasitism factors of endoparasitoids are located in the ovaries and venom apparatus, while the gut appears to be the main barrier that must be circumvented for transmission ,33. Ther viruses .2, 104, 105, 106, and 107 with distilled water for the absolute standard curve construction. Three biological and two technical repeats were performed. The primers for this study are shown in p < 0.05).For quantification, the total RNA  extracted from the above tissues was subjected to the Thermo Scientific Verso cDNA kit  to synthesize the cDNA as a template, where the RT enhancers were used to remove the contaminating genomic DNA. The abundance of RdRP gene copies (copies/μL) among tissues was measured with real time quantitative PCR (qPCR) using ABI 7500 after constructing the absolute standard curve. Before qPCR, a recombinant plasmid named pEASY-Blunt-RdRP was constructed by inserting the purified PCR product of RdRP to the pEASY-Blunt cloning vector . Then, the recombinant plasmid pEASY-Blunt-RdRP was digested with the endonuclease Sal I . The digested plasmid was diluted to 10The Tetrastichus brontispae RNA virus 1 (TbRV-1), Tetrastichus brontispae RNA virus 2 (TbRV-2), Tetrastichus brontispae RNA virus 3 (TbRV-3), Tetrastichus brontispae RNA virus 4 (TbRV-4), and Tetrastichus brontispae RNA virus 5 (TbRV-5) were identified through transcriptome mining . The Bla7 of the Sigmavirus from Drosophila [Sigmavirus  were observed. These conserved Ti and TTP typically flanked the structural genes and made the genes independently transcribed [A sequence of 12,239 nts (nucleotides) flanking 98 nt from the 5’ leader and 112 nt from the 3’ trailer was assembled for TbRV-1 (GenBank accession number: MH643740), where A+T pairs comprised 69.5% of the nucleotides. By comparing the 5’/3’untranslated regions (5’ UTR and 3’ UTR) and intergenic regions of the TbRV-1 genomes, the putative transcription termination motifs (TTP: GGAACUUUUUUU) similar to the TTP motif G(U)osophila ,35 and tnscribed ,36.TbRV-1 has five putative ORFs (ORF I–V), located at nt positions 113–1393 (N), 1689–2600 (H), 2699–3355 (M), 3761–5410 (G), and 5781-12141 (L). These five ORFs, except for ORF II, have a conserved domain and are aligned to the corresponding proteins of Hubei dimarhabdovirus 2 or the Wuhan insect virus 7 by Blastp, rather than Blastn . AccordiAfter a thorough Blastn analysis against the genome of TbRV-1, three corresponding small RNA sequences were detected. The corresponding coverages of these three sequences were 3122–3163, 7790–7831, and 11037–12078, with 100% identity. In detail, the first two sequences were aligned to ORF III (putative Matrix protein), and the last two sequences were aligned to ORF V (putative RdRP Protein). The sequence of the three unigenes and the blast results are shown below .After filtration, RNA extraction with the Qiagen kit was mainly a mixture of virus RNA. After agarose gel electrophoresis, a smear was observed in the respective lane when the extracted RNA was loaded into the gel A. For PCy = −0.282x + 10.03, R2 = 0.9994, where y is the number after the log function of copies/ng of the genes, and x represents the corresponding ct. The gene copies of TbRV-1 were the two-folded reduction of the calculated values, as double-stranded plasmid was used for standard curves. The amplification efficiency in this range was 91.02%, the respective limitations of quantification of the RdRP gene copies were 18.9–1.89 × 106, and their corresponding ct values were 13.43–31.00. The quantitative analysis did not detect any significant differences in the gene copies of RdRP among the tissues  . The equation is = 0.225) .e = 1 × 10−81) in the amino acid sequence of RdRp to DlRhV, another rhabdovirus from parasitoids TbRV-1 was observed. In all, TbRV-1 is a novel virus, different from the previously reported DlRhV.The phylogenetic analysis-based RdRP showed that the TbRV-1 was clustered into a clade with two dimarhabdoviruses: Hubei dimarhabdovirus 2  and Wuhan insect virus 7  at high bootstrap values  by invertebrates . In accoSigmavirus clade are observed where the genome of TbRV-1 lacked a sigmaX gene, which is characteristic of Sigmavirus. Nonetheless, it cannot be inferred from this that TbRV-1 is classified as a novel species or genus because the sequence comparisons alone cannot be the only source in developing the classification schemes of viruses at this time, [The evolutionary challenge faced by RNA viruses is to maximize functional diversity with several constraints concerning the genome size. For dimarhabdoviruses, the evolution of genomic plasticity has been associated with that of alternative, overlapping and consecutive ORFs within the major structural protein genes as well as the insertion and loss of additional ORFs in each gene junction in a clade-specific manner . In thisis time, . Meanwhiis time, .Dinocampus coccinellae paralysis virus (DcPV), can induce changes in the lady beetle (Coleomegilla maculate), such as tremors, gait disturbance, and limitations in movement during parasitism [P. puparum negative-strand RNA virus 1 (PpNSRV-1), a novel member of Nyamiviridae, impaired several fitness parameters of the wasp, such as adult longevity and the offspring sex ratio but also affected parasitism [D. longicaudata parasitoids, and no role of DlRhV in parasitism was ascertained [Drosophila or the mosquito, with rhabdovirus to CO2 and its negative effects, such as the reduced longevity [In general, viruses or virus-like particles (VLPs) in parasitoid wasps (Hymenoptera: Apocrita), such as the well-known polydnaviruses ,10,45, Vrasitism . The P. rasitism . For rhaertained ,15. In tongevity . The exp


Populus species, a genus noteworthy for its remarkably small number of RNA editing sites compared to other angiosperms. 27 of these transcripts were subject to RNA editing in at least one species. In total, 355 RNA editing sites were identified with high confidence, their editing extents ranging from 10 to 100%. The most heavily edited transcripts were ccmB with the highest density of RNA editing sites (53.7 sites / kb) and ccmFn with the highest number of sites (39 sites). Most of the editing events are at position 1 or 2 of the codons, usually altering the encoded amino acid, and are highly conserved among the species, also with regard to their editing extent. However, one SNP was found in the newly sequenced and annotated mitochondrial genome of P. alba resulting in the loss of an RNA editing site compared to P. tremula and P. davidiana. This SNP causes a C-to-T transition and an amino acid exchange from Ser to Phe, highlighting the widely discussed role of RNA editing in compensating mutations.RNA editing occurs in the endosymbiont organelles of higher plants as C-to-U conversions of defined nucleotides. The availability of large quantities of RNA sequencing data makes it possible to identify RNA editing sites and to quantify their editing extent. We have investigated RNA editing in 34 protein-coding mitochondrial transcripts of four  RNA editing is the term for a post-transcriptional process by which the RNA is altered resulting in a sequence deviating from its corresponding genomic template , compensating mutations in the genomic sequence that would otherwise lead to the exchange of highly conserved amino acids in the encoded proteins by restoring the original transcript sequence  present in the organelles es e.g., .In this approach, RNA-seq reads are mapped to genomic sequences  to identify editing sites and to quantify their editing extent. This strategy is challenging because RNA editing site detection can be distorted by genomic reads that might still be present in RNA-seq data and by RNA-seq reads that may originate from nuclear loci in case of dual transcription of homologs  were used as the input for the Map Reads to Reference tool. The resulting read mappings were used as the input for the Local Realignment tool. The Reads Track output was then used by the Low Frequency Variant Detection tool to produce a Variant Track. The SNP tables contained within the Variant Track output files and the detailed mapping coverage reports were exported from CLC-GWB. Both mappings for single reads and mappings combining multiple reads from species, accessions, etc. were carried out this way. At this stage, coverage and count filters were kept deliberately relaxed in order to investigate as much of the dataset as possible. More stringent filtering was applied at later steps of the analysis (see below).Detection of RNA editing sites relied on SNP detection comparing sequencing reads of transcriptomic experiments (RNA-seq) with a genomic template. The sequencing runs used for this study (RNA-seq runs downloaded from SRA at NCBI or newly generated runs available at the SRA of NCBI: PRJNA514029) are listed in Table S1. The genomic template was a FASTA file containing all 78 potentially transcribed RNAs including hypothetical genes, rRNAs, and tRNAs derived from the annotated mitochondrial genome of The SNP tables were filtered for C-to-T polymorphisms. These filtered tables were analyzed using a custom R script (File S1) alongside with the mapping coverage report and the FASTA file containing the genomic information in order to produce graphical representations of editing sites, frequency, and coverage. Stringent filtering for coverage, count, and frequency was performed here using the following para-meters: Minimum coverage ≥ 10, Minimum count ≥ 3, Minimum Frequency ≥ 10, Probability ≥ 0.95.https://github.com/ThuenenFG/varianttools . The original version of the software Variant Tools is available on Codon positions affected by RNA editing were identified based on the position of a related RNA editing site in the CDS using simple equations. The following equations are true for the different codon positions: position mod 3 = 1 → codon position 1; position mod 3 = 2 → codon position 2; position mod 3 = 0 → codon position 3.Codon changes and amino acid changes produced by RNA editing were identified using an in-house Ruby script where the following exceptions from the standard genetic code were considered: UGA→Trp and CGG→Trp (Table S3b).P. alba clone Monrepos  according to a published protocol . If not otherwise stated, CLC-GWB  was used for data processing. Using the Trim Reads tool, all reads were trimmed including adapter trimming, quality trimming , trimming of ambiguous nucleotides , trimming of 10 nucleotides at the 5′-end and 1 nucleotide at the 3′-end and removing reads of less than 80 bp in length. All other options were set to default. In total 218,626 contigs of a length of at least 200 bp were generated by de novo assembly of all trimmed reads, using the De novo Assembly tool. The mapping mode was set to “Map reads back to contigs” (using a length fraction of 0.9 and a similarity fraction of 0.95). All other parameters were set to default. Duplicates and containments (>=98% identity) were removed using Dedupe included in BBMap (https://sourceforge.net/projects/bbmap/). The remaining 207,725 contigs were subjected to the Join Contigs tool of the Genome Finishing Module (plugin in CLC-GWB). Contig analysis type was set to “use long reads”, where all PacBio subreads were used as long reads. This step was repeated three times. The longest scaffold representing the entire mitochondrial genome was selected from the scaffolds. Overlapping sequence ends were removed from this scaffold and a N-strech inside the sequence was replaced by a related sequence obtained from one of the original MiSeq contigs.Initial quality control of the NGS reads was done with FastQC  was annotated based on the annotations of the mtDNA sequence of P. tremula W52 . This SQN-file was edited using the Sequin tool  by importing the new mtDNA sequence of P. alba (clone Monrepos) with “update sequence”. A GenBank file of the P. alba mtDNA was exported from Sequin and served as input to create the related circular gene map using the OrganellarGenomeDRAW software  were used as query in a BlastN analysis vs. the CDS sequences of P. davidiana (KY216145.1) and P. alba clone Monrepos (MK034705) extracted from the related GB accessions. All SNPs vs. the P. tremula reference identified in the alignments were listed (Table S4).The reference CDS sequences of P. tremula and P. trichocarpa generated in this study are publicly available at the SRA of NCBI (PRJNA514029). The annotated complete mtDNA sequence of P. alba clone Monrepos is available at GenBank (MK034705). Supplemental material available at Figshare: https://doi.org/10.25387/g3.7166141.The authors affirm that all data necessary for confirming the conclusions of this article are represented fully within the article, its tables, figures, and supplemental material deposited at figshare. RNA-seq data of P. tremula, P. alba, P. davidiana and P. trichocarpa .RNA editing sites (C-to-U) were identified based on mappings of RNA-seq data of Populus species. For all 29 CDS with RNA editing sites, the sites were plotted as red bars to their related nucleotide position as presented for P. tremula in Populus species). The editing extent as given by the height of the red bars was in the range of 10–100%. A value of 10% was set as threshold for the SNP frequency  when filtering original SNP data according to SNP frequency values. The coverage value at each position is plotted as a blue line allowing to check if there are regions in the CDS escaping RNA editing site detection by an insufficient coverage value (coverage threshold was set to 10 reads in SNP filtering).In total, 377 potential RNA editing sites (Table S3a) were identified in the CDS of 29 of the 34 mitochondrial genes analyzed, when considering all editing sites detected in at least one of the four rpl16 as an example in Populus species.To make comparisons between species easier, the potential RNA editing sites of all species were plotted together in individual graphical representations of the 29 genes  as shown for P. tremula W52 mitochondrial genome  and sdh4  – both annotated in forward direction – show a 72-bp overlap. No potential RNA editing site was identified in the overlapping region of both genes (Table S3a).The annotation of the complete DNA sequence of the Populus species in this study (Table S3a) were compared with RNA editing sites recently identified for P. tremula in another study  and 96% identity to P. trichocarpa chromosome 4 in a region where the gene Potri.004g049600 is annotated as “similar to sdh4”. BlastN analyses of the selected P. tremula rps4-reads provided hits with 99–100% identity to Potra185431, a nuclear P. tremula scaffold and with 96% identity to P. trichocarpa chromosome 18 in the genic region of Potri.018G031500 annotated as “rps4, mitochondrial”. These results indicate dual transcription of mitochondrial genes and their nuclear orthologs in the case of sdh4 and rps4 in P. tremula. Dual transcription of homologs in the nuclear and mitochondrial genomes has been previously reported for sdh4 in the Populus lineage . ccmFn (39 sites) and the highest density in the CDS of ccmB  or 2 . Only 14% of the editing sites (48 sites) are at the third codon position (Table S3b).Populus genes were analyzed for DNA polymorphisms to check if there is any overlap with RNA editing site positions identified in this study. Since entire mtDNA sequences with annotated genes were only available for P. tremula W52  was assembled and annotated in addition .Protein-coding sequences of mitochondrial P. davidiana and/or the P. alba individual when compared with the P. tremula individual (Table S3). Only C-to-N or N-to-C SNPs were further considered because only such SNPs may result in a loss or gain of an RNA editing site depending on the location. Only one of these SNPs is located at an RNA editing site, namely a C-to-T SNP identified in nad6 at position 146 in P. alba clone Monrepos (Table S4). The nad6-CDS sequences of more Populus individuals were compared  and in an amino acid exchange . RNA editing at C-146 detected in P. tremula and P. davidiana results in the same amino acid exchange.In total, 16 SNPs were identified in the CDS of the Populus species, 343 sites identified in at least one species were considered, which are covered by at least 10 reads in all four species analyzed (Table S3d). Among these 343 RNA editing sites, 238 sites were identified in all four Populus species (Table S3e), indicating that most of the RNA editing sites are highly conserved between the individuals analyzed and probably between the four related Populus species. The individual differences at the other RNA editing sites, especially differences observed in the P. alba individual compared to the other individuals are expected to be mainly due to a too low coverage at these positions as discussed in more detail below. In case of P. alba, only 54 GB of RNA-seq data were available, whereas for the other individuals more than 160 GB of RNA-seq data were included in the study  in at least one of the four in rpl16  previous tabacum  as well P. tremula were not identified in our study, among them one editing site in cox3 and one in rps14; both are editing sites with very low RNA editing extent. The detection of RNA editing sites with low editing extent, which is dependent on the threshold set for SNP detection, is difficult and requires sufficient coverage at the related position. Often, a very large amount of RNA-seq data is needed for obtaining enough coverage for mitochondrial genes, as most of the RNA-seq data publicly available are derived from oligo-dT-primed cDNA-libraries and include only a small fraction of mitochondrial RNA molecules (see Introduction). Moreover, contamination of genomic DNA in RNA preparations used for RNA-seq can distort (“dilute”) the values for editing extents.Some of the 329 editing sites identified by In general, some differences in identified RNA editing sites and related editing extents are not unexpected between different studies of a species especially if different RNA-seq data sets from different individuals and tissues as well as different methods for the identification of editing sites, especially different mapping parameters are used as in our and Edera’s study  of the individual of interest as reference sequences for mappings of RNA-seq data.False-positive RNA editing site detection may also arise from unspecific mapping of nuclear expressed transcripts to mitochondrial reference sequences in cases of dual transcription of nuclear and mitochondrial transcripts as known for  Lupinus  and suggFalse–positive results may also arise when C-to-U RNA editing is “mimicked” by a genomic C-to-T DNA polymorphism at an editable cytidine, which may happen if the genomic reference used for mapping RNA-seq data is from another individual/species than the RNA-seq data.Substitutions of editable cytidines with thymidines are the main cause of losses of editing sites along angiosperm evolution as shown in 17 genera .Populus RNA editing site nad6-146 in two P. alba genotypes , our results are in agreement with numerous other studies showing that editing sites are predominantly found at non-synonymous positions in protein-coding genes, most frequently at the second position  indicating that most of the RNA editing sites are highly conserved between the individuals analyzed and probably between the related species. In a recent study in the genus Leucaena, 607 conserved RNA editing positions have been identified in the mitochondrial genome when considering all three genome groups in this genus (Among the 355 RNA editing sites identified in this study, 238 sites were identified in all four As RNA-seq data sets from various tissues have been used in our study, individual differences due to tissue-specific RNA editing events may not be excluded (Populus. Furthermore, a high level of conservation has been found throughout all poplar species investigated. Interestingly, the loss of an RNA editing site by genomic substitution of an editable cytidine with thymidine was observed in two P. alba genotypes. If this finding reflects an ongoing reduction of RNA editing sites within the genus Populus cannot be clarified without deeper phylogenetic analyses in the future.In this study, the previous finding of