Here we established a protocol to obtain RNA-free N protein (N0) and successfully demonstrated the utility of a new assay for tracking assembly of N with RNA oligonucleotides into NCLPs. We discovered that the efficiency of the NCLP (N–RNA) assembly depends on the length and sequence of the RNA incorporated into NCLPs. This work provides a framework to generate purified N0 and incorporate it with RNA into NCLPs in a controllable manner. We anticipate that our assay for in vitro trackable assembly of RSV-specific nucleocapsids may enable in-depth mechanistic analyses of this process.The templates for transcription and replication by respiratory syncytial virus (RSV) polymerase are helical nucleocapsids (NCs), formed by viral RNAs that are encapsidated by the nucleoprotein (N). Proper NC assembly is vital for RSV polymerase to engage the RNA template for RNA synthesis. Previous studies of NCs or nucleocapsid-like particles (NCLPs) from RSV and other nonsegmented negative-sense RNA viruses have provided insights into the overall NC architecture. However, in these studies, the RNAs were either random cellular RNAs or average viral genomic RNAs. An in-depth mechanistic understanding of NCs has been hampered by lack of an  Nonsegmented negative-sense (NNS)A). Both the genome and antigenome RNAs are encapsidated entirely by the nucleoprotein (N) to form helical nucleocapsids (NCs) during RNA synthesis, and each RSV N protein covers 7 nucleotides (nt) (–The RSV genome contains 10 genes that encode 11 proteins. The RSV genome (negative-sense) RNA acts as the template for transcription to synthesize 10 viral mRNAs as well as the template for replication to produce antigenome (positive-sense) RNA. The antigenome RNA, in turn, serves as the template to generate genome RNA A. Both tdes (nt) –9. For eA).RSV shares a common strategy for genome replication and gene expression with all NNS RNA viruses , 12. DurFig. 1in vitro  is the first step to reconstitute a trackable NC in vitro. The crystal structure of the RSV N–RNA  pseudoring revealed that the N protein has two core lobes, with the RNA bound in the central groove (B).Obtaining a sufficient quantity of recombinant RNA-free N . Previous studies suggested that P not only binds to N0 monomers and delivers them to nascent RSV RNA genomes or antigenomes but also interacts with N–RNA to bridge the polymerase to the RNA template (NTD) interacts with N0, and the C-terminal domain of P (PCTD) interacts with assembled N–RNA , Nipah virus, and vesicular stomatitis virus suggest a potential chaperone role of PNTD, preventing N from self-aggregating or binding to cellular RNA (25–The RSV phosphoprotein (P) exists as a homotetramer with a tetramerization domain and is intrinsically flexible in both N- and C-terminal domains C. Previotemplate . Specified N–RNA , 24. Strular RNA , 25–27.0 and in vitro assembly of trackable NCLPs. We used PNTD as a chaperone to prevent nonvirus-specific N–RNA interactions by coexpressing PNTD and N together, and we established a protocol to obtain large-scale soluble RSV N0–P complex. We showed that the purified N0–PNTD could be stimulated and assembled into NCLPs by adding RSV-specific RNA oligonucleotides and that PNTD is removed upon addition of RNA (D). We then used size exclusion chromatography (SEC) and negative stain EM to characterize and visualize the resulting NCLPs. We examined the length preference and nucleotide selectivity of the RNA in NCLP assembly. We discovered that NCLP assembly with purified N0 depends on the length and sequence of the RNA. We also found that, although both length and sequence are critical for assembly of NCLPs, the longer length of RNA can compensate for the efforts of nonpreferred nucleotide-type RNA in NCLP assembly. We identified that the shortest poly(A) RNA that could stimulate assembly of N–RNA is 7 nt. To understand initial NCLP assembly, we examined the position specificity of the first 7 nt using various 7-nt RNA oligos. This study established a powerful tool to further perform in-depth mechanistic studies of RSV RNA synthesis; in particular, cis-acting RNA signals and trans-acting viral proteins.In this study, we demonstrated the feasibility of large-scale preparation of Nn of RNA D. We the0–P complex. We coexpressed the N and PNTD proteins with a His10 tag on the N protein in Escherichia coli. The binding of P and RNA to N protein seemed competitive, and we usually obtained a mixed population of N–RNA and N0–PNTD. We optimized the purification strategy to shift the equilibrium and copurified higher yields of N0–PNTD. We further purified N0–PNTD using ion exchange and SEC (A). We showed, using SDS-PAGE, that N0–PNTD (peak 1) contained both N and PNTD but did not contain RNA, based on the UV absorbance A260/A280 ratio (A and B). We also imaged the purified N0–PNTD (peak 1) using negative stain EM, and the particles appeared as single white dots on a gray background (C). The class averages showed soluble N0–PNTD complex as white dots ∼50 Å in diameter (D).In this study, we established a protocol to obtain large-scale soluble heterodimeric RSV N and SEC A. We sho80 ratio , A and Bckground C. The cldiameter D.0–PNTD could be stimulated and assembled into NCLPs by adding RSV-specific RNA oligos. We assembled NCLPs by incubating N0–PNTD with an excess molar ratio of RNA (the molar ratio of protein:RNA is 1:1.5) and isolated the NCLPs (N–RNA) by running gel filtration chromatography. When the N–RNA complex formed, we observed the appearance of peak 0 (N–RNA) and reduced levels of peak 1 (N) and peak 2 (RNA) in the SEC profile. Depending on whether N–RNA formed and the efficiency of assembly, we could evaluate and compare the value of peak 0 alone and the ratio of (peak 0)/(peak 2). The same procedure applied to other N–RNA assemblies in this study.We then demonstrated that the purified NA). N–RNA formation (peak 0) was also confirmed by the disappearance of the P protein (PNTD) band in the SDS-PAGE gel (B), and phenol extraction followed by RNA PAGE denaturing gel showed that Tr45 RNA in complex with N was intact compared with RNA alone (C). As shown in the raw micrograph, the larger complexes (N–RNA) formed upon RNA addition (D). Class averages revealed that most classes were ring-shaped particles resembling the N–RNA pseudoring structure (E) .For example, in the case of N–Tr45, we observed the appearance of peak 0 (N–Tr45) and an almost depleted level of peak 1 (N) A. N–RNA PAGE gel B, and phNA alone C. As shoaddition D. Class tructure E 9, 28), 28A). N0–PNTD. The results were analyzed using gel filtration SEC and negative stain EM. We used 21 nt, 14 nt, and 7 nt of the RSV genomic trailer sequence  and found that only Tr21 and Tr14 formed stable complexes with N (peak 0) but not Tr7 (A–C). The negative stain EM images of peak 0 of N–Tr21 and N–Tr14 revealed that most of the particles were similar to the particles shown in D and E).We then examined the length preference of NCLP assembly. We used RNAs of different lengths and sequences to incubate with purified N not Tr7 , A–C. ThA260/A280 ratio was slightly higher for N–Tr14 (86.27 mAU/64.20 mAU = 1.34) than for N–Tr21 (87.51 mAU/73.08 mAU = 1.20). The crystal structure of the N–RNA complex suggested that every N protein binds to 7 nt of RNA . In that case, because of the space restraint, for the 14-nt RNA, only one N protein is allowed to bind to RNA, but for 21-nt RNA, there will be two N proteins that are allowed to bind to RNA. This may explain a higher A260/A280 ratio of N–Tr14 (1N:14mer) than of N–Tr21 (2N:21mer).It is interesting that the t of RNA . Theoret0–PNTD but not SH7 . We also examined three other terminal genome sequences (Le or Tr) of 14-nt RNAs , all of which could form N–RNA complexes. After careful examination of these results, it seemed that Tr14 and LeC14 had more efficient formation of N–RNA than TrC14 and Le14 . Therefore, we decided to examine the nucleotide selectivity of the RNA in NCLP assembly.To validate the observation of length preference, we also used 21-nt, 14-nt, and 7-nt RNA sequences from the SH gene (derived from the RSV RNA genome): SH21, SH14, and SH7. It showed similar results; SH21 and SH14 could form N–RNA in the presence of N0–PNTD but not polyC14 or polyU14  with purified N polyU14 . We noti polyU14 . These rA and BFig. S3, ). To examine whether shorter AG RNA could form NCs, we tested AG7, which also formed N–RNA complexes . We concluded that purine (A and G) are likely to be the preferred nucleotide types of RNA to stimulate assembly with N0–PNTD into N–RNA. Our observation is consistent with other tested NCLPs in related NNS RNA viruses . We also tested a nonpreferred nucleotide (pyrimidine) pair, C and U, with different lengths. Even though CU14 did not assemble into NCLPs in the presence of N0–PNTD, a longer CU pair (CU21) could readily form N–RNA . We conclude that both length and sequence are critical for NCLP assembly but that the longer length of RNA can compensate for formation of NCLPs even though the nucleotide type of RNA is not preferred.To further examine the length and sequence dependence of NCLP assembly, we used nonpreferred nucleotide types. For a nonpreferred nucleotide U, polyU14 did not form NCLPs. Interestingly, when we used a longer RNA with nonpreferred U, polyU28, it gave rise to a nearly complete formation of NCLPs (F and G). In general, the longer the RNA oligo, the more complete the assembly. For example, polyA11 is better than polyA10 and polyA10 is better than polyA9 to assemble into NCLPs. Interestingly, we found that polyA8 formed a much smaller peak 0 than polyA7 or polyA9. We interpreted this as the potential sticky end of the RNA (1N:7-nt RNA) that interferes with the additional N incorporation into the NCLP assembly. Despite this, the trend of length dependence has also been shown using varying lengths of LeC RNAs. For example, there was a clear trend that LeC10 formed a fraction of N–RNA, and LeC9 formed a less efficient NCLP assembly, but LeC8 failed to form N–RNA .Because nucleotide A is a preferred nucleotide type for NCLP assembly, we then used poly(A) to define the minimum length requirements of RNA. We tested the length of poly(A) ranging from 11 nt to 5 nt . We founFig. S5). Interestingly, the completeness of the N–RNA assembly of polyA8 is worse than that of polyA9 and polyA7. We interpreted this to be due to the sticky end of one extra nucleotide (A) that hinders efficient NCLP assembly.Importantly, these results suggest that continuous RNA is not a necessity for NCLP assembly. In contrast to the rule of six in the Paramyxovirus family, the length of RNA is not needed to be a multiple of 7  to form the N–RNA complex in RSV –35. Nega–Figs. S6 and S7). For example, the RNAs that contain three, four, or five consecutive As, such as 3A4C-1 (5′-AAACCCC), 4A3C-1 (5′-AAAACCC), and 5A2C-1 (5′-AAAAACC), could not stimulate N0–PNTD to assemble into N–RNA (A–C). The SEC profiles of other tested 7-nt RNA oligos showed that most of them failed to assemble into N–RNA .Now we knew that 7 nt is the minimum length of RNA that could assemble into NCLPs and that N can assemble with any types of RNA when the length is sufficient. We then tested the specificity of the first seven positions of the 7-nt RNA oligos. Because A is a preferred nucleotide and C is not, we used A as a positive indicator and C as a negative indicator to reveal the importance of a specific position. We swapped the A in polyA7 with specific positions converted to C. As expected, the majority of the 7-nt RNAs could not assemble into NCLPs. There are a few exceptions . The results suggest that position 1 is critical because conversion of A to C (at position 1) completely abolished N–RNA assembly, whereas position 7 is less critical because, even with the negative indicator C (at position 7), it could still form peak 0 (N–RNA). We also observed a clear trend when we added a positive indicator (A). For example, 7AC-A1467 (5′-ACCACAA) could not form an N–RNA complex, 7AC-C23 (5′-ACCAAAA) could form a slight peak 0 of N–RNA complex, and 7AC-C2 (5′-ACAAAAA) could generate a minor N–RNA peak 0 (F–H). The results of other 7-nt RNA oligos are shown in A–HFig. S7, . We also tested 8AC-A1 (5′-ACCCCCCC), which did not generate N–RNA assembly . We also used completely scrambled sequences 7M  and 7N  . As expected, both 7M and 7N formed a slight peak 0 (N–RNA), indicating that positions and nucleotide types are important for assembly into N–RNA when the length of the RNA is at a minimum. Collectively, these results suggest that, among the first seven positions, positions 1, 4, 5, and 6 have more effects on NCLP assembly than positions 2, 3, and 7.We also demonstrated that different positions have different effects on N–RNA assembly. For example, 6A1C-1 (5′-AAAAAAC) could generate a minor peak 0 but 6A1C-2 (5′-CAAAAAA) could not , D and EA peak 0 , F–H. Thcis-acting signals came from the RSV minireplicon (e.g. luciferase). The marker genes are under the control of RSV GS and GE signals. This strategy has been valuable for mapping cis-acting signals. However, minireplicons are in vivo systems; they do not offer a tractable in vitro system for detailed analysis of enzymatic reactions and unstable intermediates. Recently, an in vitro RSV RNA polymerization assay using short naked RNA templates for initiation of RNA synthesis was developed  may be deployed for primarily stacking effects with RNA (i+1), and this may change how position 6 of the nucleotide is packed with N (i). Of course, these are speculations; further studies of in vitro assembled NCLPs are needed to understand the mechanism.In contrast to NCs from Paramyxovirus (such as MeV), which follow the rule of six , for RSin vitro assembled NCLPs were similar to the ring-like particles, a small portion appeared as short filaments by negative stain EM . When taking a close look at these filaments, most of them are not straight (curved). Judging by the size and overall shape, this observation suggests that the ring-shaped particles likely stack to each other loosely but are not packed into a higher-order filament oligomer. We interpreted this to mean that the RNAs used in this study are short RNA oligos and not continuous to support a higher-order assembly. These observations are consistent with NCLPs from other viruses, such as MeV (Although most of the h as MeV .0–P of MeV was reconstituted by fusing N and the PNTD with a cleavable linker (0–PNTD) of RSV using PNTD as a chaperone without fusing PNTD with N. Therefore, it is easier to separate N and PNTD. With purified N0–PNTD, we successfully assembled NCLPs using short RNA oligos and characterized the NCLPs using gel filtration and negative stain EM.As a comparison with previous studies, where the Ne linker , 39, in We defined the length and sequence requirements of RNA for assembly. Using polynucleotides of a specific nucleotide type, we found that A is a preferred nucleotide over C or U. Interestingly, we found that Tr14 (5′-ACGAGAAAAAAAGU) has more efficient N–RNA formation than TrC14 (5′-ACUUUUUUUCUCGU) and that LeC14 (5′-ACGCGAAAAAAUGC) is better than Le14 (5′-GCAUUUUUUCGCGU). We noticed that Tr14 and LeC14 had more preferred A in their sequences than TrC14 and Le14. Importantly, Tr14 and LeC14 are the first sequences that are encapsidated by the N protein during replication by RSV polymerase to synthesize genome (negative-sense) RNA and antigenome (positive-sense), respectively. Therefore, it makes sense that Tr14 and LeC14 can efficiently assemble with N. Nucleotide selectivity could play an important evolutionary role in promoting a desired N–RNA assembly and inhibiting an unwanted N–RNA assembly.In further examinations, we found that the shortest poly(A) RNA oligo that could stimulate assembly of N–RNA is 7 nt. With specially designed 7-nt RNA, we observed that positions 1, 4, 5, and 6 are essential for N–RNA assembly. In contrast to long filaments of MeV NCLPs, RSV NCLPs generally did not yield long filaments in our experiments. This may be due to the fact that MeV RNAs follow the rule of six, but RSV RNA does not seem to follow such a rule. Alternatively, it may be due to the assembly conditions.in vitro trackable assembly of RSV N–RNA is a powerful tool to dissect the mechanism involved in RNA synthesis. Importantly, we identified an unexpected dependence of N–RNA assembly on the length and sequence of RNA. So far, for many determined structures of NCLP assemblies from NNS RNA viruses, the RNA bases were generically modeled as pyrimidine (uracil or cytosine) to represent the backbone of different nucleotides of random cellular RNA  were subcloned into a polycistronic plasmid. A His10-tobacco etch virus cleavable tag was fused to the N terminus of the N protein, and there was no tag for PNTD. The genes encoding for N and P were first amplified separately with an overlap region between their 3′ and 5′ end, respectively. The two DNA fragments were then joined by another round of PCR amplification, which yielded a coexpression construct, His10–tobacco etch virus–NPNTD.The full-length of the RSV N (residues 1–391) and the PE. coli BL21(DE3) strain was used for protein production. Cell cultures were grown at 37 °C in LB (Miller's Luria Broth) medium until A600 reached 0.8. The temperature was then lowered to 16 °C, and expression was induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside overnight. Cells were lysed by sonication in lysis buffer A (50 mm sodium phosphate (pH 7.4), 500 mm NaCl, 5 mm imidazole, 10% glycerol, and 0.2% NP40), followed by centrifugation. The supernatant was loaded on a cobalt column pre-equilibrated with lysis buffer. The cobalt column was washed with buffer B (50 mm Tris-HCl (pH 7.4), 1 m NaCl, 10% glycerol, and 5 mm imidazole) and buffer C (50 mm Tris-HCl (pH 7.4), 500 mm NaCl, 10% glycerol, and 5 mm imidazole). The protein was eluted from the beads in buffer D (50 mm Tris-HCl (pH 7.4), 500 mm NaCl, and 400 mm imidazole). The protein was further purified by a Q column and then isolated by gel filtration (Superdex 200 column) equilibrated with buffer E (20 mm HEPES (pH 7.4) and 200 mm NaCl).The 0–PNTD and RNA oligos were mixed and incubated with a molar ratio of 1:1.5 in buffer E (20 mm HEPES (pH 7.4) and 200 mm NaCl) for 1 h at room temperature. The resulting assembly was analyzed by gel filtration using a Superdex® 200 Increase 10/300 GL  pre-equilibrated with buffer E (20 mm HEPES (pH 7.4) and 200 mm NaCl). The N0–PNTD (molecular mass = 58 kDa) was usually concentrated to a final concentration of 40–60 mg/ml and stored as the stock. The RNA oligos were purchased from IDT and saved as 0.1 or 1 mm stock. The final concentrations of the protein (N0–PNTD) and RNA oligos used in the assembly were 13.3 μm and 20 μm, respectively. The sequences of the RNA oligos used in this study are summarized in The purified NThe peak fractions of N–RNA were collected after gel filtration SEC. RNA was extracted using phenol–chloroform . Briefly, the N–RNA samples were mixed and vortexed with phenol–chloroform and centrifuged at high speed  for 15 min. Then the upper aqueous solution was carefully transferred to 100% isopropanol, inverted, and stored at −80 °C overnight. After high-speed centrifugation at 15,000 rpm for 15 min, the supernatant was discarded. Then the pellets were washed twice by adding 70% EtOH and centrifuged. Then the RNA samples were air-dried and dissolved in nuclease-free water upon use.Samples were prepared on continuous carbon films supported by 400-mesh copper grids (Ted Pella). 4 μl of the sample was applied to a freshly glow-discharged grid, blotted to a thin film with filter paper, and immediately stained with 1% (w/v) uranyl formate. EM was performed using an FEI Talos L120C electron microscope operating at 120 keV and equipped with an FEI Ceta 4k x 4k charge-coupled device camera. Micrographs were collected at nominal magnifications of ×73,000 (1.97 Å/pixel). The images were acquired at a defocus value of −1.2 ∼ −2.0 μm and electron doses of ∼25 e−/Å2. 2D class averages of negative stain EM micrographs were processed using EMAN2 .Y. G., D. C., and B. L. conceptualization; Y. G., H. M. A., A. S., M. K., and T. R. data curation; Y. G., D. C., and B. L. formal analysis; Y. G. validation; Y. G., D. C., H. M. A., S. H., C. O., M. K., and T. R. investigation; Y. G., D. C., and B. L. methodology; Y. G. and B. L. writing-original draft; B. L. supervision; B. L. funding acquisition; B. L. visualization; B. L. project administration; B. L. writing-review and editing.


This method has successfully identified the cell type-specific transcriptome in three different tissues: endothelial cells in brain, epithelial cells in intestine and adipocytes in white adipose tissue. As this method does not require isolation of cells or RNA prior to the transcriptomic analysis, SLAM-ITseq provides an easy yet accurate snapshot of the transcriptional state in vivo.Cell type-specific transcriptome analysis is an essential tool for understanding biological processes in which diverse types of cells are involved. Although cell isolation methods such as fluorescence-activated cell sorting (FACS) in combination with transcriptome analysis have widely been used so far, their time-consuming and harsh procedures limit their applications. Here, we report a novel  Highlighted Article: A novel in vivo metabolic RNA sequencing method, SLAM-ITseq, enables cell type-specific transcriptome analysis without time-intensive cell or RNA sorting steps, making it accessible to a broader research area. Animals consist of various organs, which are further composed of heterogeneous populations of highly specialised cells. Thus, it is important to look at transcriptomic changes at the cellular level to understand animal physiology. To capture the transcriptome of a specific cell type, mechanical cell isolation methods such as fluorescence-activated cell sorting (FACS) or laser-capture microdissection (LCM) prior to RNA quantification have widely been used so far. Moreover, combined with such cell-isolation methods, the recent advance in high-throughput RNA sequencing (RNA-seq) methods now enables us to quantitate transcripts at single-cell resolution . Howeverin vivo metabolic RNA-labelling method, TU-tagging, to study cell type-specific transcriptomes, using a uracil analogue 4-thiouracil (Toxoplasma gondii (T. gondii) uracil phosphoribosyltransferase (UPRT) converts 4-thiouracil to 4-thiouridine monophosphate (4-thio-UMP), which is incorporated into newly synthesised RNA molecules to generate thiol-containing RNA (thio-RNA). As UPRT homologs in many organisms, including mice, are enzymatically inactive  in a specific cell type and exposed them to 4-thiouracil to ‘label’ newly synthesised RNA. Then, they pulled-down the thio-RNA using a biochemical isolation method and quantified by RNA-seq to determine enrichment level of labelled RNA over the total RNA. Even though similar methods have been tested in various model organisms : we redesigned the experiment to use a different control to account for background labelling, used the RNA-seq method called thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAMseq) to directly identify thiol-containing uracil at single-base resolution  with previously developed UPRT transgenic mice, which express haemagglutinin(HA)-tagged UPRT in a Cre-inducible manner. From a cross of homozygous UPRT mice (uprt/uprt) and hemizygous Cre mice (cre/0), both uprt/0; cre/0 (Cre+) and uprt/0; +/+ (Cre−) mice were obtained. When Cre+ mice are exposed to 4-thiouracil, the RNA synthesised in the cells expressing UPRT is labelled. To identify the labelled transcripts, RNA extracted from the whole tissue was treated with iodoacetamide (IAA) to alkylate the thiol group of the thio-RNA and then subsequently used as RNA-seq input. During the reverse transcription step of RNA-seq library preparation, a guanine (G), instead of an adenine (A), is base-paired to an alkylated 4-thiouracil leading to the thymine to cytosine base conversion (T>C) at the corresponding T position in the reads generated from the thio-RNA. T>C mismatch-aware alignment and T>C counting per gene were performed  under a cell type-specific promoter  was performed on complementary DNA (cDNA) obtained from each animal. As expected, we confirmed that UPRT was expressed only in Cre+ animals , suggesting that neither Cre nor UPRT expression has considerable effect on the transcriptome itself.After exposing both Cre animals B, and si animals C. ImportTable S1), genes that are significantly labelled in Cre+ were identified at FDR<0.05 . Although slightly higher proportion of labelled genes were observed in the bins of higher T content, considering there are labelled genes with fewer than 50 T bases and those genes represent minor fraction of the population, it suggests that the paucity of T bases in a gene has little effect on the discovery of labelled transcripts.Next, we employed a statistical approach to identify the labelled transcripts. As nucleotide conversion is a binomial process and the probability of it occurring at a given T position can be modelled by beta distribution for biological replicates, beta-binomial distribution can describe the T>C fraction per gene among biological replicates. Based on the variance estimate of T>C fraction , 11 were called as significant, all apart from Tie1 and Cd34 . As these genes should be expressed globally in brain, it is suggestive that SLAM-ITseq is sensitive enough to detect small shift in the labelled fraction despite the majority of a given gene being unlabelled. Next, to compare SLAM-ITseq with the conventional FACS-based method, we used a published RNA-seq dataset from multiple cell types that were sorted from mouse cortices by FACS for transcriptome analysis (Tie2-GFP) was used to isolate endothelial cells in this study. Based on the FPKM value in the dataset, we have defined the genes that are expressed in endothelial cells, and genes that are expressed only in non-endothelial cells in the brain . As shown in the Euler diagram . Together, these results indicate the successful labelling of endothelial transcripts without labelling the transcripts synthesised in surrounding cells.To comprehensively analyse the functional profile of significantly labelled genes, gene ontology (GO) enrichment analysis was performed on the labelled gene list. The biological process GO terms in the list were enriched for a cluster represented by ‘cardiovascular system development’, which is known to be related to the endothelial functions in addition to all the other general terms . For comprehensive analysis of labelled genes in each strain, GO term enrichment analysis was performed . Although +Adipoq-Cre cells revealed to enrich for GO terms represented by ‘fat cell differentiation’ , which is related to adipocytes, no such specific cluster was observed in +Vil-Cre results . This could be because the +Vil-Cre cells consist of heterogeneous types of cells, which have distinct roles, and thus did not enrich for specific terms as a whole. These results demonstrate that SLAM-ITseq can identify cell type-specific transcripts using Cre lines specific to a wide range of tissues, regardless of the proportion of the cell type of interest in a tissue.Next, we examined whether this method is applicable to study transcriptomes of other cell types, using different promoters for the Cre expression control. Vil-Cre  and Adipipoq-Cre  mice werhe brain A and 4A,he brain B and 4B. in Cre+ D and 4D.in vivo in a wide range of tissues by circumventing laborious and potentially disturbing cell isolation steps. The results above show that this method is applicable to different murine tissues by simply using an existing cell type-specific Cre line. Its sensitivity and specificity were confirmed by examining successful labelling of genes that are known to be specific for each cell type in the tissues examined. Extended exposure time could potentially increase the labelling level and the number of labelled transcripts, which could be of benefit when the steady-state transcriptome is sought.SLAM-ITseq dramatically broadens the application of SLAMseq to the study of cell type-specific transcriptomes Since TU-tagging was reported, there have been several other papers describing cell type-specific transcriptome analysis independent of cell isolation. One potential problem with the use of 4-thiouracil for tagging is that it could potentially be incorporated to RNA independent of external UPRT expression either by negligible endogenous Uprt activity or by an unknown alternative salvage pathway. To overcome this problem, 5-ethynylcytosine (EC) was suggested as an alternative to 4-thiouracil, because the incorporation of EC to RNA requires cytosine deaminase (CD) in addition to UPRT, and thus might increase the specificity . Howeverin vitro  Act of 1986 under a UK Home Office license that approved this work (PF8733E07), which was reviewed regularly by the Wellcome Sanger Institute Animal Welfare and Ethical Review Body.All mice were maintained in a specific pathogen-free facility with sentinel monitoring at standard temperature (19-23°C) and humidity (55±10%), on a 12 h dark/12 h light cycle (lights on 0730–1900) and fed standard rodent chow . Both food and water were available UPRT transgenic mice (uprt/uprt) were crossed with hemizygous Cre mice  (cre/0), and then uprt/0; cre/0 (Cre+) and uprt/0; +/+ (Cre−) animals were obtained as resulting offspring. To confirm the genotype, DNA from mouse ear-clips was isolated using the Sample-to-SNP kit (Life Technologies), and products amplified using a Viia7 qPCR machine (Life Technologies). Results were analysed against known calibrator controls using the ddCt method (All mice used were crossed with C57BL/6NTac at least once before used for further crossings. Homozygous nic mice  (uprt/up#010803)  (cre/0),t method . TaqMan + and Cre− mice using a 25 G×⅝” needle (Terumo). Number of animals used (>2) was determined based on the minimum number required for the statistical test used . Mice were culled and tissues harvested 4 h after the injection. The collected tissues were cut into small pieces (less than 5 mm in thickness) and submerged in RNAlater (Sigma-Aldrich) for storage at −20°C.4-Thiouracil administration was performed using the previously reported methods , 2014. BAfter removing RNAlater using clean Kimtech (Kimberly-Clark), around 30 mg of tissue was homogenised in 1 ml TRIsure (Bioline), using TissueLyser LT (Qiagen) and 7 mm stainless steel beads (Qiagen). RNA extraction was performed following the manufacturer's instruction with minor modifications: the isopropanol precipitation step was carried out with the addition of 20 µg/ml glycogen and 0.1 µM DTT and the isopropanol precipitation at −20°C was extended to 2 h. Purified RNA was subsequently treated with 1 U of TURBO DNase (Invitrogen) to eliminate potential DNA contamination. After the DNase reaction, RNA was cleaned up using RNA Clean & Concentrator-5 (Zymo Research) and stored at −80°C with 1 mM DTT in order to prevent oxidation.T. gondii UPRT  and M. musculus Hprt . Technical triplicates were prepared and the mean CT value was calculated for each biological replicate.Reverse transcription was performed using SuperScript II Reverse Transcriptase (Invitrogen) and random hexamers (Invitrogen) following the manufacturer's instruction. Around 10 µg of the synthesised cDNA was used as an input for 10 µl qPCR reaction using PowerUp SYBR Green Master Mix (Applied Biosystems) with the gene-specific primer pairs targeting DNase-treated RNA was reacted with IAA to alkylate the thiol group following the protocol previously described . BrieflyMus musculus primary genome assembly GRCm38 was used. For counting T>C per gene, 3′UTR annotation data from Refseq and Ensembl was used. The other parameters used were as follows: −5 12 −n 100 −m −mv 0.2 −mts −rl 100.Demultiplexed fastq files were first analysed with FastQC (version 0.11.5) for quality check. These sequence data were then analysed by the software designed for SLAMseq analysis, SLAM-DUNK , to quantify how many T>Cs are detected per gene in each sample. For the mapping and SNP calling, P-value, and FDR <0.05 was set to determine the significantly labelled transcripts.To identify significantly labelled transcripts, first, genes that had zero coverage on T in any sample were excluded. Next, the number of T>Cs and the total T coverage of the genomic sequence in each annotated gene were used to perform two-sided beta-binomial test by the R package ibb (version 13.06) . To contA published data table containing FPKM value for each gene in multiple cell types isolated from brain has been further analysed . Zhang eP-value from beta-binomial test in ascending order and then used as input for PANTHER 13.1 (pantherdb.org/)   to perfodb.org/)  with all


Translational Advisory Boards (TABs) are select groups of researchers, practitioners, and, in some cases, service recipients  that convene regularly to advise researchers on ongoing studies. TABs provide direction and support, knowledge and insight, alternative points of view, and suggestions for overcoming obstacles and improving research functions. TABs are especially valuable in applied research in which “real world” conditions create challenges ranging from the anticipated  to the unanticipated . In this presentation, the researchers evaluate one TAB involved with an ongoing pragmatic research trial of Adult Day Services Plus (ADS Plus), an intervention for family caregivers to persons with dementia. Including individuals with diverse points of view was critical in the composition of the TAB. The TAB consisted of four seasoned researchers, four ADS industry professionals, and six program directors from the ADS Plus treatment and control groups. Creating meetings that were productive was imperative. TAB sessions were held bi-annually and had highly structured agendas soliciting guidance on specific issues. Qualitative analysis of the TAB sessions revealed three themes: assistance with recruitment; fidelity of the intervention; and sustainability of the intervention. This guidance helped the researchers to shift directions in recruitment and to consider further refinement of the intervention to increase sustainability. In looking at costs, the annual research personnel, TAB personnel, and overhead expenses were estimated at $2,500-$3,000 and $4,000-$5,500, respectively. In conclusion, TABs can be an effective tool to support research and should be considered for use in large-scale projects.


The acquisition of somatic mutations is the most common event in cancer. Neoantigens expressed from genes with mutations acquired during carcinogenesis can be tumor-specific. Since the immune system recognizes tumor-specific peptides, they are potential targets for personalized neoantigen-based immunotherapy. However, the discovery of druggable neoantigens remains challenging, suggesting that a deeper understanding of the mechanism of neoantigen generation and better strategies to identify them will be required to realize the promise of neoantigen-based immunotherapy. Alternative splicing and RNA editing events are emerging mechanisms leading to neoantigen production. In this review, we outline recent work involving the large-scale screening of neoantigens produced by alternative splicing and RNA editing. We also describe strategies to predict and validate neoantigens from RNA sequencing data. Since the immune system recognizes tumor-specific peptides, they are potential targets for personalized neoantigen-based immunotherapy. They are presented by the major histocompatibility complex (MHC) on the surface of tumor cells, which can be recognized and killed by T cells. These antigenic peptides are largely classified into three groups: viral antigens, cancer germline antigens, and mutation-derived neoantigens . Among tRecent progress in sequencing technologies has enabled the profiling of RNA processing events caused by various forms of post-transcriptional regulation. In particular, alternative splicing and RNA editing have drawn considerable attention since they promote proteome diversity through changes in amino acid sequences. In addition, the transcript isoforms are easily detectable utilizing conventional RNA sequencing (RNA-Seq) data if proper computer-based methods are applied. Accordingly, analyses of large-scale RNA-Seq data have shown the genome-wide prevalence and regulation of alternative splicing and RNA editing ,5. Data Of note, like somatic mutations, alternative splicing and RNA editing can produce cancer-specific antigens . GeneticAlternative splicing can produce multiple transcripts according to the patterns through which exons and introns are joined . Alternacis- and trans-acting regulatory factors. Aberrant splicing patterns can be induced by genetic variants in splicing regulatory sequences around splice sites. The variants near splice sites are more likely to have functional effects on splicing by disrupting existing splice sites or creating new sites [trans-acting splicing regulators can activate or repress splicing of their target exons [Numerous studies have revealed cancer-specific splicing changes, suggesting their applicability for cancer diagnosis and therapy -19. Receew sites ,21. In aew sites ,23. Lastet exons .With growing interest in cancer immunotherapy, two recent studies evaluated the contributions of alternative splicing to neoantigen production. The first study performed a comprehensive analysis of alternative splicing with the development of an integrated workflow utilizing large-scale genomics datasets . Kahles In this context, a recent study was meaningful because it focused on intron retention, a splicing type that was neglected in the previous study. Intron-containing peptides are often subject to degradation by NMD, and the cleaved peptides can be presented on MHC class I molecules. Smart et al.  developeRNA modifications change the biochemical composition of RNA molecules and affect their structure and metabolism. RNA modifications are called the “epitranscriptome,” meaning that they are forms of post-transcriptional alterations that do not affect the RNA sequence, in analogy to how the term “epigenome” describes functionally relevant changes that do not involve changes in the DNA sequence . Recent As mentioned, A-to-I editing is detectable from RNA-Seq data by modifying the analysis pipeline. A prediction should be carefully made due to frequent false positives arising from single-nucleotide polymorphisms or sequencing errors. This limitation has been overcome in recent years by developing bioinformatics methods for accurate predictions. By comparing genomic DNA and RNA sequencing data from the same individuals, the biological noise from genomic polymorphisms could be reduced. Technical noise caused by sequencing errors and incorrect alignment can be removed by focusing on high-quality reads. Public databases collecting well-annotated edited sites are also useful for reliable screening and functional annotation ,32. In tRecent advances in RNA editing research have contributed to scientists’ understanding of the mechanisms involved in human cancers through the accumulation of evidence of mutated peptides induced by RNA editing ,35. ThroImprovement of neoantigen prediction from patient samples is an important issue for developing effective immunotherapy. Current approaches to neoantigen prediction have focused on somatic mutations, even though genetic changes affecting protein production can occur at many different levels. Most computational tools developed so far have aimed to evaluate the effect of non-synonymous DNA variants on producing mutated peptides. Representative analysis pipelines such as pVAC-Seq  and NeopThe recent studies described herein suggest that alternative splicing and RNA editing can serve as important sources of neoantigens. The challenge in utilizing RNA-derived neoantigens is the development of bioinformatics methods with increased accuracy and performance. RNA-Seq is now a popular technique, and data on RNA-Seq have been accumulating on a daily basis. However, many researchers feel that it is not easy to detect posttranscriptional modifications, such as alternative splicing and RNA editing. Therefore, improvement of the analysis pipeline will be required to make the RNA-derived neoantigen prediction more reliable.


To characterize RNA–capsid binding sites genome-wide within mature RNA virus particles, we have developed a Next-Generation Sequencing (NGS) platform: viral Photo-Activatable Ribonucleoside CrossLinking (vPAR-CL). In vPAR-CL, 4-thiouridine is incorporated into the encapsidated genomes of virus particles and subsequently UV-crosslinked to adjacent capsid proteins. We demonstrate that vPAR-CL can readily and reliably identify capsid binding sites in genomic viral RNA by detecting crosslink-specific uridine to cytidine transitions in NGS data. Using Flock House virus (FHV) as a model system, we identified highly consistent and significant vPAR-CL signals across virus RNA genome, indicating a clear tropism of the encapsidated RNA genome. Certain interaction sites coincide with previously identified functional RNA motifs. We additionally performed dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to generate a high-resolution profile of single-stranded genomic RNA inside viral particles. Combining vPAR-CL and DMS-MaPseq reveals that the predominant RNA–capsid interaction sites favored double-stranded RNA regions. We disrupted secondary structures associated with vPAR-CL sites using synonymous mutations, resulting in varied effects to virus replication, propagation and packaging. Certain mutations showed substantial deficiency in virus replication, suggesting these RNA–capsid sites are multifunctional. These provide further evidence to support that FHV packaging and replication are highly coordinated and inter-dependent events. Icosahedral RNA viruses must package their genetic cargo into the restrictive and tight confines of the protected virions. High resolution structures of RNA viruses have been solved by Cryo-EM and crystallography, but the encapsidated RNA often eluded visualization due to the icosahedral averaging imposed during image reconstruction. Asymmetrical reconstructions of some icosahedral RNA virus particles have revealed that the encapsidated RNAs conform to specific structures , which mNodaviridae. The small bipartite genome comprising RNA 1 (3.1 kb) and RNA 2 (1.4 kb) is packaged into a 34 nm non-enveloped T = 3 icosahedral virion. Only two non-structural proteins are produced by FHV RNA 1: the RNA-dependent RNA polymerase (RdRp) and sub-genomic RNA encoded protein, B2. The B2 protein was discovered as the virus's approach to evade the invertebrate anti-viral RNA silencing machinery , genetic tractability and ability to infect Drosophila and mosquito cells in culture and insects (reviewed in (Flock House virus (FHV) is a non-enveloped, single-stranded positive-sense RNA (+ssRNA) virus from the family achinery ,6, whichachinery . FHV RNAachinery ,9. The machinery ,10. FHV iewed in ,12). Moriewed in  or the viewed in .Both authentic virions of FHV and the related Pariacoto virus have been reconstructed by cryo-EM and X-ray crystallography to reveal highly ordered dodecahedral cages of RNAs ,18. The The FHV encapsidation process remains largely unknown. One molecule of each RNA 1 and 2 is specifically encapsidated into virus particles , while sNext-generation sequencing (NGS) in combination with crosslinking techniques provides a high-throughput approach to study transcriptome-wide RNA-protein interactions (reviewed in ). A numb‘vPAR-CL’ .In an analogous fashion to PAR-CLIP, here we applied the same principle to study the interaction of FHV genomic viral RNA in the context of assembled virions. Unlike the complex cellular micro-environment, virions represent a highly simplified enclosure with few well-defined components . Therefore, we were able to identify for specific RNA–capsid interaction events in virions without interference from other cellular components. Furthermore, since viruses can be readily separated from other cellular components, we avoided the need of immunoprecipitation for RNA recovery, and thus largely simplifying the PAR-CLIP methodology. This method is hence named Here, using FHV as a model system, vPAR-CL methodology was validated by determining that the increased U to C (U–C) transition rate was highly specific to crosslink between viral RNA and capsid. We noticed that the intensity of vPAR-CL signals was subjected to the dose of 4SU and time of incubation. Triplicate FHV vPAR-CL experiments revealed significant and highly consistent vPAR-CL signals across the encapsidated genome, which implicates a clear tropism of RNA cage inside capsid shell. The multiple clusters of vPAR-CL sites suggest that FHV encapsidation may require multiple synergetic packaging sites. DMS-MaPseq  was used to chemically probe single-stranded FHV genomic RNA in virions. We thus constructed a whole genome DMS-MaPseq-imposed RNA secondary structure map for FHV. We noticed RNA–capsid interaction sites favored double stranded RNA regions. Synonymous mutations were designed to disrupt predicted vPAR-CL sties in dsRNA regions, which resulted in varied effects to virus RNA replication, propagation, and virulence. Mutations over certain vPAR-CL sites showed evidential deficiency in RNA replication, suggesting these sites serve a multifunctional role in both virus packaging and replication. This provides further evidence to support that FHV packaging and replication are highly synchronized and inter-dependent events.Drosophila melanogaster (S2) cells were regularly maintained and passaged with Schneider's Drosophila Media (Gibco) containing 10% fetal bovine serum, 1 × Antibiotic-Antimycotic (Gibco), 1 × MEM non-essential amino acids solution, and 1 mM sodium pyruvate.As described previously , wild-tyTo purify FHV, 1% Triton X-100 was added to the cell culture containing P1 viruses. Cell culture underwent one freeze-thaw cycle, and cell debris was removed with 3000 × g centrifugation. FHV in the supernatant was crudely purified with 4% polyethylene glycol (PEG) 8000 and centrifuged (6000 × g) to remove debris . This waS2 cells were maintained in T75 flask until 70–90% confluency. Cells were infected with purified Flock House virus (P1) at MOI = 1 . As an i2  RNA methylation method was described previously ,44. In t2. The reaction mix was incubated at room temperature for 10 min, followed by 57°C incubation for 1.5 h, and 75°C termination for 15 min. The terminated reaction was then digested with RNase H to remove RNA template. The purified cDNA was click-ligated with Illumina adapters and final PCR amplification with indexes. Library pooling and Illumina sequencing platform are the same as above.Methylated FHV RNAs and respective controls were processed with ClickSeq library construction method as describe above with the exception of the use of the high-fidelity and processive thermostable group II reverse transcriptase enzyme  during reverse transcription. 100 U of TGIRT-III was mixed with 250 ng of RNA template, 0.5 mM of AzNTPs/dNTPs mixture (AzNTPs:dNTPs = 1:35), and the following reaction conditions: 5 mM dithiothreitol (Invitrogen), 10 U RNaseOUT (Invitrogen), 50 mM Tri–HCl pH 8.0, 75 mM KCl and 3 mM MgClcutadapt (FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html) to remove the remaining random nucleotides from the Illumina adapter sequence and random base-pairing as a result of azide-alkyne cycloaddition from cDNA fragments (command line parameters: fastx_trimmer -Q33 -f 7). A further quality filter was applied to remove any reads that contained more than 4% nucleotides with a PHRED score <20 . The remaining reads were aligned to FHV genome . Data generated from vPAR-CL experiments were aligned for end-to-end matches with Bowtie (v1.0.1) , provided by RNAstructure suite.RNA secondary structure prediction was conducted with tructure  with 310e  and downstream primer (TAAGAGAATTATGCAGTGCTGCCATAACCATG) were used in combination with mutation primers Table  to generTo generate mutant viruses, the plasmids containing vPAR-CL site mutations were used to transfect S2 cells with above-stated methods. Each mutant transfection consisted of equal amount of one mutated RNA genome with disrupted vPAR-CL site, and wt genome of the other RNA Figure . These PThe virulence of P0 vPAR-CL mutant viruses was measured by transfecting S2 cells with plasmids containing mutant viral genomes. For this experiment, transfection was conducted in black 96-well plate, with each well seeded with 25k S2 cells. The transfection reagents and methods were similar to above, except for scaling down to adapt for 96-well plate. For each mutant virus, 100 ng plasmids of each mutant genome and the other wild type genome were used. alamarBlue was incubated with cell culture for 4 h, before detecting fluorescence with EnSpire plate reader (PerkinElmer) at 560 nm excitation and 590 nm emission. The relative fluorescence then normalized reverse-ratiometrically with mock transfection = 0% and FHV wt RNA transfection = 100%.The relative virulence of P1 vPAR-CL mutant viruses was measured via infecting S2 cells with purified P1 mutant viruses. The P1 mutant viruses were purified through sucrose cushion , PEG precipitation (4% v/v PEG8000), DNase I and RNase A treatment, buffer exchanged and concentrated with PES membrane protein concentrator (100K MWCO). The concentration of purified P1 viruses was confirmed by SDS-PAGE gel electrophoresis and densitometry. 25k S2 cells were seeded in black 96-well plate and 0.12 ng  of seri® (NEB), per manufacture's protocol. The entire RT-PCR reaction was analysed by agarose gel electrophoresis.Transfected P0 vPAR-CL mutants were sampled for RT-PCR to detect RNA production. Total RNA was extracted from transfected cells and media with Direct-zol RNA kit (Zymo Research), and DNase I in-column digestion was conducted to remove plasmids. For each mutant and control, 200 ng of total RNA was used as template for RT-PCR. RT reactions was conducted with SuperScript III reverse transcriptase (Invitrogen), per manufacture's protocol. PCR was conducted with OneTaqAfter collecting P0 transfections, the cell/virus/supernatant mix was centrifuged at 1000 × g for 10 min. Supernatant fraction was removed and collected thereafter. The cell pellet was washed once with 1× PBS and centrifuged as before. The washed cellular fraction was then resuspended in 1× PBS and 1× cOmplete (Roche). 150 μl of supernatant (of each sample) was supplemented with 1× cOmplete and then reduced with vacuum centrifuge prior to SDS-PAGE.All SDS-PAGE assays were conducted with Bolt 4–12% Bis–Tris Plus Gels (Invitrogen). Membrane transfer was conducted with iBlot 2 Dry Blotting System (Invitrogen) with standard protocol. Western blot was conducted with iBind Western Device (Invitrogen) with standard protocol. Rabbit anti-FHV polyclonal antibody was given as a kind gift from Dr Vijay Reddy (Scripps Research), which was labelled with Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen). Prior to membrane transfer, part of SDS-PAGE gel was cut and stained with Coomassie brilliant blue R-250 to highlight α-tubulin (55 kDa) as a loading control.PAR-CLIP (photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation) is a well-established method for identification of RNA–protein binding sites and can provide nucleotide-resolution through analysis of specific uridine to cytidine transitions that occur at the site of RNA-crosslinking during cDNA synthesis ,40,52. Ht-test with P = 2.82E–53). This possibly reflects a minority variant present during virus passaging. Other than this, UV irradiation alone was not sufficient to increase U–C transition rate in the absence of 4SU (4SU–/UV+). In contrast, 4SU–/UV+ exhibited lowered U-C transition rate, for an unknown reason   FHV without 4SU (4SU-) or 4SU-containing FHV (4SU+) was treated with (UV+) or without (UV–) UV irradiation. As illustrated for FHV RNA 1 in Figure  Figures  and 3B.  Figures . We also Figures . We only) Figure , D. This) Figure  and notiHistograms of the U-C transition rate frequencies at all genomic U positions are shown in Figure Our vPAR-CL method requires no immunoprecipitation to recover and enrich for crosslinked RNAs. However, this permits wild type uridine and/or uncrosslinked 4-thiouridine to persist in the RNA pool which may dilute the vPAR-CL signal. To investigate the conditions for achieving higher vPAR-CL signals, we conducted three parallel experiments Figure . S2 cellThe same results were observed when we plotted the frequency of vPAR-CL signals for these three experiments, as well as two controls Figure . This shWe noticed significant difference of vPAR-CL signal distribution between 4SU40h/4SU1.5X and 4SU1.5X and 4SU16h . To furtt-test revealed most of these sites showed significantly (P < 0.05) higher vPAR-CL signals than average. As these same sites consistently displayed significant vPAR-CL signals over parallel biological replicates, this indicates a set of consistent RNA–capsid interactions in FHV virions, which further indicates a structural tropism of FHV RNA in association with the topology of virus capsid shell.We applied the 4SU40h condition in three parallel experiments: three separate S2 cell cultures were incubated with virus and 4SU, and individually purified viruses were exposed to UV and thereafter proceeded to sequencing. As before, vPAR-CL experiments were conducted in pairs, with each vPAR-CL dataset comprising one crosslinked sample (4SU+/UV+) and one control with the same RNA but without UV irradiation (4SU+/UV–). To ensure reliable transition rate calculation, we selected for U positions with coverage of at least 10000 reads  for posin vivo  provides a reliable and high-throughput method to probe RNA secondary structures in vivo ,44,56. Tin vivo , demonstin vivo ) across in vivo . We plotin vivo . We analin vivo  and E shRNAstructure Web Server  DMS-MaPseq sites (60 sites in RNA 1 and 30 sites in RNA 2) were imposed as unpaired constraints in the b Server  with ‘Folgorithm ,50. Regalgorithm . Despiteχ2 = 4.7, P = 0.03, chi-square test), whereas three sites were 1 nt. adjacent to double-stranded stems. In the much shorter RNA 2, 8/15 of most significant vPAR-CL sites are located in dsRNA stems , whereas 3 are 1 nt. adjacent. The lower frequency of RNA 2 vPAR-CL sites found in double-stranded regions of RNA is expected as RNA 2 represents a shorter RNA with fewer vPAR-CL sites. However, these data suggest that RNA 1-capsid interactions may predominate in the dodecahedral dsRNA cage observed immediately inside FHV particles. In Table In combination with vPAR-CL data, we noticed that the significant RNA–capsid interaction sites favored double-stranded base-pairing. In RNA 1, among the 28 most significant (>2σ) vPAR-CL sites, 22 are located in double-stranded regions  on RNA 2  Supplem. This stTo determine whether the identified vPAR-CL sites have a biological function, we selected 11 vPAR-CL sites from RNA 1 and 5 vPAR-CL sites from RNA 2 as candidate sites Table , we intrTotal cellular RNA was extracted from transfected cells and in-column DNase digestion was conducted to remove remaining plasmids. From each transfection, 200ng of purified RNA was used as template for RT-PCR to detect FHV RNA Figure . We notiTo confirm capsid production, we separated cells and supernatant from the transfected cells. Western blots with anti-FHV were used to detect capsid proteins in both cellular components and supernatants Figure . In the To expand mutant viruses, we further inoculated naïve S2 cells with equal amount of transfected P0 cell mix. From the inoculated P1 cell culture, we observed different degrees of cytopathic effect (CPE) under microscope , which wWe further tested P1 mutant virus relative virulence by infecting cells with mutants at MOI = 1 . Most muPhotoactivatable nucleoside analogs were successfully utilized in the past to enhance crosslinking efficiency and hence, providing approaches to study RNA–RNA and RNA-protein interactions (reviewed in ). Thionu8 and 109 cells , which can be easily separated from cellular components. Crosslinks within purified virus particles allow us to: (i) eliminate the need for immunoprecipitation to recover crosslinked RNA; (ii) look for specific in virion interactions between viral RNA genomes and viral capsid proteins; (iii) study a reductionist and highly controlled microenvironment. The greatly simplified vPAR-CL methodology, in combination with ClickSeq library construction technology , grantedIn our vPAR-CL method, the final pool of purified viral RNA can contain large number of canonical uridines, or uncrosslinked 4SUs. As a consequence, the signal of any randomly generated, non-specific crosslinking events will be largely diluted into background level. Only the consistent crosslinking sites due to homogeneity in RNA–capsid interactions within a viral population can readily provide distinguishable vPAR-CL signals from background. Therefore, in contrast to the canonical PAR-CLIP approach where only crosslinked RNA fragments are sequenced, we are also able to identify regions of the viral genome where there is no reproducible vPAR-CL signal, either due to a lack of RNA–capsid interactions or heterogeneous interactions. This is best illustrated in Figure In both PAR-CLIP and vPAR-CL, there are intrinsic limitations of 4SU-induced crosslinking. Firstly, crosslinking is only limited to U positions. Any potential interaction between protein and other nucleotides is undiscoverable. Next, 4SU crosslinking with protein is affected by reactivity of amino acid side chains ,36, withSeveral approaches were used to ensure reliable interpretation of vPAR-CL signals on FHV: (i) to ensure reliable interpretation of transition rate, we limited our analysis to genomic positions with at least 10k coverage. For this reason, our FHV vPAR-CL experiments reliably covered U34–U3034 on RNA 1, and U9–U1337 on RNA 2  were co-expressed in plant cells, assembled virions only packed their own respective viral RNAs . IntraceOverall, our studies and vPAR-CL methodology provide insight into the nature of the encapsidated RNA genome of ssRNA viruses. Some ssRNA viruses, such as MS2 phage, have successfully yielded asymmetrical cryo-EM reconstructions that reveal the internal topology of the packaged genome . For othThe raw sequencing data for both vPAR-CL and DMS-MaPseq experiments are available in the NCBI sequence read archive (SRA) with accession number: PRJNA554838.gkz1124_Supplemental_FilesClick here for additional data file.


The astounding regenerative abilities of planarian flatworms prompt steadily growing interest in examining their molecular foundation. Planarian regeneration was found to require hundreds of genes and is hence a complex process. Thus, RNA interference followed by transcriptome-wide gene expression analysis by RNA-seq is a popular technique to study the impact of any particular planarian gene on regeneration. Typically, the removal of ribosomal RNA (rRNA) is the first step of all RNA-seq library preparation protocols. To date, rRNA removal in planarians was primarily achieved by the enrichment of polyadenylated (poly(A)) transcripts. However, to better reflect transcriptome dynamics and to cover also non-poly(A) transcripts, a procedure for the targeted removal of rRNA in planarians is needed.S. mediterranea. Our protocol is based on subtractive hybridization using organism-specific probes. Importantly, the designed probes also deplete rRNA of other freshwater triclad families, a fact that considerably broadens the applicability of our protocol. We tested our approach on total RNA isolated from stem cells (termed neoblasts) of S. mediterranea and compared ribodepleted libraries with publicly available poly(A)-enriched ones. Overall, mRNA levels after ribodepletion were consistent with poly(A) libraries. However, ribodepleted libraries revealed higher transcript levels for transposable elements and histone mRNAs that remained underrepresented in poly(A) libraries. As neoblasts experience high transposon activity this suggests that ribodepleted libraries better reflect the transcriptional dynamics of planarian stem cells. Furthermore, the presented ribodepletion procedure was successfully expanded to the removal of ribosomal RNA from the gram-negative bacterium Salmonella typhimurium.In this study, we describe a workflow for the efficient depletion of rRNA in the planarian model species The ribodepletion protocol presented here ensures the efficient rRNA removal from low input total planarian RNA, which can be further processed for RNA-seq applications. Resulting libraries contain less than 2% rRNA. Moreover, for a cost-effective and efficient removal of rRNA prior to sequencing applications our procedure might be adapted to any prokaryotic or eukaryotic species of choice. Schmidtea mediterranea are well known for their extraordinary ability to regenerate. This ability is supported by the presence of a large population of adult pluripotent stem cells, termed neoblasts p < 2.2e-In using custom ribodepletion probes, our major concern was that the utilized probes would lead to unspecific co-depletion of planarian coding transcripts. To exclude this possibility, we first mapped our pool of 88 DNA probes in antisense orientation to the planarian transcriptome allowing up to 8 mismatches and gaps of up to 3 nts. This mapping strategy requires at least 75% of a DNA probe to anneal to its RNA target. It resulted in only 11 planarian genes to be potentially recognized by 20 DNA probes from our oligonucleotide pool. Next, we carried out a differential expression analysis of these 11 potentially targeted transcripts between the ribodepleted libraries and poly(A)-selected ones. The analysis revealed that 9 out of 11 potential targets were downregulated at least 1-fold in at least two poly(A) experiments Fig. a. As theSalmonella typhimurium using a pool of organism-specific DNA probes (riboPOOL) developed by siTOOLs Biotech   between riboPOOL depleted libraries and libraries prepared with the Ribo-Zero kit  Fig. a. We comary Fig. a. Moreovkit Fig. b. Taken For samples from typical model organisms, such as human, mouse and rat, there are numerous commercial kits available for the removal of rRNA, e.g. NEBNext from New England Biolabs, RiboGone from Takara and RiboCop from Lexogen. This also applies to typical gram-positive and gram-negative bacteria (MICROBExpress from Thermofisher and Ribominus from Invitrogen). Moreover, these kits can be utilized with a certain degree of compatibility for the depletion of rRNA in organisms of distinct phylogenetic groups . However, as the breadth of molecularly tractable organisms has increased in the past decade, the necessity to develop organism-specific rRNA depletion techniques has risen as well –42. To dS. mediterranea. Our protocol is based on the hybridization of biotinylated DNA probes to planarian rRNA followed by the subsequent removal of the resulting rRNA-DNA hybrids by using streptavidin-labeled magnetic beads. We tested the efficiency and specificity of our protocol by depleting rRNA from total RNA of neoblasts, planarian adult stem cells. A comparative analysis between ribodepleted and poly(A)-selected libraries revealed that our protocol retains all information present in poly(A) selected libraries. Over and above, we found ribodepleted libraries to contain additional information on histone mRNAs and transposable elements. The abundance of histone mRNAs in neoblasts is not unexpected, as planarian neoblasts are the only dividing cells in adult animals and thus require histones for packaging newly synthesized DNA [S. mediterranea. Last, we validated the efficiency of the developed workflow by removal of rRNA in the gram-negative bacterium S. typhimurium. Therefore, the proposed workflow likely serves as an efficient and cost-effective method for rRNA depletion in any organism of interest.In this study, we describe a novel rRNA depletion workflow for the planarian flatworm ized DNA , 47. Theized DNA , 49. Plaized DNA , 48–50. ized DNA . In addiized DNA , 52. UsiS. mediterranea and related freshwater triclads. It is based on the hybridization of 40-mer biotinylated DNA oligos to ribosomal RNA followed by the subtraction of formed DNA-RNA hybrids. The protocol is very robust and ensures the efficient removal of rRNA even from low input total RNA. Moreover, we suggest the general applicability of the presented workflow to any prokaryotic or eukaryotic organisms by using organism-specific pools of probes.This study describes an rRNA depletion workflow for the planarian model system Ribosomal RNA depletion was conducted as described in the result section. To evaluate Fragment analyzer separation profiles, planarian total RNA (1000 ng for each sample) was subjected to rRNA depletion using varying concentrations of NaCl  in the hybridization buffer.https://phylot.biobyte.de). The tree was visualized using the Interactive Tree of Life (iToL) tool [The phylogenetic tree was constructed using NCBI taxonomic names at phyloT (oL) tool .Planarian RNA-seq data were processed as follows: Reads after removal of 3′-adapters and quality filtering with Trimmomatic (0.36)  were triSalmonella typhimurium SL1344 were processed with READemption 0.4.3 using default parameters [RNA sequencing data from rameters . SequencDNA probe sequences were mapped to the planarian transcriptome SMEST.1  using thAdditional file 1: Sequences of DNA oligonucleotides used for the removal of planarian rRNA. Assembled planarian rRNA sequences are indicated.Additional file 2: (A)-(D) RNA-seq coverage profile for genes potentially targeted by ribodepletion probes in rRNA depleted (ribodepleted) and poly(A) enriched  libraries. The position of antisense probes mapping to the transcripts is marked in red.


The Biochemistry of the Nucleic Acids, 10th Edition  to function, a protein partner complex, called the ‘RNA-induced silencing complex’ (RISC), is essential, in which the Argonaute is the core protein. Many enzymes and protein partners are also required to construct a mature and functional miRNA after the precursor miRNA gene is expressed in the nucleus.Because we have walked the paths of the life sciences with this experience of biochemistry, it is no exaggeration to say that we feel compelled to act as editors of the research topic ‘Current Advances in the Research of RNA Regulatory Enzymes’ in the RNA section of Although many RNA researchers are already aware of the importance of enzymes and other proteins in RNA Biology, the research efforts in this field are still heavily weighted towards the RNA side rather than toward the enzyme and protein side. Therefore, we set the editorial direction of our research topic towards the enzyme/protein side. Although we cannot list these enzymes as exhaustively as the biochemistry book mentioned above, we have collected 11 excellent review papers on this research topic. Many of them are from the field of tRNA research because we both work in this particular field. This is a pertinent direction because several important subjects, including RNA modification, RNA editing, intracellular RNA trafficking, and the regulation of RNA processing, can be exemplified in one type of RNA molecules, tRNA. Therefore, this e-book is focused on important concepts that hold true even if tRNA is replaced with another RNA molecule. The first half of the book considers enzymatic catalysis reactions in which RNAs are the substrates, and the second half considers RNA-binding proteins that control the fates of their target RNAs.Tomita and Liu introduce a unique enzyme that forms a methyl cap on the 5′-monophosphate of tRNAHis, and Yashiro and Tomita review the structure–function relationships of the uridyltransferases that act on the 3′-termini of noncoding RNAs, such as pre-miRNAs. Interestingly, the TUT4/7 uridyltransferase alternates between a protective or degradative role for the let-7 miRNA precursor according to the presence or absence of its the partner protein Lin28, respectively. Shigematsu et al. describe how the 2′-3′-cyclic-phosphate-forming RNases create a novel ‘hidden’ layer of the transcriptome. We then move on to enzymes that modify internal nucleotides, especially in tRNAs. Hori summarizes various tRNA-modification enzymes in thermophilic bacteria and their regulation. He explains the importance of the network of modified nucleotides to the stability of tRNA at high temperatures. Dixit et al. focus on the multimerization of the tRNA methyltransferase subunits to accommodate various tRNA substrates, and the interdependent actions of methyltransferase and deaminase. These modifications at or near the anticodon greatly affect decoding of a near-cognate codon corresponding to the same amino acid by a particular tRNA. Hou et al. explain the effects of m1G37, next to the anticodon, on codon-specific translation and its utilization in Mg2+ homeostasis in bacteria. Some tRNAs are encoded by intron-containing genes, and their splicing is essential for their function. Hirata reviews the structural characteristics and evolutionary relationships of various types of archaeal tRNA-splicing endonucleases. Hopper and Nostramo describe the processing steps for eukaryotic small noncoding RNAs, especially tRNAs, ranging from terminal processing to tRNA-type splicing.We begin with enzymes that modify the RNA termini. Hopper and Nostramo summarize how tRNAs move across the nuclear pore during their biogenesis in yeast, emphasizing the involvement of parallel pathways that mainly transport other classes of RNAs. We then address issues related to the transcription and translation of mRNAs, mainstream topics of RNA biology. First, Yokoyama reports that SL1, a subunit of the RNA polymerase I preinitiation complex for rRNA transcription, plays another role as a component of the super elongator complex required for transcription by RNA polymerase II, which is also related to the MLL oncogene. Beyond transcriptional control, mRNA translation itself provides another platform for regulation. Otsuka et al. describe recent progress in our understanding of the translational regulation by RNA-binding proteins, which recognize the AU-rich elements (ARE) that occur in many mRNAs. Some ARE-binding proteins regulate mRNA stability, whereas others control the initiation of translation. Organisms face various process malfunctions, and so they are equipped with sophisticated quality control systems to cope with such failures. These quality control mechanisms also function during translation. Ikeuchi et al. discuss the quality control mechanism induced by ribosomes stalled on mRNAs, and provide mechanistic insights into the ribosome-associated quality control that rescues stalled ribosomes on aberrant mRNA, preventing the production of unwanted proteins.In the second half of the e-book, the proteins that interact with RNAs and their involvement in gene expression are examined. Important aspects here are the dynamics of intracellular movement and localisation of eukaryotic RNAs in addition to their funcational regulation. We discuss some RNA-related enzymes and RNA-interacting proteins in this e-book, retaining a focus on biochemical aspects. The proteins discussed above act on a limited but diverse set of target RNAs in their biogenesis, movement, function, and regulation, but we still do not have a full substrate list for each enzyme or protein. We hope that the issues discussed in this e-book have some bearing on the scientific interests of individual readers. We will be greatly pleased if this collection of review articles provides a useful guide to novel research directions.AK and TY wrote the manuscript.This work was supported in part by a JSPS KAKENHI Grant-in-Aid for Scientific Research (C) (grant number 17K07517) and by a JSPS KAKENHI Gran-in-Aid for Scientific Research on Innovative Areas “Hadean Bioscience” (grant number 26106003) (to A.K.), as well as by a JSPS KAKENHI Grant-in-Aids for Scientific Research (C) (grant number 17KT0113 & 17K07289) and by a JSPS KAKENHI Gran-in-Aid for Scientific Research on Innovative Areas “Nascent Chain Biology” (grant number 17H05672) (to T. Y.).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.


In vivo, apoptotic endothelial cells release two types of extracellular vesicles upon caspase-3 activation: apoptotic bodies and exosome-like nanovesicles (ApoExos). Only ApoExos are immunogenic: their injection causes inflammation and autoimmunity in mice. Based on deep sequencing of total RNA, we report that apoptotic bodies and ApoExos are loaded with divergent RNA cargos that are not released by healthy endothelial cells. Apoptotic bodies, like endothelial cells, contain mainly ribosomal RNA whereas ApoExos essentially contain non-ribosomal non-coding RNAs. Endogenous retroelements, bearing viral-like features, represented half of total ApoExos RNA content. ApoExos also contained several copies of unedited Alu repeats and large amounts of non-coding RNAs with a demonstrated role in autoimmunity such as U1 RNA and Y RNA. Moreover, ApoExos RNAs had a unique nucleotide composition and secondary structure characterized by strong enrichment in U-rich motifs and unstably folded RNAs. Globally, ApoExos were therefore loaded with RNAs that can stimulate a variety of RIG-I-like receptors and endosomal TLRs. Hence, apoptotic endothelial cells selectively sort in ApoExos a diversified repertoire of immunostimulatory “self RNAs” that are tailor-made for initiation of innate immune responses and autoimmunity.Endothelial cells have multifaceted interactions with the immune system, both as initiators and targets of immune responses.  Endothelial cells are also the targets of immune processes leading to atherosclerosis, hypertension, microangiopathy and vasculitis5. Pleiotropic interactions between endothelial and immune cells find particularly vivid illustration in the context of transplantation, where endothelial cells act as initiators and critical targets of graft rejection and graft-versus-host disease (GVHD)7. Indeed, antibodies targeting the vasculature of solid allografts are associated with poor outcome in solid organ transplantation, and endothelial damage contributes to the severity of GVHD8.Endothelial cells have multifaceted interactions with the immune system, both as initiators and targets of immune responses. The endothelium can recruit immune cells to sites of inflammation or injury, trigger innate immune responses and present antigens to B and T lymphocytes12. Notably, EVs released by allogeneic immune cells were shown to trigger proinflammatory T cell responses in different models of solid organ transplantation12. Furthermore, we recently reported that exosome-like nanovesicles released by apoptotic mouse endothelial cells accelerate rejection of vascular grafts14. Consistent with the deleterious effect of autoantibodies in graft rejection and GVHD16, we observed that EVs from apoptotic mouse endothelial cells induced production of anti-nuclear antibodies and autoantibodies against the LG3 fragment of perlecan13. Autoantibody production was not associated with increased levels of anti-MHC antibodies, but correlated with increased graft infiltration by B and T cells. Notably, none of the effects of apoptotic exosome-like vesicles were observed following injection of apoptotic bodies13. Hence, apoptotic exosome-like vesicles represent a novel and immunogenic component of the paracrine apoptotic response. Moreover, our data allow for the development of a model that integrates and explains numerous reports linking tissue injury to autoantibody production and graft rejection or GVHD15. A key question is how apoptotic exosome-like vesicles (ApoExos) may trigger an autoimmune cascade. An attractive explanation is that these vesicles would contain danger associated molecular patterns (DAMP) that bind to pattern recognition receptors (PRRs) on innate immune cells. In our quest to identify such DAMPs, we elected to analyze the transcriptome of ApoExos. This choice was based on two premises. First, nucleic acids are ligands for the most diversified repertoire of PRRs, which includes toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and members of the cGAS-STING pathway17. Second, EVs have been shown to carry functional RNAs that can be sensed by PRRs18; the nature of these RNAs is cell type dependent and dictated by the metabolic state of the cells20.Extracellular vesicles (EVs) are now recognized as masters of intercellular communication and most of their functions are in the regulation of immune responses13. Our results revealed that ApoExos have a distinct transcriptomic profile and carry non-coding RNA sequences exhibiting immunostimulatory potential, including mitochondrial transfer RNAs, U1 small nuclear RNA, and pathogen-like endogenous retroelements. Moreover, we show that RNA editing by adenosine deaminases acting on RNA, an important mechanism in self vs. non-self-discrimination of nucleic acids, was reduced in ApoExos. We also observed in ApoExos a dramatic enrichment for Poly-U, AU- and GU-rich motifs, known to be TLR7 and TLR8 agonists. Finally, RNA structure modeling revealed that ApoExos displayed a higher abundance of unstably folded RNA sequences which are more prone to generate single-stranded structures, the preferred ligands of endosomal TLRs. Overall, our work demonstrates that apoptotic endothelial cells release EVs loaded with RNAs which are recognized by RLRs and endosomal TLRs , and therefore have the ability to elicit innate immune responses.In the present work, we sequenced the whole RNA content of i) apoptotic human umbilical vein endothelial cells (HUVECs) and ii) of the two types of EVs released by these apoptotic HUVECs: apoptotic bodies and ApoExos15 Likewise, injection of these EVs increased the severity of GVHD in mice  or after induction of apoptosis by 4 h serum starvation (SS). Quality control of the obtained vesicles confirmed that, as previously reported13, ApoExos contained elevated levels of LG3 , the prototypic RLR20. Non-ribosomal non-coding RNAs with the highest abundance in ApoExos  secondary structures that trigger TLR3 and the RLR Melanoma Differentiation-Associated protein 5 (MDA5)40. Other less well characterized ‟repetitive sequences” (repeats) share features of EREs41. However, it is common practice to filter out EREs and repetitive transcripts in order to simplify transcriptomic analyses. This bias is propagated by alignment tools such as BLAST that mask ‟low complexity and repetitive regions” as a default option24.Endogenous retroelements (EREs) represent about 43% of the human genome and belong to three main classes: long terminal repeats (LTRs), long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs)http://www.repeatmasker.org/). Again, ApoExos presented a very distinct profile , cells use adenosine deaminases acting on RNA (ADAR) to perform adenosine-to-inosine RNA editing and prevent the formation of RNA duplexes. More than 90% A to I RNA editing occurs in Alu regions, and its extent is most commonly evaluated using the Alu Editing Index (AEI)43. Employing REDItools45, we therefore analyzed Alu editing in STAR-mapped reads from HUVECs and EVs. After applying filters restricting the search to Alu regions covered by at least 10 reads and displaying at least 1% editing, we screened A to I (seen as G) nucleotide changes in all datasets. Two observations emerged from these analyses. First, the AEI was lower in ApoExos than in HUVECs and apoptotic bodies  and pentanucleotides (5nts) from our datasets using Jellyfish51. These analyses clearly showed that ApoExos ribonucleotide content was very significantly enriched in A- and/or U-rich 3nts and 5nts  of random RNA sequences and that of RNAs with differential abundance in ApoExos vs. apoptotic bodies (see methods for details). A low MFE corresponds to a stably folded RNA sequence, whereas a high MFE is associated with more dynamic structures. A high-energy sequence is therefore more likely to exhibit secondary structure conformations that include open linear sequences. We selected MC-FlashFold for this analysis because of its unique ability to quantitatively assess the contribution of non-canonical base pairings in a sequence56, which enables accurate folding of RNA sequences that can be challenging to other predictive RNA folding algorithms. In our analyses, we excluded ribosomal RNAs because their secondary structure is molded by associated ribosomal proteins, and we strictly focused on sequences whose abundance differed by at least 4-fold in ApoExos vs. apoptotic bodies. Since MFE is dependent on sequence length, MFEs were normalized to MFE M- scores . The notable finding was that globally, RNA sequences in ApoExos presented a higher MFE than in apoptotic bodies  were found to be enriched in exosomes released by cancer cells, to activate TLR3 and RLRs and to trigger IFN signaling61. EREs present in ApoExos can form double-stranded RNA and thereby stimulate TLR3 and MDA5 via two mechanisms62. First, EREs readily form RNA duplexes when they are transcribed from both DNA strands. Second, Alu sequences, a variety of SINEs, constitutively form RNA duplexes unless they are edited by ADAR enzymes. Since they contain huge quantities of EREs , U1 may contribute to the uracil enrichment in ApoExos , has a specific role in autoimmunity. U1 RNA stimulates TLR3, TLR7, TLR8 and RIG-I, and autoantibodies against its associated nucleoproteins are present in all patients with mixed connective tissue diseasexos Fig. . Of notexos Fig.  and othexos Fig.  and in Eet al.67 demonstrated that exosomes isolated from the plasma of lupus erythematosus patients can activate endosomal TLR7 in plasmacytoid dendritic cells. This effect was reverted by chloroquine, indicating that endosomal acidification is required for dendritic cell activation. Salvi et al. also showed that this activation was due to G- and U-rich miRNAs present in the vesicles, coherent with the increased G- and U-nucleotide content found in our ApoExo small RNAs . For EVs production, HUVECs were incubated in standard vesicles-free endothelial-growth medium or serum-free RPMI (Life Technologies) medium for 4 h. Vesicles-free medium was depleted from EVs by ultracentrifugation at 200,000 g, 4 °C for 18 h prior to cell culture. After 4 h of incubation, medium was collected and EVs were isolated through differential centrifugation as previously described13. Briefly, harvested medium was centrifuged for 15 min at 1200 g to remove apoptotic cells and cellular debris. Subsequently, the supernatant was collected and centrifuged for 15 min at 50,000 g to isolate apoptotic bodies. The remaining supernatant was collected and ultracentrifuged for 18 h at 200,000 g to pellet ApoExos. As reported previously, EVs isolated after centrifugation at 50,000 g are mostly apoptotic bodies ranging from 1 to 5 uM  but also contain some smaller membrane vesicles within microvesicle size range (0.25 to 1 uM). The fraction isolated by centrifugation at 200,000 g is enriched in smaller EVs ranging in size from 30 to 100 nm. Isolated vesicles were resuspended in Trizol for RNA extraction or in PBS for use in functional assays.Human Umbilical Vein Endothelial Cells  from two independent batches, each produced from a pool of 10 different individuals, were cultured in 175 cm73. Briefly, HUVECs were stained with 5-chloromethylfluorescein diacetate (Thermo Fisher) according to manufacturer’s protocol. Cells were then cultured in standard vesicles-free or serum-free RPMI, as described in the previous section. Harvested EVs were analyzed with a FACS Canto II equipped with a forward scatter photomultiplier tube specific for detection of small particles. Briefly, during acquisition, vesicles are compared to fluorescent Sky Blue microspheres of 40–90 nm, 400–600 nm, 700–900 nm, 1000 nm and 2500–4500 nm diameter. An exosome-like (ApoExos) gate including particles from 100 to 1000 nm, and an apoptotic body gate including particles larger than 1000 nm in diameter were used to detect exosome-like vesicles and apoptotic bodies, respectively.Quantification of HUVECs-derived exosome-like vesicles by flow cytometry was performed as previously describedTotal RNA was extracted using TRIzol® Reagent (Life Technologies) according to the manufacturer’s protocol. RNA was then purified using the miRNeasy micro kit (Qiagen) and submitted to on-column DNAse I digestion using the RNAse-free DNAse set (Qiagen) as recommended. Samples quality and quantity were determined on Agilent 2100 Bioanalyzer using RNA 6000 Pico kit (Agilent Technologies). For whole transcriptome analysis, libraries were generated from 60 ng extracted RNA using the KAPA stranded RNA-seq kit (Roche) following the manufacturer’s protocols. Paired-end (2 × 80 base pairs) sequencing was performed using the Illumina NextSeq 550 system. Eight RNA-seq libraries  were sequenced in a single run yielding a total of 800 M reads (60 M to 120 M paired-end reads per sample). For small RNA sequencing, libraries from the same 4 duplicated samples were generated from 20 ng RNA using the CleanTag kit for small RNAs (TriLink). Single-read (1 × 75 base pairs) sequencing was performed on Illumina NextSeq 550 (50 M reads per sample).The raw reads were trimmed to remove adapter sequences and low quality extremities  using Trimmomatic version 0.35. RNA-Seq data have been deposited in Gene Expression Omnibus archives under accession number GSE119108. For read distribution, reads were aligned on human genome GRCh38  using STAR version 2.5.1b. Mapped reads were then characterized as exonic, intronic or intergenic using RSeQC 2.6.3.26. For global annotated transcripts and repetitive elements analysis  to inosine (I) editing positions that were covered by at least 10 RNA sequencing reads and that presented an editing frequency ≥0.01. RNA editing positions located within Alu regions were then identified by intersecting genomic positions identified by REDItools and SINE table from repeat masker (UCSC Table Browser). AEI calculation was done as previously described43.To identify RNA editing events within 51 setting k-mer length to 3- or 5- nucleotides.For total RNA sequencing data Fig. , in orde74 software. The resulting reads were quality trimmed with Cutadapt75 then stripped from their rRNA content by mapping to a rRNA transcript index using HISAT276. Only unmapped reads were kept. Unmerged R1 reads were then reverse complemented and joined to their corresponding R2 reads with a gap of 40 uncalled nucleotides (N). The two resulting sets of reads (merged and joined) were chopped into 31-nt long sequences (k-mers) using Jellyfish ignoring unique k-mers. K-mer counts were normalized by considering read counts after rRNA removal. K-mers with a count lower than 10 in each sample were discarded. Differential k-mer occurrence analysis was performed using the software package edgeR77 in R78. A robust estimate of the negative binomial dispersion parameter was computed for each k-mer using the estimateGLMRobustDisp function79. A false discovery rate (FDR) cut-off of 5% was applied, keeping only statistically relevant k-mers that presented at minimum a 4-fold change between conditions. The dataset was separated in 2 smaller parts, each consisting of upregulated k-mers in each condition. The resulting 2 subsets of k-mers were assembled into longer sequences (contigs) separately using a de Bruijn graph assembler. All contigs ranging from 31 to 300 nucleotides were folded using MC-Flashfold56, returning an MFE value.Adapter-trimmed paired-end reads from apoptotic bodies and SS-ApoExos were extended and merged using the BBMerge80.l of contig i. The MAD value was calculated using the ‘mad’ function from the ‘statsmodels.robust.scale’ package of the Python programming language and using the default normalization constant value (approximately 0.6745). This transformation was chosen because of its property to center the median around 0 for a given distribution, making comparisons more straightforward in cases of non-normality.Because MFE values are only comparable between sequences of similar lengths, we proceeded to transform them into MFE modified z-scores (M-scores). For each RNA contig previously assembled, MFE values were transformed into M-scores as described by Iglewicz and HoaglinTo assess the statistical significance of the observed energy differences between samples, a permutation test was applied to the MFE M-scores and the binned MFE M-scores. A two-sided permutation test was performed to determine whether the MFEs in ApoExos are higher than the ones in apoptotic bodies. First, the median difference of the measures was obtained, then the two conditions were pooled together in 100,000 randomly chosen ways of dividing the data, in sets mimicking the respective size of the original samples. The p-value was defined as the proportion of the median differences for each of the randomly generated samples, whose value was more than or equal to the observed median of the original sets. If the p-value was less than 0.05, the null hypothesis that both samples come from the same distribution of energies was rejected and the alternative hypothesis that ApoExos RNAs and apoptotic bodies RNAs have a differing general tendency in folding was accepted.Supplementary Information