Drosophila and human proteins with more than 45% of them forming new interaction interfaces. The comparison of orthologous proteins enables the identification of evolutionary conserved RBDs in globular domains and intrinsically disordered regions (IDRs). By comparing the sequences of IDRs through evolution, we classify them based on the type of motif, accumulation of tandem repeats, conservation of amino acid composition and high sequence divergence.RNA-protein complexes play essential regulatory roles at nearly all levels of gene expression. Using in vivo crosslinking and RNA capture, we report a comprehensive RNA-protein interactome in a metazoan at four levels of resolution: single amino acids, domains, proteins and multisubunit complexes. We devise CAPRI, a method to map RNA-binding domains (RBDs) by simultaneous identification of RNA interacting crosslinked peptides and peptides adjacent to such crosslinked sites. CAPRI identifies more than 3000 RNA proximal peptides in  Drosophila RNA-protein interactome from the level of multisubunit complexes down to the RNA-binding amino acid.Comprehensive characterisation of RNA-protein interactions requires different levels of resolution. Here, the authors present an integrated mass spectrometry-based approach that allows them to define the  RNA–protein interactions can be studied on a high-throughput level from two angles3. The protein-centric angle relies on isolating proteins and sequencing the RNAs bound to them7. On the other hand, the RNA-centric approach detects proteins bound to RNA using mass spectrometry9. An example of an RNA-centric method is fixing RNA–protein interactions in vivo using UV irradiation and isolating polyA-containing (polyA+) RNAs using oligodT-coupled beads and highly stringent washes12. This approach was used to identify around 1800 mammalian direct RNA-binding proteins (RBPs), including hundreds of novel RBPs, many of which are involved in non-canonical pathways like energy metabolism and DNA repair16. However, proteins which interact with polyA+ RNA indirectly have not been probed on a systematic level until now.RNA–protein complexes are key components in the life cycle of RNA17. The throughput of these techniques is limited due to the heterogeneity in peptide-ribonucleotide conjugates and difficulty of identifying the conjugated peptide tandem MS spectra by standard database search engines. Even with a specialised database search algorithm and homogeneous thio-uridine-mediated UV crosslinking, the polyA+ RNA interactome capture method could only identify 133 unique crosslinking sites in 57 S. cerevisiae proteins19. Alternative approaches rely on predicting crosslinked peptides by detecting peptides adjacent to the site of crosslink21 or by comparing peptide intensities between crosslinked and non-crosslinked samples22.Most of the new RBPs discovered by the RNA-centric RBP capture methods do not possess known RNA-binding domains (RBDs). Previous RBD mapping approaches have applied mass spectrometric techniques to identify peptides covalently bound to RNADrosophila and human cells, uncovering 234 new Pfam RBDs in the two species. We identify 29 conserved RBDs with a globular structure. Furthermore, we uncover hundreds of IDRs mapped by 870 CAPRI-peptides. Based on manual screening, we find 40 pairs of IDRs whose RNA-binding function and location is conserved between Drosophila and human orthologs. We also produce exhaustive coverage of the Drosophila RNA-binding proteome (RBPome). Our study compares the RBPs captured by formaldehyde (FA) and ultraviolet (UV) crosslinking at a high-throughput level, and shows that FA crosslinking can retrieve not only direct RNA binders but also the secondary layer of RNA-interacting proteins. Our integration of RBD discovery by CAPRI with FA- and UV-mediated interactome capture provides the basis for a more complete understanding of the metazoan RNA–protein interaction network.Here, we introduce CAPRI (Crosslinked and Adjacent Peptides-based RNA-binding domain Identification), a technique which enhances RBD discovery by simultaneously identifying both crosslinked peptides (in the immediate vicinity of the RNA–protein interface) and adjacent peptides (next to the crosslinked peptide) from the same sample. We apply CAPRI to Drosophila RBPome at four levels of resolution  is limited compared to mammals (~1800 proteins)2. Previous attempts to characterise the fly RBPome have relied exclusively on UV crosslinking23, which only retrieves proteins in direct contact with RNA nucleobases24. In contrast, FA can create inter-protein and RNA–protein crosslinks, which are ideal for capturing both direct and indirect RNA–protein interactions  cells. We optimised the FA interactome capture such that comparable amounts of both proteins and RNA were captured by FA and UV crosslinking  comprising 1512 proteins and the FA-crosslinked RNA-binding proteome (FA-RBPome) encompassing 2327 proteins  enrichment analysis of molecular functions and biological processes uncovered that the most enriched terms were related to RNA functions for both RBPomes  Crosslinked and Adjacent Peptide-based RNA-binding domain Identification (CAPRI).We were interested in discovering RBDs in the hundreds of RBPs identified in our RBPomes. The current standard methodology employed for RBD mapping is detection of UV-crosslinked RNA-peptide heteroconjugates by mass spectrometry31  by the MS analysis software .In the second branch of CAPRI, we analysed crosslinked peptides using a newly developed pipeline. The CAPRI protocol permits enrichment of crosslinked peptides over non-crosslinked peptides from the same sample Fig. . This ma34. The HCD/CID fragmentation of crosslinked peptides in MS/MS preferentially cleaves the glycosidic and phosphodiester bonds but not the covalent link between the base and the amino acid side chain. Thus, we treated the four standard ribonucleotides  as if each was composed of two PTMs: the ribonucleobase  and the rest of the ribonucleotide (ribose and phosphate)   Fig. . One-hunBDs Fig. . The remBDs Fig. .We validated the XL-peptides by visualising their positions relative to RNA in seven available RNA-protein crystal structures . We performed on-bead proteolysis of oligo-dT-immobilised FA-crosslinked RBPs with Lys-C and trypsin . We implemented a visualisation based on concentric circles to show the predicted proximities of individual components of the 79-subunit spliceosomal complex to RNA  RNA in vitro together with Not2 and Not5 (Not3 in Drosophila)46. We mapped the RNA-binding region of the fly Rga/NOT2 protein to its N-terminus, thus placing it in the innermost circle of our visualisation . Briefly, UV-crosslinked cells were resuspended in highly denaturing guanidine thiocyanate-containing buffer  bind RNA  in the cell by adapting commercially available silica solid phase extraction (similar to the recently published 2C technique48 (see Methods). We first validated the assay using three known RBPs: SRRT, PPIE and RACK1 , radixin (RDX) and moesin (MSN), which had been predicted to interact with RNA in previous RBPomes21   Fig. . ALDH6A12) Fig. , while M2) Fig. . We clon2) Fig. . This fiDrosophila and humans using CAPRI  in both flies and humans  and the non-classical RBD DM15   Fig. . All the59. For example, we identify XL-peptides overlapping with the disordered YG-rich C-terminal region in human HNRNPA1  and Yps (fly). Although we could not determine any clear motifs or extensive sequence conservation between the ortholog pair, the CAPRI-identified IDRs of the two proteins exhibited conserved amino acid composition  and 1512 direct polyA+ RNA-binding proteins (UV-RBPome). Employing FA crosslinking enabled us to identify a secondary interaction layer comprising proteins involved in metabolism, endocytosis and DNA replication  were grown to exponential phase (6–8 million cells/ml) in Schneider (S2) medium in shake flasks at 70 rpm at 25 °C. The cells were crosslinked directly in the medium with 0.1% (v/v) FA (0.05–1% FA was used for optimisation) at room temperature for 10 min rotating at 8 rpm. The FA was quenched by adding glycine  and rotating for 5 min at 8 rpm. The cells were pelleted by centrifugation at 600 x 2 plates). In case of cells growing in shake flask, cells were pelleted in the medium by spinning at 500 x g for 5 min. For large-scale experiments, 450 × 106 cells were resuspended in 10 ml ice cold PBS and spread on a 25 × 25 cm2 plate. The plates were placed immediately on an ice-water mixture, 4 cm away from 254 nm wavelength UV lamps and irradiated with a total energy of 200 mJ/cm2. The crosslinked cells were immediately scraped off the plates and pelleted by spinning at 500 x g for 5 min at 4 °C. After discarding the supernatant the cell pellet was flash frozen in liq. N2 and stored at −80 °C for a maximum of 1 month.The cells were counted and plated out on cell culture dishes at 60% confluency overnight. The next day the medium was removed and cold PBS is added  cells were grown in 10% FBS in DMEM medium to 90% confluency in 15 cmDrosophila S2 DRSC and HEK293 cells. The proteins covalently crosslinked to the RNA were isolated, purified and ultimately analysed by mass spectrometry. The following protocol was adopted from two previous publications12. It has been streamlined for the particular cell line  by optimising crosslinking and, homogenisation conditions, cell number to lysis buffer ratio and washing steps.UV or FA crosslinked and non-crosslinked cells were used to isolate polyA+ RNA by oligo-dT magnetic beads from g for 10 min at 4 °C to pellet aggregates if any. The extract was incubated with a suspension of 4 ml oligo-dT magnetic beads (NEB) at room temperature for 1 h on a rotating wheel (set at 10 rpm).Non-crosslinked and crosslinked (UV or FA crosslinked) frozen cell pellets were resuspended quickly in 10 ml of OLB , 0.5 M LiCl, 5 mM DTT, 1x cOmplete protease inhibitor from Roche) by using a pipette. The lysate was immediately homogenised to shear genomic DNA with a rotor stator homogeniser for 90 s (UV) or 60 s (FA), respectively at 2000 rpm. The extracts were diluted to 40 ml with OLB. The extract was spun at 10,000 x After incubation, the beads were collected on a 50 ml NEB magnetic stand for 25 min at room temperature (RT). The supernatant was removed and flash frozen or stored on ice for a second round of purification. The beads were washed at RT with 40 ml of OLB for 5 min . All further wash steps were carried out at 4 °C. Briefly, the beads were serially washed once with 40 ml WB1 , twice with 40 ml WB2  and finally in a smaller 1.5 ml tube twice with 1 ml WB3 . The RNA was eluted in 400 μl of Elution buffer  by heating the mixture in a thermomixer at 56 °C for 4 min followed by removal of magnetic beads (magnetic stand). The beads were routinely reused for a second round of RNA isolation from the same cell extract. The RNA concentration and quality in each eluate were evaluated by employing Qubit assays , agarose gel electrophoresis. To evaluate efficiency of mRNA capture equivalent fractions of input and eluted RNA were finally purified (subsequent to complete protein degradation) and quantified by quantitative reverse transcription PCR (RT-qPCR). To check for genomic DNA contamination by qPCR, quantification was performed on equal volumes of reverse transcribed eluate (cDNA) and non-reverse transcribed eluate (genomic DNA).70. Finally, tryptic peptide mixtures were desalted using C18 reversed-phase STAGE tips71.Three biological replicates of RBPome protein samples  were subjected to in-gel tryptic digestion by reconstituting them in 1x LDS sample buffer followed by separation on 4–12% Bis-Tris gels (NuPAGE) and colloidal Coomassie staining . Entire gel lanes were sliced into six pieces and processed (Promega trypsin) by standard in-gel trypsin digestionDrosophila total RNA on the filters and subjecting them to the standard FASP31 washes with 200 μl of 8 M urea in 0.1 M Tris-HCl, pH 8.0 (UA buffer) by centrifugation , 4 M Urea in 0.1 M Tris-HCl, pH 8.0 (UB buffer) by centrifugation . Next, 50 μl of 4 M urea, 50 mM Tris-HCl, pH 7.9 was added to the filters and this solution was diluted to a final urea content of 1 M by addition of 150 μl 50 mM Tris-HCl, pH 7.9. The mixture was incubated with 0.5 µg of trypsin at 25 °C for 3 h. The RNA retained on the filter was collected by inverting the filter and centrifugation at 14,000 x g for 20 min. After centrifugation, the RNA in all the filtrates and eluates was ethanol precipitated and analysed on a 1% agarose gel.The FASP filters were selected after screening for total RNA retention. We screened Amicon 30 kD MWCO and Microcon 30 kD and 10 kD MWCO filters by adding Filters were also screened for retention of small nucleic acid oligos . Recovery of the oligonucleotides in the flowthrough was calculated by analysing input and flowthrough samples in a Nano-drop UV spectrometer.CAPRI combines on-bead digestion of covalently linked RNA–protein complexes with RNA-FASP in order to subsequently purify adjacent peptides and UV crosslinked ribonucleotide-peptide heteroconjugates.8 crosslinked (UV or FA) S2 cells or eight 15 cm2 plates of UV crosslinked HEK293 cells were resuspended in oligo-dT lysis buffer and homogenised by using a rotor stator homogeniser. The extract was cleared by spinning at 10,000 x g for 10 min and added to 4 ml suspension of oligo-dT magnetic beads. The mixture was incubated at room temperature for 1 h rotating at 10 rpm on a wheel. The beads were washed once with 40 ml OLB, once with 40 ml WB1 and twice with 40 ml WB2 at 4 °C. The beads were transferred to a 1.5 ml microcentrifuge tube and washed with 1 ml OBD buffer . The on-bead digestion was performed at 25 °C for 3.5 h in 400 µl of OBD buffer (rotating at 4 rpm) by addition of 1 µg Lys-C (Wako) per 40 µg of proteins. Next, the beads were separated (magnetic stand) and the supernatant containing released peptides was removed. The beads were transferred to a 5 ml protein low-binding tube and washed once with WB2 and three times with WB3 both at 4 °C. The RNA-polypeptide heteroconjugates were eluted from the beads in 400 μl of Elution buffer  by constant agitation  at 56 °C for 4 min.In all, 4.4 × 10g for 30 min at 20 °C. In all subsequent steps, solutions on the filter were first mixed in a thermomixer at 600 rpm for 1 min (RT). The filter units were first washed once with 200 μl of 10 mM EDTA, 0.1 M Tris-HCl, pH 7.5, and then with 200 μl of 8 M Urea in 0.1 M Tris-HCl, pH 7.9 by centrifugation . For cysteine alkylation 100 μl of 5 mM Iodoacetamide (IAA) was added and the solution was incubated at 25 °C for 15 min in the dark. IAA was washed away by centrifugation . This was followed by a wash with 100 µl of UB buffer . Lys-C (0.5 µg) was added to the filter in 100 µl UB buffer for another round of digestion and incubated at 25 °C for 6 h for Drosophila S2 cells (10 h for HEK293 cells). After digestion the released peptides were washed away with a 100ul 0.5 M NaCl wash. To estimate the efficiency of Lys-C digestion, these peptides were cleaned-up using C18 STAGE tips71 and analysed by mass spectrometry. The RNA-peptide heteroconjugates retained on the filter were further washed with 100 μl of 4 M Urea, 0.1 M Tris-HCl, pH 7.9. Next, 50 μl of 4 M urea, 50 mM Tris-HCl, pH 7.9 was added to the filters and this solution was diluted to a final urea content of 1 M by addition of 150 μl 50 mM Tris-HCl, pH 7.9. The RNA-peptide heteroconjugates were further digested overnight with 0.5 µg of trypsin at 25 °C. Next day, the released adjacent tryptic peptides (liberated from site of RNA crosslink) were collected by spinning . The filter units were further washed with 100 μl of 1 M urea, 50 mM Tris-HCl, pH 7.9. Both filtrates were combined and labelled as pool A peptides. The RNA-peptide complexes that have remained on the filter were again resuspended in 50 μl of 4 M urea, 50 mM Tris-HCl, pH 7.9 buffer. The urea was diluted to 1 M final concentration exactly as described above. To degrade RNA , 1 μl benzonase  was added and the mixture was incubated for 30 min at 37 °C. Subsequently an RNase cocktail consisting of 0.5 μl RNases A , 1.0 μl RNase-T1  and 0.5 µl RNase I  were added and incubated first for 60 min at 37 °C and second for 90 min at 52 °C with the aim of completely trimming the remaining RNA-peptide heteroconjugates to a length of one to maximally three ribonucleotides. Following a final proteolytic digestion step  the released peptide-ribonucleotide heteroconjugates were collected . The filter units were washed for the last time with 50 µl 0.5 M NaCl, 10 mM Tris-HCl, pH 7.4 and centrifuged as described above. Ultimately, the filtrates harbouring the ribonucleotide crosslinked peptides were combined and labelled as pool B peptides. All peptide samples were acidified with formic acid  prior to C18 STAGE tip column clean-up71. Both pools (A and B) were analysed separately by LC/MS (described below). To identify adjacent peptide the.raw files from pool A and B were processed together by MaxQuant. To identify crosslinked peptides only the.raw files from pool B were used. TFA was omitted from all steps throughout this protocol.Eluted RNA-polypeptide heteroconjugates were adjusted to a final concentration of 2 mM EDTA, 50 mM NaCl, and 5 mM DTT. The samples were directly concentrated on 30 kD MWCO Microcon ultrafiltration devices  by centrifugation at 16,000 x 6 S2 DRSC cells with oligo-dT beads as described before at 4 °C. Once the RNA had been captured on beads, the proteins were digested. To pursue this the beads were transferred to a 1.5 ml microcentrifuge tube and washed with 1 ml OBD buffer . The on-bead digestion was performed at 25 °C for 1 h in 400 µl of OBD buffer (rotating at 4 rpm) by addition of 1 µg Lys-C and 1 µg of trypsin. After 1 h the supernatant was removed and replaced with 400 µl of OBD buffer for another round of digestion with 1 µg trypsin for 2 h. Next, the beads were separated and the supernatant containing released peptides was removed. The beads were transferred to a 5 ml protein low-binding tube and washed once with 4 ml OLB, once with 4 ml WB2, three times with 4 ml WB3 and finally with prechilled 1 ml 200 mM ammonium bicarbonate (ABC) buffer for 2 min each. The RNA was eluted with 200 µl of 50 mM ABC buffer at 56 °C for 3 min and again with 100 µl of 50 mM ABC buffer at 56 °C for 3 min. The eluates were cleared of remnant beads by using a magnet and spinning at 2000 x g for 5 min. The RNA peptide bonds were de-crosslinked by incubating the eluates at 65 °C, 1100 rpm for 90 min. We took an aliquot of RNA for Bioanalyzer capillary electrophoresis. The remaining RNA was degraded with a mixture of 0.5 µl of benzonase, 0.5 µl of RNase A and 0.5 µl of RNase I for 90 mins at 37 °C at 1100 rpm and for another 30 min at 52 °C at 1100 rpm. The peptides were further digested with 0.5 µg trypsin and cleaned-up for liquid chromatography–mass spectrometry (LC-MS) analysis by SP3 purification72.FA crosslinked RBPs were purified from 4.4 × 1073. Briefly, Q Exactive mass spectrometer (Thermo Fisher Scientific) interfaced with an Easy nLC1000 UHPLC system (Thermo Fisher Scientific) was used for all experiments. For chromatographic separation of peptides, peptides were analysed on in-house packed fused-silica emitter microcolumns  packed with 1.9 µm reverse-phase ReproSil-Pur 120 C18-AQ beads (Dr. Maisch)). For RBPome samples, peptides were separated by a 4 h linear gradient of 5–80%  at a constant flow rate of 300 nl/min. For RBD samples, CAPRI-peptides were separated by a 1 h linear gradient of 5–80%  at a constant flow rate of 250 nl/min. For DDA acquisition, the “fast” (RBPome) and “sensitive” (CAPRI-peptides) method from Kelstrup et al.74 was adopted with the following alterations. The full scan was performed at 70,000 resolution (at m/z 200) with a scan window of 350–1650 m/z. The automatic gain control target for MS1 was set to 3e6 and for MS/MS scan it was set to 1e5. A top10 workflow at a MS/MS resolution of either 17,500 or 35,000 (depending on sample complexity) for selecting the most abundant precursor ions in positive mode for HCD fragmentation (NCE = 28) was employed. Precursor ion charge state screening was enabled, and all unassigned charge states, as well as singly charged ions, were rejected. The lowest fixed mass recorded in MS2 spectra was set to m/z 100 ensuring the detection of all RNA derived marker ions in ribonucleotide-peptide heteroconjugate spectra. Selected ions were excluded from repeated fragmentation in a time frame of 30 s (CAPRI-peptides) or 60 s (RBPome samples).General nanoLC-MS set-up was similar to one in Musa et al.32 software (version 1.5.2.8) and peak lists were searched either against the Drosophila or human Uniprot FASTA database (version September 2015) concatenated with a common contaminants database by the Andromeda search engine embedded in MaxQuant. Cysteine carbamidomethylation was set as a fixed modification and N-terminal acetylation, deamidation (NQ) and methionine oxidation as variable modifications. FDR was set to 1% for proteins and peptides, respectively and was determined by searching a reverse database. Enzyme specificity was set to trypsin , and a maximum of two (RBPome) or three (CAPRI) missed cleavages were allowed in the database search. For deciphering Lys-C missed cleavage four missed cleavages were allowed and enzyme specificity was restricted to Lys-C. Peptide identification was performed with an allowed initial precursor mass deviation up to 4.5 ppm and an allowed fragment mass deviation of 25 ppm with a minimum required peptide length of 6 amino acids and a maximum peptide mass and charge of 4600 Da and 7 + , respectively. The “match between runs” (matching time window 0.7 min), and “second peptide” features were enabled. Label-free quantification was done using the maxLFQ algorithm. Protein groups were identified with at least two peptides, wherein one of them should be unique to this protein group.DDA MS raw files for RBPome, CAPRI adjacent peptides and FA-dom-peptides were analysed by MaxQuantt-test from the Limma package (FDR < 0.01) comparing crosslinked to the respective non-crosslinked samples and subsequently filtered by an average intensity cutoff ( > eightfold). A finalised unique list of genes was selected from the first protein of the majority protein ID column in MaxQuant output tables.The MaxQuant proteinGroups.txt output file was used to identify UV and FA RBPomes. Contaminant protein groups were removed. Missing raw intensity values were imputed with minimum intensity observed. Protein groups defined by the presence of more than two unique peptides were statistically analysed by a moderated two-sided t-test (Benjamini–Hochberg FDR < 0.05) comparing crosslinked to the respective non-crosslinked samples and subsequently filtered by an average intensity cutoff ( > eightfold). Peptides mapping multiple times to the very same protein sequence (UniProt ID) were removed. An in-house python script was developed to assemble a database of adjacent peptides, uniprot sequences and Pfam domain identifications. The tryptic adjacent peptides were extended in silico to the next nearest  Lys-C cleavage site (lysine) in both directions  and were named ADJ-peptides. Finally, the longest protein isoform possessing the largest total number of mapped ADJ-peptides was chosen to represent each gene based on Ensembl gene ID. This protein list constitutes the ADJ-RBPome. Peptide coverage was calculated for each amino acid position in the protein sequence based on the number of tryptic adjacent peptides identified at the position. Comparison between full-protein peptides and ADJ-peptides was performed by treating the MaxQuant peptides.txt output file from interactome capture similar to the tryptic adjacent peptides.We used the peptides.txt output files from MaxQuant analysis to catalogue tryptic adjacent peptides that are significantly enriched in UV crosslinked samples. Peptides mapping to contaminants were removed. Missing intensity values were imputed with the minimum peptide intensity observed. Peptides were selected based on a moderated 52 and pCLAPMS (ref.40) peptides was performed similar to adjacent peptides to yield extended-RBDpeptides and extended-pCALP peptides . The RBDpeptides were extended to the next Arg-C or Lys-C digestion sites based on the enzyme used in domain enrichment. RBR-ID-ATP using T4 Polynucleotide Kinase in 20 µl PNK buffer  for 10 min at 37 °C in a thermomixer set at 900 rpm. The beads were washed twice with NDB buffer and proteins from both radiolabeled and non-labelled tudes were eluted in 1x NuPAGE LDS sample buffer at 90 °C for 5 min in a thermomixer at 1100 rpm. The unlabelled and unlabelled proteins were separated on a SDS gel and transferred separately to nitrocellulose membranes. The proteins were detected by using an anti-Flag-HRP antibody (1:1000 dilution) (Sigma A8592) and the labelled RNA-protein heteroconjugates were visualised by autoradiography.Respective proteins and domains were cloned into pcDNA5-FRT-TO plasmids with addition of a C-terminal 15 kD 3Flag-HBH tag comprising a sequential arrangement of the following epitope-tag sequences: Flag, hexahistidine, in-vivo biotinylation signal peptide, hexahistidine that are derived from the HBH tagOligos used to clone protein domains:LARP1(DM15 domain)For primer: 5ʹ AGACTTAATTAAGCCACCATGCGTACTGCTTCCATCAGCTCCAGRev primer: 5ʹ AATAGGCGCGCCTTTGGGGTCAATGTCCAAATTTTUBAP2L (IDR - RG rich)For primer: 5ʹ AGACTTAATTAAGCCACCATGGATGGTGGCCAGACGGAATCRev primer: 5ʹ AATAGGCGCGCCCTGGGAGCCTGTAGTACTGCCGHNRNPU (SPRY-AAA domains)For primer: 5ʹ AGACTTAATTAAGCCACCATGGCCAAATCTCCTCAGCCACCTGRev primer: 5ʹ AATAGGCGCGCCTTTTTGGGCTTCTTCCTTCTGAAGTTILF3 (DZF domain)For primer: 5ʹ AGACTTAATTAAGCCACCATGATTTTTGTGAATGATGACCGCCATRev primer: 5ʹ AATAGGCGCGCCCCCGTCCTCCTCCATTGGGGTPBP4 (GTP-binding domain)For primer: 5ʹ AGACTTAATTAAGCCACCATGAGAAAAGTCAAATTTACTCAACAGAATTACCRev primer: 5ʹ AATAGGCGCGCCAACTTTAATAACACCTTCCTCAGTCAGGYLPM1 (IDR)For primer: 5ʹ AGACTTAATTAAGCCACCATGAGAGGCAACAGCTCATCTTACAGAGRev primer: 5ʹ AATAGGCGCGCCTCCTCTTTCTGGATACTCTCGAATCPRRC2A (IDR)For primer: 5ʹ AGACTTAATTAAGCCACCATGATAACCAAGGGGAAGCTAGGGGRev primer: 5ʹ AATAGGCGCGCCTTTATCCTGCTGCTGAGCCCTCCUBAP2L and proteins in Fig. qPCR oligos for detection of genomic DNA contaminationDrosophila roX2For primer: 5' AGCTCGGATGGCCATCGARev primer: 5' CGTTACTCTTGCTTGATTTTGCDrosophila rpl22For primer: 5' GGCTAGCCCGAAGTTTTCTTRev primer: 5' AGCTGATCCCTTCAGTGGAADrosophila hxkFor primer: 5' AGTGTGTACCGCTTCCATCCRev primer: 5' ATCAGATCGAAGGTGATGCCDrosophila 18s rRNAFor primer: 5' CTGAGAAACGGCTACCACATCRev primer: 5' ACCAGACTTGCCCTCCAATDrosophila tubulinFor primer: 5' TGTCGCGTGTGAAACACTTCRev primer: 5' AGCAGGCGTTTCCAATCTGDrosophila actinFor primer: 5' GCGTCGGTCAATTCAATCTTRev primer: 5' AAGCTGCAACCTCTTCGTCADrosophila Rpl32For primer: 5' ATGCTAAGCTGTCGCACAAATGRev primer: 5' GTTCGATCCGTAACCGATGTHuman 18s rRNAFor primer: 5' CTCAACACGGGAAACCTCACRev primer: 5' CGCTCCACCAACTAAGAACGHuman POLGFor primer: 5' ATCATAGTCGGGGTGCCTGARev primer: 5' ATCATAGTCGGGGTGCCTGAHuman GAPDHFor primer: 5' TAGGGCCCGGCTACTAGCGGTRev primer: 5' CGCCAGGCTCAGCCAGTCCCFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Supplementary Data 12Supplementary Data 13Reporting SummarySource Data


The hepatitis C virus (HCV) genome contains structured elements thought to play important regulatory roles in viral RNA translation and replication processes. We used in vitro RNA binding assays to map interactions involving the HCV 5′UTR and distal sequences in NS5B to examine their impact on viral RNA replication. The data revealed that 5′UTR nucleotides (nt) 95–110 in the internal ribosome entry site (IRES) domain IIa and matching nt sequence 8528–8543 located in the RNA-dependent RNA polymerase coding region NS5B, form a high-affinity RNA-RNA complex in vitro. This duplex is composed of both wobble and Watson-Crick base-pairings, with the latter shown to be essential to the formation of the high-affinity duplex. HCV genomic RNA constructs containing mutations in domain IIa nt 95–110 or within the genomic RNA location comprising nt 8528–8543 displayed, on average, 5-fold less intracellular HCV RNA and 6-fold less infectious progeny virus. HCV genomic constructs containing complementary mutations for IRES domain IIa nt 95–110 and NS5B nt 8528–8543 restored intracellular HCV RNA and progeny virus titers to levels obtained for parental virus RNA. We conclude that this long-range duplex interaction between the IRES domain IIa and NS5B nt 8528–8543 is essential for optimal virus replication. Flaviviridae family . Ce. CeXbaI-The amount of HCV RNA in 1 μg of cellular RNA was performed by RT-qPCR using the HCV sense primer HCV-5UTR(130–146)-S (nt 130–146) and antisense primer HCV-5UTR(272–290)-AS (nt 272–290)  and QuanWT or pER-1bmutants in conjunction with primers HindIII-T7-HCV(1–19)-S and HCV-5UTR(272–290)-AS or the NS5B 8374–8676 (HCV-NS5B(8374–8392)-S and HCV-NS5B(8657–8676)-AS to yield 5′UTR(1–290)WT, 5′UTR(1–290)LM and 5′UTR(1–290)SM, NS5B(8374–8676)WT, NS5B(8374–8676)LM and NS5B(8374–8676)SM RNAs. RNA probes were uniformly labeled with [α-32P]UTP  and unincorporated isotope was removed using RNA Cleanup of RNeasy Mini Kit (Qiagen Canada).Target and probe RNAs were synthesized using the MEGAscript™ T7 kit and DNA templates encoding regions of interest in the 5′UTR and NS5B. DNA templates were generated using pER-1b32P-labeled RNA probes were mixed with unlabeled RNAs in 50 mM sodium cacodylate (pH 7.5), 300 mM KCl and 1 mM MgCl2, denatured by incubation at 95 °C for 3 min, followed by stepped cooling from 70 °C to 37 °C (1 °C/min). RNA–RNA complexes were allowed to form for 30 min at 37 °C followed by immediate electrophoresis in a 5% native polyacrylamide gel supplemented with 2.5 mM MgCl2. Electrophoresis was performed at 4 °C for 1 h at 120 V in TBM buffer (45 mM Tris/HCl (pH 8.3), 43 mM boric acid, 0.1 mM MgCl2). Gels were dried and band intensity seen in the autoradiographs was measured using ImageJ software . Kd and Bmax values were calculated using the nonlinear regression analysis  according to the equation Y = Bmax × X / (Kd+X), where X is the concentration of unlabeled RNA, Kd is the dissociation constant, Y is the percentage of bound probe and Bmax is the amplitude of the reaction and represents maximum binding.RNA binding assays were performed as outlined . Brieflyt-test was used when comparing replication differences between wild-type HCV and mutant virus constructs.The NS5B RNA secondary structure spanning nt 8490–8560 coding sequence was predicted using mViennaRNA package 2.4.8 ,27. RNAcIn silico analysis indicates that a highly conserved (90% 5′UTR and 70% NS5B inter-genotype sequence identity) yet uncharacterized hetero-duplex could be formed by the 5′UTR domain IIa nt 95–110 and NS5B nt 8528–8543, which may contribute to genome circularization and HCV RNA replication  18]. RN. RN18]. 32P-labeled NS5B probe was incubated with increased amounts of 5′UTR target RNA showed an average dissociation constant Kd value of 70.67 ± 7.20 SE nM (n = 3) and an average binding maximum Bmax value of 85.15 ± 2.25%   . These KB SL 3.2 .In order to determine which nucleotides within the two RNA sequences were required to form the stable duplex, we constructed RNA molecules spanning the entire 5′UTR nt 1–290 and NS5B nt 8374–8676 complex. RNA construct designated as 5′UTR(1–290) large mutant (LM) contained six altered nucleotides in 5′UTR nt 95–110 , Dbl.32P-labeled NS5B or 5′UTR in combination with large and small mutant target RNAs showed a 5.2- and 6.6-fold increase in Kd values for 5′UTR(95–110) and NS5B(8528–8543) large mutations as compared to WT HCV RNA  and NS5B(8374–8676) RNAs cause a loss of binding affinity. Moreover, binding assays realized with molecules containing compensatory mutations were able to reconstitute duplex interaction.These results demonstrate that mutations in one of the two sequences predicted to be involved in the interaction of 5′UTRLM, 5′UTRSM, NS5BLM, NS5BSM, as well as virus genomes containing the large and small complementary mutations, called Double-LM (DblLM) and Double-SM (DblSM). The former four mutant genome constructs were expected to reduce the formation or stability of the 5′UTR:NS5B complex while the latter two genome constructs (DblLM and DblSM) were expected to reconstitute the 5′UTR and NS5B complementary annealing, restore complex formation or stability, and restore virus replication. To ensure that any observed changes in virus replication were not due to altered viral RNA-dependent RNA polymerase amino acid sequences, our constructs contain only silent mutations in the second or third codon nucleotides, which were expected to weaken NS5B-5′UTR RNA duplex free energy while maintaining the native RNA polymerase amino acid sequence.In vitro binding assays showed decreased duplex affinity and lesser rates of complex formation when small changes were introduced into 5′UTR or NS5B sequences. To determine whether the changes observed in vitro would impact virus replication, we constructed HCV genomes containing 5′UTRLM, NS5BLM, 5′UTRSM and NS5BSM produced on average 5.3- (p = 0.006), 6- (p = 0.005), 2.6- (p = 0.03) and 4.2-fold (p = 0.008) less RNA as compared to parental genome   .LM, NS5BLM, 5′UTRSM or NS5BSM produced on average 4.8- (p = 0.02), 3.2- (p = 0.04), 10- (p = 0.009) and 7-fold (p = 0.01) lower virus titers compared to parental genome   .LM, NS5BLM, 5′UTRSM or NS5BSM produced on average 3- (p = 0.01), 13- (p = 0.006), 4- (p = 0.01) and 6-fold (p = 0.001) less virus RNA copies compared to parental genome  (Progeny virus cellular RNA levels from genome constructs containing 5′UTRl genome . Progeny = 0.06) .Taken together, these results show that the long-range interaction between the 5′UTR nt 95–110 and NS5B nt 8528–8543 is necessary for efficient virus production. Disruptions of the 5′UTR at nt 107 and nt 104, or of NS5B at nt 8531 and nt 8534 are able to significantly lower HCV RNA production and virus titers , and theFlaviviridae supports the importance of RNA secondary structure contact between the distal 5′- and 3′-ends of the HCV genome in the control of virus replication [Flavivirus genome secondary and tertiary RNA conformations and their distal interactions serve as a balance between the two major viral events of RNA translation and RNA replication ,36,37,38lication ,15,39. Ilication  and RNA lication . SL9326 lication ,42. The lication ,44, the lication  and the lication . This laLM/KdWT, LM/KdWT, d) values to those of unaltered HCV RNA (KdLM/KdWT, We observed that 5′UTR nt 1–290 and NS5B nt 8374–8676 RNA sequences formed a high-affinity complex at low magnesium concentrations and in the absence of cell proteins which was controlled largely by HCV domain IIa nt 95–110 and NS5B nt 8528–8543 sequences . 5′UTR nStructural studies of the HCV IRES domain II indicates a large extended SL structure  that allLong-range interaction between RNA duplexes located at the distal ends of the HCV genome are hypothesized to facilitate and regulate important stages in the virus replication cycle. The data presented here indicate that a novel hetero-duplex formed by 5′UTR nt 95–110 and NS5B nt 8528–8543 is essential for optimal virus RNA production. Virions containing silent mutations within these two sequences, and expected to disrupt duplex annealing, were shown to decrease both cellular HCV RNA levels and progeny virus titers when compared to the parental virus. Virions containing complementary silent mutations within 5′UTR nt 95–110 and NS5B nt 8528–8543, and expected to restore RNA annealing, were shown to increase virus RNA and progeny virus titers to levels comparable to the parental virus.


Racial and ethnic inequities in health among older adults in the U.S. are well documented. A substantial amount of the health literature focuses on physical and mental disparities; however, less research has examined racial and ethnic differentials in subjective, self-rated health (SRH). Prior research has documented racial and ethnic inequities in SRH, though mechanisms by which these disparities occur are still largely unknown. One potential mechanism by which these disparities may arise is through unequal access to psychosocial resources through variability in social networks. Utilizing data from the 2006 Health and Retirement Study (HRS) along with the 2006 HRS Psychosocial and Lifestyle Questionnaire, the current study seeks to explore racial and ethnic differentials in SRH from a social network perspective. Ordinal logistic regression is used to predict SRH by race and Hispanic ethnicity potentially mediated by possession and number of friends, frequency of contact with friends, and psychosocial and subjective well-being. Results indicate that older black adults are less likely to rate their health in a higher category of SRH than older white adults, and this relationship is not significant for other racial groups and Hispanics. Additionally, psychosocial and subjective well-being and frequent written-communication with friends are found to significantly predict better SRH. The results suggest that feeling well mentally is crucial for SRH, but equally important is regular contact with friends. Further research should employ more robust measures of social networks to elucidate the role that network composition plays in predicting SRH for racial and ethnic minorities.


AAF neurons also display faster and more transient responses than A1 neurons. Additionally, offset responses in AAF – unlike in A1, increase with sound duration. Local field potential (LFP) and laminar analyses suggest that the differences in sound responses between these two primary cortices are both of subcortical and intracortical origin. These results emphasize the potentially critical role of AAF for temporal processing. Our study reveals a distinct role of two primary auditory cortices in tone processing and highlights the complexity of sound encoding at the cortical level.In the rodent auditory system, the primary cortex is subdivided into two regions, both receiving direct inputs from the auditory thalamus: the primary auditory cortex (A1) and the anterior auditory field (AAF). Although neurons in the two regions display different response properties, like response latency, firing threshold or tuning bandwidth, it is still not clear whether they process sound in a distinct way. Using  Tracing experiments in cats and mice confirmed parallel connections from the thalamus to both A1 and AAF3 and showed that in cats the vast majority of thalamocortical projections to A1 and AAF originate from largely non-overlapping cell groups of the medial geniculate body (MGB)4.For sound perception, auditory signals have to travel a long way from the cochlea, through the cochlear nuclei, superior olivary complex, inferior colliculus, and auditory thalamus up to the auditory cortex where two regions, A1 and AAF, receive them in parallel. An early study already distinguished A1 and AAF as two separate auditory fields in mice based on anatomical and functional parameters7. Additionally, A1 neurons have narrower tuning, lower response thresholds, and decreased spontaneous activity in comparison to AAF neurons, consistently in many species8. On the other hand, AAF neurons show faster temporal modulation than A1 neurons9, significantly shorter receptive field durations and latencies10, better timing precision11, and fast-pass selectivity for the rate of frequency-modulated sweeps12. Despite this knowledge, it is still not clear whether their role in sound processing is distinct.A1 and AAF are both tonotopically organized, but differ in the proportion of cortical area tuned to specific frequencies. In cats and ferrets, A1 neurons tuned to different frequencies are evenly distributed but AAF is characterized by an underrepresentation of cells tuned to mid-range frequenciesin vivo electrophysiological recordings in the mouse auditory cortex, we asked whether any tone properties are processed differently in these two primary auditory fields. Our results reveal significantly stronger responses to sound offset in AAF than in A1 neurons. Offset responses were reported previously in auditory cortex (ACx) neurons14 and their strength was compared between different auditory fields17 but their distinct properties in A1 and AAF were not described. Considering several inter stimulus intervals and sound durations, we show that offset is robust in AAF, but not in A1, and is positively correlated with sound duration. LFP analysis indicates that offset responses are not emergent in the cortex, but are of subcortical origin. Comparing response strengths between input (L4) and supragranular (L2/3) layers also suggest that offset responses are equally important in both layers in AAF, unlike in A1 where offsets in the input layers are dominating. Additionally, recordings from AAF neurons revealed that offset responses are poorly tuned and have weak tonotopy. Finally, we demonstrate that offset responses, even if significantly higher in fast spiking than in regular spiking neurons, are not specifically attributed to one of these two cell types. Our study reveals a crucial role of AAF in processing tone offsets, and thereby the possibility of distinct functions in sound processing for these two primary auditory cortices.Using in vivo multi-electrode electrophysiological recordings and retrograde tracing. Both regions were identified based on their functional tonotopy. Pure frequency tones (PTs) varying in frequency from 4 to 48.5 kHz and in level from 0 to 80 dB SPL presented with randomized inter-stimulus-intervals (ISIs) (500–1000 ms) were used to determine tuning receptive fields. By simultaneous recordings with 4 shank probes (200 µm distance between shanks in a 4 × 8 electrode configuration), we identified A1 and AAF based on their caudo-rostral and diagonal ventro-dorsal increase in best frequency , respectively. We then injected retrograde tracers in both regions, identifying somas of neurons projecting to A1 or AAF. Confocal imaging of the thalamocortical slices revealed a robust signal in the MGB, confirming that both A1  and no gradient in AAF . Even more striking, responses to 50 ms long PTs revealed robust offset responses in AAF neurons, whereas less prominent offset was seen in A1  activity leading to 202 SU in AAF  and 192 in A1 . We confirmed that auditory responses have shorter onset latencies in AAF than in A1 neurons Fig. 5,6,8,10.The analysis of the response decay in A1 and AAF  and AAF , respectively. Similar to our results in anesthetized preparations, offset responses in awake mice were robust in AAF and weak in A1  repeated 10 times with varying duration 50–500 ms) and inter-stimulus-intervals (50–2000 ms) Fig. . Based o0–500 ms 19.We then estimated the forward suppression that could potentially be caused by offset responses based on the relationship between onset response amplitudes and ISIs. There was no significant difference in forward suppression between A1 and AAF neurons Fig. . We addi0, f1, f2 and f3, where f1 = 2*f0; f2 = 3*f0; f3 = 4*f0. We recorded responses to 60 dB SPL HTs  with varying duration (50–500 ms) and ISIs (50–2000 ms)  instead of PTs. HTs consisted of four frequency components: fms) Fig. . Based oms) Fig. . Compari 9, 18, 2, 36 kHz ms) Fig. . These r20; we only analysed data recorded in L4 here. Based on the protocols with varied sound duration and intervals we could see, as for the spike rate analysis  and L4 (300–500 µm)sis Fig. . LFP peasis Fig.  as previ20. This suggests that, in addition to receiving offset responses from subcortical nuclei in the input layer (L4), offset responses are further processed in supragranular layers (L2/3) of AAF, but less so in A1. An analysis of latencies indicates that AAF offset responses appear equally fast in L4 and L2/3  for all recorded AAF neurons. We found a few neurons with clear onset and offset tuning Fig. . Most ot21. FS units had significantly shorter onset  and regular (RS) spiking neurons based on their spike waveforms, and analysed their responses to 9 kHz PTs played at 60 dB SPL for 500 ms. The peak-to-trough times (p2t) of our sorted units gave a bimodal distribution  has not been previously reported. In general, there has been a significant asymmetry in the relevance of excitatory onset and offset responses in auditory processing. Previous studies reported 25–75% of offset responsive cellsion Fig. , intensince Fig. .Auditory responses are shaped by stimulus characteristics. For example, our data reflect an increase in the percentage of offset responses evoked in A1 neurons by HTs as compared to PTs. This substantial increase was not observed in AAF. HTs evoke bigger auditory responses than PTs in general, and they could therefore contribute to an increased number of offset cells in A1. The lack of increase in AAF could be explained by the fact that the percentage of offset responses to PTs is already high in this brain region. Another possibility could be that A1 and AAF display different amount of offset responses for distinct stimuli. A broader group of sounds should be tested to fully address this question.26. In our study, we measure an increase of offset response strength from anesthetized to awake conditions in A1, but this increase is even higher in AAF  L4) Fig.  processeL4) Fig. . Could i32. Our results indeed reveal higher offset responses in FS than RS cells  but not for the detection of long gaps (>100 ms)35. Our data display offset responses predominantly for longer duration signals, or for shorter signals with longer ISIs  male or female C57BL/6 J mice .2) was performed with a scalpel just above the right auditory cortex and covered with silicone oil. For awake recordings, the surgery was performed under isoflurane  and the brain was additionally covered with a silicone casting compound  during the 2 h recovery period from the anaesthesia.Mice were anesthetized with intraperitoneal injection of ketamine/xylazine  and subcutaneous injection of bupivacain/lidocain  was used for analgesia. Ketamine (45 mg/kg) was supplemented during surgery as needed. For surgery, mice were head fixed and their body temperature was kept at 37 °C with a heating pad . Craniotomy  except for the data presented in Supplementary Fig. Sounds were generated with a digital signal processor  at 200 kHz sampling rate and played through a calibrated MF1 speaker  positioned at 10 cm from the mouse’s left ear. Stimuli were calibrated with a wide-band ultrasonic acoustic sensor .8). For all figures we used units with onset response tuning quality higher than 1 (d’ > 1). For Fig. To determine BF and tuning receptive fields we used PTs  varying in frequency from 4 to 48.5 kHz in 0.1 octave increments and in level from 0 to 80 dB SPL in 5 dB increments. Tuning receptive fields, best frequency and spiking rates were calculated in fixed time windows: A1 onset: 10–60 ms, A1 offset: 60–110 ms, AAF onset: 8 58 ms, AAF offset: 58–108 ms. TRFs were smoothed with a median filter (4 × 4 sampling window) and thresholded to 0.2 of peak amplitude. d’, a parameter used to assess the tuning quality of the TRF, was calculated as the difference in mean spike count within the tuning field and mean spike count outside the boundary of tuning field divided by their arithmetic average standard deviation  irregular and poorly tuned offset receptive fields found in AAF, which did not allow to designate offset BF with high accuracy and therefore any analysis of responses as a function of offset BF would be imprecise, (2) the fact that 9 kHz PTs evoked significant offset responses in almost all tested AAF but not A1 neurons irrespectively of their tuning  or HTs (9 + 18 + 27 + 36 kHz) with 4 ms cosine on and 0.01 ms cosine off ramps, which were varied in duration , ISI  and played at 60 dB SPL. Sound level ratio (in V) of harmonic components was as follow: 1f36. Briefly, mice were anesthetized with ketamine/xylazine (80 mg/kg). Following the initial microelectrode mapping, retrograde tracers, cholera toxin beta subunit (CTB) conjugated to either 594  or Alexa 488  were injected into A1 or AAF, (N = 2 for each region). After injections, the surgical area was closed and the animal allowed to recover for approximately 96 hours before transcardial perfusion. Animals were first perfused with 0.9% saline and then with a cold (4 °C) solution of 4% paraformaldehyde prepared in 0.1 M phosphate buffer (pH 7.4). Following perfusion, the brains were removed and placed in 30% sucrose for 1 to 3 days. Blocks containing the auditory cortex were cut with a vibratome in a modified horizontal plane that included A1 and MGB. Slices (80 µm) were visualized with a confocal laser-scanning microscope (Zeiss LSM700) with a Plan-apochromat 10×/0.8 NA objective.Retrograde tracing was performed as previously describedStatistical tests were performed with GraphPad Prism software version 7.03 . The standard error of the mean was calculated to quantify the amount of variation between responses from different populations. Nonparametric, unpaired Mann-Whitney test was used to calculate whether there were any significant differences between medians of recordings in A1 and AAF. Wilcoxon paired test or paired t-test were used to compare differences between paired values obtained in different treatments which have (paired t-test) or not have (Wilcoxon test) Gaussian distribution. Spearman correlation tests were used to test for significant associations between pairs of variables measured with rankings.Supplementary Information


Nanopore sequencing from Oxford Nanopore Technologies (ONT) and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) long-read isoform sequencing (Iso-Seq) are revolutionizing the way transcriptomes are analyzed. These methods offer many advantages over most widely used high-throughput short-read RNA sequencing (RNA-Seq) approaches and allow a comprehensive analysis of transcriptomes in identifying full-length splice isoforms and several other post-transcriptional events. In addition, direct RNA-Seq provides valuable information about RNA modifications, which are lost during the PCR amplification step in other methods. Here, we present a comprehensive summary of important applications of these technologies in plants, including identification of complex alternative splicing (AS), full-length splice variants, fusion transcripts, and alternative polyadenylation (APA) events. Furthermore, we discuss the impact of the newly developed nanopore direct RNA-Seq in advancing epitranscriptome research in plants. Additionally, we summarize computational tools for identifying and quantifying full-length isoforms and other co/post-transcriptional events and discussed some of the limitations with these methods. Sequencing of transcriptomes using these new single-molecule long-read methods will unravel many aspects of transcriptome complexity in unprecedented ways as compared to previous short-read sequencing approaches. Analysis of plant transcriptomes with these new powerful methods that require minimum sample processing is likely to become the norm and is expected to uncover novel co/post-transcriptional gene regulatory mechanisms that control biological outcomes during plant development and in response to various stresses. Nevertheless, specific new bioinformatics tools have been designed for several of the applications, which have not been part of second-generation sequencing pipelines. These tools are needed to provide greater flexibility to achieve different goals as well as to address new issues, such as higher error rates and low throughput. We present currently available bioinformatics methods for PacBio and ONT read analysis, including reads-of-interest (ROI) extraction, error correction , mapping+ RNA selection with capturing of 5′ capped mRNAs using a cap-binding protein  selected to construct PacBio long-read sequencing libraries using, e.g., Clontech SMARTer PCR kit . The len protein . Full-le protein . With thplatform . Compariplatform .At present, PacBio offers two fourth-generation sequencers: the RSII was the first commercially available sequencing instrument and the recently improved Sequel device provides much higher throughput (up to 20 Gb per SMRT Cell). PacBio’s sequencing strategy is based on the usage of zero-mode waveguide (ZMW) technology, which consists of tiny nano-wells initially described in 2003 . The ZMW1.Subsequently, the RSII system and the Sequel system store the base-call data and associated quality metrics in HDF5 and BAM files format, respectively. The bax2bam tool can convert HDF5 file format into BAM formatThe SMRT Analysis module from SMRT Link from PacBio is adopted for obtaining effective subreads . Then ex2. Compared to NGS or PacBio, the MinION is portable (weighs 90 g), real-time, long-read, and low-cost device. It is also possible to use the SMARTer protocol for full-length cDNA synthesis , which includes one MinION sequencer, two flow cells, one sequencing kit, and a wash kitynthesis , which i channel . Then ch4. Any computer with above minimal requirements can run a MinION without deterioration of performance during sequencing. Desktop or laptop computer with a MinKNOW and EPI2ME Desktop Agent installation provided by Oxford Nanopore and Metrichor Ltd., respectively, need to be connected with MinION  is one of the methods for error correction that relies only on long reads . Compare5. These are fast and accurate alignment tools for PacBio and Nanopore long reads with high insertion and deletion error rate. Transcriptome Analysis Pipeline from Isoform Sequencing (TAPIS)   also per (TAPIS)  and STAR (TAPIS)  are two  (TAPIS) .6. The majority of clustering strategies used for species without reference genome have been developed for ESTs, which appeared before the age of PacBio and ONT. Clustering programs designed for ESTs, such as UCLUST   and ISOdDrynaria roosii , alternative cleavage and alternative polyadenylation (APA), and alternative splicing (AS) events are three major processes that contribute to transcriptome diversity. AS of precursor mRNAs (pre-mRNAs) can potentially increase the number of protein isoforms produced from multiexon genes and regulate gene expression through multiple mechanisms such as altered translational efficiency of splice isoforms, non-sense-mediate decay, and miRNA-medicated mRNA degradation . Though Zea mays , >42,000. edulis , and >16iorrhiza . Additioauvignon , and ove. Moench . For 35.. Moench . In the  spliced .S. bicolor , skipped exon (SE), alternative 5′ splicing site (A5SS), alternative 3′ splicing site (A3SS), and mutually exclusive exons . In addi bicolor , Z. mays fusion  fusion  also pre) fusion .Nanlin 895”  including head-to-head, tail-to-tail, and fully overlapping types have been shown to function in transcriptional and post-transcriptional gene regulation . In totanscripts . Furthernscripts . At the nscripts .de novo assembling of grass Oropetium thomaeum  and 5-methylcytosine (m5C) profiles were reported using the m6A- or m5C-targeted antibodies, respectively, for RNA immunoprecipitation (RIP) followed by high-throughput sequencing (m6A-Seq/m5C-Seq) (Since full-length native RNA-Seq (nRNA-Seq) of ONT provides multiple benefits compared to NGS, this method has been applied for detecting viral transcriptomes , 16S rRN animals . In Arabm5C-Seq) . This RIm5C-Seq) .Saccharomyces cerevisiae (Glycine max) seed transcriptome has been sequenced using MinION sequencing. However, this study adopted cDNA sequencing method, which could not be used for characterization of RNA modifications (+ RNA (+ RNA(A)+ RNA uncovered a large number of novel isoforms   in mRNA from direct RNA-Seq data (The library construction protocol for direct RNA-Seq was designed for poly(A) transcripts . Steps irevisiae  revealedrevisiae . Howeverrevisiae . Thus, trevisiae . Also, bications . So far,s (+ RNA  and one  (A)+ RNA – have be novel) . The autSeq data .6A, m5C, 5-hydroxymethylcytosine  in RNA has been shown to alter the kinetics of nucleotide incorporation at the modified site. Based on this, it was suggested that by monitoring cDNA synthesis in real time in ZMWs modifications in RNA can be identified using the altered kinetic signature (From the Iso-Seq library construction step, it becomes apparent that the RNA modification information will be removed. Thus, common Iso-Seq libraries cannot be used for detecting RNA modification. Beside direct RNA-Seq, the PacBio reads from genome sequencing without any PCR amplification step can be used to detect DNA methylation marks, such as mcytosine , and 4-mcytosine , respecte manner . Howeversolution . ONT seqhia coli  and humahia coli . It can fication . By usinfication . Furtherignature .Previous studies have shown that it is difficult to reconstruct splice isoforms and quantify differential expression of isoforms using short reads obtained with second-generation sequencing . In compLZ, HZ, MK, KP, LG, and AR wrote, discussed, and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We found that m1A was present in the tRNA, but not in the genomic HIV-1 RNA and the abundant 7SL RNA. We were able to calculate that an HIV-1 virion contains per 2 copies of genomic RNA and 14 copies of 7SL RNA also 770 copies of tRNA, which is approximately 10 times more than thus far expected. These new insights into the composition of the HIV-1 virion can help in future studies to identify the role of nonprimer tRNAs in retroviruses. Moreover, we present a promising new tool for studying the compositions of virions.The mechanism of action of various viruses has been the primary focus of many studies. Yet, the data on RNA modifications in any type of virus are scarce. Methods for the sensitive analysis of RNA modifications have been developed only recently and they have not been applied to viruses. In particular, the RNA composition of HIV-1 virions has never been determined with sufficiently exact methods. Here, we reveal that the RNA of HIV-1 virions contains surprisingly high amount of the 1-methyladenosine. We are the first to use a liquid chromatography-mass spectrometry analysis (LC/MS) of virion RNA, which we combined with m Chemical RNA modification expand the repertoire of four natural nucleosides and influence RNA structure and function. The majority has been found in highly concentrated RNA moieties such as ribosomal (rRNA) or transfer RNA (tRNA). Nevertheless, there is growing evidence that coding messenger RNA (mRNA) and regulatory RNA also contain various chemical modifications2. Their existence in mRNA is complicated to be proved by common LC/MS technique but special and selective profiling or capturing techniques for various RNA modification have been developed lately. In particular, pseudouridine has been found in the mRNA thanks to the development of the selective profiling techniques Pseudo-seq.3 and Ψ-seq.4. Also, the widespread presence of 5-methylcytosine across the human transcriptome has been demonstrated by a bisulphite technique combined with deep sequencing5. A to I editing of the mRNA has been confirmed by Inosine chemical erasing6. The NAD captureSeq.7 allowed identification of RNAs with covalently attached Nicotinamide adenine dinucleotide (NAD) at 5′ end. So far, this method has enabled the detection of NAD in sRNA, and mRNA in E. coli7 and in eukaryotes9.To date, more than 140 chemical RNA modifications are known6A) in mRNA11 paved the way for the emerging field of epitranscriptomic. Another common epitranscriptomic mark in mRNA was supposed to be 1-methyladenosine (m1A)13. As well as for m6A detection the specific antibodies have been used to detect m1A in the human transcriptome. The problem of immunoprecipitation methods is that they do not allow for single base resolution of particular RNA modification. The authors claimed identification of thousands of transcripts bearing m1A in eukaryotic cells. A subsequent study indicated that the m1A is only present in 15 sites of the mRNA, in the previously known T-loop of tRNAs, and in rRNA from HEK293T cells14. Apart from immunoprecipitation, the authors of the later method employed two types of reverse transcriptases enabling single-base resolution.The development of a profiling technique for 6-methyladenosine , dengue, Zika, yellow fever and West Nile virus. The m6A modulated infection by HCV and the depletion of m6A methyltransferases or m6A demethylase was found to increase or decrease the HCV particle production, respectively. A photo-crosslinking-assisted m6A sequencing technique was used to precisely map m6A editing sites on the HIV-1 genome16. The m6A sites were found to cluster in the HIV-1 3′ untranslated region and to recruit YTHDF “reader” proteins. The m6A editing and the recruitment of YTHDF proteins were identified as the positive regulators of HIV-1 mRNA expression.So far, m6A can be employed by viruses. At first, we focused on the viral packageome of HIV-1. It was believed that HIV-1 packs in virion not only two copies of genomic RNA, but also 12–14 copies of guiding 7SL RNA18, 8 copies of tRNALys3 that serve as primer for its reverse transcriptase21, around 12 copies of tRNALys1,2, around 50 copies of rather randomly packed tRNAs and some fragments of other mRNA, rRNA etc26. In total, the host cell-derived RNA should comprise up to half of the virion RNA by mass28.As new layers of RNA modifications are constantly being uncovered in the transcriptome, we can presume that other RNA modifications besides m1A  in the packageomic RNA that corresponded to approximately 340 1-methyladenosines , 2′-O-methyladenosine (Am) and 2-methylthio-N6-threonylcarbamoyladenosine (mS2t6A) adenosine, N6-isopentenyladenosine, Supplementary Table 29). Analysing the synthetic standards of m6A, m1A, Am and t6A, we were able to extrapolate their proportional representation in the digest .Using LC/MS analysis, we observed methylated adenosines. menosine mt6A 12 that is normally read as A by reverse transcriptase. We prepared three deep sequencing libraries using SuperScriptTM III reverse transcriptase, which should either misread m1A or arrest meeting m1A. One deep sequencing library was prepared with TGIRTTM reverse transcriptase, which should confirm the m1A position at base resolution.To investigate the exact positions of mIn the deep sequencing protocol Fig. , we used1A in position 58 of tRNALys TTT-3.1., tRNALys CTT-2.1., tRNALys CTT-1.1. and tRNAAsn GTT-2.1., as well as in position 9 of tRNAAsp GTC-2.1.. Based on a previous report30, we expected the reverse transcriptase to be paused (observed arrest) when meeting m1A. However, pattern of misreading for m1A in artificial sequences read by reverse transcriptase was also reported32. We observed this behavior of reverse transcriptase in the position 58 of some tRNAs. Approximately 70% of the m1A in position 5834 was misread . In the sample that was treated with basic conditions, the misreading of position 58 was only approximately 30%  was observed only in the experiment with TGIRTTM. In order to verify that the m1A profiling method works in our hand also when long RNAs are used, we tested the protocol on 28S rRNA from HEK293T cells. In this case, we observed arrest of the reverse transcriptase meeting m1A in position 1317 in 80%. This is contrary to the reverse transcriptase TGIRTTM, which misread the m1A mainly as G in 28S rRNA  the misreading pattern, ii) the arrests of reverse transcriptase, and iii) a similar motif to GUUCNANNC  in 7SL RNA and the genomic HIV-1 RNA  as well as for the 5′ end of tRNALys TTT-3.1. As the 5′ end of tRNALys TTT-3.1 and tRNALys CTT-1.1 (2.1) is similar, the 5′ end probe hybridize to all three tRNAs. We also prepared ladder with the sequence of tRNALys TTT-3.1 by in vitro transcription. The ladder helped us to estimate the size of observed bands. To rule out that the observed fragment in the deep sequencing data was caused by the fragmentation step, we analysed the RNA isolated from the viral particles and from the MT4 cells infected by HIV-1 before and after fragmentation. We observed the presence of 3′-tRNA fragments only in the RNA from viral particles after fragmentation 1A in its sequence, we were able to quantify the number of all tRNAs in the viral packageome to be around 770. It is, however, important to mention that others often studied viral particles produced from different cells such as COS-7 cells38 or CEM-SS cells40, or produced by transfection and not infection22. Those factors can influence the composition of viral particles.In other works, the quantification of further tRNAs co-packed by an HIV-1 virion was usually omitted. Based on the knowledge that every tRNA contains one m1A, 35 other RNA modifications41. The authors, however, conclude this fact only based on MS data, which means that the samples could be contaminated with heavily modified tRNA molecules. tRNALys TTT-3.1., for example, can be bound so tightly to gRNA that it is not possible to separate the pure genomic RNA. Another error could have been introduced in the preparation of the viral samples. As the authors used only transfection, the plasmid containing the HIV-1 genomic sequence could have been fully transcribed into RNA. Based on our screening of HIV-1 sequence, we know that the additional plasmid sequence pNL4-3 contains the original motif GUUCNANNC, which might be methylated. However, we cannot exclude that a certain position in the genomic HIV-1 is methylated from less than 5% as our profiling technique would not be able to track it. However, this amount of m1A would not significantly contribute to the observed LC/MS signal. It is also arguable, whether such level of methylation would have any biological significance.A recent report on RNA modifications in viral RNA that was purified via selective probes indicates that the genomic RNA of HIV-1 can contain besides m1A in LC/MS analysis. Pyrosequencing and recently also deep sequencing methods of small non-coding RNAs in HIV-1 infected cells have proven the existence of a very abundant 18-nt tRNA fragment from tRNALys TTT-3.1. that is complemental to the HIV-1 primer binding site43. Rather than being a primer of reverse transcription, this tRNA fragment when overexpressed led to a decrease in viral replication. Nevertheless, the concentration of these tRNA fragments was so low that we were neither able to observe the fragments in isolated RNA from infected cells nor in packageomic RNA. Other types of 3′ tRNA fragments were observed only in Northern blot analysis of fragmented RNA. Therefore, we also ruled out the possibility that m1A can come from any type of tRNA fragments.In our work, we also considered the existence of 3′ tRNA fragments that could contribute to observed high amount of mLys TTT-3.1. is quite well understood, the function of nonprimer tRNAs such as tRNALys CTT-2.1., tRNALys CTT-1.1., tRNAAsp GTC-2.1. or tRNAAsn GTT-2.1. is still very enigmatic. One of the hypothesis suggests that the matrix domain of the HIV-1 Gag protein binds almost exclusively to specific tRNAs in the cytosol44. tRNAs shield the membrane-binding surface of matrix and regulate the interaction with intracellular membranes prior reaching the plasma membrane27. Cellular tRNAs might also help to import HIV-1 intracellular reverse transcription complex to nucleus45. Another theory suggests that the enrichment of particular tRNAs in virion can manipulate with tRNA pool in cells and enhance production of the Gag polyprotein and virus production in general46. In any case, the importance of nonprimer tRNAs in virion is very high. In this work, we bring new tool – LC/MS in combination with deep sequencing –to study the virion RNA composition. Specific RNA modifications such as m1A can bring us missing information layer and in future, we can calculate the RNA composition of other viruses. Moreover, these techniques can be used for studies of an impact of viral infection on various RNA modification in host transcriptome.As the role of primer tRNA2/95% air. The MT4 cell line was cultured in RPMI 1640 w/o L-Glutamine and 25 mM Hepes supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 µg/mL) . The HEK293T cell line was cultured in DMEM high glucose supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 µg/mL) .The human CD4 + T-cell line MT4  and the human embryonic kidney HEK293T cell line  were cultured under standard conditions at 37 °C under a humidified (>90%) atmosphere of 5% COMT4 cells were collected by centrifugation , HEK293T cells were collected by trypsinization and subsequent centrifugation as above. Cells pellets were washed with PBS and cells were lysed with RNAzol reagent (Sigma-Aldrich). Large and small RNA fractions were purified according to the RNAzol manufacturer’s protocol. The RNA concentration was determined on NanoDrop ONE (ThermoFisher Scientific) and the RNA sample quality control was performed on a 4200 TapeStation System (Agilent).5 cells per mL) at a ratio of 1:9. The co-culture was synchronized by three successive additions of infected culture supernatant to uninfected MT4 cells .MT4 cells were initially infected with a cell-free HIV-1 strain NL4-3, which was generated by transient transfection of HEK293T cells with a pNL4-3 plasmid . The infected cultures were subsequently expanded by co-cultivation. 48 h post-infection, cell culture supernatants containing viral particles and infected cells were added to uninfected MT-4 cells  and filtration (0.45 µm pore size cellulose-acetate filter (VWR)), and stored at −80 °C.47.Infectious titres were determined as 50% tissue culture infectious dose by endpoint titration using serial 10-fold dilutions of the virus on TZM-bl cells2 25 mM, CaCl2 25 mM) with DNase I , RNase I (200 U/mL) and RNase A  and incubated 2 h at 37 °C. RNase/DNase treatment was stopped by adding RNAzol (Sigma-Aldrich).Virus particles were concentrated from cleared culture medium by centrifugation through a cushion of 20% (wt./wt.) sucrose in phosphate-buffered saline (PBS) . The pellet was resuspended in RNase/DNase buffer  and ultracentrifuged . HIV-containing fractions were detected by western blot analysis using the anti-HIV capsid protein antibody. Selected fractions were collected, diluted with PBS five times and the virus was pelleted by ultracentrifugation . The virus-containing pellet was resuspended in RNase/DNase buffer and RNase/DNase treatment was performed as described above.Samples from each fraction of the Optiprep gradient were mixed with loading buffer in the ratio of 5:1 and denatured for 5 min at 95 °C. Separation was carried out at 15% SDS PAGE at constant voltage 150 V for 90 min. Separated proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad) and blocked at casein . HIV-1 CA protein was detected with polyclonal anti HIV-1 CA antibody  and as secondary antibody was used Goat anti Rabbit IgG labelled with horseradish peroxidase . After washing, the membranes were incubated with SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) and the intensity of chemiluminescence was detected using the CCD camera   according to the manufacturer’s protocol. The quality of RNA samples was checked by HS RNA ScreenTape.  were fully digested by Nuclease P1  in 40 µL of 50 mM ammonium acetate buffer (pH 4.5) for 1 h at 37 °C. After addition of Alkaline phosphatase  and CutSmart buffer , the samples were incubated for another 1 h at 37 °C. Digested RNA samples were diluted in 200 µL and purified over Microcon® – 10 kDa centrifugal filters (Merck). The flow-through was concentrated using a SpeedVac system to the volume of 20 µL for LC-MS analysis.48. The gradient was 0–6 min, 100% A; 6–7.5 min, 100–99% A; 7.5–9.5 min, 99–94% A; 9.5–15 min, 94% A; 15–25 min, 94–50% A; 25–27 min, 50–20%; 27–29.5 min, 20% A; 29.5–30 min, 20–100% A; 30–40 min, 100% A. The flow rate was 0.05 mL/min. The autosampler cooled the samples to 8 °C. The LC system was coupled on-line to a mass spectrometer  to acquire masses of nucleosides by electrospray ionisation. Ions were scanned in a positive polarity mode over full-scan range of m/z 100–1200. The source parameters were as follows: capillary voltage, 3 kV; source temperature, 150 °C; sampling cone, 40; extraction cone, 5; desolvation temperature, 450 °C; desolvation gas flow, 600 L/h.All samples were measured in technical duplicates. The separation of the digested RNA samples (8 µL injection volume) was performed by an LC system  on a C18 column  at 40 °C using a gradient of water (A) and acetonitrile (B), each containing 0.1% (v/v) formic acid1A, m6A, Am, A, t6A; Jena Bioscience, Sigma-Aldrich, CarboSynth) at three different amounts: 64, 320 and 1600 fmol each were injected on a column to compare the response of each nucleoside under defined ionization conditions. Mixtures were measured in the technical triplicates. For each standard, an extracted ion chromatogram (EIC) was generated using a major fragment observed in its full scan spectrum (fragmentation occurs in the ion source). The chromatographic peaks in EICs were integrated. The standard peak area  was used to calculate the ionization efficiency ratio of the tested nucleosides in this concentration range  was used to achieve size distributions of the fragments from 50–200 nt. RNA samples (1–2 µg) were incubated with 100 mM ZnCl2CO2, 2 mM EDTA, pH 10.4) for 1 h at 60 °C to convert m1A into m6A via Dimroth rearrangement. Samples were purified by RNA Clean & Concentrator columns (Zymo) and analysed on HS RNA Screen Tape®.One half of each sample was incubated with alkaline buffer  for 30 min at 37 °C. The whole procedure was repeated, followed by final heat deactivation of the enzyme at 75 °C for 5 min.After denaturation of the RNA samples at 90 °C for 30 s, followed by cooling down on ice, dephosphorylation was performed by 0.5 U of FastAP alkaline phosphatase (ThermoFisher Scientific) in dephosphorylation buffer  was performed in the First-Strand Buffer, containing RT primer (5 µM), dNTP mix , DTT (5 mM), BSA (50 µg/µL) in 30 µL reaction mixture for 1 h at 50 °C. RT primer excess was digested by a combination of Lambda exonuclease, Exonuclease 1 and FastAP alkaline phosphatase  after reaction. RNA was degraded by NaOH and samples were ethanol precipitated.2, 20 mM Tris-HCl, pH 7.5), with DTT (5 mM) and RT primer (5 µM) for 30 min at room temperature. After 30 min dNTPs  were added and the reaction was incubated at 60 °C for 50 min. The reaction was stopped by the addition of 1 µL of 5 M NaOH and was incubated for 3 min at 95 °C. Samples were cooled down at room temperature, neutralized with 1 µL of 5 mM HCl and ethanol precipitated.Reverse transcription with TGIRT™-III  was performed in 19 µL of reaction buffer  was done in 50 mM Tris-HCl buffer (pH 7.4), containing 10 µM ATP and 20 mM MgCl2 with T4 DNA ligase (1.5 U/µL) at 4 °C for 72 h. The enzyme was heat deactivated at 65 °C for 10 min. Samples were ethanol precipitated and dissolved in 12 µL of water (molecular biology grade).3′-Tailing of cDNA was performed using 1 U/µL of Deoxynucleotidyl transferase  in 1x TdT buffer containing 1.25 mM CTP for 30 min at 37 °C with heat deactivation . Double stranded DNA anchor was prepared as described previouslyTaq DNA Polymerase (NEB) in 20 µL of total reaction mixture. Initial denaturation was performed at 95 °C for 60 s, following by annealing for 60 s at 54 °C, elongation for 60 s at 68 °C and denaturation for 30 s at 95 °C. Final extension was performed at 68 °C for 5 min.PCR amplification was performed with barcoded PCR primers (Supplementary Table PCR reaction mixture was loaded on 1.3% agarose gel (140 V for 2 h). Fractions between 100–400 nt were cut and DNA was extracted from the gel by NucleoSpin® Gel and PCR Clean-up (Macherey –Nagel).50 and Atropos51. Clean sequences longer than 20 bp were mapped to human genome and HIV-1 genome using bwa aligner v0.7.10-r78952. All statistics and graphs were generated by custom scripts (github:).Libraries were trimmed using cutadapt2PO4, 180 mM Na2HPO4, 7% SDS, 1% BSA, 1 mM EDTA, pH 7.2) at 45 °C for 1 h using a ProBlot hybridization oven (Labnet). Meanwhile, 5 μL of 100 μM probe (Sigma-Aldrich) was end-labelled using 20 U of T4 polynucleotide kinase (NEB), 2 μL of γ-32P-ATP  in 20 μL of supplemented kinase buffer. The labelling was performed at 37 °C for 30 min. The enzyme was inactivated at 65 °C for 5 min and the probe was purified from unincorporated nucleotides using Micro Bio-Spin P-30 columns (BioRad) according to the manufacturer’s instructions. The probe was added to the membrane in 10 mL of fresh Church buffer and hybridized at 45 °C overnight. The probe was washed twice for 10 min each with low stringency buffer (2x SSC + 0.1% SDS) and once with high stringency buffer (0.1x SSC + 0.1% SDS), all at 45 °C. The membrane was sealed in foil, incubated with phosphor imaging plate  and read using Typhoon FLA 9500 .Isolated RNA from HIV-1 particles and total RNA isolated from infected MT4 cells were each divided in two parts. One part was chemically fragmented as described for the deep sequencing libraries. Denaturing acrylamide gel  was prepared from 19:1 acrylamide:bis-acrylamide in 0.5x MOPS buffer  and 7 M urea. The polymerized gel was pre-run at 100 V for 30 min in 0.5x MOPS buffer. 15 µL of the RNA samples  were denatured for 15 min at 55 °C and loaded into the gel wells. The gel was initially run at 50 V for approximately 15 min to concentrate the samples in wells, and then at 150 V until the bromphenol blue reached 90% of the gel length. The gel was blotted onto a charged nylon membrane  by capillary transfer in 20x SSC buffer  overnight. The membrane was crosslinked twice on a default setting  using electronic ultraviolet crosslinker . The crosslinked membrane was hybridized with 10 mL of Church buffer (70 mM NaHSupplementary Information


The exchange of extracellular vesicles (EV) between immune cells plays a role in various immune regulatory processes. EV are nano-sized lipid bilayer-enclosed structures that contain a multitude of proteins and small non-coding RNA molecules. Of the various RNA classes present in EV, miRNAs have been most intensively studied because of their known gene-regulatory functions. These miRNAs constitute only a minor part of all EV-enclosed RNA, whereas other 20–200 nt sized non-coding RNAs were shown to be abundantly present in EV. Several of these mid-sized RNAs perform basic functions in cells, but their function in EV remains elusive. One prominent class of mid-sized extracellular RNAs associated with EV are the Y-RNAs. This family of highly conserved non-coding RNAs was initially discovered as RNA component of circulating ribonucleoprotein autoantigens in serum from Systemic Lupus Erythematosus and Sjögren's Syndrome patients. Y-RNA has been implicated in cellular processes such as DNA replication and RNA quality control. In recent years, Y-RNA has been abundantly detected in EV from multiple different cell lines and biofluids, and also in murine and human retroviruses. Accumulating evidence suggests that EV-associated Y-RNA may be involved in a range of immune-related processes, including inflammation, immune suppression, and establishment of the tumor microenvironment. Moreover, changes in plasma levels of extracellular Y-RNA have been associated with various diseases. Recent studies have aimed to address the mechanisms underlying their release and function. We for example showed that the levels of EV-associated Y-RNA released by immune cells can be regulated by Toll-like receptor (TLR) signaling. Combined, these data have triggered increased interest in extracellular Y-RNAs. In this review, we provide an overview of studies reporting the occurrence of extracellular Y-RNAs, as well as signaling properties and immune-related functions attributed to these RNAs. We list RNA-binding proteins currently known to interact with Y-RNAs and evaluate their occurrence in EV. In parallel, we discuss technical challenges in assessing whether extracellular Y-RNAs are contained in ribonucleoprotein complexes or EV. By integrating the current knowledge on extracellular Y-RNA we further reflect on the biomarker potential of Y-RNA and their role in immune cell communication and immunopathology. Extracellular vesicles (EV) are 50–300 nm sized lipid bilayer-enclosed vesicles containing proteins and nucleic acids , which aIt has been shown that EV-enclosed RNAs can be functionally transferred to target cells –11. ManyEV constitute a unique way in which molecular messages are exchanged between cells. Upon transfer, the lipids, proteins, and RNA associated to EV can modify the function of recipient cells , 26–28. It is important to realize that not all extracellular RNA is associated with EV. Other macromolecular structures in the extracellular milieu, such as ribonucleoproteins (RNPs) and lipoprotein particles, also contain RNA , 29–31. It has been demonstrated that both the protein- and miRNA composition of EV can change upon exogenous stimuli imposed on EV-producing cells , 13, 16.in vitro and in vivo studies have demonstrated that intercellular transfer of EV-associated miRNA and mRNA leads to changes in recipient cell function   . In the  exosome . It was  exosome .Interestingly, it was recently discovered in neuronal cells that Y3-RNA can act as a molecular sponge for the enhancer protein HuD (ELAVL4) . HuD canIn cells, Y-RNA does not only occur in its full length form, but has also been shown to be cleaved into specific fragments of 25–35 nt. This cleavage, which is carried out by the enzyme RNase L  Figure , box 7 oTaken together, the highly conserved family of Y-RNAs interacts with, and regulates the localization and activity of various RNA-binding proteins involved in basic cell functions.in vitro cultured primary and immortalized cell types, as well as in various biofluids  to 4 in Table The data in Tables ** . Additionally, protein binding may shield motifs in Y-RNA which may trigger cellular RNA sensors. For instance, the La-protein potentially shields the triphosphate moiety , which m** . As ment** . Similar** , 96. Twowww.microvesicles.org), a repository for extracellular vesicle proteomics data , for example, dissociates miRNA-122 from AGO2/mRNA complexes in hepatocytes and drives subsequent expulsion of miR-122 from the cell via EV . Furthernditions . FurtherIn conclusion, a large number of proteins known to interact with Y-RNA have been detected in EV. Some of these proteins may be involved in sorting of Y-RNA into EV, but the underlying mechanisms should be further explored Figure , box 10.Extracellular Y-RNA has not only been detected in EV and RNP, but also in various retroviruses such as murine leukemia virus (MLV) and human immunodeficiency virus (HIV) –108 Fig, 110. InRetroviruses use specific host tRNAs to prime reverse transcription, which is a key step in the retroviral life cycle . In addiThe high abundance of Y-RNA in EVs and RNPs raises the question whether extracellular Y-RNAs have signaling functions when transferred to target cells Figure , box 12.Table in vivo induced IL10 release and reduced damage in a myocardial infarction model in rats  into target cells as a model for EV-mediated transfer of these RNAs. Although this may currently be the most feasible approach, several limitations can be identified. The naked, synthetic RNAs employed in these studies are not complexed to proteins, whereas RBP may play a role in the function of truly EV-associated Y-RNAs. Additionally, the route of uptake of lipofected RNA complexes may be different from EV-enclosed RNAs, resulting in delivery of the Y-RNA to subcellular locations other than those reached after EV-mediated delivery.Heligmosomoides polygyrus contain high levels of nematode stem-bulge RNAs (which are related in sequence to Y RNAs) and suppress cytokine release in mice  , which a in mice . Further in mice .While EV-associated Y-RNAs seem to induce a range of different immune-related effects, circulating RNP containing Y-RNA predominantly induce immune activation. Many of these effects reportedly depend on TLR-mediated triggering Figure , box 13.In vitro cultured macrophages treated with lipids release increased levels of fragmented Y-RNA into the medium (Besides triggering inflammation in SLE and SS, extracellular Y-RNA complexes have also been demonstrated to induce apoptosis in atherosclerosis  and cance medium . Hizir ee medium , box 16.e medium . Not onlThe studies described above suggest that the functional effects of extracellular Y-RNAs depend on both the macromolecular structure to which it is associated and the conditions under which the Y-RNA is released. In addition to the TLR-mediated effects of Y-RNA that have been reported to date Figure , box 17,The abundance of circulating Y-RNA in body fluids has triggered interest in the potential use of Y-RNA as biomarker for disease. Increased levels of Y-RNA have been observed in the circulation of cancer patients , 25, 31.−/−mice used as a model for atherosclerosis (Increased levels of circulating extracellular Y-RNA have also been observed in the context of atherosclerosis and coronary artery disease. Repetto and colleagues observed a higher number of 5′ Y1-fragments in the blood of ApoEclerosis . Likewisclerosis . This raclerosis  and platclerosis . Howeverclerosis , therebyclerosis . Thus, tIn conclusion, differences in circulating Y-RNA may be further explored as biomarkers for disease, but it is critically important to evaluate and standardize the various methods used to isolate different carriers of Y-RNA in body fluids. Additionally, acquisition of knowledge on how disease-associated changes in cells affect the release of Y-RNAs will help to better understand their biomarker potential.Current data suggest that the family of Y-RNAs does not only play a role in intracellular processes to maintain cell function, but also acts as versatile intercellular messengers. Various studies have indicated that extracellular transport of Y-RNA is a highly efficient process employed by many different cell types. Additionally, Y-RNA is one of the most abundant extracellular non-coding RNAs in human plasma. Such extracellular RNA can occur in RNP or in EV. One potential trigger that regulates extracellular release of Y-RNA is TLR signaling. Moreover, currently available data suggest functional involvement of extracellular Y-RNA in various immune-related processes. Y-RNAs can bind to several different proteins. We here provided an overview of Y-RNA binding proteins that occur inside cells and in Y-RNA-containing RNP or EV released by cells into the extracellular milieu. We propose that binding to these proteins not only determines how Y-RNA regulates cellular processes, but may also drive their sorting into EV and could be essential for functional effects of Y-RNA transferred to recipient cells Figure . Partly EN performed literature research, drafted the manuscript, and edited the text. TD performed literature research, made the inventory of Y-RNA binding proteins in EV by searching data repositories, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Oreochromis mossambicus) larvae along 12 days of development, from pre-hatching to the completion of yolk sac absorption. Gene expression analysis revealed differential and complimentary time-dependent expression of the PepT1 variants and PepT2 along the larval development period. Immunofluorescence analysis showed differential protein localization of the three peptide transporters (PepTs) along the gastrointestinal tract, in a similar pattern to the adult. In addition, PepT1a was localized in mucosal cells in the larvae esophagus, in much higher abundance than in the adults. The results of this study demonstrate specialization of intestinal sections and absorbance potential of the enterocytes prior to the onset of active exogenous feeding, thus pointing to an uncharacterized function and role of the gastrointestinal tract and its transporters during the larval period.Fish larvae differ greatly from the adult form in their morphology and organ functionality. The functionality of the gastrointestinal tract depends on the expression of various pumps, transporters, and channels responsible for feed digestion and nutrients absorption. During the larval period, the gastrointestinal tract develops from a simple closed tube, into its complex form with differentiated segments, crypts and villi, as found in the adult. In this study, we characterized the expression of three peptide transporters  in the gastrointestinal tract of Mozambique tilapia ( Dicentrarchus labrax)  (Oreochromis mossambicus) (Oreochromis niloticus) (Sparus aurata) (Gadus morhua)   (Danio rerio) has been established as a model organism for various developmental studies. However, when addressing feeding and nutritional physiology, this species is not an ideal model  , Senegalalensis) , Mozambiambicus) , Nile tiloticus) , 2002, g aurata) , Atlanti morhua) , Califorornicus) , and Sumentatus) . In the entatus) . The zebal model . Unlike Tilapia is an important group in the cichlid family with increasing research interest in its physiology, genetics, and regulatory processes . FujimurProtein is an important nutrient in fish diets, as it supports energetic supply for physiological processes and growth, in addition to tissue and protein construction . In addislc15a genes. These transporters are mainly known for their important role in di- and tri-peptides absorption into the enterocytes – the epithelial cells of the intestine. In mammalians, two peptide transporters have been found, PepT1 and PepT2, but in fish, there are three transporters, with two PepT1 paralogs . PepTs are solute carriers-proton-dependent transporters, members of the POT family and coded by the paralogs . All thrparalogs .In recent years, there has been increased interest and research on PepTs’ participation in protein absorption in fish under different environmental and dietary conditions . HoweverMozambique tilapia fish used in this study were derived from a stock maintained at the aquaculture facility of the Agricultural Research Organization (ARO). This stock originated from Natal, South Africa, and was brought to Israel in the 1970s.This study was approved by the Agricultural Research Organization Committee for Ethics in Experimental Animal Use, and was carried out in compliance with the current laws governing biological research in Israel .Tissue samples of gills, esophagus, stomach, liver, spleen, muscle, heart, intestine, kidney, skin, brain, pituitary, and fat were taken from four males Mozambique tilapia (54 ± 3 g). The intestine from each fish was divided into three segments; anterior intestine (AI), middle intestine (MI), and posterior intestine (PI). The tissues were kept in 1 ml of RNA-save buffer  at −20°C until RNA extraction procedure.Breeding families consisting of one male and 4–6 females, in 200 L aquaria, were constantly monitored to observe spawning. Two days after spawning and fertilization (2 dpf), eggs were removed from the females’ buccal cavities and transferred to hatching jars. Each experiment was conducted on full-sibs from a single spawn.In order to determine the main tissues expressing the peptide transporters in the larvae, 60 larvae (full sibs from a single spawn) were sampled at 9 dpf and dissected using microsurgery under binocular. The yolk sac was removed and the gastrointestinal tract was separated from the larvae body. Tissue samples (GI tract and Larvae body) from 10 larvae were pooled (six replicas per tissue), and stored in 1 ml of RNA-save buffer at −20°C until RNA extraction procedure.In order to track the expression of the PepT transcripts along the embryonic period, six embryos from an additional spawn were sampled daily, commencing from 3 dpf to 14 dpf. Each larva served as an individual biological replica. The larvae were kept in 1 ml of RNA-save buffer at −20°C until RNA extraction procedure.For immunofluorescence staining, a second time-course experiment was conducted. Larvae from a single spawn were sampled each day, between 6 to14 dpf. The larvae were fixed in 4% PFA for 10 min and then dried on paper. The yolk sac was removed using microsurgery under binocular while making sure that the gastrointestinal tract remained untouched. Following the yolk sac removal, the larvae were incubated in 4% PFA for 24 h at 4°C, followed by two washes in PBS, 50% ethanol, and stored in 70% ethanol at 4°C. The larvae were then dehydrated through a series of graded ethanol baths to displace water . Samples were then embedded in paraffin and 5-μm sections were cut using a microtome and placed on microscope slides. The slides were incubated overnight on a 39°C heated plate and were stored at 4°C until staining.Total RNA was extracted using Trizol reagent, purified from DNA contamination using TURBO DNA-free Kit (Ambion), quantified with Nano-Drop spectrophotometer (Thermo Scientific), and then reversed transcribed into cDNA using Verso cDNA Synthesis Kit (Thermo Scientific).For each gene, forward and reverse primers for qPCR analysis  were tesTissues sections slides from larvae and adults were stained according to the protocol described by Post hoc comparisons among groups were performed using the Tukey-Kramer HSD for tissue distribution and larvae time-course real time results, and Student’s t-test for the ratio between the GI tract and the larvae body (α = 0.05). Data are presented as means ± SEM.Statistical analyses were conducted separately, for each experiment and each expressed gene, using one-way analysis of variance (ANOVA). p < 0.0001), while PepT2 expression was detected in the MI and PI segments. PepT2 was also found to express in the kidney but in lower level compare to the MI segment (p < 0.0001). The expression level in the rest of the tissues was significantly lower (p < 0.0001) than in the intestine (p < 0.0001) of the PepT variants in comparison to the larvae body. The ratio between expression levels in the GI tract and the larvae body was approximately 565 for PepT2, 46,000 for PepT1a, and 140,000 for PepT1b transcripts  (Fundulus heteroclitus)  , killifioclitus) , and Euroclitus) . These toclitus) . The conImmunofluorescence localization of the PepT1a in the esophagus was unexpected, as PepT1 expression was reported mainly in the intestine , while tIn fish, the esophagus has an important role in maintaining salt and water balance. It has been shown that the desalination of seawater starts at the esophagus, which exhibit a low membrane permeability to water together with active and passive absorption of ions [reviewed by The localization of PepTs on the apical membrane of the enterocytes, as seen in the immunofluorescence staining, demonstrates that the intestine can uptake peptides even in the pre-feeding larvae. Our results indicate that in addition to the yolk sac absorption there might be another source of nutrients supplementation, even at early developmental stages. These nutrients might be of exogenous , endogenous, or of microbial source. This hypothesis, as well as the nutritional and developmental significance of intestinal nutrient absorbance for the larva, needs further study.Cyprinus carpio), the addition of small peptides to the feed, increased the expression of PepT transcript and the abundance of cholecystokinin (CCK) secreting cells in the intestine .PC and AC conceived and designed the experiments. PC, TN, and TS bred, sampled and handled the fish. PC performed the microsurgery, gene expression, and immunofluorescence analyses. AC and SH secured funding and supervised the project. PC and AC wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.