Wolbachia-Drosophila system, this bacterial mRNA enrichment yielded a 3-fold increase in Wolbachia mRNA abundance and a concomitant 3.3-fold increase in the percentage of transcripts detected. More specifically, 70% of the genome could be recovered by transcriptome sequencing compared to 21% in the total RNA. Sequencing of similar bacterial mRNA-enriched samples generated from Ehrlichia-infected canine cells covers 93% of the Ehrlichia genome, suggesting ubiquitous transcription across the entire Ehrlichia chaffeensis genome. This technique can potentially be used to enrich bacterial mRNA in many studies of host-microbe interactions.Despite numerous advances in genomics and bioinformatics, technological hurdles remain to examine host-microbe transcriptomics. Sometimes the transcriptome of either or both can be ascertained merely by generating more sequencing reads. However, many cases exist where bacterial mRNA needs to be enriched further to enable cost-effective sequencing of the pathogen or endosymbiont. While a suitable method is commercially available for mammalian samples of this type, development of such methods has languished for invertebrate samples. Furthermore, a common method across multiple taxa would facilitate comparisons between bacteria in invertebrate vectors and their vertebrate hosts. Here, a method is described to concurrently remove polyadenylated transcripts, prokaryotic rRNA, and eukaryotic rRNA, including those with low amounts of starting material (e.g. 100 ng). In a  Rickettsia pathogens that cause Rocky Mountain spotted fever, typhus, and other spotted feversOrientia tsutsugamushi that causes scrub typhusEhrlichia chaffeensis that causes EhrlichiosisAnaplasma phagocytophilum that causes AnaplasmosisRickettsia endosymbionts3Wolbachia endosymbionts5Ehrlichia chaffeensis and Anaplasma phagocytophilum may be endosymbionts of ticks but pathogenic to humansWolbachia endosymbiont of Drosophila ananassae wAna and its invertebrate fruit fly host, Ehrlichia chaffeensis Wakulla infected canine cell culture, and the Wolbachia endosymbiont of Brugia malayi wBm and its invertebrate nematode host.Many important biological interactions and diseases arise from a diverse variety of obligate intracellular bacteria. This may be best epitomized by the bacteria in the order Rickettsiales, which includes several Category ABC Wolbachia endosymbionts are intracellular endosymbionts of many arthropods and filarial nematodes5Wolbachia strains can induce parthenogenesis, male killing, feminization, and cytoplasmic incompatibility in arthropods and can be mutualistic in some insects and filarial nematodes5Wolbachia strains have led to several studies on their use as biocontrol agents targeting arthropod vectors8Wolbachia hosts have also been found to have extensive lateral gene transfer from the bacteria to the host11Drosophila ananassae line from Hawaii13Ehrlichia chaffeensis, the causative agent of Ehrlichiosis, is a bacteria found in the lone star tick, Amblyomma americanumEhrlichia is found in tick vectors and in vertebrate reservoirs, such as white-tailed deerDrosophila ananassae Hawaii lineWolbachia-colonized Drosophila ananassae has only a very small amount of bacterial 23S rRNA that can be detected using an Agilent Bioanalyzer  in the three replicates examined  D. ananassae reads mapping in the total RNA, 1,923 (0.02%) Wolbachia reads and 9,318,954 (99.98%) D. ananassae reads mapping in the polyA-enriched RNA, and 62,089 (1.0%) Wolbachia reads and 6,029,306 (99%) D. ananassae reads mapping in the bacterial mRNA-enriched samples, as reported by IDXSTATS in MPILEUP.Following the successful assessment of bacterial mRNA enrichment by qRT-PCR and microfluidics, three samples were prepared for Illumina MiSeq sequencing: total RNA, polyA-selected RNA, and the bacterial mRNA-enriched RNA. The MiSeq reads were mapped with BWA MEMWolbachia reads is not higher in the bacterial mRNA-enriched sample when compared to total RNA, it is important to remember that the bacterial rRNA has been depleted, indicating more of this percentage should arise from bacterial mRNA than in the other samples. Thus, while the reads from the total RNA sample and polyA-enriched RNA samples span 298 kbp (21%) and 66 kbp (4.7%) of the 1.4 Mbp wRi reference Wolbachia genome, respectively, the reads spanned 977 kbp of the wRi genome (70%) in the bacterial mRNA-enriched sample. Therefore, while the relative abundance of Wolbachia reads are the same in the polyA-enriched and bacterial mRNA-enriched samples, the latter is the only sample where bacterial transcripts can be detected across the majority of the genome.While the percentage of D. ananassae rRNA was found in all samples, but relative to the total RNA was 5.2-fold decreased in abundance in the polyA-enriched RNA and 2.1-fold decreased in abundance in the bacterial mRNA-enriched sample  yielded a ΔCt of −1.0 following bacterial mRNA enrichment, or a reduction of 47–55%, which we attribute to general loss of RNA with the low input method  and the insubstantial amount of personnel hours added upon inclusion of this step means that it will be cost effective.Wolbachia mRNA or Ehlrichia mRNA is estimated from the list price to be $563/sample. While this is substantially more than the cost of the bacterial mRNA enrichment method presented here, it may have benefits for samples where the rRNA depletion is less than ideal, like capturing bacterial mRNA from insect hosts. One study suggests a 1670-fold enrichment of RNAWolbachia samples described here substantially to the levels of sequencing conducted on Ehrlichia and make the SureSelect the more cost effective option.Recently, Agilent has introduced custom capture systems designed for capturing RNA, analogous to its systems for capturing DNA. For comparison, the cost of a custom capture of Wolbachia-Drosophila system. Sequencing of a bacterial mRNA-enriched sample isolated from a canine-Ehrlichia system results in 20% of the sequence reads arising from the bacterial transcriptome. In both cases, these methods enabled more cost-effective transcriptional profiling of host-bacteria samples than conventional methods. While both of the bacteria are from the order Rickettsiales, it is likely that this technique will be widely applicable for studying host-bacteria transcriptomics or host-microbiome metatranscriptomics.Here, we describe a method to efficiently remove eukaryotic host mRNA from 100 ng and 5 μg of starting material through polyA depletion. Combined with Ribo-Zero reductions, which efficiently remove rRNA from bacteria and eukaryotes, more bacterial mRNA transcripts can be identified with higher coverage in a D. ananassae from Klang, Selangor, Malaysia (UCSD Stock No. 14024–0371.33). Insects were reared on Jazz-Mix Drosophila food  in plastic bottles in an insect growth chamber  at 25 °C and 68% humidity. Natural infection by Wolbachia endosymbiont wAna was confirmed with Wolbachia-specific fluorescence in situ hybridization on ovariesTo examine the contribution of different RNAs in pre- and post-enrichment samples, we used wild-type Ehrlichia chaffeensis Wakulla strain  was propagated in a canine macrophage cell line DH82 as described previously6 cells) were harvested, and total RNA was extracted using an RNeasy Mini Kit  with the optional on column DNase-digestion per the manufacturer’s protocol.The Using 5 μg total RNA, the Ribo-Zero removal protocol , was carried out with the standard amount of magnetic beads (225 μL per reaction), followed by the Invitrogen Dynabeads polyA enrichment protocol  keeping the polyA depleted material in the supernatant and discarding the polyA-enriched material. The similarity in composition, pH, and ionic strength of Epicentre’s and Invitrogen’s reaction buffers allowed us to follow the protocols consecutively. Since the total RNA mixture was assumed to have a higher ratio of host rRNA compared to endosymbiont rRNA, we added 8.5 μL Human/Mouse/Rat Removal Solution to 1.5 μL Gram Negative Bacteria Removal Solution at the Ribo-Zero rRNA removal step for the standard removal. For the low input removal, we added 1.7 μL Human/Mouse/Rat Removal Solution to 0.3 μL Gram Negative Bacteria Removal Solution. The Ribo-Zero Magnetic Kit procedure was followed and, after removal of the magnetic beads, the eluate was processed with the standard Dynabeads protocol. Samples taken before and after bacterial mRNA enrichment were analyzed on an Agilent Bioanalyzer.Using both the Agilent Bioanlayzer and qRT-PCR (below), the template added reflects the same amount of starting material. For example, if the input volume of starting material was 50 μL and the final output supernatant was 100 μL, 1 μL of the starting material was used per qRT-PCR reaction or Bioanalyzer well for the total/starting RNA sample while 2 μL of the supernatant was used for the bacterial mRNA enriched sample. This was done to reflect the dilution between the starting material and the final supernatant volumes. As a consequence, these areas of integration and the Ct values described below can be compared directly in order to evaluate the composition of the samples.http://www.clontech.com/JP/Products/cDNA_Synthesis_and_Library_Construction/Next_Gen_Sequencing_Kits/ibcGetAttachment.jsp?cItemId=75952&fileId=6660677&sitex=10025:22372:US), the Ribo-Zero rRNA removal protocol  was carried out using a smaller amount of rRNA removal beads (90 μL), followed by the Invitrogen Dynabeads polyA enrichment protocol  keeping the polyA-depleted material in the supernatant and discarding the poly-enriched material. Like the higher input method, the similarity of Epicentre’s and Invitrogen’s reaction buffers allowed us to follow the protocols consecutively. Since the total RNA mixture was assumed to have a higher ratio of host rRNA compared to endosymbiont rRNA, we added 1.7 μL Human/Mouse/Rat Removal Solution to 0.3 μL Gram Negative Bacteria Removal Solution at the Ribo-Zero rRNA removal step, maintaining the same removal solution ratio as the 5 μg method. The modified Ribo-Zero Magnetic Kit procedure was followed and, after removal of the magnetic beads, the eluate was processed with the standard Dynabeads protocol.Using 100 ng total RNA and a protocol distributed by Clontech  per the manufacturer’s protocol. For the bacterial RNA-enriched sample, the polyA isolation step was omitted. The total RNA sample was prepared from all RNA present in the sample, while the polyA-selected library had the polyA isolation step performed. Adapters containing seven nucleotide indexes were ligated to the double-stranded cDNA. The DNA was purified between enzymatic reactions and the size selection of the library was performed with AMPure XT beads . The libraries were pooled and sequenced on an Illumina MiSeq or Illumina HiSeq sequencer paired end run, as specified in the text.D. ananassae and wRi genomes. The alignments were sorted and duplicates removed using Picard v.1.129. Coverage across the genome was measured using SAMTOOLS MPILEUP implemented in v.0.1.19 with the options “–BQ0 –d10000000”Forward and reverse reads were aligned with the BWA MEM command implemented in BWA v0.7.12 using the –M optionForward and reverse reads were aligned with Bowtie2D. ananassae datasets are available in SRP061993 http://www.ncbi.nlm.nih.gov/sra/?term=SRP061993) and the E. chaffeensis data set as SRX487088 (http://www.ncbi.nlm.nih.gov/sra/?term=SRX487088).The data set(s) supporting the results of this article are available in the Sequence Read Archive (SRA) repository. The How to cite this article: Kumar, N. et al. Efficient Enrichment of Bacterial mRNA from Host-Bacteria Total RNA Samples. Sci. Rep.6, 34850; doi: 10.1038/srep34850 (2016).


RNA editing is an important mechanism that expands the diversity and complexity of genetic codes. The conversions of adenosine (A) to inosine (I) and cytosine (C) to uridine (U) are two prominent types of RNA editing in animals. The roles of RNA editing events have been implicated in important biological pathways. Cellular RNA editing activity in response to influenza A virus infection has not been fully characterized in human and avian hosts. This study was designed as a big data analysis to investigate the role and response of RNA editing in epithelial cells during the course of infection with various subtypes of influenza A viruses.Using a bioinformatics pipeline modified from our previous study, we characterized the profiles of A-to-I and C-to-U RNA editing events in human epithelial cells during the course of influenza A virus infection. Our results revealed a striking diversity of A-to-I RNA editing activities in human epithelial cells in responses to different subtypes of influenza A viruses. The infection of H1N1 and H3N2 significantly up-regulated normalized A-to-I RNA editing levels in human epithelial cells, whereas that of H5N1 did not change it and H7N9 infection significantly down-regulated normalized A-to-I editing level in A549 cells. Next, the expression levels of ADAR and APOBEC enzymes responsible for A-to-I and C-to-U RNA editing during the course of virus infection were examined. The increase of A-to-I RNA editing activities in infections with some influenza A viruses (H1N1 and H3N2) is linked to the up-regulation of ADAR1 but not ADAR2. Further, the pattern recognition receptors of human epithelial cells infected with H1N1, H3N2, H5N1 and H7N9 were examined. Variable responsive changes in gene expression were observed with RIG-I like receptors and Toll like receptors. Finally, the effect of influenza A virus infection on cellular RNA editing activity was also analyzed in avian hosts.This work represents the first comprehensive study of cellular RNA editing activity in response to different influenza A virus infections in human and avian hosts, highlighting the critical role of RNA editing in innate immune response and the pathogenicity of different subtypes of influenza A viruses.The online version of this article (10.1186/s12864-017-4330-1) contains supplementary material, which is available to authorized users. Influenza A viruses are among the most common and the most significant causes of respiratory diseases in a broad range of animals from avian to human hosts because of the high morbidity and mortality of some subtypes of influenza A viruses –3. InfluInfluenza A virus invasion triggers a number of cellular responses and alters the host transcriptome –16. RNA During viral infection, the innate immune system of the host is meant to act as a first line defense against viral invasion. The host innate immune system has the ability to recognize viral nucleic acids as invader. For example, viral double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) with a 5′-triphosphate, which are typical products of viral replication, can be detected by the cytoplasmic retinoic acid-inducible gene I (RIG-I) like receptors (RLRs) –34 and TThe development of high-throughput sequencing technology , 41 and To characterize A-to-I and C-to-U RNA editing activities in human epithelial cells upon infection of influenza A viruses, we first sought to compile and collect transcriptome sequencing data from human samples with infected influenza A virus of various types. We identified from NCBI  database RNA-seq data from human lung and tracheobronchial epithelial cells infected with different subtypes of influenza A viruses, H1N1, H3N2, H5N1 and H7N9 , which was similar to the methods described previously [R = 0.91, p = 6.16e-11, Fig. R = 0.92, p = 4.5e-11), which was consistent with previous results [To call A-to-I and C-to-U RNA editing events from mapped RNA-seq data, we used a modified pipeline that applied a series of filters to remove the noises  with median level from ~70% at 3 h post-infection changing to ~80% at 24 h post-infection  was used to quantify the gene expression level measured by FPKM (fragments per kilobase of transcript per million mapped reads) and to identify the differentially expressed genes between control and virus-infected groups. For expression of ADAR and APOBEC genes in HTBE cells infected with H1N1, H3N2, or H5N1 Fig. , we founDifferential expressions were observed for APOBEC3 family of enzymes during the course of influenza virus A infections, but not for APOBEC1 or APOBEC2 Fig. . SimilarThen we analyzed the expression ADAR and APOBEC enzymes using RNA-seq data we generated from H7N9 infected A549 cells. No significant change was observed with the expression of ADAR1–3, APOBEC1, APOBEC2, and APOBEC3A 3D, 3G, and 3H, whereas APOBEC3B, and 3C had a significant decrease, and APOBEC3F had a significant increase in A549 cells during the course of infection with H7N9 Fig. . Again tSince detection of viral RNAs by Pattern Recognition Receptors (PRRs) is the first step for initiating anti-viral responses, we further examined the expression changes of of PRR genes triggered by different subtypes of influenza A viruses in human lung and tracheobronchial epithelial cells. In comparison with control group, there was a general increase in the gene expression of RIG-I like receptors, including DDX58/RIG-I, IFIH1/MDA-5, DHX58/LGP-2 in human lung and tracheobronchial epithelial cells infected with H1N1, H3N2, H5N1, or H7N9 , and in avian hosts (ileum and lung of chicken and quail), highlighting the important roles of RNA editing in innate immune response. A striking difference of cellular RNA editing activities in response to various influenza A virus subtypes were observed. The increase of A-to-I RNA editing activities in infections with some influenza A virus (H1N1 and H3N2) is linked to the up-regulation of ADAR1 but not ADAR2, through RIG-I like receptors  and Toll-like receptors  in innate immune system. Our study gives a strong functional implication of A-to-I RNA editing on the pathogenicity of different subtypes of influenza A viruses.The transcriptome sequencing data for the A549 cells infected with H7N9 were obtained from our laboratory. A549 cells were infected with H7N9 (A/Anhui/1/2013) at MOI of 1 for 1.5 h, 3 h and 7 h. Total RNA was isolated from the infected cells using the RNeasy Mini Kit . The sequencing library was prepared using Illumina TruSeq RNA sample preparation kit v2. The mRNA was derived from the total RNA using poly-T oligo-attached magnetic beads. Then the mRNA was fragmented and converted into cDNA. The adapters were ligated to the cDNA and then the fragments were amplified by PCR. Paired-end sequencing (101 × 2) was performed using Illumina Hiseq 2000. Each condition of the samples was sequenced in duplicate. The RNA-seq data have been deposited in NCBI’s Gene Expression Omnibus under accession code GSE97949.Coturnix japonica) Genome Sequencing Project” website (http://viewer.shigen.info/uzura/download.php), the reference genome and the gene annotation data for human (hg19) were downloaded from Illumina’s iGenomes project (https://ccb.jhu.edu/software/tophat/igenomes.shtml). The reference genome data for different subtypes of influenza A viruses are downloaded from GISAID  (http://platform.gisaid.org/epi3/).The public transcriptome sequencing data used in this study were mainly from NCBI’s GEO (Gene Expression Omnibus) and SRA (Sequence Read Archive). The RNA sequencing data for HBE cells infected with H1N1 (PR/8/34) at MOI of 1 for 8 or 24 h were obtained from NCBI’s SRA under accession number SRP066992   using pa.2.1  usiP values lower than 0.05 were considered to indicate a statistically significant difference or correlation.Statistical analysis was performed using the computing environment R (Version: 3.3.2). Data were expressed as mean ± SE. The one way ANOVA test was used to compare the RNA editing levels of the common editing sites at different time points. Pearson correlation test was used to measure the strength of a linear association between two variables. Additional file 1: Table S1.The sources of deep-sequencing transcriptome data of hosts infected with different subtypes of influenza viruses. (XLSX 14 kb)Additional file 2: Figure S1.Information of common editing sites. (XLSX 42 kb)Additional file 3: Figure S2.The A-to-I and C-to-U RNA editing levels of common editing sites of HBE cells infected with H1N1. (DOCX 72 kb)Additional file 4: Figure S3.Figure S4. Expression profiles of Pattern Recognition Receptors in HBE cells infected with H1N1. (DOCX 631 kb)Expression profiles of ADAR and APOBEC enzymes in HBE cells infected with H1N1. Additional file 5: Table S2.Table S3. Expression profiles of ADAR and APOBEC enzymes in quail infected with H5N1 and H5N2. (DOCX 23 kb)Expression profiles of ADAR and APOBEC enzymes in chicken infected with H5N1 and H5N2. Additional file 6: Table S4.Work flow of this study. (DOCX 76 kb)


Caenorhabditis elegans , in germlines. These phasiRNAs, known as one of PIWI-interacting RNAs (piRNAs), mainly repress transposable elements. Similarly, reproduction-specific phasiRNAs have been identified in the family Poaceae, although DICER LIKE (DCL) protein-dependent phasiRNA biogenesis in rice is distinct from piRNA biogenesis in animals. In plants, phasiRNA biogenesis is initiated when 22-nt microRNAs (miRNAs) cleave single-stranded target RNAs. Subsequently, RNA-dependent RNA polymerase (RDR) forms dsRNAs from the cleaved RNAs, and dsRNAs are further processed by DCLs into 21 to 24-nt phasiRNAs. Finally, the phasiRNAs are loaded to ARGONAUTE (AGO) proteins to induce RNA-silencing. There are diverse types of phasiRNA precursors and the miRNAs that trigger the biogenesis. Their expression patterns also differ among plant species, suggesting that species-specific combinations of these triggers dictate the spatio-temporal pattern of phasiRNA biogenesis during development, or in response to environmental stimuli.It has been almost 30 years since RNA interference (RNAi) was shown to silence genes via double-stranded RNAs (dsRNAs) in  Caenorhabditis elegans, double-stranded RNAs (dsRNAs) induce RNA silencing, called RNA interference (RNAi)  bound to AGO4 cause RNA-directed DNA methylation (RdDM) and TGS. TGS, mediated by rasiRNAs via DNA methylation, suppresses many transposable elements (TEs) and repeat regions to prevent their transposition and transmission to the next generation , which are one type of phased small interfering RNAs (phasiRNAs) in plants.RNA silencing is classified into two types: transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). In Arabidopsis thaliana, play a role in leaf phase transition from juvenile to adult phases of vegetative stages  family, pentatricopeptide repeat (PPR) genes, and MYB transcription factors that include complement sequences recognized by AGO1/7-tasiRNAs complexes  identified as precursor RNAs are single-stranded. They are transcribed with poly(A) tails without introns during early reproductive stages prior to meiosis in rice  is a rice AGO protein that functions in the development of pre-meiotic germ cells and the progression of meiosis in both male and female organs. MEL1 binds dominantly to 21-nt phasiRNAs bearing a 5′-terminal cytosine, although AGOs interacting with 24-nt phasiRNAs remain unknown  encodes the 1.2-kbp non-coding RNA that regulates PSMS. The abundant LDMAR transcript induces male sterility during long days, and is processed into small RNAs  of pms3 mutants, although the male sterility phenotype in pms3 is distinct from that of mel1 in both male and female tissues.  is a phenomenon where particular rice strains show male sterility under long-day conditions, but are fertile under short-day conditions. Long-day-specific male-fertility-associated RNA , related to rice MEL1, is believed to function in female gametogenesis, since a semi-dominant ago5-4 mutant is defective in the initiation of mega-gametogenesis  degradome, a modified RACE that detects cleaved transcripts, and a transcriptome analysis of mel1 and dcl4 mutants, have failed so far to identify any cleaved target transcripts with complementary sequences of these phasiRNAs, indicating that MEL1-phasiRNAs may not trans-regulate targets  modifications are depleted in early meiosis I  and AGO3 in Medicago truncatula (Fabaceae), the 22-nt miR1507, miR2109, and miR2118 act as triggers to initiate generation of 21- and 22-nt phasiRNAs from mRNAs that encode nucleotide binding and leucine-rich repeat (NB-LRR) pathogen-defense genes and these 22-nt miRNAs are conserved in other legumes and nonlegumes, members of the family Solanaceae . The functions of phasiRNAs derived from the large gene families of MYB and PPR remain unknown. In contrast, NB-LRR gene families are regulated by both triggered-miRNAs and phasiRNAs generated from NB-LRRs, and the phasiRNAs also regulates in trans at other NB-LRR loci in legumes. Thus, NB-LRR-phasiRNA regulation shows a self-reinforcing network involving cis- or trans- regulation , over 2000 phasiRNA-produced loci (PHAS loci) have been identified, including ~800 loci derived from NB-LRRs and ~600 from non-coding RNAs, recognized by 41 species of miRNAs. Interestingly, miR418/miR2118 super-families in gymnosperms target both NB-LRR RNAs and reproductive non-coding RNAs, resulting in production of phasiRNAs derived from both NB-LRR RNAs and reproductive non-coding RNAs  and 21-nt phasiRNAs accumulate in pollen sacs, and miR2118 localizes in epidermis of premeiotic anthers in maize and African rice. In contrast, lincRNAs (24-nt PHAS), miR2275, and 24-nt phasiRNAs are expressed in tapetum and meiocytes in meiotic anthers  How are numerous lincRNAs regulated during specific developmental stages and pathogenesis? (2) What is the molecular mechanism of lincRNA cleavage by 22-nt miRNAs? (3) What is the primary role of phasiRNAs in plants? Answering these questions will require experiments exploiting technologies such as genomics/epigenetics using non-coding RNA mutants, cell sorting, biochemistry, and structural analysis. Such studies are expected to help us to understand the significance of non-coding regions in both plants and animals.


Bamboo mosaic virus (BaMV), is a member of the Potexvirus genus of Flexiviridae. BaMV can infect at least 12 species in 3 genera of bamboo. Symptoms include chlorotic mosaic on infected leaves and necrotic streaking on shoots and culms. With a high incidence of more than 90% infection in many bamboo plantations, the virus causes great economic loss, particularly in Taiwan, in terms of the quality and quantity of bamboo shoot production, a popular food.Bamboo mosaic was first reported in Brazil in 1975, then in Taiwan, Australia, USA, India, China, etc. The causal agent, However, study of BaMV did not start extensively until BaMV was identified as a member of the Potexvirus genus at the molecular level, in the early 1990s. Furthermore, the satellite RNA (satRNA) associated with BaMV (satBaMV) was found to be the only satRNA in Potexvirus. After completion of the full-length sequencing of genomic RNA of BaMV and generation of the BaMV infectious cDNA clone, study of BaMV stepped into a new dimension. Since then, BaMV has been extensively studied at the molecular level, covering different both basic and applied research, by a group of researchers in Taiwan. To date, more than 100 BaMV papers have been published in SCI international journals.Like all potexviruses, BaMV has a flexuous morphology with a single-stranded positive-sense RNA genome. By investigating BaMV, the filamentous structure of flexible viruses was first determined at the near-atomic level by cryo-electron microscopy. This finding solved the mystery of how flexible virus particles maintain structural integrity as mechanical forces deform their structure. In addition, two types of subviral agents, defective RNA and satellite RNA, were found associated with BaMV in the field. Besides studying the viral RNA genomic structure and function, BaMV research has also focused on the virus-host interaction.Meng and Lee). The authors also detail the interactions between replicase and the host proteins identified in Nicotiana benthamiana. Chen et al. cover the location of cis-acting elements in the viral RNA and their specific functions, including the recognition of the replicase complex for minus-strand, plus-strand and subgenomic RNA syntheses, viral RNA packaging, and viral movement. Moreover, the authors reveal the host factors that might be involved in delivering BaMV cargoes during intracellular movement, including the delivery of viral RNA to the chloroplast for replication and viral RNA complex to move cell to cell (Cheng). Huang et al. describe the strategies used to identify the host proteins positively and negatively regulating the BaMV RNA replication and viral movements. One of the host proteins reported to be downregulated after BaMV infection is now revealed as carbonic anhydrase . Nuclear-encoded chloroplast carbonic anhydrase may be involved in the re-initiation step of BaMV RNA replication.In this Research Topic, we summarize the BaMV research with 6 reviews and 4 research articles. To uncover the functional activity of replicase encoded by a positive-sense RNA virus, Meng and Lee describe the enzymatic activities associated with each of the functional domains of the BaMV-encoded replicase, including its unique capping mechanism, which may be conserved across the alphavirus superfamily (Alazem and Lin). BaMV is unique in some isolates carrying satBaMVs, particularly the interfering satBaMV, with crucial roles in modulating BaMV replication and viral symptom development. Lin and Lin summarize the molecular mechanisms underlying the interaction of interfering satBaMV and BaMV (Lin and Lin).In addition to host factors revealed in replication and movement, the study of BaMV-plant interactions explores a role for abscisic acid (ABA), a plant hormone, in the antiviral silencing pathway that could lead to interference in virus accumulation. Alazem and Lin describe how ABA affects the accumulation of BaMV and other viruses via the gene silencing pathway  could stimulate effective neutralizing antibodies against JEV infection in mice. This study demonstrates an alternative way to produce an effective vaccine candidate against JEV in plants by the BaMV-based vector system.Finally, We thank our colleagues and friends, in total 27 authors, for their valuable contributions to this eBook and cohesive collaboration in the study of BaMV biology in the past 2 decades. We are also grateful to Dr. Anne Simon (University of Maryland), who encouraged us to initiate this Research Topic. We hope the knowledge we have gained from BaMV can serve as the groundwork and biotechnological application for Potexvirus research and RNA research as well.All authors listed have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Triple-negative breast cancer (TNBC) is an operational term for breast cancers lacking targetable estrogen receptor expression and HER2 amplifications. TNBC is, therefore, inherently heterogeneous, and is associated with worse prognosis, greater rates of metastasis, and earlier onset. TNBC displays mutational and transcriptional diversity, and distinct mRNA transcriptional subtypes exhibiting unique biology. High-throughput sequencing has extended cancer research far beyond protein coding regions that include non-coding small RNAs, such as miRNA, isomiR, tRNA, snoRNAs, snRNA, yRNA, 7SL, and 7SK. In this study, we performed small RNA profiling of 26 TNBC cell lines, and compared the abundance of non-coding RNAs among the transcriptional subtypes of triple negative breast cancer. We also examined their co-expression pattern with corresponding mRNAs. This study provides a detailed description of small RNA expression in triple-negative breast cancer cell lines that can aid in the development of future biomarker and novel targeted therapies. Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer, representing ~15% of cases, but ~25% of all breast cancer deaths. TNBCs are defined by their absence of estrogen and progesterone receptor expression and HER2 amplifications. Lacking these biomarkers, TNBCs are insensitive to current targeted therapies. Molecular heterogeneity, and a lack of high-frequency “driver” alterations amenable to therapeutic intervention have hindered the development of new treatments for TNBC . These tSmall RNAs (sRNAs) are short non-coding RNAs (ncRNA) of 200 nucleotides or less in length. The discovery of novel sRNAs and their annotations have substantially enhanced our understanding of the complex regulation of the genome. MicroRNAs (miRNAs) are, by far, the most studied small RNA, with over 10,000 publications to date. Discovered in 1993 , miRNAs Earlier studies of sRNA have relied on low-throughput real-time quantitative polymerase chain reaction (RT-PCR) or hybridization-based microarrays. The advancement of HTS technology has substantially increased the detection limit of miRNAs, and more importantly, enabled the examination of miRNA at a single nucleotide resolution, in addition to quantifying abundance. Furthermore, HTS enables a global assessment of sRNAs and not limited to a set of previously known targets. Subsequent bioinformatics analysis of sRNA sequencing data can identify, quantify and determine the differential expression of a variety of small non-coding RNAs. Since size-selection sequencing methods are agnostic to sRNA species, it has the potential to capture many species of sRNAs that include miRNAs, miRNA isoforms (isomiRs) ,14, tranThe present study provides an in-depth characterization of small non-coding RNA species in TNBC, and identifies subtype-specific differences in major non-coding RNA species detectable: micro RNA (miRNA), miRNA isoforms, transfer RNA (tRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), Y RNA (yRNA), single recognition particle RNA (7SL RNA), and 7SK RNA. These data validate previously identified pathways, and highlight potential biomarkers for future studies.6) were harvested and sRNA isolated  using a standard spin protocol. RNA quality assessment and RNA-seq was performed by the Vanderbilt Technologies for Advanced Genomics core . Libraries were prepared using the TruSeq Small RNA sample preparation kit . The sRNA protocol specifically ligates RNA adapters to mature miRNAs harboring a 5′-phosphate and 3′-hydroxyl group as a result of enzymatic cleavage by RNase III processing enzymes, e.g., Dicer. In the first step, RNA adapters were ligated onto each end of the sRNA, and reverse transcription was used to create single-stranded cDNA. This cDNA was then PCR amplified for 18 cycles with a universal primer, and a second primer containing one of 20 uniquely indexed tags to allow multiplexing. Size-selection of the cDNA constructs was performed using a 3% gel cassette on the Pippin Prep  to include only mature miRNAs and other sRNAs in the 5–40 bp size range, and to remove adapter–adapter products. The resulting cDNA libraries then underwent a quality check on the Agilent Bioanalyzer HS DNA assay  to confirm the final library size, and on the Agilent Mx3005P quantitative PCR machine, using the KAPA library quantification kit  to determine concentration. A 2 nM stock was created, and samples were pooled by molarity for equimolar multiplexing. From the pool, 10 pM of the pool was loaded into each well of the flow cell on the Illumina cBot for cluster generation. The flow cell was then loaded and sequenced on the Illumina HiSeq 3000 to obtain at least 15 million single end (1 × 50 bp) reads per sample. The raw sequencing reads in BCL format were processed through CASAVA-1.8.2 for FASTQ conversion and de-multiplexing. The chastity filter from Illumina’s Real-Time Analysis software was applied, and only PF (pass filter) reads were retained for further analysis. Raw data files available at Gene Expression Omnibus under the accession GSE108286.We cultured 26 TNBC cell lines  for this study; the cell culture procedures were previously described [We also performed total RNA-seq on the 26 TNBC cell line for comparative purpose. Total RNA was isolated with the Aurum Total RNA Mini Kit . All samples were quantified on the QuBit RNA assay . RNA quality was checked using Agilent Bioanalyzer. RNA integrity number (RIN) for both sRNA and total RNA was 10. The ribosome RNA reduction was performed using the Ribo-Zero Magnetic Gold Kit (Human/Mouse/Rat) . The RNA libraries were sequenced on Illumina High HiSeq 3000 with paired-end 100 base pair long reads.HTS data processing was performed using a custom in-house data analysis pipeline Tiger  for sRNAThe total RNA-seq data was quality controlled following standard RNA-seq quality control protocols  using tohttps://portal.gdc.cancer.gov/) to validate the correlation patterns identified in this study.Differential sRNA expression analysis among subtypes of TNBC cell lines were performed by MultiRankSeq  . Due to We performed TNBC subtyping as previously described ,39, usinTo determine if different species of small RNAs species are enriched in TNBC subtypes, we performed sRNA sequencing of 26 TNBC cell lines . After qTo identify uniquely enriched non-coding RNAs, we conducted differential expression analysis between each of the TNBC subtypes . AdditioO-methylation of target RNA. yRNAs are components of the autoantigenic Ro ribonucleoproteins were uniquely enriched in the LAR subtype. yRNAs are overexpressed in various cancers, [There were differences in the expression of small nuclear RNA (snRNA) components of spliceosomes, transcription elongation, and signal recognition particle complexes across TNBC subtypes. Spliceosomes are integral to eukaryotic precursor messenger RNA maturation. BL1 subtype cell lines were enriched for U7 small nuclear RNAs that are involved in histone pre-mRNA processing (RNU7-19P and RNU7-3P), while the LAR subtype displayed lower levels of U2 snRNAs . The BL1 subtype was enriched in the 7SK snRNA component of the positive transcription elongation factor P-TEFb . The seccancers,  and implcancers, ,43.Transfer RNAs (tRNAs) are adaptor molecules, typically 76 to 90 nucleotides in length that physically link mRNA and the amino acid sequence of proteins. tRNAs deliver individual amino acids to ribosomes for protein translation. Val, Gly, Lys, Glu, and His were by far the most abundant tRNAs across all TNBC cell lines, representing ~95% of all tRNAs A. tRNA dSince miRNA were the most abundant non-coding RNA species and have the ability to regulate diverse pathways by targeting mRNAs, we performed pathway analysis on miRNAs exclusive to each TNBC subtype . As antiWe performed correlation analyses between the expression of sRNA and mRNA in TNBC cell lines. All correlations were computed using Spearman’s correlation to minimize outlier and scaling effects between datasets. We found that there is a slight bias toward positive correlation for miRNA vs. mRNA in our TNBC cell lines; this bias increased with higher absolute correlation values . To ensuTo further study the correlations in detail, we constructed a list of known tumor suppressor genes and oncogenes . The notA summary network of tumor suppressor and oncogenes correlated with all sRNA species is provided in The functions of non-coding RNAs are continuously being uncovered, and are implicated in epigenetic, transcriptional, and post-transcriptional regulation. NGS has expanded our ability to investigate RNA expression outside of the coding genome. While many studies have implicated miRNAs that associate with prognosis, little is known about the expression pattern of non-coding RNAs in triple-negative breast cancers ,53. UsinN-glycan biosynthesis [Using differential expression analysis, we identified non-coding RNAs that are associated with biological TNBC subtypes. The majority of the differentially expressed small non-coding RNAs were miRNAs, and correlation with mRNA validated several targets. The BL1 and BL2 subtypes were enriched in mRNA targeting members of the ErbB receptor tyrosine kinase family. However, the BL2 subtype displayed enrichment in miRNAs regulating Wnt signaling and pathways regulating pluripotency of stem cells. Furthermore, the BL2 subtype was enriched in miRNAs regulating calcium and cyclic GMP protein kinase G signaling, suggesting modulation of this pathway made be potentially therapeutic as in head and neck squamous cancer . The LARynthesis ,57. As eynthesis .In addition to miRNA, we identified several other species that were differentially enriched in TNBC subtypes. The BL1 subtype cell lines were enriched in U7 snRNAs that are involved in histone pre-mRNA processing, likely due to the increased cell cycle and proliferation associated with this subtype . The BL1


RNA-dependent RNA polymerases (RdRp) are very ancient enzymes and are essential for all viruses with RNA genomes. We reconstruct the origin and evolution of this polymerase since the initial stages of the origin of life. The origin of the RdRp was traced back from tRNA ancestors. At the origin of the RdRp the most ancient part of the protein is the cofactor-binding site that had the capacity of binding to simple molecules as magnesium, calcium, and ribonucleotides. Our results suggest that RdRp originated from junctions of proto-tRNAs that worked as the first genes at the emergence of the primitive translation system, where the RNA was the informational molecule. The initial domain, worked as a building block for the emergence of the fingers and thumb domains. From the ancestral RdRp, we could establish the evolutionary stages of viral evolution from a rooted ancestor to modern viruses. It was observed that the selective pressure under the RdRp was the organization and functioning of the genome, where RNA double-stranded and RNA single-stranded virus formed a separate group. We propose an evolutionary route to the polymerases and the results suggest an ancient scenario for the origin of RNA viruses. Unlike strategy . These estrategy , which astrategy . The 3D strategy . In all strategy . With thstrategy , it was strategy . Anotherstrategy . In bothstrategy . Severalstrategy , 2016. Fstrategy , 2016a sstrategy , the tRNstrategy . The oristrategy , 2016a. strategy . This reHerein, we used the sequences derived from RNA ancestor molecules that when translated had matches with the RdRp, as suggested by 1 to predict by homology the best structural model. For a better structural resolution of the ancestral proteins, each structural ancestral model was submitted to the program ModRefiner ; RNA Polymerase Rabbit Hemorrhagic Disease Virus (PDB: 1KHV); RNA Polymerase Sapporo Virus (PDB: 2CKW); Hepatitis C RNA Polymerase (PDB: 2D41); Neurospora Crassa RNA Polymerase (PDB: 2J7N); RNA Polymerase Birnavirus (PDB: 2PGG); RNA Polymerase Infectious Bursal Disease Virus (PDB: 2PUS); RNA Polymerase Rotavirus (PDB: 2R7T); RNA Polymerase Infectious Pancreatic Necrosis Virus (PDB: 2YI8); RNA Polymerase Cypoviruses (PDB: 3JA4); Enterovirus A RNA Polymerase (PDB: 3N6L); RNA Polymerase Norwalk Virus (PDB: 3UQS); RNA Polymerase Rotavirus A (PDB: 4AU6); RNA Polymerase Thosea Assigns Virus (PDB: 4XHA); Rhinovirus A (PDB: 1XR7); Enterovirus C (PDB: 3OL6); Foot-and-Mouth Disease Virus (PDB: 1U09); Cardiovirus A (PDB: 4NZ0); Japanese Encephalitis Virus (PDB: 4HDH); Dengue Virus (PDB: 2J7U); Bovine Viral Diarrhea Virus 1 (PDB: 1S48); Qbeta Virus (PDB: 3MMP); Reovirus (PDB: 1MUK); and La Crosse Bunyavirus (PDB: 5AMQ). In Supplementary Table b server 5.Figure 1, the sequences and the structural models generated by homology to ancestral sequences, as suggested by Figure 1A tRNA1, Figure 1B tRNA 2, and Figure 1C tRNA 3 showed similarities among them, with small differences in the amino acids composition. The sequence in Figure 1D tRNA 4 is the most divergent and can represent a second module in the formation of the primordial protein. Figure 2A, note the structure of the ancestral control based in the three dimensional structure of proteins as obtained from the Protein Data Bank, and in Figure 2B, the structural alignment between the control and the tRNA3. The RMSD value of the structural alignment was 2.75, despite the high value of the structural conformation, indicating that either the tRNA 3 and the control can have the same ancestor or that they are at different points of the same evolutionary history. The results suggest that the ancestral sequence and structure are convergent, which in turn indicates that the origin of the RdRp can be traced back to tRNA ancestors.In Table 1, the analysis of substrate and cofactor binding to the ancestral sequences derived from ancestral tRNAs is shown. The data indicate that the ancestral structures have the capacity of binding to simple molecules as magnesium, calcium, ribonucleotides, among others ligands. The most frequent cofactor found in this analysis was magnesium that is a cofactor in the modern RdRp. These analyses indicated that at the origin of the RdRp the most ancient part of the protein is the cofactor-binding site. In modern proteins, the cofactor-binding site is located at the palm domain, which is considered by evolutionary analysis the most ancient domain in the modern RdRp ; between the tRNA 3 translated and the palm domain of RNA Polymerase DSRNA Bacteriophage with RMSD value of 1.93 (1HHS); and the thumb domain of RNA polymerase Rotavirus with RMSD value of 1.44 (2R7T). In Figure 4, it is shown the structural tree constructed by Neighbor Joining (NJ). The result indicates that the ancestral structures derived from translated ancestral tRNAs and the control behave like a root, although the tree is not rooted. This condition can reflect the antiqueness of this protein domain. Notice the proximity between the ancestor sequences and the virus family Leviviridae, Reorividae, and Cystoviridae, which are described as very ancient viral families  and evolved to a DS-RNA (Reoviridae/Cystoviridae). These families of viruses are described as very ancient families (Flavoviridae, Picornaviridae, Calciviridae, Burnyavoridae, and Permutatetraviridae. A third reversion of the genome organization, from SS-RNA to DS-RNA, occurred with the emergence of Birnaviridae, being this condition more recent (Figure 4. Evidently, the dependence/independence of a primer emerged more than once during the evolution of RNA viruses (The evolution of RdRp is intrinsically linked with the evolution of RNA viruses, mainly because this enzyme is responsible for the replication of RNA viral genomes. In  viruses . An impofamilies . This ore recent . The ind viruses .Figure 4.The evolutionary position of RNA viruses in the tree of life is a controversial subject in modern biology . Its abuBased in the present results, we suggest a hypothetical scenario where initially a ribozyme with polymerase activity could have enhanced its activity by the binding of a simple cofactor, as magnesium , thus, eSdF, AdSJ, and MVJ conceived and designed the experiments; SdF, AdSJ, and TGR performed the experiments. SdF, AdSJ, TGR, and MVJ analyzed the data; SdF, TGR, and MVJ contributed to analysis tools; and SdF, AdSJ, and MVJ wrote the paper.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 encapsidated potexviral RNA to share commontranslational features in vitro and the MP1 to be potent overhomological virions of its “own” species and over heterologicalvirions of other species, as well exhibiting selective specificity. Reciprocalcross-activation is observed among viral species phylogenetically either closeor distant. There is direct evidence that MP1 binding to the end of the virionis necessary, but not sufficient, for translational activation of encapsidatedRNA.We had shown the genomic RNA of potexviruses potato virus X and thealternanthera mosaic virus to be inaccessible  We suggest that two mechanisms oftranslation activation operate at different stages of the infection. Theencapsidated RNA becomes accessible to ribosomal translation in primaryinfected cells following the phosphorylation of the PVX coat protein. The MP1produced during the course of the infection binds to the PVX virions’end, producing the virus transport form and, thus, activating the encapsidatedRNA  strain was transformed with theconstructs. The recombinant proteins were purified following expressioninduction by chromatography on a Ni-NTA agarose. The SDS PAGE analysis of theprotein samples using a 8-20% gel revealed a single band corresponding toeither NMV MP1 (26.7 kDa) or PAMV MP1 (27.2 kDa).The NMV MP1 and PAMV MP1 expressing plasmids were constructed using the pQE30vector (Qiagen). The corresponding coding regions, supplemented with His6 tags,were amplified on the the NMV and PAMV viral RNA templates using the followingprimers: NMV-forward-BamHI(+) 5’-acacggatccatggactgtaagta-3’,NMV-reverse-PstI(-) 5’-acacctgcagcgtagttaacaggtg-3’Auc-forward-BamHI(+) 5’-acatggatccggaatggaatat-3’, andAuc-reverse-PstI(-) 5’-acacctgcagatcagtctaaat-3’. TheTranslation in vitroThe translation was performed in a wheat germ extract cell-free system(Promega), following a modified manufacturer protocol as described. The RNAImmune electron microscopyThe immune electron microscopic observation was done as described in. PolycloPotexvirus encapsidatedRNA, we propagated, harvested, and purified NMV and PAMV viral preparations,accompanied by these viruses’ recombinant MP1 samples. We found thetranslational properties of encapsidated NMV and PAMV RNA to not differ fromthose of PVX and AltMV. Our findings show virion-packed NMV and PAMV RNA to benontranslatable in vitro,but it could be rendered translatable following exposure to its own MP1.The same had been demonstrated for AltMV and PVX RNA.To study the translational features of Fig. 2, 5).Worthwhile, the treatment of encapsidated PAMV RNA with PVX MP1does not render it translationally active contrary to AltMV RNA[Fig. 2, 6).We reported previously on the translational cross-activation of encapsidatedAltMV and PVX RNA after the interaction of PVX with AltMV MP1 and vice versa, 10.We ltMV RNA. The samFig. 3, 4),whereas PVX MP1 activated encapsidated NMV RNA.No reciprocal cross-activation exists between NMV and PVX, as is the case for NMV andAltMV; i.e., NMV virion-packed RNA remains nontranslatable when exposed to AltMV MP1;AltMV virion-packed RNA, in contrast, does undergo activation under treatment with NMV MP1 or PVX MP1[A different pattern was observed when analyzing the translation activation ofNMV and PVX virion-packed RNA, while NMV MP1 appeared incapable of activating PVX RNA. These oWe also tested whether PAMV MP1 is capable of activating encapsidated NMV orAltMV RNA. This movement protein proved to activate the translation of NMVviral RNA but not AltMV viral RNA. Similarly, the PAMV virion-packed RNA doesnot translationally become activated upon treatment with AltMV MP1 (data notshown).The results described above are summarized inTable). Reciprocalcross-activation was discovered in pairs: PVX – AltMV and NMV – PAMV.The findings of cross-activation studies point out that NMV MP1 is capable oftranslationally activating encapsidated PAMV and AltMV RNA but not encapsidatedPVX RNA. Furthermore, PAMV MP1 can translationally activate NMV virion-packedRNA but not AltMV and PVX RNA. We have already observed cross-activation ofencapsidated AltMV and PVX RNA. Now, we have found evidence of PVX MP1activating NMV virion-packed RNA, as well as encapsidated AltMV RNA, but notPAMV RNA. Moreover, AltMV MP1 was found to lack the capability of activing PAMVand NMV virion-packed RNA.Potexvirus movement proteins are known to belong to superfamilyI helicases, which contain seven highly conservative NTPase/RNA helicase motifsconstituting a NTPase/RNA helicase domain[The amino acid sequence of the corresponding MP1 was compared to shed somelight on the data obtained.Additional experiments were performed to clarify this question.The MP1 motif IV sequences of PVX, AltMV, PAMV, and NMV show ahigh degree of variability: hence, the prediction that these MP1s bind to theend of heterologous virions in the absence of cross-activation is questionable and AltMV MP1that render encapsidated PVX RNA translatable, as well as the PAMVMP1 that lacks such capability.In a control experiment,no binding of gold beads was observed in the absence of MP1. Similar resultswere obtained when studying AltMV viral particles:virion end binding was observed for PAMV MP1, AltMV MP1, and NMV MP1, while onlyAltMV MP1was able to activate AltMV virion-packed RNA.We used immune electron microscopy employing primary antibodies against the MP1of PVX, AltMV, and PAMV and gold-conjugated secondary antibodies. This approachhelped us clearly visualize the binding to the PVX virion end of PVX MP1. The PAMV andNMV clustering in the same subgroup, Ib, tracks well with the fully reciprocalcross-activation observed for this pair. The observed cross-activation of PVXand AltMV virion-packed RNA corresponds probably to a lesser branching betweenthe subgroups Ia and Ic compared to the one between Ia and Ib[et al. showed that the full-lengthmovement proteins of potexviruses can bind to heterologous virions but fail toactivate the translation of its genomic RNA. We consider phylogeneticallyrelated movement proteins to share conformational features and, thus, able toloosen the helical proteinaceous virion of either virus, hence exposing the5’-end of genomic RNA to ribosomes [We have demonstrated in this study a reciprocal cross-activation of translationin the pairs PAMV-NMV and AltMV-PVX, while other pairs show eithernon-reciprocal cross-activation (NMV-PVX and NMV-AltMV) or a total absence ofcross-activation (PAMV-PVX) or closely related subgroups . The movement proteins 1 capableof translationally activating heterologous potexviruses encapsidated RNA arelikely to share protein molecule conformation; this suggestion, however, is amatter for further research.Our results clearly indicate that encapsidated potexviral RNAs sharetranslational features


The RNA that is packaged into exosomes is termed as exosomal-shuttle RNA (esRNA); however, the players, which take this subset of RNA (esRNA) into exosomes, remain largely unknown. We hypothesized that RNA binding proteins (RBPs) could serve as key players in this mechanism, by making complexes with RNAs and transporting them into exosomes during the biosynthesis of exosomes. Here, we demonstrate the presence of 30 RBPs in exosomes that were shown to form RNA–RBP complexes with both cellular RNA and exosomal-RNA species. To assess the involvement of these RBPs in RNA-transfer into exosomes, the gene transcripts encoding six of the proteins identified in exosomes  were silenced by siRNA and subsequent effect on esRNA was assessed. A significant reduction of total esRNA was observed by post-transcriptional silencing of MVP, compared to other RBPs. Furthermore, to confirm the binding of MVP with esRNA, a biotinylated-MVP was transiently expressed in HEK293F cells. Higher levels of esRNA were recovered from MVP that was eluted from exosomes of transfected cells, as compared to those of non-transfected cells. Our data indicate that these RBPs could end up in exosomes together with RNA molecules in the form of RNA–ribonucleoprotein complexes, which could be important for the transport of RNAs into exosomes and the maintenance of RNAs inside exosomes. This type of maintenance may favor the shuttling of RNAs from exosomes to recipient cells in the form of stable complexes. Extracellular vesicles (EVs) represent a heterogeneous population of nano- and micro-sized vesicles secreted by virtually all cell types studied so far–ranging from prokaryotes to eukaryotes. Among these, the best characterized are exosomes which are 30–150 nm in diameter, originate from the endocytic compartments and are matured in the multivesicular bodies (MVBs) of the late endosomes (for detailed mechanisms see ). Upon mi.e., 18S and 28S rRNA [Notably, we for the first time, and later on other researchers have shown that exosomes obtained from various sources contain a substantial amount of RNA –24, and 28S rRNA , 19, 28,28S rRNA , 28–32. 28S rRNA , 32, 33.It is important to note that almost all RNA in cells exists as ribonucleoprotein (RNP) complexes. As such, proteins capable of interacting with RNA  are key players in the regulation of the post-transcriptional processing and transport of RNA molecules. Through specific interactions with their cognate RNA molecules, RBPs regulate RNA processing, nucleocytoplasmic transport and maturation, intra-compartmental localization and turnover –36. In fNot only within the cell, but also in the extracellular space the presence of RNA in association with RBPs is increasingly being recognized. This implies the secretion of RNA in EVs as EV-associated forms , 27, 37,In this study, we sought to identify whether the RBPs extracted from exosomes could interact with RNAs extracted from different sources i.e. with exosomal RNA (esRNA), as well as cellular mRNA and cellular miRNA and their interactions can be detected in the extracellular environment. We hypothesized that these proteins could be involved in (a) the packaging of RNA into exosomes, (b) maintenance of RNA molecules inside exosomes when secreted to extracellular environment. We present a method  showing the binding of exosomal proteins to cellular RNA  and esRNA. Further to this, we developed a method for the purification of RNA-RBP complexes from exosomes for the identification of RBPs in exosomes. The extracted exosomal proteins were allowed to bind with biotinylated RNAs from cells and exosomes with added streptavidin-coated Dynabeads. The bound proteins were re-extracted and were then loaded onto an SDS-PAGE gel, and RNA-RBP bands were excised, trypsinised and analyzed using LC-MS/MS allowing us the identification of 30 RBPs in exosomes that bind to different RNA species . Further, we validated the effect of RPBs on esRNA by silencing some of these RBPs in the cytoplasm and observed their consequence on RNA amount in exosomes. The post-transcriptional gene silencing of transcripts encoding six of the identified proteins  show that down-regulation of RBPs (in particular Major Vault Protein (MVP)) caused a significant reduction of the total RNA present in exosomes. Furthermore, to validate the binding of MVP with esRNA, cells were transfected with a plasmid encoding the full-length human MVP. After transiently expressing the biotinylated-MVP in transfected cells, the exosomes were isolated and the biotinylated MVP was eluted/purified from exosomes of transfected cells to assess the MVP binding with esRNA. Results showed that the biotinylated-MVP was detected in transfected cells and was partitioned to exosomes. Moreover, the higher levels of esRNA were recovered from the MVP eluted from exosomes of transfected cells as compared to non-transfected cells. In light of our findings, we propose (a) these RBPs could end up in exosomes together with exosomal-RNA (esRNA) molecules in the form of complex of RNA and ribonucleoproteins (RNP-complexes), and (b) these exosomal-RBPs could be important for the fate of the exosomal RNAs, including the transport of RNAs into exosomes and the maintenance of RNAs inside exosomes. This type of maintenance and stability may favor the shuttling of RNAs from exosomes to recipient cells , when cells are required to package RNA into exosomes and deliver to other cells.2 culture flasks  according to the manufacturer’s instructions. The fetal bovine serum  was depleted of exosomes by centrifugation for 70 minutes at 120000g followed by filtration through 0.2 μm filters and was added to culture medium. Cells were cultured for 72 hr and were harvested at 80% confluency. Cells were trypsinized and pelleted at 300g for 5 minutes and cell pellets were collected for cell lysate analysis. Exosomes were isolated from culture supernatant as described previously [g on a Beckman Optima LE80K ultracentrifuge by using Ti70 rotor for 70 minutes. Exosome pellets were resuspended in 300 μl PBS or directly lysed for RNA isolation.Human epithelial (HTB-177) cells  were cultured in 175 cmeviously , with slThe short RNA fractions, composed of RNAs smaller than 200 nucleotides and enriched in miRNAs from HTB-177 cells, were isolated using miRNeasy Mini kit followed by miRNeasy Cleanup Kit , according to the manufacturer’s instructions. MRNA from HTB-177 cells was extracted from total RNA using the Oligotex mRNA Mini Kit . Total RNA from HTB-177 exosomes was isolated by Trizol reagent , following the manufacturer’s instructions. The pattern of RNA  was evaluated using the Agilent RNA 6000 Nano Kit on an Agilent 2100 Bioanalyzer (Agilent Technologies).The native proteins from cells and exosomes were extracted using ProteoJET Mammalian Cell Lysis Reagent  together with Protease Inhibitor Cocktail  according to manufacturer’s instructions.The biotinylation of cellular miRNA, cellular mRNA, and exosomal total RNA was performed using Pierce RNA 3' End Biotinylation Kit  following the manufacturer’s instructions with some modifications. For each reaction cellular miRNA (50 pmol), cellular mRNA (1.6 pmol), RNA template control (50 pmol) and exosomal RNA (10 to 20 pmol) were biotinylated. Before incubating the reactions, the cellular miRNA/mRNA and total exosomal RNA were heated at 85°C for 5 minutes to relax the RNA secondary structure. Biotinylation reactions were incubated at 16°C for 2h (cellular miRNA), overnight (cellular mRNA), and 3h , and determination of the biotinylation efficiency was performed according to the manufacturer´s protocol. Briefly, serial dilutions of the biotinylated cellular short RNA and total exosomal RNA were dotted onto a nylon membrane  using a biotinylated IRE (Iron Response Element) RNA control. The nylon membrane was UV cross-linked with biotinylated RNA for 2 minutes followed by detection with enhanced chemiluminescent substrates for horseradish peroxidase (HRP) from Chemiluminescent Nucleic Acid Detection Module , as specified in the manufacturer’s protocol.Measurement of RNA:protein interactions was performed with the LightShift Chemiluminescent RNA EMSA Kit . The IRE RNA with cytosolic liver extract included in the kit was used as positive control according to the manufacturer’s instructions with some modifications. The incubated reactions included 4 nM of cellular miRNA with 4 μg lung cellular (HTB-177) protein extract, 4 nM of cellular mRNA with 8 μg lung cellular protein extract, 5 nM of cellular miRNA with 20 μg exosomal protein extract, 2 nM of cellular mRNA with 10 μg exosomal protein extract, 5 nM exosomal RNA with 20 μg exosomal protein extract. The specific unlabeled RNA competitors were added in excess (200 fold) in RNA-protein binding buffer (1X REMSA-buffer) for each of above-mentioned assays. Regarding the IRE RNA control, 6.25 nM biotinylated IRE RNA control was incubated with 4 μg of cytosolic liver extract and with 6 μg lung cellular (HTB-177 cell line) protein extract. Before adding the biotinylated RNA, the reaction components were pre-incubated for 15 minutes at room temperature (RT), and the binding reactions were incubated for 30 minutes at RT before adding the loading buffer. The binding reactions for IRE RNA and cytosolic liver extract and lung cells (HTB-177) proteins were electrophoresed on 4% and 6% DNA Retardation Pre-cast gels . The gels were transferred to a nylon membrane using the XCell II™ Blot Module  in 0.5X TBE, at 300 mA for 45 minutes. After UV crosslinking of biotinylated RNA to the nylon membrane, the biotinylated RNA was detected with the Chemiluminescent Nucleic Acid Detection Module , following the manufacturer’s protocol. The excess of unlabeled RNA (200 fold) was added to the binding reactions, and specificities of the RNA-protein interactions were examined (to observe if the labelled and unlabeled RNA sequences competed for binding to the same protein).2O, respectively, were added in order to reach the final concentration 1X for the binding buffer, scaling the relative amount of RNA/proteins from the REMSA experiments. The samples were incubated for 30 minutes at RT, with gentle rotation. To remove unbound proteins, the samples were washed twice with 1X binding buffer. The RBPs bound to the RNA were isolated by the addition of extraction buffer (50 mM Tris-HCl pH 7.5 + 0.1% SDS) to the complex and incubated at 85°C for 5 minutes. Samples without RNA were treated equally and used as negative controls to identify unspecific proteins bound to the beads.For the purification of RNA Binding Proteins (RBPs), paramagnetic Dynabeads M-280 Streptavidin  were used. The beads were first washed in Wash and Binding Buffer (W&B) three times. For RNA application, the beads were washed twice in the same volume of Solution A, once in Solution B, and re-suspended in W&B buffer, following the manufacturer’s instructions. 100 pmol of cellular biotinylated miRNAs were mixed with 1 mg Dynabeads, 40 pmol of biotinylated exosomal RNA were mixed with 400 μg Dynabeads, and 5 pmol of labelled mRNA were incubated with 500 μg of Dynabeads. All reactions were incubated for 20 min at RT with gentle rotation. The beads were separated on a MPC-S magnet , and after washing a small fraction (2 μl) of the sample was used for evaluating the binding after UV crosslinking of biotinylated RNA to the nylon membrane, by chemiluminescence. Biotinylated RNA/Dynabeads complexes were resuspended in the 2X RNA-proteins Binding Buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 M NaCl), and one volume of cellular and exosomal proteins + Hhttp://www.proteomics.cf.gu.se/). Briefly, for the liquid chromatography an Agilent 1100 binary pump was used, together with a reversed phase column, 200 x 0.075 mm, packed in-house with 3 μm particles Reprosilpur C18-AQ. To separate the peptides, the flow through the column was reduced by a split followed by a 50 min gradient of 0–50% CH3CN. The nano-flow LC-MS/MS was done on a 7-Tesla LTQ-FT mass spectrometer (Thermo Electron) equipped with a nanospray source modified in-house. The spectrometer was operated in a data-dependent mode, automatically switching to MS/MS mode. MS-spectra were acquired in the FTICR, while MS/MS-spectra were acquired in the LTQ-trap. For each scan of FTICR, the three most intense, doubly or triply charged, ions were sequentially fragmented in the linear trap by collision induced dissociation. All the tandem mass spectra were searched by MASCOT  program to identify proteins. The analysis was repeated two times.For protein identification, the isolated RBPs  or proteins from negative samples (without RNA) were collected in 5% SDS gel, stained with RuBPs, bands were excised, trypsinised and analyzed with LC-MS/MS by the Proteomics Core facility at the University of Gothenburg  for Gene Ontology (GO) analysis relative to RNA-related processes and cellular localization, and by literature search. To identify conserved RNA-binding domains, the specialized blast from NCBI was used, searching against Conserved Domain Database (CCD) database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml), which includes NCBI curated 3D protein domains, as well as data from 5 among the major sources of protein families databases [http://rbpdb.ccbr.utoronto.ca).Bio-computational analysis of the proteins identified by LC-MS/MS was carried out based on their biological functions and cellular localization by using the DAVID functional annotation tool (atabases . This anhttp://www.ingenuity.com) was used to build a biological network of identified RBPs in exosomes and their parental cells. Individual network analysis was performed for the identified-exosomal-RBPs interacting only with miRNAs and mRNA. Similarly, the network analysis for identified RBPs in parent cells interacting with both cell-mRNAs and cell-miRNAs was performed and biological functions were retrieved.Finally, the Ingenuity software (5 cells per well and grown in the presence of Opti-MEM I Reduced Serum Media (Thermo Fisher Scientific), 10% FBS (Sigma) previously depleted of exosomes, 1% Penicillin-Streptomycin (Gibco) and 1% L-glutamine (Gibco). HiPerFect transfection reagent was mixed with siRNA and added to the cells at a final concentration of 10 nM. The list of siRNAs and QPCR-primers used in this study is presented in Table 1. AllStars negative control siRNA  was used as non-targeting irrelevant siRNA. The transcripts were silenced in duplicate experiments except for hnRNPA2B1 and MVP, which were performed in 4-replicates.Gene silencing in HTB-177 cells was achieved by transfecting corresponding siRNA against the mRNA transcripts of each of the six genes using HiPerFect transfection reagent from Qiagen according to the manufacturer´s protocol. Cells were plated in 6-well tissue culture plate (Sarstedt) at a density of 6 x 10After 48 hours of incubation at 37°C, the supernatants were harvested and pooled; exosomes were extracted from the conditioned cell culture supernatant from siRNA transfected cells by centrifugation at 300g for 15 minutes to pellet the cell debris; then the supernatant was ultracentrifuged at 120000g on a Beckman Optima LE80K ultracentrifuge by using Ti70 rotor for 70 minutes. The isolated exosomes were resuspended in 30μl of PBS, and were used for both their quantification, as proteins, by using the Qubit spectrofluorometer (Thermo Fisher Scientific) and the isolation of total RNA using the miRCURY™ RNA Isolation Kit–Cell & Plant (Exiqon) according to the manufacturers’ instructions. Exosomal RNA was quantified using the Quant-iT™ RiboGreen® RNA Assay Kit (Thermo Fisher Scientific) and taking advantage of a FLUOstar Omega Microplate Reader (BMG LABTECH). To express the relative amounts  of RNA present in exosomes originated from HTB-177 cells, transfected with siRNA against MVP for 48 hours in comparison with the correspondent non-targeting negative control siRNAs, for each sample, the ratio between total exosomal RNA (ng) / total exosomes (μg) was calculated.After 48 hours of incubation at 37°C, the siRNA transfected cells were directly lysed in the wells for RNA isolation using the miRCURY™ RNA Isolation Kit–Cell & Plant (Exiqon), according to the manufacturers’ instructions. The RNA was quantified by Qubit spectrofluorometer (Thermo Fisher Scientific).https://www.graphpad.com/), the significant p-value threshold was >0.05.The statistical significance of exosomal and cellular RNA variation after MVP silencing was calculated by applying an unpaired t-test with Welch`s correction using the software GraphPad Prism v7.02 (RNA from transfected HTB-177 cells was reverse transcribed with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For the assessment of MVP silencing efficiency, 1 μg of total RNA from transfected HTB-177 cells was reverse transcribed with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Q-PCR was performed on an Applied Biosystems Real-Time PCR System Viia 7 using 100 ng of the resultant cDNA, TaqMan Fast Advanced Master Mix (Applied Biosystems) and gene specific TaqMan probe. The same procedure was used for the quantification of other silenced five genes except the use of Fast SYBRgreen PCR Master Mix (AppliedBiosystems) and gene specific primers. Gene expression was normalized to the housekeeping gene GAPDH as below:2CT-ΔΔ:In Q-PCR, the relative quantification of each silenced gene, after 48h siRNA-silencing, is calculated in comparison to the relative negative control samples  and normalized to the housekeeping gene GAPDH. We used the method of Where RQ stands for relative quantification and represents the fold change of silenced gene, e.g. the transcripts of MVP gene. The data is presented as average fold change values (RQ). The average of four biological replicates for hnRNPA2B1 and MVP, and two biological replicates for other four transcripts were presented.HEK293F cells (Life Technologies) carrying a stable integration of the BirA (biotin ligase) coding sequence were transiently transfected with a plasmid encoding the full-length human MVP protein with a C-terminal avitag and a 10x histidine tag. Two hundred and seventy mL of cell suspension at a density of 1.1 x 106/mL were resuspended in Freestyle media in a 2L Erlenmyer flask and placed in a shaking incubator for three hours at 37°C, 8% CO2, 140 rpm. Transfection reactions were set up by incubating 200ug plasmid DNA in Optimem and 300μl 293fectin (life Technologies) in Optimem separately for 5 minutes at room temperature, before mixing and incubating for a further 20 minutes at room temperature. The DNA/293fectin complex was added to the cells that were supplemented by 300mL of Freestyle media three days later.2, 2 mM DTT, 160 U/mL RNasin (Promega), 2x proteases inhibitor , and 0.4% Triton x-100) were added to cell pellets and resuspended well to create the cell lysate.Six days post-transfection, the cells were harvested by centrifugation at 2500xg for 20 minutes. The cell pellets were resuspended in RIPA buffer + protease inhibitors (100ul), spun down and kept for MVP analysis from cell lysate). 1 volume of 2x lysis buffer [100 mM Tris-HCl pH 7.5, 300 mM NaCl, 3.6 mM MgClThe supernatants were then run through a stericup filter (0.2 micron) and concentrated using tangential flow filtration with a 300kDa cut-off filter to a volume of 50mL. Ultracentrifugation was then performed to isolate exosomes at 38,000 rpm for 90 minutes at 4°C (Beckman: Ti45 rotor) and the exosome pellets were resuspended in 500μl PBS.2, 2 mM DTT, 160 U/mL RNasin (Promega), 2x proteases inhibitor , and 0.4% Triton x-100) were added to exosome pellets and resuspended well to create the exosome lysate. The resultant exosome lysate was then used for the MVP analysis. The biotinylated MVP from exosome lysates was captured onto streptavidin-coated Dynabeads M-280 that had been blocked with BSA and yeast tRNA to prevent the non-specific binding of proteins and RNA, respectively. For the MVP capture, 1.4 mg of total protein was incubated with 450 μL of paramagnetic Dynabeads M-280 Streptavidin , at 4°C for 3 hours with gentle agitation. Previously, the Dynabeads were washed and prepared for RNA manipulation following the manufacturer’s specifications, and a blocking step was performed by adding the yeast tRNA  and incubated for 1 hour at 4°C with agitation, followed by two washes with 0.01% PBS-BSA. Exosomes from untransfected HEK293F cells were processed in parallel and were included in the analysis as negative control.Exosomes (from transiently transfected HEK293F cells) were used for MVP analysis. 1 volume of 2x lysis buffer [100 mM Tris-HCl pH 7.5, 300 mM NaCl, 3.6 mM MgClAfter the MVP capture incubation, 511ng of denatured RNA extracted from un-transfected exosomes was added to the dynabeads-exosome samples and incubated for 1 hour at 25°C. Finally, samples were washed 4 times with PBS-BSA (0.01%), and MVP/RNA complexes were eluted from the Dynabeads adding 350 uL of lysis solution buffer , vortexing 15 seconds and incubating 5 min at room temperature with gentle agitation. 20 uL of eluted sample was kept for protein detection and the RNA was extracted from the remaining volume following kit specifications. Protein samples from different stages (elutes) of RBP purification were separated by SDS-PAGE and the protein bands were detected by Comassie staining. The presence of MVP in the elution buffer from different elutes/fractions was confirmed by SDS-PAGE (pull-down). RNA was quantified by Quant-iT™ RiboGreen® RNA Assay Kit (ThermoFisher Scientific).https://www.graphpad.com/), considering the significant p-value threshold as >0.05.The statistical analysis was performed applying an unpaired t-test with Welch`s correction using the software GraphPad Prism v7.02 . The results revealed that the biotinylation reaction for all RNA species used  was highly efficient, with biotinylation rates greater than 75%.The physical interactions between exosomal-RBPs with RNAs from exosomes and RNA from their parental cells were investigated using RNA electrophoretic mobility shift assays (REMSAs). RNA from exosomes (esRNA) and RNA from their parental cells (miRNA and mRNA) were enzymatically labelled with biotin at their 3’ terminus. A 43 nucleotide synthetic RNA template and a biotinylated iron response element (IRE) RNA were used as an internal reference and positive control, respectively for the assessment of biotinylation rate. Dot blots were performed to evaluate the efficiency of the RNA labelling reaction and to calculate the amount of the biotinylated RNA obtained . The specificities of RNA-protein interactions were determined using competition assays, in which the excess (200 fold) of unlabeled RNA was added to the binding reactions, resulting in a decrease in the shifted signal if the labelled and unlabeled RNA sequences competed for binding to the same protein. The control samples behaved as expected (first group). Although, the same amount (200-fold) of competitor RNA was used, there was a difference in band shifts between IRE RNA + liver proteins  and IRE RNA + cellular proteins . This difference in the shift i.e. partial can be explained by following possibility; there was an excess of cellular proteins for which the loaded competitor RNA  was not enough to compete with IRE RNA  and we see the partial band shift from this labeled RNA. Moreover, the liver extracts are abundant in IRP (iron-responsive protein) and bind more specifically to IRE RNA, whereas the proteins from our cell extracts are heterogeneous and not enriched in IRP as compared to liver extracts which is enrich in IRP. In addition to shift assays of esRNA and cell miRNA, although, although we identified mRNA-binding proteins in exosomes, but the gel did not show the shift for mRNA .To perform REMSA, the protein extracts from exosomes, HTB-177 lung cells, and liver were obtained and incubated with the biotinylated RNA species described above. A reaction containing the IRE RNA and cytosolic liver protein extract was used as a positive control because the IRE and iron-regulatory protein interaction is ubiquitous in cells. The native proteins from cells and exosomes were incubated with biotinylated iron-responsive element RNA (IRE RNA), cellular miRNA, and esRNA in separate assays. The presence of RNA-protein complexes for the different RNA species Fig 2A). Samples without RNA were processed in parallel and were used as negative controls to identify proteins that bound non-specifically to the beads. The experiment was performed twice and proteins identified in both biological replicates, but not in the negative controls, were taken forward . For each assay, we compared the proteins detected in negative control and the actual sample. We detected some common proteins both in negative sample (this might be due to non-specific binding to beads) and actual sample. We excluded the common proteins that were also detected in negative sample. It is likely that they might be RBPs but we excluded them to avoid any misinterpretation of data. In total, 30 RBPs were identified from exosomes from the individual RNA species assays . From 30 exosomal-RBPs, 20 RBPs were identified in complex with esRNA. Out of these 20 esRNA-binding exosomal RBPs, 12 were exclusive to esRNA and rest of the 8 were shared (Table 2 and S1 Table). 9 RBPs were identified in complex with cellular miRNA (Table 2 and S2 Table), and 14 RBPs were identified in complex with cellular mRNA (Table 2 and S3 Table). The overlapped proteins between the lists of exosomal RBPs that bind with different RNA species are shown in Venn diagrams . Notably, four RBPs  were common to all three subsets . 1 exosomal-RBP was common between cell-miRNA and cell-mRNA samples. 2 exosomal-RBPs were in complex with cell-miRNA only, and 7 were in complex with cell-mRNA only.To detect and isolate the RBPs in cells and exosomes, the biotinylated esRNAs, cellular miRNAs, and cellular mRNAs were attached to streptavidin-coated paramagnetic Dynabeads M-280 and were incubated with native protein extracts of exosomes in separate RNA species assays i.e. . The protein-RNA complexes were eluted from the beads. The RBPs bound to different RNAs were then loaded onto an SDS-PAGE gel and target bands were excised, trypsinised and analyzed using LC-MS/MS . As expected, the number of RBPs identified in the parental cells was higher than the number of RBPs in exosomes. In total, 122 RBPs were identified from cells (S4 Table), of which 72 were in complex with miRNA (S5 Table), 82 were in complex with mRNA (S6 Table), and 33 were common to both samples (RBPs identified in complex with cell-miRNA and cell-mRNA) (S4 Table). The overlap between the lists of identified RBPs in cells and exosomes are shown in Venn diagrams .The assays were also performed for protein extracts from cells i.e.  and the protein-RNA complexes were eluted from the beads and RBPs were then identified by LC-MS/MS (same method adopted in Table 2). We screened the candidate proteins for the presence of known functional RNA-binding domains  using the NCBI tool CDS. In addition, we performed a gene ontology (GO) term molecular function analysis of all identified proteins using the DAVID annotation tool (https://david.ncifcrf.gov/), considering the GO terms related to RNA binding, processing, splicing, mRNA/ncRNA metabolic processing, and the RNP complex. The protein classification was improved further by a literature search to identify experimental evidence of the involvement of the proteins in direct or indirect RNA binding.To classify the tentative functions of these RBPs (of particular interest those found in exosomes), the list was subsequently filtered based on the predicted abilities of the proteins to interact with RNA either directly or indirectly . The network included 35 nodes (gene products), 27 of which were among the identified exosomal-RBPs including MVP (indicated in grey). Individual network analysis was performed for the identified-exosomal-RBPs interacting only with miRNAs and mRNA ( respectively). The retrieved functions showed that these RBPs are involved in ‘RNA post-transcriptional modification, protein synthesis, and RNA-transport. Similarly, the network analysis of identified RBPs in parent cells interacting with both cell-mRNAs and cell-miRNAs was performed and biological functions were retrieved (S5 Table). The network retrieved for the cellular-RBPs identified in this study comprised 35 proteins (including MVP) involved in ‘RNA post-transcriptional modification, protein synthesis, and gene expression’. The proteins’ MS data (http://microvesicles.org). Vesiclepedia is an integrated and comprehensive compendium of molecular data  identified in different classes of extracellular vesicles derived from different species ranging from archaea, bacteria, and eukarya, including human.To understand the direct and indirect functional associations between the query protein sets, we used Ingenuity Pathway Analysis (IPA) software to build a biological network of the RBPs in exosomes and their parental cells. A key function of the exosomal-RBPs identified in this study was ‘RNA post-transcriptional modification and protein synthesis’ ( MS data  Tables hS7 Table).To evaluate the possible role of exosomal-RBPs in the transport of RNA into exosomes (esRNA), gene transcripts encoding six of the identified exosomal-proteins  were silenced in cells using short interfering RNAs (siRNAs). XPO5 was identified only in one of the replicates but still it was included among the genes that were silenced  . Conversely, the down regulation (silencing) of MVP caused a significant reduction in total RNA present in exosomes, approximately by 50%  . However, after silencing of hnRNPH1 the amount of RNA in exosomes was increased as compared to negative control. Interestingly, the silencing of hnRNPA2B1, an RBP that has previously been shown for sorting of RNA into exosomes [Fig 4B). The reduction of RNA amount in exosomes after the silencing of RBPs in cytoplasm indicates the possible involvement of RBPs (particularly MVP) in RNA transport into exosomes.To further determine whether the selected RBPs are involved in the transport of RNA into exosomes, the amount of RNA present in exosomes was determined after silencing. The results showed that post-transcriptional silencing of HSP90AB1, XPO5, hnRNPH1, or hnRNPM does not reduce significantly the amount total RNA present in exosomes . Next, we captured the biotinylated MVP (from lysed cells and exosomes) onto streptavidin-coated Dynabeads M-280, and eluted by pulldown assay to confirm the expression of biotinylated MVP in transfected cells and subsequent detection in exosomes. After the pull down assay, the transient expression of MVP in the cytoplasm of transfected cells was confirmed, whereas shown absent in untransfected cells ( PD (pull-down lane). After the pull down assay, the presence of MVP within exosomes from these transected cells was confirmed ( PD (pull-down lane)), whereas absent in exosomes from untransfected cells ). Further, we investigated whether the exosomal-MVP was coupled with RNAs. RNAs that had co-eluted with MVP was subsequently isolated and quantified which showed that the amount of RNA present in the MVP elute (i.e. from MVP-RNA complex) was significantly higher than untransfected control .Based on our results that transcriptional-silencing of MVP causes down regulation of the RNA content in exosomes, we further investigated (i) whether, the Major Vault Protein (MVP) is in complex with RNAs within exosomes, as RNA-protein (RNPs) complexes. We transiently expressed biotinylated MVP in HEK293F cells stably expressing biotin ligase (BirA) and purified exosomes from these cells. A Western blot analysis for the detection of MVP in HEK293F cell lysate and exosomal protein extracts shows that plasmid (MVP-biotin) was successfully expressed in cells and was partitioned into exosomes when overexpressed in their parent HEK293F cells . Because the predominant cellular content i.e. rRNA (80–85%) [Using high-throughput protein identification and characterization, other investigators have identified nucleoproteins in exosomes listed in Vesiclepedia . Based o(80–85%) , is almo(80–85%) , we sele(80–85%) ; and . However, there is no clear picture how many RBPs may exist in exosomes and we may have missed identifying all RBPs in these EVs, especially RBPs that are expressed slightly in exosomes or they bind to few number of RNA molecules. Among the specific exosomal-RBPs that we identified, approximately 90% have already been reported in proteomic studies of different exosome types [e.g., hnRNPA2, hnRNPH1, hnRNPK, hnRNPE1, and hnRNPUL1) may play a role in nucleocytoplasmic RNA transport under specific conditions [In this study, we identified 30 RBPs in exosomes that were able to bind to cellular RNA and esRNA species. Out of these 30 RBPs, twenty exosomal-RBPs bound to esRNA ; 14 to cell-mRNA, and 9 to cell-miRNA, whereas four RBPs were common between the three groups  maturation. We detected ELAC2, which catalyzes the removal of the 3’ trailer from precursor tRNAs, leading to the production of tRF-1001 , as wellFig 3), recent evidence show that post-transcriptional modifications of RNA could be relevant for the sorting of RNA into exosomes [In the context of networking and post-transcriptional modifications  could collaborate (could work cooperatively) to determine above-mentioned roles in RNA sorting into exosomes. Regarding post-transcriptional modification and gene synthesis in the exosome context, there are already some evidences that the exosomal proteome is enriched in proteins involved in such processes [The networking and presence of post-transcriptional modifier RBPs identified in our exosomes . This protein is a multi-subunit RNP that together with other proteins forms a large macromolecular complex, a characteristic hollow barrel-like shape . BecauseThe differences between the presentation of cellular and esRNAs indicate that esRNAs are not randomly packaged into exosomes; rather there must be a strictly regulated sorting mechanism that provides these vesicles with a subset of RNA, during their biosynthesis. It has been shown that all RNAs within cells are not naked; rather they are coupled to RBPs, as well as their associated proteins (binding partners), that affect their metabolism as well as intra-compartmental translocations. This suggests that esRNA must be coupled to proteins (i.e. they must exist as RNP complexes).We identified RBPs in exosomes that were able to interact with different RNA species. Our assessment is that: (a) since RBPs are found bound to their related RNAs, these RBPs do not end up in exosomes alone, but together with RNA molecules as a complex of RNA and ribonucleoproteins, and (b) the presence of RBPs in exosomes could be for purpose such as the maintenance of RNAs inside exosomes and shuttling of this RNA into other cells–so called exosome shuttle RNA (esRNA) .Table 2) and are known to be involved in different stages of RNA metabolism, including transport, splicing, and maturation. Notably, our results revealed that selective silencing of cytoplasmic MVP significantly reduced the amount of total RNA in exosomes , indicating the possible role of MVP in RNA transport from cells to exosomes. Moreover, after the pull down assay the quantification of RNA that had co-eluted with MVP showed that the amount of esRNA present in the MVP-elute was significantly higher than esRNA from untransfected cells. Interestingly, this indicates that MVP is complexed with higher level of exosomal RNAs than exosomes from non-transfected cells . In the current scenario, our results indicate that MVP is important in RNA transport into exosomes, however, which RNA species interact with MVP in these exosomes still remains to be answered. Studies have shown that the most abundant RNA species in exosomes is vault RNA (vRNA) and that this RNA binds to MVP [Fig 4D). However, in addition to vault RNA found in exosomes, the presence of several other coding and ncRNA species have been reported in exosomes [Although, RBPs have been reported to regulate exosome biogenesis, their role in RNA sorting into exosomes is only recently beginning to be explored , 44, 45.s to MVP . In lineexosomes , 88. MVPexosomes . It has exosomes . Given tFig 4B). This inverse correlation of hnRNPH1 to the amount of esRNA can be hypothesized in a way that this protein could be important in feedback-loop to keep the cytoplasmic RNA levels (intracellular retention of RNA) by preventing RNA transport into exosomes. However, the current data regarding hnRNPH1 is obtained from two biological replicates and whether this protein has a possible role in cytoplasmic RNA retention would need further validation.Surprisingly, after silencing of hnRNPH1 the amount of RNA in exosomes was increased as compared to negative control . Recently, a study identified the role of sumoylated hnRNPA2B1 in the transfer of specific subset of miRNAs into exosomes from Jurkat cells [Fig 2C). This discrepancy of hnRNPA2B1 in the transfer of specific subset of miRNAs into exosomes could be due to steric hindrance caused by sumoylated residues in hnRNPA2B1 that prevent the protein/RNA interaction at the surface of the Dynabeads. If miRNAs are transported to the exosomes by this modified form of the protein, which is predominant but not exclusive in exosomes, we might have identified the interactions taking place between RNAs and the regular form of the protein (non-sumoylated). Alternatively, the interaction between hnRNPA2B1 and its target RNAs may be specific and targeted to certain miRNA molecules that cannot be detected by the method used here. However, further studies may rule out these discrepancies.In addition to reduction of total exosomal RNA after MVP silencing, the silencing of hnRNPA2B1 also caused a slight reduction of total RNA present in exosomes, but at non-significant level ≈13% . However, in which stage RNP-RNA complexes are inserted into exosomes–during the biosynthesis of vesicles and what are the underlying mechanisms remains unclear.After having shown the interactions of exosomal-RBPs with different RNA species, it indicates that the RNA found in exosomes are coupled to RBPs (i.e. they exist as RNP complexes) that we detected in our study . In the first model, RNA-protein-complexes (RNPs) could use a budding mechanism from nuclear envelop (NE) to be packaged into vesicles; termed “esRNA–protein” granules. The esRNA-protein granules could fuse and enter into MVBs forming intermediate vesicles, which later mature into intraluminal vesicles of MVBs. A similar process has been described in Drosophila for the existence of RNP-granules from the nucleus at the neuromuscular junction. Specifically, RNP-granules fuse with the inner nuclear membrane and bud into the nuclear envelope. Subsequently, the RNP-containing buds could migrate into the cytoplasm by fusing with the outer nuclear membrane [ right side), RNA–protein complexes (RNPs)—transported via nuclear pore complex (NPC)—are attracted to the internalization site / receptor-proteins that could mediate inward budding of MVBs, to form intraluminal vesicles. Upon fusion with the plasma membrane, the MVBs release the intraluminal vesicles as exosomes containing RNA–RNPs complexes into the extracellular environment.Here, we postulate two plausible paths for the transport of RNAs into exosomes during biosynthesis of these vesicles from multivesiclur bodies (MVBs)  in exosomes and opens further avenues to explore the underlying mechanisms. However, our data does not show the specific sequence motifs of MVP and esRNA species. Since the formation as well as the silencing of MVP-RNA complexes have been validated and verified in all independent assays, and at least in two cells lines (HTB-177 and HEK293F), the absence of RNA sequencing to find binding motif does not alter our conclusion that MVP plays important role in RNA transport into exosomes. Shurtleff and colleagues have proposed that different cell types may use RBPs with distinct binding preferences to secrete miRNAs, and perhaps other RNA classes, in exosomes. Authors have also proposed that some cell types may deploy multiple RBPs to sort RNAs into exosomes . Our datS1 FigAlthough we identified mRNA-binding proteins in exosomes, but the gel did not show the shift for mRNA.(TIF)Click here for additional data file.S2 FigVenn diagrams that present the overlap between the lists of identified RBPs in cells (A) and exosomes (B) capable of binding to different species of RNAs.(TIF)Click here for additional data file.S3 FigThe RBPs in this network are involved in “RNA post-transcriptional modification, carbohydrate metabolism, and lipid metabolism”.(TIF)Click here for additional data file.S4 FigThe main function of this network is “RNA post-transcriptional modification, gene expression, and protein synthesis”.(TIF)Click here for additional data file.S5 FigThe proteins of this network are involved in “RNA post-transcriptional modification, protein synthesis, and gene expression”.(TIF)Click here for additional data file.S6 Fig(A) Western blot showing the confirmation of MVP-biotin expression in transfected HEK293F cells after the pull down assay (PD (pull-down lane). (B) The presence of MVP within exosomes from biotinylated MVP-transfected cells PD (pull-down lane), whereas it was absent in exosomes from untransfected cells.MVP-Bio: Biotinylated MVP. Samples: W = proteins from different wash steps during the MVP purification, and PD = pull-down proteins (MVP) after elution, I = input, and UB = unbound after RBP capture.(TIF)Click here for additional data file.S1 TableIn total, 47 proteins were identified, including 20 RBPs (bold), according to GO terms and data retrieved from literature. Proteins in common with negative controls (13 proteins) are listed separately below. None of the proteins present in the negative control were RBPs.(PDF)Click here for additional data file.S2 TableIn total, 64 proteins were identified of which 9 proteins were RBPs (bold) according to the GO terms. Proteins in common with negative controls (12 proteins) are listed separately below. None of the proteins present in the negative control were RBPs.(PDF)Click here for additional data file.S3 TableIn total, 26 proteins were identified of which 14 proteins were RBPs (bold) according to the GO terms. Proteins in common with negative controls (81 proteins) are listed separately below. None of the proteins present in the negative control were RBPs.(PDF)Click here for additional data file.S4 Table122 known RBPs were identified in total: 72 RBPs in complex with miRNA and 82 in complex with mRNA. 32 RBPs were in common in both the samples (miRNA and mRNA).(PDF)Click here for additional data file.S5 TableIn total, 157 proteins were identified, including 72 RBPs (bold), according to GO terms. Proteins in common with negative controls (33 proteins) are listed separately below. None of the proteins present in the negative control were RBPs.(PDF)Click here for additional data file.S6 TableIn total, 238 proteins were identified of which 83 proteins were RBPs (bold) according to the GO terms. Proteins in common with negative controls (56 proteins) are listed separately below. None of the proteins present in the negative control were RBPs.(PDF)Click here for additional data file.S7 TableThe RQ values have been calculated accordingly to: (1) endogenous control GAPDH and (2) calibrator sample negative control (HTB-177 cells treated with NC-siRNA). Data from four biological replicates are shown.(PDF)Click here for additional data file.


The highly abundant, small stable RNAs that are synthesized by RNA polymerase III (RNAPIII) have key functional roles, particularly in the protein synthesis apparatus. Their expression is metabolically demanding, and is therefore coupled to changing demands for protein synthesis during cell growth and division. Here, we review the regulatory mechanisms that control the levels of RNAPIII transcripts and discuss their potential physiological relevance. Recent analyses have revealed differential regulation of tRNA expression at all steps on its biogenesis, with significant deregulation of mature tRNAs in cancer cells. The basic machinery for RNA transcription evolved prior to the last common ancestor of all extant organisms. Bacteria and Archaea retain a single RNA polymerase (RNAP), whereas most Eukaryotes use three distinct polymerases for different classes of RNA, with partially overlapping sets of components . TranscrSRP1) and RNase P (RPR1)  and a Wellcome Trust Fellowship [077248] to D.T. Work in the Wellcome Trust Centre for Cell Biology is supported by Wellcome Trust core funding [092076].