RNA/DNA hybrids form when RNA hybridizes with its template DNA generating a three-stranded structure known as the R-loop. Knowledge of how they form and resolve, as well as their functional roles, is limited. Here, by pull-down assays followed by mass spectrometry, we identified 803 proteins that bind to RNA/DNA hybrids. Because these proteins were identified using in vitro assays, we confirmed that they bind to R-loops in vivo. They include proteins that are involved in a variety of functions, including most steps of RNA processing. The proteins are enriched for K homology (KH) and helicase domains. Among them, more than 300 proteins preferred binding to hybrids than double-stranded DNA. These proteins serve as starting points for mechanistic studies to elucidate what RNA/DNA hybrids regulate and how they are regulated. RNA/DNA hybrids are abundant in human cells. They form during transcription when nascent RNA is in close proximity to its DNA template. The resulting RNA/DNA hybrids and the displaced single-stranded (ss) DNA are called R-loops. RNA/DNA hybrids are structurally different and more stable than the corresponding double-stranded DNAs .RNA/DNA hybrids are found in origins of replication , immunogBecause the number and location of hybrids are critical to maintaining cellular function, most likely there are regulatory proteins that distinguish RNA/DNA hybrids from their double-stranded (ds) DNA counterparts. The structures of RNA/DNA hybrids with different sequences have been studied alone  and in cThe roles of RNA/DNA hybrids are beginning to be recognized, but much remains unknown. It is not clear what regulates the formation and resolution of RNA/DNA hybrids. It is also not known how hybrids affect processing of RNA and what transcriptional steps they regulate. Naturally occurring mutations and yeast mutant collections have facilitated much of the mechanistic studies of R-loops. But the mutant screens alone have yet to yield a comprehensive view of R-loops. High-throughput methods to identify proteins that interact with nucleic acids have provided valuable information on gene regulation . A comprBAMBI gene, and the second is in the 3′ end of the DPP9 gene. We synthesized 600-mer and 90-mer RNA/DNA hybrids that correspond to sequences underlying R-loops in BAMBI and DPP9, respectively. BAMBI and DPP9. The BAMBI and DPP9 regions are GC-rich, with GC content of 76% and 62%, respectively, consistent with findings that regions with G-rich RNA and complementary C-rich DNA are prone to hybrid formation . Among the proteins identified in our BAMBI and DPP9 pull-down assays are RNase H1 (RNASEH1) and XRN2, which are known to bind to and modify RNA/DNA hybrids . This provides evidence that the hybrid-binding proteins we identified interact with R-loops in vivo. Second, we validated the hybrid-protein interaction by reverse immunoprecipitation. Using antibodies specific for DDX1 and FUS, we pulled down the protein-nucleic acid complexes, then by quantitative PCR, we showed enrichment of DNA and RNA corresponding to the BAMBI and DPP9 hybrids  complex that comprises the non-POU domain containing octamer binding protein (NONO) and paraspeckle protein component 1 (PSPC1). DBHS proteins form heterodimer and oligomers with multiple domains for RNA binding . The ubiquitin transferase, UHRF1, interacts with and recruits DNMT1 . There are 14 proteins that bound to only the hybrid forms of BAMBI and DPP9 but not to the corresponding dsDNA. These include several members of the nuclear exosomes such as DIS3L, EXOSC3, and EXOSC6. In addition, there are 350 proteins that favor the hybrid forms of BAMBI and DPP9 compared to the corresponding dsDNA. We validated the affinity of some of these proteins to RNA/DNA hybrids versus dsDNA by biolayer interferometry. BAMBI and DPP9 hybrids are distinct, the proteins that bind to both of them likely recognize their hybrid structures rather than sequences. Some of these hybrid-binding proteins, such as nucleolin  that is known to bind to hybrids, but most of the proteins were not known to interact with hybrids. We classified these hybrid-binding proteins into those (84) that are repelled by hybrids and those (364) that are attracted to hybrids relative to the underlying dsDNA. Although the hybrid-binding proteins were identified through in vitro studies, we provide evidence that they interact with R-loops in vivo.In this study, we identified more than 800 proteins that bound to RNA/DNA hybrids. R-loops are three-stranded structures that comprise an RNA/DNA hybrid and a displaced ssDNA. Here, we focused on the hybrid because the ssDNA has been the focus of many studies in the DNA repair field. We used two biotinylated hybrids corresponding to R-loops in the promoter of Cellular functions rely on proteins and their interactions with each other and with nucleic acids. Although binding does not imply function, this set of hybrid-binding proteins helps to narrow down where one should focus further investigations. Such efforts are particularly useful at the beginning of studies in which preliminary results suggest many pathways could be involved and targeted analysis may miss critical pathways. Studies are elucidating the functions of R-loops, but much remains unknown. The 803 hybrid-binding proteins described in this paper suggest that proteins involved in RNA processing from splicing to unwinding RNA are involved in hybrid-mediated regulation. RNA/DNA hybrids are also key components in DNA replication as Okazaki fragments. Molecular studies of the hybrid-binding proteins identified in this study can elucidate how proteins divide their roles (or not) between transcription and DNA replication. These hybrid-binding proteins serve as a starting point for studying how proteins recognize RNA/DNA hybrids and the functional consequences of these interactions. Delineation of how these proteins interact with RNA/DNA hybrids in transcription, DNA replication, and other cellular processes will deepen our understanding of these crucial biological pathways.5 cells/mL in RPMI 1640 supplemented with 15% fetal bovine serum, 2 mM L-glutamine, and 100 units/mL penicillin-streptomycin.Immortalized B-cells (Coriell) were cultured to a density of 5 × 10DPP9 3′ UTR  were synthesized by Integrated DNA Technologies . Oligos were dissolved in Annealing Buffer . To generate the RNA/DNA hybrid, 10 µM of each oligo was mixed and heated for 5 min at 95°C and cooled down gradually to room temperature.Ninety-mer RNA and DNA oligos corresponding to BAMBI promoter  was generated as previously described . The 600-nt RNA transcript was synthesized from this BAMBI dsDNA template using MEGAscript T7 Transcription kit . The T7 promoter sequence in dsDNA was then removed by SfcI  digestion. The dsDNA and the transcribed ssRNA were dissolved in 10 mM Tris HCl at pH 8.0, 1 mM EDTA, 50 mM NaCl, and incubated for 5 min at 95°C and slowly cooled down to room temperature. Reannealed dsDNA was removed by HpaII digestion , and the RNA/DNA hybrid was purified using agarose gel electrophoresis.Six hundred-mer RNA/DNA hybrid corresponding to escribed . dsDNA wTo confirm the integrity of RNA/DNA hybrids, each hybrid was digested with RNase H1 (a gift from Dr. Robert Crouch at the NIH) or RNase T1  for 1 h at 37°C, extracted with phenol/chloroform, and precipitated with ethanol. The digested hybrid was analyzed by agarose gel electrophoresis and used in protein precipitation experiments.Cultured B-cells were lysed in lysis buffer  supplemented with 1× Complete protease inhibitors (Roche), 1× phosphatase inhibitors II and III (Sigma), and 0.1 unit RNase inhibitor (Thermo Fisher Scientific). Cell lysates were precleared for 2 h at 4°C using streptavidin beads . Thirty picomoles of biotinylated RNA/DNA hybrid or dsDNA were conjugated with streptavidin beads and incubated with precleared lysates containing 240 µg total protein for 2 h to overnight at 4°C. Protein-nucleotide complexes were pulled down with streptavidin beads and washed three times in 20 mM Tris at pH 7.5, 10 mM NaCl, 0.1% Tween-20. Proteins were eluted in 1× LDS sample buffer  containing 1× sample reducing reagent  for 5 min at 95°C.Hybrid-binding protein was validated by Western blot using the following antibodies: anti-NONO , anti-NPM1 , anti-RECQL , and anti-GAPDH .Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis was performed. Each eluted sample from protein precipitation was divided into five fractionated by one-dimensional SDS-PAGE. Each fraction was digested in gel, and tryptic peptides were injected onto a UPLC Symmetry trap column . A blank gel slice was digested and injected as a background control. Tryptic peptides were separated by reversed phase HPLC on a nanocapillary analytical column . Eluted peptides were analyzed on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific).MS/MS spectra were searched against the UniProt human database  with theBAMBI and DPP9 hybrids, respectively, and 1092 and 995 proteins were pulled down by BAMBI and DPP9 dsDNA, respectively. We focused on proteins that were reproducibly pulled down by both BAMBI and DPP9 in downstream analysis. To exclude the possibility that the hybrid-binding proteins are pulled down through nonspecific binding to nucleic acids, we pre-incubated B-cell extract with biotinylated ssRNA and dsDNA sharing the same sequence with BAMBI hybrid and depleted nonspecific proteins using streptavidin beads. We then repeated pull down and LC-MS/MS using BAMBI hybrid and showed that all hybrid-binding proteins were still specifically pulled down. Fold enrichment of proteins in pull down is calculated as the ratio of MS/MS counts of a detected protein from two samples. We set the threshold of fold enrichment ≥1.2 for a protein to be considered enriched.To identify proteins that specifically bind to hybrids or dsDNA in the pull-down experiments, we required at least four peptides with unique sequences from a given protein to be included. Using this criterion, 1460 and 1018 proteins were pulled down by 6) were lysed in 400 µL cell lysis buffer , and nuclei were collected by centrifugation. The nuclei pellet was resuspended in 200 µL nuclear lysis buffer . Genomic DNA containing R-loops was then extracted by phenol:chloroform and precipitated by ethanol. Purified material was resuspended in 200 µL IP dilution buffer  and sonicated at 4°C in Bioruptor (Diagenode) at Hi setting (30 sec on/30 sec off) for 5 min, three times, to fragments with an average size of 500 bp. Three micrograms of S9.6 monoclonal antibody (a gift from Dr. Stephen H. Leppla at NIH) or nonspecific mouse IgG  was used for each immunoprecipitation. Input and precipitates were analyzed by quantitative PCR using specific primers .DRIP was adapted from a previous report  with modhttp://hannonlab.cshl.edu/fastx_toolkit/). Low-quality sequences at the ends of reads represented by stretches of “#” in the quality score string in FASTQ file were also removed. Reads shorter than 35 nt after trimming were excluded from analysis. Sequencing reads were aligned to human reference (hg19) using GSNAP (version 2013-10-28)  and sequenced on an Illumina HiSeq 2500 platform. An average of 40 million 100-nt reads per sample was generated. Sequencing reads were preprocessed to remove adapter sequences from the end of reads using the program fastx_clipper from FASTX-Toolkit (3-10-28)  using th3-10-28)  and requ32P-ATP and 1 unit/µL of T4 PNK kinase for 30 min at 37°C. Beads were washed five times with PNK wash buffer  and resuspended in 100 µL 2× sample buffer and separated on a 4%–12% SDS-PAGE and transferred to a nitrocellulose membrane. The SRSF1 ribonucleoprotein complex was visualized by autoradiography, and the band corresponding to SRSF1 was isolated. RNA was extracted by Proteinase K digestion, purified by phenol-chloroform extraction, and precipitated with three volumes of ethanol. The purified RNA from each cellular fraction was ligated with a unique 3′ adapter with Rnl2(1–249) K227Q ligase (NEB) overnight at 4°C. The RNA was loaded onto a 15% Urea-PAGE, and the ligated RNA cut out and extracted from the gel with 400 µL 0.3M NaCl for 45 min at 60°C with vigorous shaking. The gel pieces were filtered away and RNA in the flow-through precipitated with three volumes of ethanol. The RNA pellet was dissolved in water and ligated with 5′ adapter using Rnl1 ligase (NEB) for 1 h at 37°C. The RNA was loaded onto a 12% Urea-PAGE, and the ligated RNA cut out and extracted from the gel with 400 µL 0.3 M NaCl for 45 min at 60°C with vigorous shaking. The gel pieces were filtered out and RNA in the flow-through precipitated with three volumes of ethanol. The RNA was reverse transcribed using SuperScript III reverse transcriptase (Thermo Fisher Scientific) with 3′ RT primer for 2 h at 50°C, according to the manufacturer's instructions. Next, the generated cDNA was PCR amplified using Taq DNA polymerase (Thermo Fisher Scientific). The primers used for PAR-CLIP are listed in Supplemental Table S4. The PCR band corresponding to the correct size of amplification (143–153 bp) was purified using a 3% PippinPrep gel according to the manufacturer's instructions and quantified. PAR-CLIP cDNA libraries were sequenced on an Illumina HiSeq 3000 instrument. Clusters of overlapping reads uniquely mapped to the human genome hg19 were generated using the PARalyzer software (http://www.gencodegenes.org)  . The hg17 cultured human B-cells or primary skin fibroblasts were harvested and lysed in 100 µL lysis Buffer with protease and RNase inhibitors. Five micrograms of anti-SRSF1 antibody , anti-FUS antibody , and anti-DDX1 , negative control IgG  were conjugated to Magnetic Protein A/G beads. One hundred microliters of cell lysate was added into 900 µL Immunoprecipitation Buffer with RNase inhibitor and incubated with 50 µL beads-antibody complex overnight at 4°C. Bead-bound immunoprecipitates were then washed six times using cold Wash Buffer with RNase inhibitor and incubated with protease K in the presence of 1% SDS for 30 min at 55°C. RNA and DNA were then extracted from supernatants using phenol:chloroform:isoamyl alcohol and precipitated using ethanol. Precipitated RNA was digested by DNase I . cDNA was synthesized using random hexamer primer by TaqMan Reverse Transcription Reagent kit (Applied Biosystems). Quantitative PCR was carried out to quantify cDNA and DNA with primers annealing to BAMBI and DPP9 hybrids using Power SYBR Green PCR Master Mix . Primer sequences are listed in Supplemental Table S4. RNA from anti-SRSF1 immunoprecipitate and input RNA were prepared into RNA-seq libraries using Illumina TruSeq Stranded Total RNA Library Prep kit  and sequenced on HiSeq 2500. Sequencing reads were preprocessed and aligned as described above. Enrichment of transcripts in the immunoprecipitate was analyzed using the program described by Antanaviciute et al. (Reverse immunoprecipitation was carried out using Magna RNA-Binding Protein Immunoprecipitation Kit (Millipore) following the manufacturer's protocol. Briefly, for each immunoprecipitation reaction, 2 × 10e et al. . TranscrDPP9 dsDNA 90-mer was generated as described above. dsDNA and RNA/DNA hybrid were loaded onto the sensors until saturation. The nucleotide-labeled sensors were then washed with buffer, followed by addition of DDX5 at concentrations of 2 and 8 µg/mL, NONO at concentrations of 1 and 4 µg/mL, SUPT5H at concentrations of 2 and 8 µg/mL, and RNase H1 (RNASEH1) at 3.8 and 19 nM. All reactions were tested in TBS buffer . A reference sample of buffer and protein alone did not show any signal drift. Association and dissociation were monitored for 10 min each. All experiments were conducted in the Octet instrument with agitation at 1000 rpm.Analysis of dsDNA or RNA/DNA hybrid binding to candidate proteins was carried out using the Octet RED96 system (ForteBio) with sensor detection of the change in wavelength (nm shift). Purified candidate proteins DDX5 , NONO , SUPT5H , and human RNase H1  were evaluated. Biotinylated dsDNA or RNA/DNA hybrid at concentrations of 5 nM was immobilized onto a Streptavidin-SA biosensor. The biotinylated Fibroblasts were fixed with 4% paraformaldehyde for 15 min at room temperature, then washed three times with phosphate-buffered saline (PBS). Slides were then placed in blocking solution  for 1 h at room temperature. Primary antibody staining was done overnight at 4°C in PBS with 1% bovine serum albumin and 0.3% Triton X-100 using 1:500 S9.6 antibody, 1:500 DDX18 antibody , or 1:500 nucleolin antibody . Slides were then washed three times with PBS for 5 min each, incubated with 1:500 secondary antibody  for 2 h at room temperature in the dark, and then washed three times with PBS for 5 min each before DAPI nuclear staining. Imaging was performed with a Leica DMI 6000CS laser confocal microscope with a Leica HCX PL APO 63× oil objective.IUPred  was usedhttps://www.ncbi.nlm.nih.gov/geo/) under accession number GSE117671. The mass spectrometry data from this study have been submitted to the PeptideAtlas database (http://www.peptideatlas.org/) with the identifier PASS01169.The PAR-CLIP, RNA-IP-seq, and DRIP-seq data from this study have been submitted to the NCBI Gene Expression Omnibus (GEO;


Human telomere RNA performs various cellular functions, such as telomere length regulation, heterochromatin formation, and end protection. We recently demonstrated that the loops in the RNA G-quadruplex are important in the interaction of telomere RNA with heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). Here, we report on a detailed analysis of hnRNPA1 binding to telomere RNA G-quadruplexes with a group of loop variants using an electrophoretic mobility shift assay (EMSA) and circular dichroism (CD) spectroscopy. We found that the hnRNPA1 binds to RNA G-quadruplexes with the 2’-O-methyl and DNA loops, but fails to bind with the abasic RNA and DNA loops. These results suggested that hnRNPA1 binds to the loop of the RNA G-quadruplex by recognizing the base of the loop’s nucleotides. The observation provides the first evidence that the base of the loop’s nucleotides is a key factor for hnRNPA1 specifically recognizing the RNA G-quadruplex. Human telomeric RNA has been reported to form G-quadruplex structures ,7,8,9,10We first examined the role of 2′-OH in the loop’s nucleotide on hnRNPA1 binding to the G-quadruplex using a G-quadruplex bearing the DNA loop (DNA-loop-Gq), which lacked the 2′-OH group in the loop . We obse32P-labeled RNA) (1–100-fold)  . Predict00-fold) a. The re00-fold) b,c. On t00-fold) d,e. ThesCircular dichroism (CD) spectroscopy was used to examine the G-quadruplex structure formed by all of the oligonucleotides ,15 Figu. DNA-loo+. Given the K+ ion being dominant inside cells, the study of the interaction of the protein and G-quadruplex in buffers containing K+ will draw intense interest.The introduction of abasic sites into the G-quadruplex is a powerful tool to detect the key part of the G-quadruplex for the interaction between the protein and G-quadruplex. In this study, we used an intermolecular dimeric G-quadruplex with two equivalent loops. Alternatively, the number and location of abasic parts in different loops can be introduced in intramolecular G-quadruplexes. Such an approach is useful for investigating the recognition mechanism and binding affinity of the RNA G-quadruplex with protein. It is known that metal cations can influence the G-quadruplex topology ,17,18. WRecent studies have suggested that telomere RNA G-quadruplexes may dimerize to form higher-order G-quadruplexes comprising two stacked G-quadruplex subunits ,5,9. WheIn conclusion, the base at the loop of the RNA G-quadruplex is a structural basis for the preferential recognition of hnRNPA1. This finding provides new insights into the hnRNPA1 binding to the RNA G-quadruplex.32P]ATP  by T4 polynucleotide kinase  and were purified using Mini Quick Spin Oligo Columns, Sephadex G-25 .Oligonucleotide and protein: All oligonucleotides were synthesized with a 1 μmol scale by using an automatic DNA/RNA synthesizer . Then the oligonucleotides were separated from the support and deprotected according to the manufacturer’s protocol. All oligonucleotides were purified by using reverse-phase HPLC . All oligonucleotides were labeled with [γ-E. coli strain C41 (DE3) and was expressed as previously described [E. coli by using Bensonase endonuclease digestion , the repurification process was performed with Ni-NTA affinity resin, and the protein was concentrated by using a VIVASPIN filter .The hnRNPA1 was cloned into the pET-15b vector (Novagen) for transformation into escribed . Then thv/v) glycerol at room temperature for 30 min; these were measured by a 1 cm path-length cell.CD measurements: CD spectra were recorded on a JASCO model J-820 CD spectrophotometer. The CD spectra of the oligonucleotides with or without hnRNPA1 were carried out in a 10 mM Tris-HCl buffer (pH 7.0), 100 mM NaCl, 0.1 mg/mL BSA, 5 mM dithiothreitol, and 10%  glycerol). Afterwards, the samples were incubated for 30 min at room temperature and then loaded onto a native polyacrylamide gel (8%). Electrophoresis was carried out in a 1 × TBE buffer supplemented with 20 mM NaCl at 80 V and 4 °C. All gels were exposed in a phosphorimager cassette and imaged using a FLA-7000 bioimager .Gel-shift assay: The binding of


His anticodon-like structure. Introducing point mutations that are expected to disassemble this putative anticodon mimic alleviated self-association, concomitant with increased synthesis of the protein. Finally, we found that increased cellular levels of uncharged tRNAHis lead to reduced self-association and increased HisRS translation, in a manner that depends on the anticodon-like element. Together, these results reveal a novel post-transcriptional autoregulatory mechanism that exploits binding mimicry to control mRNA translation according to tRNA demands.Aminoacyl-tRNA synthetases (aaRSs) are well studied for their role in binding and charging tRNAs with cognate amino acids. Recent RNA interactome studies had suggested that these enzymes can also bind polyadenylated RNAs. Here, we explored the mRNA repertoire bound by several yeast aaRSs. RNA immunoprecipitation (RIP) followed by deep sequencing revealed unique sets of mRNAs bound by each aaRS. Interestingly, for every tested aaRSs, a preferential association with its own mRNA was observed, suggesting an autoregulatory process. Self-association of histidyl-tRNA synthetase (HisRS) was found to be mediated primarily through binding to a region predicted to fold into a tRNA Better known for their enzymatic role in charging tRNAs with their cognate amino acids, this study shows that tRNA synthetases also bind mRNAs, regulating translation in order to balance the production of a tRNA synthetase with the level of its cognate tRNA. RNA-binding proteins (RBPs) encompass a significant fraction of an organism proteome and are implicated in many cellular processes . The groRecent high-throughput studies applied an unbiased approach whereby polyadenylated RNAs and their associated proteins were isolated through an oligo dT column and proteins were identified by mass spectrometry ,5. TheseEscherichia coli threonyl-tRNA synthetase (ThrRS) can bind its own mRNA in a region upstream to the start codon (“operator” region)  above the average) . Gene OnGluRS also shares 11 target mRNAs with MetRS, including the mRNAs encoding GluRS and MetRS themselves  see bel. The gromRNAs bound exclusively by MetRS appeared to be highly enriched for those encoding proteins that bind ATP (32 of the 115 mRNAs) . MetRS wS. cerevisiae aaRSs.A common feature that appeared for each aaRSs that we tested was a strong association with its own mRNA (“self-association”) . HisRS . Self-asTo validate these sequencing results by an alternative approach, we performed semiquantitative reverse transcription PCR (RT-PCR) to RNA samples from the Input or Bound samples . cDNA frGluRS and MetRS are known to charge their cognate tRNAs while in a trimeric complex with Arc1 (Arc1:MetRS:GluRS [AME] complex) ,27. The 32P and resolved in a gel. HisRS proteins of increased sizes are apparent due to their associated RNA fragments  to HisRragments . Importaragments . Thus, Hragments . Similarragments . These rThermus thermophilus revealed that these are the primary contacts with tRNA  revealed that both CD-GFP and ABD-GFP were isolated at similar levels. However, co-IP of HisRS-TAP was observed in the CD-GFP IP and not in the ABD-GFP IP . Thus, cWe next wished to identify the site(s) along the mRNA that HisRS binds in vivo. To this end, we devised a fragmentation RIP procedure (fRIP) . ExtractS. cerevisiae, HisRS recognizes only a single tRNAHis, with a GUG in its anticodon loop  was tagged at its 3' UTR with multiple MS2 loops and expressed together with a plasmid expressing MS2-binding protein fused to GFP (MS2BP-GFP). Transcripts were isolated on GFP-Trap columns by virtue of their association with the MS2BP-GFP fusion . Western254) profile of polysomal distribution in sucrose gradients did not reveal any general difference between the WT and GAG-containing strains (35S]-methionine (35S-met) to the growth medium of both strains. Aliquots were collected at different time points after addition, lysed, and subjected to PAGE autoradiography either directly  or after isolation of HisRS-GFP by GFP-Trap beads , a stress condition known to increase the levels of uncharged tRNAHis G2) mature sequence ). Lysate was cleared by centrifugation at 10,600 g for 10 min at 4°C. For isolation of TAP-tagged proteins, lysates were loaded on proteinA-Sepharose beads  and rotated at 4°C for 2 hr. Samples were washed four times in Buffer C  and subjected to cleavage with 80 U of TEV (Invitrogen 12575–015) for 2 hr at 16°C in TEV buffer. Cross-linking was reversed by heating at 65°C for 2 hr in reverse cross-linking buffer , and RNA was precipitated after phenol:choloroform extraction. For isolation of GFP-tagged proteins, lysates were loaded on GFP-Trap_A (ChromoTek) and rotated at 4°C for 2 hr. Samples were washed four times in Buffer C and eluted by 0.2 M Glycine buffer (pH 2.5). Cross-linking reversal and RNA isolation was done as described above for TAP-tagged sample.2 of 254 nm) on ice. Cells were lysed using bead beater in buffer B and immunoprecipitation was conducted as described above. Cross-linking was reversed by 0.8 U/ μl proteinase K (NEB P8107S) at 50°C for 1 hr in PK Buffer , and RNA was precipitated after phenol:choloroform extraction.GFP-tagged strains were grown in YPD to mid logarithmic phase, subjected to UV cross-linking  according to the manufacturer's instructions. The polyA selection step was excluded for the Bound samples to minimize losses. All samples were sequenced on Illumina HiSeq 2500 platform, yielding 10 to 18 million reads per sample. The reads were mapped to the S288c Saccharomyces cerevisiae version R64-2-1 genome using Tophat2 version 2.1.0 with up to three mismatches allowed per read. Only uniquely mapped reads were counted to genes using HTSeq-count package version 0.6.1.For semiquantitative PCR, cDNA was prepared from 500 ng RNA by High-Capacity cDNA Reverse Transcription Kit (Maxima) according to the manufacturer’s instructions, and subjected to PCR with gene-specific primers . AliquotStrains expressing HisRS mRNA variants  tagged at the 3' UTR with multiple MS2 loops and expressing pCP-GFP  were groHisRS-GFP strain was grown in YPD to mid logarithmic phase and subjected to cross-linking by addition of formaldehyde  for 10 min at room temp. Cross-linking was terminated with 0.125 M glycine for 3 min, and cells were lysed in Buffer B. Lysate was cleared by centrifugation for 10 min at 10,600 g at 4°C and fragmented by 0.2 U/μl RNase I , with shaking for 4 min at 37°C. Samples were loaded on GFP-Trap_A (ChromoTek) for 2 hr at 4°C, and washed four times in Buffer C. Proteins were eluted by 0.2 M Glycine buffer (pH 2.5). Cross-linking was reversed by heating at 65°C for 16 hr in reverse cross-linking buffer and RNA was precipitated following phenol:choloroform extraction. For semiquantitative PCR, cDNA was prepared by High-Capacity cDNA Reverse Transcription Kit (Maxima) according to the manufacturer’s instructions and subjected to 14 PCR reactions with specific primers that designed to cover the entire HisRS transcript. Aliquots were set aside every five cycles, run on agarose gel, and stained with ethidium bromide.Point mutations by the CRISPR/Cas9 were introduced according to protocol . BrieflyPlasmid expressing guideRNA recognizing GFP (S65T) sequence in bRA66 (Addgene #100952) backbone was transformed into either HisRS-GFP WT, GAG, Stem disrupt, or Stem unchanged strains together with PCR product amplified from pSL-MS2-12X (Addgene #27119) using HisRS MS2L F and HisRS MS2L primers . Note th35S methionine  was added, and 2-ml aliquots were set into cycloheximide  at the indicated times. Labeled HisRS-GFP was immunoprecipitated with GFP-Trap_A (ChromoTek) by incubation at 4°C for 2 hr and washed four times in Buffer C. Proteins were eluted from the beads with LSB, resolved on 10% PAGE, and exposed to phosphorimager. Radioactive signals were quantified by ImageJ.Cells (10 ml) were grown to mid-log phase in SC, washed, and resuspended in 10 ml SC-met. Growth was continued for another 5 min, 2, 0.5 mM DTT, 100 μg/ml cycloheximide,1 mg/ml heparin, 1% Triton X-100). Lysates were cleared by 10 min centrifugation at 9,000 g, and loaded on 10%–50% linear sucrose gradient. Polysomal complexes were resolved by 160 min centrifugation at 35,000 rpm in SW41 rotor and separated into 12 fractions with ISCO UA6 device. RNA was extracted from each fraction and analyzed by northern blot with probes for HisRS or Actin mRNAs [Polysome analysis was done as described . Brieflyin mRNAs .S1 FigYeast strains expressing tagged aaRSs were subjected to RIP using the TAP or GFP-Trap beads. Protein samples from the total cellular lysate (Input), unbound FT, LW step, and the eluted fraction (Bound) were resolved on PAGE followed by western analysis with the indicated antibodies (A) or stained with Silver stain (B). Equivalent amounts were loaded except of the Bound sample that was three times larger. Size markers are shown to the left. aaRS, aminoacyl-tRNA synthetase; FT, flow-through; GFP, green fluorescent protein; LW, last washing; RIP, RNA immunoprecipitation; TAP, Tandem Affinity Purification.(PDF)Click here for additional data file.S2 FigVenn diagrams were generated from the mRNAs that appeared within the 1.5 IQR region among all strains that were subjected to the TAP protocol (A), GFP-Trap protocol (B) and for HisRS tagged with either TAP of GFP (C). Shared genes or GO terms are indicated. aaRS, aminoacyl-tRNA synthetase; GFP, green fluorescent protein; GO, Gene Ontology; HisRS, histidyl-tRNA synthetase; IQR, InterQuartile Region; TAP, Tandem Affinity Purification.(PDF)Click here for additional data file.S3 FigStrain expressing HisRS-TAP was subjected to UV illumination, to covalently cross-link protein–RNA interactions. RNA was then digested by three different concentrations of RNaseI . The TAP-tagged HisRS were purified together with the bound RNA fragments, and RNAs were radioactively labeled. Cross-linked HisRS–RNA complexes were resolved on SDS-PAGE and transferred to a membrane. A) Ponceau red protein staining of the membrane. Arrow indicates the band corresponding to HisRS-TAP, showing similar amounts of isolated protein in all samples. B) Autoradiography of the membrane, revealing bound HisRS–RNA complexes. An increase in HisRS apparent size is observed due to association with RNA. C) HisRS–RNA complexes of each sample were cut from the membrane, and RNA was recovered from the membrane by digesting the protein with proteinase K. RNA was separated using denaturing TBE Urea Polyacrylamide Gel and exposed to autoradiography. RNA species longer than 100 nts are clearly observed at the low RNaseI treatment. HisRS, histidyl-tRNA synthetase; TAP, Tandem Affinity Purification; TBE, Tris/Borate/EDTA.(PDF)Click here for additional data file.S1 TableNotably, none of the exclusively mitochondrial aaRSs was detected in any of these studies. This is likely due to low polyadenylation of mitochondrial mRNAs, which is necessary for the RNA interactome isolation. Nevertheless, it confirms that mRNA association occurs while the cell is compartmentalized , hence no binding of mitochondrial aaRSs to cytosolic, polyadenylated transcripts occurs. aaRS, aminoacyl-tRNA synthetase.(PDF)Click here for additional data file.S2 Tablearc1Δ MetRS background-corrected RIP efficiency, calculated as in Sheet 3. GlnRS, glutamine-tRNA synthetase; HisRS, histidyl-tRNA synthetase; RIP, RNA immunoprecipitation; RPM, reads per million; ValRS, valyl-tRNA synthetase.Mapped unique reads for each GeneID in the S288c genome. The file includes several sheets. Sheet 1: uniquely mapped reads for all RIP-seq experiments. Sheet 2: raw read counts normalized to RPM. Sheet 3: HisRS background-corrected RIP efficiency. Transcripts with normalized read coverage <15 in the Input samples, and noncoding RNAs were filtered out. Bound sample were then divided by its corresponding Input sample to calculate RIP efficiency, and HisRS RIP efficiency was corrected to the Untagged RIP efficiency. Sheet 4: GlnRS background-corrected RIP efficiency, calculated as in Sheet 3. Sheet 5: ValRS background-corrected RIP efficiency, calculated as in sheet 3. Sheet 6: GluRS background-corrected RIP efficiency, calculated as in Sheet 3. Sheet 7: MetRS background-corrected RIP efficiency, calculated as in Sheet 3. Sheet 8: (XLSX)Click here for additional data file.S3 TablemRNAs that appeared larger than 1.5 IQR region among all strains that were subjected to the TAP RIP protocol were selected and names and their gene ID are presented. Sheet 1 includes the genes that were used to generate the Venn diagram in (XLSX)Click here for additional data file.S4 TableTranscripts bound by a single aaRS or bound by two aaRSs were used to generate GO Term (using SGD GO Term Finder Version 0.86). aaRS, aminoacyl-tRNA synthetase; GO, Gene Ontology.(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 TableData are split into sheets according to the relevant figure. RT-qPCR, reverse transcription quantitative PCR.(XLSX)Click here for additional data file.


Escherichia coli mRNAs by cellular RNases. These protected mRNA fragments fold into energetically stable RNA structures, reminiscent of those of intergenic regulatory ncRNAs. In addition, a subset of these ncRNAs coprecipitate with the major ncRNA chaperones Hfq and ProQ and display evolutionarily conserved sequences and conserved expression patterns between different bacterial species. Our data suggest that protein-coding genes can potentially act as a reservoir of regulatory ncRNAs.Bacteria and archaea utilize regulatory small noncoding RNAs (ncRNAs) to control the expression of specific genetic programs. These ncRNAs are almost exclusively encoded within intergenic regions and are independently transcribed. Here, we report on a large set ncRNAs that are “carved out” from within the protein-coding regions of  Prokaryotic genomes encode a plethora of small noncoding RNAs (ncRNAs) that fine-tune the expression of specific genes. The vast majority of known bacterial ncRNAs are encoded from within intergenic regions, where their expression is controlled by promoter and terminator elements, similarly to protein-coding genes. In addition, recent studies have shown that functional ncRNAs can also be derived from gene 3′ untranslated regions (3′UTRs) via an alternative biogenesis pathway, in which the ncRNA segment is separated from the mRNA via RNase cleavage. Here, we report the detection of a large set of decay-generated noncoding RNAs (decRNAs), many of which are completely embedded within protein-coding mRNA regions rather than in the UTRs. We show that these decRNAs are “carved out” of the mRNA through the action of RNase E and that they are predicted to fold into highly stable RNA structures, similar to those of known ncRNAs. A subset of these decRNAs is predicted to interact with Hfq or ProQ or both, which act as ncRNA chaperones, and some decRNAs display evolutionarily conserved sequences and conserved expression patterns between different species. These results suggest that mRNA protein-coding regions may harbor a large set of potentially functional small RNAs. Small noncoding RNAs (ncRNAs) control gene expression in prokaryotes, regulating processes such as nutrient acquisition, stress responses, virulence, and biofilm formation –5. Gener–7–cis-encoded antisense transcript repertoires (–cpxP membrane stress response chaperone is transcribed into an mRNA containing a conserved ∼60-nucleotide (nt) 3′UTR, which can independently act as an Hfq-dependent ncRNA. This ncRNA, CpxQ, is separated from the mRNA by RNase E, enabling its activity as a regulator of multiple genes  to track mRNA decay dynamics in bacteria. By exposing solution . We reposolution .E. coli mRNAs displayed uniform decay profiles across the entire transcript body, our analysis recovered 58 mRNA subsegments, 80 to 300 nt in size (average of ∼160 nt), that were preferentially stabilized compared with the rest of the transcript  is functional at 30°C but is inactive at 44°C (P  < 10−9 [Wilcoxon]).To independently validate that these stabilized RNA fragments are liberated from mRNAs through RNase activity, we analyzed total RNA from  RNase E . In thes at 44°C . We sequ at 44°C . We foun10.1128/mBio.01730-18.1FIG S1P values are shown in the figure. Download FIG S1, TIF file, 0.1 MB.decRNA generation is dependent on RNase E activity. Data are presented as box plot distributions showing the fold differences in expression between decRNAs and their respective host genes (decRNA expression/host gene expression), calculated for all decRNAs reported in this study . Values Copyright © 2018 Dar and Sorek.2018Dar and SorekCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the P  < 10−10 [Wilcoxon]) and of stability similar to that of known, intergenic ncRNAs  followed by RNA-sequencing analysis . RNA-seqCopyright © 2018 Dar and Sorek.2018Dar and SorekCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01730-18.3FIG S3FIG S3, TIF file, 0.3 MB.Putative decRNA interactions with Hfq. Interactions between decRNAs and Hfq were evaluated using recently published data from Hfq immunoprecipitation followed by RNA sequencing . IP sampCopyright © 2018 Dar and Sorek.2018Dar and SorekCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the ftsI-decRNA, embedded within penicillin-binding protein-3, is predicted to bind both Hfq and ProQ , which is one of the major elements required for interaction with Hfq  and possand ProQ  and S3; eriments .ftsI-decRNA revealed that it is part of the mraZ-rsmH-ftsL-ftsI-murE-containing operon, which includes 3 additional decRNAs, encoded within mraZ, ftsL, and murE  of 0.5. Rifampicin was added to the cultures to reach a final concentration of 500 µg/ml, and samples were collected at different time points as noted previously (E. coli and the temperature-sensitive RNase E mutant were grown at the permissive temperature of 30°C overnight in LB and were diluted 1:100 in fresh LB media the next day. The cultures were then grown at 30°C until an OD600 of 0.5 was reached and were then incubated at 44°C for 10 min. The samples were mixed with ice-cold stop solution (90% ethanol and 10% saturated phenol) to deactivate cellular processes and were then collected via centrifugation at 4°C. RNA extraction and whole-transcriptome sequencing were performed as described in reference Previously published rifampicin RNA-decay time-series experiments were taken from reference eviously . For theCP009273, NC_016810.1, and NC_015663.1 RefSeq genomes , using NovoAlign (Novocraft) V3.02.02 with default parameters, discarding reads that were nonuniquely mapped as previously described  downloaded from NCBI and that of decRNAs (n = 58) and random E. coli genomic sequences  were compared using a two-sided Wilcoxon rank sum test. The length of the random sequence set was selected with a length distribution representative of the 121 ncRNAs described above.Data corresponding to the level of RNA structure stability per nucleotide  were calculated using the RNA fold energetic stability score  divided sibA, sraB, and istR  under accession no.


Adenosine to inosine RNA editing in protein-coding messenger RNAs (mRNAs) potentially leads to changes in the amino acid composition of the encoded proteins. The mRNAs encoding the ubiquitously expressed actin-crosslinking proteins Filamin A and Filamin B undergo RNA editing leading to a highly conserved glutamine to arginine exchange at the identical position in either protein. Here, by targeted amplicon sequencing we analysed the RNA editing of Filamin B across several mouse tissues during post-natal development. We find highest filamin B editing levels in skeletal muscles, cartilage and bones, tissues where Filamin B function seems most important. Through the analysis of Filamin B editing in mice deficient in either ADAR1 or 2, we identified ADAR2 as the enzyme responsible for Filamin B RNA editing. We show that in neuronal tissues Filamin B editing drops in spliced transcripts indicating regulated maturation of edited transcripts. We show further that the variability of Filamin B editing across several organs correlates with its mRNA expression. With reWhile the majority of editing events occurs in untranslated, repeat-containing regions of mRNAs, about 100 editing events are found in coding regions of mRNAs that lead to recoding of the mRNAs. Their evolutionary conservation further underscores the importance of these editing events . In mammThe majority of protein recoding editing events have been identified in the nervous system, where the editing-induced amino acid exchanges modulate the activity of the encoded proteins . OutsideFlna gene causes lethal heart and vascular defects [Flnb gene leads to lethal skeletal malformations with bone fusions [In humans, mutations in filamin A and B give rise to developmental disorders. While Filamin A mutations show cardiac, neurological, skeletal and intestinal impairments, Filamin B mutations only affect joint and skeletal development . This difference is mirrored in the mouse, where the deletion of the  defects ,17 while fusions ,19.Flna RNA is conserved from birds to humans and is located in a highly interactive region of Filamins seemingly affecting multiple cellular functions. Our previous spatio-temporal analysis showed that Flna RNA editing in the mouse is highest outside of the nervous system, particularly in lungs, blood vessels and gastrointestinal tract [The pre-mRNAs encoding FLNA and FLNB undergo RNA editing leading to a glutamine (Q) to arginine (R) exchange at the identical position in either protein . Editingal tract .Flnb transcripts, a second editing event has been observed upstream of the known Q/R site [Flnb protein (S/G site).In human and murine Q/R site ,22. In hFlnb editing in mature mRNAs across several organs during post-natal development. Together, we show that editing of Flnb is highest in the musculoskeletal system where FLNB has been found to be most essential. Seemingly Flnb editing is independently regulated from editing of Flna. Furthermore, we confirm that ADAR2 is the enzyme responsible for Flnb RNA editing and that the Flnc transcript does not undergo A-to-I editing.Here, we profile 2.Flnb Q/R editing site was obtained from cDNAs generated from different mouse tissues at birth (P0), 21 days (P21) and 120 days after birth (P120) in three biological replicates for each time point. The tissue origin and time point of every sample were marked by a combination of two 8 nucleotide-long barcodes, both on the 5ʹ- and 3ʹ-ends. A total of 200 samples was sequenced. Replicas of 19 tissue-derived samples at P0, 24 tissues at P21 and P120 together yielded 1.49 million reads. On average, a coverage of 7,450 reads was obtained per sample, varying from a minimum of 247 to a maximum of 7,990 reads per sample . Sequencing quality was assessed in every sample. Even at the lowest coverage the observed background sequencing error rate – in the range of one-tenth of a percent – did not affect the quantification of editing rates, which is higher by at least one order of magnitude .To determine the distribution, abundance and developmental change of ADAR-mediated editing of filamin B mRNA we comprehensively quantified editing levels of this RNA in mouse organs at different time points during post-natal development using Illumina sequencing technology. The strategy applied to uniquely identify up to 96 multiplexed samples per sequencing round is outlined in Suppl. Figure 1. Briefly, a short PCR amplicon containing the The A-to-I editing rate was determined as the number of reads with a guanosine (G) instead of the expected genomic adenosine (A) over the total number of reads in the sample. To verify the editing levels obtained by amplicon sequencing, a few samples were also tested by direct Sanger sequencing. As expected, amplicon sequencing was more sensitive to detect low editing rates while similar editing levels were detected at higher rates .Flnb Q/R site  varied greatly amongst different mouse tissues, ranging from a minimum of 1.8% in small intestine in young mice (P21) to a maximum of 77.2% in femur in adult mice (P120)  where editing levels decreased or stayed constant with increasing age (The A-to-I editing rate at the e (P120) . As showsing age .10.1080/Flnb editing levels were detected in cerebellum at any given time point increasing from 22% in newborns to 53% in P120 mice  and skin (51.3%) in adult mice, tissues previously appreciated to display no significant level of editing (Within the nervous system highest Flnb editing levels measured by us are highly similar to those reported in the corresponding mouse Genotype-Tissue Expression (GTEx) data set ). However, the mouse editing levels differ greatly from those reported for human tissues, where Flnb RNA editing is in general lower than in the mouse ).The recent publication from Tan et al. . provideFlnb editing levels we observed in brain tissues differ from those of a previous study, where 95% Flnb editing was observed by RNA sequencing of whole-brain samples from several mouse strains [Flnb mRNA, whereas the RNA sequencing also captures unspliced and partially spliced pre-mRNA transcripts. To check whether the lower levels of Flnb editing detected by us could be due to the enrichment of mature transcripts, we compared A to G transitions by Sanger sequencing on both unspliced and spliced Flnb RNA transcripts amplified from the same tissue using specific primers. In all tested P120 brain samples editing at the Q/R site was above 80% in Flnb pre-mRNA transcripts, while editing frequency decreased to ~50% in spliced Flnb mRNA . These RNA only ) which iFlnb editing. While in ADAR1 KO cortex Flnb editing was detected in about 30% of the reads, no A-G transitions were detected on Flnb RNA in the cortex of ADAR2 KO mice  mice allowed us to determine which of the two functional RNA editing enzymes is responsible for  KO mice . To conf samples .10.1080/Flnb Q/R editing level and ADAR2 expression correlate, quantitative PCR was performed on representative samples from adult wild type mice. As shown in Flnb edited transcripts and ADAR2 mRNA expression in the same tissue. Next, Flnb mRNA expression was measured by quantitative PCR in samples from adult mice. A positive correlation is apparent (R2 = 0.7113) between the expression level of Flnb mRNA and its editing frequency  amplicons from wild type adult mouse tissues where Flnc is mainly expressed [Despite a low degree of sequence homology, murine Filamin C (Flnc) mRNA and amino acid sequences overlap the Q/R editing site on murine Flna and Flnb. To check if xpressed ,12 were 3.Flnb editing increase with age in both nervous and non-nervous tissues.Previous studies have shown a gradual increase of A-to-I editing levels for protein-coding transcripts during mammalian neuronal development ,27 in thFlnb editing rates were detected outside the nervous system where most other protein-coding ADAR targets had been identified [Flna [Flna and Flnb RNA editing profiles can be found. For instance, Flna RNA is highly edited in stomach and colon while Flnb editing is very low in all segments of the digestive tract. This suggests a likely functional role for Flna editing in the gastrointestinal apparatus.The highest entified . Howevered [Flna . InteresFlnb editing is high in both the vascular and musculoskeletal systems. This finding is interesting as Flnb knockout mice show impaired development of the microvasculature and skeletal system with bone and joint fusions [Flnb transcript and the main function of the encoded protein. It should also be noted that all components of the musculoskeletal system show high Flnb editing levels.Instead,  fusions ,19. ThisThe developmental origin of bones is heterogeneous. Craniofacial bones develop from the embryonic ectoderm cell layer while the limb skeleton generates from the mesoderm layer. Still both types of bones showed comparable high Flnb editing levels.Flnb editing frequency in white abdominal fat, especially in adult mice. To our knowledge this is the first time Flnb editing is measured in adipose tissue. Further investigation would be needed to clarify the role of Flnb editing in fat where it might be linked to both metabolism and hormonal production.Surprisingly, we observed a high Flnb editing levels are detected in the cerebellum in agreement with recently published data [Flnb pre-mRNA and mRNA. In the present study editing levels were determined exclusively on spliced Flnb mRNA. Indeed, we could verify that Flnb editing was elevated in pre-mRNA compared to mRNA in all samples tested.Within the central nervous system the highest hed data . Intereshed data . Beyond Flnb pre-mRNA. Therefore, we speculated that the presence of two editable nucleotides in the splice site consensus sequence could affect splicing efficiency. Comparing the intron-exon junction of unedited, partially edited or fully edited Flnb transcripts to the consensus 5′ splice site sequences clearly showed that RNA editing decreases the probability of a Flnb transcript of being spliced. It seems therefore plausible that edited Flnb transcripts are less efficiently spliced, a phenomenon already reported by us and others [Moreover, apart from the already known S/G and Q/R sites we observed an editing site located in the intronic 5ʹ splice site of the d others ,33.Flnb Q/R site [Flnb editing is catalysed by both ADAR enzymes [Flnb transcript is ADAR2. In fact, no edited adenosine and no compensatory activity from ADAR1 enzyme were detected at the Flnb Q/R site in ADAR2 knockout samples. Next, by Sanger sequencing we confirmed that ADAR2 is the enzyme responsible for Flnb RNA editing in other tissues of both neuronal and non-neuronal origin. Nevertheless, we observed no direct correlation between ADAR2 mRNA expression and Flnb editing at the Q/R site, suggesting that Flnb editing does not depend on the stoichiometry of the enzyme-substrate ratio. Instead, Flnb mRNA expression seemingly correlates with the variability of Flnb editing levels across mouse tissues. This finding is in agreement with previous studies where similar variabilities in editing levels were observed [Flnb editing and ADAR2 expression, editing of Flnb could possibly be affected by competing RNA-binding proteins or editing regulators [Flna and Flnb RNAs which are both edited by ADAR2. Our data also show that editing levels in pre-mRNA and mRNA differ. Thus, levels of editing in mature mRNAs may also be regulated by differential splicing. Moreover, Flnb editing has been shown to be altered in esophageal and liver carcinomas due to the increased activity of ADAR1 indicating that this enzyme may also target Flnb, at least under specific conditions [Flnb RNA editing will be required.Terajima et al. recently reported that in the mouse liver ADAR2 is the enzyme responsible for editing of the Q/R site . This co enzymes . In thisobserved . At thisgulators ,30,31,37nditions ,39. CleaFlna and Flnb editing sites, no adenosine to guanosine transition was detected in Flnc mRNA.Eventually, despite a high degree of transcript sequence homology in the region corresponding to the In summary, with this study we confirmed that in mouse not only filamin A but also filamin B mRNA is highly edited outside the nervous system. The tissue specificity of the editing profiling of these two filamins highlights both common and different traits, suggesting a distinctive role for the RNA editing of these two cytoskeletal proteins. In particular, Flnb editing is elevated in the very same musculoskeletal tissues which are mostly affected in Flnb-associated human diseases and in Flnb-deficient mice. Indeed, uncovering the functional role of Flnb editing will be the next experimental challenge.4.4.1.Flnb RNA editing profiling male wild type (C57Bl/6J) mice were sacrificed and dissected organs were snap frozen in liquid nitrogen.For Cerebral cortices of rescued ADAR1 knockout  and ADAR4.2.Flnb amplicon sequencing, triplicates of tissues from three C57BL/6J mice per time point  were analyzed.Tissues were homogenized in peqGOLD TriFast™ reagent  and total RNA was extracted following the manufacturer’s instructions. For 4.3.Aliquots of total RNA were digested with DNase I (Thermo Fisher Scientific) in the presence of Ribolock RNase inhibitor (Thermo Fisher Scientific) following the manufacturer’s instructions. cDNAs were synthesized using the Revert Aid reverse transcriptase (Thermo Fisher Scientific) in the presence of random hexamers. To detect possible genomic DNA contamination a control reaction lacking the reverse transcriptase was performed in parallel for every sample.4.4.Filamin B fragments were amplified in a 25 cycle Polymerase Chain Reaction (PCR) using the Q5® High-Fidelity DNA Polymerase (New England Biolabs Inc.). Primers were designed on different Filamin B mRNA exons, close to the editing site (underlined sequence) and contained part of the Illumina Adaptor Sequence .ACACGACGCTCTTCCGATCTTCGCTGCCTCACTGTTCTGAG-3ʹForward primer: 5ʹ-CAGACGTGTGCTCTTCCGATCTCGCTGGCTGGTTAACTTTTAATC-3ʹReverse primer: 5ʹ-PCR products (100 nucleotide long) were purified on silica columns . The eluted samples were used for a second round of PCR (15 cycles) with the NEB Next® Multiplex Oligos for Illumina® – Dual Index Primers Set 1 (New England Biolabs Inc.). PCR products were run on a 2% agarose gel and bands at the expected height (194 nucleotides) were cut and purified on silica columns. The DNA concentration of the eluted samples was quantified by fluorimetric assay  and samples were pooled accordingly.www.vbcf.ac.at).The overall process of cluster generation, sequencing, image processing, demultiplexing, and quality score calculation was performed at the VBCF NGS Unit (https://github.com/genome/bam-readcount) with the minimal base quality parameter and the minimal read mapping quality set to 20. All mouse genome coordinates mentioned refer to the genome assembly GRCm38/mm10.Obtained reads were mapped against the full-length amplicon sequence using bwa mem  with def4.5.The expression of ADAR2 mRNA or Flnb pre- and mRNA was quantified by real time PCR (qPCR) on a selection of P120 tissues. qPCR was performed using the GoTaq qPCR master mix (Promega), the CFX Connect™ Real-Time PCR Detection System  and the following primers:Flnb mRNA forward: 5ʹ-CAGCCCTTACCTGGTGCCCGT-3ʹFlnb mRNA reverse: 5ʹ-CTTTGCATCAATCTTCCCTTTCG-3ʹFlnb pre-mRNA forward: 5ʹ-GTAACTACGAGGTGTCTATCAAGT-3ʹFlnb pre-mRNA reverse: 5ʹ-GATGGATGCTTCCTTGTGCC-3ʹADAR2 forward: 5ʹ-CTTGCCCTGAAGGAGTTTTG-3ʹADAR2 reverse: 5ʹ-TCAGTGCTGCTGGAACTCAT-3ʹGAPDH forward: 5ʹ- AATGTGTCCGTCGTGGATCT-3ʹGAPDH reverse: 5ʹ- CCCTGTTGCTGTAGCCGTAT-3ʹExpression levels were calculated by the ΔCt method using GAPDH as a reference gene.A-to-I editing of Flnc mRNA was analyzed by Sanger sequencing of PCR amplicons obtained with the following primers:Flnc forward: 5ʹ- CTCCCTCTCAGACGACGCTC-3ʹFlnc reverse: 5ʹ- GCATCAATCACACCACGTGC-3ʹ4.6.Splicing scores were obtained using the MaxEntScan::score5ss software available here:http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.htmlAccording to the model based on the maximum entropy (MaxENT) principle  a log-od


C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both sense and antisense transcripts exist at the C9orf72 locus but the function of the antisense lncRNA is unknown. RNA toxicity of the transcribed repeat expansion has been implicated in the pathogenesis of C9orf72-related ALS/FTD, not only through direct sequestration of important RNA binding proteins but also indirectly through non-ATG dependent translation into dipeptide repeats. Formation of RNA/DNA hybrid R-loops may also play a key role in the pathogenesis of this condition and this mechanism could provide a link between the repeat expansion, DNA damage, repeat instability and deficiency of RNA binding proteins. Non-coding C9orf72 antisense transcripts could also act to epigenetically regulate gene expression at the locus. The potential effects of such non-coding RNAs should be considered in the design of antisense oligonucleotide therapeutics for C9orf72-related ALS/FTD. Furthermore, the mechanisms of RNA dysregulation exemplified by C9orf72-related disease may help illustrate more broadly how a “perfect storm” of dysfunction occurs in ALS/FTD and how targeting these factors could lead to corrective or preventative therapies.A hexanucleotide repeat expansion in the first intron/promoter region of  It is typically a disease of late adulthood, with onset peaking between 50 and 75 years of age, and in most cases it has a rapidly progressive course with average survival after diagnosis of only around 3–5 years . Around Despite the clinical phenotypes of ALS and FTD being markedly different from each other, both conditions share key features at the pathological level, including in most ALS cases and in many FTD cases a characteristic cellular pattern of TDP-43 proteinopathy ,12. In an expansion (>30 repeats being classed as pathogenic) within the first intronic region of the C9orf72 gene .,20.C9orfC9orf72 expression is that transcript V2, which starts downstream of the expansion, accounts for the vast majority (92.6%) of all C9orf72 transcripts as determined by ENCODE CAGE-seq data [C9orf72 cases, and it is known that TDP-43 loss of function tends to lead to the retention of long introns [A notable observation with regards seq data . Some stseq data . Howeverseq data . Yet othseq data . The causeq data . Similarseq data . An intrseq data . Misloca introns .n and antisense (CCCCGG)n RNA foci are seen within cell nuclei, confirming that antisense transcription takes place [C9orf72 antisense lncRNA is unknown. However, a similar antisense transcript has been found in mice (Gm12367) and analysis of comparative genomic alignments points towards a degree of conservation of the region immediately upstream of C9orf72 among eutherian mammals  that lies downstream of exon 1b . Furtheres place . The fun mammals .Fig. 2ThC9orf72 using the search function of the miRBase database of miRNAs (www.mirbase.org) reveals a putative miR-511-5p binding site 518–535 nucleotides 5’ to the exon 1a start site  loops, while the RNA version of the repeat has been shown to adopt a parallel quadruplex topology again with stacks of four G-quartets but this time linked with three propeller  loops [n is transcribed and forms antisense foci but these antisense RNA repeats cannot form G-quadruplexes and their structural conformation has not yet been formally resolved [Cellular toxicity of transcribed repeat expansions is an increasingly recognised disease mechanism, particularly among neurogenetic conditions such as myotonic dystrophy . Repetitxpansion ,20,58. Tequences . G-quadrl) loops . It is kresolved . While tresolved .C9orf72 expansion-positive cells. This is likely to partially reflect the relative promiscuity with which RNA-binding proteins (RBPs) bind to RNA. Reported binding factors include ADARBP, hnRNP-H, SRSF1, hnRNP-A3, hnRNP-A1, Pur-α, SRSF2, hnRNP-F, ALYREF and nucleolin [C9orf72 RNA foci. Another particular RBP that has been identified by multiple studies to bind the C9orf72 repeat expansion is heterogeneous ribonucleoprotein H (hnRNP-H) [C9orf72 patients [Despite multiple attempts, it has proved somewhat difficult to consistently identify the factors binding to RNA foci in ucleolin . Among thnRNP-H) . hnRNP-Hpatients . FurtherC9orf72 expansion-positive neurones, where RNA foci sequester hnRNP-H, one might expect increased levels of TRF2-S during neuronal development, which might in turn lead to dysregulation of mRNA transport, a mechanism that has been implicated previously in ALS pathogenesis. Similarly, should a corresponding reduction in TRF2 occur, this might lead to activation of the DNA damage response due to telomere instability, with the possibility of premature neuronal senescence.If sequestration and cellular depletion of hnRNP-H is indeed a key factor in ALS/FTD pathogenesis, how could this effect be mediated? Looking at the known targets of hnRNP-H, one intriguing possibility may relate to alternative splicing of telomere repeat-binding factor 2 (TRF2) . TRF2 isC9orf72 repeats have been shown to undergo RAN translation leading to the aggregation of dipeptide repeats, which themselves have toxic properties and have been linked to a neurodegenerative eye phenotype in fruit fly models [In addition to direct RNA toxicity, y models . Argininy models . The roly models .5C9orf72 repeat locus, such R-loops can in principle form in either or in both directions. Indeed, R-loop formation has even been shown to promote antisense transcription itself [There is a propensity for expanded repeats within DNA to form so-called “R-loop” structures during transcription . This isn itself .C9orf72 and hnRNP-H discussed previously, there is likely to be a more generalisable connection between RBP dysregulation and R-loop formation in ALS/FTD; one which concerns genome integrity and DNA repair [C9orf72 RNA foci, appears to help prevent R-loop formation in this way and it has been shown that depleted SRSF1 levels are associated with increased numbers of R-loops [Notwithstanding the potential mechanism linking A repair . Aside fA repair . Such daA repair . Stallin R-loops .SETX and a known cause of juvenile-onset ALS, is an RNA helicase that actively resolves R-loop structures [ATXN2, whose polyglutamine repeat expansion can be a modifying factor in ALS, might be involved in R-loop resolution in a manner akin to its yeast orthologue Pbp1 [MATR3 and a rare cause of ALS) is a target of ATM, an important kinase involved in the repair of double-strand breaks in DNA [Intriguingly, a number of other ALS-predisposing genes also have links to either R-loop biology or to DNA repair processes. Senataxin, encoded by ructures ,103. It gue Pbp1 . SFPQ, igue Pbp1 . Matrin s in DNA . Finallys in DNA .6C9orf72 promoter remains unknown, there are several possibilities that may be considered. For example, a number of such antisense transcripts have been found to play a regulatory role in the expression of their sense counterparts [While the function of the antisense lncRNA transcribed from the terparts . How couterparts ,116. Secterparts . This coterparts . These lC9orf72 expansion-positive patients) and PRC1 is involved in chromatin compaction through monoubiquitination of lysine 119 of histone H2A (H2AK119ub). Interestingly, heterochromatin formation has also been linked to R-loop formation, with repressive H3K9me2 marks being formed in HeLa cells at the sites of transcriptional pausing in association with recruitment of components of the RNA interference system [The mechanism by which natural antisense transcript (NAT) expression can lead to chromatin modifications has not been fully elucidated. However, lncRNAs are known to be involved in recruiting polycomb repressive complexes 1 and 2 (PRC1 and PRC2) to specific gene loci . PRC2 ree system .7SOD1 transcripts have also now commenced clinical trials following favourable first-in-man phase I studies [Antisense oligonucleotides (ASOs) are short  synthetic nucleic acid analogues that mimic the molecular structure of DNA and RNA, allowing them to base-pair with complementary native RNA target sequences. ASOs are a highly versatile class of molecules, whose sequence, chemistry and overall design can be engineered to suit multiple different circumstances and to engage multiple different cellular pathways ,120. To  studies .C9orf72 repeat expansion was identified, multiple groups immediately set about trying to knock down the repeat-containing transcript using ASOs to see whether this could provide a viable therapeutic avenue for an otherwise incurable disease. A number of studies demonstrated that the burden of RNA foci within cells could be reduced by treatment with antisense gapmer-style ASOs targeting the repeat-containing C9orf72 transcripts [C9orf72 transcripts may not adequately treat the condition. Furthermore, it has been reported that higher levels in frontal cortex and cerebellum of transcript V1, whose pre-mRNA will contain the repeat expansion and could therefore potentially be knocked down by repeat-targeting ASOs, is in fact correlated with a survival advantage following onset of disease [C9orf72-targeting ASOs have yet to be fully commenced.As soon as the nscripts ,42,127.  disease . This fiC9orf72 therapeutics have focussed on the direct approach of knocking down (GGGGCC)n-containing repeat transcripts, relatively little attention has been paid to alternative ASO approaches. If aberrant R-loop formation turns out to be a key pathogenic factor in C9orf72-related ALS/FTD, then ASO chemistries capable of preventing or otherwise abrogating such R-loop formation should be investigated for their therapeutic potential, as has been seen previously in myotonic dystrophy not only with LNA-gapmer but also with LNA-mixmer oligonucleotides [C9orf72. Furthermore, if natural antisense transcript (NAT) expression at the C9orf72 locus is indeed involved in regulating expression of the gene's sense transcripts, whether by dsRNA formation, transcriptional interference or induction of epigenetic repressive marks, ASOs designed to knock down the antisense transcript may in fact prove to be of therapeutic benefit via normalisation of previously haploinsufficient expression levels.Whilst most investigators into leotides . Intrigu8C9orf72 has been a case of the gene confounding those studying it at every turn. Firstly, the repeat expansion itself is hard to model owing to its large size, G-C content and inherent instability. Secondly, the disease it causes (ALS/FTD) is only partially understood in terms of its aetiology and therefore it is hard to interpret the contribution of the expansion to disease pathogenesis. Furthermore, the behaviour of the repeat expansion and its clinical effects do not appear to conform to what we have learnt from other repeat expansion disorders, either in terms of genetic anticipation or in relation to a correlation between expansion size and age of onset. However, as more becomes known about the molecular underpinnings of ALS/FTD in all its forms and about C9orf72 in particular, a fuller and more rounded pathogenetic picture is starting to become clear  will be treatable or preventable by correction of just one of its many pathogenic components.Finally, it is worth noting that although


RNA-binding proteins (RBPs) may play a critical role in gene regulation in various diseases or biological processes by controlling post-transcriptional events such as polyadenylation, splicing and mRNA stabilization via binding activities to RNA molecules. Owing to the importance of RBPs in gene regulation, a great number of studies have been conducted, resulting in a large amount of RNA-Seq datasets. However, these datasets usually do not have structured organization of metadata, which limits their potentially wide use. To bridge this gap, the metadata of a comprehensive set of publicly available mouse RNA-Seq datasets with perturbed RBPs were collected and integrated into a database called RBPMetaDB. This database contains 292 mouse RNA-Seq datasets for a comprehensive list of 187 RBPs. These RBPs account for only ∼10% of all known RBPs annotated in Gene Ontology, indicating that most are still unexplored using high-throughput sequencing. This negative information provides a great pool of candidate RBPs for biologists to conduct future experimental studies. In addition, we found that DNA-binding activities are significantly enriched among RBPs in RBPMetaDB, suggesting that prior studies of these DNA- and RNA-binding factors focus more on DNA-binding activities instead of RNA-binding activities. This result reveals the opportunity to efficiently reuse these data for investigation of the roles of their RNA-binding activities. A web application has also been implemented to enable easy access and wide use of RBPMetaDB. It is expected that RBPMetaDB will be a great resource for improving understanding of the biological roles of RBPs.http://rbpmetadb.yubiolab.orgDatabase URL:  A lack of fully structured metadata limits the wide use of valuable RNA-Seq datasets in public repositories such as Gene Expression Omnibus (GEO)  and ArraRBPs play a critical role in multiple cellular processes in eukaryotes. RBPs bind to double- or single-stranded RNA molecules and are potential key factors in biological processes, such as pre-mRNA splicing, RNA methylation and protein translation . BesidesFMR1)  . AlternaFMR1) . For insFMR1) . And a dFMR1) .To investigate the functions of RBPs in biological processes or diseases such as the ones mentioned above, a large number of studies have been conducted, resulting in exponential growth of RBP-related papers in recent years . For exaTo address this challenge, we worked to curate RNA-Seq datasets from GEO and ArrayExpress with one or more RBPs being perturbed, e.g. by knock-out, knock-down or overexpression. Important dataset annotations such as genotypes and PubMed references were manually curated to ensure high accuracy. Curated datasets can be used in gene expression analysis ,19 and aIn this paper, we describe our main curation methods used in constructing RBPMetaDB and the statistics of the database. To demonstrate the use of RBPMetaDB, a number of promising candidate RBPs have been identified by comparing RBPs with RNA-Seq datasets and all the RBPs annotated in Gene Ontology (GO). In addition, a web application has been developed to host the database to broaden the use of curated metadata and the original raw datasets among biomedical communities.NOTSuperSeries[Description]ANDgse[EntryType]AND“Musmusculus”[porgn:__txid10090]AND(“expressionprofilingbyhighthroughputsequencing”[DataSetType]OR“noncodingrnaprofilingbyhighthroughputsequencing”[DataSetType]) and against ArrayExpress using the query . These queries resulted in 1194 unique datasets in mice. Due to the limitations of the search functions of GEO and ArrayExpress, many of these datasets do not have perturbed RBPs despite the official symbols of some RBPs being mentioned in the titles or descriptions of the datasets. To retain the datasets with perturbations of RBPs, we manually curated each dataset  ( dataset  and retahttp://rbpmetadb.yubiolab.org). RBPMetaDB is implemented using Flask (http://flask.pocoo.org), a microframework for web development in Python. The MySQL database is used for data storage. The website of RBPMetaDB is freely available, and it presents the GEO/ArrayExpress accession numbers, descriptions, number of samples, associated curated RBPs, perturbation and PubMed references for each RNA-Seq dataset.To facilitate access to these datasets, we launched a database called RBPMetaDB  were retrieved using the R package GO.db . Using tP-value <Ezh2 is the most-studied gene, with 35 RNA-Seq datasets in RBPMetaDB. However, most studies of EZH2, as a catalytic subunit of polycomb repressive complex 2, focus on its capacity for mono-, di- and tri-methylation of histone H3 on lysine K27 (H3K27me1/2/3)  has been demonstrated to be an important stage-specific regulator of Crem mRNA processing that controls Crem polyadenylation in mouse testis. Cstf2t can lead to an overall decrease of the Crem mRNAs generated from internal promoters in Cstf2t−/− mice (What’s more, among the RBPs without related RNA-Seq datasets, 140 RBPs already have one or more mouse models  on the I−/− mice ,31. TherMettl3 loss- or gain-of-function studies (Mettl3. On the contrary, RBPMetaDB does not return irrelevant datasets of a given RBP, and it returns more accurate results than GEO.To facilitate the use of RBPMetaDB, a user-friendly website has been launched. The website allows users to access all the key information related to the curated RNA-Seq datasets, including the GEO/ArrayExpress accession numbers, dataset titles, numbers of samples, associated RBPs, perturbation types and PubMed IDs . The con studies . GEO ret studies , but it RBPMetaDB provides the first comprehensive, manually curated database of mouse RNA-Seq datasets with specific RBPs being perturbed. At the time of writing, it consists of the annotation of 292 mouse RNA-Seq datasets. These datasets provide valuable information for studying RNA-binding activity of RBPs. To keep RBPMetaDB updated, every six months, we will extract an updated list of RBPs from GO annotation to search GEO and ArrayExpress for newly released mouse RNA-Seq datasets and curate them. The results will be added to RBPMetaDB. RBPMetaDB will provide a valuable resource for many different research communities to understand how RBPs are involved in a variety of biological or disease processes.Database Online.Supplementary DataClick here for additional data file.


In this paper we report a new vaccine/gene delivery platform that demonstrates the benefits of using a self-amplifying (“replicon”) mRNA that is protected in a viral-protein capsid. Purified capsid protein from the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is used to in vitro assemble monodisperse virus-like particles (VLPs) containing reporter proteins  or the tandem-repeat model antigen SIINFEKL in RNA gene form, coupled to the RNA-dependent RNA polymerase from the Nodamura insect virus. Incubation of immature DCs with these VLPs results in increased activation of maturation markers – CD80, CD86 and MHC-II – and enhanced RNA replication levels, relative to incubation with unpackaged replicon mRNA. Higher RNA uptake/replication and enhanced DC activation were detected in a dose-dependent manner when the CCMV-VLPs were pre-incubated with anti-CCMV antibodies. In all experiments the expression of maturation markers correlates with the RNA levels of the DCs. Overall, these studies demonstrate that: VLP protection enhances mRNA uptake by DCs; coupling replicons to the gene of interest increases RNA and protein levels in the cell; and the presence of anti-VLP antibodies enhances mRNA levels and activation of DCs in vitro. Finally, preliminary in vivo experiments involving mouse vaccinations with SIINFEKL-replicon VLPs indicate a small but significant increase in antigen-specific T cells that are doubly positive for IFN and TFN induction.Many mRNA-based vaccines have been investigated for their specific potential to activate dendritic cells (DCs), the highly-specialized antigen-presenting cells of the immune system that play a key role in inducing effective CD4 Accordingly, a gene delivery platform that includes self-amplifying mRNA inside a protective capsid allowing for cell targeting and uptake[in vitro self-assembled virus-like particles (VLPs) for the protection, specifically using the RNA-dependent RNA polymerase (RdRp) from Nodamura virus (NoV) and capsid protein from Cowpea Chlorotic Mottle virus (CCMV).The delivery of RNA genes has great potential in a range of therapeutic applications–5, with nd uptake–11 couldNoV is a positive-sense RNA insect virus with a bipartite genome whose two molecules are co-packaged in the same virion. The larin vitro into wildtype capsids, as long as the length lies in the range 2500-4200nt so that it does not significantly differ from that (3200nt) of the largest of the CCMV RNAs[in vitro assembled capsids, known as virus-like particles (VLPs) – and in particular ones containing RNA replicons – have been shown[Tobacco Mosaic Virus (TMV) and RNA replicons from Semliki Forest Virus, with the replicons requiring that a TMV “origin-of-assembly”/packaging sequence be inserted into them[CCMV is a positive-sense RNA plant virus with a tripartite genome of four genes contained in three single-stranded RNA (ssRNA) molecules. Like NoCCMV RNAs–21. Theseen shown to both een shown–21, the into them.It is a feature of our replicon gene delivery platform that the gene of interest is coupled to the replication and translation of the NoV viral components, so that it has the capability of being used for a large array of possible therapies. Here we use the reporter proteins luciferase and enhanced yellow fluorescent protein (eYFP) to characterize and quantify the coupling of RNA replication and protein expression by the replicon. We then evaluate the efficacy of this platform for delivering antigens in RNA form by incorporating into the replicon a model antigen sequence . Since unpackaged mRNA or replicon mRNA show no perceptible change in maturation or RNA levels compared to negative controls, pointing up the fact that the VLP effectively protects the mRNA and helps it gain entry into the DCs.In this paper we demonstrate that amplification of reporter genes by the NoV replicase results in much greater protein expression than an equal copy number of the reporter gene mRNA alone. Further, when immature DCs are incubated with VLPs containing this replicon mRNA significant increases in DC maturation and RNA levels of the gene of interest (eYFP or the tandem OVA SIINFEKL epitope) are observed. The samples with Because many vaccines need to be administered incrementally in doses, an additional important factor is how the drug/vaccine is affected by existing immunity against the exposed outer portion of VLPs. Accordingly, antibodies were generated against CCMV capsid protein (CP) by immunizing mice with CCMV VLPs containing a non-translatable RNA. VLPs containing eYFP-replicon or OVA-replicon were incubated with these polyclonal anti-CCMV CP antibodies before being added to immature DCs, leading to enhanced DC maturation and RNA levels of eYFP or OVA compared to those of “naïve” VLPs, i.e., ones that have not been exposed to antibodies.Finally, in preliminary in vivo experiments, we have measured the T-cell response to a series of mouse vaccinations with VLPs containing the SIINFEKL antigen in replicon form, and find a small but significant population of SIINFEKL-sensitive T cells that are doubly-positive in INF and TFN cytokine production.in vitro packaging[in vitro, and includes other important features such as a self-cleaving peptide sequence (T2A peptide) derived from Thosea asigna virus, which separates the viral and exogenous proteins to render them functional, and a Hepatitis-Delta-Virus (HDV) ribozyme for providing monodisperse RNA transcripts.In the present work we dispense with RNA2 and add a gene of interest (GOI) to the subgenomic sequence of RNA see , as showpackaging–21 of thin vivo experiments assaying T-cell response in mice.Depending on the goal of our assay, we use one of several GOIs  injections consisting of 100μg of VLP in a 100μl volume were administered, separated by an interval of one week, and on the 4th week ex vivo analyses of spleen T cells were carried out by FACS. The negative control experiment (“Ctr”) was a corresponding series of injections of buffer solution, and the positive control was the T-cell analysis of a 96:4 mixture of T-cells from wildtype mice and from OT-1 mice engineered with a SIINFEKEL-specific T-cell receptor. As seen in In a preliminary in vitro packaged into VLPs. This enhancement of marker protein expression has been correlated with a much stronger amplification of RNA replication of the replicons, corresponding to RNA levels as much as ten-thousand-fold greater than those of the housekeeping protein beta-actin. Further enhancement of DC activation and RNA replication – likely facilitated by Fc- receptor-mediated[We have shown that levels of DC activation are significantly enhanced by incorporation of reporter genes and model antigens into self-replicating RNA molecules – replicons – that have been -mediated VLP uptaThe modest T-cell response reported here provides a proof-of-principle for our RNA replicon virus-like particle vaccine platform. In contrast with plasmid-DNA and viral-vector delivery systems, in particular those involving lentiviruses, adenovirus, and adeno-associated virus, there is no need for genetic information to access the nucleus of the host cell and no possibility of integrating genes into the DNA of the host cell. There are also no viral-specific immune responses that interfere with efficacy of vaccine boosts. mRNA vaccines have none of these drawbacks because they are translated directly in the cytoplasm and gene expression is only transient. They also have the advantage that they act simultaneously as adjuvants, because they interact with pattern-recognition receptors that elicit innate immune response enhancing the maturation of DCs and induction of adaptive immune response. Further when combined with the right RNA replicase gene, the mRNA vaccine is amplified strongly before being translated, resulting in a high level of in situ protein expression. Several mRNA replicon vaccines of this kind have been reported  the insect virus Nodamura provides the self-replicating RNA molecule (with its associated RNA-dependent RNA polymerase) into which genes of interest (such as the OVA antigen) can be inserted; and (2) the plant virus CCMV provides the capsid protein ensuring the Recombinant human GM-CSF and IL-4 were purchased from R&D Systems . PE-labeled antibodies (Abs) against human CD80, CD86 and MHC II were from BD Pharmingen  and isotype control Abs from R&D Systems . Complete Freund's Adjuvant, Incomplete Freund's Adjuvant and Lipopolysaccharide  were obtained from Sigma . The goat anti-mouse HRP-conjugated secondary ab  was purchased from Santa Cruz Biotechnology . The BioFXr TMB super-sensitive one-component HRP microwell substrate was from SurModics  and the BioStack Microplate Stacker from BioTek Instruments . RPMI-1640 (-L glutamine) and L glutamine were from Thermo Fisher  and heat-inactivated fetal bovine serum (FBS) from Sigma .2. Standard passaging was performed with the involvement of PBS for washing, and 0.25% trypsin-EDTA for cell detachment. All the above cell culturing reagents and medium were from ThermoFisher. The cells were passaged to 60-90% confluence in 24-well cell-culture-treated plates and, prior to transfection, were washed with PBS. For each well, 5 μl of lipofectamine-2000 (Thermo Fisher) was diluted with Opti-mem medium (Thermo Fisher) before mixing with 0.5 μg of RNA that is also diluted to an equal volume in Opti-mem. Since the replicon is so effective, the masses of mRNA prescribed in the lipofectamine-2000 manufacturer’s protocol for the replicon were making the cells sick. By diluting the replicon RNA, a similarly large luciferase activity compared to higher concentrations of transfected mRNA is observed . Masses of 12.5 ng of luciferase replicon mRNA and 3 ng of luciferase mRNA were diluted with uncapped carrier RNA of similar length up to 0.5 μg, in order to achieve a similar number of liposomes per transfection.BHK-21 cells were cultured in medium composed of high-glucose DMEM medium with 10% FBS and penicillin/streptomycin. The incubation conditions were 37˚C and 5% CORenilla Luciferase Assay System from Promega , and activity was measured using a Monolight 2010 luminometer. Cells were washed with PBS before directly lysing using passive lysis buffer and a cell scraper. 100 μl of Renilla luciferase assay reagent was added to a polycarbonate tube and luminescence was measured with 10 seconds of integration and a 2-second delay directly after adding 20 μl of cell lysate and mixing. Luciferase activity was performed in biological and assay duplicates for each time point.The luciferase assay was performed using the The plasmids constructed for these experiments were derived from plasmids kindly provided by Dr. Leonid Gitlin, from his work in Prof. Raul Andino’s lab at UCSF. Synthesis was performed from Nodamura virus strain Mag115, cDNA using random hexamers and SuperScriptIII (Invitrogen). The eYFP-replicon used was the same construct entitled pNoda-B1-FPG in their paper. The luciferase-replicon and OVA epitope-replicon plasmids were constructed from their construct pNoda-PolT2A-GFP. The luciferase gene for the luciferase-replicon was PCR-amplified from the plasmid pSP64-Ren Luc-Poly(A) – denoted as SP6 luciferase in 2, 4 mM μl DTT, and 2 mM spermidine. The rNTP concentration was 7.5 mM. Incubation was for 3 hours at 37°C, followed by a 30-minute incubation with turbo DNase at the same temperature. The resulting RNA was purified using the RNeasy mini kit from Qiagen, and then capped using the Vaccinia Capping System from New England Biolabs . This process added a 7-methylguanylate cap structure to the 5’ end of RNA in the presence of the capping enzyme, reaction buffer, GTP, and the methyl donor, SAM. The in vitro transcription and capping were both done in the presence of RNase inhibitors to preserve the RNA, and the capping mixture was again purified by an RNeasy mini column (Qiagen). Generating VLPs first involves isolation of CCMV CP from CCMV. The CCMV was purified from infected Cowpea plant leaves. To purify the CP, the virion was loaded into 6 kDa dialysis bags, and dialyzed in disassembly buffer . After dialysis, the disassembled CP-RNA mixture was pipetted into polycarbonate tubes and centrifuged in a TLA 110 rotor at 95k for 2 hours.  Fractions were taken after centrifugation, and a threshold A260/280 ratio of 0.6 was used to verify that they did not contain RNA. The capsid protein was dialyzed into Buffer B . After determining the CP concentration with a Nanodrop Spectrophotometer, CP was combined with mRNA at a ratio of 12.9 μg of CP to 3μg mRNA (representing the minimum excess of CP necessary for complete encapsidation of mRNA) in buffer B but without DTT or PMSF, for a total volume of 100μl. This mixture was then dialyzed into RNA Assembly Buffer, RAB , after which the pH was dropped to form capsids by dialyzing into Virus Suspension Buffer . Finally, the sample was transferred to RAB without DTT in order to have a VLP sample at the necessary neutral pH for biological experiments. Once VLPs have been verified (methods for which are described in the following two sections), the mg/ml concentration of VLPs is measured using the Nanodrop by dividing the 260nm absorbance by 5.8, the same factor used when determining the concentration of purified virus. Because of the small excess of CP used for complete packaging of RNA, the recovery of VLP mass is about 80% of the total mass of mRNA and CP used in the assembly.The replicon constructs were linearized with XbaI and subsequently purified using QIAquick PCR purification columns from Qiagen . The linearized DNA was transcribed with T7 RNA polymerase. The buffer composition of the reaction was 40 mM Tris pH 7.5, 25 mM MgClAfter assembly, the first step to verify the formation of homogeneous VLPs is agarose gel electrophoresis. A native agarose gel was run at 50 V for 1.5 hours, with the running buffer and the 1% agarose gel being made of 100 mM Sodium Acetate and 1 mM EDTA; the staining is with ethidium bromide. For reference, wild-type CCMV virion is included in a separate lane – the middle, in The preparation of well-formed intact VLPs was confirmed by negative-stain electron microscopy. 6 μl of VLPs is applied to glow-discharged copper grids (400-mesh) coated with Parlodion and carbon. After 1 min, the solution is blotted with Whatman filter paper. 6 μl of 1% uranyl acetate is then added to the grid. After 1 min, the excess uranyl acetate is removed by blotting. The grids are stored overnight in a desiccator and analyzed with a JEM 1200-EX transmission electron microscope equipped with a wide-angle (top mount) BioScan 600-W 1×1K pixel digital camera operated at 80 keV. The diameter of the VLPs is estimated by taking the geometric mean of two orthogonal measurements of the capsids obtained with ImageJ  software from recorded images. The diameter of 100 VLPs is measured in this way and compared to the diameter of wtCCMV that had been previously imaged.- monocytes negatively isolated from human peripheral blood mononuclear cells were cultured in a 24-well plate (1.0 × 106 cells/ml/well) with RPMI-1640 (-L glutamine) medium containing 1 × L glutamine and 10% heat-inactivated fetal bovine serum for 5 days. The donors were anonymous, but the number designation of PBMC for the figures included in this article are as follows: To generate immature DCs, CD146 cells/ml/well) were pulsed with naked eYFP mRNA, CCMV VLPs carrying eYFP mRNA, naked eYFP replicon mRNA, CCMV VLPs carrying eYFP replicon mRNA, CCMV VLPs carrying OVA-replicon mRNA, OVA-replicon-carrying CCMV VLPs pre-incubated with anti-CCMV VLP abs or naïve serum abs, and eYFP-replicon-carrying CCMV VLPs pre-incubated with anti-CCMV VLP abs or naïve serum abs. DCs stimulated with LPS and media were used as a positive and negative control, respectively. Following 24 hours of incubation, expression of activation markers for human DCs such as CD80, CD86 and MHC II was analyzed using flow cytometry.On day 5, immature DCs . They were immunized subcutaneously with CCMV VLPs containing a non-translated RNA (20 μg/per mouse/per immunization) on weeks 0, 2, and 4 using Complete Freund's Adjuvant for priming and the subsequent boost with Incomplete Freund's Adjuvant. The animal use protocol was approved by IACUC committee of Boehringer Ingelheim Pharmaceuticals. Serum samples collected one week after the 32SO4. Plates were read on a microplate reader (BioStack Microplate Stacker) at 450 nm. All experiments were performed in duplicate.To determine the end-point titers, CCMV VLPs were coated onto Immulon 2 HB 96-Well Microtiter plates  at 0.5μg/mL using PBS overnight at 4°C. Uncoated surface was blocked using phosphate-buffered saline (PBS) containing 3% Bovine Serum Albumin for 1 hr at 37°C. The plates were subsequently washed 5× with 0.05% Tween 20 in PBS. Mouse serum serially diluted (three-fold) in the blocking buffer was added to each well and incubated for 2 hrs at 37°C. The plates were washed 5×, and horseradish peroxidase (HRP)-conjugated secondary ab  was added to each well and incubated for 1 hr at 37°C. Plates were washed 5× and developed by adding 100 μl BioFXr TMB super-sensitive one-component HRP microwell substrate for 5 min. Reactions were stopped with 50 μl of 2 N HoC. To isolate RNA, the plate was thawed rapidly and an equal volume of ice-cold 1 × PBS was added to the well(s). The plate was spun at 1400 rpm for 5 min at 4°C. The fluids were removed carefully and the cell layer was washed again twice with ice-cold 1 × PBS. The cells were lysed for 5 min at room temperature in 50 μl of Lysis solution supplemented with DNase I . The reactions were then stopped with 1/10 volume stop solution and left at room temperature for 2 min. 10 μl of the lysate was added to the reverse transcription reaction cocktail that contained 1× SYBR RT buffer with 1× RT Enzyme mix. The reaction was run at 37°C for 1 hr followed by a 5-min 95°C heat kill of the enzyme.The Power SYBR Green Cells-to-CT kit was used for the detection of OVA and eYFP mRNA uptake by DCs. Briefly, the passively pulsed dendritic cells were washed twice with ice-cold 1 × PBS. RNALater (200 μl) was added to the well(s). The plate was sealed with PetriSEAL and placed at -80Four microliters of the reverse-transcribed material was added to 16 μl of gene-specific forward and reverse primers at 400 nM in the PowerSYBR Green PCR master mix. The samples were run using a Viaa7TM cycler . The CT values were generated by the Viaa7 program. Relative expression of the pulsed mRNA was calculated by finding the differences between pulsed mRNA CT values and the CT value for the ACTB housekeeping gene generated from the same cells by using the relation: RelExp = POWER ).For quantifying luciferase RNA in BHK cells in the ad libitum. Mice were group-housed (8 mice per cage) under pathogen-free and controlled environmental conditions  and handled according to the institutional, governmental and European Union guidelines . Animal studies were approved by the internal ethics committee and the local governmental committee.C57BL/6Tac female mice were provided by Taconic Denmark. The mice were transferred to the local AAALAC accredited animal facility at Boehringer Ingelheim RCV GmbH & Co KG at an age of 6-8 weeks, and immediately after arrival were allowed to adjust to conditions for at least 5 days before they were used for experiments. Standardized diet (PROVIMI KLIBA) and autoclaved tap water were provided Two different vaccination schedules were tested. In the first scenario mice were vaccinated subcutaneous (s.c.) only once, with either buffer as control, with the Ova Protein (50μg), with the SIINFEKL Replicon (100μg), or with the VLP containing the SIINFEKL replicon (100μg). The volume of all applications was 100μL, and after one week the mice were euthanized and spleens were harvested for FACS analysis. In the second vaccination scenario mice were vaccinated with buffer as control or with buffer containing VLPs containing the SIINFEKL replicon RNA (100μg). The vaccinations were administered s.c. with a volume of 100μL for 3 consecutive weeks (q7d), and one week after the last injection the mice were sacrificed and their spleens were harvested for FACS analysis. In addition one C57BL/6Tg(TcraTcrb)1100Mjb/Crl (OT-1) mouse provided by Charles River Laboratories (France) and one C57BL/6Tac mouse were euthanized and their spleens harvested to collect T-cells as control for the FACS analysis.+ T cells, present in the cell suspensions, were stimulated with 8μg/ml of SIINFEKL and TEWTSSNVMEERKIKV(EMC microcollections) peptides together with 10μg/ml anti-CD28 antibody (BioLegend) for 6 hours. One hour after start of stimulation, a protein-transport-inhibitor cocktail (eBioscience), containing Brefeldin A and Monensin, was added to block cytokine release. Before surface-marker staining, dead cells were labeled with a fixable viability stain and Fc receptors were blocked  to avoid nonspecific binding. Key surface markers were stained with fluorescently labeled antibodies, followed by fixation/permeabilization  and intracellular cytokine staining .Splenocytes from vaccinated and control mice were obtained by tissue homogenization followed by red blood cell lysis (ammonium-chloride-potassium lysis). Ovalbumin specific CD8oC. After thorough removal of unbound pentamer, key surface markers were labeled fluorescently with specific antibodies.For immunophenotyping including the detection of H-2Kb/SIINFEKL specific CD8 T cells, spleens were dissociated using a spleen dissociation kit from Miltenyi biotec, together with a gentle MACS Dissociator. After red blood cell lysis, labeling of dead cells and Fc Receptor blockade, single cell suspensions were incubated with H-2Kb/SIINFEKL pentamer (ProImmune) for 30 minutes at 4 to 8Flow data were collected with a BD LSRFortessa cell analyzer and further analyzed using FlowJo 10 and GraphPad Prism 7.03.S1 FigAntibody titers from mice immunized with CCMV VLPs containing a non-translated RNA.(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 FigAmount of INFγ and TNFα double-positive cells when stimulated with SIINFEKL peptide.(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.