It is governed by multiple regulatory factors, including RNA-binding proteins (RBPs), that bind to specific RNA sequences embedded in a specific structure. The ability to predict RNA-binding regions recognized by RBPs using whole-transcriptome approaches can deliver a multitude of data, including false-positive hits. Therefore, validation of the global results is indispensable. Here, we report the development of an efficient and rapid approach based on a modular hybrid minigene combined with antisense oligonucleotides to enable verification of functional RBP-binding sites within intronic and exonic sequences of regulated pre-mRNA. This approach also provides valuable information regarding the regulatory properties of pre-mRNA, including the RNA secondary structure context. We also show that the developed approach can be used to effectively identify or better characterize the inhibitory properties of potential therapeutic agents for myotonic dystrophy, which is caused by sequestration of specific RBPs, known as  AS leads to the formation of different mRNA variants from the same precursor transcript due to conditional inclusion or exclusion of alternative exons or their parts depending on the tissue type, developmental stage or disease conditions4. The process is controlled by a group of tissue-specific trans-elements that act as alternative splicing regulators, including muscleblind-like proteins (MBNLs), CUG triplet repeat binding, elav-like family proteins (CELFs) and RNA-binding fox homolog proteins (RBFOXs). They define the splicing pattern of regulated alternative exons by binding to specific RNA sequence/structure motifs (cis-elements)7. The inclusion or exclusion of alternative exons is primarily determined by the interplay of several alternative splicing regulators and other types of regulatory proteins, including splicing enhancers and silencers  or serine/arginine-rich splicing factors (SRSFs))8.Alternative splicing (AS) is a co-transcriptional process that leads to a significant increase in proteome diversity12 or Fuchs endothelial corneal dystrophy13. Interestingly, the MBNL1, MBNL2 and MBNL3 paralogs possess both redundant and unique functions in RNA processing and disease pathogenesis14. The best studied MBNL function is alternative splicing regulation15. All paralogs recognize RNA targets through an RNA-binding domain that contains four zinc fingers (ZF) organized into two tandems11. The MBNL-binding RNA motif is specified as a 5′-YGCY-3′ sequence (Y represents a pyrimidine)17 with preferentially single-stranded pyrimidines18. Depending on the location of the MBNL-binding motifs, whether upstream or downstream of the alternatively spliced exon, MBNLs serve either as repressors or activators of alternative exon inclusion, respectively19. A similar mode of action was previously described also for RBFOXs20 and CELFs21. Although many functional MBNL-binding motifs within pre-mRNAs  have been well described, most remain unknown25.MBNL proteins are key developmental regulators that are involved in different human diseases—e.g., myotonic dystrophy (DM)26, especially cross-linking and immunoprecipitation combined with deep sequencing (CLIP-seq)27 with subsequent modifications: individual-nucleotide resolution (iCLIP)28 and photoactivatable ribonucleoside-enhanced (PAR-CLIP)29. In general, CLIP-seq is a multistep method that starts from the physical cross-linking of RBPs with RNA sequences in situ using UV light. The protein/RNA complexes are subsequently purified by immunoprecipitation and molecular mass-based separation. Eventually, short RBP-bound RNA fragments are high-throughput sequenced31, followed by data filtration. The obtained overlapped reads form CLIP-seq clusters, which are considered to be RBP-bound regions. This method generates an enormous amount of data, enabling deeper insight into the proteins’ regulatory mode of action but also provides a high percentage of non-functional RNA binding sites and/or false-positive results. Thus, the potential RNA targets and RBP-binding motifs must still be verified in individual tests.The last decade has introduced several technologies for high-throughput analysis of protein/RNA interactions, including MBNL/RNACUG repeats (CUGexp) associated with myotonic dystrophy type 1 (DM1) development.Herein, we described a new verification strategy that uses antisense oligonucleotides (AONs) and highly redundant hybrid splicing minigenes as a genetic construct carrying an alternative exon as well as interchangeable MBNL-sensitive regulatory elements within an upstream intron. The strategy is designed for the rapid, simple, reproducible, and efficient determination of functional protein/RNA interactions based on CLIP-seq or other whole transcriptome experiments. It also allows evaluation of the regulatory properties of MBNL-binding sites. Moreover, we show that the hybrid minigenes can be used to reveal and elucidate potential crosstalk between different RBPs in the regulation of alternative splicing. This method was tested for MBNL-specific RNA-binding regions but could be applicable for other RBPs. Finally, we emphasize the usefulness of hybrid minigenes to study the efficacy of potential therapeutic agents for inhibiting the interaction between MBNLs and expanded Atp2a1 transcript and tested their inhibitory properties in vitro and in cellulo. Atp2a1 pre-mRNA contains alternative exon 22 (ex22), which as confirmed by mutagenesis, is positively regulated by the binding of all three MBNL paralogs to two YGCY motif-containing regions (region #1 and #2) that are localized within intron 22, ~110 nucleotides (nt) downstream of ex22 . First, we confirmed the sensitivity and specificity of this assay for a few RNA fragments that have previously been described in the literature as targets of MBNL133. We did not observe any interaction of this protein with four negative control RNAs (RNA-Ctrl1-4), with or without YGCY motifs  or 2′-O-methyl (2′OMe) modification of the RNA structure with or without a phosphorothioated backbone (PS) and DNA-based AONs. The LNAs (LNA(-PS)#1 and #2) contained 10-nt residues and were complementary to individual regions #1 or #2, while the 2′OMe- and DNA-type AONs were 21-nt-long and covered both regions (2′OMe(-PS) and DNA)  Fig. . Having old Fig. . Consist #2 Fig. , confirmAtp2a1 minigene, which encompasses a pre-mRNA sequence from exons 21 to 23 (Atp2a1WT), and the MBNL1-overexpressing vector. RT-PCR analysis showed substantial MBNL1-dependent enhancement of alternative ex22 inclusion  the presence of an MBNL-dependent alternative exon adjacent to putative binding motifs with CLIP-seq clusters either within the exon or upstream or downstream intron up to 250 nt from the exon; (ii) overlapping of at least three YGCY sequence motifs that are at least partially conserved in human and mouse.To confirm the applicability of AONs for the verification of RBP/RNA interactions, we selected five additional pre-mRNAs, besides ing Fig. . To datePphln1-, NASP-) and exonic  RNA fragments and designed AONs that were complementary to these regions (Mbnl1-RNA described in38)  or two  regions composed of a string of YGCY motifs as the most essential for MBNL1/RNA interactions  or upstream (NASP) introns and induced MBNL-dependent alternative exon promotion or repression, respectively. In each case, the direction and strength of alternative splicing changes upon AON application were similar to the effect of siRNA-mediated MBNL1,2 silencing  responsible for 3′ splice site definition. Depending on the binding site localization within introns, either downstream or upstream of alternative exon, MBNLs may play a role as an enhancer for U2AF65 binding40 or its competitor by modifying the RNA secondary structure41, respectively. The other possibility is that MBNLs associated with RNA affect binding of other trans-factors such as CELF142 and RBFOX43.It remains unclear what mechanism drives alternative exon regulation by MBNL proteins bound to individual RNA cis-elements tested in this study could affect U2AF65 recruitment. Therefore, AONs blocking the RNA/MBNL interaction may contribute indirectly to observed alternative splicing changes. On the other hand, binding of AON itself could directly disrupt spliceosome organization via RNA structure rearrangements. However, due to a relatively long distance between MBNL-binding sites and alternative exons (~110–270 nt) it seems to be less possible. Blocking intronic or exonic MBNL-binding sites by AONs might also interfere with binding of other trans-factors pivotal for regulation of the alternative exon inclusion. Exon skipping induced by AON targeting either the 5′ and 3′ splice sites and/or exonic splicing enhancers (ESEs) has often been reported in studies on splicing manipulation in genetic, aberrant splicing-based disorders, including Duchenne Muscular Dystrophy (DMD)44. Deutekom and others showed that utilizing a set of AONs that cover many regions along with an exonic sequence leads to exon exclusion45.Analogously, the binding of MBNLs to intronic Atp2a1WT minigene (Atp2a1Δ). Removal of this cassette resulted in a strong reduction of the ex22 splicing response to MBNL1 overexpression   and exonic  RNA fragments representing the sequences inserted into the hybrid minigene, as described above for Atp2a1-RNA  or within partially single-stranded regions . Only in Nfix-RNA the predicted MBNL-binding motifs were located within a stable hairpin structure with two symmetrical internal loops that were not recognized by any of the enzymatic structure probes.Previous observations led us to design hybrid minigenes as an alternative and complementary tool to test the sensitivity of RNA regulatory elements to MBNLs. To prepare the hybrid minigenes, we removed a natural 111-bp-long MBNL-binding cassette, which contains regions #1 and #2 with YGCY motifs, from intron 22 of the ion Fig. . A similrl) Fig. . Subsequtte Fig. . The seqtes Fig. . To bettRNA Fig. . RNA strAtp2a1Δ-Pphln1, Atp2a1Δ-NASP, Atp2a1Δ-Nfix, Atp2a1Δ-Ldb3 and Atp2a1Δ-Mbnl1, and the MBNL1 overexpressing vector. As anticipated, we observed that for all of the precursor RNAs synthesized from hybrid minigenes, inclusion of ex22 was promoted by MBNL1, similar to that for the original Atp2a1WT pre-mRNA , the endogenous MBNLs already strongly elevated ex22 inclusion compared with Atp2a1WT . This result sheds light on the question regarding the actual mechanism of MBNL-dependent exon repression. We hypothesize that MBNLs, when bound to exonic cis-elements, can affect the binding or activity of other trans-factors that act as alternative splicing enhancers. This may explain the perplexing splicing effects that we observed when testing AONs that targeted the exonic sequences of Nfix, Ldb3 and Mbnl1 pre-mRNAs , we demonstrated that recombinant SRSF1 bound to the studied exonic Mbnl1-RNA and Nfix-RNA with high affinity, ranging from 13 to 60 nM, respectively 20 and (AGG)20 . Overexpression of MBNL1 led to the exclusion of ex1  or Fuchs endothelial corneal dystrophy.To emphasize the versatility of the CTG repeats located in the 3′-untranslated region of the myotonic dystrophy protein kinase gene (DMPK)56. Expression of mRNA with CUG expansion ((CUG)exp) leads to mRNA retention in the nucleus as well as pathogenic recruitment and sequestration of nuclear proteins, including MBNLs, to an RNA hairpin-like structure formed by the repeats55. The longer the repeats, the more severe the missplicing of hundreds of genes due to the functional insufficiency of MBNLs57. On the other hand, in Fuchs endothelial corneal dystrophy, toxic (CUG)exp RNA repeats occur within intron 2 of a transcription factor 4 pre-mRNA (TCF4)58.DM1 is a neuromuscular, autosomal dominant disease that is caused by the expansion of three-nucleotide CUG)exp, antisense oligonucleotides and small compounds have been frequently employed to specifically bind to repeats and prevent MBNL sequestration62. We have previously shown that an 8-nt-long oligonucleotide composed of LNA-PS moieties (LNA-PS-CAG-8)63 and erythromycin (EM)64 prevent MBNL sequestration on this toxic RNA by complementarily hybridizing to or potentially binding to U/U mismatches within the structure formed by (CUG)exp, respectively. These molecules diminish adverse molecular outcomes, mainly abnormalities in alternative splicing in the cell and mouse models of DM1.Among the therapeutic strategies aimed at ameliorating the toxic effect of 17 (Atp2a1Δ-(CUG)17) 17. In support of our hypothesis, hybrid minigenes expressing the pre-mRNAs containing (CUG)4 or (CUG)8 undergo MBNL-mediated splicing with lower rate of ex22 inclusion compared to Atp2a1Δ-(CUG)17  Fig. . Our preCUG interaction and used them in our minigene assay. Cells co-transfected with the Atp2a1Δ-(CUG)17 minigene and MBNL1 overexpressing vector were treated with different concentrations of either LNA-PS-CAG-8 or EM. We observed a strong and gradual decrease in ex22 inclusion induced by LNA-PS-CAG-8 but not by the control LNA oligonucleotide, reaching below the level established by endogenous MBNLs at a concentration of 125 nM exp interaction in both in vitro and in vivo models64.Next, we chose LNA-PS-CAG-8 and EM as examples of efficient inhibitors of the MBNL/ nM Fig. . The EM  nM Fig. . This reAtp2a1Δ-(CUG)17 hybrid minigene is a very sensitive tool for characterizing different MBNL/(CUG)exp inhibitors and that after certain modifications, such as reporter gene inclusion, the hybrid minigene can be successfully applied in high-throughput screening. A related screening system, based on the detection of alternative splicing pattern changes within the Clcn1 minigene’s pre-mRNA and including luciferase system for high-throughput analysis, has been described previously65. The advantage of our Atp2a1Δ-(CUG)17 minigene is its sensitivity, reproducibility and simplicity because it can be introduced into non-DM1 cell models. Moreover, the assay provides not only qualitative but also quantitative information about the potency of the studied compounds and additionally enables to monitor the differences in the number of MBNLs associated with CUG repeats. Investigation of potential therapeutic agents for diseases mediated by toxic RNA that disrupts the activity of RBPs, including myotonic dystrophies, is an ongoing process. Our assay could facilitate the screening of molecules on in vitro stages.These results show that the All of the DNA sequences were purchased from oligo.pl®  and are listed in Supplementary Tables. All RNA-based AONs were synthesized by RiboTask™ and are listed in Table Atp2a1WT minigene, a fragment of the Atp2a1 gene between exons 21 and 23 containing MBNL-sensitive alternative exon 22 was amplified from mouse genomic DNA using Platinum® high fidelity Taq DNA polymerase (Invitrogen™) according to the manufacturer’s protocol with a SrcMres_F/SrcMres_R primer set (Ta = 55 °C) whose sequences are provided in Supplementary Table XhoI and BamHI. To obtain the Atp2a1Δ minigene, a 111-bp sequence within intron 22 in the Atp2a1WT minigene that included the MBNL-binding regions was substituted with a 34-bp linker sequence containing unique restriction sites for NotI and SalI. First, two fragments of the final Atp2a1Δ insert were amplified using the pEGFP_F/NotSal_R and NotSal_F/pEGFP_R primer sets (Ta = 60 °C) to introduce the 34-bp linker in both PCR products. Second, based on their complementarity to 34-nt linker, the obtained PCR fragments constituted the template for PCR with the pEGFP_F/pEGFP_R primer set (Ta = 60 °C) to provide a full-length Atp2a1Δ sequence. To obtain Atp2a1-hybrid minigenes, the sequences of interest from the Pphln1, NASP, Nfix, Ldb3, Mbnl1 and Ctrl (Capzb) genes were amplified from human (NASP) or mouse (others) genomic DNA with the Pph1insF/Pph1insR, NASPinsF/NASPinsR, NfixinsF/NfixinsR, Ldb3insF/Ldb3insR, Mbnl1insF/Mbnl1insR, CTRLinsF/CTRLinsR primer sets . To obtain the Atp2a1-(CUG)17 minigene, oligodeoxynucleotides CTG17F and CTG17R with restriction sites for either NotI or SalI, were used. Prior to their hybridization for one hour at RT, they were purified on a denaturing 10% polyacrylamide (PA) gel, followed by precipitation and denaturation at 95 °C for 5 min. All of the amplified DNA fragments of interest were ligated into the pEGFP-C1 vector (CloneTech) prior to transformation into DH5α bacteria cells. To obtain the Mbnl1 minigene, a DNA sequence including the 3′ part of the intron (53 bp) and 5′ part of exon 1 (110 bp) of the Mbnl1 gene was amplified from human genomic DNA using the Mbnl1F/Mbnl1R primer set (Ta = 55 °C), introducing restriction sites for NotI or SalI  was a gift from Dr. Tom Misteli (Addgene plasmid # 17990)66.To obtain the 2. HeLa, C2C12 or HSkM cells plated in 12-well plates were transfected at 50–60% confluence with Lipofectamine® 2000 (Invitrogen™) according to the manufacturer’s protocol. Single transfection was conducted with an siRNA mix against MBNL1 and MBNL2 (25 nM each) , 50 nM AllStars negative control siRNA (Qiagen), or a specific AON at 125 nM, in which AON-Ctrl was 2′OMe-PS unspecific to a tested transcript. Co-transfection was conducted with 200 ng of the minigene and 500 ng (or as indicated in the figures) of the MBNL1, SRSF1 or GFP expressing vector. For verification of the MBNL-binding regions and inhibition of the MBNL/(CUG)exp interaction, the co-transfection was followed by a 4-hour incubation and transfection with selected AONs. Erythromycin was added directly to the medium. The cells were harvested 48 hours after transfection.Human HeLa and mouse C2C12 cells were grown in high-glucose DMEM medium (Lonza) supplemented with 10% fetal bovine serum (Sigma) and 1x antibiotic/antimycotic (Sigma). Human skeletal myoblast (HSkM) cells were grown in HAM F-10 medium (Sigma) supplemented with 20% FBS, 1x antibiotic/antimycotic, 0.39 µg/ml dexamethasone (Sigma) and 10 ng/ml epidermal growth (Sigma). All cells were grown at 37 °C and in a 5% atmosphere of COTotal RNA was isolated from cells using TRI-reagent (Sigma) according to the manufacturer’s protocol. cDNA was synthesized using GoScript™ Reverse Transcriptase (Promega) with Random Primers (Promega) according to the manufacturer’s protocol. RT-PCR was performed using GoTaq® Flexi DNA Polymerase (Promega) with the primer sets listed in Supplementary Table Atp2a1-RNA mutants were prepared in two rounds of PCR similar to that for the Mbnl1Mut minigene. In the first step, specific Atp2a1mt_F/Atp2a1_TR and Atp2a1mt_R/Atp2a1_TF were used. In the second round of PCR, only _TF and _TR were used. The transcription reaction was performed in 50 µl containing 10 µl of DNA template (~0.5 μg), 0.15 mM of each ribonucleotide (Invitrogen™), 0.45 mM guanosine (Sigma-Aldrich), 50 U of T7 RNA Polymerase (Ambion), 1 × T7 transcription buffer (Ambion), and 40 U of RNasin®Plus RNase Inhibitor (Promega). The transcripts were purified on a denaturing 8% PA gel (19:1 acrylamide:bisacrylamide), followed by ethanol precipitation. For 5′-end radiolabeling, 2 pmol of RNA was incubated with 2 pmol (12 mCi) of [γ-P32] ATP, 1 U of RNasin®Plus RNase Inhibitor (Promega) and 10 U of OPTI Kinase (Affymetrix) in 1 x reaction buffer (Affymetrix) at a total volume of 10 µl and at 37 °C for 45 min. The 5′-32P-labeled RNAs were subsequently run on a denaturing 8% PA gel. The bands of RNAs were visualized via Storage Phosphor Screen BAS-IP (IP) and a laser scanner FLA-1500 (FujiFilm) and were then excised, followed by ethanol precipitation and resuspension in 20 µl of double-distilled water.The templates for the transcription reactions of RNA fragments were obtained in two rounds of PCR. In the first PCR, longer products were amplified with genomic mouse or human DNA templates using specific _F/_R primer sets  are listed in Supplementary Table 38 with slight changes. Briefly, 5′-32P-labeled RNA was first subjected to a denaturation and renaturation procedure in either buffer N  for ribonucleases T1 and T2 or in a commercial S1 buffer (Fermentas) for endonuclease S1. Limited RNA digestion was initiated by mixing 5 µl of the RNA sample (~0.5 pmol) with 5 µl of a probe solution containing S1 and T2 at the concentrations indicated in Supplementary Figs 2+ at 55 °C for 2.5 min. The bands of RNAs were visualized via IP and the laser scanner FLA-1500 after overnight (O/N) exposition. In silico prediction of the secondary structure was performed using the mfold program with experimentally defined constraints.Analysis was conducted as previously described69.The GST fusion construct with a truncated 41-kDa MBNL1 isoform sequence encoded by exons 1–4 of the MBNL1 gene with a His6 tag at the C-terminus was prepared as previously describedBamHI and NotI. Sequencing of the obtained construct was followed by transformation of the E. coli BL21(DE3)pLysS strain (Novagen). Production of 23-kDa SRSF1 containing a His6 tag at the C-terminus was performed as previously described70.To generate the construct for recombinant SRSF1, 558 bp of the SRSF1 coding sequence was amplified using the pEGFP-SF2 vector (Addgene) as a template and SRSF1_F/ SRSF1_R primer set (Supplementary Table 32P-labeled RNAs (0.05 nM) were subjected to a denaturation and renaturation procedure by heating the samples at 95 °C for 1 min followed by a 10-min incubation at RT. Subsequently, the RNAs were incubated with the indicated protein concentrations (ranging from 0 to 200 nM) in buffer FB  at 37 °C for 15 min. Studying the inhibitory property of AONs, 0.05 nM of 5′-32P-labeled RNAs mixed with 0.5 µM of RNA-based AONs or 20 µM of DNA-AONs (Supplementary Table via IP and laser scanner FLA-1500 after O/N exposition. The dissociation constant (Kd) of the MBNL1/RNA complexes treated or non-treated with different AONs was calculated in the GraphPad program using the following equation: one site specific binding curve (Y = Bmax*X/(Kd + X)).The filter binding assay (FBA) was performed to assess the affinity of MBNL1 to different RNAs. 5′-32P-labeled RNA fragments with the indicated SRSF1 concentrations (ranging from 0 to 800 nM). The reactions were performed in a volume of 10 µl in buffer A ) at 37 °C for 20 min. To identify SRSF1-binding regions, 0.5 µM of 2′OMe-PS-Mbnl1 or -Nfix along with 0.05 nM of 5′-32P-labeled RNAs were subjected to denaturation and renaturation, followed by incubation at 37 °C for 20 min in buffer A. The samples were mixed with loading buffer  and run on a non-denaturing 8% PA gel. The gel was subsequently dried, and the signal was visualized via IP and the laser scanner FLA-1500 after O/N exposition. The Kd of the SRSF1/RNA interaction was calculated based on the signal of free RNA and using the equation: one phase decay curve equation (Y = (Y0 − Plateau)*exp(−K*X) + Plateau) in Graph Pad program.The electrophoretic mobility shift assay (EMSA) was carried out by incubation of 0.05 nM of 5′-in vitro and in cellulo are expressed as the means ± standard deviation (SD). The statistical significance of the RT-PCR and EMSA results was determined by two-tailed Student’s t-test using Microsoft Excel . Statistical analysis was calculated using 2–4 biological repeats. Similar results were obtained from at least two independent experiments.Group data obtained All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).Supplementary Information


Diabrotica virgifera virgifera. The RNA sequence consists of 9,823 nucleotides (nt) with a 3′ polyadenylated tail, containing a single open reading frame that encodes a 3,028-amino-acid polyprotein.The genome sequence of a novel iflavirus was identified from the transcriptome of the western corn rootworm,  Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is the worst insect pest of maize in North America and is invasive in Europe (Bacillus thuringiensis (Bt) insecticidal toxins, but populations resistant to Bt toxins have been reported (–Western corn rootworm (WCR), n Europe . Managemreported . Hence, reported , Lepidopreported , and Hymreported –7. Here,de novo assembled using Trinity (Sacbrood virus (accession number NP_049374).WCR adults were collected from cornfields in the United States , and Europe . Total RNA was extracted from ~100 individuals from different locations using TRIzol reagent . Additionally, putative viral RNA was isolated from virions, which were crudely extracted from WCR using the PEG virus precipitation kit (BioVision). Eight RNA sequencing (RNA-seq) libraries of either total RNA or viral RNA were prepared using the TruSeq RNA sample preparation kit version 2 (Illumina). All libraries were sequenced using an Illumina HiSeq 2000 platform, generating 3.7 to 21 million single-end 100-bp reads. After trimming adaptor sequences, the Illumina reads were  Trinity . ContigsDiabrotica virgifera virgifera virus 1 (DvvV1), contains an open reading frame , with a 317-nt 5′-untranslated region (UTR) and a 422-nt 3′-UTR, followed by a polyadenylated tail. The G+C content of the virus is 39.45%. The entire ORF encodes a 3,028-amino-acid polyprotein. Conserved domains include four structural protein domains at the N terminus  and two nonstructural protein domains at the C terminus, the RNA_helicase domain (nt 1423 to 1530) and RNA-dependent RNA polymerase domain (nt 2632 to 2965). Although the primary cysteine protease sequence motif of GxCG was identified (amino acids 2382 to 2385), the protease-C3 domain was not, suggesting divergence of the DvvV1 protease domain sequence. The presence of the virus in WCR adults was confirmed by reverse transcription-PCR (RT-PCR) using SuperScript III , with overlapping primers, and rapid amplification of cDNA ends . Based on the arrangement of conserved domains, we conclude that this virus is a novel insect iflavirus. Relatively few iflaviruses are associated with overt symptoms or acute disease (This new virus, tentatively named KY064174.The genome sequence was deposited in GenBank under accession no.


Exosomal RNA extraction was compared using four different RNA purification methods. We determined the minimal starting volume of serum required for exosome preparation and showed that high quality exosomal RNA can be isolated from sera stored for over a decade. Finally, RNA-Seq libraries were successfully prepared with exosomal RNAs extracted from human cell-free serum, cataloguing both coding and non-coding exosomal transcripts. This method provides researchers with strategic options to prepare RNA-Seq libraries and compare RNA-Seq data quantitatively from minimal volumes of fresh and archival human cell-free serum for disease biomarker discovery.Exosomes are endosome-derived membrane vesicles that contain proteins, lipids, and nucleic acids. The exosomal transcriptome mediates intercellular communication, and represents an understudied reservoir of novel biomarkers for human diseases. Next-generation sequencing enables complex quantitative characterization of exosomal RNAs from diverse sources. However, detailed protocols describing exosome purification for preparation of exosomal RNA-sequence (RNA-Seq) libraries are lacking. Here we compared methods for isolation of exosomes and extraction of exosomal RNA from human cell-free serum, as well as strategies for attaining equal representation of samples within pooled RNA-Seq libraries. We compared commercial precipitation with ultracentrifugation for exosome purification and confirmed the presence of exosomes  RNA was extracted using the RNeasy Mini Kit, according to the manufacturer’s instructions. RNA was quantified using the Nanodrop ND-1000 (Thermo Fischer Scientific).The RNeasy Mini Kit was used to extract exosomal RNAs according to the manufacturer’s protocol. RNA was quantified using the Nanodrop.Exosomal RNAs were phase-separated as described above. The upper aqueous phase was collected and 10% volume of sodium acetate  was added to each sample. Four microliters glycogen  and 2.5 times the acquired aqueous volume of absolute ethanol were added. Samples were mixed, incubated overnight at -80°C and then centrifuged at 16,000 g at 4°C for 30 min to pellet RNA. The pellet was washed with 70% ethanol and centrifuged at 16,000 g at 4°C for 5 min. Ethanol was aspirated and the pellet was centrifuged again to remove additional supernatant. Pellets were dried for 5 min before being resuspended in 32 μL of nuclease-free water and purified RNA was quantified using Nanodrop.The AllPrep DNA/RNA Mini kit  was used to extract exosomal RNAs following the manufacturer’s protocol. RNA was quantified using the Nanodrop.5'-AATGATACGGCGACCACCGA-3' and 5'-CAAGCAGAAGACGGCATACGA-3'. Libraries were sequenced to generate single-end 75 bp reads on NextSeq 500 platform (Illumina) in high output running mode. Sequencing was performed at the Molecular Genomics Core facility at the University of Southern California.Twenty nanograms exosomal RNA was used to prepare each RNA-Seq library. External RNA Controls Consortium (ERCC) spike-ins  were added as control for normalization of the samples. Strand-specific RNA-Seq libraries were constructed using the NEBNext Ultra Directional RNA Library Prep Kit  with the following adjustments to the manufacturer’s instructions: fragmentation was performed for 1 min at 94°C given the size range of our exosomal RNA was typically smaller than 1000 bp (data not shown), and PCR was set to 15 cycles. A unique index was ligated to each sample using the NEBNext Multiplex Oligos for Illumina Index Primers Sets 1–4 . The resulting library concentrations were quantified using the Nanodrop, a Library Quantification Kit , and the Bioanalyzer 2100 (Agilent). The KAPA Kit contains Library Quantification DNA Standards 1–6  and a Library Quantification Primer Premix. The sequences of primers are Raw data were downloaded as FASTQ files and QC performed using MultiQC . Around t tests were used to calculate P-values when comparing methods for exosomal RNA extraction. One-way ANOVA was applied to calculate a P-value when contrasting exosomal RNA extraction from different serum volumes. Spearman’s rank correlations were utilized to decide significance of the correlations between RNA yield or OD260/OD280 with storage time. P-values smaller than 0.05 were considered statistically significant.Paired Student’s https://www.ncbi.nlm.nih.gov/sra/SRP142765.Exosome RNA-Seq data are publicly available at: A workflow for exosomal enrichment and validation, RNA extraction, and RNA-Seq library generation is illustrated in 260/OD280 ratios were therefore compared for exosomal RNA isolated using four different RNA extraction methods: the RNeasy Mini Kit combined with TRIzol LS, the RNeasy Mini Kit alone, conventional RNA precipitation, and the AllPrep DNA/RNA Mini Kit , gene expression microarrays or RNA-Seq), both RNA quantity and quality are critical. RNA yield and ODMini Kit . CompariMini Kit , suggestMini Kit . NanodroMini Kit , each ofpitation . ExoQuicpitation . In geneTo determine the minimum volume of cell-free serum required for exosomal RNA isolation, 63 μL, 125 μL, 250 μL and 500 μL aliquots of cell-free serum were subjected to exosomal purification using both ultracentrifugation and ExoQuick . ExosomaWe then tested the performance of our optimized exosomal RNA extraction on a cohort of clinical samples collected over the last 25 years. One hundred and five serum samples were processed using the ExoQuick reagent followed by RNA extraction using TRIzol LS combined with the RNeasy Mini Kit. The serum samples were stored at -80°C immediately after removing blood cells see , and hadTo demonstrate that exosomal-derived RNA isolated using our optimized method can generate high quality RNA-Seq data, RNA-Seq libraries were constructed using 20 ng exosomal RNA from 5 unique sera specimens from healthy women. The average size of inserted exosomal RNAs was around 450 bp . The conAlthough next-generation sequencing has transformed biomarker research, RNA-Seq profiling of liquid biopsy specimens is a relatively underexplored frontier. Our optimized protocol demonstrates that exosomes can be rapidly enriched from a very limited amount of fresh or archival human cell-free serum (under 63 μL) with particularly high efficiency when using a commercially available reagent–ExoQuick, followed by RNA extraction performed using TRIzol LS and the RNeasy Mini Kit. The exosomal RNA isolated using this protocol can be used for many downstream applications–RT-qPCR, gene expression microarray analysis, and, as demonstrated here, RNA-Seq analysis. RNA-Seq provides the most comprehensive characterization of exosomal transcriptomes, and can be used in functional modeling studies or in the discovery of biomarkers that could be used in the clinic for diagnosis, prognostication, or for assessing therapeutic response.In contrast to RNA-Seq libraries built from RNA extracted from other sources (e.g. whole cells), the number of mapped reads in sera exosomal RNA-Seq libraries showed poorer correlations with quantifications performed using the Nanodrop, qPCR, and the Bioanalyzer. This could be because the exosomal RNA concentrations are typically lower, and near the lower limit of detection for Nanodrop. Nanodrop (or Qubit) measurements tend to overestimate nucleic acid concentrations and have greater variability while Bioanalyzer analyses typically underestimate concentration and demonstrate less variability. The accepted ‘gold-standard’ is an RT-qPCR measurement approached based on a standard curve of known DNA concentrations. Somewhat surprisingly, our results showed that for exosomal RNA-Seq libraries, Nanodrop quantification correlates higher with RT-qPCR measurements than Bioanalyzer. We therefore recommend estimating library concentration using Nanodrop and performing low-depth sequencing (e.g. using the Illumina MiniSeq) to accurately quantify the ratio of each library in the pool, and then adjusting the ratios as required before proceeding with deeper sequencing. This strategy will provide equal number of reads for each library within the pool, which is essential for quantitative analyses of exosomal RNAs across samples .260/OD280 of 2.5, suggesting that small RNAs may be over-represented, as observed in previous studies [The ultracentrifugation protocol also performed well in our comparisons, but is only amenable to smaller studies, as substantially more serum samples can be processed in a drastically shorter time (at similar cost) using ExoQuick method  compared with ultracentrifugation . Additionally, use of the ExoQuick reagent retrieves more exosomes and exosomal RNAs than ultracentrifugation. Several methods of exosomal RNA extraction can be implemented, and it should be noted that precipitation of RNA by absolute ethanol recovered more RNA from ExoQuick-purified exosomes, but not ultracentrifugation-purified exosomes. Those exosomal RNAs had an average OD studies , 24. TheIn the past decade, exosomal components have been examined extensively to identify potential prognostic and diagnostic biomarkers for many human diseases, including cancer, but exosomal transcriptomes remain poorly characterized , 30. As


Influenza A virus nucleoprotein (NP) association with viral RNA (vRNA) is essential for packaging, but the pattern of NP binding to vRNA is unclear. Here we applied photoactivatable ribonucleoside enhanced cross-linking and immunoprecipitation (PAR-CLIP) to assess the native-state of NP–vRNA interactions in infected human cells. NP binds short fragments of RNA (~12 nucleotides) non-uniformly and without apparent sequence specificity. Moreover, NP binding is reduced at specific locations within the viral genome, including regions previously identified as required for viral genome segment packaging. Synonymous mutations designed to alter the predicted RNA structures in these low-NP-binding regions impact genome packaging and result in virus attenuation, whereas control mutations or mutagenesis of NP-bound regions have no effect. Finally, we demonstrate that the sequence conservation of low-NP-binding regions is required in multiple genome segments for propagation of diverse mammalian and avian IAV in host cells. Influenza A virus packaging depends on interactions between nucleoprotein (NP) and viral RNA (vRNA), but the pattern of NP binding is unclear. Using PAR-CLIP, Williams et al. here show that NP binds vRNA non-uniformly and that RNA structures in low-NP binding regions are important for packaging. NP is thought to coat viral RNA (vRNA) uniformly in cells and virus particles; however. uniform coating likely would preclude the possibility for RNA structure formation in RNPs. Numerous virus families utilize structured RNA elements for specific biological processes throughout infection, including genome packaging4. For example, viral RNA elements are required for efficient replication, mRNA splicing, and genome packaging of IAV7. Structure formation has been demonstrated with in vitro folded IAV vRNA and engineered genome segments, but the structural constraints imposed by nucleoprotein on vRNA generated during infection is not known9. Elucidation of the physiological interaction between NP and viral genomic RNA may provide novel insights into how IAV is capable of coordinating its lifecycle. Thus, we set out to determine the in vivo landscape of NP–vRNA interactions.Influenza A virus (IAV) possesses a segmented, negative-sense RNA genome that is bound by the viral nucleoprotein (NP) throughout replication. Recent cryo-electron microscopy studies of the IAV ribonucleoprotein complex (vRNP) provide evidence for a NP–RNA complex structure with a corkscrew-like morphology and the tripartite polymerase complex at one end and a loop at the other end. However, current models conflict with each other and yield no information about RNA conformation, binding, or NP–RNA association11. Interaction between vRNAs has been demonstrated in vitro and disruption of packaging signals or interacting segment regions attenuated virus replication at the stage of genome packaging14. In many cases, mutation of a single segment leads to a significant decrease in the packaging efficiency of other segments15. Additionally, viral particles largely package only one copy of each genome segment18. Together, these results suggest that genome segments function as a multipartite, cooperatively packaged entity, possibly potentiated by segment–segment interactions, rather than a stochastically generated particle20.Infection and complete replication of IAV requires delivery of all eight genome segments into a recipient cell. All IAV segments require packaging signals derived from the termini on each segmentIn this study, we set out to determine how IAV NP interacts with vRNA during infection in cells. We show that the NP of IAV binds the vRNA non-uniformly and that regions of low-NP binding are enriched for predicted RNA secondary structures. Synonymous mutations designed to destabilize the predicted RNA structure attenuate IAV replication, whereas synonymous mutations that maintain the predicted RNA structure or mutations in NP-bound RNA regions have no effect on virus replication in vitro or in vivo. Viral attenuation is associated with an increase in defective virus production, suggesting that low-NP-binding regions and the predicted RNA structures are required for viral genome packaging.21. We infected human cells with WT-PR8 virus for 16 h in the presence of 4-thiouridine (4-SU) to enhance cross-linking of NP–RNA complexes and then generated Illumina 1 × 50 sequencing libraries of the NP-bound RNA  coupled to next-generation sequencing was used to resolve the interaction between the negative-sense RNA genome of IAV and NP during infection of human 293T cellsRNA Fig. . The impRNA Fig. . AdditioRNA Fig. . These rTo determine the sensitivity and specificity of the PAR-CLIP assay, we performed Western blotting analysis for IAV NP and cellular ß-actin on input lysate and immunoprecipitated proteins Fig. , bottom.PAR-CLIP identified both human and virus-derived RNAs that interacted with NP. The procedure enriched for IAV RNA sequences relative to RNA-seq libraries Fig. , and theQ < 0.01, and ≥ 18 nucleotides long, we identified 24 regions in the viral genome that were low in NP binding relative to RNA-seq and 18 regions that met two of these three criteria that we did not investigate further . The NP1410–1495 region contains a previously hypothesized vRNA pseudoknot5. Additional regions of NP intermediate binding  were also included in the following studies. Computational prediction of RNA structures using RNAfold in low-NP-binding regions guided further mutational analysis, and these regions were mutagenized to either disrupt (NP22–68:A and NP1410–1495) or maintain (NP22–68:B) the predicted secondary and tertiary structure formation  formation and destabilizing a predicted stem-loop structure (NP22–68:A) in the 3′ region of the vRNA segment resulted in reduction of focus area, as a measure of multi-cycle replication and spread , also did not affect focus size  of NP1410–1495 and NP22–68:A mutant viruses was significantly lower (P < 0.05) compared to all other viruses when normalized to HA-units, indicating a greater production of non-infectious particles  or stem-loop structure (NP22–68:A) resulted in aberrant genome constellation stoichiometry. Consistent with the viral replication assays, mutation of intermediate NP-bound regions or mutations designed to maintain the predicted 3′ stem-loop structure (NP22–68:B) result in unaltered ratios of genome segments in all but one segment-specific instance , segment 2 (PB1497-561:A) and segment 8 (NS23–86:A) reduced the focus area of mutant viruses in MDCK cells , did not alter focus size  did not  and 2 (PB1) mutants did not display altered reporter activity  and segment 2 (PB11828–1858 and PB12032–2058) did not alter focus size or TCID50 per HA-unit  resulted in decreased packaging of segments 3, 4, 5, and 7 relative to segment 1  mutant viruses packaged reduced levels of segment 6 vRNA (NA)  was different from that of the WT RNA oligomer and the RNA containing synonymous mutations that maintained the RNA structure (PB1497–561:B)  indicated a lower thermal stability. In contrast, the thermal profile of the PB1497–561:B RNA was similar to that of WT RNA. Combined these data show that stable RNA structures are required for IAV replication and genome packaging.Low-NP-binding regions in segments 1, 2, and 8 also were evaluated for their contribution to viral replication by mutagenesis designed to disrupt predicted secondary and tertiary RNA structures  Fig. . The PB2nce Fig. . FinallyPR8 Fig. . Togethe1823–1944 and NP1410–1495), identified in WT-PR8, to the replication of a North American avian IAV (A/shorebird/Delaware/22/2006 (IAV-H7N3)) and the 2009 pandemic H1N1 IAV ). The PB2 and NP gene-segment of IAV-PR8 and IAV-H7N3 are divergent and representative of mammalian and avian viruses, respectively 30. However, additional low-NP-binding regions were identified throughout the segment body and outside of traditional packaging signals31. Our findings agree with studies assessing structure-guided mutagenesis of hypothetical vRNA structures (segment 7) or biochemical analyses of vRNA (segment 8) that largely overlap with low-NP-binding regions assessed here32. Additionally, we identified specific nucleotides required for co-packaging of segment 2 (PB1) and segment 6 (NA), in a region recently implicated in directing reassortment outcomes and co-segregation of these segments in vitro34. Therefore, NP PAR-CLIP allowed us to directly identify potentially functional RNA regions in IAV genomes required for coordinated genome packaging.We found many low-NP-binding RNA regions that overlap with previously predicted packaging and bundling signals near segment termini , and penicillin–streptomycin (Gibco). Human embryonic kidney cells (293-T) were maintained in Opti-MEM (Life Technologies) with 10% FBS, l-glutamine, and penicillin–streptomycin. MDCK and 293T cells were a kind gift from Dr. Richard Webby at St. Jude Children’s Research Hospital.Madin–Darby Canine Kidney (MDCK) cells were maintained in minimal essential medium (MEM) with 5% fetal bovine serum , MEM-vitamins (Gibco), 35. Viruses were generated by transfection of all eight plasmids into co-cultures of 293T and MDCK cells (1 μg per plasmid) with polyethylenimine . Wild-type (herein IAV-PR8 (WT-PR8), IAV-pH1N1 (WT-pH1N1) or IAV-H7N3 (WT-H7N3) and mutant viruses were generated in the same manner with the exception of substituting individual plasmids harboring mutagenized DNA for the single indicated wild-type plasmid. The next day, the transfection mixture was removed and replaced with Opti-MEM containing MEM-vitamins, l-glutamine and penicillin–streptomycin. Forty-eight hours post-transfection, an additional 1 ml of the same media containing 1 μg ml−1 TPCK-Trypsin (Worthington) was added to the co-culture. Seventy-two hours after addition of TPCK-Trypsin, culture supernatant was collected and clarified by centrifugation . Viral stocks were generated by infection of MDCK cells in a T75 flask. Cells were washed once with PBS, and 200 μl of transfection supernatant was mixed with 25 ml of infection media (M0.1B) composed of MEM containing vitamins, l-glutamine, penicillin–streptomycin, 0.1% Bovine Serum Albumin (Gibco) and 1 μg ml−1 TPCK-Trypsin for 48 h. Stocks were aliquoted and stored at −80 °C until use, and all studies were conducted with passage 1 stocks following verification of mutant sequence identity. All viruses were generated at least twice independently. Sequences for primers utilized during mutagenesis of IAV plasmids are available in Supplementary Table Eight bidirectional pHW2000 plasmids containing cDNA for A/Puerto Rico/08/1934 (H1N1), A/California/04/2009 (H1N1), or A/shorebird/Delaware/22/2006 (H7N3) were previously described21 coupled to next-generation sequencing to discern nucleotide resolution maps of protein–RNA interaction across the IAV genome. Confluent 293-T cells were infected at an MOI of 1 for 16 h in the presence of 100 μM 4-thiouridine (4-SU) and then cross-linked with ultraviolet light ). PAR-CLIP was performed essentially as described before21 with the exception of antibodies used to immunoprecipitate NP–RNA complexes: monoclonal antibodies HB65 (ATCC) or MAb8258 (EMD Millipore). On-bead Calf Intestinal Alkaline Phosphatase  treatment was used to dephosphorylate RNA ends followed by 5′ end-labeling with 32P catalyzed by T4 Polynucleotide Kinase . Briefly, protein–RNA complexes were separated on 4–12% SDS-PAGE gels and transferred to nitrocellulose membranes. 32P-labeled RNA was identified by autoradiography, excised, and extracted following proteinase K (New England Biolabs (NEB)) digestion. To account for potential bias introduced during adaptor ligation and sequencing, we also generated RNA-seq libraries using RNA extracted from uncross-linked cell lysates of influenza infected 293T cells from which RNA was isolated by TRIzol (Invitrogen) extraction, then 10 μg RNA was fragmented by Mg2+ at 95 °C for 12 min . 32P-labeling of this RNA was performed in solution with T4 PNK (NEB), and size-selection of 10–100 nt fragments by Urea-Page (15%) was performed. In both cases, total RNA was precipitated with ethanol, and used to prepare Illumina sequencing libraries. 3′-OH RNA previously prepared by CIP treatment was ligated to a 3′ adaptor using T4 RNA Ligase 2, truncated K227Q (NEB), according to manufacturer’s instructions. The 5′ adaptor sequence contains NNN-degenerate nucleotides in addition to a sequence used for demultiplexing to facilitate collapsing of redundantly generated PCR products and ascertain the frequency of individual NP–RNA interaction events. This adaptor was ligated to the 5′-end of RNA using T4 RNA Ligase 1 (NEB). Following isolation of adaptor ligated and radiolabeled RNA, all subsequent library generation steps were identical. Independent PAR-CLIP or RNA-seq libraries were pooled and cDNA synthesized using a primer complementary to the 3′ adaptor . Libraries were amplified by PCR for 9–15 cycles using Phusion DNA polymerase  and primers annealing to the 5′ and 3′ termini of the DNA that enable flow-cell binding. Size selection of libraries was conducted by extraction of amplicons from 6% urea gel electrophoresis, and then libraries were precipitated. Molarity of libraries was determined by qPCR  and size by Bioanalyzer (Agilent). Illumina sequencing on the HiSeq 2500 instrument (1 × 50 bp reads) was performed by the Genome Technology Access Center at Washington University in St. Louis.To identify interactions between viral RNA and IAV nucleoprotein, we adapted the protocol for PAR-CLIP21. PAR-CLIP and RNA-seq data were generated from four independent experiments. For PAR-CLIP and RNA-seq libraries we used an analysis pipeline that collapsed unique barcoded reads, removed adaptors, and aligned them to the viral genome. Strandedness was determined post-alignment and all subsequent analysis was performed on negative-sense viral genomic RNA (vRNA). For reads that mapped to the viral RNAs, we normalized the number of reads per nucleotide to the total number of reads per genome segment to yield a normalized coverage ratio for both PAR-CLIP and RNA-seq libraries. We then compared the normalized coverage for each preparation at nucleotide resolution using an unpaired t test with False Discovery Rate (FDR) correction (Benjamini–Hochberg)36. vRNA nucleotides with a FDR minima Q < 0.01 and fold-change > 3 were identified and these regions were extended to the final nucleotide of each regionm, where Q ≤ 0.1. These areas represent low-NP or high-NP-binding regions of interest (ROI). ROI ≥ 18 nt were subjected to RNA structure analysis with mFold, RNAfold, and vsFold5 to determine computationally the theoretical minimal free energy (ΔG) of each region as well as the potential secondary or tertiary RNA structure formation38. Nucleotide composition of ROIs was determined by calculating the percent of A, G, C, or U in each region and comparing them to the percent of each nucleotide in the IAV genome. PAR-CLIP data sets were compared to RNA-seq data sets at each nucleotide position on a per-segment basis and positions using unpaired t test with FDR correction as above with a threshold of Q = 0.05. Sites of statistically enriched transition in PAR-CLIP data sets were tabulated and the sequence distance between these positions was calculated. Transition events with a step-size of 1, i.e. sequential nucleotides both with high transition rate, and were excluded from analysis because they likely represent single NP–RNA interactions of adjacent 4-SU nucleotides in vRNA. Transition rate for four PAR-CLIP libraries were averaged prior to distance calculation.All NGS data were analyzed on the Washington University in Saint Louis School of Medicine’s McDonnell Genome Institute (MGI) cluster using publically available analysis programs  and in-house scriptsE. coli, plated on selective LB Agar. Colonies were selected and grown overnight in LB broth and selective antibiotics. All plasmids were grown in 200 ml LB Broth prior to preparation by Qiagen HiSpeed Endotoxin-Free MaxiPrep and verified by Sanger Sequencing (Genewiz).Mutant selection was performed after analysis of open reading frames to assess potential nucleotide degeneracy while retaining coding sequence. Subsequently, we selected five underrepresented ROIs in four segments  for functional assessment of manipulating predicted RNA structures on viral replication. We also identified four regions in segment 5, amenable to extensive silent mutagenesis that were either highly bound or represented at the same frequency in PAR-CLIP and RNA-seq data sets. We introduced between 2 and 7 synonymous mutations simultaneously in silico to the identified regions and reassessed structural stability or pseudoknot formation using the same structure prediction programs. We selected variant codon combinations that would disrupt or maintain the predicted vRNA structure but not change the encoded amino acid, alter codon usage, or disrupt alternative reading frames or splicing events. Once predicted destabilizing mutations were identified, mutant viruses were generated. Mutations were introduced into pHW2000 bidirectional plasmids by inverse PCR with primers . Foci were visualized by addition of HRP-conjugated streptavidin and TruBlue streptavidin substrate and imaged on a Biospot reader (Cellular Technology Limited). Individual foci were counted and area was calculated using the Analyze Particles extension within Fiji39. The area of the foci formed by mutant viruses was normalized to the average of WT-PR8 foci per experiment. A minimum of 60 foci was analyzed per virus.Six-well plates of MDCK cells were plated and inoculated with 1 ml of 10-fold serially diluted virus stock for 1 h in M0.1B. After 1 h, the inoculum was removed, and replaced with an overlay of 1% Low Melting Point Agarose in MEM supplemented with 1 μg mld lysine overnight. Cells were infected with WT-PR8 for 16 h then fixed with 4% methanol-free PFA in PBS (pH 7.4), washed with PBS, permeabilized with saponin, then NP staining was performed using anti-NP MAb HB65 (1 µg ml−1) and goat-anti-mouse Alexa-488 (Invitrogen). Nuclei were counterstained with DAPI. Coverslips were mounted to slides with ProLong® Diamond Antifade Mountant (Molecular Probes). To determine if viral protein production was impacted by synonymous mutations, we utilized confocal microscopy to visualize the distribution of NP during infection as a marker of viral protein production and a proxy for trafficking of vRNPs. MDCK cells were infected with WT-PR8 and indicated mutant viruses (MOI of 0.2) for 8 h, then fixed and permeabilized as before. NP staining was performed as above using anti-IAV-NP MAb HB65 and Goat-anti-Mouse Alexa-488 (Invitrogen). Slides were imaged with Zeiss LSM 880 Confocal Laser Scanning Microscopy with Airyscan and analyzed with Zeiss Zen Black Software performed within the Molecular Microbiology Imaging Facility at Washington University in Saint Louis.We infected 293T cells in the presence or absence (mock) of 4-SU with WT-PR8 (MOI of 1) for 16 h. Briefly, 293T cells were seeded on glass coverslips coated with poly-35. A vRNA-like firefly luciferase reporter plasmid and a Renilla luciferase expression plasmid also were included. Briefly, 293T cells were seeded into 24-well plates and transfected with equal amounts of all six plasmids  in Opti-MEM containing TransIT-LT1 (Mirus). Cells were maintained at 37 °C for the duration of the experiment. Forty-eight hours later, cells were lysed for analysis of luciferase activities (Promega). Each combination of polymerase proteins (set of plasmids) was examined in duplicate. The relative light units (RLU) of firefly luciferase activity were normalized to the RLU for Renilla luciferase activity within the same sample to account for differences in transfection efficiency between wells and experiments. The activity of each plasmid set containing a mutant segment was normalized to the activity of WT-PR8.To assess the impact of silent mutations on polymerase complex activity, pHW2000 plasmids encoding WT or mutant PB2, PB1, PA, and NP were utilized in a dual-luciferase reporter assay as previously described5 MDCK cells (MOI of 0.2). Eight hours post-infection, cells were collected and fixed. Intracellular staining of IAV antigen was performed as above using an anti-IAV NP primary antibody (HB65) (1 µg ml−1) and an Alexa-488-conjugate goat-anti-mouse secondary antibody (Invitrogen). The mean fluorescence intensity (MFI) of NP+ cells was plotted and calculated from two experiments performed in duplicate. Co-expression of NP and M in MDCK cells was determined 16 hpi with an MOI of 5 and 0.05. Infected cells were fixed and stained as above with the additional staining step of utilizing mouse-anti-M primary antibody (M2-1C6) (2 µg ml−1) and goat-anti-mouse Alexa-647 secondary. A second intracellular staining step utilizing biotinylated mouse-anti-NP (mAb8258b) (1 µg ml−1) and Alexa-488 conjugated streptavidin was then performed. The frequency of co-expression was calculated by determining the number of cells expressing NP or M as well as those expressing both. The percentage of infected cells co-expressing NP and M was calculated by dividing co-expressing cells by all cells expressing one or more viral protein as previously described23. Analysis of viral proteins was determined using a flow cytometer and FlowJo software (Tree Star).To determine the amount of NP generated at a fixed time during infection, we infected 2 × 105) were seeded in 24-well plates and inoculated the next day with 200 TCID50 of IAV. Cells were washed once with PBS before addition of inoculum in M0.1B and incubated for 1 h at 37 °C. Subsequently, the cells were washed twice with PBS and 1 ml of M0.1B supplemented with 1 μg ml−1 TPCK-Trypsin was added to each well. Culture supernatants were collected at 24 and 48 hpi and the amount of infectious virus was quantified by titration on MDCK cells.MDCK cells  were bred in-house in a barrier facility at Washington University School of Medicine, St. Louis, MO or purchased from Jackson Laboratories. Mice received food and water ad libitum and all experiments were conducted in accordance with rules of the Institutional Animal Care and Use Committee. Mice were anesthetized with isoflurane in an airflow chamber and then inoculated intranasally with 30 μL of sterile PBS containing 1000 TCIDg for 5 min, passed through a 0.22 µM filter, and pelleted on 30% sucrose cushion by ultracentrifugation . Pelleted virus particles were directly resuspended in 350 μl TRK lysis buffer, and RNA purified immediately and eluted in 30 μl DEPC H2O . cDNA was synthesized from 5 µl of RNA with SSIII Reverse transcriptase (Invitrogen) and a vRNA specific primer22. Total cDNA was diluted 1:10,000 and used to quantify each of the 8 genome segments by SYBR Green qPCR (PowerUpTM SYBR® Green Master Mix) with primer pairs previously published22. Relative abundance of each genome segment was calculated as before, except with normalization to Segment 1 or 7 depending on the virus assessed.MDCK cell derived stocks of WT-PR8 or mutant viruses were clarified by centrifugation at 1200 × 497–561, PB1497–561:A, and PB1497–561:B :θn the minimum ellipticity at 210 nm or the maximum ellipticity at 260 nm, θd the maximum ellipticity at 210 nm or the minimum ellipticity at 260 nm, and θobs the observed ellipticity at Fd. The ellipticity, θobs, when Fd = 0.5 was calculated and used to calculate Tm from the linear fit of the transition region. For comparisons between different RNA samples, the first derivative of the ellipticity, θobs, the change in molar ellipticity as a function of temperature (dθ/dT), was calculated and plotted.The RNA oligomers corresponding to PB1t tests with the Benjamini–Hochberg correction to identify areas in which these sequencing preparations were statistically different from each other (Q < 0.1 and Q < 0.01). Transition-distance was determined using FDR (Q < 0.05). For analysis of the focus-forming assay, luciferase assay, and lung viral titers, we used one-way ANOVA with multiple comparison corrections . TCID50 per HA-unit was analyzed using an unpaired t test.All statistical analyses were performed using GraphPad Prism 7.0. For comparison of PAR-CLIP and RNA-seq data sets, we used multiple unpaired The unprocessed PAR-CLIP and RNA-seq files have been uploaded to the NCBI Sequence Read Archive . All other data supporting the findings of this study are available within the article and its Supplementary Information files, or are available from the authors upon request.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1


Circular Dichroism (CD), UV-visible, FTIR spectroscopic methods, zeta potential and Dynamic Light Scattering (DLS) were used to analyze the particle formation, particle charge, particle size, aggregation, RNA conformation, binding constant and mode, and the effect of polymer complexation on RNA stability.HBCD/RNA= 1.25 × 103.The results indicate that the interaction of RNA with HBCD leads to the formation of a linear dendritic supramolecule biopolymer with an overall binding constant of KThe small sized synthesized polymer can be considered as an appropriate system for preventing RNA aggregation and protecting the gene by host-guest interaction. CDs, cyclic oligosaccharides are well-known structures in supramolecular polymers in which the monomers are held together by reversible and highly directional non-covalent bonds 8. These biocompatible and nontoxic compounds 9 possess a cage-like supramolecular structure, with hydrophilic outer surfaces and lipophilic inner cavities. Various hydrophobic drugs can be embedded in their hydrophobic cavities via non-covalent interactions 14. However, CDs can only cause a minor enhancement in the solubility of hydrophobic drugs 16.Recently, a variety of macromolecule structures such as Cyclodextrins (CDs) and dendrimers have been examined for the formation of stable complexes with genes19. Polyglycerol dendron functionalized cyclodextrins (β-CD-g-PG) are novel carriers which can be synthesized by controlled anionic polymerization of glycidol. The Hyperbranched Cyclodextrins (HBCD) are small sized, well-defined polymers with higher water solubility, oxidative stability and biocompability. They exhibit high potential of encapsulating hydrophobic guest molecules due to the molecules binding to interior or exterior part of their structure 22.Recent advances have been focused on functionalizing CDs to obtain small sized macromolecules with higher free cavities and water solubility et al studied the host-guest interaction between β-CD-g-PG with long alkyl chain adamantines 23. The incorporation of insulin into HBCD polymers significantly caused enhanced drug absorbance across the nasal barrier and decreased blood glucose concentration 24. Paclitaxel was effectively encapsulated by HBCD for targeted drug delivery 25. Recently, β-cyclodextrine-polyethyleneimine polymers (PEI-CDs) were examined for in vitro transport of miRNA(microRNA) and siRNA  26. CDs have also been studied as topical drug delivery systems. Application of CDs derivatives in transdermal drug delivery has improved drug release/permeation, decreased druginduced local irritationand hasoptimized systemic and local dermal drug delivery. Recently, Loftsson et al reviewed the role of cyclodextrins on drug delivery through biological membranes 27.Tao 29, there is no precise information on the molecular aspects of interaction between HBCD and RNA in aqueous solution. Thus, HBCD interaction with RNA was studied in aqueous solutions with HBCD/RNA molar ratios of 1/16 to1/1 at pH=6.5–7.5 utilizing CD, UV, and FTIR measurements. The complexes were analyzed to study the particles size and charge parameters. The structural analyses of RNA secondary structures, binding site of HBCD to RNA, and HBCD-RNA binding constant were provided by spectroscopic results.Although several experiments report the interaction of CDs with guest molecules nm was utilized to check the protein content of RNA solutions. The A260/A280 ratio for RNA was 2.10, indicating that the RNA samples were nearly free from protein 30. Other chemicals were of reagent grade and used without further purification.Sodium methoxide, methanol and acetone were purchased from Merck. β-cyclodextrin, glycidol and Baker’syeast RNA from Sigma Chemical  were employed without further purification. The absorbance band at 280 20. Briefly, β-CD (0.5 g) was deprotonated by dissolving sodium methoxide (14.4 mmol) in dried methanol. Mixture was stirred at room temperature using a polymerization ampule equipped with a magnetic stirrer for 1 hr. Methanol was vaporized via vacuum oven at 60°C for 2 hr. Glycidol (92 mmol) was added to deprotonated β-CD and temperature was gradually increased up to 120°C. The mixture was stirred at 120°C for 12 hr and allowed to cool. It was dissolved in pure methanol, and precipitated upon addition of aceton. It was neutralized by filtration over cation-exchange resin. The precipitate was dried via vacuum oven at 80°C. Finally, a yellow viscous product was obtained with 87% yield.HBCD (β-CD-g-PG) was synthesized by anionic ring opening multi branching polymerization method mg/ml) were prepared by dissolving RNA in 10 ml of phosphate buffer (pH=7.4) with occasional stirring at 5°C for 24 hr. Using the molar extinction coefficient of 6600 cm−1M−1 which was represented as molarity of phosphate groups, the final concentration of RNA stock solution was assessed spectrophotometrically at 260 nm32. UV absorbance of the diluted RNA solution (40 μM) at 260 nm was 0.11 (with the path length of 1 cm), and the final concentration of RNA stock solution was 25 mM in phosphate. For infrared spectroscopic measurements, various amounts of HBCD in phosphate buffer (pH=7.4) were added dropwise to RNA solutions to achieve favorable HBCD contents of 0.625, 1.25, 2.5, 5 and 10 mM with an ultimate RNA concentration of 10 mM. Infrared spectra were recorded 1 hr after the mixing of HBCD solutions with RNA solutions. For UV measurements, various concentrations of HBCD (500–1200 μM) were used with constant RNA concentration (40 μM).Homogeneous sodium-RNA solutions -(polynucleotide solution)], a sharp RNA band at 968 cm−1 was used as internal reference 34. This band is assigned to sugar C-C and C-O stretching vibration and does not change (intensity or shifting variation) upon HBCD complexation to RNA, so it will be deleted upon spectral subtraction.To record the infrared spectra, a Nicolet FTIR spectrometer (Magna 550) equipped with a liquid-nitrogencooled HgCdTe (MCT) detector was used. The spectra of the HBCD/RNA solutions were collected utilizing a cell assembled with ZnSe windows and treated by OMNIC software. The spectra of solutions were recorded after 1 cm−1 (RNA) as a function of HBCD concentrations, the intensity ratios of RNA in-plane vibrations related to phosphate stretching vibrations and A-U and G-C base pairs were measured with an error of ±0.9%. In order to determine the ligand binding to RNA bases and backbone phosphate groups, similar intensity variations have been used 36. After peak normalization, the plot of relative intensity (R) of various RNA in-plane vibrations including 1698 (guanine), 1650 (uracil), 1606 (adenine), 1489 (cytosine) and 1241 cm−1 (phosphate groups) versus polymer concentrations were gained. The peak normalization was performed using Ri = Ii/I968 where Ii is the intensity of the absorption peak for pure RNA in the complex with i as ligand concentration, and I968 as the intensity of the peak at 968 cm−1  (By considering the reference band at 968 ference) .−4–12×10−4M and constant RNA concentration of 40×10−6M. Binding constants of HBCD-RNA complexes were calculated as reported 37. The interaction between HBCD as ligand [L] and RNA as substrate [S] is assumed to be 1:1 which forms a single complex SL (1:1). 0 from the mass balance expression St=[S]+ [SL], [S]=St/(1+K11[L]) is obtainedA LKB model 4054 UV-Visible spectrometer was used to record the absorption spectra; quartz cuvettes of 1 cm were used with various HBCD concentrations of 5×10As the double reciprocal plot of 1/ΔA versus 1/[L] is linear, the binding constant can be estimated from the following equation:μM) were mixed with constant concentration of RNA (40 μM). The spectra of free baker’s yeast RNA and HBCD-RNA adducts were recorded using a Jasco J-720 spectropolarimeter at pH=7.3. A quartz cell with a path length of 1 cm was used for far-UV region (200–260 nm) measurements. Six scans were accumulated at a scan speed of 50 nm/min, with data being collected at every nanometer from 200 to 260 nm. The temperature of sample was kept at 25°C using a Neslab RTE-111 circulating water bath connected to the water-jacketed quartz cuvette. Using Jasco Standard Analysis software, spectra were corrected for buffer signal, and a conversion to Mol CD (Δε) was performed.Various HBCD concentrations  and free RNA (40 μM), a Dynamic Light Scattering (DLS) particle size analyzer (Brookhaven-model: 90plus USA) was used. A zeta potential meter (Brookhaven-model: ZetaPALS) was used to measure zeta potential of HBCD-RNA complexes, free HBCD (1000 μM) and free RNA (40 μM) at 37°C.To determine polydispersity and the average diameter of HBCD-RNA polymer, free HBCD  and major participation of bases mainly with guanine, cytosine and backbone phosphate groove at higher concentrations. Evidence for this comes from the spectral changes of free RNA upon HBCD complexation.FTIR spectral studies have identified weak interaction of HBCD with phosphate and bases of RNA at low HBCD concentrations (cm−1 (asymmetric PO2 stretch) , no significant interaction was observed between HBCD and RNA bases as represented by small shifting and intensity changes of RNA in-plane vibrations at 1698 , 1650 , 1606 (A), 1489 (C) and 1241 stretch)  and 3. Tbrations . This iscm−1 (1/1); C band at 1489 cm−1 to 1474 (r=1/4) was observed and the intensities of the adenine, guanine, cytosine and uracil bands strongly augmented. The intensity increase was characterizedby the presence of several positive features at 1715 (guanine), 1650 (uracil), 1510, 1490 cm−1(cytosine) and 1245 (phosphate) in the difference spectrum of HBCD-RNA (At higher HBCD concentrations (r=1/4), major shifting of the guanine and cytosine bands (G band at 1698 to 1680 HBCD-RNA . The specm−1 (1/1); cytosine band at 1489 to 1460 (r=1/2), 1477 cm−1 (r=1/1)) were observed. The spectral changes for the bases (r=1/2 and 1/1) can be attributed to the interaction of OH branches of HBCD with mainly G and C and to a lesser extent with A and U bases , drastic shifting of the guanine and cytosine bands (guanine band at 1698 to 1706  U bases  and 3. Scm−1 in the spectra of HBCD-RNA complexes (HBCD-PO2 interaction was evident from intensity increase and shifting of the PO2 antisymmetric band at 1241 and symmetric band at 1085 omplexes  and 3.cm−1 to 1243 (r=1/8), 1245 (r=1/4), 1253 (r=1/2) and 1249 cm−1 (r=1/1) for the complexes can be ascribed to minor phosphate interaction with HBCD at lower concentrations and strong interaction at higher concentrations . Similar spectral changes were observed in PAMAM-tRNA complexes 36.For the phosphate group, shifting of the band at 1241 nm (positive) (41.CD spectra of RNA and its complexes with different HBCD concentrations are shown in ositive)  is consiμM), no significant shifting was observed; however, the molar ellipticity of the band at 209 nm was strongly enhanced by increasing the polymer concentration (500 to 2000 μM). In the spectra of HBCD-RNA complexes, no shifting was observed for the band at 269 nm (cm−1 (ribosephosphate) and in HBCD-RNA complexes at 1689–1688 (G), 1241–1234 (PO2), 864–868 and 812–810 cm−1 (ribose-phosphate) in the complexes  were determined by subtracting the absorbance of the free RNA at 260 nm from those of the complexed. The concentration of free ligand was determined by the subtraction of the complexed ligand from total ligand. The data of 1/[ligand complexed] almost proportionally augmented as a function of 1/[free ligand] (0) vs. 1/(HBCD concentration) was linear, binding constant (K) was calculated from the ratio of the intercept to the slope  was indicative of a weak stability through attraction forces between HBCD and RNA. Similar bindings were reported for G3.0 PAMAM dendrimers conjugated to 1-pyrene carboxaldehyde G3.0-PY (as guest) and HBCD (as host) complexes 42 in which the enhanced λMax of the guest molecule was attributed to the host-guest interactions. Besides, UV-Vis spectrum of RNA at 260 nm , 867–861 (ribosephosphate), 1247–1241 (phosphate), and 1700–1688 (guanine) indicates that RNA remains in a conformation upon HBCD complexation  (nm at pH=7.4 and at 37°C) is in good agreement with previous literature reports , the value of ζ for the supramolecular complex (HBCD-RNA) decreases to −9.22 mV. Decrease in ζ-value can be resulted from the intermolecular forces which led to HBCD-RNA supramolecular assembly.The size distribution profile of small particles in suspension or (macro) molecules in solution can be determined by Dynamic Light Scattering (DLS) mplexes) . The obt reports  28. Afte3) indicated weak HBCD interaction with RNA which easily dissociates in aqueous solution. Strong bonds between RNA and HBCD were formed at higher HBCD concentrations. The small sized synthesized complex (5 nm) successfully prevented RNA aggregation and protected RNA by host-guest interactions between the cavity of HBCD polymers and the bases of gene. No RNA conformational change occurred upon HBCD interaction and RNA remained in A-family structure.An unprecedented type of gene delivery system was successfully developed based on linear dendritic supramolecular biopolymer. RNA-HBCD complexes can be used to gain insight into the new strategies for gene delivery. Our study provided substantial quantitative data on the effect of low and high HBCD concentrations on polynucleotide structure and the binding affinity of RNA to HBCD. According to the spectroscopic results, the following points are considerable: RNA binds HBCD through non-covalent binding (host-guest interactions) between the gene bases as guest and β-cyclodextrine cavities in HBCD as host. The binding constant (K=1.25×10


Highly mutable, infinitely malleable, and all-powerful: this is often the underlying assumption for how spontaneous mutations fuel RNA virus adaptation. Though essential for adaptation, mutations within RNA virus genomes can exact significant fitness costs. Without the capacity to detect and repair mismatched or damaged nucleotides, viral RNA genomes are prone to mutations introduced by mechanisms intrinsic and extrinsic to viral replication. However, large population size, complementation, cellular chaperones, and recombination can buffer viral populations against deleterious and lethal mutations. As such, viral replication is a rapid, tenuous dance between the generation of sufficient genetic diversity on which natural selection can act and the production of less-fit variants. The purpose of this article is not to describe how RNA viruses evolve. Rather, it aims to provide an introduction to some of the mechanisms by which mutations arise during RNA virus replication, as viral mutation rates are the ultimate source of genetic diversity.−6 and 10−4 substitutions per nucleotide per cell infection [Storage and transmission of genetic information depends upon the correct formation of hydrogen bonds between nucleobases. Mutations arise when mismatches are introduced during RNA virus replication or as a result of postreplicative base modification. Host RNA-modifying enzymes and nitration or oxidation of nucleobases can alter hydrogen bonding and increase the probability of point mutations during subsequent rounds of replication. A mutation rate describes the rate (not frequency) at which spontaneous mutations arise during a single infection and reflects both cell- and virus-dependent mechanisms. Because most mutations are likely lethal or deleterious ,2, naturnfection . Such hipol) demonstrate that RdRp replication fidelity is similar to DNA polymerases lacking exoribonuclease (ExoN) activity [One of the main contributors to viral mutation rates is the intrinsic fidelity of the viral RNA-dependent RNA polymerase (RdRp) or retroviral reverse transcriptase (RT). Replication fidelity specifically describes how accurately an RNA or DNA genome is copied relative to the template strand. Cellular DNA replication fidelity can be simplified to three major conceptual steps: (i) nucleotide selection and extension by the polymerase; (ii) removal of mismatched nucleotides by polymerase-associated 3ʹ-to-5ʹ exonuclease (proofreading) activity; and (iii) postreplicative repair of mismatched or damaged nucleotides. RNA viruses lack postreplicative repair, and the key difference between the intrinsic fidelity of the viral RdRp or RT and most replicative cellular DNA polymerases is the lack of associated proofreading activity . Error-pactivity . Hence, activity . Whetherpol provided the first descriptions of high- and low-fidelity RdRp variants [pol variant was isolated following passage in the presence of the antiviral nucleoside analog ribavirin, and resulted in a 3-fold increase in fidelity [pol decreased fidelity by 14-fold and provided early evidence for the existence of a lower limit for replication fidelity [The intrinsic fidelity of viral RdRps is governed by multiple biochemical and biophysical checkpoints, such as conformational changes, mediated by amino acids proximal to and distal from the RdRp active site (reviewed in ). Studievariants ,10. The fidelity . Though fidelity  for a refidelity . RdRps wfidelity . Though fidelity , and mutfidelity ,16. BecaBecause viruses are dependent upon the host cell, replication is inexorably linked to the cellular microenvironment. As such, viral mutation rates reflect multiple events intrinsic and extrinsic to viral replication. Vesicular stomatitis virus (VSV) replicates with a similar mutation rate across multiple mammalian cell lines but has a lower mutation rate during replication in insect cells . CucumbeAltered concentrations of host ribonucleotide triphosphate (rNTP) pools could influence viral mutation rates, as viruses exclusively use host nucleotides. Inhibitors of enzymes critical for de novo purine and pyrimidine biosynthesis, such as mycophenolic acid and brequinar, can exert a potent antiviral effect during RNA virus replication ,21. Low-Both reactive oxygen and nitrogen species  can be generated during viral infection and damage all classes of biomolecules, including nucleobases. Watson-Crick base pairing is predicated on hydrogen bonding, and the oxidation or nitration of cellular nucleobases effectively modulates hydrogen bonding capacity. The resulting nucleobases, such as 8-oxoguanine or 8-nitroguanine, can be mutagenic. Nitric oxide (NO), a physiologically relevant RNS produced during viral infection, is mutagenic during Sendai virus replication but is not antiviral . SimilarHost-encoded protein families such as apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) and adenosine deaminase acting on RNA (ADAR) can modify viral nucleic acid. APOBEC3G is packaged into HIV-1 virions and deaminates cytosine bases to uracil within viral complementary DNA (cDNA) . This reConstant adaptive evolution is essential for RNA viruses to overcome intrinsic, innate, and adaptive host immunity, antiviral therapeutics, and for intra- and inter-host transmission. As the ultimate source of all genetic variation, mutations are key to this adaptive process. Without this phenotypic diversity, adaptation via natural selection cannot occur. Elucidating how spontaneous mutations are generated during viral replication is critical for understanding virus evolution. The capacity to generate RNA viruses with altered fidelity could yield new paradigms for antiviral treatment and live-attenuated vaccine development, assuming sufficient levels of attenuation can be achieved. Most of these studies are based on mutation frequency assays, highlighting the need to determine mutation rates across virus families. Beyond these human health applications, RNA viruses are highly tractable systems that could be used to answer fundamental questions about evolutionary processes in cellular organisms.


There is an urgent need for an improved empirical understanding of the relationship among biodiverse marine resources, human health and development outcomes. Coral reefs are often at this intersection for developing nations in the tropics—an ecosystem targeted for biodiversity conservation and one that provides sustenance and livelihoods for many coastal communities. To explore these relationships, we use the comparative development contexts of Haiti and the Dominican Republic on the island of Hispaniola. We combine child nutrition data from the Demographic Health Survey with coastal proximity and coral reef habitat diversity, and condition to empirically test human benefits of marine natural resources in differing development contexts. Our results indicate that coastal children have a reduced likelihood of severe stunting in Haiti but have increased likelihoods of stunting and reduced dietary diversity in the Dominican Republic. These contrasting results are likely due to the differential in developed infrastructure and market access. Our analyses did not demonstrate an association between more diverse and less degraded coral reefs and better childhood nutrition. The results highlight the complexities of modelling interactions between the health of humans and natural systems, and indicate the next steps needed to support integrated development programming. Coastal ecosystems provide valuable ecosystem services such as protection from storms and beach erosion, habitat grounds for commercially important fish species and other mariculture, and hotspots for marine biodiversity  that shoAn estimated 80% of the world’s fish catch that provides these benefits is provided by the 22 to 30 million people employed in small-scale artisanal fisheries , 5. For Associations between human health and marine ecosystems has led many to attribute a causal relationship between biodiversity and human welfare (e.g. ), but thIn the tropics, coral reefs are particularly diverse and an important source of food and income . An estiAs one of society’s most marginalized groups , 24, smaWe selected the island of Hispaniola, which consists of Haiti and the Dominican Republic, because of the availability of quality population, health and environmental data in two contrasting national contexts. The large coastline of this landmass increases the probability of geographic proximity between population/health data and marine data. While the population size of the two countries is about the same, their socio-economic characteristics differ vastly. Haiti is the poorest country in the Western Hemisphere and amongst the poorest in the developing world . In 2007The state of natural resources in both countries differs considerably–Haiti’s natural terrestrial and marine resources are far more degraded than those of the Dominican Republic. According to the Food and Agriculture Organization , about 9Hispaniola is in a priority ecoregion, the Greater Antillean Marine, which includes the coral reef of Haiti and the Dominican Republic, and is considered one of the 43 priority marine ecoregions crucial to the conservation of global biodiversity . HoweverCoastal resources can play an important role in the nourishment and livelihoods of populations in Hispaniola. In Haiti, coastal resources are the primary source of income and food for an estimated 50,000 fishers and their families ; of thatIntense and poorly regulated fishing, in addition to other factors, are threatening fish stocks; the International Union for the Conservation of Nature has classified 21 fish species as threatened in Haiti and 22 in the Dominican Republic . High-vaThe socioeconomic and biophysical environmental differences described above enable us to explore whether biodiverse marine resources play a vital role in fostering positive child health and nutrition outcomes under differing developmental conditions.In this study, we explore the association between distance to coastline and proxies for marine biodiversity and child health and nutrition outcomes in two countries with different levels of development . HealthyWhile we are not able to address all of the proposed connections in these pathways , this stWe hypothesize that coastal proximity and marine biodiversity will be less important in shaping child health and nutrition outcomes in more advanced development contexts. Developed road and transportation systems, modern modes of agricultural production and fishing can influence food production, commercialization and consumption in ways that facilitate access to fortified foods, imports, and alternative proteins in addition to influencing household wealth. The consumption of fish and other wild resources may become more influenced by the availability and price of substitutes  rather tWe used health and nutritional data from the Haiti DHS 2005–2006 and the Dominican Republic DHS 2007 to evaluate the association between biodiverse marine resources and childhood fish and shellfish consumption, dietary diversity and incidence of severe stunting. Procedures and questionnaires for the DHS surveys have been reviewed and approved by ICF International Institutional Review Board in accordance with the United States Department of Health and Human Service requirements for the “Protection of Human Subjects (45 CFR 46) Any country-specific DHS survey protocols are additionally approved by an ethics review board in the host country. The research complies with the current laws of the country in which they were performed. We did not use more recent datasets to avoid confounding issues caused by the destructive 2010 earthquake and subsequent distribution of aid. DHS surveys are conducted using a two-stage stratified cluster sampling design. The majority of DHS since 2000 are geo-referenced to enable spatial analysis and linking with other geo-referenced datasets. In order to ensure respondents’ confidentiality, all of the GPS coordinates are randomly displaced prior to the public release of the data so that urban clusters are displaced up to 2 kilometers and rural clusters are displaced up to 5 kilometers, with a further 1 percent of rural clusters displaced up to 10 kilometers. To minimize the effect of the displacement of GPS coordinates on our analyses, we include all clusters with a centroid falling within 5km of the coastline which would account for the majority of cases.A total of 9,998 households participated in the 2005–2006 round of the DHS in Haiti . In the We focus on dietary diversity among children aged 6–35 months and severe stunting among children under the age of five years. We include a measure of fish and shellfish consumption as a dependent variable as well due to the direct linkage to marine resource access. The United Nations Children’s Fund uses various indicators for child nutritional status, such as low birth weight, underweight, wasting, and vitamin A intake . We seleData on dietary intake, including fish and shellfish consumption, were collected for most recently born children under 3 years of age. Responses are based on a 24 hour recall period. Our definition of dietary diversity is restricted to children between 6–35 months and parallels that of Johnson et al.  and AlvaAnthropometric data were collected for a subsample of children who are under the age of five. Based on WHO growth standards , childreTo avoid unobserved intra-household correlation in our outcomes of interest, we restrict our study sample to the most recently born child. The sample sizes for the portion of the analyses on dietary diversity are,1,111 children in Haiti and 4,375 children in the Dominican Republic and on stunting are 1,782 for Haiti and 7,204 for the Dominican Republic. The unweighted percent distribution of children across the categorical variables is presented in Table A in This study explores the association between marine resources and child health and nutrition outcomes using three proxy measures: (1) distance to coast; (2) the diversity of coral reef geomorphology (habitat); and (3) a coral reef threat index near the index child’s community. Distance to coastline provides a measure of access to marine resources in general. For an initial assessment of the role of biodiversity, we use the number of different reef habitats as our proxy of coral reef diversity, as in , and useTo determine a cluster’s access to the coastline and coral reefs, distances were calculated from the displaced cluster locations to the closest segment of coastline and the closest coral reef. GPS clusters were assigned a coastal tangent location at the closest point on the coastline from the displaced cluster location according to the U.S. Department of State's Office of the Geographer (INR/GGI)—Small Scale International Boundaries (SSIB). The distances are classified into categories in order to reduce the error introduced by the GPS displacement. Of the 332 survey clusters in Haiti, about 50 percent are within 5 km of the coast line in contrast to the Dominican Republic where 25 percent of the 1,425 sampled clusters are within 5 km of the coastline  , the mosCoral reef geomorphology was classified into the following categories: barrier island, barrier continental, barrier atoll-bank, fringing island, fringing continental, patch island, patch continental, patch atoll-bank, shelf island and shelf continental. From the reef geomorphology maps, we created a habitat diversity index ranging from 0–3 such that zero corresponds to coastline with no coral reef; 1 corresponds to the least diverse reef (1 type); 2 corresponds to moderately diverse reef (2 types) and 3 corresponds to the most diverse reef (3 or 4 types). Landsat 7 images, used to delineate the reef and classify the geomorphology, were collected in 2001–2002. While dredging and other destructive practices can significantly alter the reef geomorphology used here to define habitat types, geomorphology was not likely to significantly change between image acquisition and the DHS surveys .We use the World Resources Institute’s coral reef threat indicator as a proxy for the degree of coral reef degradation . The corChildhood health and nutrition is influenced by a number of individual , household (size and composition), and community-level (e.g. water and sanitation) factors , 67. BecThe extent of growth faltering may vary by age, and the nutrition of children during the first two years is critical for their long-term cognitive and physical development. Additionally, nutritional outcomes such as stunting, which reflect chronic or long-term malnutrition, may be more prevalent among older children. Education and income are likely to determine where households (re)locate. Educated and wealthier families possess both the knowledge and resources to move families closer to locations that have better options for both sustenance and livelihoods. Although households are likely to purchase food in daily and weekly local markets, access to a mode of transport should facilitate access to food not available in the immediate vicinity of where households are located. Urban areas are more likely than rural areas to offer alternative food crops and animal protein, as well as livelihoods other than fishing. Additionally, larger communities are also more likely to offer alternatives to fish consumption. DHS urban and rural classifications are both country and survey specific. To standardize the classification of clusters as urban or rural between the two countries, Landscan 2011 data was used to determine the surrounding population counts and population density. Clusters were buffered by the maximum potential displacement distance  to ensure that the buffered area was certain to contain the true original cluster location. The buffering also accounts for the error in the Landscan data, including the slight shift of the raster due to the applied projection. Clusters were reclassified according to their mean population density.β0 is the intercept; βDistance is the estimated effect of distance to coastline on the outcome of interest of child i; βcoralreef is the estimated effect of coral reef health on the outcome of interest of child i; and Because our outcome variables are dichotomous, we use a multivariate logistic regression model to estimate the association between coastal proximity, coral reef diversity and coral reef degradation. We model these associations independently for each country because the DHS surveys were independent and because of the distinct socioeconomic and biophysical country contexts. Thus, we focus on comparisons of the relative benefits of marine resources. The model specification is as follows:We include the variables sequentially into the analysis, building progressively complex models. The first model includes distance to coastline, the second incorporates coral reef diversity and the third through sixth models include household level characteristics that are likely to have a confounding effect . We do not include coral reef diversity and coral reef threat in the same model because of overlapping categories between the two . We are unable to include an interaction of coral reef diversity and threat because this would result in a loss of statistical power due to few cases , in contrast to the Dominican Republic where the comparable group of children have the highest levels of dietary diversity  nor with reef threat   are about 40 percent In both countries, coral reef threat is not a statistically significant predictor of child dietary diversity (Tables E and F in We explore associations with fish and shellfish consumption (hereto referred to as fish consumption), as it is a dietary factor that might connect access to coastal resources and coral reefs with human health and nutrition. In Haiti, children at all distances greater than 5 km from the coastline were over 50% less likely to have consumed fish (p < 0.05) , whereasTables The results for coral reef habitat diversity run counter to our expectations that more diverse coral reef are associated with better child health outcomes. Haitian children near very diverse (3 or 4 types) coral reef habitat are about 4.6 times more likely to be severely stunted than children living in communities in which reef habitat is the least diverse (1 type of reef) p<0.01, . HoweverIn contrast to Haiti, the association between distance to coastline and the likelihood of stunting is negative in the Dominican Republic. Net of reef habitat diversity and confounding variables  inclusion of household wealth Click here for additional data file.