Human TATA binding protein associated factor 2 N (TAF15) and Fused in sarcoma (FUS) are nucleic acid binding proteins belonging to the conserved FET family of proteins. They are involved in diverse processes such as pre-mRNA splicing, mRNA transport, and DNA binding. The absence of information regarding the structural mechanism employed by the FET family in recognizing and discriminating their cognate and non-cognate RNA targets has hampered the attainment of consensus on modes of protein-RNA binding for this family. Our study provides a molecular basis of this RNA recognition using a combination of solution-state NMR spectroscopy, calorimetry, docking and molecular dynamics simulation. Analysis of TAF15-RRM solution structure and its binding with stem-loop RNA has yielded conclusive evidence of a non-canonical mode of RNA recognition. Rather than classical stacking interactions that occur across nitrogen bases and aromatic amino acids on ribonucleoprotein sites, moderate-affinity hydrogen bonding network between the nitrogen bases in the stem-loop RNA and a concave face on the RRM surface primarily mediate TAF15-RRM RNA interaction. We have compared the binding affinities across a set of single-stranded RNA oligonucleotides to conclusively establish that RNA binding is dependent upon structural elements in the RNA rather than sequence. Human TATA binding protein associated factor 2N TAF15/TAF2N), also known as RNA binding protein 56 (RBP56), belongs to a conserved FET family of proteins. Other members of this protein family include EWS (Ewing’s sarcoma) and FUS/TLS (fused in sarcoma/translocated in liposarcoma) proteins5/TAF2N, 34TAF15 is normally found associated with RNA polymerase II in transcription pre-initiation complexes13144 RanBP2 type Zinc finger (ZnF) with interspersed unstructured elements rich in arginine and glycine, known as RGG motifs. The RRM exhibits highest degree of sequence conservation among the members of the FET family.Architecturally, TAF15 is composed of an intrinsically unstructured N-terminal QGSY rich activation domain which is retained in the oncogenic fusions and possesses trans-activating properties16β-tropomyosin pre-mRNA with its C-terminal region having high affinities to GGUG sequence motifs20222311FUS and TAF15 are functionally more related to each other than EWSSON  stem-loop cluster with higher affinities than that for RNA with GGU repeatTAF15, relative to its counterpart, is less characterized. TAF15 regulates gene expression of cell cycle regulatory genes through a pathway involving miRNAsThough there is plethora of literature pertaining to the functional roles of FET protein family and their implications in various disease conditions, a thorough biophysical or structural characterization is still lacking. This can be possibly attributed to its high intrinsic disorder making it not easily amenable for such studies. So far, only the RRM domain of FUS has been structurally characterized15N-{1H} Het-NOE measurements. 15N-{1H} Het-NOE values, corresponding to an average of ~0.75 in the RRM and ZnF domains, and ~0.38 in the RGG linker, indicated the presence of highly dynamic linker between the structured RRM and the RanBP2 type ZnF domain in the multi-domain protein  consisting of four anti-parallel β-strands and two α-helices arranged as an α/β sandwich. T235-Q239 forms the β1, I271-T275 forms the β2, P282-S289 forms the β3 and K314-F317 forms the β4 strands. β1 and β2 strands are connected by α1-helix (T247-Q256) while β3 and β4 strands are connected by α2-helix (P293-F303). The structure contains several loops; loop L1 (G240-S246) between β1 and α1, loop L2 (I257-I271) between α1 and β2, loop L3 (D276-K281) between β2 and β3, loop L4 (F290-P293) between β3 and α2 and loop L5 (D304-I313) between α2 and β4. The two highly conserved ribonucleoprotein (RNP) sequence motifs, namely RNP2 (I236-L241) and RNP1 (K283-F290); are located on β1 strand and proximal part of β1-α1 loop, and on β3 strand and distal part of β2-β3 loop, respectively.An ensemble of 20 lowest energy minimized structures had a global backbone RMSD of 0.8 Å and an all atom RMSD of 1.2 Å . Furtherα1–β2 loop, which is much less predominant in case of other RRMs . The absence of significant number of surface exposed aromatic side chains in the β1 and β3 strands and the presence of an extended α1-β2 loop in all the members of FET protein family  cluster24SON stem-loop RNA recognition is still unknown. To determine the RNA binding regions, different constructs of TAF15 and FUS expressing the RRM alone  and RRM along with the disordered RGG motif and RanBP2 type zinc finger  were analyzed for binding using ITC and solution-state NMR spectroscopy.RNA targets for the FET family of proteins have been identified recently, which highlight the importance of RNA secondary structure in recognition, specifically the AU-rich stem loops and the SON stem-loop RNA was confirmed by measuring the 1D 1H spectrum in water, which showed 8 imino proton resonances between 10.9 and 14 ppm indicating eight hydrogen bonded nucleotide pairs, corresponding to the stem region.Structural integrity of the Kd = 10 μM), TAF15-RRM-RanBP2 (Kd = 6 μM), FUS-RRM (Kd (avg) = 11 μM) and FUS-RRM-RanBP2 (Kd = 8 μM) with the SON stem-loop RNA . Upon titrating TAF15-RRM against the rest . Signifithe rest . The resthe rest .SON stem-loop RNA  could be identified for several hydrogen-bonded bases. Assignments were confirmed using the increased resolution offered by 2D  HSQC and 2D  HSQC spectra. Resonances arising from loop protons were highly overlapped and exhibited broad resonance lines in both free and bond state. These resonances were left unassigned after assignment of H6, H8, H2, H5 and H1′ resonances of bases in the stem belongs to loop region.Partial sequence-specific resonance assignments of sonances . SubsequSON resonances in bound and unbound states. Highest perturbations were observed for H1′-H2′ cross-peaks of loop resonances , β2 strand and the proximal part of β2-β3 loop (loop L3). This region appears to forms the primary binding interface, mainly because, all the residues that exhibit very strong CSPs (averaged CSP + 2σ) are confined to this region indicating a much larger role played by these loops and β2 strand in recognizing the RNA. Interestingly, all the threonines that showed maximum CSPs are part of this region, indicating that they play a crucial role in recognition through possible extensive H-bonding with the SON stem-loop RNA.Residues of TAF15-RRM showing significant CSPs upon interacting with the nterface . Interesβ1 and β3 strands respectively 3435β1 strand, RNP2) and A286-T287 , it looks like the two RNP sites in TAF15-RRM play a less prominent or no role in SON stem-loop RNA recognition possibly due to the absence of critical aromatic residues. The N- and C-termini comprising the residues N233-N234 and R320-R321 showing significant CSPs also appear to contribute to binding. The significant CSP of Cδ/Hδ and Cε/Hε resonances of surface exposed aromatic side-chains of Y274 on β2 strand and F237 on β1strand  using the experimentally derived CSP information that provided initial clues as to the role of loops L2 and L3 as well as a portion of the β2 strand in RNA recognition. The lowest energy docked structure after a 10 ns molecular dynamics simulation showed a 591 Å2 net surface area of the RNA loop (A11-A14) stably docked into a 564 Å2 positively charged cavity formed by K264, K265 and K268 on loop L2, K277, K281, K283 on loop L3, β2 and β3 strands as well as R320 and R321 on the carboxy terminus  of the L2 loop and a larger, outward movement of L3 (6.0 Å), along with minor rearrangements of F237 and Y274 aromatic rings and the α2 helix , U13 (76 Å2) and A14 (104 Å2) exhibited high solvent inaccessibility. Protein residues that underwent major dehydration were F237 (46 Å2), T266 (36 Å2), K268 (48 Å2), Y274 (57 Å2), K277 (89 Å2), K283 (40 Å2) and T319 (82 Å2). Despite the robust nature of this interface, loops L2 and L3, along with A11-C15 of the RNA showed relatively high root mean squared fluctuations (RMSF) in the simulation  was analyzed for binding to TAF15-RRM using CSPs from solution-state NMR spectroscopy . Only fe-β3 loop . This isα1-β2 loop (Loop L2) of TAF15-RRM remained unperturbed, which was showing significant CSPs in FUS-RRM upon binding to GGUG containing RNAThe RRM domains of TAF15 and FUS are highly similar, with a sequence similarity of >95% and a RMS deviation of ~0.6 Å between both the structures. Therefore, it is logical to assume similar RNA targets and similar binding preferences for both the RRMs of TAF15 and FUS, based on the aforementioned similarities and broadly considering the non-specific nature of RRM-RNA interactions. However, most of reported residues and regions in FUS-RRM  binding to GGUG containing 12-mer RNA showed no binding in TAF15-RRM against GGUG containing 6-mer RNA. Further, the unique expanded in-vivo RNA binding sites of TAF15 and a battery of best scoring RNA targets of different lengths reveals a recurring CUG motifβ2-β3 loop (Y274-T275)  . Similar74-T275) . To asce74-T275) . These rSON stem-loop RNA and several CUG and GGUG containing linear ssRNA oligomers. Titrations of RNA against the RRM in solution have enabled us to narrow down to the SON stem-loop RNA as the most likely binding partner of TAF15 and FUS, given that other RNAs show little or no association in vitro. Also, we show that the SON stem-loop RNA recognition is predominantly mediated by the RRM with little or no influence by the downstream RGG and ZnF domains. Structural analysis of NMR structure of TAF15-RRM alone and its experimentally derived docked model with SON RNA has also provided sufficient evidence of a largely hydrogen-bonding mediated interface, which is in contrast to canonical RRM-RNA interactions that mainly involve amino acid-nitrogen base π-π stacking. While our docked model has provided crucial input regarding the non-canonical nature of RNA binding by this RRM, the mechanistic insights into base recognition and discrimination still remains elusive. This study, therefore, provides a platform for understanding mechanism of RNA recognition particularly by TAF15, and in general the FET family of proteins, which is likely to aid in understanding the molecular basis of neuromuscular disease progression.RNA-binding specificities of the FET family of protein have ostensibly, been widely spread out over several sequence and secondary structure motifs as evidenced by past studies. Our study provides a biophysical basis to this observation, centered on the interaction of RRM domain of TAF15 with http://dnasu.org/DNASU/Home.do)The c-DNA clones of full length human TAF15 and FUS were obtained from DNASU plasmid repository  measured on 1 mM U-13C,15N-labeled TAF15-RRM in 20 mM sodium phosphate buffer pH 6.2, 50 mM NaCl, 5% D2O (v/v), 0.02% NaN3 (w/v) were used for structure calculation. Unlabeled SON stem-loop RNA was used at a concentration of 1.5 mM for homonuclear and heteronuclear experiments. For NMR titration experiments, the corresponding unlabeled RNAs were titrated against the 0.5 mM U-15N-labeled proteins at increasing molar equivalents of the RNAs until no further changes in the 2D  HSQC spectrum were observed, signifying saturation. The final NMR samples contained 1:1.2 stoichiometric complexes of 0.5 or 1 mM U-15N or U-13C,15N-labeled TAF15-RRM/FUS-RRM and corresponding unlabeled RNA in 20 mM sodium phosphate buffer pH 6.2, 25 mM NaCl, 5% D2O (v/v), and 0.5 or 1 mM U-15N or U-13C,15N or U-2H,13C,15N-labeled TAF15-RRM-RANBP2 and corresponding unlabeled RNA in 20 mM sodium phosphate buffer pH 6.2, 25 mM Na2SO4 and 5 mM β-mercaptoethanol. Buffers were prepared in DEPC-treated water. The NH backbone resonances of TAF15 in complex were unambiguously assigned using 3D HNCA, 3D HNCACB, 3D CBCAcoNH and 3D HNCO spectraSON stem-loop RNA were carried out at 273 K and subsequently at 298 K using 2D  NOESY  in H2O and D2O, 2D  TOCSY (tmix = 50 ms) in D2O, 2D  HSQC in H2O and 2D  HSQC spectra in D2O at natural abundance. Two separate 2D  HSQC spectra were measured with frequency offsets and 1JHC coupling constants optimized for base and sugar HC resonances, respectively. All NMR spectra were measured on Bruker Avance III spectrometers equipped with 5 mm cryogenic triple resonance TCI probes, operating at field strengths of 500 and 700 MHz. All spectra were referenced to DSS15N,1H] HSQC spectra using the following equationThe 3D δ(1HN) and Δδ(15N) are the changes in backbone amide chemical shifts for 1HN and 15N respectively upon RNA binding. For determining regions of the RNA involved in interaction with TAF15-RRM, 2D 13C--filtered  NOESY (tmix = 350 ms) spectrum in D2O and 2D  NOESY  spectra in H2O and D2O were acquired at 298 K for SON stem-loop RNA in complex with U-13C,15N TAF15-RRM in a 1:1 stoichiometric ratio. 2D  NOESY  spectra of the 15N labeled TAF15-RRM protein in D2O were also measured as a reference.where Δ200  calorimeter. TAF15-RRM and FUS-RRM were prepared in 10 mM HEPES pH 7.0, 20 mM Na2SO4 while TAF15-RRM-RanBP2 and FUS-RRM-RanBP2 were prepared in 10 mM HEPES pH 7.0, 20 mM Na2SO4 and 5 mM β-mercaptoethanol. Use of DEPC treated double distilled water and buffers eliminated RNase contamination. 10-fold higher concentrations (1 mM in DEPC treated water) of various RNAs were titrated against 100 μM aforementioned protein constructs in the cell chamber with a stirring speed of 1000 rpm and 1 μl or 2 μl of regularly spaced (150 sec) sequential injections. The heat of dilution control (i.e. titration of RNA into buffer) was subtracted from the integrated peak area and the binding constant (Ka), molar binding stoichiometry (N), molar binding entropy change (ΔS), and molar binding enthalpy change (ΔH) were determined directly from the fitted curve. Curve fitting and data analysis were carried out using the software Origin 7 supplied with the calorimeter.All ITC experiments were carried out at 298 K on a Microcal iTC47. UNIO’10 protocol was used wherein seven cycles of NOE assignments and structure calculations were iteratively performed which resulted in gradual reduction of target function scoresφ and ψ) restraints were obtained from the backbone and 13Cβ chemical-shift values using the program TALOS-N15N-{1H} nuclear Overhauser enhancements (NOEs) ratios were calculated using signal intensities from NOE and reference spectra (INOE/Iref).We previously reported the sequence-specific resonance assignments of TAF15-RRM and TAF15-RRM-RanBP2SON stem-loop RNA were generated ab initio using Assemble2+ and Cl- ions up to a concentration of 150 mM. The AMBER99SB-ILDNPreliminary coordinates of http://haddock.science.uu.nl/services/HADDOCK/haddock.php) using CSP information derived from NMR titration experiments. ‘Active residues’ were defined as those exhibiting perturbations greater than one standard deviation from the mean and ‘passive residues’ as amino acids exposed more than 50% to solvent in the vicinity of active residues. Given that existing structures of RRM-stem-loop RNA complexes involve binding to the RNA loop region , bases of the SON loop (A11-A14) were defined as active residues with the passive residues being left as default. The lowest energy cluster representative was chosen as the docked model.Experimentally-restrained docking of the modeled RNA on to TAF15-RRM was carried out on the High Ambiguity Driven Protein-Protein Docking (HADDOCK) web serverhttp://www.ebi.ac.uk/pdbe/pisa/).Since RNA coordinates used for docking in HADDOCK assume a largely rigid backbone, we performed simulation MD runs on the representative complex structure in GROMACS. Using the AMBER99SB-ILDN force field, the complex was solvated, neutralized and minimized in a cubic box (8.7 × 8.7 × 8.7 nmAccession codes: Coordinate of the Human TATA binding protein associated factor 2 N (TAF15) has been deposited in the Protein Data Bank (PDB) under the accession code 2MMY. The sequence-specific resonance assignments of TAF15-RRM and TAF15-RRM-RanBP2 have been deposited in the BioMagResBank database (www.bmrb.wisc.edu) with the accession numbers, 19320 and 19561 respectively.How to cite this article: Kashyap, M. et al. Structural delineation of stem-loop RNA binding by human TAF15 protein. Sci. Rep. 5, 17298; doi: 10.1038/srep17298 (2015).


Structured portions within an mRNA transcript typically interact with specific ribosomal proteins to prevent expression of the entire operon, thus balancing levels of ribosomal proteins across transcriptional units. Three distinct RNA structures from different bacterial phyla have demonstrated interactions with S15 to regulate gene expression; however, these RNAs are distributed across a small fraction of bacterial diversity.Autogenous Rhizobium radiobacter specifically interacts with S15 in vitro, and allows in vivo regulation of gene expression in an E. coli reporter system. In addition, structural probing and nuclease protection assays confirm the predicted secondary structure and indicate nucleotides required for protein interaction.We used comparative genomics in combination with analysis of existing transcriptomic data to identify three novel putative RNA structures associated with the S15 coding region in microbial genomes. These structures are completely distinct from those previously published and encompass potential regulatory regions including ribosome-binding sites. To validate the biological relevance of our findings, we demonstrate that an example of the Alphaproteobacterial RNA from de novo ncRNA identification and reveals a diversity of distinct natural RNA regulators that support analogous biological functions. Furthermore, this work indicates that many additional uncharacterized RNA regulators likely exist within bacterial genomes and that the plasticity of RNA structure allows unique, and likely independently derived, solutions to the same biological problem.This work illustrates the importance of integrating comparative genomic and transcriptomic approaches during The online version of this article (doi:10.1186/1471-2164-15-657) contains supplementary material, which is available to authorized users. Over the last two decades numerous RNA regulators have been discovered that range in function from directing development in eukaryotes , to contS-adenosyl methionine (SAM)-binding riboswitch architectures )/([S15] + KD) where [S15] corresponds to the concentration of S15 in the reaction. The residuals were minimized using the Solver function in Microsoft Excel to find both the Max% and the KD. KD values given in the text represent the mean of 3 or more independent binding assays ± the standard deviation.RNA binding capability was examined by filter-binding assay (FBA). A fixed amount of 5′-labeled RNA  was renatured for 15 minutes at 42°C. The nuclease was inactivated with inactivation/precipitation buffer (Life Sciences) and RNA fragments recovered by ethanol precipitation. Precipitated RNAs were suspended in 10 uL Urea Loading solution (Life Sciences) and incubated for 5 minutes at 95°C. Five uL of each reaction was loaded on 10% denaturing Acrylamide/Bis-acrylamide gel. The gel was dried and examined using a GE Healthcare STORM 820 phosphorimager and ImageQuant software. Partial hydroxyl cleavage reactions were generated by incubating RNA in Reaction Buffer  at 95°C for 7 minutes. Denaturing T1 reaction was conducted according to manufacture′s protocol (Ambion). For in-line probing, 5′-labeled RNA was incubated for 40 hours at 25°C in Reaction Buffer . The reaction was stopped using Urea loading solution .The RNA-protein binding reaction described above was used for RNAse probing assays. After incubation, 1 uL RNAse A  or RNAse VI  was added and the reaction incubated for 15 minutes at 25lac promoter was replaced with a trc promoter. The protein expression plasmid was constructed by amplifying the DNA fragment encoding the rpsO gene containing a SacI restriction site and a ribosome-binding site on the 5′ terminus (5′-caagagctcaggaggttttaaaatgtcgattactgcagagcgcaaag) and XbaI site on the 3′ terminus (5′- caatctagattagcggcgaatgccgagagc) from genomic DNA extracted from R. radiobacter (ATCC 23308). The PCR product was digested with SacI and XbaI enzymes and inserted into the pBAD33 expression vector (ATCC 87402) digested with the same enzymes.The ptrc-RNA-GFP plasmid was constructed from pLac-thiMwt-tetA-gfpuv plasmid  as outlirpsO E. coli cells  were co-transformed with an RNA and protein plasmid . Overnight cultures were grown +/- L-arabinose (15 mM), then diluted the next day to OD600 = 0.150 in fresh media (LB + 100 ug/mL AMP + 34 ug/mL CHL +/- 15 mM L-arabinose). At log phase, IPTG  was added to induce GFP expression and cells were grown an additional 5 hours. Cells were collected, washed with PBS, then stored in PBS overnight. GFP expression was measured using a SpectraMax M5 fluorimeter . Fluorescence was calculated by normalizing GFP to cell density (GFP/OD600).K12: Δhttps://github.com/jsa-aerial/gaisr. Sequence alignments corresponding to the structured RNAs in Figure http://snrnasm.bio.unc.edu.The software described in Figure Alignment of Alphaproteobacterial RNA.(TXT 86 KB)Additional file 1:Alignment of Actinobacterial RNA.(TXT 54 KB)Additional file 2:Alignment of Chlamydia RNA.(TXT 5 KB)Additional file 3:Supplementary Figures and Methods.(PDF 272 KB)Additional file 4:


The 3′ untranslated region (3′UTR) of human astroviruses (HAstV) consists of two hairpin structures (helix I and II) joined by a linker harboring a conserved PTB/hnRNP1 binding site. The identification and characterization of cellular proteins that interact with the 3′UTR of HAstV-8 virus will help to uncover cellular requirements for viral functions. To this end, mobility shift assays and UV cross-linking were performed with uninfected and HAstV-8-infected cell extracts and HAstV-8 3′UTR probes. Two RNA-protein complexes (CI and CII) were recruited into the 3′UTR. Complex CII formation was compromised with cold homologous RNA, and seven proteins of 35, 40, 45, 50, 52, 57/60 and 75 kDa were cross-linked to the 3′UTR. Supermobility shift assays indicated that PTB/hnRNP1 is part of this complex, and 3′UTR-crosslinked PTB/hnRNP1 was immunoprecipitated from HAstV-8 infected cell-membrane extracts. Also, immunofluorescence analyses revealed that PTB/hnRNP1 is distributed in the nucleus and cytoplasm of uninfected cells, but it is mainly localized perinuclearly in the cytoplasm of HAstV-8 infected cells. Furthermore, the minimal 3′UTR sequences recognized by recombinant PTB are those conforming helix I, and an intact PTB/hnRNP1-binding site. Finally, small interfering RNA-mediated PTB/hnRNP1 silencing reduced synthesis viral genome and virus yield in CaCo2 cells, suggesting that PTB/hnRNP1 is required for HAstV replication. In conclusion, PTB/hnRNP1 binds to the 3′UTR HAstV-8 and is required or participates in viral replication. Astroviridae family, consisting of non-enveloped viruses whose plus-sense, single-stranded polyadenylated genome of approximately 7 kb, is organized in three open reading frames (ORFs) ORF1a, ORF1b and ORF2 Human astroviruses (HAstVs) are a major cause of acute gastroenteritis in children constituting an important public health problem. The astroviruses belong to the Viral 3′UTRs participate in the interaction with host-cell and viral proteins, and may interact with other parts of the viral genome In other single-stranded positive RNA viruses, the interaction between the UTRs and cellular factors such as Polypyrimidine Tract Binding protein , several heterogeneous nuclear ribonucleoproteins (hnRNPs), La autoantigen (La), poly (A) binding protein (PABP), poly-r (C)-binding proteins (PCBP1/2) also known as hnRNPE2, cellular protein eukaryotic elongation factor 1A (eEF1A) and Nucleolin, might be involved in viral replication The 3′UTR of all HAstV serotypes is highly conserved. As predicted in silico, the RNA structures these 3′UTRs are conformed by two hairpins joined by a single stranded linker, and a putative PTB binding site located either within or next to the linker. This structural conservation suggests that the 3′UTR might be involved in the regulation of viral activities, such as replication. To explore the role of the HAstV-8 3′UTR RNA in the regulation of viral functions, we studied the interaction of human CaCo2 cellular factors with RNA probes of this region with or without the PTB binding site. Mobility shift assays evidenced the formation of two RNA-protein complexes, CI and CII, and the supershift of complex CII by PTB antibodies. UV cross-linking assays showed that seven proteins of 35, 40/45, 50, 52, 57/60 and 75 kDa were directly bound to this 3′UTR, and immunoprecipitation assays identified PTB in the 57/60-kDa bands. Also, immunofluorescence analysis showed perinuclear PTB signals localized mainly in the cytoplasm of HAstV-8 infected cells. Moreover, recombinant PTB was cross-linked to 3′UTR probes containing intact helix I, and PTB binding sites. As expected, siRNA-mediated PTB knockdown in HAstV-8 infected CaCo2 cells showed reduced RNA viral genome synthesis and viral yield. Our results suggest that PTB binds to the cognate UCUU site, localized in the linker of the 3′UTR of HAstV-8, that helix I is also required for such binding, and that it is required and participates in viral replication.2 in Advanced-Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 5% fetal calf serum (FCS) , antibiotics (penicillin and streptomycin) and 2 mM L-Glutamine (Invitrogen). CaCo2 cells were infected with HAstV-8 strain Yuc 8 . The Infections and the virus were obtained as previously described Monolayers of CaCo2 cells  were grown at 37°C, 5% COCytoplasmic S10 fractions or 1% NP-40 cytoplasmic membrane extracts were obtained from uninfected and HAstV-8-infected CaCo2 cells as previously described Thirty or sixty micrograms of cell extracts were monitored by western-blotting. To rule out cross-contamination between cytoplasmic, membrane and nuclear fractions, a monoclonal anti-Actin (Millipore), polyclonal anti-Lamin A and polyclonal anti-hnRNP1 (Santa Cruz Biotechcnology) were used. PTB was monitored with a polyclonal anti-hnRNP1 and monoclonal anti-carboxy-terminus PTB (Invitrogen). The cytoplasmic membrane-associated proteins were monitored with polyclonal anti-PDI . Proteins were developed by chemiluniscense following the manufacturer’s instructions .TAATACGACTCACTATAGGGGAAGGAGGGTACAGCTTCCTATCCTCGAATTC)/HI/mPTB-rev (GAATTCGAGGATACGAAGTGTACCCTCCTTC). These products were used as templates to amplify the PCR templates RNA synthesis, including the full-length 3′UTR and mPTB , as well as the 32 bp-long HI  oligonucleotides. To maintain the same structure of the wt probe, nucleotides T6676C6677 were substituted for AG in the HI/mPTB template. The T7 RNA polymerase promoter (underlined) was included in all forward oligonucleotides used. DNA duplexes were visualized on 8% native acrylamide gel before use for PCR amplification. PCR reactions were done in presence of 10 µM of DNA duplex for full-length 3′UTR, HI/mPTB, and mPTB, whereas HI used the full-length 3′UTR as a template. The PCR mix contained 10 µM of each oligonucleotide, 10 mM dNTPs (Roche), 25 mM MgCl2, and 2 U of Dream Taq polymerase in the appropriate buffer (Fermentas). Incubation times and temperatures were: 2 min at 94°C, followed by 35 cycles of 1 min at 94°C denaturation, annealing for 1 min at 52°C and extension 1 min at 72°C, and a final extension at 72°C for 5 min. The same conditions were used for the unrelated RNA (REST) control template with the primers reported elsewhere Oligonucleotides  were used amplify by PCR the full-length 3′UTR and mPTB templates, and their short versions HI and HI/mPTB. DNA templates for the full length-3′UTR, mPTB and HI/mPTB were generated by DNA duplexes according to the protocol recommended by Sigma-Aldrich, using the respective primer sets HAstV-8-F/HAstV-8-R , mPTB-fwd/mPTB-rev (UCUU PTB binding site within nt 6700 to 6704 changed to CGAA), and HI/mPTB-fwd (32P (NEN-Dupont)-labeled RNA transcripts with RiboProbe T7 Transcription System (Promega) following the conditions described by the manufacturer. One unit of DNase I- RNase-Free (Promega) was added for 30 min at 37°C and transcripts were purified with NucAway columns (Ambion) as recommended by the manufacturer. The RNA transcripts were measured in a Beckmann scintillation counter and stored at −20°C until used. The unlabeled probes were synthesized with MegaShort-Script T7 Transcription kit (Ambion), as recommended by the manufacturer.DNA templates were used to synthesize UTP-4 cpm) was added and the mixture was incubated at 4°C for 15 min to allow complex formation. Gels were dried and autoradiographed. For the competition assays, unlabeled RNAs were included in the preincubation reaction. For super mobility shift assays, polyclonal antibodies to hnRNP1 and goat-IgG anti-rabbit-HRP (Zymed) non-related antibodies were preincubated for 15 min in the binding reaction after the addition of labeled RNA.Five and ten micrograms of S10 extracts from uninfected and HAstV-8 infected CaCo2 cells were use for mobility shift assays following protocols previously described 32P-labeled RNA probe (1×105 cpm) was incubated at 4°C in crosslink binding buffer  with S10 extracts (20 µg) from the uninfected and HAstV-8 infected CaCo2 cells or purified recombinant PTB (0.50 and 1 µg). RNA-protein complexes were irradiated with UV light (254 nm) for 20 min on ice and the unbound RNA was digested with RNase A and RNase T1  for 30 min at 37°C. Samples were analyzed by 10% SDS-PAGE. Gels were dried and autoradiographed. UV crosslinking- immunoprecipitations were carried out as described previously The UV crosslink assays were carried out using protocols previously described CATCTCTGCCCCCTCTGCTGA) and GAPDH-R (GGATGACCTTGCCCACAGCCT). Incubation times and temperatures were: 10 min at 95°C, followed by 30 cycles at 95°C for 30 sec, 50°C for 30 sec and 72°C for 30 sec. The reaction was performed three times for each sample using a Rotor Gene 6000 (Corbett Research). The data were analyzed with the comparative threshold cycle (2-ΔΔCT) method. The virus titer was reported as focus-forming units/ml t test was used to compare between two treatment groups. The P-values<0.005 were considered as statically significant. All statical analyses were done using GraphPad Prism 6 .For siRNA-mediated knockdown of PTB expression, CaCo2 cells were transfected using the Nucleofector device and solution T, as recommended by the manufacturer . For each siRNA, the optimal concentrations were determined experimentally and all were obtained from Ambion. Mock-transfected cells were treated with transfection reagent only, and a nontarget siScrambled (160 nM) was used as a negative control. The concentrations used for siPTB were 2.5 µM and 5 µM. 72 h post- transfection, the cells were infected with HAstV8, and samples were harvested for RNA isolation, western-blotting and virus titer determination at 15 h p.i. For RNA and Proteins were isolated with AllPrepRNA/Protein kit (Qiagen) as recommended by the manufacturer. Proteins were quantitated with 660 nm Protein Assay Reagent (Pierce) following manufacturer’s instructions and cellular and viral protein expressions were determined by western-blotting with anti-hnRNP1, viral RdRp anti-1b-2  as previously described 5) were seeded in an 8-well chamber slide plate  and grown at 37°C, 5% CO2, 72 h. The anti-CQFG antibody, which recognizes the VP34 capsid protein , was used (1∶100 dilution) to monitor HAstV8 infection of CaCo2 cells. PTB silencing was monitored with the anti-hnRNP1 antibody. For immunofluorescence, cells were washed in PBS- 50 mM NH4Cl, incubated with secondary antibodies anti-rabbit coupled to Alexa-Fluor 594 at 1∶700 dilution (Invitrogen), Fluorescein (FITC)-conjugated donkey anti-goat IgG at 1∶75 dilution (Jackson ImmunoResearch), respectively 1 h at room temperature. Finally, the cells were incubated with Vectashield with DAPI mounting media , and visualized under lasser confocal microscopes LSM710 (Carl Zeiss) and Olympus Fv300.Immunofluorescence assays were carried out as previously described The secondary structure modeling of the full-length 3′UTR sequences of HAstV-1 to −8  were carried out with the RNA Mfold software release (version 3.2) The integrity of the secondary structure of the 3′UTR facilitates the recruitment of cellular and viral factors to the VRC and is also involved in viral viability, RNA stability, translation initiation, or intracellular localization Cellular proteins bound to the 3′UTR in positive-strand RNA viruses, play important roles in assembling the viral RNA replication complex, selecting and recruiting viral RNA replication templates to perform the synthesis of the minus-strand RNA When labeled HAstV-8 3′UTR (nt 6674 to 6759) was UV cross-linked to uninfected and infected CaCo2 cell extracts we detected that seven proteins of 35, 40, 45, 50, 52, 57/60 and 75 kDa from both cell extracts were bound to the probe. However, stronger signals of individual proteins of infected cell extracts were observed . As expeSupershift assays were conducted to confirm the identity of the 57/60 kDa UV cross-linked protein . IncubatTo confirm that PTB/hnRNP1 binds to HAstV8 3′UTR, UV cross-linking immunoprecipitation experiments were carried out using infected S10 CaCo2 extracts and labeled RNA. A 57/60 kDa polypeptide was precipitated with protein G-Sepharose and hnRNP1 antibody , lane 7.Many positive-strand RNA viruses replicate and translate in the cytoplasm, which take place in association with intracellular membranes. For example, viral replicase complexes from other positive-strand RNA viruses assemble on intracellular membrane surfaces PTB/hnRNP1 is one of the most frequently IRES-specific cellular trans-acting factors implicated in RNA stability, translation and replication of several human viruses as poliovirus, dengue, hepatitis C (HCV) and other positive-strand RNA viruses 6 ffu/ml) with siPTB in comparison with siScrambled-transfected cells (1.8×107 ffu/ml)  and helix I of the 3′UTR structure. PTB/hnRNP1 is redistributed to the cytoplasm during HAstV-8 infection in membrane-associated fractions, and it is required, directly or indirectly, for HAstV-8 replication. We are currently investigating other factors involved in such process.Figure S1UV-cross linking was carried out with radiolabeled an unrelated RNA was incubated with 1 µg of recombinant His-PTB (A) or 0.25, 0.50, and 1 µg of BSA were incubated with the wild type 3′UTR RNA (B). Free probes appear in lanes 2 and 3 respectively. The molecular mass markers (MWM) are shown.(TIF)Click here for additional data file.Figure S2Caco-2 cells were mock-transfected (M) or transfected with the indicated siRNA (A). Total protein was harvesting to demonstrate PTB knockdown by western blot, Actin was used as loading control. The reduction of PTB/hnRNP1 expression was quantitating by densitometry (B) taking actin or/and mock transfected cell as the reference, arbitrary density units  was plotted against the siRNA concentration . The PTB/hnRNP1 knockdown was verified by confocal immunomicroscopy Olympus Fv300 (C).(TIF)Click here for additional data file.


RNA quantification is often a prerequisite for most RNA analyses such as RNA sequencing. However, the relatively low sensitivity and large sample consumption of traditional RNA quantification methods such as UV spectrophotometry and even the much more sensitive fluorescence-based RNA quantification assays, such as the Qubit™ RNA HS Assay, are often inadequate for measuring minute levels of RNA isolated from limited cell and tissue samples and biofluids. Thus, there is a pressing need for a more sensitive method to reliably and robustly detect trace levels of RNA without interference from DNA.To improve the quantification limit of the Qubit™ RNA HS Assay, we spiked-in a known quantity of RNA to achieve the minimum reading required by the assay. Samples containing trace amounts of RNA were then added to the spike-in and measured as a reading increase over RNA spike-in baseline. We determined the accuracy and precision of reading increases between 1 and 20 pg/μL as well as RNA-specificity in this range, and compared to those of RiboGreen®, another sensitive fluorescence-based RNA quantification assay. We then applied Qubit™ Assay with RNA spike-in to quantify plasma RNA samples.RNA spike-in improved the quantification limit of the Qubit™ RNA HS Assay 5-fold, from 25 pg/μL down to 5 pg/μL while maintaining high specificity to RNA. This enabled quantification of RNA with original concentration as low as 55.6 pg/μL compared to 250 pg/μL for the standard assay and decreased sample consumption from 5 to 1 ng. Plasma RNA samples that were not measurable by the Qubit™ RNA HS Assay were measurable by our modified method.The Qubit™ RNA HS Assay with RNA spike-in is able to quantify RNA with high specificity at 5-fold lower concentration and uses 5-fold less sample quantity than the standard Qubit™ Assay.The online version of this article (doi:10.1186/s12867-015-0039-3) contains supplementary material, which is available to authorized users. Recent studies utilizing trace amounts of RNA present in biospecimens such as biofluids, single cells and minute clinical samples have revealed their novel functions and biomedical potentials -14. RNA An alternative approach is fluorescence-based RNA quantification that utilizes the fluorescent property of nucleic acid binding dyes. Unbound dyes are nearly non-fluorescent, but upon binding to nucleic acid, the complex exhibits a large increase in fluorescence, thereby greatly amplifying nucleic acid signal for detection at concentrations much lower than that required by UV spectrophotometry ,16,18-21The Qubit™ RNA HS Assay is highly selective for RNA over DNA  and provHere we used an RNA spike-in to set a baseline reading of the Qubit™ Assay and measured RNA sample as an increase over RNA spike-in. This method was validated to accurately measure RNA at lower concentrations and require less sample compared to standard Qubit™. We tested the utility of this spike-in approach by measuring plasma RNA samples that fell below the detection limit of the standard Qubit™ Assay. We named the modified assay the Spike-in Qubit™ RNA HS Assay because this optimization takes advantage of an RNA spike-in.The Qubit™ RNA HS Assay Kit , Qubit™ 2.0 Fluorometer  and Axygen PCR-05-C tubes (Axygen) were used for all measurements. The Qubit™ working solution was made according to manufacturer’s instructions. We added 180 μL of working solution to each assay tube, up to 20 μL of RNA, and water to bring the final volume to 200 μL. 10 μL of the Qubit™ RNA Standard solutions were used for standard tubes. “RNA spike-in” was made by diluting the Qubit™ RNA Standard #2 (10 ng/μL rRNA) included in the Qubit™ RNA HS Assay kit to 2.5 ng/μL and 2 μL was added into each tube. 18 μL of water was added into one tube for RNA spike-in alone reading. “RNA sample” was made by diluting the Qubit™ RNA Standard #2 to 250 pg/μL and increasing volumes of RNA sample were added into remaining tubes to create expected reading increases of 1, 2, 3, 4, 5, 7.5, 15 and 20 pg/μL over RNA spike-in alone. Assay tubes were vortexed for 2–3 s, centrifuged briefly (~5 s), and then incubated at room temperature for 2 min to allow the assay to reach optimal fluorescence before measuring with the Qubit™ Fluorometer. Each tube was measured three times to obtain the average reading. Reading increase was calculated by subtracting RNA spike-in reading from that of spike-in plus sample RNA reading. The experiment was repeated four times using independently prepared RNA spike-in, RNA sample, and working solution. In addition, the total RNA from human trophoblast cells was used as “RNA sample” and tested as described above for a total of four independent experiments.Based on the original concentration measured by NanoDrop® ND-1000 (Thermo Fisher Scientific Inc.), the trophoblast total RNA was diluted to 60 pg/μL in water. A mixture of RNA and DNA (60 pg/μL each) was prepared by diluting in water the trophoblast total RNA and the Qubit™ DNA Standard #2 (10 ng/μL DNA) included in the Qubit™ DNA HS Assay kit. 18 μL of each sample was measured by the Spike-in Qubit™ as described above and by the Quant-iT™ RiboGreen® RNA Assay  following manufacturer’s instructions using a BioTek Synergy™ 4 Multi-Mode Microplate Reader (BioTek). Four independent repeats were performed with each method.The standard Qubit™ RNA HS Assay was performed according to manufacturer’s instructions. The Spike-in Qubit™ RNA HS Assay was performed as described above. For the Spike-in Qubit™ Assay, RNA sample concentration was calculated as: [Sample] = reading increase (pg/μL) x assay volume (μL) ÷ sample volume for Spike-in Qubit™ (μL). Three independent measurements using separately prepared RNA spike-in and Qubit™ reagents were made for each sample. A detailed step-by-step protocol for the Spike-in Qubit™ RNA HS Assay is provided in the Supplementary Methods.After obtaining informed consent, 8.5 mL of peripheral blood samples were collected into Vacutainer™ tubes containing acid citrate dextrose solution A (BD) and immediately inverted eight times to mix anticoagulant additive with blood. Blood samples were centrifuged at 1,900 g for 10 min at 4°C in a swinging-bucket centrifuge (Eppendorf) and plasma was aspirated using disposable transfer pipets (VWR), aliquoted into 2 mL polypropylene microcentrifuge tubes (Sarstedt) and centrifuged at 16,050 g for 5 min at 4°C in a benchtop centrifuge (Eppendorf). The final plasma was pooled and mixed in 15 mL tubes , then aliquoted into 1.5 mL DNA LoBind tubes (Eppendorf), snap frozen in liquid nitrogen and stored at −80°C. Plasma RNA isolation was performed using three commercial RNA isolation kits: miRCURY™ RNA Isolation Kit – Biofluids (Exiqon), mirVana™ PARIS™ Kit  and miRNeasy Micro Kit (Qiagen) following manufacturer recommended protocols. The miRCURY™ kit is designed to isolate RNA shorter than 1000 nucleotides (nt) and the mirVana™ and miRNeasy kits isolate total RNA.After informed consent was obtained, tissues were collected from manual vacuum aspiration. To isolate trophoblast cells, chorionic villi were identified and floated in DPBS supplemented with 10 mg/ml Gentamycin, minced with sterile scalpel blades, placed in 35 ×10 mm dishes and incubated with 3.3 mg/ml Collagenase (Sigma) for about 2 hours at 37°C in the cell incubator. Cells were further washed twice with Amniomax complete medium  and cultured in Amniomax complete medium for 7 days. RNA from trophoblast cells was extracted with All Prep® DNA/RNA/Protein mini kit (Qiagen) according to the manufacturer’s protocol. All aspects of these studies were reviewed and approved by the Institutional Review Board at Albert Einstein College of Medicine.Data analyses were performed using Excel® for Mac 2011 (Microsoft) and GraphPad Prism 6 (GraphPad Software). Relative error (RE) was calculated as RE = (average measured reading − expected reading) ÷ expected reading. Coefficient of variation (CV) was calculated as CV = standard deviation ÷ average measured reading. Deviation from ideal was calculated as the sum of the absolute value of RE and CV. A two-way ANOVA interaction analysis was performed to determine whether the differences between the measurements of the 60 pg/μL RNA sample and those of the mixed RNA and DNA sample (60 pg/μL each) were consistent for the Spike-in Qubit™ RNA HS Assay and the Quant-iT™ RiboGreen® RNA Assay.The lower reading limit of the Qubit™ RNA HS assay for accurate quantification is 25 pg/μL . In order to quantify samples that fall below this limit, prior to adding the RNA sample that was to be measured, we added 5 ng of RNA spike-in (2.5 ng/μL Qubit™ RNA standard #2) into a 200 μL Qubit™ assay to generate an expected baseline reading of 25 pg/μL. Then, the RNA sample was added to the baseline and the increase over baseline would correspond to the reading of RNA sample Figure .Figure 12 of 0.99837 after four independent experiments . To represent a typical RNA sample that might introduce additional influences on fluorometric measurement, the same experiment was repeated with a 250 pg/μL human trophoblast total RNA sample. Strong linear correlation was also evident with slope of 0.9723 and R2 of 0.99469  was added at increasing volumes into Qubit™ assay tubes containing RNA spike-in to create expected reading increases ranging from 1 to 20 pg/μL. As shown in Figure We assessed the accuracy and precision of the Spike-in Qubit™ Assay for all tested reading increases in order to determine the lower quantification limit that passed accuracy and precision requirements. Accuracy is inversely correlated to the relative error (RE) and precision is inversely correlated to the coefficient of variation (CV). The Qubit™ RNA HS Assay used “deviation from ideal”, the combined value of the absolute value of RE and CV, to evaluate the precision and accuracy and set deviation from ideal of <20% for its lower quantification limit in the core quantification range. Using the same criterion, we determined that Spike-in Qubit™ Assay achieved a lower quantification limit of 5 pg/μL for both Qubit™ Standard #2 RNA and trophoblast total RNA, which is 80% less than that of the standard Qubit™ Assay (25 pg/μL) Tables  and 2. TWe also cross-compared the precision and accuracy of the Spike-in Qubit™ Assay with those of the Quant-iT™ RiboGreen® RNA Assay that has a lower quantification limit of 1 pg/μL. A 60 pg/μL trophoblast total RNA sample was measured in four independent repeats with both methods . As shown in Table RNA samples purified from serum or plasma are present at low concentration (~30 ng per 1 mL of plasma), making their quantification challenging ,23,24. TWe speculate that the Spike-in Qubit™ approach may work because the readings of RNA spike-in alone and with additional RNA samples fall into the linear and high-precision quantification range of the Qubit™. It ensures that the reading increase over RNA spike-in baseline is of high precision and linear therefore of high accuracy. There are limitations to the Spike-in Qubit™ RNA HS Assay. First, it requires extra steps to prepare and add RNA spike-in. However, these steps only consist of diluting the Qubit™ RNA Standard #2 (that comes pre-made in the kit) and adding it into a master mix. Therefore, the time and risk of introducing error are minimal. In addition, because an excess amount of Qubit™ RNA Standard #2 is provided in the Qubit™ Assay kit, there is no need to purchase additional reagents. As the validation for reading increases between 1 and 5 pg/μL was performed by adding RNA samples in 0.8 to 4 μL volumes, Pipetting of small volumes could have contributed to variation in measurements due to pipetting error.The Spike-in Qubit™ RNA HS Assay reported here achieved accurate and precise RNA quantification at a new lower quantification limit of 5 pg/μL, 5-fold lower than that of the standard Qubit™, while maintaining the RNA specificity of the original assay. This improvement lowers minimal RNA concentration measurable from 250 pg/μL to 55.6 pg/μL and reduces minimal RNA consumption from 5 ng to 1 ng. As demonstrated in the successful quantification of plasma RNA samples, the Spike-in Qubit™ RNA HS can be readily used to quantify RNA samples having low concentrations and limited quantities.


The replication enzyme of RNA viruses must preferentially recognize their RNAs in an environment that contains an abundance of cellular RNAs. The factors responsible for specific RNA recognition are not well understood, in part because viral RNA synthesis takes place within enzyme complexes associated with modified cellular membrane compartments. Recombinant RNA-dependent RNA polymerases (RdRps) from the human norovirus and the murine norovirus (MNV) were found to preferentially recognize RNA segments that contain the promoter and a short template sequence for subgenomic RNA synthesis. Both the promoter and template sequence contribute to stable RdRp binding, accurate initiation of the subgenomic RNAs and efficient RNA synthesis. Using a method that combines RNA crosslinking and mass spectrometry, residues near the template channel of the MNV RdRp were found to contact the hairpin RNA motif. Mutations in the hairpin contact site in the MNV RdRp reduced MNV replication and virus production in cells. This work demonstrates that the specific recognition of the norovirus subgenomic promoter is through binding by the viral RdRp. Noroviruses are the major causative agents of nonbacterial epidemic gastroenteritis worldwide –4. DespiCaliciviridae family. The majority of noroviruses express three conserved open reading frames named ORF1–3 while MNV has an additional ORF4  to express the recombinant MNV RdRp with a six-histidine tag at the N-terminus. Site-specific mutations in the MNV RdRp were introduced using the Quick-Change site-directed mutagenesis kit (Agilent Technologies). All constructs used in this study were confirmed by sequencing with the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems).The cDNA encoding the MNV RdRp was amplified by polymerase chain reaction (PCR) from the cDNA clone of MNV-1 strain CW1 (GenBank accession number: DQ285629.1) to contain a 5′ NdeI restriction endonuclease cleavage site and a BamHI site at the 3′ terminus. Following restriction enzyme digestion, the cDNA was cloned into the Table Escherichia coli Rosetta cells harboring the pET15bMNVRdRp plasmid were grown at 37°C until the optical density at 600 nm reached between 0.6 and 0.8. Isopropyl thiogalactoside (IPTG) was added to a final concentration of 1 mM to induce protein expression for 16–18 h at 16°C. The cells were harvested by centrifugation and resuspended in lysis buffer A  and sonicated on ice three times for 15 s each. The lysate was clarified by centrifugation at 11 000 g for 30 min and the supernatant was mixed with Ni-NTA resin according to the manufacturer's instructions (Qiagen). The slurry was packed in a column, washed extensively with lysis buffer containing 30 mM imidazole and eluted with lysis buffer containing 200 mM imidazole. The eluted protein was stored in storage buffer ).E. coli cells transformed with pBAD GII.4RdRp plasmid were cultured at 37°C until OD600 was around 0.6. l-Arabinose was added to a final concentration of 0.02% to induce the protein expression. The cells were continually grown at 37°C for 5 h and then harvested by centrifugation and suspended in buffer A for cell lysis and purification using the Talon metal affinity resin (Clontech Laboratories) using the manufacturer's instructions. The recombinant protein was washed with lysis buffer containing 20 mM imidazole and eluted with the same buffer that contained 100 mM imidazole. The eluted protein was stored in buffer containing 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.9, 150 mM NaCl, 20% glycerol, 5 mM DTT. All recombinant proteins were quantified by using the Bradford assay and the purity assessed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie brilliant blue. The recombinant proteins were stored at −80°C for future use.Recombinant GII.4 norovirus RdRp was expressed and purified as previously described . Brieflyet al. with minor modifications (32P-CTP during RNA synthesis in vitro. All RNA synthesis reactions were of 20 μl and contained 20 mM sodium glutamate (pH 8.2), 12.5 mM dithiothreitol, 4 mM MgCl2, 1 mM MnCl2, 0.5% Triton X-100 (v/v), 0.2 mM guanosine triphosphate (GTP), 0.1 mM adenosine triphosphate (ATP) and uridine thriphosphate (UTP), 33.3 nM [α-32P]- cytidine triphosphate (CTP) , 50 nM of template RNA and 250 nM of recombinant RdRp unless indicated otherwise. The reaction was incubated at 30°C for 2 h, and then stopped by the addition of ethylenediaminetetraacetic acid (EDTA) (pH 8.0) to a final concentration of 10 mM. RNA products were directly loaded onto a 24% polyacrylamide gel containing 7.5 M urea. The radiolabeled RNA products were visualized and quantified by using a PhosphoImager  and ImageQuant software.RdRp assays were performed as previously described by Yi ications . All RNA4HCO3 (pH 7.8) to digest the sample overnight at 37°C. 3 M LiCl was used to precipitate the RNA–peptide complex followed by two 70% ethanol washes. RNA–peptide crosslinks were reversed by incubating the samples for 1 h at 70°C in a buffer containing 50 mM Tris pH 7.5, 200 mM NaCl, and 0.1% trifluoroacetic acid. The samples were purified and concentrated using a Ziptip column (Millipore) and analyzed by MALDI-ToF mass spectrometry (MS)  in positive-ion mode. Assigned peaks all corresponded to within 0.5 Da of the theoretical masses of peptides from MNV RdRp.The RCAP assay was carried out as previously described . Briefly2, 1 mM MnCl2 and 1 mM DTT. The samples were transferred to microplates on ice and ultraviolet (UV) crosslinking was performed at 365 nm for 10 min. Trypsin digestion and precipitation of the peptide–RNA complexes were as described above. The pellet was suspended in a buffer containing 50 mM Tris pH 7.5, 200 mM NaCl and digested with RNAse A (1 μg) at 37°C for 30 min. Peptides and peptide–nucleotide conjugates were purified using a C18 column and analyzed by LC–ESI–MS/MS using an nanoliquid chromatography system (AB SCIEX) interfaced to an externally calibrated LTQ Orbitrap hybrid mass spectrometer equipped with a nanospray ion source (Thermo Scientiﬁc). A 4 μl aliquot of the peptide digest dissolved in 12 μl of mobile phase A was washed with buffer A for 5 min at a flow rate of 5 μl min−1 using a reversed-phase C18 trap column made from a 15 mm × 100 μm integrafrit column and packed with Magic C18 resin . The desalted peptides were then separated with Buffer B gradient using a reversed-phase C18 column  packed in a 150 mm × 75 μm, 15 μm tip (New Objective) and analyzed in positive ion mode. Buffer A consisted of 0.1% formic acid in acetonitrile:water  and Buffer B consisted of 0.1% formic acid in acetonitrile:water . The flow rate was 300 nl min−1 with the following gradient: 3% B from 0–1 min, ramp to 10% B from 1–9 min, ramp to 30% B from 9–28 min, ramp to 40% B from 28–31 min, ramp to 80% B from 31–35 min, hold at 90% B from 36–43 min, ramp to 3% B from 43–44 min, then re-equilibrate the column at 3% B for 14 min . The mass spectrometer was operated in an automated data dependent mode, alternating between an FT-MS scan in the Orbitrap and five collision-induced dissociation (CID) scans in the linear ion trap. The Orbitrap monitored precursor ions from m/z 300 to m/z 2000 at 15 000 resolving power. The five most intense precursor ions in each scan were sequentially isolated in the linear trap with a 2 m/z isolation width and activated at 35% normalized collision energy. The cycle was continuously repeated throughout the entire separation with the dynamic exclusion set to 45 s with a repeat count of 2. Identification of peptides covalently attached to 4SU was performed using the Proteome Discoverer™ software by searching the raw LC–MS/MS data using SEQUEST® against the protein sequence and allowing for variable modifications corresponding to possible 4SU structures attached to the protein.RdRp crosslinking reactions were performed in clear-bottom 96-well microplates. The reactions were of 25 μl in volume and contained 2 μM purified MNV RdRp and 4 μM of 4SU-labeled RNA in a buffer containing 20 mM HEPES pH 7.5, 4 mM MgCl3V 1420 multi-label counter (Perkin Elmer) was used to read fluorescence polarization data from the plates. The excitation and emission wavelengths were set at 485 and 535 nm, respectively. The data were plotted against MNV RdRp concentration and the data were fit to a hyperbola (Y = min + (max − min) × S/(S + Kd)).All assays were performed in 20 μl reactions. 0.1 μM of the FL-MNV-proscript was mixed with MNV RdRp at various concentrations in the reaction buffer containing 10 mM Tris–HCl pH 8.0, 30 mM NaCl, 1% glycerol and 0.5 mM 2-mercaptoethanol. A Victor2 and 1 mM MnCl2. Recombinant MNV RdRp or mutant R411A was mixed with Mps at a 1:2 molar ratio. All reactions used a ramp rate of 0.5°C/min and a temperature range from 25 to 75°C. The results were analyzed and plotted using GraphPad Prism.Differential scanning fluorimetry (DSF) was used to measure the thermodenaturation profiles of proteins. DSF used a Stratagene MX3005P real-time PCR machine. Each sample was prepared in a total volume of 25 μl that contained a 5× final concentration of SYPRO Orange (Molecular Probes) in buffer containing 20 mM sodium glutamate (pH 8.2), 12.5 mM dithiothreitol, 4 mM MgCll-glutamine, 0.075% sodium bicarbonate, 100 units/ml penicillin and 100 μg/ml streptomycin  supplemented with 10% fetal calf serum (FCS), 2 mM ptomycin . BHK celptomycin .50) titrations were performed on BV-2 cells using 10-fold serial dilutions. The viral TCID50 was determined by scoring for signs of cytopathic effect at 5 days post-infection by visual inspection. Western blot for the viral RdRp used rabbit anti-MNV NS7 serum  and mouse anti-GAPDH (Applied Biosystems) as a loading control. HRPO-conjugated primary and secondary goat antibodies recognizing rabbit or mouse immunoglobulins were detected by chemiluminescence . Passage 1 stocks of the viable viruses were generated by inoculating BV-2 cells (seeded at a density of 1 × 105 in six-well plates) with 700 μl of recovered virus for 1 h. RNA was extracted from the stocks using the GenElute™ System (Sigma–Aldrich) for RT-PCR amplification and verification of the mutations by DNA sequencing.The R411A, R411K, R416A and R411/416AA mutations were introduced into the MNV-1 infectious clone (pT7:MNV 3′RZ) by overlapping mutagenic PCR. Virus was rescued from cDNA clones using the reverse genetics system based on recombinant Fowlpox expressing T7 RNA polymerase, previously described in . Brieflywww.cgl.ucsf.edu/chimera).All RNAs secondary structures in this study were predicted by using the program Mfold . Moleculde novo initiated and primer-dependent RNA synthesis by the Hepatitis C virus RdRp  and terminated at the end of the template sequence in the proscript. The 10-nt product likely arose from correct de novo initiation from the T + 1 cytidylate but terminated one nucleotide before the RdRp reached the final 3′ nucleotide of the template RNA. Premature termination of RNA synthesis has been reported for several recombinant RdRps in vitro . Fluorescence polarization assays generated the binding isotherms that were fitted to a one site binding model. The MNV RdRp had a s Figure .50 for Mps and Mhp of 2.36 and 16.2 μM, respectively , or Chp (the hairpin from the Chikungunya virus proscript) Figure . RNA syno Figure .in vitro, but neither could direct de novo initiated RNA synthesis , which has the single-stranded sequence from MNV and the hairpin from GII.4, directed the GII.4 RdRp to produce 20% of the de novo initiated RNA when compared to Gps . Following crosslinking to MNV RdRp and trypsin digestion, the peptide-RNA complexes were precipitated with LiCl. The RNAs in the complexes were then thoroughly digested to release nucleotides from the peptides, leaving only the crosslinked nucleotide. The identities of the cross-linked peptides were then determined by high-resolution and collision induced dissociation (CID) mass spectra using the LTQ-Orbitrap instrument. Kramer et al. have shown that crosslinking of proteins and 4SU substituted RNAs could undergo the loss of H2S, the loss of the double bond in 4SU, and the loss of phosphates from either or both the 3′ or the 5′ positions  and WDSTQQRAILK  were identified to bind 24-4SU and 2-4SU, respectively . However, some signals for both peptides were also observed with 2-SU and 24-4SU, suggesting that the bulged region in the hairpin and the base of the hairpin RNA could contact the RdRp at two different locations. The three 4SU cross-linked peptides identified here were also identified in the RCAP assay discussed previously  analogs and amino acids . Three vositions . All of U Figure . CID datTo assess the functional relevance of these residues that contact the Mhp, we examined their degree of sequence conservation in the RdRps from multiple noroviruses. Residues R185, R255, and R416 are highly conserved among noroviruses while K259 and R411 were conserved only in murine noroviruses Figure . We reasde novo initiated RNA synthesis and produced only a fraction of the primer product using Mps as the template  of 43.5°C. R411A had a TMapp of ca. 2°C lower than that of the WT and the R416A mutant had a TMapp that was 1°C higher than that of the WT. Thus, both substitutions affected the conformation of the RdRp even before template binding.The observed defect in RNA binding exhibited by R411A and R416A could be due to a change in the RdRp conformation needed for proscript binding or an effect on the affinity for the proscript. To examine whether RdRp conformation was affected, we used differential scanning fluorimetry (DSF) to measure the thermodenaturation profile of the WT and mutant RdRps. This assay measures the binding of the dye SYPRO orange by hydrophobic regions of proteins that will become increasingly exposed with temperature . Bound STMapp with either the WT RdRp or the mutants. Mps did reduce the total fluorescence of the proteins, but the same effect was observed for the WT RdRp and the mutants , a lysine substitution (R411K), an alanine substitution at R416 (R416A), or alanine substitutions of both R411 and R416 (R411AR416A). Each infectious clone was transfected into BHK cells, and cell lysates were collected. The TCID50 titer in the lysate was determined in BV-2 mouse microglial cells that can be infected by MNV. Mutants R411A and R411K had 10- and 3-fold lower viral titers than the WT MNV (Figure We sought to determine whether substitutions in the MNV RdRp that affected sgRNA initiation V Figure . SequencV Figure . ConstruV Figure . Interes50 of the WT and mutant viruses and the amount of virus released determined over a 48 h period (Figure We characterized mutant viruses R411A and R411K for infection of BV-2 cells. The cells were infected with a 1 TCIDd Figure . Mutant d Figure . At 9 h d Figure . AltogetE. coli to investigate the specific recognition of the MNV subgenomic promoter. Previous studies have identified a highly conserved RNA stem-loop structure as the core promoter for calicivirus sgRNA synthesis (In this study, we expressed and purified recombinant MNV RdRp in ynthesis . Howeverde novo initiated RNA synthesis and the affinity of RNA binding. The two residues in the NS7 protein are also required for wild-type levels of MNV RNA synthesis in the context of MNV infection. Notably, substitutions at R411 were less detrimental for RNA synthesis in vitro and mutant virus with substitutions at residue R411 retained infectivity in cells (Figure The residues in the MNV RdRp that contact the subgenomic promoter hairpin were mapped Figure . Mutatios Figure . Mutant s Figure . It is pet al. found that residue R185 near motif F was involved in binding to the RNA in all available viral RdRp structures including distantly related HIV reverse transcriptase (de novo RNA synthesis (de novo initiated RNA synthesis. Mutant RdRp R255A near motif A also showed defective polymerase activity for both primer extension and de novo initiated RNA synthesis. It is likely that R185 and R255 serve to stabilize core promoter binding.Three regions of the MBV RdRp were identified to contact the hairpin within the MNV subgenomic promoter: a sequence near motif F, residues near motif A within the finger subdomain, and the α-helix near motif E. Lee criptase . The F1 ynthesis . For thede novo initiation but fully competent for primer extension. Mutant R416A was defective for de novo initiation but also for the synthesis of the full-length product, likely due to an effect the termination of RNA synthesis. R411A and R416A both had similar defect in binding Mps. It is likely that R411 and R416 maintain contact with the proscript and the template throughout the process of RNA synthesis.Mutations in the α-helix near motif E had more complex effects on RNA synthesis. Mutant R411A was specifically defective for in vitro RNA synthesis assays did not result in a significant amount of protein-primed product, possibly due to the lack of RNA elements that will promote the formation of the VPg-nucleotide complex (Lin, data not shown; Our results show that the norovirus RdRp can specifically recognize the subgenomic promoter and the initiation cytidylate in the absence of other viral proteins. This was somewhat surprising given that the norovirus sgRNA is likely to be VPg-linked to facilitate translation . Additioet al. (de novo initiated RNA synthesis. This level of specificity for the template sequence could prevent the viral replication complex from misrecognition of the RNAs which could lead to defects in the process of RNA synthesis and possibly increase the detection of the RNA products by innate immune receptors (The norovirus subgenomic promoter consists of two components: the RNA hairpin and the template sequence. The hairpin motif has been previously demonstrated to serve as the promoter for RNA synthesis by plant viruses –53. In tet al.  have ideeceptors .et al. (RdRps are one of the four major classes of template-dependent polymerases that also include DNA-dependent DNA polymerases, the reverse transcriptases, and the DNA-dependent RNA polymerases. It is worth noting that core promoter recognition by the recombinant norovirus RdRps is a feature shared with bacteriophage DNA-dependent RNA polymerases ,58–59. Iet al.  had propSupplementary Data are available at NAR Online.


Caenorhabditis elegans mutants deleted for TDP-1, an ortholog of the neurodegeneration-associated RNA-binding protein TDP-43, display only mild phenotypes. Nevertheless, transcriptome sequencing revealed that many RNAs were altered in accumulation and/or processing in the mutant. Analysis of these transcriptional abnormalities demonstrates that a primary function of TDP-1 is to limit formation or stability of double-stranded RNA. Specifically, we found that deletion of tdp-1: (1) preferentially alters the accumulation of RNAs with inherent double-stranded structure (dsRNA); (2) increases the accumulation of nuclear dsRNA foci; (3) enhances the frequency of adenosine-to-inosine RNA editing; and (4) dramatically increases the amount of transcripts immunoprecipitable with a dsRNA-specific antibody, including intronic sequences, RNAs with antisense overlap to another transcript, and transposons. We also show that TDP-43 knockdown in human cells results in accumulation of dsRNA, indicating that suppression of dsRNA is a conserved function of TDP-43 in mammals. Altered accumulation of structured RNA may account for some of the previously described molecular phenotypes  resulting from reduction of TDP-43 function. The reduction of dsRNA accumulation is likely to be a conserved function of TDP-43 proteins because knockdown of mammalian TDP-43 in HeLa cells and M17 neuronal cells also causes dsRNA accumulation.In this study, we investigated the role of TDP-1 on the transcriptome. We discovered that TDP-1 functions to maintain the amount of mature RNA transcripts originating from potentially double-stranded precursor RNAs and to limit nuclear dsRNA accumulation in multiple tissues. Immunoprecipitation using a dsRNA-specific antibody revealed that tdp-1 mutant animals to identify changes in RNA metabolism or abundance. While two deletion alleles exist for tdp-1, tdp-1(ok803) and tdp-1(ok781), only the tdp-1(ok803) allele appeared to be a clear null ; therefore, our study is focused on this allele. We created and sequenced poly(A)-selected cDNA libraries from wild-type and tdp-1(ok803) animals. Mapping of sequenced reads showed over 50% of all amplifiable annotated genes were well represented . A differential gene expression comparison (RPKMs) between tdp-1(ok803) mutants and wild-type revealed over 1,700 transcripts aberrantly represented with close to an equal number of transcripts underrepresented as overrepresented  changes in splice site representation  animals.Because TDP-43 functions in alternative splicing, we also asked whether transcripts with altered abundance in the mutant had splicing abnormalities. Splicing analysis identified ∼350 genes with significant  for common characteristics within the RNA molecule. Interestingly, we noticed that a large proportion of altered transcripts had potential double-stranded structure. Specifically, many over/underexpressed transcripts contained either antisense overlap with another gene or multiple inverted repeats within intronic regions.Mammalian TDP-43 maintains the abundance of transcripts with long introns. One interpretation of this observation is that transcripts require TDP-43 because of an inherent characteristic of the RNA transcript itself and not due to its involvement in a common functional pathway. To look for common features in RNA transcripts affected by TDP-1, we examined the most dramatically altered transcripts in the P = 5.3 × 10−181, hypergeometric distribution (hgd)) , 40% of underrepresented genes in tdp-1(ok803) RNA-seq contained inverted repeats, representing a significant enrichment   mutant worms by immunostaining with a dsRNA-specific antibody, J2. The J2 antibody is specific to dsRNA duplexes greater than 40 bp  worms with the J2 antibody. As shown in Figtdp-1(ok803) worms had a significant increase in nuclear inclusions reactive with the J2 antibody. This increase was observed in multiple tissues including: gut  mutants. In order to determine whether identified A-to-I edits represented true physiological targets, we sequenced RNA in parallel from a strain deleted for the only functional worm ADAR, adr-2(gv42). Using a compiled list of all nucleotide positions that showed a discrepancy between the RNA and DNA sequence , we hand-annotated 210 regions containing convincing evidence of A-to-I RNA editing (Supplementary adr-2(gv42) mutants . The majority (> 90%) of editing events occurred in introns and 3′ UTRs, and all observed editing was consistent with promiscuous editing, which occurs in long dsRNA , our analysis did successfully identify eight of the ten published transcripts characterized as edited due to preferential digestion with T1 RNAse  Bass, , we hypol, et al . To confl, et al . Transcrtdp-1 maintains A-to-I RNA editing within these regions, we calculated the percent editing across all identified edited regions that were well represented  in both wild-type and tdp-1 mutants. Roughly 50% of well-represented regions showed significantly (P < 0.05) altered editing  RNA-seq are shown , increased amounts of A-to-I editing indicate that the accumulation of dsRNA in tdp-1(ok803) mutant worms is not due to a reduction in RNA editing. Rather, increases in the structure or stability of dsRNA could result in a slightly higher frequency of editing in tdp-1 mutants.To determine whether  mutants . While tet al, tdp-1(ok803) mutants. Because J2 immunoprecipitation was done in untreated lysate, entire transcripts containing 40 bp or more of dsRNA anywhere within the molecule will be precipitated. J2-immunoprecipitated RNA and total RNA were sequenced in a strand-specific manner. Pairwise correlation of J2-IP RNA-seq replicates indicated that the J2-IP replicates were highly similar .The J2 antibody can be used to immunoprecipitate dsRNA, as shown for Alu transcripts in mammalian cell lysates  as an effective method to isolate dsRNA. Comparison between input and J2-immunoprecipitated RNA confirmed that J2-IP selected for a subset of transcripts FigA. As a pP < 0.05, FDR < 0.1, > 1.5-fold) over input were considered “selected” in the IP. We identified 2,875 transcripts enriched in the wild-type J2-IP, representing 27% of all genes expressed in the experiment . Most (95%) of the enriched RNAs originated from protein-coding genes. Both antisense-overlap genes and A-to-I edited genes were significantly enriched (P < 1 × 10−15 hgd) by J2-IP  mutants, we used DESeq software to compare normalized (to input) expression levels of all genes expressed in the J2-IP between tdp-1 mutants and wild-type . After filtering out low expressed genes and genes not enriched by J2-IP over input in either sample , we identified 1,422 genes with increased J2-IP enrichment  in tdp-1 mutants compared to wild-type, representing about 40% of all transcripts selected by J2-IP. In contrast, only 166 genes  were identified as having less dsRNA structure/stability in tdp-1 mutants  J2-IP by RT–PCR and showed this enrichment is abolished by pretreatment with dsRNA-specific RNAse .To identity transcripts with more dsRNA structure/stability in tants Fig. This retdp-1 mutant J2-IP did not indicate that any specific class of dsRNA (e.g. antisense to another gene or target of siRNA) was being affected in tdp-1 mutants. Rather, we found that most transcripts that form dsRNA structure simply have more dsRNA structure/stability in the absence of TDP-1 . However, the exception to this observation was A-to-I RNA edited transcripts, which were significantly under represented  among genes increased in tdp-1(ok803) J2-IP. This result suggests that hyper-editing of these transcripts could counteract any potential increases in dsRNA structure in tdp-1 mutants.Analysis of transcripts increased in tdp-1 controls the structure of a specific functional class of transcripts, we performed a gene ontology analysis of genes whose transcripts have altered dsRNA structure/stability in tdp-1 mutants. Among genes with increased recovery in tdp-1(ok803) J2-IP, we found an enrichment for genes functioning in nucleosome assembly  as well as genes involved in translation and protein modification/turnover (Supplementary tdp-1 mutants showed that both 9 and antisense transcripts originating from histone genes were observed in tdp-1(ok803) and wild-type total RNA-seq data. However, J2-IP recovered the antisense histone-region transcripts only in tdp-1(ok803) , suggesting that these transcripts form intermolecular hybrids whose structure or stability is preserved in the absence of TDP-1 function. Interestingly, genes with decreased recovery in tdp-1(ok803) J2-IP were enriched for neuronal-specific genes, many of which are highly edited  of annotated repeat regions (taken from wormbase) indicated that most repeat regions showed increased dsRNA structure/stability in tdp-1 mutants compared to wild-type  J2-IP compared to only 5% in wild-type. An example of a tandem repeat increased for dsRNA structure/stability is shown in FigA major fraction of transcripts with increased enrichment in -type Fig, with thtdp-1(ok803) animals were significantly enriched  for transcripts isolated by J2-IP from tdp-1(ok803) (Supplementary col-119 (encodes a collagen protein), which is transcribed antisense to another expressed transcript. In the mature poly(A) RNA pool, this transcript is decreased. However, col-119 transcript is overrepresented twofold in total RNA, suggesting that the reduction in poly(A) RNA results from defective maturation, as opposed to decreased expression/transcription. J2-IP enriched this transcript almost threefold in tdp-1(ok803) compared to wild-type controls, indicating this transcript has a high degree of aberrant dsRNA structure/stability that interferes with its maturation.If excess dsRNA structure/stability results in altered levels of mature transcripts, we would predict transcripts that are over/under-expressed in the poly(A)-selected pool would be preferentially recovered by J2-IP. Indeed, transcripts aberrantly represented in poly(A) RNA-seq from et al, tdp-1 mutant animals, we compared normalized expression levels (RPKMs) for all intronic regions (> 250 bp) represented in the J2-IP RNA-seq. Among enriched regions, 1,287 introns were significantly different  between tdp-1(ok803) and wild-type, with 75% showing an increased abundance in tdp-1(ok803) J2-IP  mutants indicated that ∼31% contained anecdotal evidence of A-to-I editing  and 80% overlapped with annotated complex and/or simple structural elements (see Supplementary tdp-1(ok803) J2-IP were highly enriched for A-to-I editing  and conversely, regions with increased structure were unenriched for editing , suggesting that hyper-editing in tdp-1 mutants may result in a decrease in dsRNA in some intronic regions.TDP-43 homologs sustain the expression of transcripts with long introns presumably by binding the intronic sequences and allowing expression or stabilization of the transcript  mutants revealed a significant enrichment  for intronic regions with differential recovery in tdp-1(ok803) J2-IP among transcripts altered in splicing. As a representative example, Figpqn-41 transcript, which displays enhanced inclusion of exon 18 in tdp-1(ok803) poly(A) RNA-seq and enrichment by J2-IP of the intronic regions bracketing that exon in tdp-1(ok803) mutants. A-to-I RNA editing was also observed in these introns consistent with the formation of dsRNA. Additional examples are provided (tdp-1(ok803) splicing abnormalities could be due to excess dsRNA formation and/or A-to-I editing within the intronic regions of transcripts altered in splicing.Double-stranded RNA structure, as well as A-to-I RNA editing, is known to modulate alternative splicing  mutants. As TDP-1 orthologs bind primarily intronic regions, we suspected that TDP-1 might associate with nascent pre-mRNAs co-transcriptionally in order to limit dsRNA structure or stability in these regions. Chromatin immunoprecipitation (ChIP) sequencing is widely used to determine the association site of RNA-binding/processing factors that co-transcriptionally process nascent RNA  TDP-1 binding sites . Further, TDP-1 association increased with increasing UG-repeat size . While this result indicates TDP-1 association is enriched in UG-rich regions, many TDP-1 bound regions did not contain UG repeats and not all UG repeats were bound by TDP-1, mirroring results reported for mammalian TDP-43 binding.To determine whether TDP-1 acts directly on transcripts with potential double-stranded structure, we asked whether TDP-1 protein associates with genes containing excessive dsRNA in tdp-1 mutants (as assayed by J2-IP enrichment) showed about 40% of these genes contained clear TDP-1 binding sites . For J2-IP enriched intronic regions, TDP-1 binding showed a more significant enrichment . A large fraction (37%) of TDP-1 ChIP peaks also overlapped with annotated repeat regions such as transposons and tandem repeats , confirming that TDP-1 associates with potentially double-stranded transcripts and mirroring mammalian studies showing TDP-43 binds transposable elements  animals under standard conditions . However, TDP-1 might function in other neuronally mediated processes, such as chemotaxis. We assayed the response of tdp-1 mutant animals to attractive volatile compounds  that are sensed by the AWCR and AWCL neurons  olfactory deficits in tdp-1(ok803) mutant animals   .tdp-1 mutants were due to increased dsRNA accumulation or an alternate function of tdp-1. If aberrant dsRNA results in chemotaxis defects, it could either be because dsRNA itself is intrinsically deleterious or because the dsRNA leads to the hyper-activation of downstream processes, such as inappropriate production of endogenous siRNAs. To ask whether the tdp-1(ok803) olfactory defects were due to the RNAi pathway, we created tdp-1(ok803) strains that also had a loss of function mutation for a gene essential to the RNAi pathway, rde-1(ne219). Chemotaxis assays on this strain showed that mutation of rde-1 completely suppressed the olfactory defect in tdp-1(ok803) animals , rde-1(ne219) double-mutant as compared to the tdp-1(ok803) single-mutant worms  in TDP-43 siRNA-treated cells than in control cells  not necessarily the function of its resultant protein. To ask whether TDP-43 knockdown in human cells resulted in increased dsRNA in the same transcript types as worm tdp-1 deletion, we performed J2-IPs followed by RT–PCR of RNA isolated from TDP-43 knockdown cells and mock-treated controls. As our analysis in worms indicated that repetitive sequences and intronic regions were the most affected by tdp-1 loss of function, we focused on these transcript types. As a representative example of a repetitive sequence, we assayed the Herv-K retrotransposon, which is misregulated in ALS pathological samples  at a level similar or greater than the decrease observed for human La  allele renders worms hypersensitive to oxidative stress and interacts with the C. elegans insulin-like signaling (ILS) pathway. Interestingly, daf-16 (the key transcription factor controlling ILS in C. elegans) is hyper-edited in tdp-1(ok803)  worms could contribute to the altered stress sensitivity.While worms lack an interferon response, our data indicate that processes downstream of dsRNA  result in the neuronal phenotypes of ro et al  demonstrtdp-1ok80 allele rin vivo, in contrast to protocols that rely on isolation and refolding of RNA in vitro  due to innate characteristics of those transcripts. Importantly, expression of tandem array transgenes in C. elegans, which is known to produce dsRNA , and some do not (replicative histone transcripts and ncRNAs). Similarly, ncRNAs and replicative histone transcripts have unique 3′-end formation pathways that differ from the canonical cleavage and polyadenylation machinery used by mRNAs  RNA pool. These same transcripts are associated with TDP-1 and contain increased amounts of dsRNA structure/abundance in tdp-1 mutants. We do not know the exact mechanism by which increased dsRNA formation/stability alters the abundance of potentially structured mRNAs in tdp-1 mutants. However, increased structure could either increase or decrease transcript abundance depending on the individual regulatory pathways acting on each transcript. For example, increased structure could trigger siRNA production, which is known to down-regulate transcript abundance of antisense-overlap transcripts in multiple systems . Body bends of 4 L4 worms thrashing in 1× S basal were captured for 30 s. Three independent biological replicates were analyzed for wild-type and tdp-1(ok803) animals, resulting in a total of 12 worms per group. All the movies were filtered as suggested in the manual prior to body bend analysis.Liquid thrashing assays were performed using synchronized 1-day-old adults grown at 20°C. Thrashes were counted for 30 s under a dissecting microscope by hand. For automated thrash counting, et al, Immunoblotting of worm and human proteins was done according to Ash et al, et al, Extracts for J2-IP experiments were made from young adult animals grown on solid plates at 15°C. Worms used for TDP-1 immunoprecipitation followed by RT–PCR were cross-linked according to Zisoulis et al, Immunoprecipitations were preformed as described  , and when confluent, 1.75 × 10RNA for poly(A) and total RNA sequencing libraries was extracted from whole animals by TRIzol extraction. Genomic DNA was removed using TURBO DNase (Invitrogen). For total RNA libraries, 5 μg of RNA was run through a Ribo-Zero column  to remove ribosomal RNA. For poly(A)-selected libraries, RNA was selected using sera-mag magnetic oligo dT beads (Therma Scientific). Libraries were created using Illumina TruSeq kits (RS-122-2001). RNA recovered by immunoprecipitation with the J2 antibody  of young adult worms as well as input material (as a loading control) was converted into strand-specific total RNA libraries using V2 Scriptseq (Epicenter #SSV21106) kits following manufacturer's instructions, except reverse transcription was done with SuperScript III (Invitrogen #18080-044) using incrementally increasing temperatures from 42 to 59°C to allow for transcription though structured RNAs. rRNA was not removed from J2-IP RNA samples. Immunoprecipitated DNA from ChIP samples was converted in sequencing libraries using ChIP-Seq DNA Sample Prep Kit (IP-102–1001). Libraries were sequenced on Illumina HiSeq 2000 platforms. Data were deposited under GEO accession number GSE61581.C. elegans genome. Differential expression in poly(A) selected libraries between wild-type and tdp-1(ok803) mutant was calculated using the Bioconductor package edgeR (release 2.12)  increased in abundance > 1.5-fold over input were considered enriched by J2-IP. To determine genes, repeat regions and introns differentially selected by J2-IP in wild-type versus tdp-1(ok803) extract , read counts for well-expressed regions were normalized to input (see Supplementary Methods) and read into DESeq v1.14.0. A significance cut-off of P < 0.05 and FDR < 0.1 was used.Detailed algorithms and specifications for sequencing analysis including read filtering, mapping, and differential expression calculations are provided in the Supplementary Methods and Supplementary Fig S14. In brief, reads were trimmed and filtered, and rRNA reads were removed. Reads were aligned to the WS220 adr-2(gv42), and tdp-1(ok803) total RNA samples, the alignments were piled up using an in-house program. Positions in wild-type and tdp-1(ok803) samples with a discrepancy between the genomic sequence and mRNA sequence consistent with A-to-I editing (A-G and C-T transitions) were identified. Potential edited positions were compared to the adr-2(gv42) sample and sites also showing A-to-G or C-to-T changes were discarded. To identify editing in introns enriched by J2 immunoprecipitation , reads mapping to all J2-IP enriched introns were inspected visually on the IGV genome browser and hand-annotated as edited if editing was well represented and was present in all three biological replicates. Additional details in Supplementary Methods.For wild-type, tdp-1(ok803). The total number of edited nucleotides was divided by the total number of potentially edited nucleotides (determined by evidence of editing in either sample) for each location. Significant changes were determined by performing a two-proportion z-test between wild-type and tdp-1(ok803) percent edited ratios; P < 0.05 for significant changes.The Integrative Genome Viewer (IGV) tool, count, was used to transform alignment files into pileups for downstream analysis. Well-expressed (> 20 reads) edited regions were used to compare percent editing between wild-type and et al, C. elegans genes expressed in our RNA-seq samples.David (Huang da tdp-1(ok803) mutants, poly(A)-selected RNA from each strain was resequenced 100 bp paired-end at a high read depth (∼280 million reads per sample) to achieve the depth and mapping specificity to analyze alternative splicing. The Cuffdiff (v1.30) differential splicing test was used for splicing analysis.To capture splicing changes between wild-type and et al, P-value cut-off was set at P < 0.01. Regions positive for TDP-1 peaks were counted only if an overlapping peak existed in both biological replicates, and there was no peak in the RNase-treated sample.Filtered and trimmed ChIP-seq samples were aligned to WS220 using Bowtie. Significant TDP-1 ChIP peaks were identified using MACS software (version 1.42) (Zhang et al, Strand displacement assay was done as described in Rajkowitsch & Schroeder  with the