Saccharomyces cerevisiae, results in transcription initiation of antisense RNAs embedded within body of protein-coding genes. These RNAs are distinct from the previously identified non-coding RNAs and cover 11% of the yeast genome. These RNA species have been named Set2-repressed antisense transcripts (SRATs) since the co-transcriptional addition of the H3K36 methyl mark by Set2 over their start sites results in their suppression. Interestingly, loss of chromatin resetting factor Set2 or the subsequent production of SRATs does not affect the abundance of the sense transcripts. This difference in transcriptional outcomes of overlapping transcripts due to a strand-independent addition of H3K36 methylation is a key regulatory feature of interleaved transcriptomes.Maintenance of a regular chromatin structure over the coding regions of genes occurs co-transcriptionally via the ‘chromatin resetting' pathway. One of the central players in this pathway is the histone methyltransferase Set2. Here we show that the loss of Set2 in yeast,  Maintenance of chromatin structure in coding regions is partially dependent on transcription, with histone methyltransferase Set2 playing a role in this process. Here, the authors provide evidence that Set2 regulates repression of a specific set of antisense RNAs embedded within the coding genes. Advances in genomics technologies have led to the system-wide identification of transcription units in several organisms. One feature common to transcript profiles from different organisms is its pervasiveness—a consequence of transcription occurring from large parts of the genome in addition to the protein-coding genesTo offset the limited improvements in the sensitivity to which low abundant RNA can be confidently identified, current strategies are designed to enhance RNA stability, so that they fall within the detection range of the latest next-generation sequencers. One method is to disrupt the RNA-degradation pathways, leading to increased abundance of these transcripts, aiding subsequent sequencing using the highly sensitive microarray or Next-generation sequencing techniques456Along with degradation, a number of other mechanisms work to limit pervasive transcription. A recent screenThe process of chromatin dynamics over promoters is well understood, with the identification of key factors and elucidation of molecular mechanisms15161720Antisense transcripts are a class of ncRNA that are transcribed from the strand complementary to protein-coding genes or non-coding transcription units SET2 deletion mutant (set2), indicating strand-specific effects of strand-independent addition of histone modifications. Identification of these transcription units would serve as a valuable resource to understand both the function and regulation of ncRNA.Using deep sequencing methods, we have identified and annotated a novel group of antisense transcripts, called Set2-repressed antisense transcripts (SRATs), which are de-repressed upon loss of Set2. These transcription units are usually buried within the coding region of genes. We show that elongation over the promoters of overlapping transcription units prevents chromatin dynamics, suppressing transcription initiation from these promoters. Despite an increased expression of these internal antisense units, sense transcription elongation is largely unaffected in the SET2, we purified total RNA from the deletion and wild-type yeast strains in replica. With the goal of discerning RNA production from both strands of DNA, we prepared libraries from ribodepleted total RNA using the strand-specific RNA sequencing kits (Illumina), and subjected the libraries to deep sequencing (Methods). High level of correlation between the replicates attested the reproducibility of the samples.To identify the transcripts produced upon the deletion of YDR452W  of SRAT expression upon SET2 deletion (On the basis of a bioinformatics identification pipeline (Methods), we identified a list of 853 antisense transcripts that were de-repressed upon loss of the Set2 histone methyltransferase. We named these SRATs. SRAT282 is an example, found associated with YDR452W . We compdeletion , left.SET2 deletion  tail at the 3′ end of a nascent transcript. To measure of the stability of the SRATs, we determined whether some of these transcripts were polyadenylated (Methods). The poly-A-enriched fraction was subjected to strand-specific RNA-seq, with a similar genomic coverage as for the ribodepleted total RNA samples. Interestingly, 532 SRATs are polyadenylated, accounting for almost 50% of the SRATs identified . These sdeletion , right, deletion .set2 mutant, it was detected at very low levels in the wild-type samples as well , with radiolabelled riboprobes against four specific SRATs generated from our annotation. All four SRATs demonstrated distinct Set2-dependent expression , right b as well . Further as well , right. SET2, we transformed both wild-type and the set2 mutant strains with either an empty vector or a full-length Set2 yeast expression cloneset2 strain resembles the wild-type strain (To confirm that the identified SRATs were indeed the consequence of the deletion of e strain . The denset2 mutant with the empty vector (similar to the set2) compared with the deletion strain with the full-length expression vector  when compared to the BY4741 wild-type e strain . This ine strain . Compari clearly . HoweverWe wanted to compare the genomic spread and transcript lengths of the SRATs with previously identified coding and non-coding transcripts . Protein4In contrast to the unstable non-coding transcripts, which can only be identified by enhancing RNA stability or production, SUTsSET2-mediated H3K36 methylation, initiate from the 3′ end of genes and terminate within the coding region.On the basis of the per cent of the genome covered by each class of transcript, we can conclude that most of these transcripts overlap with each other, giving rise to an interleaved transcriptome. Given that the SRATs are predominantly antisense to previously identified transcripts, we wanted to decipher their organization with respect to these transcription units. For this purpose, we focused on three parameters as shown in Given a critical role of H3K36 methylation in preventing the initiation of antisense transcription, we wanted to determine whether the levels of this methyl mark was altered in the genes with buried antisense transcripts compared with all the genes in the yeast genome. For this purpose, we analysed the ChIP-microarray data of the distribution of H3K36 trimethylation normalized to the H3 levels that we published previouslyset2 mutant  at the 3′ end of genes, while the other half initiates from within the body of genes  in the wild-type strain were plotted against the fold-change in ncRNA expression in the ild-type . We thenild-type . This suild-type .set2 . Interestingly, the expression of the SUT is confirmed by the K4–K36 methylation distributionAZR1 gene. Interestingly, loss of functional Set2 in the deletion mutant, results in the de-repression of the AZR1 gene, despite the presence of the SUT  by the co-transcriptional addition of the H3K36 methyl mark. Similarly, ncRNA transcription over the promoter of a protein-coding genes also suppresses its expression through a similar mechanism. To validate the mechanism of transcription-mediated regulation of overlapping transcripts, we used the SUT569-anscript . We delensferase . This esSet2-mediated H3K36 methylation is a repressive mark added over gene bodies during transcription elongation. However, it is a selective mark, suppressing transcription initiation from promoters within overlapping transcription units, without affecting ongoing transcription elongation. Artificially tethering Set2 methyltransferase enzyme to an activator and directing its localization to gene promoters has been shown to result in transcriptional repressionSET2 deletion mutant enhances the stability of SRATs.Organizing the yeast genome into overlapping and interleaved transcription units utilizes the process of RNAPII transcription to recruit the Set2 methyltransferase to add the H3K36 methyl mark and regulate gene expression. An interesting consequence of linking H3K36 methylation-dependent transcriptional suppression on one strand to transcription on the other is the removal of regulatory burden from the synthesized RNA. Interestingly, the SRATs are targeted for immediate degradation. Loss of the nuclear Rrp6 or nuclear Xrn1 RNA-degradation pathways in a in cis, we cannot speculate on the possible effect of the ncRNA in trans based on our data.Our observation that a subset of CUTs, SUTs and XUTs are suppressed by H3K36 methylation suggests that ncRNA production is regulated both at the level of transcription—by chromatin regulatory factors—and RNA stability—determined by the access of RNA to the various RNA-degradation machineries. While our observation that a significant number of SRATs are polyadenylated would suggest longer half-lives and possible cytoplasmic localization, most are targeted for degradation. The function of the polyadenylated SRATs in the cytoplasm is currently unknown, but they may be subjected to degradation by the nonsense-mediated decay pathway, as is the case with some CUTs and SUTsOur analysis has identified a number of novel transcripts that are suppressed by Set2-mediated H3K36 methylation. The annotation of these novel transcripts is a crucial first step in determining their function in the regulation of various cellular processes in yeast. Since these novel non-coding transcripts have the potential to be a part of gene regulatory circuits35H3K36 methylation has been shown to be involved in a number of nuclear processes including DNA damage response3738392047S. cerevisiae strains used in this study are listed in All g. The supernatant was poured into a 50 ml Phase Lock gel tube, mixed with an equal volume of chloroform and spun at 3,000 r.p.m. for 10 min. The aqueous supernantant containing the RNA was precipitated using isopropanol and sodium acetate. The RNA was washed with 70% ethanol and dissolved in an appropriate amount of 10 mM Tris pH8.0 prepared in DEPC water. The RNA was quantitated on a NanoDrop machine and its purity estimated using the absorbance ratio at 260 nm over 280 nm.Total RNA was prepared using the hot-phenol method as described previouslyFor polyA selection, 4 μg of total RNA in 750 μl of RNAse-free water was incubated at 65 °C for 10 min and added to 75 mg of oligo dT cellulose , pre-washed and resuspended in an equal volume of 1 × NETS buffer , for 1 h on a rotator at room temperature. This mixture was poured into a disposable Bio-Rad column and allowed to settle. The mixture was gently washed five times with 1 × NETS buffer. The mRNA was eluted twice with 1 × ETS buffer  heated to 65 °C. The eluates were pooled and the RNA was precipitated by standard techniques, dissolved and quantitated.Total RNA isolated from the specified strains was ribodepleted using the Ribo-ZeroTM Magnetic Gold Kit (Yeast)  according to manufacturer's instructions. The depleted RNA was then used for strand-specific library construction utilizing the TruSeq Stranded Total RNA LT . The libraries from eight samples were pooled together and run on a single lane of HiSeq2500 machine for sequencing 50 bp single end reads. Poly A+ libraries were generated using the TruSeq Stranded mRNA Kit .Since identification of low-abundance novel transcripts leads to discontinuity, fragmented annotation and ambiguous end definitionSET2 deletion strain to the yeast genome (based on sequence dated April 2011 in the Saccharomyces Genome Database (http://www.yeastgenome.org/) and was obtained from the site ftp://ftp.ncbi.nlm.nih.gov/genbank/genomes/Eukaryotes/fungi/Saccharomyces_cerevisiae/SacCer_Apr2011) and identified all transcripts arising from each strand using the Cufflinks program. A five-nucleotide read gap was used to separate different transcripts. We used the BEDtools bioinformatics suite to refine this list of novel transcripts to remove known coding transcripts and their untranslated regions4SET2 deletion strain. This requirement also ensured that transcripts with breaks in the reads were removed from the analysis. Second, transcripts with a FDR of <5%  were selected. Finally, we instituted a cutoff at twofold increase in the abundance of the transcripts in the SET2 deletion mutant over the wild-type, to select ones that are upregulated upon loss of Set2. These parameters helped us obtain a list of 853 transcripts that we named SRATs.We aligned the unique reads obtained from the set2 mutant were grown in 200 ml of YPD, crosslinked and treated with zymolase to spheroplast the yeast cells. The spheroplasts were digested with MNase as described earlier−1 in buffer Z) was added to a final concentration of 0.5 mg per ml and incubated at 30 °C with continuous mixing in a water bath. After >90% of the cells have spheroplasted, the mixture was pelleted at 7,000 g, and resuspended in 2 ml of NP buffer  with 500 μM spermidine, 1 mM B mercaptoethanol and 0.075% NP-40. Three 600 μl aliquots of cells were used to titrate MNase  amounts and incubated at 37 °C for 20 min. to achieve the formation of 80% of mononucleosomes. The reactions were stopped with 1 × STOP buffer (1% SDS and 10 mM EDTA). The samples were treated with Proteinase K (20 mg per ml) at 65 °C, removed the RNA and purified the DNA by phenol:chloroform extraction and ethanol precipitation. The purified DNA was subjected to paired-end 50-base reads using HiSeq2500 sequencer. The reads were aligned to the sacCer3 genome using bowtie2 with default settings. The paired reads were filtered (100- to 200-base fragments only) and centred. The reads were visualized using ngsplot. Normalized coverage genome browser tracks were generated in R.Wild-type and Data were analysed using the R package. Differential expression analysis was carried out using the limma package in R. All plots and graph were created using inbuilt codes in R.The SRATs were sorted according to their chromosomal location, irrespective of the strand they are on and numbered chronologically. The full list of SRATs is provided in NET-Seq raw data were downloaded from public databases and aligned to the yeast genome. Reads for SRATs and protein-coding genes were extracted and used for analysis using existing codes in R.set2 mutant.Coordinates of genomic regions 500 bp upstream of the SRAT start site on the sense strand were used to score the NET-Seq data. The data were filtered for a twofold change in NET-seq signals and a minimum log2 NET-Seq signal of 0.5 RPKM in the SET2 mutant compared with the wild-type and an SRAT length >1,500 nt. These parameters were chosen to ensure that these low-abundance transcripts could be reproducibly detected in the northern blots. Multiple PCR primers were generated for each SRAT, SUT or XUT, such that the length of each probe ranged from 500–800 bp. Depending on the strand targeted by the riboprobe, a T7 promoter primer was added to the ends of the either the forward or reverse primer. The probe was amplified using yeast genomic DNA using the Taq DNA polymerase. The amplicons were purified and estimated. 1 μg of the probe DNA used to prepare the riboprobes using the MAXIscript Kit  with 32P labelled UTP (Perkin Elmer). The labelled probed were purified by using microspin G50 columns to remove unincorporated radionucleotides. The primers used to prepare the riboprobes are listed in For validating the production and confirming SRAT expression, we carried out strand-specific northern blot analysis using riboprobes. We selected a few SRATs based on a fold change in expression greater than twofold in a −1) was separated on a 1.5% agarose gel running in 1 × MOPS buffer  with 6.6% formaldehyde. After separating the RNA, the gel was visualized with ultraviolet light and washed thrice with distilled water remove formaldehyde. The gel was then soaked in 10 × SSC (1.5 M NaCl and 150 mM sodium citrate) and the RNA transferred to a Zeta probe membrane overnight. The blots were incubated at 50 °C for 2–4 h in prehybridization buffer . The riboprobes were added to the buffer after heating at 95 °C for 2 min and incubated overnight at 50 °C. The blots were washed twice at 50 °C using the wash buffer  and exposed on phosphorimager cassettes from GE. The images were scanned on a Typhoon phosphorimager and quantified using the IQ software. The abundance of the SRAT was normalized to either ACT1 or SCR1 transcripts. Original uncropped blots of all northern blots used for the figures and the quantitation are shown in Northern blots were carried out with either 20 μg of total RNA or 2 μg of polyadenylated RNA, as described previouslyWhole-cell extracts for yeast were prepared according to protocol published elsewhereHHF2-HHT2 is provided on plasmid covering the deletions of both genomic copies of the histone genes. An auxotrophic marker switch was used to replace the wild-type copy of histones with the H3K36A mutant. The H3K36A-pDM18 plasmid was obtained from the Stowers Molecular Biology Facility as a part of the Shima mutant libraryThe FY2162 strainH3 K36 trimethylation ChIP was analysed from our previous publicationhttp://trace.ncbi.nlm.nih.gov/Traces/study/?acc=SRP089706&go=go) under accession number SRP089706. The NET-Seq data were downloaded from GEO database with the accession number GSE 25107. The H3K36 trimethylation ChIP-Seq data were downloaded from ENA database with the accession number ERP007035. Original data underlying this manuscript can be accessed from the Stowers Original Data Repository at http://www.stowers.org/research/publications/libpb-1115. All other original data can be obtained from the authors upon reasonable request.The Sequencing data generated during this study are available at NCBI SRA Browser .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-9.List of significant SRATs that are poly A+ enriched.List of genes upregulated upon loss of Set2 in the poly A+ enriched RNA-Seq.List of Age related genes with SRATs .SRATs upregulated in the aging yeast cells based on the Hu et al. 2014 study.List of yeast strains and their genotypes used in the study.Chromosome co-ordinates of the identified SRATs and their nomenclature.List of primers used in the study.


Emerging coronaviruses  pose serious health threats globally, with no specific antiviral treatments available. These viruses are able to faithfully synthesize their large genomic RNA. We report, however, that their main RNA polymerase, nsp12, is not accurate. To achieve accuracy, CoVs have acquired nsp14, a bifunctional enzyme able to methylate the viral RNA cap [methyltransferase (MTase)] and excise erroneous mutagenic nucleotides inserted by nsp12. Strikingly, ribavirin can be excised from the viral genome, thus showing no antiviral activity. The crystal structure of nsp14 shows that it is unique, having been replaced by other MTase types during evolution. This unprecedented RNA correction machinery has allowed RNA genome size expansion, but also provided potential nucleoside drug resistance to these deadly pathogens. Coronaviruses (CoVs) stand out among RNA viruses because of their unusually large genomes (∼30 kb) associated with low mutation rates. CoVs code for nsp14, a bifunctional enzyme carrying RNA cap guanine N7-methyltransferase (MTase) and 3′-5′ exoribonuclease (ExoN) activities. ExoN excises nucleotide mismatches at the RNA 3′-end in vitro, and its inactivation in vivo jeopardizes viral genetic stability. Here, we demonstrate for severe acute respiratory syndrome (SARS)-CoV an RNA synthesis and proofreading pathway through association of nsp14 with the low-fidelity nsp12 viral RNA polymerase. Through this pathway, the antiviral compound ribavirin 5′-monophosphate is significantly incorporated but also readily excised from RNA, which may explain its limited efficacy in vivo. The crystal structure at 3.38 Å resolution of SARS-CoV nsp14 in complex with its cofactor nsp10 adds to the uniqueness of CoVs among RNA viruses: The MTase domain presents a new fold that differs sharply from the canonical Rossmann fold. Faithful transmission of genetic information is central to all living organisms. In the RNA virus world, RNA-dependent RNA polymerases (RdRps) lack co- and postreplicative fidelity-enhancing pathways, and final RNA genome copies incorporate mutations at a much higher rate than that observed for DNA genomes . The lowCoVs include two highly pathogenic viruses responsible for severe acute respiratory syndrome (SARS)  and MiddOutside CoVs and other members of the Nidovirales order, 3′-5′ ExoN activity is only found in the Arenaviridae family . For exaNsp14 can bind to several viral partners. It can associate with the polymerase complex (nsp12/nsp8/nsp7), and the resulting complex retains all associated enzymatic activities  . It can In the present study, we have solved the SARS-CoV nsp14 crystal structure. We show that the molecule flexibility is essential in the interaction with the viral polymerase. We visualize conformational changes responsible for nsp10-mediated ExoN stimulation using X-ray crystallography and small-angle X-ray scattering (SAXS) methods. We have reconstituted the CoV RNA proofreading pathway, by which a ribonucleotide misincorporated into RNA is excised by an active replicase complex. Rbv 5′-monophosphate is incorporated at the 3′-end of RNA by the SARS-CoV polymerase. It is also efficiently excised by the nsp14-ExoN activity, which acts as a proofreading activity. Because of its dual role in synthesis and repair of viral RNA, this multifunctional replicase complex represents an attractive target for designing novel compounds active against CoVs. Finally, the crystal structure reveals that the nsp14 N7-MTase domain, strikingly, does not belong to the canonical Rossmann fold MTase family, making nsp14 a unique RNA processing enzyme and giving it a central role in large RNA genome evolution.12121, with cell dimensions a = 185.97 Å, b = 189.74 Å, c = 195.93 Å, and α = β = γ = 90°, and containing four heterodimers in the asymmetrical unit  using the anomalous scattering signal of zinc A. The crcal unit . The foucal unit A, while cal unit B.i) a flexible N terminus forming the major part of contacts with nsp10 (cyan), followed by (ii) the exonuclease domain (orange), (iii) a flexible hinge region formed by a loop and three strands (gray) (iv) a C terminus N7-MTase domain (pink). The surface defined by the C terminus end of the ExoN domain  is reminiscent of a saddle, which may accommodate a protein partner (discussed below). The nsp14 structure compared with the one published  a s (gray) C, and . The most conserved feature of the Rossmann fold family, the βαβ structural motif, is absent, as are standard MTase motifs in the protein sequence, explaining why it had escaped detection upon sequence alignment-based investigations -binding residue  and 1/135,000  (We compared the SARS-CoV polymerase complex (formed of nsp12/nsp8/nsp7) and Dengue virus (DENV) NS5 for their efficiency of single “correct” or “incorrect” nucleotide incorporation into RNA . Both pomispair) . In contmispair) B. Using mispair) . U:G, C:mispair) .In conclusion, the SARS-CoV polymerase exhibits a nucleotide insertion fidelity in vitro that is surprisingly lower than that of DENV RdRp. It shows a general tendency to be error-prone, despite the low mutation rate observed at the virus level .P < 0.001). In line with reverse genetics findings  is a primary determinant of mutation rates for RNA viruses. Overall, a figure of 10al RdRps . Drake  the acquisition of nsp14-ExoN as an active part of the replication complex to increase fidelity has allowed relaxation of nucleotide selection by the viral RdRp, and (ii) as a consequence of this compensated low fidelity, larger RNA “coronavirus-like” genomes but encoding for a RdRp with much better intrinsic fidelity may exist; together with an associated ExoN, there might be further evolutionary space to satisfy Drake’s rule of approximately one mutation per replicated “larger” genome. Clearly, the origin and fate of nidovirus nsp14 sequences, structures, and activities may be significantly informative in the history of RNA-to-DNA genome evolutions.Our results unveiled a second unexpected finding: The SARS-CoV polymerase complex exhibits a significantly lower fidelity compared with that of DENV RdRp, a virus with a genome one-third of its size. From this observation, we hypothesize that  are compelling targets for antiviral therapy, designing nucleoside analogs and specific nuclease inhibitors concomitantly might prove a valid option against viruses endowed with RNA repair systems.2+-bound proteins were eluted with 200 mM imidazole. A second chromatography step was performed on a Hiload 16/60 Superdex 200 column .All plasmids used in this study are described by Subissi et al. , except 6-tag (The SARS-CoV nsp14-coding sequence was cloned into expression vector pDEST14 (Thermo Fisher Scientific) to produce a recombinant protein carrying an N terminus His6-tag . Site-di6-tag .Rwork = 19.6% and Rfree = 23.4% and was confirmed to have good stereochemistry according to MOLPROBITY ] of protein complex nsp10/nsp14 (4 mg/mL) in gel filtration buffer versus 600 μL of reservoir solution containing 0.1 M trisodium citrate (pH 5.5), 8% PEG 8000, and 30% hexanediol. Crystals were cryoprotected with a reservoir solution supplemented with 20% PEG 200, prior to being flash-cooled in liquid nitrogen. Diffraction data were collected at beamline Proxima1 at the Soleil synchrotron. The MAD data were collected to 3.5 Å resolution at the peak (λ = 1.28242 Å) and inflection (λ = 1.28282 Å) wavelengths for zinc. Higher resolution (3.38 Å) data were collected from a single crystal of the native complex (λ = 1.28348 Å). Datasets were processed and analyzed with the autoPROC toolbox , and theLPROBITY . Data coLPROBITY .32P]ATP (PerkinElmer), and are hereafter termed LS2*, LS2-Rbv*, and LS3*. LS2* was then annealed to either LS1, LS15, LS16, or LS17; LS2-Rbv* was annealed to LS15; and LS3* was annealed to LS8 by heating at 70 °C for 10 min and then cooling down to room temperature (with a primer/template ratio of 1.2:1).Synthetic RNAs  were purchased from Biomers (HPLC grade), and LS2-Rbv was synthesized by Dharmacon/Thermo Fisher Scientific (HPLC grade). 2] supplemented with the SARS-CoV polymerase complex . The percentage of active enzyme was estimated at <10% using titration of primer/template with increasing enzyme concentrations and manual burst analysis. Reactions were performed at 30 °C using indicated concentrations of either one NTP, Rbv-TP (Jena Bioscience), or all NTPs (at 500 μM each). For incorrect nucleotides, product formation followed a linear increase of incorporation product up to 20 min, and 18 min was chosen as a suitable time for steady-state kinetic studies. Reactions were quenched by the addition of an equal volume of loading buffer (formamide with 10 mM EDTA). RNA polymerization products were analyzed in polyacrylamide/7 M urea gels. RNA products were visualized using photostimulated plates and a phosphorimager (Fuji).All polymerase reactions were preincubated for 30 min at 30 °C, containing 0.5 μM radiolabeled primer annealed to a template in reaction buffer [20 mM Tris (pH 8), 10 mM KCl, 1 mM DTT, 2 mM MgCl2O, and RT was performed with a forward primer (5′-GTATCCCCATCTCATTTTA-3′) using a OneStep RT-PCR Kit (QIAGEN). After heating to 95 °C , the reverse primer was added (5′-GTCATTCTCCTAAGAAGC-3′). Cloning of the PCR products was performed with the CloneJET PCR Cloning Kit (Thermo Fisher Scientific), and plasmid DNAs were sequenced using a third primer (5′-CGATGAGTTTTCGGTATTATC-3′) by Eurofins Genomics. The reported P value was calculated using a Z-test for comparison of two proportions.RNA products to be sequenced were excised and electroeluted using a GEBAFLEX extraction kit (Interchim), following the manufacturer’s instructions. Recovered RNAs were precipitated and resuspended in HSupplementary File


Site-directed RNA editing is an approach to reprogram genetic information at the RNA level. We recently introduced a novel guideRNA that allows for the recruitment of human ADAR2 to manipulate genetic information. Here, we show that the current guideRNA design is already able to recruit another human deaminase, ADAR1, in both isoforms, p110 and p150. However, further optimization seems necessary as the current design is less efficient for ADAR1 isoforms. Furthermore, we describe hotspots at which the guideRNA itself is edited and show a way to circumvent this auto-editing without losing editing efficiency at the target. Both findings are important for the advancement of site-directed RNA editing as a tool in basic biology or as a platform for therapeutic editing. Site-directed RNA editing is a method to recode genetic information at the RNA level . The appHuman cells express three different forms of ADAR (adenosine deaminase acting on RNA), called ADAR1, 2, and 3, which are potentially re-addressable for site-directed RNA editing A 2,8]. . 8]. BesHere, we demonstrate that a trans-acting guideRNA that has recently been developed for the recruitment of ADAR2 is also capable of recruiting both ADAR1 isoforms. Furthermore, we studied the auto-editing of the guideRNA itself, and present novel guideRNA sequences that are less prone to auto-editing but still allow for the recruitment of ADAR2.6 cells were seeded on a 10 cm dish. After one day, 1 µg of the respective ADAR1-form in a pcDNA5 vector under control of the tet-on CMV promoter, and 9 µg of pOG44 expressing the Flp recombinase were transfected with lipofectamine 2000 (30 µL). One day later, the medium was changed for at least two weeks to selection medium . Cells were kept in selection medium prior to the editing experiment, which was then performed in the absence of antibiotics.The 293 Flp-In T-REx system (Life Technologies) was used for stable integration of a single copy of the cDNA of ADAR1p110 or ADAR1p150 at a genomic FRT-site in engineered 293 cells. Briefly, 4 × 10 2. Cells (3 × 105/well) were seeded into poly-d-lysin-coated 24-well plates and induced with doxycycline (10 ng/mL). Transfection was carried out 24 h later with lipofectamine 2000, applying 300 ng of W58x GFP and 1300 ng of guideRNA per well. Editing was evaluated 72 h after transfection by isolation and sequencing analysis of the target mRNA. For the latter, total RNA from the cells  was DNaseI-digested, followed by reverse transcription, Taq-PCR amplification, and Sanger sequencing.Protocol for experiments shown in cis was in-vitro-transcribed with T7 RNA pol as described earlier [2+) = 3 mM, no heparin, no spermidine, and incubation in editing buffer  for 180 min while cycling between 30 °C and 37 °C.Protocol for the experiment shown in 5/well) were seeded into 24-well plates. Transfection was carried out 24 h later with lipofectamine 2000, applying 300 ng of wt eGFP-guideRNA and 600 ng of the respective ADAR per well. RNA was isolated for sequencing 48 h after co-transfection as described above.Protocol for experiments shown in In this study, we started from a recently developed guideRNA architecture  that is The original guideRNA design is relatively AU-rich. Editing could suffer from the auto-editing of the guideRNA itself thus destabilizing the hairpin. We tested this in an initial editing experiment on an in-vitro transcribed RNA substrate that contained the eCFP W66amber sequence in frame with a cis-acting guideRNA inside the PCR tube . Under tTo reduce auto-editing of the guideRNA, 3, 6, or 13 A/U base pairs in the original R/G-motif have been substituted by G/C base pairs, creating variants 2, 3, and 4 A. To assUnder these assay conditions, the starting guideRNA architecture, version 1, gives only subtle auto-editing, and only at positions A8 by ADAR2 and A13 by ADAR1p110. With ADAR2, we found an additional off-target editing at A52, which is part of the mRNA binding sequence. For ADAR1p150, no auto-editing was detectable. It is reasonable to infer that the 36 bp RNA substrate , given in The G/C substitutions do not only remove editable adenosines and put adenosines into contexts non-preferred for editing , but they rather increase the free energy gain of folding from −18 kcal/mol up to −42 kcal/mol as estimated by the software mfold  Figure A. We tesThis is the first report about site-directed RNA editing with human ADAR1 isoforms, the constitutive (p110) and the inducible one (p150). As the R/G-guideRNA was developed from a classical ADAR2 substrate for the recruitment of ADAR2, it could be expected that the recruitment of ADAR1 isoforms is less efficient. However, we achieved site-directed RNA editing with the R/G-guideRNAs with both ADAR1 isoforms, in particular with the p150 form; thus, the general strategy is feasible with ADAR1, and our R/G-guideRNA provides a starting point for the development of more appropriate guideRNAs for ADAR1.cis, we found full conversion at the targeted adenosine even in cell culture (trans, editing levels above 60% are difficult to obtain. It is likely that there is still space for further optimization of the guideRNA′s design to obtain better editing results with all three enzymes. In summary, both findings are important for the advancement of site-directed RNA editing as a tool in basic biology or as a platform for therapeutic editing [Furthermore, we show the first results on auto-editing of the guideRNA itself. We have defined the hotspots for auto-editing and show a simple strategy to circumvent it by minimal substitution in the hairpin secondary structure. The respective optimized guideRNAs are much less auto-edited; however, our results show that a high substitution degree may interfere with editing efficiency at the target. Even though the confinement of auto-editing was not improving the overall editing yield under the given continuous expression of the guideRNA, it could improve editing when drug-like, chemically stabilized R/G-guideRNAs are explored in the future. Notably, when defining the hotspots of auto-editing by applying an artificial eCFP mRNA containing the guideRNA in  culture . In cont editing ,28.


A report on the Wellcome Trust Conference on Computational RNA Biology, held in Hinxton, UK, on 17–19 October 2016. Recent years have witnessed a profound shift in our understanding of RNA biology. Several novel biochemical and sequencing techniques are producing vast amounts of data that fundamentally challenge the textbook view of RNA as a simple intermediate step of gene expression, revealing a wealth of unexpected new roles and shedding light on the complexity of the RNA world. While the emerging picture unequivocally points to the centrality of RNA as a mediator of most cellular functions, the richness and heterogeneity of modern datasets pose significant interpretative challenges and call for an interdisciplinary approach where statistical and computational methods will play an increasingly important role.The Wellcome Trust Conference on Computational RNA Biology provided a good opportunity to overview the state of the art in this up-and-coming interdisciplinary field. Organised by the scientific committee of Alex Bateman , Ivo Hofacker , Karissa Sanbonmatsu  and Mihaela Zavolan , the conference was held at the Wellcome Genome Campus in Hinxton, near Cambridge (UK) on 17–19 October 2016. Featuring two keynote talks by Christine Mayr  and Ben Blencowe , thirteen invited talks and fourteen short contributed talks, the conference provided a very broad survey of quantitative and computational RNA biology. These were further complemented by two lively poster sessions, where participants had an opportunity to engage with over 40 posters during evening drinks receptions.In this report, we briefly recount the content of the conference by providing condensed, headline-style summaries of the research described in the talks and some posters. Within the scope of this brief report, we cannot possibly do justice to the wealth and breadth of material presented and we will not be able to mention much interesting research, particularly within the poster sessions. We would like to stress that omissions in this report are not based on quality, but simply on a personal judgement as to what material could be most coherently presented in a very limited space.Perhaps the most remarkable discovery in modern RNA biology is the realization of the diversity of the transcriptome. Technologies based on next-generation sequencing (NGS) have demonstrated the existence of many novel classes of transcripts and the great variety of protein-coding transcripts, in terms of both isoforms and synonymous variants. The diversity of the transcriptome and its interaction with phenotypes was the main theme of both keynote talks. Ben Blencowe (University of Toronto) introduced the concept of alternative splicing regulatory networks and their role in development and autistic spectrum disorders. Blencowe illustrated how analysis of NGS data has enabled the discovery of a novel class of micro-exons (3–27 nucleotides) that are strongly conserved and whose alternative exclusion is associated with the autism phenotype. Isoform quantification methods were discussed by Eduardo Eyras , who explained how the SUPPA method achieves high computational performance by decoupling read mapping from transcript annotation. Methodologies for isoform quantification from time series RNA-seq data using the DICEseq method were also presented in the poster session by Yuanhua Huang . Naturally, the presence of isoform RNA molecules does not immediately imply isoform expression at the protein level, as translational regulation may preferentially select only a subset of isoforms. This question was addressed by Lorenzo Calviello , who used ribosome profiling data and the Splice-aware Translational Annotation (SaTAnn) tool. This analysis revealed that almost 55% of genes (in human HEK293 cells) translate a single isoform, and highlighted widespread translational control. SaTAnn also received the Best Acronym Award, beating stiff competition from CRAC and BUM-HMM (see below).′ UTRs can give rise to dramatically different functions in the protein they code for. A prominent example is given by the CD47 transcript in human: variants with a long 3′ UTR are preferentially bound by the HuR protein (due to the abundance of HuR binding sites on the long UTR), which then leads to membrane localization of the nascent protein, while CD47 proteins synthesized from a short 3′ UTR variant remain in a perinuclear localization. Shorter transcript variants can also arise from alternative use of polyA sites, the presentation topic of Christina Leslie , although in this case the shorter transcript mostly results in a truncated protein or in a non-coding RNA (ncRNA).While alternative splicing has long been recognized as a major determinant of the diversity of the transcriptome, recent research is also shedding light on the functional significance of synonymous variants, i.e. transcripts that differ only in the non-coding region. Christine Mayr  described how transcript variants with different 3The discovery of a great variety of novel ncRNAs was also one of the major breakthroughs of NGS technologies; ncRNAs remain, however, largely mysterious in their biological function. Albin Sandelin  described data from cap analysis of gene expression (CAGE) experiments illustrating the pervasiveness of bidirectional transcription, often giving rise to mRNA–ncRNA pairs. He further explained how genomic features such as density of polyA sites or closely spaced transcription start sites influence ncRNA expression. Igor Ulitsky  used synteny to elucidate the function and origin of lincRNAs (long intergenic ncRNAs), highlighting a modest level of sequence conservation, partly explained by the presence of enhancers within lincRNAs. Sequence comparison methodologies, initially developed to study paralog genes, were also discussed by Jana Hertel  to address ncRNA evolution. Finally, Todd Lowe  used chromatin data from the Encyclopedia Of DNA Elements (ENCODE) project to discover widespread epigenetic regulation of the human tRNA transcriptome.RNAs in vivo fold in complex secondary and 3D structures. It is widely believed that RNA structures play a major regulatory role in determining the possible interaction partners of RNAs, and ultimately, their function. The computational biology community has long had a sustained interest in predicting RNA structures and the conference witnessed several interesting presentations on the matter.While in principle feasible configurations could be computed by minimizing free energies derived from microscopic physical principles, the computation is in general prohibitively complex. Simon Poblete  presented a novel approach to coarse-graining the state space of possible configurations, leading to considerable accelerations in molecular dynamics simulations. Other talks described approaches that instead use auxiliary data to bypass the difficult step of molecular simulations. Craig Zirbel  described JAR3D, a set of probabilistic models parametrized on the RNA 3D Motif Atlas, that infer new 3D motifs from sequences. Debora Marks  described how evolutionary couplings can be used within global probability models to improve the predictive power of optimisation algorithms. Evolutionary arguments can also be invoked to exploit pairwise covariations in multiple RNA alignments to deduce the conservation of RNA secondary structures. This line of reasoning was used by Elena Rivas  to argue against the conservation of secondary structures in long ncRNAs, stirring a certain level of debate within the conference. Mutation patterns underlying structure conservation were also employed by Zasha Weinberg (University of Leipzig) to discover a new group of riboswitches (metabolite-binding RNAs) and by Martin Smith  to cluster evolutionarily conserved RNA structural patterns.A major source of excitement within the RNA structure community is the development of novel sequencing-based techniques for structure probing in vivo. High-throughput experiments using a variety of probing agents are being performed at an increasing pace and Yiliang Ding  described FoldAtlas, a curated repository for such data that is likely to become a precious resource. In poster sessions, Krishna Choudhary  highlighted the importance of quality control by presenting metrics for rapid quality assessment of structure probing data. Alain Laederach  showed how structure probing techniques led to the discovery of riboSNitches, mutations in the non-coding part of a transcript which can alter the secondary structure of the UTR, leading to functional changes with often dramatic associations with disease phenotypes. Alina Selega (University of Edinburgh) described BUM-HMM, a novel probabilistic model for controlling for biological variability within high-throughput structure probing data. Mirko Ledda  presented a probabilistic model to incorporate structure probing data into a pseudo-free energy term used in folding prediction algorithms.Another major focus of the conference was the discussion of the RNA interactome. Indeed, many of the exciting discoveries in recent RNA biology are due to the exceptional flexibility of RNA as an interacting molecule, acting on DNA, other RNAs and proteins. Characterizing these interactions quantitatively is a primary avenue of research in RNA biology, both experimentally and computationally.Caenorhabditis elegans. He identified a large number of proteins (>600 in both species) with a very high degree of conservation. Remarkably, a large fraction of RNA-binding proteins (RBPs) turned out to be metabolic enzymes interacting with RNAs belonging to the same metabolic pathways. Guido Sanguinetti (University of Edinburgh) discussed computational models of cross-linking and cDNA (CRAC) data (another UV cross-linking technique), which was used to model the changes in polymerase post-translational modifications during transcription, and the fast kinetics of co-transcriptional degradation during stress induction. Bojan Zagrovic (University of Vienna) presented data supporting the hypothesis that translation originated by direct interactions between codons and amino acids, an old idea from Carl Woese that is now being tested with modern technologies.Protein–RNA interactions are primarily identified via cross-linking with ultraviolet (UV) light using the cross-linking and immunoprecipitation (CLIP) protocol. Rolf Backofen  described GraphProt, a computational approach to detect RNA–protein binding motifs from CLIP data in a supervised learning pipeline using sequence and (predicted) structural features. Andre Gerber  used cross-linking to determine the entire mRNA-interacting proteome in yeast and Escherichia coli by measuring interactions of sRNAs bound to the Hfq protein, enabling the detection of several hundred new RNA–RNA interactions. Paul Gardner  discussed new results indicating that mRNA sequences appear to be under selection in order to avoid random interactions with ncRNAs. Surprisingly, avoidance of ncRNAs seems to be a better predictor of protein abundance than codon usage.RNA–RNA interactions also play a central role in many regulatory processes; Yair Gatt  described RIL-seq, a modification of the cross-linking, ligation and sequencing of hybrids (CLASH) method, to identify targets of small RNAs in ′-O-methylation, a common epi-transcriptomic modification that may contribute to ribosome diversity, eventually tuning translation. Jorg Morf  described a new bead-based method for assaying RNA–RNA proximity, yielding highly reproducible results. Fabian Amman (University of Vienna) presented a computational approach to optimize cell-free translation in synthetic biology applications. In poster sessions, Qi Liu  demonstrated the first secondary structure measurements of pre-mRNAs in vivo, obtained with a high-throughput nuclear RNA structure probing method. Michael Clark  outlined a novel technique combining full-length cDNA sequencing and targeted RNA sequencing, which aims to assist isoform quantification. Stefanie Ebersberger  presented an in vitro derivate of individual-nucleotide resolution CLIP (iCLIP) for generating the intrinsic binding landscape of an RBP, and the computational tools for comparison with iCLIP.A number of talks in the workshop reported the development of new methodologies for both novel experiment types and technological applications. Martin Jansson  described how RiboMeth-seq enables measurement of 2The wealth of material presented in the talks and posters naturally stimulated lively discussions about the outstanding challenges in the field. We try here to capture the spirit of these discussions, naturally from our own (biased) perspective. One major impression is that, while many of the data-generating technologies are increasingly becoming quantitative, many of the interpretations still rely on qualitative models, rather than predictive mathematical models. A related topic is the almost complete absence of modeling efforts towards illustrating the dynamical aspects of RNA life. One exception was the talk by Nacho Molina  on transcriptional control by transcription factors in single cells, which heavily drew on modeling the stochastic dynamics of transcription initiation. Similar models for understanding the post-transcriptional dynamics of RNAs are in short supply and represent a major area of future development. Finally, most computational talks address single data types; most likely, novel biological insights could arise from joint modeling of multiple data types. Integrative models featured in the posters of Philipp Boss  in the context of integrating different CLIP protocols and Ronny Lorenz (University of Vienna), who described generic methods to integrate auxiliary data in structure prediction. We expect that such approaches will become more widespread in the future, and look forward to hearing more about them at the next Computational RNA Biology conference in 2 years time.


Lily virus X (LVX), which infects lilies, was determined for the first time from lilies in Japan. As with previous reports, the genome of the Japanese LVX isolate lacked an AUG start codon for the triple gene block protein 3-like region.The complete genome sequence of  Lily virus X (LVX) is a member of the genus Potexvirus in the family Alphaflexiviridae (Lilium formosanum) from England , one of the movement proteins  were collected in the Chubu District of Japan in 2016. Total RNA was extracted from leaves using Isogen reagent . A paired-end sequencing cDNA library was constructed from the extracted RNA using a TruSeq RNA sample prep kit v.2  and sequenced using a MiSeq instrument (Illumina) and a MiSeq reagent kit v.2 (500 cycles). After quality control and trimming using Trimmomatic v.0.36 software  was 5,824 nucleotides (nt) long, excluding the poly(A) tail at its 3′ end. It contained four open reading frames (ORFs) encoding RNA-dependent RNA polymerase (RdRp) (nt 73 to 3969), TGBp1 (nt 3999 to 4649), TGBp2 (nt 4649 to 4975), and coat protein (CP) (nt 5110 to 5715). The TGBp3-like region (nt 4893 to 5129) lacked an AUG start codon, as in the case of the previously reported isolate (Lily virus X isolate J has been deposited in the DNA Data Bank of Japan and GenBank under the GenBank accession number LC335818.The complete genome sequence of


Here, we determine crystal structures of tandem RRM domains of hnRNP A2/B1 in complex with various RNA substrates, elucidating specific recognitions of AGG and UAG motifs by RRM1 and RRM2 domains, respectively. Further structural and biochemical results demonstrate multivariant binding modes for sequence-diversified RNA substrates, supporting a RNA matchmaker mechanism in hnRNP A2/B1 function. Moreover, our studies in combination with bioinformatic analysis suggest that hnRNP A2/B1 may mediate effects of m6A through a “m6A switch” mechanism, instead of acting as a direct “reader” of m6A modification.Human hnRNP A2/B1 is an RNA-binding protein that plays important roles in many biological processes, including maturation, transport, and metabolism of mRNA, and gene regulation of long noncoding RNAs. hnRNP A2/B1 was reported to control the microRNAs sorting to exosomes and promote primary microRNA processing as a potential m 6A 'reader'. Here, the authors determine crystal structures of RRM domains of hnRNP A2/B1 in complex with various RNA substrates and determine that hnRNP A2/B1 may function as an auxiliary factor in 'm6A switch' instead.RNA-binding protein hnRNP A2/B1 is suggested to promote miRNA processing as a m Each hnRNP contains at least one RNA-binding domain (RBD), such as RNA recognition motif (RRM), K-Homology (KH) domain, or an arginine/glycine-rich box3. Sequence-specific association between hnRNPs and their RNA targets are typically mediated by one or more RBDs, which usually bind short, single-stranded RNA5, but in some instances, also recognize structured RNAs6.Heterogeneous nuclear ribonucleoproteins (hnRNPs) play a variety of roles in regulating transcriptional and post-transcriptional gene expression, including RNA splicing, polyadenylation, capping, modification, export, localization, translation, and turnoverHNRNPA2B1 gene encodes two protein isoforms, A2 and B1, through alternative splicing. The B1 isoform contains an insertion of 12 amino acids (aa) at its N terminus8. Both isoforms have an RNA-binding domain (RBD) composed of tandem RRMs separated by a 15-aa linker, and a C-terminal Gly-rich low complexity (LC) region that includes a prion-like domain (PrLD), an RGG box, and a PY-motif containing a M9 nuclear localization signal (PY-NLS)11. These domains are represented schematically with residue numbers based on the hnRNP B1 isoform , through promoting excess incorporation of hnRNP A2/B1 into stress granules and driving the formation of cytoplasmic inclusions in animal models14. A transcriptome-wide analysis of hnRNP A2/B1 targets in the nervous system identified a clear preference for UAG(G/A) motifs confirmed by three independent and complementary in vitro and in vivo approaches16. This is consistent with previous studies indicating that hnRNP A2/B1 binds specifically to UAGGG, GGUAGUAG, or AGGAUAGA sequences18. Another recent study demonstrated that hnRNP A2/B1 recognizes a consensus motif containing UAASUUAU (S = G or C) in the 3′ UTR of many mRNAs and helps recruiting the CCR4-NOT deadenylase complex19. In addition to participating the regulation of mRNAs, hnRNP A2/B1 is also involved in the activities of many other RNA species. For example, hnRNP A2/B1 can promote association of the long noncoding RNA HOTAIR with the nascent transcripts of HOTAIR target genes, thus to mediate HOTAIR-dependent heterochromatin initiation20.hnRNP A2/B1 has multiple effects on RNA processing through binding specific sequence. It can bind to HIV-1 RNA, causing nuclear retention of the vRNA, as well as microRNAs, sorting them into the exosomes through binding “EXO-motifs”N6-methyladenosine, a widespread nucleotide modification in mRNAs and noncoding RNAs22. hnRNP A2/B1 was found to mediate m6A-dependent nuclear RNA processing events by binding G(m6A)C-containing nuclear RNAs in vivo and in vitro, in which hnRNP A2/B1 associates with a subset of primary microRNA transcripts through binding m6A, promoting primary microRNAs processing by recruiting the microprocessor complex Drosha and DGCR823.Recently, hnRNP A2/B1 was proposed to bind RNA transcripts containing 6A “reader,” since no significant preference for m6A modification by either tandem RRMs or full-length protein of hnRNP A2/B1 are observed.Although a series of studies with different approaches pointed out the multitudinous functions of hnRNP A2/B1 mediating by diverse RNA motifs in vivo, no detailed mechanism for the binding specificities has been determined. Thus, more biochemistry and structural studies are essential to understand the RNA-binding properties of hnRNP A2/B1 at the molecular level. Here, we report the crystal structures of hnRNP A2/B1 in complex with variant RNA targets to unveil the RNA-binding specificities and multivariant characteristics. Moreover, our structural data along with RNA-binding and bioinformatic analysis do not support that hnRNP A2/B1 functions as an mTo elucidate the RNA-binding properties of tandem RRMs of hnRNP A1/B1, we purified a number of truncations of the hnRNP A2/B1 protein. Using isothermal titration calorimetry (ITC) method, we characterized the RNA-binding activities of each construct with a set of RNA oligonucleotides of sequences according to previously determined binding motifs. ITC results showed that the construct containing the N-terminal fragment (aa 1–11) and two RRM domains, i.e., aa 1–195, can bind target RNAs with high affinity. In addition, deletion of the N-terminal fragment had no obvious impact on the RNA binding. This suggested that the construct containing the tandem RRMs of hnRNP A2/B1 is sufficient for binding target RNAs.1G2G3A4C5U6G7C8-3′ (termed 8mer RNA), which is derived from a recent individual-nucleotide-resolution CLIP study23. ITC analysis showed that binding of the 8mer RNA occurs at a 1:1 ratio with a Kd of 276.2 nM  of hnRNP A2/B1 bound to the 8-nt RNA oligonucleotide 5′-A nM Fig. . The cry2–195 of  nM Fig. ; both RR 24 Fig. . Each RN 24 Fig. .1), the 2′-OH group forms a hydrogen bond and π–π interactions with the side chain of His108. Besides hydrogen bonding interactions, base stacking with the Phe24 on the other side also contributes to the definition of the binding environment  has a higher affinity  in complex with the 8mer RNA did not provide insight into specific RNA recognition by RRM2. We thus designed another RNA oligonucleotide shown in Supplementary Table ity Fig. . The 10mity Fig. . Unlike ity Fig. . When thity Fig. . Howeverity Fig. . Moreove1 and G2 in the 10mer RNA complex structure are observed. N1 of A1 is recognized by the main chain amine group of Val97, whereas N6 and N7 form hydrogen bonds with the Lys94 side chain either directly or mediated by a water molecule  is hydrogen bonded to the Glu92, and the N1 and N6 of A4 form hydrogen bonds with the side chain of Lys120 and Asn181 mediated by water, respectively  and RRM2 (111–195) can be superimposed with a root-mean-square deviation of 0.901 Å with a high sequence identity , the AGG motif is shifted to 5′-end and recognized by RRM1 in a manner almost identical to the wild-type 10mer RNA structure  in complex with different 10mer RNA mutants. Three complex structures containing RNA mutants A1G, U6G, and A7U were determined at high resolution Fig. . For theure Fig. . In addiion Fig. . Therefoion Fig. .Fig. 4RN6 is substituted with G in the U6G complex (5′-A0A1G2G3A4C5G6A7G8C9-3′), G6 lost two hydrogen bonds from Arg99 and Arg185, only keeping hydrogen bonds with Glu182. However, the recognition of the AG core motif by RRM2 is still well maintained , U7 forms hydrogen bonds with the side chain of Lys113 and Glu142 and forms a π–π stacking interaction with Phe157. More interestingly, U6 adopts a sandwich-like interaction mode with Phe115 and the A4 base, which is exactly same as the U6 in the 8mer RNA substrate  were reduced onefold and tenfold, respectively, compared to the non-methylated RNA . This domain may also contribute in some way to RNA binding, but was not included in our structural study , and used them to measure the binding affinities with various RNA substrates by EMSA and ITC experiment 34. In this analysis, we quantified binding at each of these 168 mapped m6A residues by assigning each m6A residue an “intensity value”, which was the normalized number of miCLIP reads that overlapped each m6A residue34. The intensity value is influenced by transcript abundance and m6A stoichiometry. We next determined the binding of hnRNP A2/B1 at each of these m6A sites based on the normalized number of mapped hnRNP A2/B1 HITS-CLIP tags at the m6A site. For most m6A residues, there was no correlation between m6A intensity and hnRNP A2/B1 binding, although 12 m6A sites showed proximal hnRNP A2/B1 binding, which might be the result of coincidental proximity between m6A and a non-methylated consensus site recognized by hnRNP A2/B1. As a control, we analyzed YTHDC1, a nuclear YTH domain-containing m6A reader36. A similar analysis of YTHDC1 showed increasing YTHDC1 binding with increased m6A levels for essentially all m6A sites  RNA . Thus, YTHDC1 has a considerably higher overlap with m6A than hnRNP A2/B1. Therefore, this analysis again supports the idea that YTHDC1 is the predominant nuclear m6A reader compared to hnRNP A2/B1.Our finding that only a small subset of nuclear mRNA Fig.  showed a38. Recent CLIP-Seq data further showed that hnRNP A2/B1 prefers to bind A/G-rich sequences17. However, there was no molecular basis for the recognition of different RNA substrates of hnRNP A2/B1. Here, we determined the crystal structures of the tandem RRMs of hnRNP A2/B1 in complex with various RNA substrates, revealing the molecular details of specific target RNA recognition and shedding light on the mechanism for hnRNP A2/B1 in various RNA-mediated biological functions.The RNA-binding domain of hnRNP A2/B1 comprises two RNA recognition motifs, RRM1 and RRM2, which is followed by a C-terminal glycine-rich region. hnRNP A2/B1 was previously demonstrated to bind UUAGGG and UAG RNA motifs through various analyses39 and provides an explanation for how sumoylated hnRNP A2/B1 directs the loading of specific EXO-miRNAs into exosomes by binding GAGG, the so-called EXO motif14. Furthermore, our results provide the structural basis for hnRNP A2/B1 binding to the UA-rich UAASUUAU motif in the 3′ UTR of some mRNAs, which was shown to be necessary for loading the CCR4-NOT complex to mRNAs19. Taken together, our structures illustrate the sequence-specific RNA-binding properties of hnRNP A2/B1 and give support to previously reported diverse binding sites18  by promoting local unfolding of RNA. This unfolding is due to the weaker base pairing of U with m6A compared to A41. m6A induced RNA unfolding and increases the accessibility of hnRNP C to single-stranded RNA, and is therefore termed an “m6A-switch”42. HuR, also known as ELAVL1, has been found to preferentially bind to the 3′-UTR region of mRNAs that lack m6A43. In this case, m6A could impede the formation of a structured RNA motif needed for HuR binding. Our structural study, combined with biochemistry and bioinformatic results, suggest that m6A switches may account for the previously seen enhanced hnRNP A2/B1 binding adjacent to m6A. Instead of direct binding to m6A, m6A may promote accessibility of hnRNP A2/B1 to certain binding sites, thereby explaining how m6A can facilitate the ability of hnRNP A2/B1 to enhance nuclear events such as pri-miRNA processing. Further in vitro and in vivo investigations will be required to uncover the details of this mechanism.It has recently been demonstrated that hnRNP A2/B1 specifically recognizes mBamHI and XhoI, then ligated into a modified pET-28a plasmid carrying the Ulp1 cleavage site. Mutations were generated based on the overlap PCR. Recombinant plasmids were confirmed by DNA sequencing and transformed into Escherichia coli BL21 (DE3) to produce target proteins with N-terminal hexahistidine-sumo fusions. E. coli cells were cultured in LB medium at 37 °C with 50 mg/l kanamycin until the OD600 reached 0.6–0.8, then the bacteria were induced with 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) at 18 °C for 16 h. Bacteria were collected by centrifugation, resuspended in buffer containing 20 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM imidazole pH 8.0, and lysed by high pressure. Cell extracts were centrifuged at 38,758 × g for 1 h at 4 °C. Supernatants were purified with Ni-NTA (GE), the target protein was washed with lysis buffer and then eluted with a buffer containing 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 500 mM imidazole. Ulp1 protease was added to remove the N-terminal tag and fusion protein of the recombinant protein and dialyzed with lysis buffer 3 h. The mixture was applied to another Ni-NTA resin to remove the protease and uncleaved proteins. Eluted proteins were concentrated by centrifugal ultrafiltration, loaded onto a pre-equilibrated HiLoad 16/60 Superdex 75-pg column in an Äkta-purifier , eluted at a flow rate of 1 ml/min with the same buffer containing 10 mM Tris-HCl pH 8.0, 100 mM NaCl. Peak fractions were analyzed by SDS-PAGE  and stained with Coomassie brilliant blue R-250. Purified fractions were pooled together and concentrated by centrifugal ultrafiltration. The concentration was determined by A280. The protein was concentrated to 10 mg/ml for crystallization trials.Plasmids encoding different fragments of hnRNP A2/B1 were PCR amplified from the human cDNA. PCR products were double digested with restriction endonuclease 6A: 5′-AGGm6ACUGC-3′, 10mer-m6A: 5′-AAGGm6ACUAGC-3′) with m6A modification were ordered from Dharmacon (Thermo Scientific.), and the other unmodified or unlabeled RNA oligonucleotides were synthesized by the IDT-394 synthesizer in our own lab. The 5′-FAM-labeled RNA chains (5′-FAM-GGACU-3′ and 5′-FAM-GGm6ACU-3′) were ordered from Bioneer Corporation. All the RNA oligonucleotides used for crystallization and biochemical experiments in this study are summarized in Supplementary Table The RNA oligonucleotides (8mer-mhnRNP A2/B1 RRMs (12–195) in complex with 8-nt-RNA 5′-AGGACUGC-3′ was crystallized using the hanging drop vapor diffusion method by mixing 1 μl of protein–RNA mixture (molar ratio 1:1.2) and 1 μl of reservoir solution at 20 °C. The crystals suitable for X-ray diffraction were grown in reservoir solution consisting of 0.1 M Tris pH 8.5 and 25% (w/v) polyethylene glycol 3,350 (Hampton Research). hnRNP A2/B1 RRMs (12–195) in complex with 10-nt-RNA 5′-AAGGACUAGC-3′ was screened as above. The crystal suitable for X-ray diffraction was grown in reservoir solution consisting of 0.2 M Tri-sodium citrate and 20% (w/v) polyethylene glycol 3,350 (Hampton Research). A1G, U6G, and A7U were crystallized as the methods mentioned above in solution containing 20% PEG 3,000, 0.1 M sodium citrate pH 5.5; 20% PEG 3,350, 0.2 M lithium sulfate, 0.1 M Bis–Tris pH 6.5; 25% PEG 1,500, 0.1 M MMT pH 9.0, respectively. Data collection were performed at 100 K with cryo-protectant solution  glycerol). Diffraction data were collected using a wavelength of 0.97776 Å at beamline BL18U1 of the Shanghai Synchrotron Radiation Facility (SSRF).44. Cycles of refinement and model building were carried out using REFMAC5 and COOT until the crystallography Rfactor and Rfree converged to 19.16% and 23.62%, respectively46. Ramachandran analysis showed that 96.0 of the residues were in the most favored region, with 4.0% in the additionally allowed region. For hnRNP A2/B1-10-nt complex, the diffraction data set was processed and scaled using the HKL3000 package. The phase was determined by molecular replacement using the program Phaser with the hnRNP A2/B1(12–195) model collected before as the search model. Cycles of refinement and model building were carried out using REFMAC5 and COOT until the crystallography Rfactor and free Rfree converged to 18.39% and 22.27%, respectively. Ramachandran analysis showed that similarly to hnRNP A2/B1-8-nt, 99% of the residues were in the most favored region, with 1% in the additionally allowed region. For another three complex structures, the same methods were used to solve the structures as mentioned above. The details of data collection and processing are presented in Supplementary Table For hnRNP A2/B1 RRMs (12–195)-8-nt complex, the diffraction data set was processed and scaled using HKL3000. The phase was determined by molecular replacement using the program Phaser with the structure of UP1 (PDB code: 1U1Q) as the search modelGo of protein–RNA binding was computed as RTln(1/KD), where R, T, and KD are the gas constant, temperature and dissociation constant, respectively.ITC assays were carried out on a MicroCal ITC200 calorimeter  at 25 °C. The buffer used for proteins and RNA oligomers was 10 mM HEPES pH 8.0, 50 mM KCl, 1 mM EDTA, and 1 mM BME. The concentrations of proteins were determined spectrophotometrically. The RNA oligonucleotides were diluted in the buffer to 5–15 μM. The ITC experiments involved 20–30 injections of protein into RNA. The sample cell was loaded with 250 μl of RNA at 5 μM and the syringe with 80 μl of protein at 100 μM; for weak complexes, the measurement was repeated with increased concentrations. Reference measurements were carried out to compensate for the heat of dilution of the proteins. Curve fitting to a single binding site model was performed by the ITC data analysis module of Origin 7.0  provided by the manufacturer. ΔKd from three independent experiments.Aliquot of 0.5 μM of FAM-labeled RNA was mixed with increasing concentrations of full-length hnRNP A2/B1 proteins in a buffer containing 10 mM HEPES pH 8.0, 50 mM KCl, 1 mM EDTA, and 5 mM beta-mercaptoethanol in a total volume of 10 μl and incubated at room temperature for 30 min. The electrophoresis was performed with 6% native-PAGE at 4 °C in running buffer containing 0.5× Tris-borate-EDTA (TBE) buffer. The gel was visualized by using a Typhoon FLA-9000  using a method for FAM (Laser 488 nm). Bound and free RNA were quantified using ImageJ. Binding curves were fit individually using GraphPad Prism 6.0 software fitting with “One site – Specific binding with Hill slope” (GraphPad Software). Curves were normalized as percentage of bound oligonucleotides and reported is the mean ± SD of the interpolated Proteins of RRM1 and RRM2 domains of hnRNP A2/B1 were purified using the same procedure as hnRNP A2/B1 (12–195). Analytical gel filtration chromatography was performed at 4 °C using a Superdex 75 10/300 global column  pre-equilibrated in 20 mM Tris, pH 8.0, 100 mM NaCl. Aliquots of 100 μl of samples including various hnRNP A2/B1 constructs were injected at a flow rate of 0.3 ml/min. To study complex formation, RRM1 and RRM2 proteins were mixed and incubated on ice for at least 1 h prior to loading.23 . In addition to the raw data, the author uploaded sequence alignment files, GSM1716539_A2B1_HITS_CLIP_1.bedgraph.gz and GSM1716539_A2B1_HITS_CLIP_2.bedgraph.gz were also obtained from the GEO database for comparison purposes. Robust crosslinking-induced mutation sites (CIMS) (FDR ≤0.001) in hnRNP A2/B1 HITS-CLIP data were called using a method published elsewhere47. UV-induced deletion sites48 were used as hnRNP A2/B1-binding sites.Nuclear hnRNP A2/B1HITS-CLIP sequence data were obtained from a previously published study6A-seq data from MDA-MB-231 cells were obtained from a previously published study49 . Adapter-free, high-quality sequence reads were aligned to the hg19 genome build using bowtie2 according to the source publication49. RPM (reads per million mapped reads) was calculated using bedtools.Nuclear m6A sites in HEK293T cells, miCLIP sequencing data34 (GEO accession number: GSE63753) and YTHDC1 iCLIP data35 (GEO accession number: GSE78030) were obtained from the GEO database. Sequence alignments were carried out according to the respective publications. Images of genome alignments were prepared using IGV genome browser and Adobe Illustrator.For YTHDC1 binding at m6A stoichiometry at 10 bp flanking miCLIP sites34 on nuclear RNAs such as MALAT1, NEAT1, and XIST was compared using an XY-scatter plot in R. Only m6A sites conforming a non-BCANN consensus were considered for this analysis. These represent unique sites obtained from merging (mergeBed -s -d 2) of CIMS- and CITS-based m6A site calls from ref. 34. All the rRNA, tRNA, and mitochondrial genomic miCLIP sites were removed. Tag counting was performed using the bedtools suite. Tag counts (uTPM + 1) were compared using scatter plots and Pearson correlation coefficients (r) were determined in R. Cluster overlap analysis was carried out using bedtools intersect tool (intersectBed -s -u).hnRNP A2/B1 or YTHDC1 binding and mThe coordinates that support the findings of this study have been deposited in the Protein Data Bank with accession codes 5EN1 for RRMs-8mer-RNA complex and 5HO4 for RRMs–10mer RNA structures. A1G, U6G, and A7U are 5WWE, 5WWF, and 5WWG, respectively. Other data in this study are available from the corresponding author on reasonable request.Supplementary InformationPeer Review File


The k-turn is a powerful building block in the construction of nano-scale molecular objects, exemplified by a quasi-circular six-k-turn object. D3 symmetry with a shape reminiscent of that of a cyclohexane molecule in its chair conformation. This work demonstrates that the k-turn is a powerful building block in the construction of nano-scale molecular objects, and illustrates why k-turns are widely used in natural RNA molecules to organize long-range architecture and mediate tertiary contacts.k-Turns are widespread RNA architectural elements that mediate tertiary interactions. We describe a double-kink-turn motif comprising two inverted k-turns that forms a tight horse-shoe structure that can assemble into a variety of shapes by coaxial association of helical ends. Using X-ray crystallography we show that these assemble with two (dumbell), three (triangle) and four units (square), with or without bound protein, within the crystal lattice. In addition, exchange of a single basepair can almost double the pore radius or shape of a molecular assembly. On the basis of this analysis we synthesized a 114 nt self-complementary RNA containing six k-turns. The crystal structure of this species shows that it forms a quasi-cyclic triangular object. These are randomly disposed about the three-fold axis in the crystal lattice, generating a circular RNA of quasi  One of 26 In principle such objects can enclose molecular pores of different sizes. k-Turns suggest themselves as a very natural choice of construction unit for this purpose, and Saito and colleagues28 have explored this previously.Thus k-turns may be regarded as a natural nano-scale building block in RNA. RNA has previously been used as a material for engineering on the molecular scale, and helical bulges have been used to create regular structures of square or triangular shape.i.e. six k-turns in total, with three loops on each strand) within one double-stranded RNA molecule would close to form a triangular structure. We have synthesized this structure and demonstrated its quasi cyclic structure by X-ray crystallography. This illustrates the precision with which the k-turn organizes three-dimensional RNA structure, and introduces the two-k-turn unit as a valuable construction unit in nano-technological engineering of RNA structure.In this work we have closely examined the packing of a double-stranded RNA containing two k-turns related by two-fold rotational symmetry in different crystal lattices . These t2 where the loops lie on opposite strands (29 These are connected through their NC helices (i.e. the helices with the G·A pairs), with ten basepairs separating the two loops, including the G·A pairs .29 Within the crystal lattice two-k-turn units interact through their termini end-to-end, giving an overall dumbell shape with two-fold rotational symmetry symmetry  and S2†.29The end-to-end association of helices by which the two-k-turn units associate is closely similar in both structures . The ter30 and conformation (whether they adopt the N3 or the N1 conformation).29 We have solved new crystal structures for two further forms of the two-k-turn unit in which the 3b,3n sequences of the Kt-7 k-turns were changed either to U·U or to GC. Both sequences confer the N3 conformation,29 but neither is well folded in the presence of metal ions alone.30 These were therefore co-crystallized in the presence of Archeoglobus fulgidus L7Ae protein (AfL7Ae) which drives the folding of k-turns33 irrespective of 3b,3n sequence.30We have shown that the 3b,3n sequence of k-turns plays a critical role in determining both folding characteristics P212121) with a resolution of 2.65 Å . Three two-k-turn units bound to six L7Ae proteins formed a triangular assembly in the lattice with three-fold rotational symmetry  with a resolution of 2.87 Å . In contrast to the 3b,3n = U·U structure, four two-k-turn units have associated in the lattice to form a square structure with four-fold rotational symmetry , covalently linked through their C helices. The 3′ cytosine nucleotides of the two-k-turn units were changed to uridine, so that the connected NC helices contain central U·G and G·U pairs. In our experience this adds torsional flexibility, improving the probability that a regular helical conformation could be maintained in the connection between the NC helices, and we found that our crystals diffracted to significantly higher resolution when these were included  and solved the structure at a resolution of 2.75 Å  (Sigma-Aldrich) and incubated at 65 °C for 2.5 h to remove t-BDMS protecting groups. All oligonucleotides were purified by gel electrophoresis in polyacrylamide in the presence of 7 M urea. The full-length RNA product was visualized by UV shadowing. The band was excised and electroeluted using an Elutrap® Electroelution System  into 45 mM Tris-borate (pH 8.5), 5 mM EDTA buffer for 8 h. at 200 V at 4 °C. The RNA was precipitated with ethanol, washed once with 70% ethanol and suspended in water.Ribooligonucleotides were synthesized using A. fulgidus L7Ae was cloned into a modified pET-Duet1 plasmid (Novagen).37 A hexahistidine-L7Ae fusion protein was expressed in E. coli BL21-Gold (DE3) pLysS cells (Stratagene). The protein was purified by chromatography on HisTrap . After removal of the N-terminal six-histidine tag the protein was further purified by chromatography on heparin, followed by gel filtration on Superdex 200.The gene encoding full-length The crystallized construct of the RNA had the sequence : GGCGAAGAUCCGGUGAGCC. This self-complementary sequence forms the secondary structure identical to that in 2 was incubated for 5 min at 37 °C. Crystals were grown by vapor diffusion using drops prepared by mixing 1.0 μL of the RNA–protein complex with 1 μL of a reservoir solution comprising 1.6 M tri-sodium citrate dihydrate (pH 6.5) at 18 °C. Crystals appeared after 5 days. The crystals were flash frozen by mounting in nylon loops and plunging into liquid nitrogen. The crystals were characterized in-house with a MicroMax HF007 copper rotating anode X-ray generator equipped with an ACTOR sample changer system and a Saturn 944HG+ CCD detector (Rigaku). Suitable crystals were stored and full datasets subsequently collected on beamline I02 at Diamond Light Source .A mixture of 0.25 mM RNA and 0.25 mM L7Ae in 5 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM MgClP212121 and unit cell dimensions a = 261.3 Å, b = 69.2 Å, c = 85.4 Å. From the crystal density,39 three RNA–protein complexes were expected to be present in the asymmetric unit.The crystals had space group 40 Ramachandran analysis shows that 98.7% of amino acid residues are in the most favored and additionally allowed regions. Model geometry and the fit to electron-density maps were monitored with MOLPROBITY41 and the validation tools in COOT.The structure was determined by molecular replacement, with PDB 4BW0 as the search model using the program PHASER.The crystallized construct of the RNA had the sequence: GGCGAAGAGCCGGCGAGCC. This self-complementary sequence forms the secondary structure identical to that in 2 was incubated for 5 min at 37 °C. Crystals were grown by vapor diffusion using drops prepared by mixing 1.0 μL of the RNA–protein complex with 1 μL of a reservoir solution comprising 0.2 M ammonium acetate, 10 mM calcium chloride, 50 mM sodium cacodylate (pH 6.5) and 10% w/v polyethylene glycol 4000 at 18 °C. Crystals appeared after 7 days. They were transferred to a solution containing an additional 30% w/v polyethylene glycol 200 for ∼2 s. The crystals were flash frozen by mounting in nylon loops and plunging into liquid nitrogen. Suitable crystals were stored and subsequently used to measure full datasets on beamline I02 of Diamond Light Source .A mixture of 0.3 mM RNA and 0.3 mM L7Ae in 5 mM Tris·HCl (pH 8.0), 100 mM NaCl, 10 mM MgClC121 and unit cell dimensions a = 118.7 Å, b = 70.6 Å, c = 92.8 Å. From the crystal density,39 three RNA–protein complexes were expected to be present in the asymmetric unit.The crystals had space group 40 Ramachandran analysis shows that 99.8% of amino acid residues are in the most favored and additionally allowed regions. Model geometry and the fit to electron-density maps were monitored with MOLPROBITY41 and the validation tools in COOT.The structure was determined by molecular replacement with PDB 4BW0 as the search model using the program PHASER.The crystallized construct had the sequence: GGCGAAGAACUGGGGAGCUGGCGAAGAACUGGGGAGCUGGCGAAGAACUGGGGAGCU.2 was added to a final concentration of 10 mM. The hanging-drop vapor diffusion method was used for crystallization. A volume of 1.0 μL of RNA was mixed 1 : 1 with well solution comprising 3.5 M Na formate, 0.1 M Na acetate (pH 4.6) at 20 °C. Crystals (approximate dimensions 100 × 40 × 40 μm) with space group P6322 grew in about 10 days. Crystals were briefly washed in well solution supplemented with 30% glycerol. The crystals were flash frozen by mounting in nylon loops and plunging into liquid nitrogen. A 2.75 Å resolution dataset was collected on beamline ID23-1 of European Synchrotron Radiation Facility.This self-complementary sequence forms the structure shown in 43The resolution cutoff for the data was determined by examining both CC1/2 and difference map of the magnesium ions, as described previously.P6322 and unit cell dimensions a = 70.3 Å, b = 70.3 Å, c = 47.8 Å. From the crystal density,39 only one sixth of the duplex was expected to be present in the asymmetric unit.The crystals had space group H. marismortui Kt-7  as the search model using the program PHASER.40The structure was determined by molecular replacement with 44 and RCrane.45 The structure was refined with Refmac5 46 from the CCP4 suite of programs47 and Phenix refine.48 Model geometry and the fit to electron-density maps were monitored with MOLPROBITY41 and the validation tools in COOT.Structural models were built in CootAtomic coordinates and structure factor amplitudes have been deposited with the PDB with accession codes 5G4U, ; 5G4V and ; 5G4T.The authors declare no competing financial interests.


Influenza polymerase replicates, via a complementary RNA intermediate (cRNA), and transcribes the eight viral RNA (vRNA) genome segments. To initiate RNA synthesis it is bound to the conserved 5΄ and 3΄ extremities of the vRNA or cRNA (the ‘promoter’). 5΄-3΄ base-pairing in the distal promoter region is essential to position the template RNA at the polymerase active site, as shown by a new crystal structure with the 3΄ end threading through the template entry tunnel. We develop fluorescence polarization assays to quantify initiation of cap-primed (transcription) or unprimed (replication) RNA synthesis by recombinant influenza B polymerase bound to the vRNA or cRNA promoter. The rate-limiting step is formation of a primed initiation complex with minimally ApG required to stabilize the 3΄ end of the template within the active-site. Polymerase bound to the vRNA promoter initiates RNA synthesis terminally, while the cRNA promoter directs internal initiation at a significantly lower rate. Progression to elongation requires breaking the promoter 5΄-3΄ base-pairing region and favourable compensation by the emerging template-product base-pairs. The RNA synthesis assay is adaptable to high-throughput screening for polymerase inhibitors. In a pilot study, we find that initiation at the cRNA promoter is unusually susceptible to inhibition by 2΄F-2΄dNTPs. Whilst influenza A is more variable and potentially more dangerous to humans due to its pandemic potential, influenza B is also clinically important and recently some vaccines have been augmented to contain two A strains and two B strains to take into account the divergence in the influenza B lineage (de novo) producing a full-length complementary, positive-sense anti-genomic RNA (cRNA). The cRNA co-transcriptionally assembles with polymerase and NP to form a cRNP . Furthermore, especially virulent strains of influenza virus originate sporadically and unpredictably, either by spontaneous mutations or by genome segment re-assortment between existing viruses of human and/or animal origin . Influen lineage . One pro lineage ,8 show t lineage ,10. In t lineage –14 to gem a cRNP  which ism a cRNP .in vitro RNA synthesis assay to quantitatively characterize primed and unprimed RNA synthesis by influenza polymerase using a model system comprising full-length recombinant influenza B polymerase, a promoter consisting of the separated conserved 5΄ and 3΄ (anti-) genome ends and no NP. Using the assay, we determined the enzymatic parameters of influenza B virus polymerase. We also address the mechanistic question of how the same polymerase initiates RNA synthesis either terminally or internally, directed only by the respective cRNA or vRNA promoter bound. By analyzing RNA synthesis from chimeric promoter RNAs, a cooperation of the 5΄-3΄ base-pairing region and the template-directed initial base-pairs was found to be responsible for allowing or preventing terminal initiation to proceed to elongation while an appropriate primer by-passed the rate-limiting formation of the first phosphodiester bond that yields ApG. This work is complemented by a new X-ray structure of influenza B polymerase co-crystallized with the vRNA promoter and a capped primer where we see for the first time 3΄ end of the template within the active site cavity. The assay we developed is easily adaptable to high-throughput-screening of compound libraries for polymerase inhibitors. By characterizing the inhibition profile of a set of nucleotide-analogues, we demonstrate the unusual susceptibility of polymerase initiating RNA synthesis from the cRNA promoter to 2΄F-2΄dNTPs and provide evidence that early initiation is more prone to inhibition than elongation.Here, we establish a fluorescence polarization based d-desthiobiotin in buffer A. Fractions containing the target protein were pooled and diluted with two volumes of buffer B  glycerol, 2 mM Tris(2-carboxyethyl)phosphine (TCEP), pH 7.4) prior to loading on a heparin column . Proteins were eluted by a gradient of buffer B supplemented with 1 M NaCl. Homogeneous monomeric influenza B polymerase was concentrated  and stored in aliquots at −80°C. Protein concentration was determined by measuring the absorbance at 280 nm and using the extinction coefficient 287 300 M−1 cm−1.The influenza B/Memphis/13/03 polymerase heterotrimer was expressed as a self-cleaving polyprotein and purified as described  with min7GpppAAUCUAUAAUAG-3΄ (for enzymatic studies) or 5΄-m7GpppAAUCUAUAAUAGC-3΄  and the 2΄F-2΄dNTPs were purchased from TriLink BioTechnologies . All other RNAs used were purchased from IBA  with sequences listed in Capped-RNAs with the sequence 5΄-m7GpppAAUCUAUAAUAGC-3΄) in a buffer containing 500 mM NaCl, 50 mM HEPES pH 7.5, 5% glycerol and 2 mM TCEP. The best diffracting crystals appeared in 100 mM sodium acetate pH 3.8–4.0 and 150 mM di-ammonium phosphate and had a diamond shape. They were frozen with an additional 25% glycerol for cryo-protection. Data were collected on ID23-1 beam line at the ESRF and structure solved by molecular replacement, using the known FluB polymerase structure (PDB 4WRT)  equipped with polarizers with excitation and emission wavelengths set to 495 and 515 nm, respectively. Total fluorescence intensity was corrected for the volume change due to addition of protein and the observed fluorescence anisotropy corrected for the change in fluorescence intensity according to Equation , dissociation constants were derived by fitting the binding isotherms to a 1:1 binding model of RNA and protein according to Equation  in binding buffer  glycerol, 2 mM Tris(2-carboxyethyl)phosphine (TCEP), pH 7.4) at the concentrations indicated. After attaining equilibrium, total fluorescence intensity and fluorescence anisotropy were measured at Equation :(1)\docT = 25°C. After attaining equilibrium (∼10 min), fluorescence intensity and polarization were recorded with excitation and emission filters of 485 and 520 nm, respectively . Observed fluorescence polarization was corrected for changes in intensity and normalized according to Equation  in binding buffer  glycerol, 2 mM Tris(2-carboxyethyl)phosphine (TCEP), pH 7.4) with 3΄ RNAs labelled at their 5΄ end by Cy3 at the concentrations indicated. After attaining equilibrium (∼0.5 h), the FAM's fluorescence intensity was recorded as averaged technical triplicates . The decreasing fluorescence intensity with increasing Cy3-labelled 3΄ RNA is plotted relative to its initial value as mean values of at least duplicate experiments with the standard deviation indicated. Mean values were then analysed according to Equation , yielding the corresponding dissociation constants and error of the fitting routine stated. 0.2 μM FAM-Ex-5-labelled v5΄ nt 1–10 were used to study its weak interaction with either Cy3-labelled v3΄ nt 1–9 or v3΄ nt 1–18.RNA–RNA interactions were quantified by a simple FRET-assay. Briefly, ∼0.01 μM short 5΄ RNAs labelled at their 3΄ ends by FAM-Ex-5 were incubated at 2, 2 mM TCEP, pH 7.4) at T = 24°C to initiate the RNA synthesis reaction. RNA synthesis from the cRNA promoter was performed analogous to the vRNA promoter by using the corresponding cRNA-sequences. To monitor RNA-primed polymerization, reactions were supplemented by 0.5 mM ApG  or by 1 μM of capped-RNA  if not indicated otherwise. The reactions were incubated in polypropylene reaction tubes and at indicated times aliquots  transferred to reaction tubes containing the quenching solution (80 μl 4.5 M NaCl) to reach 4 M NaCl final. After attaining equilibrium, fluorescence polarization (FP) signals were recorded with a CLARIOstar microplate reader  in 384-well plates  at T = 25°C with excitation and emission filters of 485 and 520 nm, respectively. Progress curves were fitted to pseudo-first order reactions according to Equation , MK the Michaelis constant, x the concentration of capped RNA and A the maximal RNA-synthesis reaction.Changes of spectroscopic properties associated with FAM-labelled short single-stranded RNA becoming double-stranded are the principle for the new assay monitoring RNA synthesis activity in solution. If not indicated otherwise, 0.25 μM influenza B polymerase pre-incubated with a 1.2 molar excess of v5΄ RNA 1–14 (5΄-pAGUAGUAACAAGAG-3΄) were added to 0.15 μM v3΄ FAM-labelled template RNA (5΄-FAM-Ex-5-UAUACCUCUGCUUCUGCU-3΄) and 0.5 mM NTPs in assay buffer  glycerol, 5 mM MgClEquation :(4)\docIn vitro, we quantified these RNA–RNA interactions by a fluorescence resonance energy transfer (FRET) assay using short model 5΄ and 3΄ RNAs labelled at their distal ends by fluorescence donor and acceptor, respectively  of ∼0.75 while the corresponding 18-mers of the complementary c5΄ and c3΄ RNAs, the replication intermediate cRNA ends, interacted weaker . Consistent with this, the RNA-folding prediction program Mfold/DINAMelt . However, the proximal v5΄ (nt 1–10) and v3΄ (nt 1–9) strands interacted much more weakly under the same experimental conditions (DK (v5΄ nt 1–10 + v3΄ nt 1–9) = DK (v5΄ nt 1–10 + v3΄ nt 1–18) > 1 μM). To quantify the interaction of the promoter RNA strands with the polymerase in vitro, we used fluorescently-labelled RNAs and measured the change in fluorescence anisotropy upon polymerase promoter complex formation. Influenza B polymerase interacted with each of the 18-mer v3΄, v5΄, c3΄, c5΄ or the v5΄ nt 1–10 separately with a dissociation constant in the low nanomolar range and in a one-to-one stoichiometry  > 1 μM), a simultaneous binding to the polymerase and in a one-to-one-to-one ratio could be demonstrated genome segment and this interaction pseudo-circularizes the vRNA (or cRNA). During initiation, the 3΄ end serves as the template RNA and the 5΄ end as a crucial allosteric co-factor. Due to partial complementarity, the conserved 5΄ and 3΄ extremities in solution can, independently of the polymerase, anneal to adopt the so-called ‘panhandle’ conformation –22. In vy Figure . The vRNDINAMelt –25 prediDINAMelt . As expey Figure . The v5΄y Figure , which cy Figure . Using vnstrated . These rin vitro RNA synthesis assay was developed that allows monitoring of the RNA-dependent RNA polymerization activity of influenza polymerase. It is based on two principles: (i) the active polymerase incorporates nucleotides into a nascent product RNA based on complementarity to the template RNA thus potentially producing a template-product duplex; (ii) the fluorescence polarization (FP) of FAM attached to the 5΄ end of the model template RNA changes significantly between its initial single-stranded state and when it is annealed to the complementary product strand. While the total fluorescence intensity essentially remains constant, the observed change in FP displays a direct proportionality to the ratio of complementary product RNA to labelled template RNA at the experimental conditions ([RNA] >> DK (template + product RNA)) and is sensitive to full-length or full-length-like products only  (kobs), from which the initial rate of RNA synthesis is derived by multiplying kobs with the signal-amplitude, in turn proportional to the amount of RNA synthesized. The rate of RNA synthesis showed the expected linear dependency over a wide range of concentrations of active polymerase (i.e. a polymerase bound to the conserved 5΄ and 3΄ extremities of the vRNA), the slope corresponding to the turnover number (kcat ∼ 0.25 min−1 at 0.5 mM NTPs) (An emplate) . To faciemplate) . The higemplate) . At an eemplate)  yielded mM NTPs) . This FPmM NTPs) . As a prmM NTPs) .de novo (unprimed). Employing our new assay, kinetics of both modes of initiating RNA synthesis were recorded with or without a capped 12-mer RNA primer ending in the favored AG-3΄ , indicating that a rate-limiting step of RNA synthesis is bypassed by providing a suitable primer . By comparing this result with the MK of non-capped RNA  and ApG alone , it is clear that both the cap and the RNA moiety of the primer contribute to enhance efficiency. To obtain insights into the enthalpic and entropic contributions of the initiation of RNA synthesis, the unprimed and primed reactions were performed at different temperatures  and with the polymerase activated by the v5΄ 1–14. Shortening the v3΄ template RNA below 12 nucleotides or truncating the v5΄ to nt 1–12 or shorter resulted in inactivation of RNA synthesis . T,8. T7,8)er Table , resultide novo replication initiation mechanisms have been proposed for vPol and cPol , respectively at capped RNA primed conditions and 0.025 mM NTPs, Figure k = 0.47 ± 0.12 min−1 ); or (ii) the distal 5΄-3΄ base-pairing region of the cRNA promoter being swapped for the corresponding vRNA promoter ). In addition, swapping of the proximal cRNA promoter RNAs for the corresponding vRNA promoter ) ) . Only th n = 5)) . BrieflySince both the 5΄-3΄ base-pairing region and the template-directed first few base-pairs are responsible for fast, terminal or slow, internal initiation, we propose that breaking promoter base-pairs is energetically compensated by making new template-product base-pairs in the active site Figures  and 5. TIn previous structures of influenza B polymerase with bound promoter the 3΄ extremity (nt 1–6) was not observed in the internal active site cavity but was either not visible, due to disorder  or bound on the polymerase surface  . We attein vitro and cell culture experiments, 2΄F-2΄dGTP had been established to inhibit influenza polymerase reversibly and competitively with respect to GTP (Using t to GTP . By detet to GTP . The purt to GTP . Initiatt to GTP . An explt to GTP ), more at to GTP . Substitt to GTP . Only thDuring initiation of RNA synthesis, influenza A polymerase in the context of a native RNP produces small abortive transcripts of a capped RNA (ending with … AG-3΄) elongated by only one or, less efficiently, by two nucleotides . Only suin vitro (Escherichia coli RNA polymerase (Fluorescence polarization (FP) is widely used to study protein-nucleic acid-interactions ,35,36. Fin vitro ,40. It sin vitro , hence tin vitro ,43, poliin vitro ,44,45 anlymerase , a high lymerase . Indeed,lymerase . Whilst DK (MK), pseudo first-order conditions are fulfilled and the reaction progresses single-exponentially. Determining the FP-signal at one defined reaction time within linear range of the assay is sufficient for preliminary screening of compound libraries. As a proof of principle, we demonstrated the assay's HTS-capabilities by characterizing a set of nucleoside-analogues for their potential to inhibit RNA synthesis  and substrate NTPs, as well as catalytic rates for the differently primed RNA synthesis reactions, were important for development of an optimized and robust screening assay. Briefly, maintaining an excess of primer over active polymerase over template RNA at concentrations exceeding the is Table .in vitro enzymatic parameters of influenza B polymerase that we measured (Table kcat) but also higher affinity for the substrate NTP (MK) and the capped RNA primer (Table −1) and the cPol-like internal initiation mechanism involving prime-and-re-align with slow rates (∼0.006 min−1) in vitro. The crystal structure of influenza polymerase bound to the vRNA promoter in the pre-initiation conformation shows a 5΄-3΄ base-pairing region in the distal promoter region involving 5΄ nucleotides 11–13 base-pairing with 3΄ nucleotides 10–12 . It remains to be seen what degree of mechanistic coupling exists between the making and breaking of these compensating base-pairs. Only the wild-type cPol promoter with its combination of a strong 5΄-3΄ base pairing region  and a weak initiation signal  resulted in slow, internal initiation  5΄-3΄ base-pairing region in the promoter distal part. Since varying the strength of the 5΄-3΄ base-pairing region directly impacted the observed rate of RNA synthesis, we conclude that this step (breaking the 5΄-3΄ base-pairing region) is the next rate-limiting step of the complete reaction. We speculate that this step separates initiation from elongation. If elongation, or any step proceeding breaking the 5΄-3΄ base-pairing region would be slower, varying the 5΄-3΄ base-pairing region should not result in changes of the rate constants of full-length product RNA formation observed. Mechanistically, the only difference between primed and unprimed initiation of RNA synthesis is the accelerated frequency of the former reaction to proceed to final product-formation. With a catalytic rate about 300-fold improved for the primed over the unprimed initiation mechanism, our data argue that with a suitable capped RNA primer available, the influenza polymerase would almost exclusively perform transcription. So how does the polymerase ‘switch’ from transcription (capped RNA primed) to replication (unprimed) at the same vRNA promoter? A plausible explanation is based on the fact that influenza polymerase is known to be associated with host Pol II CTD  is in relatively low abundance  or that viral and/or host factors might be recruited, accounting for elevated vRNP over cRNP levels (Although our methodology allows detailed analysis of certain aspects of RNA synthesis, notably the differences between vPol and cPol, there are limitations to l II CTD ,51, whicl II CTD . It is tl II CTD  and, in bundance ,55 compaP levels .In conclusion, the strengths of our newly developed, FP-based RNA synthesis assay are the defined conditions, reliability and effectiveness in high-throughput screening for inhibitors. As a proof of principle, we screened a set of compounds targeting RNA synthesis and found that cPol and vPol are differently inhibited by the 2΄F-2΄dNTP nucleoside analogues. This result could be exploited to develop novel influenza polymerase inhibitors .Co-ordinates and structure factors for the influenza B polymerase co-crystallized with vRNA promoter and capped RNA primer are deposited in the wwwPDB with accession number 5MSG.Supplementary DataClick here for additional data file.