Lys3 for its annealing to the viral RNA at the primer binding site (PBS). Despite the fact that the result of this rearrangement is thermodynamically more stable, there is a high-energy barrier that requires the chaperoning activity of the viral nucleocapsid protein. In addition to the nucleotide complementarity to the PBS, several regions of tRNALys3 have been described as interacting with the viral genomic RNA. Among these sequences, a sequence of the viral genome called PAS for “primer activation signal” was proposed to interact with the T-arm of tRNALys3, this interaction stimulating the initiation of reverse transcription. In this report, we investigate the formation of this additional interaction with NMR spectroscopy, using a simple system composed of the primer tRNALys3, the 18 nucleotides of the PBS, the PAS (8 nucleotides) encompassed or not in a hairpin structure, and the nucleocapsid protein. Our NMR study provides molecular evidence of the existence of this interaction and highlights the role of the nucleocapsid protein in promoting this additional RNA-RNA annealing. This study presents the first direct observation at a single base-pair resolution of the PAS/anti-PAS association, which has been proposed to be involved in the chronological regulation of the reverse transcription.The initiation of reverse transcription of the human immunodeficiency virus type 1 (HIV-1) requires the opening of the three-dimensional structure of the primer tRNA Lys3 is the natural primer of all immunodeficiency viruses, including the type 1 human immunodeficiency virus (HIV-1). Indeed, the primer tRNA is strongly bound to the genomic RNA through Watson–Crick base-pairing of its 18 3′-terminal nucleotides with the complementary viral primer binding site (PBS)  that acts as an RNA chaperone In retroviruses, the initiation of reverse transcription is primed by a cellular tRNA that is encapsidated in viral particles. tRNALys3 as the primer of reverse transcription is strictly maintained in HIV-1 evolution. Earlier experiments from several groups have shown that altering the PBS sequence alone is not sufficient to stably switch tRNA usage Lys3 as primer by HIV-1, the search for the determinants of specific tRNALys3 selection, other than the PBS, was the subject of extensive studies Lys3 and the viral RNA have been naturally considered as potential secondary determinants and were proposed to play a role in reverse transcription Lys1,2 as the primer of reverse transcription in vitroLys3-primed reverse transcription initiation in the HIV-1 HXB2 and NL4.3 strains Lys3 . This recombinant tRNA  and the ability to mimic the natural pairing occurring in both the MAL and HXB2 isolates. We also worked with a single-stranded sequence of PAS .We performed our analysis on a model system involving critical fragments of the U5 region of HIV-1 viral RNA. The RNA molecules used to investigate the formation of the PAS/anti-PAS complex will be described below. Interestingly, the PAS sequence is involved in base-pairing within the viral RNA whatever the isolate. In the MAL isolate, the PAS sequence is part of a stem-loop structure S PASss;  and 2C tS PASss;  and 2D cLys3 imino groups were previously performed ds in NOESY experiments, we first assigned their resonances. The PASds hairpin contains six base-pairs and seven imino protons. ds. Briefly, a GU base-pair gives rise to an intense NOE as the imino protons of the guanine and the uridine are facing each other in such a base-pairing at around 2.5 Å. In addition, in a GU pair, the imino proton of the guanine is the most shielded. Therefore, the imino proton of G129 was assigned to 10.21 ppm and that of U135 to 11.60 ppm. From this starting point, the assignment of all imino protons was straightforward and is summarized in Assignments of tRNALys3, we first devised two different strategies described in the materials and methods section. Briefly, the first procedure is a heat annealing-based experiment whereas the second one uses the chaperone activity of the NCp7 protein. The 1H-15N correlation NMR spectra recorded after performing these procedures on a 15N-labelled tRNALys3/PASds mixture are identical to that of the free tRNALys3 . Noticeably, the presence or the absence of NCp7 did not change the ratio between these two species (data not shown). Moreover, the comparison of the results obtained with PASss or with PASds strongly suggests that the embedment of the PAS sequence in base-paired region prevents it from annealing to the anti-PAS sequence of tRNALys3. In addition, the feature that NCp7 did not affect the ratio of the different RNA entities as compared with the heat annealing procedure is in complete accordance with NCp7 being an RNA chaperone We subsequently used the single-stranded PAS sequence to conduct the two procedures in parallel. All the signals of the free tRNAeatments  but the eatments . We attrLys3 to the PBS is strongly favoured . In addition, the annealing of tRNALys3 to the PBS will partially open the tRNA structure and thus increase the accessibility to the anti-PAS sequence. Therefore, in order to investigate whether the PBS could influence the annealing of the PAS sequence with tRNALys3, we decided to conduct the heat-annealed procedure in the presence of the PBS. Lys3/PBS imino groups was previously published Lys3  was assigned to G51 since its imino proton gives an NOE to both imino protons of the G52-U126 pair. The imino groups carried by the PAS sequence  were more difficult to assign since they are not labelled and therefore do not give rise to peaks in the TROSY experiment. We proceeded using a stepwise strategy: first we identified in the NOESY experiment of the mixture , we did not succeed in obtaining more than 50% of PAS/anti-PAS complex. We could not rule out that the amount of NCp7 added in our samples is not sufficient to reach higher amount of the PAS/anti-PAS complex. Briefly, we want here to mention that no exchange cross-peaks between individual imino protons in the free and annealed form of the PAS RNA are observed in the different NOESY spectra  is annealed to tRNALys3.It is interesting to notice that in addition to the signals of the PAS annealed to tRNAound PAS . These c spectra  and S3. ss/anti-PAS complex that is formed, since the free single-stranded PASss has no observable imino proton. However, we investigated the possibility to make this complex with the heat-annealing or the NCp7-mediated procedures. Interestingly, the heat-annealed complex is present in sufficient amount to give observable NMR signals when 1 equivalent of PASss is mixed with equimolar quantity of tRNALys3 and PBS . As a result, the tRNA/PBS complex can easily be promoted by both the heat-annealed and the NCp7 procedures Thermodynamic considerations are important to properly describe and understand these large structural rearrangements involving three different RNA molecules. The formation of the complex between tRNALys3 and the PAS is very different. tRNALys3 on its own is more stable than paired with the PAS sequence. Indeed, the free energy (ΔG) reaches −22 kcal/mol with a melting temperature Tmc calculated by UNAFold above 80°C whereas for the PAS/anti-PAS complex, ΔG is only −8.8 kcal/mol with a Tmc around 40°C . Indeed, in this case, the thermodynamic parameters of the tRNA/PAS complex have not changed, but the PAS sequence cannot fold on its own and only 6 bp  that further stabilizes this region. This could explain why the single-stranded PAS can only be partially annealed to the primer tRNALys3  that was shown to stimulates the initiation of reverse transcription Lys3 are of comparable stability  the annealing of tRNALys3 to the PBS, which results in the partial opening of the tRNA structure and therefore increases the accessibility to the anti-PAS sequence; (ii) the presence of the nucleocapsid chaperone protein, which might destabilize the initial PAS base-pairing within the viral RNA and favour RNA strand exchange. On the other hand, it is disfavoured by the molecular inaccessibility of the PAS sequence as a result of its embedment in a base-paired region within the viral RNA. Altogether, the PAS/anti-PAS interaction appears to be a new illustration of the chaperone activity of the NCp7 protein. In addition, our data highlight the power of NMR experiments to map global RNA folds and demonstrate that NMR is a powerful tool to characterize annealing processes in detail.We showed in this report that base-pairings between the PAS in the viral RNA and the anti-PAS in tRNA15N-labelled tRNALys3 was expressed in E. coli (JM101TR strain) from a recombinant plasmid and purified as previously described ds (17 nucleotides) and PASss (8 nucleotides) were purchased from Dharmacon research with 2′-O-bis (acetoxyethoxy)-methyl (ACE) protection. The samples were deprotected following manufacturer recommendations and lyophylised. They were then dissolved in a phosphate buffer  with 50 mM KCl. The PBS and the PASds hairpin were folded by heating at 90°C for 5 min and snap-cooling on ice. The sequences of RNA samples used in this study are indicated in ds hairpin corresponds to the sequence and secondary structure context of the MAL isolate, and was chosen as a compromise between the size of the RNA (to be kept to a minimum for the NMR study) and the ability to mimic the natural pairing occurring in both the MAL and HXB2 isolates pLysE and the overexpressed protein was purified as previously described The recombinant NCp7 protein  was overexpressed from the bacterial expression vector pRD2, which contains the NCp7 coding region from HIV-1 strain NL4-3 subcloned into pET-3a . This plasmid was kindly provided by M.F. Summers Lys3 (0.44 mM), PASds and PBS at ratio 1∶1∶1 was first heated at 70°C in the NMR spectrometer for the time necessary to acquire 1D 1H NMR spectrum . Then the temperature of the sample was progressively decreased to 50°C, 40°C, 25°C and 15°C in the NMR spectrometer and a 1D 1H NMR spectrum was acquired at each temperature. At 15°C, a NOESY and a TROSY NMR experiments were recorded to analyse the RNA complex in the tube.In the standard heat-annealed procedure, RNA samples were heated at 85°C for 2 minutes, next incubated at 65°C for 10 minutes before a slow cooling to room temperature and putting them in the NMR tube. In the experiment of In the NCp7-mediated procedure, two equivalents of NCp7 were added to RNA samples and the mixture was then heated at 37°C for 30 min to several hours. Both procedures were performed in the NMR buffer (10 mM KPO4 pH 6.5) containing 50 mM KCl or 125 mM KCl. The same results were obtained at both salt concentration.http://mfold.rna.albany.edu/) ds was measured by UV spectroscopy measuring the absorbance at 260 nm from 15°C to 80°C (Thermodynamic stabilities and melting temperatures reported in  to 80°C .1H NMR spectra were recorded using a watergate sequence for solvent suppression 1H-15N TROSY experiments http://www.ccpn.ac.uk/).NMR data were measured on a Bruker Avance DRX600 spectrometer equipped with a TCI cryoprobe. 1D 15N-tRNALys3 and others partners were carried out at a concentration of 0.45 mM when we needed to acquire a NOESY experiment and at a concentration of 0.1 mM when only a TROSY experiment was necessary to analyze the RNA complex in the NMR sample.The interaction between Figure S1RNA sequences used to model the secondary structure context of the PAS sequence within A) the MAL isolate and B) the HXB2 isolate. These RNA fragments were used to derive the thermodynamic parameters for the PAS sequence within the two different viral RNA isolates (See ates See .(TIF)Click here for additional data file.Figure S2ds to tRNALys3.Tests of annealing of PAS A) Schematic drawing of the experiment, B) Superimposition of two TROSY experiments recorded at 15°C showing the imino groups of tRNALys3 (0.1 mM) alone  and after the heat-annealed procedure with 1 equivalent of PASds (red spectrum).(TIF)Click here for additional data file.Figure S3Superimposition of three NOESY experiments recorded at 15°C with a mixing time of 150ds, in red for a mixture of tRNALys3, PASds and PBS for which the annealing between the tRNALys3/PBS was promoted, in black for a mixture of tRNALys3, PASds, PBS, NCp7 for which the annealing tRNALys3/PBS/PASds was promoted . The NOESY cross-peaks for GU base-pairs within PASds, and tRNALys3/PAS complex are indicated. The assigment of the imino groups within the PAS/anti-PAS complex is indicated in purple for 15N-signals and the assignment for black cross-peaks corresponding to non-labelled imino groups are in orange.(TIF)Click here for additional data file.Figure S4ss to tRNALys3 in the presence of PBS.Annealing of PAS Superimposition of two TROSY experiments recorded at 15°C showing the imino groups of tRNALys3 (0.1 mM), in black: in complex with PBS  and PASss  after the heat-annealed procedure; and in red: in complex with PBS  and PASss  after the NCp7-mediated procedure. Only the GC regions are shown since the T54 imino group was not observed in these experiments.(TIF)Click here for additional data file.Figure S5ds.UV melting curve of the PAS(TIF)Click here for additional data file.Figure S61H NMR spectra of the PASds alone (0.16Region of the imino protons of the ds (0.16mM) and of the PASmM) mixed with NCp7 at 1∶1 ratio. The spectra were recorded at 15°C using a watergate sequence to (TIF)Click here for additional data file.


Arabidopsis thaliana. Our analyses revealed that the midi RNA fraction contained mostly 20–70-nt-long fragments derived from various RNA species, including tRNA, rRNA, mRNA and snRNA. The majority of these fragments could be classified as stable RNA degradation intermediates (RNA degradants). Using two dimensional polyacrylamide gel electrophoresis, we demonstrated that high copy number RNA (hcn RNA) degradants appear in plant cells not only during stress, as it was earlier suggested. They are continuously produced also under physiological conditions. The data collected indicated that the accumulation pattern of the hcn RNA degradants is organ-specific and can be affected by various endogenous and exogenous factors. In addition, we demonstrated that selected degradants efficiently inhibit translation in vitro. Thus, the results of our studies suggest that hcn RNA degradants are likely to be involved in the regulation of gene expression in plants.It is becoming increasingly evident that the RNA degradome is a crucial component of the total cellular RNA pool. Here, we present an analysis of the medium-sized RNAs (midi RNAs) that form in The online version of this article (doi:10.1007/s11103-013-0079-3) contains supplementary material, which is available to authorized users. These 19-nt-long molecules corresponded to the 5′ ends of tRNAs and constituted approximately one-third of the small RNA pool in phosphate-deficient roots. Interestingly, these RNAs accumulated to much lower levels in the shoots. Based on this observation, the authors postulated that these molecules were products of a specific cleavage rather than of random RNA decay. In addition, Hsieh et al.  plants were grown under short-day, standard conditions as described previously , SALK_018392 , SALK_005512  and GK-160G05  . Subsequently, 1 μg of RNA was labeled with γ32P ATP using T4 PNK kinase (Fermentas) and subjected to 2D-PAGE.RNA samples, which were enriched in molecules that were shorter than 200 nt, were isolated from Arabidopsis  using the 2D-PAGE was performed under semi-denaturing conditions (4 M urea) as previously described  were normalized. To this end, for each pair of images to be compared three spots that displayed invariable intensity in relation to the background were selected (three corresponding spots for both images) and for each image the mean intensity of these three spots (mean count) was calculated. Then, for every spot its relative intensity (relative spot count) was determined by dividing initially quantified intensity by mean count. Finally, the changes in individual degradants’ accumulation were determined by comparison of the relative spot counts of matching spots. The accumulation was considered significantly different when twofold change of relative spot count was observed.Two cDNA libraries of RNAs from Arabidopsis leaves were prepared based on the protocols that were elaborated by Lau et al. , ChappelThe 2D-library was constructed using RNAs that were extracted from individual spots that were visualized on the 2D-gels. For the proper selection of RNA spots, 9 experiments of 2D-separation were performed, each of which involved harvesting Arabidopsis rosette leaves from 3 independent plant cultivations. Subsequently, 3 independent RNA extractions followed by RNA separations in 2D-gels were performed from each plant harvest. Finally, after analyzing the obtained images, gel fragments corresponding to the 70 most intensive spots that were present on all images were chosen and excised. To optimize cloning, fragments that were excised from the gel were divided into three fractions, and each fraction contained pooled gel fragments of similar spot intensity. RNA was eluted from the gel fragments, and equivalent (radioactivity in cpm) portions of each RNA fraction were utilized for cloning.The second cDNA library  was prepared using the RNA that was eluted from 1D-gel slices . The resulting RNA fraction, which was subsequently used for cloning, contained molecules that migrated slower than 18-nt RNA size marker but faster than the main band corresponding to tRNA. Accordingly, most of tRNA molecules were not included in this library.For both libraries, RNAs that were isolated from the gels were ligated with a 3′ End Donor oligonucleotide, which contained the BanI restriction site, a blocked 3′-hydroxyl terminus and a pre-activated 5′ terminus and was commercially available from IDT, as previously described . ORFs, pseudogenes and intergenic sequences were generated using the TAIR9 release of the Arabidopsis genome annotation, and the sequences for which there were no matches in the TAIR9-annotated portions of the genome were used to search the non-redundant set of Arabidopsis sequences that was derived from GenBank.The sequences of the molecules from both cDNA libraries were mapped onto the reference Arabidopsis genome using BLAST  was used for in vitro translation of Luciferase Control RNA (Promega) that encodes functional firefly luciferase. Translation reactions were prepared according to the manufacturer’s recommendations with some modifications. All reactions were carried out in 10 μl final volume with 50 % Wheat Germ Extract, 80 μM Amino Acid Mixture, 8 units of ribonuclease inhibitor RNaseOUT (Invitrogen) and 2 μg Luciferase Control RNA. 10 pmol of chemically synthesized, 5′ monophosphorylated RNA oligonucleotide (IBA) corresponding to the degradant was added to a translation reaction and incubated for 1.5 h at 30 °C. A control reaction with no short RNA added was performed in parallel. After incubation, luciferase activity was quantitated (three independent measurements) with Victor X4 Multilabel Plate Reader (PerkinElmer) and Luciferase Assay System (Promega). For a single measurement 2.5 μl of a translation reaction mixture were used. All reactions were carried out in triplicates.32P and subjected to 2D-PAGE analysis. After 1D-separation, the fraction of RNA that migrated slower than the 18-nt-long radiolabeled RNA marker but faster than the majority of tRNAs was subjected to 2D-separation. Upon autoradiography, hcn-midi RNAs and si/miRNAs were visualized . Of 59 putative degradants, 32 molecules were derived from tRNA (47.8 %) and 25 were derived from rRNA (37.3 %)  represented fragments of larger RNA species. Accordingly, these molecules could be classified as intermediates of RNA degradation that accumulated in plant cells (see Online Table 1). Three molecules were classified as unknown transcripts (or fragments of those transcripts) from intergenic regions and 5 were identified as intact tRNA molecules (tRNALeu and 4 tRNAGly isoacceptors and fragments of 3 tRNAGlu and 3 tRNAArg isoacceptors (Table Gly and tRNALeu) were identified in the 2D-library.Further analysis revealed that tRNA degradants were derived from only 12 different types of nuclear tRNAs and 10 types of organellar tRNAs . We also analyzed how particular tRNA isoacceptors contributed to the degradome. We found fragments of 4 tRNAThe second most abundant class of RNA degradants in the 2D-library (25 molecules) consisted of nuclear and organellar rRNA fragments. The vast majority of rRNA fragments were derived from 25S rRNA (10 molecules) and 18S rRNA (9 molecules). The remaining fragments corresponded to 5S rRNA (3 molecules), 23S rRNA (1 molecule) and 16S rRNA (2 molecules) . One hcn-midi RNA from the 2D-library was identified as a derivative of the U2.3 small nuclear RNA, and only one sequence was identified as a fragment of an mRNA. The latter was a fragment of the 5′ untranslated region (UTR) of the mRNA that encodes a RING/U-box superfamily protein (AT4G39140).Each of the 350 sequenced concatemers from the 1D-library contained 2–7 individual molecules. As a result, 154 unique RNAs were identified and the size variants were grouped as described above for the 2D-library. The length of these molecules ranged from 15 to 47 nt, and as expected, nearly all of the cloned molecules (except for 7 (4.5 %) si/miRNA molecules) were the putative products of RNA degradation (Online Table 2). Once again, most of these fragments were classified as tRNA  or rRNA  derivatives . To verify this presumption we compared the structures of both libraries. More than half of the RNA degradants from the 2D-library  was also found in the 1D-library. Among these RNAs were fragments of 19 tRNAs, 15 rRNAs and 2 intergenic sequences (IGS). Two types of RNA: fragments of transcripts of transposable elements and si/miRNAs were present only in the 1D-library  and tRNAMet . The former fulfilled both structural criteria mentioned above and was shown to be an effective translational inhibitor in rabbit reticulocyte lysate, while the latter lacked a 5′ TOG motif and was unable to block translation in the same system  and the 5′ fragment of human tRNAMet  did not block translation in the plant in vitro system. Among the Arabidopsis degradants no. 112A (tRNALeu) was the strongest repressor and caused approximately 70 % decrease in translation efficiency. The other two degradants reduced translation by c.a. 32 %  and 26 % . One of the functions proposed for tRNA fragments is the inhibition of translation. Ivanov et al.  demonstrHs-5Ala, 0 nt and After characterizing the specific accumulation patterns of hcn-midi RNAs in leaves, we attempted to determine the extent to which this pattern was organ-specific. To this end, RNA samples isolated from Arabidopsis roots and flowers were subjected to 2D-PAGE analysis in the same way as the rosette leaf samples. Subsequently, the obtained gel images were compared . In all performed comparative analyses the pattern of the previously selected 70 spots, which corresponded to hcn-midi RNAs from rosette leaves, was applied as a reference.Quantitative analysis of leaf and root samples revealed that only 11 degradants displayed a significantly altered level of accumulation. All of them were more prevalent in leaves Fig. a. Compardcl1-5, dcl2-1, dcl3-1 and dcl4-2) and wild type plants  of these degradants displayed higher level of accumulation in mutant leaves than in wild type. Interestingly, in case of other analyzed mutants, all but one differentiating hcn-midi RNAs were less abundant than in wild type leaves. The only exception was one degradant more prevalent in dcl3-1 than in wild type. Moreover, although only a limited number of changes reached the threshold of significance, it was evident that in dcl2-1, dcl3-1 and dcl4-2 the majority of hcn-midi RNAs accumulated to lower levels than in wild type.We also attempted to determine if the RNAi machinery is involved in the generation of hcn-midi RNAs. Therefore, we examined whether the biogenesis of these molecules is dependent on the presence of Dicer-like (DCL) ribonucleases. We performed 2D-PAGE analyses of RNA samples that were isolated from rosette leaves of four Arabidopsis mutants, each lacking one of the DCLs. Earlier, it was shown that these mutants display an aberrant accumulation of miRNAs, siRNAs and tasiRNAs because of defects in small RNA biogenesis pathways  certain types of RNA molecules are not completely digested  that are uniquely found at the 5′ ends of these tRNAs. In both libraries from Arabidopsis we identified several tRNA fragments that had 5′-TOG-like motifs. Out of them we selected tRNA degradants with 5, 4 or 3 guanine residues  and tested their influence on in vitro translation. Taking into account the proposed mechanism of translational inhibition by tRNA fragments . In case of dcl3-1, dcl2-1 and dcl4-2 only several changes reached the significance threshold and were associated with a reduced prevalence of hcn-midi RNAs in comparison to wild type (with one exception in dcl3-1). Despite evidence showing that Dicer can be involved in the cleavage of tRNAs and snoRNAs Supplementary material 2 (XLSX 77 kb)Supplementary material 3 (PDF 4080 kb)Below is the link to the electronic supplementary material.


Δ21) and a peptide of 60 amino acids (P60) encompassing the molecular recognition element (MoRE) of P that binds RNA-free N (N0). The complex crystallized in a decameric circular form, which was solved at 3.0 Å resolution, reveals how the MoRE folds upon binding to N and competes with RNA binding and N polymerization. Small-angle X-ray scattering experiment and NMR spectroscopy on the soluble complex confirms the binding of the MoRE and indicates that its flanking regions remain flexible in the complex. The structure of this complex also suggests a mechanism for the initiation of viral RNA synthesis.Replication of non-segmented negative-strand RNA viruses requires the continuous supply of the nucleoprotein (N) in the form of a complex with the phosphoprotein (P). Here, we present the structural characterization of a soluble, heterodimeric complex between a variant of vesicular stomatitis virus N lacking its 21 N-terminal residues (N 0) to encapsidate the newly synthesized (+)RNA intermediate antigenomes and (−)RNA genomes. In this process, the viral phosphoprotein acts as a chaperone, forming a heterodimeric complex, named N0-P, which prevents nascent N molecules from self-assembling and from binding to cellular RNAs. We reconstructed the N0-P complex of the prototype rhabdovirus, vesicular stomatitis virus, and characterized its structure by crystal X-ray diffraction and solution experiments. Our results show how the N-terminal region of the phosphoprotein folds upon binding to the RNA-free nucleoprotein and how it prevents the non-specific encapsidation of host-cell RNA. This complex is soluble and heterodimeric, but by forcing it to polymerize into a crystal it associated into a circular decamer of heterodimers very similar to the previously crystallized decameric N-RNA ring. On the basis of our results, we propose a model that explains the role of the phosphoprotein in the encapsidation of newly synthesized RNA and in the initiation of RNA synthesis by the viral polymerase.The negative sense RNA genome of the rhabdoviruses is encapsidated by the nucleoprotein, and the replication of the genome requires a continuous supply of RNA-free, monomeric nucleoprotein (N Rhabdoviridae and Paramyxoviridae, N is assisted by the viral phosphoprotein (P). P binds to nascent RNA-free N, forming a N0-P complex (the superscript 0 denotes the absence of RNA) that prevents the polymerization of N and the non-specific encapsidation of host cell RNAs 0-binding region is localized in the N-terminal disordered region Negative-sense RNA viruses include numerous major human pathogens such as influenza virus, rabies virus, measles virus and respiratory syncytial virus. The (−)RNA genome of these viruses is condensed by a viral nucleoprotein (N) into a helical nucleocapsid NTD) and C-terminal (NCTD) domains CTD, and by the exchange of N- and C-terminal sub-domains between adjacent N protomers  NT-arm may sufficiently destabilize the N-RNA complex so that P or a peptide fragment of P containing the MoRE that binds N0, could displace the RNA molecule.In rhabdovirus nucleocapsids, every N molecule binds nine nucleotides in a positively charged cavity at the interface between its N-  combined with static light scattering (MALLS) demonstrates that both NΔ210-P60 and NΔ210-P dimer complexes are free of RNA in solution, forming soluble heterodimers or heterotrimers, respectively. Therefore, P60 fulfills both chaperone activities of full-length P. The heterodimer NΔ210-P60 crystallized, but under the crystallization conditions, it assembled into a circular decamer of heterodimers very similar to the previously crystallized decameric N-RNA ring. The crystal structure of the decameric form of the NΔ210-P60 complex reveals the molecular mechanisms by which the N0-binding MoRE of P attaches to N. NMR spectroscopy confirms that the MoRE of P binds to N in the NΔ210-P60 complex in solution as in the crystal structure and shows that the regions of P flanking this MoRE remain flexible in the complex. Finally, these results suggest mechanisms for the encapsidation of newly synthesized RNA and for the initiation of RNA synthesis by the viral polymerase.In this study, we report the reconstitution of complexes between a recombinant N of VSV lacking the 21 N-terminal residues, NΔ21) in Escherichia coli led to the formation of inclusion bodies and of poorly soluble complexes, which could not be purified. In order to improve the solubility of the NΔ21 mutant, it was produced in E. coli in fusion with an N-terminal maltose binding protein (MBP) tag. The purified MBP-NΔ21 formed soluble, oligomeric N-RNA complexes, which eluted next to the exclusion volume of a Superdex S200 column  +8,053 Da (P60) = 96,379 Da). The co-elution of MBP-NΔ21 and P60 was confirmed by denaturing 4–20% gradient PAGE  , and the molecular mass of 104±4 kDa was consistent with that of a heterotrimer composed of one NΔ21 and an intact P dimer +2×30,976 Da = 107,329 Da) (0-P complex by native mass spectrometry S) of 5.8±0.1 nm is about 1.5 fold larger than that for a globular particle of the same molecular mass  reflecting the elongated shape of the complex and the existence of a long N-terminal disordered region (aa 1–106) The molecular mass of the N,329 Da)  in accorΔ210-P60 complex contained no RNA (S of 3.2±0.1 nm and molecular mass of 53±3 kDa indicated a globular 1∶1 complex (60 does not contain the dimerization domain of P g) of the NΔ210-P60 complex determined from SAXS data (2.7±0.1 nm)  (60 were visible in a groove formed by residues of the hinge region of N (aa 200–300) at the junction between NNTD and NCTD   [15]. Thand NCTD . Previou17 to 31 . The theΔ210-P60 complex shows how the N0-binding MoRE of P prevents both the interaction with RNA and the self-assembly of soluble RNA-free N. The binding site of P is different from that of RNA but both sites do overlap (60 (aa 27–31) together with the following residues (aa 32–35) block the RNA binding cavity  binds to the central hinge region of N mainly through hydrophobic interactions. The amphipathic α-helix of P, together with residues 14 to 16, inserts into a hydrophobic groove of N. Tyr14 perfectly fits into a small hydrophobic pocket . In addiCTD harbors a negative surface potential  spectroscopy. Initially, spectra of 15N, 13C, 2H-labeled P60 in complex with unlabeled NΔ21 were recorded. In a complex of this size (53 kDa) NMR signals are significantly broadened, precluding their detection, but in the HSQC spectrum of the NΔ210-P60 complex, resonances corresponding to the last 28 amino acids of P60 (aa 41–60+linker+His6 tag) are clearly visible  suggesting weak interactions of these residues with N. The amide backbone 15N transverse relaxation rate constant (2R) is sensitive to rapid fluctuations at the pico- to nanosecond time scale, as well as to chemical shift exchange on the micro- to millisecond time scale. Transverse relaxation of the visible resonances increased from the C-terminus to the region containing the bound helix, indicating a corresponding increase in rigidity of the backbone .In the crystal structure of the N visible , suggestbackbone . Very weΔ21 attached to MBP (to maintain solubility) to 15N-labeled P60 resulted in an overall reduction in the intensity of the peaks in the HSQC spectrum, in proportion of the amount of added MBP-NΔ21 indicating that a fraction of P60 formed a complex with MBP-NΔ21. Spin relaxation measurements of equilibrium mixtures of free and bound peptide refine our understanding of the dynamics of the system. Systematically larger 2R values observed for residues 1 to 17 reveal additional contributions from chemical shift exchange (exR) that are not present in the free form of the peptide and that increase significantly upon increasing the molar ratio of MBP-NΔ21 to P60  behaves as a flexible tail and shows little evidence of interaction with N. The flanking N-terminal region (aa 1–16) interacts with N but undergoes conformational exchange. These flexible regions seem dispensable for the chaperone activities of P since a shorter peptide encompassing residues 7 to 40 of P is also capable of displacing bacterial RNA from NΔ21 and of forming a soluble heterodimeric 1∶1 complex with this protein (data not shown).In a second experiment, addition of sub-stoichiometric amounts of unlabeled N1 to P60 . This inmer Ni+1 , but in 0-binding MoRE of P bound to N and demonstrated that the regions of P flanking the MoRE conserve some flexibility in the N0-P complex. Because the N0-P complex is required for the replication of the virus 60) from rabies virus P inhibits viral replication In the current study, we have determined the structure of the NΔ210-P60 complex is a suitable model for the viral N0-P complex in agreement with previous studies. Both the crystal structure and the NMR spectroscopy experiments show that the MoRE of P, which adopts a stable conformation upon binding to N, includes residues 6 to 35 and corresponds closely to the fragment that was previously identified as essential and sufficient for maintaining N in a soluble form (aa 11–30) Our results demonstrate that the reconstituted N60, which is monomeric Δ210-P60 complex (1∶1) shows that one MoRE of P is capable of binding one N molecule. The stoichiometry of the complex formed between NΔ21 and full-length P (1∶2) in the concentration range used here suggests that a single NΔ21 is bound to P dimer. The remaining part of the P dimer is tethered to NΔ21 through a flexible linker, in agreement with the large hydrodynamic radius measured here for the NΔ210-P dimer complex. In isolation, the N-terminal region of P contains two transient α helices (aa 2–12 and 25–38) Δ210-P60 complex into crystals and were thus selected during the crystallization process.Previous studies revealed that VSV P is a dimeric and modular protein in which the N-terminal part (aa 1–106) is globally disordered Δ21) conserves the ability of self-association in the presence of RNA and, like w.t. N, forms oligomeric N-RNA complexes when expressed in bacteria. As assumed from the structure of the oligomeric N-RNA complex NT-arm stabilizes the multimeric N-RNA complexes by linking together adjacent N protomers. The N-RNA complex formed with w.t. N could not be dissociated by the addition of full-length P or of a fragment of P encompassing the N0-binding region. However, the deletion of the N-terminal sub-domain destabilized the complex and allowed P60 to displace the RNA molecule and disassemble the multimeric N-RNA complex. In a previous study, the co-expression of P with a similar variant of VSV N lacking the first 22 amino acids (NΔ22) led to the production of complexes of different sizes containing NΔ22 and P but not of N-RNA complexes, suggesting a role for the N-terminal region of N in the encapsidation of the RNA 0-P complex and the multimeric N-RNA complex, with w.t. N, the stabilization brought by the NNT-arm to the multimeric assembly would displace the equilibrium towards the formation of the N-RNA complex. In the absence of the NNT-arm, the truncated N molecules assemble onto cellular RNAs as seen in our expression system, but in the presence of co-expressed P, like upon addition of P60 to our purified MBP-NΔ21-RNA complexes, the equilibrium is displaced towards the formation of the N0-P complexes. The absence of N-RNA complex in cells co-expressing NΔ22 and P may not result from a default of encapsidation but rather from the displacement of the equilibrium towards N0-P.The truncated form of N  is involved in a stable complex with N in solution and in the crystal, arguing that crystallization has no major effect on the structure of the more rigid part of the complex. In solution, the N-terminal part of the MoRE of P (aa 1–16) appears to be in conformational exchange and could thus exist in different conformers including those observed in the crystal in which residues 6 to 16 are docked onto the backside groove of N. Crystallization of the NΔ210-P60 complex may thus select the more compact conformers and therefore not reproduce the conformational diversity of this region that is found in solution. In addition, the high conservation rate of residues of N forming the binding surface for the MoRE of P, both within VSV serotypes and between the evolutionarily more distant VSV and RAV, supports the localization of the interface between the two proteins and hints at the formation of a similar complex in RAV.This raises the question of the effect of crystallization on the structure of the NΔ210-P60 complex is RNA-free heterodimer and that, therefore, P60 or a shorter fragment of P (aa 7–40) fulfill both chaperone activities of P. The crystal structure of the NΔ210-P60 complex clearly shows how the N0-binding region of P inhibits RNA binding by filling the RNA-binding groove of N. In solution, this part of P also forms a stable complex with N as seen by NMR spectroscopy. The structure also suggests how P prevents the self-assembly of N in the absence of RNA. In the bound form observed in the crystal structure, the N-terminal extremity of the MoRE (aa 6–16) directly competes with the NNT arm of a neighboring N molecule. Assuming this region of P fluctuates between bound and free forms in the soluble N0-P complex, the free form may act as an entropic bristle, thereby also preventing the oligomerization of N. With full-length P dimer, the flexibility and the bulkiness of the remainder of the protein may also contribute to this effect by masking the binding interfaces for RNAs or other N molecules. In addition, the MoRE of P exhibits a bipolar distribution of charges, with a positive pole at its N-terminal extremity and a negative pole at its C-terminal extremity. Binding of the MoRE of P modifies the electrostatic surface potential of N, notably reducing the positive surface potential on one side of the molecule, and may thereby affect the side-by-side interaction with another N molecule.The characterizations in solution indicate that the NNT-arm stabilizes the multimeric N-RNA complex and therefore suggest that the multimeric N-RNA complex is more stable than the N0-P complex. The transfer of N from the N0-P complex to the growing N-RNA complex could simply be driven by a higher stability of the N-RNA complex. Upon transfer of N onto the RNA and release of P, the backside groove of N of the last added N molecule is liberated and becomes available for accepting the NNT-arm of the next incoming N molecule RNA synthesis at the genome 3′ end and of (−)RNA synthesis at the antigenome 3′ end 60) was amplified by PCR and cloned into the pET28a plasmid containing a C-terminal His6-tag and a linker of two amino-acids (EL) using NcoI and XhoI restriction sites. All constructions were checked by DNA sequencing.The cDNAs encoding vesicular stomatitis virus nucleoprotein or a fragment of this protein deleted of the 21 N-terminal residues were amplified by PCR and introduced into the pET-M40 plasmid (EMBL) using NcoI and XhoI restriction sites. The resulting constructs code for chimeric proteins that comprise an N-terminal maltose binding protein tag (MBP) and a tobacco etch virus (TEV) cleavage site. The cDNA encoding the 60 first N-terminal amino acids of VSV P (PEscherichia coli Rosetta (DE3) cells and the expression of the recombinant proteins was induced with 1 mM isopropyl-1-thio-β-d-galactopyranoside (IPTG) for 18 h at 16°C. Cells were harvested by centrifugation, suspended in buffer A  containing protease inhibitors  and disrupted by sonication. The extract was centrifuged at 20,000 g during 30 min at 4°C and the supernatant was filtered (0.45 µm). The MBP-fusion proteins were purified by affinity chromatography on amylose resin (New England Biolabs) followed by size exclusion chromatography (SEC) on a Superdex S200 column  equilibrated in buffer A. P60 was purified by affinity chromatography on a Ni2+ resin column (Quiagen) followed by SEC on a Superdex S75 column  equilibrated in buffer A supplemented with 50 mM Glu and 50 mM Arg. Samples for NMR spectroscopy were produced in M9 minimal medium containing MEM vitamins (Gibco). For producing 15N-labeled P60, the medium was supplemented with 1.0 g.L−1 of 15NH4Cl and 2.0 g.L−1 of unlabeled glucose, while for producing 15N, 13C, 2H-labeled P60 the minimal medium was prepared in D2O and supplemented with 1.0 g.L−1 of 15NH4Cl and 2.0 g.L−1 of 13C glucose.The plasmids were transformed into Δ210-P60 complex was prepared by incubating overnight at 4°C an excess of P60 with the MBP-NΔ21-RNA complexes. The MBP-NΔ210-P60 complex was purified by Ni2+ chelate affinity chromatography by using the His-tag present on P60 to remove the excess of free MBP-NΔ21-RNA complex, followed by SEC on a Superdex S200 column equilibrated in buffer A and amylose affinity chromatography to eliminate unbound P60.The NΔ210-P60 complex was then purified using a Ni2+ chelate affinity chromatography followed by SEC on a Superdex S200 column equilibrated in buffer A. This procedure yielded pure NΔ210-P60 complex. The samples were checked by SDS-PAGE using denaturing 4–20% gradient PAGE (Biorad).The MBP tag was removed by incubating the protein with the TEV protease overnight at 4°C. The N protein contains the additional N-terminal tripeptide GAM. The N−1. 50 µL of a protein solution at a concentration ranging from 2.7 to 8.0 mg.mL−1 were injected. On-line multi-angle laser light scattering (MALLS) detection was performed with a DAWN-EOS detector  using a laser emitting at 690 nm. Protein concentration was measured on-line by refractive index measurements using a RI2000 detector (Schambeck SFD) and a refractive index increment dn/dc = 0.185 mL.g−1. Data were analyzed and weight-averaged molecular masses (Mw) were calculated using the software ASTRA V  as described previously S) SEC was performed with a Superdex S200 column  equilibrated in 20 mM Tris–HCl, pH 7.5, 150 mM NaCl. Separations were performed at 20°C with a flow rate of 0.5 ml.minSAXS data were collected at the European Synchrotron Radiation Facility  on beamline ID14-3. The sample-to-detector distance was 1 m and the wavelength of the X-rays was 0.931 Å. Samples were contained in a 1.9 mm wide quartz capillary. The exposition time was optimized for reducing radiation damage. Data acquisition was performed at 20°C. Data reduction was performed using the established procedure available at ID14-3 and buffer background runs were subtracted from sample runs.Δ210-P60 complex was recorded for scattering vectors, −1<q<3.5 nm−1. The profiles obtained at three different protein concentrations 2.7–8.0 mg.mL−1 had the same shape and were flat at low q values indicating the absence of significant aggregation . The radius of gyration and forward intensity at zero angle (I(0)) were determined with the programs PRIMUS g, and the pair distance distribution function, P(r), were calculated with the program GNOM max) value was adjusted so that the Rg value obtained from GNOM agreed with that obtained from the Guinier analysis. The Rg values showed no significant dependence on protein concentration, confirming the absence of aggregation or intermolecular interactions in the concentration range used in this study. The measured Rg value of 2.7±0.1 nm  and the Kratky plot  were typical of globular, folded proteins.The SAXS profile of the NAb initio low-resolution bead model reconstructions of the NΔ210-P60 complex were performed from the scattering curves using the program DAMMIF http://www.pymol.org).−1 in buffer A with 0.1 µl of Hampton Crystal Screen solutions. Hits were reproduced manually. The NΔ210-P60 complex crystallized at 20°C in 0.1 M sodium acetate buffer, pH 4.6, containing 4% (w/v) of PEG4000. Single crystals were harvested from the drop, briefly soaked in the reservoir solution supplemented with 25% glycerol and flash frozen in liquid nitrogen at 100 K before data collection. X-ray diffraction data were collected at a wavelength of 0.933 Å on the ID14-2 beamline at the ESRF .Crystallization conditions were screened by the hanging drop vapor diffusion method using a PixSys4200 Cartesian robot . The screen was performed by combining 0.1 µl of protein solution at 8 mg.mL60 was assigned and constructed with the program Buccaneer The data were processed using the program iMosflm Δ210-P60 complex was crystallized in space group P21212. The crystallographic asymmetric unit contained five protomers of NΔ210-P60 complex. Each protomer includes residues 22 to 422 of N and residues 6 to 33 of P60. In some protomers, residues 34 and 35 could also be constructed.The N1H frequency of 600 MHz. All samples contained 20 mM Tris-HCl, 150 mM NaCl, 50 mM Glu, 50 mM Arg with 10% D2O adjusted to pH 6.0. The concentration of free 15N, 13C, 2H-labeled P60 was 0.9 mM and the concentration of the NΔ210-P60 complex  was 0.28 mM. In the titration experiment, 15N-labeled P60 was initially at 0.28 mM. MBP- NΔ21 was added at final concentrations of 0.09 mM or 0.17 mM. 15N R2 (CPMG) relaxation experiments were acquired using standard pulse sequences 1H dimension, and a sweep width of 1.2 kHz and 200 complex points in the 15N dimension. The magnetization decay was sampled at 10, 30, 50, 70, 90, 130, 170, 210 and 250 ms and the peak heights were used to extract the relaxation rates. To obtain estimates of the errors on the relaxation rates, a repeat measurement of one of the relaxation delays (70 ms) was carried out.NMR experiments were performed on a Varian spectrometer operating at a Text S1This file contains seven additional figures named S1 to S7 and an additional table S1.(DOC)Click here for additional data file.


The establishment of persistent viral infection is often associated with the selection of one or more mutant viruses. For example, it has been found that an intraleader open reading frame (ORF) in genomic and subgenomic mRNA (sgmRNA) molecules is selected during bovine coronavirus (BCoV) persistence which leads to translation attenuation of the downstream ORF. Here, we report the unexpected identification of leaderless genomes, in addition to leader-containing genomes, in a cell culture persistently infected with BCoV. The discovery was made by using a head-to-tail ligation method that examines genomic 5′-terminal sequences at different times postinfection. Functional analyses of the leaderless genomic RNA in a BCoV defective interfering (DI) RNA revealed that (1) the leaderless genome was able to serve as a template for the synthesis of negative-strand genome, although it cannot perform replicative positive-strand genomic RNA synthesis, and (2) the leaderless genome retained its function in translation and transcription, although the efficiency of these processes was impaired. Therefore, this previously unidentified leaderless genome is associated with the attenuation of genome expression. Whether the leaderless genome contributes to the establishment of persistent infection remains to be determined. The families Coronaviridae (Coronavirus and Torovirus genera) and Arteriviridae, together with Roniviridae, are members of order Nidovirales in which a nested set of subgenomic RNA (sgmRNA) molecules are made that are 3′-coterminal with the genome during transcription To date, the function of a leader sequence in the genome or subgenome during the coronavirus life cycle has not been systematically established. It has been suggested that the leader sequence within the context of the mouse hepatitis virus (MHV) DI RNA containing the chloramphenicol acetyltransferase (CAT) gene is nonessential for negative-strand DI RNA synthesis Structural changes within the leader sequence of BCoV have been linked to persistent infection in cell culture due to the selection of a translation-attenuating intraleader open reading frame (ORF) In a study designed to identify potential sequence heterogeneity within the 5′ end of genomic RNA during BCoV persistent infection, we unexpectedly found a leaderless genome in addition to the leader-containing genome. Along with evidence in previous studies In an attempt to identify the 5′-terminal sequence of the positive-strand viral genomic RNA during BCoV persistent infection in cell culture, total cellular RNA was extracted at the time points indicated in cis-acting elements required for replication and translation All the viruses in the order Nidovirales contain a leader sequence at the 5′ end of the positive-strand genome In addition to the present study  were gencis-acting element critical for BCoV DI RNA replication Following translation, the first step in coronavirus replication is synthesis of the negative-strand copy of the genome that in turn serves as a template for the synthesis of the genomic RNA positive strand. The coronavirus leader sequence has been demonstrated to be a During coronavirus infection, a nested set of sgmRNAs are produced from which the coronavirus structural proteins are translated; these structural proteins have been linked to viral assembly and pathogenesis. To test whether the efficiency of sgmRNA synthesis is modulated by the leaderless genome as a template during infection, the intergenic sequence (IS), a transcription signal required for sgmRNA synthesis To ascertain whether the encoded sgmRNA from the leaderless genome is translated, fluorescing HRT-18 cells were obtained as evidence of the expression of EGFP-containing sgmRNA. With respect to the upstream ORFs the EGFP ORF in both the Δ69EGFP and DIEGFP templates is in the −1 reading frame; therefore, EGFP expression from a fusion protein was not expected and the fluorescence observed from HRT-18 cells could only have resulted from expression of the encoded EGFP-containing sgmRNA. As predicted, EGFP fluorescence was observed in BCoV-infected HRT-18 cells transfected with either DIEGFP or Δ69EGFP  in whichSynthesis of a 3′ co-terminal nested set of sgmRNAs is a common feature of members of the Nidovirales th nt of the 5′-end leader-containing genomic RNA. Interestingly, the high-frequency crossover region of leader switching during negative-strand BCoV DI RNA synthesis is from the 70th to 93th nt th nt. The 70th nt is a site located at the 5′-most crossover region with respect to the positive-strand genomic RNA and this process would result in a leaderless negative-strand genomic RNA for which the 3′-end 69-nt leader sequence is missing. The newly synthesized negative-strand leaderless genomic RNA would then serve as a template for the synthesis of a positive-strand leaderless genomic RNA (The RNA-dependent RNA polymerase (RdRp) template switching event that occurs during negative-strand synthesis to produce a negative-strand leader template on negative-strand sgmRNA molecules may yield a mechanistic explanation for synthesis of the leaderless genomic RNA. This notion is supported by the following previous findings. First, it has been suggested that, for equine torovirus sgmRNAs 3–5, the body intergenic sequences on the positive-strand genome sever as terminators of transcription during negative-strand synthesis. In turn, the intergenic sequences at the 3′ end of the negative-strand of sgmRNAs 3–5 are used as promoters for the synthesis of the sgmRNA positive strand resulting in the synthesis of leaderless sgmRNAs 3–5 omic RNA . Althougomic RNA , the finomic RNA  providesomic RNA , an alteomic RNA . FurtherThe leaderless genomic RNA identified during BCoV persistent infection is not likely to be an artifact or a cleaved product because (1) the head-to-tail RT-PCR product of the leaderless genome was observed only at 67 and 97 dpi but not at other time points and (2) RT-PCR product with a length smaller than 200 bp was not observed when extracted RNA was not treated with tobacco acid pyrophosphatase as shown in cis-acting element required for translation, replication (including negative-strand synthesis), and transcription during infection using BCoV DI RNA as a test system.The discovery of a leaderless genome during coronavirus persistent infection in HRT-18 cells has led us to attempt a systematic analysis of leaderless genome behavior. It has been shown that substitution of the α-globin mRNA 5′ UTR with the MHV leader sequence enhances the translation of α-globin mRNA With regard to the significance of the leaderless genomic RNA identified during coronavirus persistence, the 5′ UTR of many positive-strand RNA viruses has been suggested to function in translation, replication, and transcription In this study, we discovered previously unidentified leaderless genomic RNA during BCoV persistent infection in cell culture, providing additional evidence that the members of Nidovirales may share a common ancestor. Functional analyses of the leaderless genomic RNA using BCoV DI RNA revealed that the leader sequence on the genomic RNA is required for coronaviral translation, replication, and transcription. Whether the leaderless genome is selected and contributes to the establishment of persistence needs to be further determined. Many aspects deriving from this study also need to be resolved. (1) Is leaderless sgmRNA synthesized during coronavirus persistent infection in addition to the leaderless genome discovered in this study? (2) Are there other mutations or structural changes in the BCoV genome or subgenome related to the maintenance of BCoV persistent infection? (3) Are there sequence alterations selected in the replicase gene that may be associated with the synthesis of leaderless genomic RNA? The elucidation of these questions will contribute to the further understanding of coronavirus persistence.A DI RNA-free stock of the Mebus strain of BCoV (GenBank accession no. U00735) was plaque-purified three times and grown in a HRT-18 cell line, as described A head-to-tail ligation method has previously been used to identify the terminal features of the RNA genome NgoMIV and XbaI was cloned into NgoMIV- and XbaI-linearized psBM65A to produce construct pΔ69-BM65Ahis. To generate pBM65AhisΔ5, oligonucleotides TGEV 7(−) and Δ5(+) were used with pBM65Ahis DNA in the first PCR, and oligonucleotides Δ5(−) and DI reverse(+) were used with pBM65Ahis DNA in the second PCR; oligonucleotides TGEV 7(−) and DI reverse(+) were used in a third PCR with the products of the first two reactions to amplify a 1248-nt product that was cloned into the TOPO XL vector (Invitrogen). Next, an 801-nt fragment obtained by digestion with SpeI and MluI was cloned into SpeI- and MluI-linearized pBM65Ahis to produce the pBM65AhisΔ5 mutant. To construct pΔ69-BM65AhisΔ5, an 801-nt fragment obtained by digestion of pBM65AhisΔ5 with SpeI and MluI was cloned into SpeI- and MluI-linearized pΔ69-BM65Ahis to produce the pΔ69-BM65AhisΔ5 mutant. Construct pDIEGFP, formally called pDIRNA-3, with the template-switching signal  and EGFP gene was previously described XbaI- and NgomIV, and the digested fragment carrying a 69-nt deletion of the BCoV 5′ UTR was cloned into XbaI- and NgomIV -linearized pDIEGFP.Construction of pBM65Ahis has been described previously MluI, transcribed in vitro with the mMessage mMachine T7 transcription kit (Ambion) according to the manufacturer’s instructions, and passed through a Biospin 6 column (Bio-Rad), followed by transfection 5 cells/dish) were infected with BCoV at a multiplicity of infection of 5 PFU per cell and transfected 2 h later with 3 µg of transcript RNA using Lipofectin (Invitrogen) The DNA constructs were linearized with 8 to 10 copies of each plasmid). The efficiency of the negative-strand RNA synthesis was normalized to the levels of the internal controls including 18S rRNA, DI RNA, and helper virus N sgmRNA; the primers used for these internal controls are described in To analyze the synthesis efficiency of negative-strand DI RNA 5 cells/dish) were infected with BCoV at a multiplicity of infection of 5 PFU per cell. After 2 h of infection, 3 µg of transcript were transfected into the BCoV-infected HRT-18 cells. The supernatant was harvested at 48 hpt, and 500 µl was used to infect freshly confluent HRT-18 cells in a 35-mm dish . Total cellular RNA was extracted with TRIzol (Invitrogen) at 48 hpi (VP1), and 10 µg of total RNA was electrophoresed through a formaldehyde-agarose gel. RNA was transferred from the gel to a Nytran membrane by vacuum blotting, and the blots were probed with the TGEV8(+) oligonucleotide, which was 5′-end labeled with 32P. The probed blot was exposed to Kodak XAR-5 film for 16 h at −80°C. For quantitating DI RNA replication, the probed blots were analyzed using a Packard InstantImager Autoradiography System.HRT-18 cells in 35-mm dishes at ∼80% confluency (∼8×10MluI-linearized pDIEGFP and pD69EGFP in vitro with the mMessage mMachine T7 transcription kit (Ambion) according to the manufacturer’s instructions. For transfection, HRT-18 cells in 35-mm dishes at ∼80% confluency (∼8×105 cells/dish) were infected with BCoV at a multiplicity of infection of 5 PFU per cell and then transfected 2 h later with 3 µg of transcript using Lipofectin (Invitrogen) The transcripts used for the detection of sgmRNA from DI RNA were synthesized from Figure S1Identification of negative-strand leaderless sgmRNA 7 during BCoV persistent infection. (A) Total cellular RNA extracted from BCoV-persistently infected cells was treated with tobacco acid pyrophosphatase and ligated with T4 RNA ligase I. The RT-PCR product was synthesized with primers BCV3′UTR1(−) (for RT) and RYN(+). RT-PCR products with a larger size of ∼300 bp  and with a smaller size of ∼200 bp  were observed. (B) The upper panel shows part of the first 88-nt sequence of the 5′ UTR in the negative-strand BCoV sgmRNA 7. The positions (1 and 70) are given on the top of the sequence, and the intergenic sequence (IS) AGAUUUG is underlined. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of ∼300 bp from lane 7, as indicated with a black arrowhead in (TIF)Click here for additional data file.Table S1Oligonucleotides used for this study.(TIF)Click here for additional data file.


Next generation DNA sequencing technologies are driving increasingly rapid, affordable and high resolution analyses of plant transcriptomes through sequencing of their associated cDNA (complementary DNA) populations; an analytical platform commonly referred to as RNA-sequencing (RNA-seq). Since entering the arena of whole genome profiling technologies only a few years ago, RNA-seq has proven itself to be a powerful tool with a remarkably diverse range of applications, from detailed studies of biological processes at the cell type-specific level, to providing insights into fundamental questions in plant biology on an evolutionary time scale. Applications include generating genomic data for heretofore unsequenced species, thus expanding the boundaries of what had been considered “model organisms,” elucidating structural and regulatory gene networks, revealing how plants respond to developmental cues and their environment, allowing a better understanding of the relationships between genes and their products, and uniting the “omics” fields of transcriptomics, proteomics, and metabolomics into a now common systems biology paradigm. We provide an overview of the breadth of such studies and summarize the range of RNA-seq protocols that have been developed to address questions spanning cell type-specific-based transcriptomics, transcript secondary structure and gene mapping. Arabidopsis thaliana  is underpinning an ongoing revolution in the life sciences and it is now difficult to identify areas of biology that are not already being profoundly affected by the massive amounts of high quality DNA sequence information that has been generated cost-effectively and efficiently, thanks to the rapid advancement of sequencing technologies. Plant biology is naturally no exception to this revolution; indeed the ease of genetic analyses in many plant species and the value of crop species have made plant science an especially fertile area for many of the “omics” technologies. Plant scientists are rapidly moving on from a decade where the first genome sequence of a plant, that of thaliana , provide2, which aims to sequence the transcriptomes of 1,000 plant species, and is just one of many current initiatives that are radically expanding the breadth and depth of our understanding of plant gene expression and evolution. Due to its accuracy and the ease of meaningful comparisons of samples not necessarily generated together, or even as part of the same experiment, RNA-seq is replacing other methods of quantifying transcript expression, including cDNA- and expressed sequenced tag (EST)-based microarray platforms  technologies, which apply the principles of NGS to the complementary DNAs (cDNAs) derived from transcript populations, were first used to study plants only a few years ago  and now latforms , as it olatforms . For exalatforms . Moreovelatforms .Nonetheless, RNA-seq has emerged as a remarkable enabling technology that is increasingly being adopted by plant researchers from a broad range of disciplines and examples of some of the associated applications and fields of research are presented in this review.A. thaliana genome sequence  genes show alternative splicing patterns . It has been reported that polymorphisms between different A. thaliana accessions is relatively high, with one single nucleotide polymorphism (SNP) every ~200 bp , splice variants and expression variation. Furthermore, with its greater sensitivity, RNA-seq enables the detection of antisense transcripts and transcribed intergenic regions; topics that are discussed further in Section “Identifying and Characterizing Novel Non-Coding RNAs.”For many heterozygous and out-crossing species, genome sequencing and annotation can only be considered complete once the breadth of intra-species polymorphism is also considered. The high quality reference genome of  ~200 bp . The com ~200 bp . To thisde novo sequencing of the transcriptome an attractive alternative to generate genetic resources for species that are of considerable biological interest for reasons that relate to factors such as their evolutionary significance or economic importance. Examples of recent such initiatives include fern  in a single RNA-seq analysis of sesame (f sesame  increasede novo assembly of 454-based ESTs derived from the pollen of tomato (Solanum lycopersicum) and two wild relatives. Approximately the same number of proteins was identified when using either the RNA-seq-derived database, generated through a few 454 pyrosequencing runs, or a highly curated community database of tomato sequences generated over more than a decade. This suggests that RNA-seq will be invaluable in facilitating protein identification and that proteome studies need no longer be so taxonomically restricted.Another striking example of the value of RNA-seq as an enabling technology is its application to advance the field of proteomics. High-throughput mass spectrometry-based protein identification relies on the availability of an extensive DNA sequence database in order to match experimentally determined peptide masses with the theoretical proteome generated by computationally translating transcripts. Indeed, the lack of extensive plant DNA sequence information and related resources is likely a contributing factor in the relatively slow progress in the arena of plant proteomics compared with proteome studies of other organisms for which high quality sequence has long been available. Vitis vinifera) berries during three stages of development identified >6,500 genes that were expressed in a stage-specific manner (Raphanus sativum) roots at two developmental stages and found >21,000 genes to be differentially expressed, including genes strongly linking root development with starch and sucrose metabolism and with phenylpropanoid biosynthesis. The radish genome has yet to be sequenced, but comparative sequence analysis of the radish RNA-seq data and the Brassica rapa genome sequence lead to the discovery of 14,641 SSRs.As with previous large-scale transcript profiling platforms, including microarrays, RNA-seq is increasingly being adopted to examine transcriptional dynamics during various aspects of plant growth and development. For example, an analysis of the transcriptome of grape  driven by various root cell type-specific promoters, digesting entire roots with cell wall degrading enzymes and fractionating the resulting protoplasts into distinct pools using an automated cell sorter (Zea mays) shoot apical meristems using RNA-seq coupled with Illumina-based NGS  plants treated with abscisic acid (ABA) or polyethylene glycol, in conjunction with published transcriptome analysis for Arabidopsis, maize, and rice, Chlamydomonas reinhardtii when the cells become deprived of sulfur, and to suggest molecular mechanisms that are used to tolerate sulfur deprivation  play important roles in gene post-transcriptional regulation  and therNA genes  but RNA-NA genes .Arabidopsis root tissues. Some sRNAs were expressed in all five tissues while others were tissue-specific, and some fluctuations in miRNA expression were also observed across developmental zones. In addition, growing numbers of RNA-seq studies are revealing the spatial and temporal differential expression of sRNAs in plant organs  are also implicated in abiotic stresses and many miRNAs and other sRNAs have been shown to be differentially expressed under phosphate starvation in Arabidopsis roots and shoots , especially in plants, and few plant lncRNAs have been characterized to date  information to help highlight important gene candidates as critical components of functional networks. Many such “guilt-by-association” gene co-expression networks have been constructed based on microarray data  and are Gene ontology enrichment analysis of RNA-seq data often illustrates the complexity of interacting pathways. For example, in a study of abiotic stress responses in maize, transcripts associated with numerous GO classifications were affected by drought treatment, including the categories “carbohydrate metabolic process,” “response to oxidative stress,” and “cell division,” among others . The autPapaver somniferum) cultivars with known differential levels of benzylisoquinoline alkaloids (BIAs) and used a combination of RNA-seq and mass spectrometry to pinpoint key regulatory steps of the almost completely defined morphine biosynthetic pathway, leading to the discovery of candidate genes implicated in BIA metabolism.Functional networks can be made more robust by integrating multiple data types and various studies have coupled RNA-seq with proteomics and/or metabolomics, characterizing the apparent downstream consequences of transcript level variation. An example of such a “systems” study involved a comparative analysis of the transcriptome, proteome, and targeted metabolome of soybean seeds from transgenic lines with suppressed expression of the storage proteins glycinin and conglycinin . This stThese examples show that the integration of transcriptomics, proteomics, and metabolomics can expose complex biological and biochemical interactions, paving the way to elucidate relationships between genotype and phenotype. Even greater resolution can be achieved by targeting tissues instead of whole organs .RNA-seq technologies can be adapted to answer-specific biological questions. Four different adaptations or applications are described here.Figure 1). As an example of the value of strand information, a study of tomato gene expression showed that while the majority of genes in the tissues analyzed had effectively the same expression profiles when analyzed by either double-stranded or strand-specific RNA-seq, approximately 5% of transcripts were associated with misleading results when assayed by double-stranded RNA-seq (dsRNA-seq) alone  alone .gl3. Transcriptome profiling is applied to a pool of two samples generated by mixing a bulk of mutant and wild-type (WT) plants . The mapping of the mutated gene is based on genetic linkage where linkage disequilibrium between markers and the causal gene is determined by quantifying the allelic frequencies between the two samples, giving the map position of the gene responsible for the mutant phenotype. Fine mapping of the mutated gene is facilitated by the RNA-seq data as its expression will often be down-regulated compared to the WT pool. Additionally, the SNPs linked to the mutated gene can be used for chromosome walking. Using RNA-seq for this purpose has therefore numerous advantages: (i) having a reference genome is not a prerequisite as de novo assembly of the transcriptome based on the RNA-seq data is sufficient; (ii) markers can be generated from the experimental data; and (iii) differential expression profiles between the mutant and the WT are generated at no extra cost. Furthermore, this approach can be modified to perform genome-wide association (GWAS) studies, accelerating breeding initiatives by providing markers targeting both genetic sequence  and gene expression, using them to identify the genomic regions associated with the traits of interest  and the double-stranded segments of RNAs by digesting the single-stranded RNAs with a ribominus treatment prior to library construction . As expected, highly structured RNA classes  were highly represented in the reads but, interestingly, other regions of various mRNAs, including introns, exons, and 5′ and 3′ UTRs were also present, indicating the presence of mRNA secondary structures. Moreover, the double-stranded regions of the introns, 3′ and 5′ UTRs appeared to be conserved, suggesting a common function. Notably, certain regions of the genome appear to be responsible for producing more dsRNAs than others, with transposable elements representing nearly 60% of these “hotspots.”Secondary structures of RNAs are central to their function, maturation, and regulation; however, little is known about the double-stranded features of most RNAs. Figure 4). The treatment removes the processed transcripts by degrading 5′ monophosphate RNAs, which are characteristic of prokaryotic RNAs, and the primary unprocessed transcripts are not affected due to the presence of a 5′ triphosphate. By comparing the maps of the reads derived from each sample, TSSs of operons are identified. dRNA-seq was first used to examine the transcriptome of the human pathogen Helicobacter pylori  is based on a comparison of a terminator exonuclease treated RNA sample with its non-treated counterpart (r pylori  and subssyringae . This mesyringae  and was RNA-sequencing is now well-established as a versatile platform with applications in an ever growing number of fields of plant biology research. Ongoing developments in sequencing technologies, such as increased read lengths, greater numbers of reads per run, and advanced computational tools to facilitate sequence assembly, analysis, and integration with orthogonal data sets will further accelerate the breadth and frequency of its adoption by plant scientists. An important issue that still needs to be addressed is the inherent bias introduced by the different steps of library construction and so the tantalizing prospect of direct RNA-seq  has greaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sturnus vulgaris) to integrate ecological reasoning with decision models, linking and testing hypotheses for value acquisition and choice mechanism. We hypothesise that options' values depend jointly on absolute attributes, learning context, and subject's state. In simultaneous choices, preference could result either from comparing subjective values using deliberation time, or from processing each alternative independently, without relative comparisons. The combination of the value acquisition hypothesis and independent processing at choice time has been called the Sequential Choice Model. We test this model with options equated in absolute properties to exclude the possibility of preference being built at the time of choice. Starlings learned to obtain food by responding to four stimuli in two contexts. In context [AB], they encountered options A5 or B10 in random alternation; in context [CD], they met C10 or D20. Delay to food is denoted, in seconds, by the suffixes. Observed latency to respond (Li) to each option alone  ranked thus: LA≈LC<LB<<LD, consistently with value being sensitive to both delay and learning context. We then introduced simultaneous presentations of A5 vs. C10 and B10 vs. C10, using latencies in no-choice tests to predict sign and strength of preference in pairings. Starlings preferred A5 over C10 and C10 over B10. There was no detectable evaluation time, and preference magnitude was predictable from latency differentials. This implies that value reflects learning rather than choice context, that preferences are not constructed by relative judgements at the time of choice, and that mechanisms adapted for sequential decisions are effective to predict choice behaviour.Foraging animals typically encounter opportunities that they either pursue or skip, but occasionally meet several alternatives simultaneously. Behavioural ecologists predict preferences using absolute properties of each option, while decision theorists focus on relative evaluations at the time of choice. We use European starlings ( We present a theoretical and experimental investigation of animal choice that links foraging ecology, state- and context-dependent learning and hypothetical cognitive mechanisms for choice. Foraging animals search, detect, pursue, catch, and consume prey of diverse kinds, each time experiencing the consequences of the whole cycle of events. Throughout these sequences they store information about prey physical attributes  and about experienced consequences of their decision to pursue such prey .From a learning point of view, experienced consequences constitute biologically programmed signals that retrospectively give value to (reinforce) earlier links of each typical sequence e.g.,  and hencOptimal choice in simultaneous encounters should be free from such effects, but simultaneous encounters may be too infrequent to select for learning processes immune to these fallacious effects. The structure of most species' ecology means that state- and context-dependent reinforcement, evolved as adaptations to serial cycles, are likely to dominate learning-dependent valuation e.g., . Thus, wmust take time to weigh evidence for alternative interpretations and/or possible ramifications before settling on a verdict.’ .Learning, choice and decision making attract multidisciplinary interest, and it is not surprising that theories follow differing rationales and make different predictions. For instance, a comprehensive recent review  includesThe view that decisions take time and deliberation time reflects a tradeoff between speed of action and accuracy of decision is a logical expectation, but it is still subject to empirical research. We have argued that because sequential encounters are frequent and simultaneous choices rare, the latter are handled by mechanisms selected for their performance in the former, and this may mean an absence of deliberation time. The mechanism we propose is that in the (rare) simultaneous encounters, each option elicits its own typical latency to act reflecting its encoded subjective value, and a “choice” results from responding towards the option eliciting the shorter latency Sturnus vulgaris) in two contexts, [AB] and [CD], thus generating context-dependent differential utilities across foraging options 5 or B10 in random alternation; in context [CD], they met C10 or D20 in a similar fashion. Suffixes indicate delays to food, in seconds. The animals then faced infrequent simultaneous choice trials between options that belonged to different contexts, namely between A5 and C10 and between B10 and C10  in sequential encounters stimuli whose outcome causes more favourable state changes relative to their context will cause shorter latencies to respond. Notice that options equal in absolute properties but experienced in different contexts can then have different value; (ii) preferences in simultaneous encounters should be predictable by assuming cross-censorship between the latencies observed in sequential encounters, and (iii) decisions should not take extra time; responding to an option in a simultaneous choice should not take longer than when the same option is encountered alone.The study was approved and carried out in strict accordance with the recommendations of the Ethical Review Committee of the Department of Zoology of the University of Oxford. The starlings that participated in this experiment were obtained and kept under Natural England license No. 20083718 and were reintroduced into a communal aviary after the experiment, before being released back into the wild. All efforts were made to minimize suffering.Sturnus vulgaris) participated in the experiment. All had laboratory experience unrelated to the contingencies they encountered here. The birds were kept outdoors before starting the experiment, when they were transferred to individual indoor cages (160 cm×45 cm×40 cm) that served as housing and testing chambers. Indoor temperatures ranged from 15 to 18°C and lights followed a 12∶12 dark schedule with light from 0700 to 1900. Starlings were rewarded with 20 mg BioServ® precision pellets throughout the experimental sessions and provided with three hours of free access to turkey crumbs (14∶30 to 17∶30) and 10 mealworms daily. This regime allows the starlings' body weights to remain stable at approximately 90% of their free-feeding weight Eight wild-caught adult European starlings , and two side subpanels (each 10 cm wide) attached to the cage at a 110 degree angle from the centre subpanel. Each panel had a response key in the centre (11 cm from the bottom) that could be illuminated with a variety of hues and black and white symbols. The middle subpanel also had a food hopper (2 cm from the bottom) that was connected to a pellet dispenser (Campden Instruments®). Trials were controlled by custom software running on the Microsoft® Windows® operating system and attached to an Animal Behavior Environment Test System (Campden Instruments®) via WhiskerServer 5 or B10 and in context [CD] with options C10 or D20.After brief preliminary training . The order of presentation of contexts alternated across subjects and days. Half of the subjects started the first session with context [AB] and the other half with context [CD]. On each particular session, the subjects that began with context [AB] ended with context [CD], and on the next day, they began with context [CD] and ended with context [AB]. Contexts were separated by an interval of either 45 (first and third transition) or 60 minutes (second transition). Sessions ended after 176 trials or 6.5 hours from the session start, whichever came first.Within each context, birds experienced 40 single-option trials (20 per option) and 4 peak trials (2 per option), all separated by a 30-s inter-trial-interval (ITI). Single-option trials emulated sequential encounters in natural settings and provided the birds information about the properties of each option. These trials began with the green centre key flashing . A single peck to this key switched its light off and caused one of the side keys to start flashing one of four possible symbols at the same rate used for the centre key. A peck to this key turned it steadily on and initiated the delay associated with the displayed symbol . The time between pecking the green centre key and pecking the flashing side key is hereafter referred to as the latency to respond. The first peck after the delay elapsed turned the side key symbol off and produced two food pellets followed by the ITI. Peak trials were identical to single-option trials except that once the flashing side key was pecked, the symbol remained continuously on for 60 s, and then extinguished without reinforcement. These trials were used to determine the animals' knowledge of the delay to reward associated with each particular option 5 and C10 or between B10 and C10. Half of the subjects were first tested with choices between A5 and C10 and the remaining half with choices between B10 and C10. When preference reached stability, choice types were swapped: subjects that previously chose between A5 and C10 faced choices between B10 and C10, and vice-versa. Both tests continued until the stability criteria were met: a standard deviation less than 10 for choice percentages and no visible trend in choice percentages, both during the last three sessions.This phase was structurally similar to the training phase except that choice trials were added. All such trials were cross-context, involving choices either between ASixteen simultaneous choice trials were presented daily. These were presented in pairs and separated by the ITI. Each pair of choice trials was presented at the beginning and the end of each context substituting the first and last two single-option trials that would normally occur during training. Choice trials were similar to single-option trials with the exception that pecking the flashing centre key caused two different symbols, one on each side key, to begin flashing simultaneously. A single peck to one of these keys turned the selected symbol continuously on, triggered the appropriate delay, and extinguished the unselected option. Thus, a bird committed itself to one of the options with its first peck to a flashing symbol. Again, the time between pecking the centre key and pecking one of the flashing side keys will be considered the as latency to choose. Once an option was selected, the trial proceeded as a regular single-option trial.5 vs. C10 and of B10 vs. C10, we used latencies from single-option trials in the same test sessions in which choice proportions were measured. A separate Monte Carlo simulation using a nonparametric estimate of the cumulative distribution functions of the data yielded extremely similar predictions and thus is not reported.Only latencies from single-option trials collected at stability were used to run the simulations. To compute predictions for preferences of A5 vs. C10 and 48 B10 vs. C10 trials]. For any given trial, the simulation drew one latency for each option [A5 and C10 or B10 and C10] from the population of observed latencies during single-option trials and predicted the option to be chosen in the simultaneous presentation as the one yielding the shortest latency.Each simulation involved 10,000 experiments of 96 trials each , starlings chose C10, the option with lower immediacy, 19.8% of the time (range: 2.1–47.9%). Conversely, when immediacy was the same but within-context ranking differed [B10 vs. C10], starlings preferred the option with higher ranking, C10, 69.7% of the time (range: 54.2–85.1%). One-sample t tests of magnitude of preference against indifference revealed that both these findings deviated significantly from chance . When given a choice between two options with the same within-context ranking but signalling different delays to reward , time  and their interaction . Of particular interest are the pecking patterns for options B10 and C10 as they provide a test for the hypothesis that contextual effects on preference were mediated by distortions of subjective temporal estimates. Post-hoc pairwise comparisons revealed that pecking rates did not differ systematically between options B10 and C10 (p = .289), but did differ for all other comparisons (largest p = .018), thus suggesting that the effect of within-context ranking was not mediated by distortions in interval timing. The analyses based on the location of peaks returned similar findings . For the B10 vs. C10 tests, the regression line falls close to the theoretical optimum whereas for the A5 vs. C10 tests the regression deviates considerably. One way to read the results of the A5 vs. C10 tests in 5 stronger than 80%, but the data do show such cases. We will refer to a potential explanation for this dissociation in the ability of the SCM to quantitatively predict choice across tests in the discussion.According to the SCM, preferences in simultaneous choice trials should be predictable from the latencies observed in sequential encounters. Predicting choice preferences from performance in single-option (no-choice) trials is an exclusive feature of the SCM, and for this reason it is of paramount importance for this study. The simulations predicted average preferences for CF = 20.042, p = .002]. Post-hoc pairwise comparisons showed that latencies differed significantly between options (largest p = .019) except for the comparison between options A5 and C10 (p = .081). Further, these latencies reflected both the immediacy and ranking of each option in its own context: greater immediacy and better ranking both shortened latency to accept an option. In particular, the within-context ranking effect is demonstrated because latencies to accept option C10 were significantly shorter than latencies to accept option B10 despite both options signalling the same delay to reward. In contrast, despite A5 and C10 signalling different delays in this case the difference between median latencies was short of conventional significance. These findings show that ranking did participate in the valuation of the options. The absence of a conventionally significant difference between A5 and C10, does not imply that absolute attributes do not have any effect, because these latencies were sufficiently short to approach psychophysical reaction times  in choice trials.t-tests failed to reveal significant difference in latencies depending on whether an option was encountered by itself or chosen in a simultaneous choice situation .10 , but the observed shortening for this option, while having the expected trend, did not reach conventional statistical significance in a two-tail test , even though it was the strongest shortening we observed  was either predicted by the Monte Carlo simulations or observed for any option.This central tendency analysis was complemented by analyses of the complete frequency distributions of latencies  and whether the translation of these subjective values into preferences in simultaneous encounters involved a relative comparison at the time of choice or resulted from the expression of the same mechanisms used in no-choice encounters. We found support for our learning hypotheses because when facing simultaneous choices between options differing in either delay to reward or in within-context ranking, starlings' choices were better explained by remembered context-dependent utility than by remembered objective properties. Analysis of temporal responding showed that preferences were not driven by distorted memory for temporal properties of the options.Consistently with the assumptions of the SCM, latencies to accept options presented singly were affected both by the options' objective properties and their within-context ranking: the higher the immediacy and the better the ranking the shorter the corresponding latencies. This supports the view that latencies expressed in sequential encounters reflect the operation of a valuation process and justifies their use in predicting simultaneous choice 10 vs. C10] but were directionally correct but less extreme than observed when options had the same ranking and different immediacies [A5 vs. C10]. This pattern of findings relates to our latency findings: both the analysis based on median latencies and that based on cumulative frequency distributions indicated that the latencies recorded for A5 in single-option trials were somewhat shorter (but not reliably so) than those found for C10. Thus, the results from the Monte Carlo simulations (that used latencies from the same distributions) did not reach the strength of preference for A5 that was observed, despite predicting preference in the correct direction. Conversely, we did find significant differences between the distribution of latencies for B10 and C10, which in turn yielded predictions that virtually matched the observed preferences.One of the unique predictions of the SCM is that we ought to be able to predict preferences in simultaneous choices from latencies observed during sequential encounters. The present experiment tested this prediction in a situation where valuation was context-dependent and thus significantly influenced by background alternatives. The model was qualitatively accurate in both choice tests and remarkably accurate quantitatively in one of them. Specifically, the model predicted preferences in simultaneous choices were almost identical to those observed when the options have the same immediacy and different rankings [B10 and C10 . In no case was there any indication for an increase in latencies consistent with a deliberation process at the time of choice. Taken together these results favour the SCM cross-censorship mechanism rather than of a deliberative process at the time of choice, particularly because the task both families of models face is asymmetric: latencies have floor but not ceiling and thus lengthening should be easier to observe than shortening.In short, then, the confluence of ecological and mechanistic hypotheses that we endorse argues that because in natural circumstances animals typically meet feeding opportunities sequentially and seldom simultaneously, their learning and decision processes do not include cognitive mechanisms evolved due to their performance in simultaneous choice, and our observations endorse this view. In sequential encounters animals assign subjective value to prey types according to their state and the contrast with the background, and in simultaneous encounters, when by necessity state and background are unique, the learning past still controls choice. Preference thus is not built at the time of choice, by a cognitive deliberation that evaluates options' differences, but by the shadow of past state-dependent valuation learning.Figure S1Average cumulative frequency distribution of latencies for each option during sequential encounters. The greater the area below each function the shorter the overall latencies to accept that particular option.(TIF)Click here for additional data file.Figure S2Average cumulative frequency distribution of latencies for each option in single-option and simultaneous choice trials. Triangles and dots represent data from single-option and simultaneous choice trials. Latencies are separated according to collection time: either at stability in the A5 vs. C10 preference tests or at stability in the B10 vs. C10 preference tests. (A) Distributions for A5 in the A5 vs. C10 preference tests. (B) Distributions for B10 in the B10 vs. C10 preference tests. (C) Distributions for C10 in the A5 vs. C10 preference tests. (D) Distributions for C10 in the B10 vs. C10 preference tests.(TIF)Click here for additional data file.Text S1Preliminary training.(PDF)Click here for additional data file.Text S2Preliminary analyses.(PDF)Click here for additional data file.Text S3Analyses of peak location.(PDF)Click here for additional data file.Text S4Analyses of complete latency distributions.(PDF)Click here for additional data file.