Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10–L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD) spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocket which is not optimized for high activity in the case of Pab21.Multiple RNA-guided pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs) which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA) is anchored by the pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from  Pseudouridine (Ψ) is one of the most common modified ribonucleotide found in functional regions of non-coding RNAs The majority of information on the structure and function of H/ACA RNPs derives from experiments performed on archaeal particles. The H/ACA sRNAs identified in archaeal genomes share common features with eukaryotic snoRNAs, but differ in their heterogeneous architectures in vitro using recombinant proteins purified from E. coli and in vitro-transcribed guide sRNAs, and reconstituted sRNPs pseudouridylate substrate RNAs consisting of simple target oligoribonucleotides Information on the role of the core proteins and the conserved RNA elements for sRNP assembly and activity has been obtained by studies performed with reconstituted particles Crystal structures have been obtained for i) the RNA-free and -bound forms of L7Ae Here, we investigated the respective conformations in various sub-RNPs or in RNPs lacking the L7AeaNOP10 interaction of two sRNAs exhibiting different efficiencies in guiding pseudouridylation. The analyses were performed in solution by chemical probing and circular dichroism (CD) spectroscopy. Our data demonstrate that the dynamic RNA conformation within RNPs can be minutely explored by CD. Moreover, our results clearly show that the H/ACA sRNA conformation depends on helix P1, which represents an important determinant for the efficiency of H/ACA RNP assembly.in vitro transcribed 32P 5′ end-labeled sRNA (∼50 fmol) with purified recombinant proteins (200 nM each) in buffer D  at 65°C for 10 min. Association of the sRNA present in the sRNP with its substrate RNA was analyzed by two approaches in vitro transcribed sRNA (2 pmol) with radiolabeled oligoribonucleotide S (150 fmol). Complexes were visualized by autoradiography following fractionation by non-denaturing polyacrylamide gel electrophoresis.As previously described 32P]CTP or [α-32P]UTP labeled substrate RNA were mixed at 65°C with the various sets of protein. Aliquots were collected at several time points and the reaction was stopped by phenol-chloroform extraction followed by ethanol precipitation. The recovered substrate RNAs were digested with RNase T2. The resulting 3′-mononucleotides were fractionated by monodimensional thin layer chromatography, and the radioactivity in the resulting bands was quantified with a phosphorimager using ImageQuant software. The quantities of Ψ residues formed were determined by taking into account the total number of U residues in the sRNA’s substrate RNA.Conditions for time course analysis of the RNA:Ψ-synthase activities of the reconstituted sRNPs were as detailed previously 32P-end labeled sRNA using the same conditions as described in the preceding paragraph. sRNAs were labeled at their 5′ ends using T4 polynucleotide kinase and [γ-32P]ATP. For lead (II) footprinting, the reactions were initiated by addition of 40 mM Pb(II) acetate (Merck) freshly prepared in sterile water and incubated for 2 min at room temperature. Reactions were stopped by addition of 0.1 M EDTA and precipitated with 1 ml of ethanol at –80°C. Next, a phenol-chloroform extraction was performed and the treated RNA samples were ethanol precipitated in the presence of 0.3 M sodium acetate and 2 μg total yeast tRNAs. After centrifugation, the pellet was washed with 80% ethanol, and the dried RNA pellets were dissolved in RNA loading dye . RNA alkaline hydrolysis ladders (cleavage after each nucleotide) were generated by incubating the labeled RNA in 1 M sodium carbonate at pH 9 for 3 min at 96°C. In order to generate RNase T1 ladders (cleavage after each guanosine), the labeled RNA was incubated in 1 M sodium hydroxide citrate in the presence of 2 µg tRNAs for 5 min at 65°C, and then treated with 1 U of RNase T1 for 10 min at 65°C. The cleavage products were separated on 10% polyacrylamide (acrylamide:bis ratio 38∶1) gels containing 8 M urea, and visualized by phosphorimaging.sRNPs were assembled on −6 for the apparatus, the nucleotide concentration and the optical path-length of the cuvette, measurements were obtained at a precision of δ (Δε) = +/−0.02 M−1.cm−1 per nt.The CD spectra were recorded using a Jobin-Yvon Mark VI circular dichrograph at a scan speed of 0.2 nm/s. Quartz spare split-compartment cuvettes with 0.435 cm path length per compartment were used and spectra were scanned in the range of 200–320 nm. The final protein and/or RNA concentration was 2 µM, and the assays were carried out at 25°C in 10 mM Tris-HCl (pH 7.5) containing 150 mM NaCl. The protein and RNA spectra alone were recorded by placing the relevant solution in one compartment of the cuvette and buffer solution in the other. Studies of the protein-RNA interaction and sub-sRNPs formation were achieved using a sequential analysis. First, the protein solution (aCBF5) was placed in one compartment and the sRNA in the second one, and CD spectra were recorded before and after mixing the cuvette contents. Proteins aNOP10 and L7Ae were then added sequentially, and after five minutes of mixing, the spectrum of each sub-sRNP was measured. Blanks were obtained for each spectrum or sequential interaction, and the values subtracted from the raw data. In each case, two spectra were averaged to increase the signal-to-noise ratio. The results are presented as normalized Δε values on the basis of the mean residue mass of 330 Da. Taking into account a sensitivity of δ(ΔA) = 101 and Pab91P1 respectively, were built by assembling a canonical A-form RNA helix corresponding to the lower stem (P1) with a terminal loop mimicking the internal loop of the H/ACA sRNA, and the ACA box. The terminal loop was extracted from the 23S rRNA structure of Deinococcus radiodurans (PDB ID: 1J5A): it corresponds to the dodecaloop found at positions 2187 to 2198 (5′-AAAAAUCACUUU-3′) which best fits the size (11-mer or 12-mer) and sequence (proportion of purines and pyrimidines) of the peudouridylation pockets from Pab21 and Pab91. The RNA models were assembled using Mc-Sym +). The full system of nearly 20,000 atoms was equilibrated for 0.25 ns before a production run of 10 ns. The simulations were performed in the NVT ensemble at 298 K, using the CHARMM27 force field The Pab21 and Pab91 RNA hairpin models, Pab21PThe MD trajectories were analyzed using: (1) the 3DNA software 2 and 180 mM Na citrate. The substrate RNA was added at a concentration of 1 µM and the molar ratio in reconstituted RNP subcomplexes was 1∶1∶5 for substrate RNA:guide RNA:proteins. The fluorescence intensity was recorded in kinetic mode until a plateau was reached, using an excitation wavelength of 325 nm and an emission wavelength of 366 nm. The samples were incubated for 10 minutes before each measurement.The fluorescence studies were carried out by adapting the assay developed by Li and colleagues Pyrococcus abyssi .The chemical probing results suggested that the guide sRNA undergoes change in conformation during the sequential reconstitution of sub-sRNPs. We used far UV circular dichroism (CD) spectroscopy to analyze in more detail such conformational changes . This ch max) at a wavelength referred as the λmax wavelength  and Pab91 (blue line) are respectively 263.8 nm and 265.6 nm. The lower λmax measured for Pab21 suggests a more marked curvature in the global structure of this sRNA as compared to Pab91. Moreover, we measured a higher Δεmax for Pab21 compared to Pab91 . This difference indicated the presence of a higher number of unstacked nucleotides in the Pab91 RNA helices compared to those of Pab21.The CD spectra of free Pab21 and Pab91 displayed a positive signal in the 250–300 nm range with a maximal Δε value  = –0.78±0.02 M−1.cm−1 per nt, max was displaced from 263.8 to 265.6 nm (±0.1 nm) although the amplitude of the peak was unchanged as compared to the spectrum obtained with the naked sRNA  for Pab21, and 265.6 to 267.6 nm (±0.2 nm) for Pab91, and a reduction in the Δε value (δ(Δε) = –1.28±0.02 M−1.cm−1 per nt for Pab21, and –0.57±0.02 M−1.cm−1 per nt for Pab91). Interestingly, Pab21 and Pab91 have comparable Δε value (7.2 M−1.cm−1 per nt) in the aNOP10-aCBF5-sRNA complex  was measured, but more remarkably, this effect was coupled with a further decrease in the RNA signal (δ(Δε) = –1.11±0.02 M−1.cm−1 per nt), compared to the spectrum of the aNOP10–aCBF5–sRNA complex (−1.cm−1 per nt). In addition, CD spectra obtained after addition of the wild-type L7Ae to the aCBF5–aNOP10(Y41A/Y44A)–Pab21 complex and upon addition of the L7Ae mutant (H70A/L74A/E77A) to the wt aCBF5–aNOP10–Pab21 complex showed that the mutant sRNPs displayed lower δ(Δε) values than the wild type sRNP (–0.82 and –0.825 vs. –1.21±0.02 M−1.cm−1 per nt)  . In view121), helix P1 of Pab91 was replaced by that of Pab21, and in the second (Pab21P191), the original P1 was replaced by that of Pab91  and Pab21P191 (λmax = 265.6 nm), and sRNA Pab21 (λmax = 263.6) and Pab91P121 (λmax = 263.8 nm). These results confirmed that the wild-type sRNA Pab91 and Pab21 do not have the same curvature, and that helix P1 has a strong impact on the global curvature of the sRNAs. Concerning the Δε values, both are modified in the chimeric RNAs and are intermediate to the values of Pab21 and Pab91 . Hence, the helix P1 alone cannot restore the structure of the parental sRNAs, but contributes significantly to the global nucleotide stacking.We next compared the CD spectra obtained with the two chimeric RNAs with those of the parental RNAs . We meas1 and Pab91P1 were built  distorts the base stacking with a positive buckle at all base-pairs, while the alternation of purines and pyrimidines in Pab21P1 (5′-GGGCCCGGC-3′) provokes a smaller incidence of buckling, with a slight negative buckling on average, deriving essentially from the large deformation of the first base-pair of the stem, a wobble pair. We hypothesize that the more pronounced buckle  relative to Pab91P1 (22.9°)  with respect to Pab21P1 (72%) during the time of simulation (10 ns) (The molecular dynamics (MD) simulations performed on these two models show some subtle differences in the conformations of helix P1 associated with sequence-specific features . The pred buckle  would we (22.9°) 4. These  (10 ns) .121 and Pab21P191 retain the ability to guide Ψ formation into their RNA substrates with an efficiency equivalent to their respective parental Pab91 and Pab21. Hence, activity of the RNP relies on a determinant distinct from helix P1, and is not always related to the efficiency of recruitment of aCBF5 on the guide sRNA.We next tested whether replacement of helix P1 modified the efficiency for guiding pseudouridylation . Time coThe data presented here show the potential of using CD in the 240−320 nm range to compare the conformation of RNAs, such as the two sRNAs Pab21 and Pab91. Importantly, this approach appears well-adapted to monitor the conformational changes occurring in RNAs upon protein binding . This apmax observed upon aCBF5 binding indicate a modification in the global conformation of Pab21 − most likely a change in its curvature. This effect was not a general feature for all sRNAs, however, as we did not detect modification in the λmax value upon binding of aCBF5 to Pab91 (data not shown). In the archaeal sRNP H/ACA, the PUA domain of aCBF5 is anchored to the lower part of the sRNA; the stability of the aCBF5–sRNA complex also arises from contacts between the catalytic domain and helix P2, whose basal region is wrapped around the catalytic domain in the structure of the H/ACA sRNP Our data show that the core proteins have different impacts on the conformation of the guide RNAs during the stepwise assembly of the box H/ACA sRNP LCN. Binding of L7Ae to the Pab21 sRNA has a low impact on the global conformation of the guide sRNA. The slight effect on RNA base stacking  likely occurs in the K-turn motif, which is folded upon L7Ae binding max and a reduction of Δε for both Pab21 and Pab91 -NOP10 in absence of the guide RNA 1 is more prone to be bent upon aCBF5 binding and would facilitate the RNP assembly. However, the conformation of P1 in the fully assembled RNP complex (including the RNA guide and RNA substrate) is very close to a canonical A-form, where the substrate should be in some optimal binding mode for catalysis (PDB ID: 3LWR) 121 is less efficient than Pab21, the more rigid P1 helix can thus contribute to increase the catalytic efficiency  Secondary structure models of P. abyssi tRNAAsp. Residue U55 is circled. The CCA sequence at the 3′ end is boxed. The CCA deletion in variants tRNAAsp-ΔCCA is shown. (B) Time course analysis of Ψ55 formation in tRNA. The tRNAAsp−ΔCCA substrate was radiolabeled during in vitro transcription by incorporation of [α−32P]CTP. It was incubated at 65°C with the protein set aCBF5–aNOP10 (CN). A mutant of protein aNOP10 Y41A/Y44A (Y) was used for the reaction. After T2 RNase digestion, the amount of Ψ formation was estimated by 2D-TLC analysis.(TIF)Click here for additional data file.Figure S2Effect of mutations in proteins L7Ae and aNOP10 on substrate RNA positioning within the sRNP. The fluorescence intensity at 366 nm was monitored while the substrate RNA labeled with both 5-FU and 2-AP bound with the Pab21 guide RNA was first titrated with saturating amounts of the sRNP proteins. All proteins were present at 5× molar excess relative to the RNA to ensure full binding. Comparison of the fluorescence intensity profiles of titration by the wild type proteins (dark blue trace) to the profiles recorded with mutant proteins aNOP10 Y41A/Y44A (green trace) (A), and with L7Ae H70A/L74A/E77A (pink trace) (B). The arrows indicate the time points at which each specific protein was added, where CN denotes the aCBF5–aNOP10 complex, CY denotes the aCBF5–aNOP10 Y41A/Y44A mutant complex, L denotes L7Ae and HLE denotes the L7Ae mutant H70A/L74A/E77A.(TIF)Click here for additional data file.Figure S3Chemical probing of sub-complexes formed by the association of L7Ae with various sRNA. (A and C) Secondary structure models of P. abyssi Pab19 and Pab160 sRNAs. (B and D) Footprinting of protein L7Ae (L) on the various sRNAs. Reactions with lead were carried out on 5′-32P end-labeled sRNA as in − and T1 correspond, respectively, to an alkaline hydrolysis ladder, and RNase T1 digestion ladder obtained under denaturing conditions.(TIF)Click here for additional data file.Figure S41 and Pab91P1.Average h-Twist versus time of the two RNA hairpin models Pab21P The plotted values are calculated by averaging the helical twist on the nine base-pairs of P1. The plots are annotated by indication of the average values of the buckle during the simulation between Pab21P1 (red) and Pab91P1 (blue). The histograms are also annotated by indication of the median values.(TIF)Click here for additional data file.


In recent years there has been a growing interest in the field of non-coding RNA. This surge is a direct consequence of the discovery of a huge number of new non-coding genes and of the finding that many of these transcripts are involved in key cellular functions. In this context, accurately detecting and comparing RNA sequences has become important. Aligning nucleotide sequences is a key requisite when searching for homologous genes. Accurate alignments reveal evolutionary relationships, conserved regions and more generally any biologically relevant pattern. Comparing RNA molecules is, however, a challenging task. The nucleotide alphabet is simpler and therefore less informative than that of amino-acids. Moreover for many non-coding RNAs, evolution is likely to be mostly constrained at the structural level and not at the sequence level. This results in very poor sequence conservation impeding comparison of these molecules. These difficulties define a context where new methods are urgently needed in order to exploit experimental results to their full potential. This review focuses on the comparative genomics of non-coding RNAs in the context of new sequencing technologies and especially dealing with two extremely important and timely research aspects: the development of new methods to align RNAs and the analysis of high-throughput data. Caenorhabditis elegans [In recent years, the non-coding RNA (ncRNA) field has rapidly expanded  with a r elegans  and Xist elegans . Since tcis [trans [This wealth of data has allowed the identification of thousands of novel short ncRNAs, including PIWI interacting RNAs  and smalcis ,35 and is [trans , some acs [trans –39, whils [trans  lncRNAs s [trans .Although the conservation level of different lncRNAs may be not always directly comparable , overall approximately half of reported human lncRNA exhibit significant conservation across mammals . These li.e., miRNAs). Previous reports have shown that at least some miRNA genes are well conserved across species [i.e., is co-varying to restore the base pairing and keep the structure unchanged.In general, a major obstacle for ncRNA detection is the difficulty to perform informative sequence comparisons. Standard primary sequence alignment is hampered by the low complexity of the nucleic alphabet, making it difficult to produce statistically meaningful RNA alignments. Ribonucleic acid chemistry relies on just four primary residues: two purines and two pyrimidines. Consequently, RNA gene sequences do not have strong statistical signals, unlike protein-coding genes. For instance two RNA sequences must share an identity of at least ~60% to be considered significant in homology relationships prediction . Below t species –61, rein species ,63. ThisFor most aligners these features of RNA are hard to account for when using standard alignment procedures that postulate positional independence and seek only to maximize identity. Furthermore, RNA can hold functional pseudo-knots. These are structural configurations where at least two RNA stem-loops are interposed one into the other. Although some comparative approaches including pseudo-knots exist ,65, thesAs mentioned, generating meaningful ncRNA alignments is a challenging task and at least in some cases, the best accuracy could be achieved by exploiting RNA structural information. However, in many situations using such information is complicated. In spite of the development of aligners that take into account the RNA secondary structure information, one major issue is the poor availability of high quality structures. The problem is at least in part due to the difficulties encountered at experimental level in crystallization. Getting crystals from RNA molecules is complicated because of their chemical specificity. The accumulation of crystals is prevented by the high RNA flexibility. RNAs have flexible structures adopting inter-domain movements and with respect to proteins have weaker tertiary interactions . The polin vivo molecule structure. The prediction is performed using primary RNA sequence data. Another possibility is including other sources of statistical information to constrain structure prediction, for instance an alignment of structurally homologous RNA sequences. Regarding single sequence RNA secondary structure predictions, there are two main groups of approaches: empirical free-energy parameters [i,j is given by the sum of the probabilities of all structures containing that base pair i,j divided by the sum over all structures [Because of these limitations, RNA structure is usually computationally predicted without any experimental support ,71. RNA rameters  and knowrameters –75. The rameters –80 the nrameters –79. The rameters  and RNAframeters  packagesrameters , an apprructures . Knowledructures  represenin vivo can be influenced by RNA chaperones [Prediction consistency is the main limit of both MFE and knowledge based methods . See th. The peraperones , RNA ediaperones , and eveaperones . At presaperones ,90, wheraperones ,92. Thiraperones .in vivo, but rather it is a model that captures the common, relevant structural aspects conserved within the family.The best secondary structure prediction accuracy can be achieved using comparative methods . These aN3m), where m is the number of sequences and N their length. This means that a pairwise comparison has the tremendous CPU cost of O(N6) which makes this algorithm inapplicable most of the times and calls for simplified heuristics. Several banded modifications of the Sankoff algorithm impose limits on the size or shape of substructures, like Dynalign [x and y be two nucleotides matching in a secondary structure in seq1, and x′ and y′ be two nucleotides matching in a secondary structure in seq2. If x aligns to x′ then implicitly y should be driven to align to y′. For example, R-Coffee uses RNAplfold [i.e., ClustalW [Due to the close relationship between sequence and structure, structure prediction and sequence alignment can be described as interdependent problems . As theoDynalign ,104, FolDynalign ,106, SteDynalign , Consan Dynalign . AnotherDynalign , a SankoDynalign  or RNAcaDynalign . In thesNAplfold  to compuClustalW ). Plan CThe situation is radically different when experimental 3D structure information is available. In this case the RNA alignment problem becomes similar to the protein structural alignment problem. This problem is nondeterministic polynomial-time complete (NP-complete) and it involves the alignment of two distance matrices. In most cases the problem can be solved in a rather satisfying way by using heuristics making the best of the geometric information contained in the PDB models. Examples of pairwise structural alignment methods for RNA are SARA , DIAL 1, iPARTS Giving an exhaustive overview of the methods available for folding and aligning structured RNA sequences is well beyond the scope of this review. Over the last twenty years, more than 30 methods have been described that deal with these issues which is an indication of the complexity of this problem, despite 25 years of research following its formal description by Sankoff.ad hoc algorithms [E-values) to avoid losing sensitivity as achieved in Rfam [In the ncRNA field another critical step is the collection of homologues to genes of interest. Homologues can be used in several situations, such as the detection of functional motifs, inference of possible evolutionary steps or the design of laboratory experiments. For instance, the conservation across species of a certain ncRNA can be used to estimate how likely a gene is to be functionally important. Such information can be used to prioritize experiments, e.g., knockdown experiments of the orthologous gene in a model organism. Over the last few years many different methods have been developed to approach the problem of RNA homology detection. As previously shown , homologgorithms , profilegorithms  or BLAST in Rfam . A numbe in Rfam ,137 the  in Rfam , BlastR i.e., syntenic regions), coordinate transfer from one gene to its homolog is straightforward and implies the projection of corresponding positions. This has been made possible thanks to the availability of genome sequences [i.e., loci that proceeded from the same genomic position in the ancestral genome [Together with sequence-based, profiles and structure-based methods, another possibility for detecting inter-species homologs involves the use of multiple genome alignments . Once esequences –142 and l genome . Althougi.e., define start and end of a transcript. Such information is extremely valuable in situ ations where the first and last exons can be respectively 5′ and 3′ associated with other transcript isoforms, thus making it difficult to define gene boundaries. Similarly, the cap analysis of gene expression (CAGE) [Recent technological advances have allowed the collection of an unprecedented amount of RNA sequence data coming from a wide range of organisms and conditions. For many years the main strategy for transcript discovery had been the sequencing of cloned complementary DNA (cDNA) of expressed sequence tags (ESTs) –146. ESTn (CAGE) ,180 is an (CAGE) . This apn (CAGE)  RNA Captgenebuild process including ab initio gene predictions and release 64 supported a total of 61 species [genebuild system is adapted to every species in the set according to the data that is available. For instance Ensembl imports and merges high quality HAVANA annotations exclusively for human and mouse. The FANTOM consortium aims to provide functional annotations to the full-length cDNAs [http://www.gencodegenes.org/archive_stats.html) to version 7 , the number of protein-coding transcripts increased from 68,880 to 76,052, and the number of lncRNAs jumped from 10,457 to 15,512. In terms of gene annotations, the number of known protein-coding genes has remained almost unchanged, while the ncRNA gene annotations expanded tremendously  represent an exception and there is no need to reassemble them, as they are small enough to be entirely covered by the read length. Unfortunately the process of reassembling transcriptomes starting from short reads is difficult. Normally RNA-seq dataset are big (gigabases to terabases), and thus need to be handled by sufficient large memory and by multi-CPU computers able to execute the algorithms in parallel with sufficient high-performance storage to store primary, temporary and output data. Although various short-read assemblers [To make the most out of the extraordinary possibilities that NGS offers, it is essential to understand the current limitations. One important point is that the reads returned by standard NGS platforms are usually short (35–500 base pairs ) and as semblers –204 weresemblers .de novo assembly or combinations of each [i.e., annotating exon-intron transcript structures. These methods include Cufflinks [It is possible to work out the transcriptome assembly following a reference-based approach, a  of each . The fir of each –211. Oth of each ,213 and  of each ,215. The of each ,217, indufflinks , Isolazoufflinks  and Scriufflinks . In [220ufflinks ,219. Traufflinks  with theufflinks . Such diufflinks . In othede novo assembly of transcripts. This strategy does not require any reference genome and is therefore independent on the correct alignment of the reads to the splice sites. Advantages of this approach are that it is less reliant on accurate genome annotation and can be applied to organisms without sequenced or fully annotated genomes. Examples of applications adopting this strategy are described in [de novo assembly to complex transcriptomes  is complicated by the dataset sizes and the dense network of alternatively spliced variants. Furthermore, de novo transcriptome assemblers need much deeper sequencing than reference-based assemblers and are largely affected by sequencing errors [Another possibility to assemble a transcriptome from-short reads is ribed in –223. Nevg errors .in-silico assignment of a transcript to one of these two groups is not always trivial and may require dedicated expert curation [i.e., generating small peptides. There are further ambiguities for coding transcripts whose untranslated structured molecules are also functional as ncRNAs [i.e., showing distinctive mutation patterns, as are expected in coding and non-coding loci. A coding region is expected to embed prevalently conservative amino acid substitutions and synonymous codon substitutions, while showing low occurrence of nonsense and missense mutations. Although CSF has been successfully applied in various research projects [Once transcriptome dataset is generated, there are additional complications in the downstream analysis if trying to distinguish genuine ncRNAs from mRNAs. Currently, this issue is becoming increasingly important as many researchers are only interested in one or the other. The most straightforward procedure would be to compare a newly generated transcriptome against existing gene annotations. However in most cases annotations are far from complete and the great majority of genes they include are protein-coding. As a consequence, in a normal RNA-seq experiment a substantial fraction of read contigs map outside of annotated exons . Previoucuration . Some trs ncRNAs  and for s ncRNAs . A commos ncRNAs ,227. Thiprojects ,228,229,projects  many putprojects ,230 thatprojects  and UniPprojects . Other aprojects , PORTRAIprojects  and CPATprojects . UnfortuCaenorhabditis elegans ncRNAs. incRNA combines predicted and experimental data for a total of nine different information sources. These include the expression data coming from various developmental stages and conditions, as well as the GC content, the predictions of RNA secondary structure folding energy, the prediction of evolutionary conserved DNA sequence and secondary structure. These results illustrate how the integration of multiple information sources ends in highly accurate predictions of novel ncRNA genes.Over the last few years, other approaches alternative or complementary to RNA-seq have been attempted to generate high-throughput ncRNA annotations. In 2009, Mitchell Guttman and co-workers published the first of a series of analysis that recently came out linking lncRNA detection to histone modifications . In thisRecently, a number of works reporting a massive quantity of novel ncRNA genes in various species have been published ,237,238.i.e., functional molecules longer than 200 nucleotides that do not show any coding potential. Some of these molecules are spliced, capped, differentially expressed in tissues/cells or developmental stages and tend to be more conserved across species than would result from neutral evolution. For these reasons and because of the increasing number of transcripts whose function has been experimentally validated, it is believed that many of these new ncRNAs belong to an important, relatively unexplored class of regulatory elements. Thanks to ongoing improvements in sequencing technologies it has become possible to collect a significant amount of these uncharacterized transcripts. The latest generation of sequencing technologies makes it possible to perform large scale sequencing of entire transcriptomes. This technique, known as RNA-seq has already had a dramatic impact on our perception of the human transcription landscape [a priori speculation regarding the genomic loci being transcribed [ncRNA functional characterization is a rapidly expanding research area. In the past few years, it has become clear that the majority of the transcripts in cells are more than mere intermediates between the hereditary information encoded in DNA and the mechanical operative component represented by proteins. Indeed, it appears that numerous transcripts may not be translated at all while still being involved in critical biological functions such as cell differentiation and chromatin remodeling. Taking together 15 human cell lines, the cumulative coverage of transcribed regions is ~62% and ~75% of the whole human genome for processed and primary transcripts, respectively . This “pandscape ,239. Simandscape ,151, plaandscape , insectsandscape , worms [andscape  and yeasandscape . In [243nscribed . If the nscribed ,245.i.e., the secondary structures are interrupted by introns). It has been shown [From a functional perspective, much remains to be done for accurate characterization and functional analysis of ncRNAs. To infer the function of novel ncRNAs one possibility is looking for functional motifs. This can be done by running motif finders algorithms to predict structurally conserved and potentially functional motifs –252. Furen shown ,237 thaten shown  or GeneWen shown . This sten shown –260. As en shown  or when en shown , offer ben shown . This reen shown  where then shown  the authen shown ,30 and cen shown ,262 the Despite the difficulties encountered when comparing ncRNAs, homology search of ncRNAs can be successfully used to detect new genes. New and ever more sophisticated algorithms will help addressing the challenges brought by NGS technologies. The ultimate goal is the creation of thorough transcriptome annotations and unbiased expression profiling of each individual transcript. It is still too early to tell. However, if they live up to their promises and expectation, the discovery of this new large class of RNAs may well define one of the turning points of modern biology.


How animals make choices in a changing and often uncertain environment is a central theme in the behavioural sciences. There is a substantial literature on how animals make choices in various experimental paradigms but less is known about the way they assess a choice after it has been made in terms of the expected outcome. Here, we used a discrete trial paradigm to characterise how the reward history shaped the behaviour on a trial by trial basis. Rats initiated each trial which consisted of a choice between two drinking spouts that differed in their probability of delivering a sucrose solution. Critically, sucrose was delivered after a delay from the first lick at the spouts – this allowed us to characterise the behavioural profile during the window between the time of choice and its outcome. Rats' behaviour converged to optimum choice, both during the acquisition phase and after the reversal of contingencies. We monitored the post-choice behaviour at a temporal precision of 1 millisecond; lick-response profiles revealed that rats spent more time at the spout with the higher reward probability and exhibited a sparser lick pattern. This was the case when we exclusively examined the unrewarded trials, where the outcome was identical. The differential licking profiles preceded the differential choice ratios and could thus predict the changes in choice behaviour. The way animals make decisions that have uncertain variable outcomes can be investigated through various approaches. Optimal foraging theory presents the question from the Darwinian perspective – animals ought to optimise food yield, or maximum energy gained per unit of time In spite of the substantial literature on how animals make choices in various experimental paradigms, in terms of patch selection or in terms of reinforcement rates, little is known about the way animals assess a choice after it has been made in terms of the possible outcome and its timing. Here we quantified aspects of the behavioural response to variability both before a choice is made and after. In particular, we identified an explicit representation of the animal's confidence in the outcome, as manifested in the post-choice behaviour. We used a discrete trial choice paradigm where rats chose between two options on every trial. Critically, the outcome of their choice was revealed after a variable time window. This allowed us to characterise the behaviour of the rats during that time window and search for behavioural correlates of reward expectancy.Rats initiated a trial and received a go-signal which was then followed by a free choice between two options – a right or left drinking spout each of which could deliver a reward (a sucrose solution). The key behavioural manipulation in the current experiment was the introduction of a variable time between the behavioural choice (i.e. first contact at one of the two spouts) and the disclosure of the outcome (i.e. presence or absence of reward). This allowed us to measure the time spent at the spout as a behavioural indication of the confidence in the outcome. For this analysis we exclusively examined the unrewarded trials where the experience of the outcome itself was identical. To further quantify the overlap between the sampling time distributions, we employed a quantile analysis see . Figure c and It, to track the local choice ratios as well as the relative sampling times respectively. It captures the normalised difference in sampling time between the two spouts while Ic captures the relative choice employing a similar formula for direct comparison (see c is negative) and they spend longer times on the low reward spout (average It is negative). Interestingly, during session 1, the time ratios cross zero indicating the rats have begun to spend more time on the high reward spout. Note that the It is calculated based on the unrewarded trials only. For all successive sessions, the time index remained positive. In contrast, the choice index remained negative throughout the first session and became positive in the middle segment of the second session. These data indicate that differential sampling or waiting time occurs in advance of a change in the choice between the two locations. Across all sessions, the choice and time indices showed a significant level of correlation (Pearson Correlation Coefficient ranging from 0.57 to 0.67 across rats).Thus far the key finding, confirmed across multiple measures, was that rats adapted both their choice ratios and the time spent at spouts dynamically across trials. We next asked whether the two behavioural measures were correlated across trials and whether one of the two systematically preceded the other. In order to do this, we defined two behavioural indices, Iison see . Figure The key finding was that sampling or waiting times on unrewarded trials robustly indicated the relative reward probabilities across days, and that this differential pattern developed before any changes in the choice between the two locations. In light of this observation, we examined other aspects of the licking profile to search for further behavioural correlates of outcome probability. We used a discrete trial choice paradigm, where two options gave rewards at two distinct contingencies of 70% and 30%. Rats started by selecting the two options equally and eventually converged on nearly exclusive choice of the high probability option. After performing at a stable level of choice, the contingencies were reversed. All rats switched their choices back to the optimum behaviour (i.e. nearly exclusive choice of the new high probability reward) during 6 to 8 sessions. During every session, an analysis of the time spent at the spout revealed a systematic pattern whereby the rats spent more time at the high reward probability spout. This was the case when we exclusively examined the unrewarded trials, where the experience of the outcome itself was identical. Critically, the differential sampling times preceded the differential choice ratios. This was most evident in the examination of choice and time indices during the first session after reversal. This measure could thus be a predictor of the changes in choice and a potential correlate of a neuronal mechanism mediating the switch between the two options. Besides the total time duration spent at either spout, the profile of licking showed systematic differences across the high and low probability reward spouts. There were more licks for the high reward spout but individual licks were denser for the low reward spout.One interpretation for the denser lick patterns at the low reward spout can be based on a property of ratio schedules. Such schedules make a reward contingent on the emission of either a fixed or variable number of responses. These schedules thus arrange a positive relation between the rate of responding and the rate of reward. Here, the contingencies were such that lick density also determined  how quickly the outcome was procured (the outcome was presented at the first lick after the designated delay) and, hence, how quickly the rat could detect the absence of the outcome and leave the spout to initiate the next trial. The denser lick patterns exhibited by the rats when they selected the spout with the lower reward probability may have been reinforced more strongly compared to the high reward as they provided quicker feedback about the non-occurrence of the reward which occurred more frequently (70% non reward versus 30% non reward). This would thereby allow the rat to rapidly abandon the low probability spout and initiate the next trial.matching or maximisingTypically, when given a choice among possible options, animals exhibit two distinct modes of behaviour described as In settings where outcome probabilities are fixed and independent from sample to sample, maximising earns the highest reward. However, it has been argued On average, only 9.6% of trials were concluded at or before 600 msec (the latest time at which reward could become available) with the time spent at the spout being 700–1100msec (interquartile range). The time spent at the spout was therefore suboptimal and did not reflect learning of the precise onset of the reward across trials. To further investigate this pattern of behaviour, we studied the times spent at the spout during the rewarded trials. The average distribution of times spent at the spout for the rewarded trials was 2200–2900 msec (interquartile range) with no systematic differences across the two sides. Median time spent at the spout for rewarded trials was 2537 msec. The longer duration is due to the running of the sucrose pump during which the rat experiences the reward. When the conclusion time of the unrewarded trials were compared with the median time spent on the rewarded trials, 99% of the unrewarded trials were abandoned by that time. It is therefore possible that the moment by moment experience of reward during the rewarded trials, and not the reward onset drives the suboptimal sampling durations for unrewarded trials.In the current paradigm we have demonstrated that higher probabilities of reward cause the animals to sample the reward spout for longer durations indicating higher expectation of reward and confidence of an outcome. This phenomenon can be exploited in paradigms where uncertainty is not only governed by introducing an explicit probabilistic reward but affected by the uncertainty innate in sensory systems. For instance, in a tactile task involving rat whiskers The experiments were conducted in accordance with the Australian and the international guidelines for the treatment of animals and were approved by the Animal Care and Ethics Committee at the University of New South Wales (ACEC number 10/47B).Subjects were four adult male 250–400 g Wistar rats. Rats were maintained on a 12:12 hour light-dark cycle (with lights on at 7 am) in a climate-controlled colony room. Rats were maintained on a mild food/water deprivation  and were rewarded with a 5% sucrose solution during the experiment.The experiment was performed in a Plexiglas chamber (30 cm in length×20 cm in width×50 cm in height) with a flooring of metal bars spaced at 1 cm. An aperture (40 mm×40 mm) was located in the front wall of the chamber. Nose-pokes into the aperture were detected by an infra-red optical sensor. Two light emitting diodes (LEDs) at the back wall of the nose-poke chamber were lit after a variable delay (100–600 ms uniform distribution) to indicate the go-signal. The reward was delivered through two drinking spouts located at either side of the aperture in the front wall . The behAfter the familiarisation to the set up and the initial shaping of the behaviour, an acquisition period was conducted over 5 days. During the acquisition the contingencies were fixed for each rat (70% rewarded on one side and 30% on the other) and were fully counterbalanced across rats. Rats performed an average of 275 trials (218.5–329.5 inter-quartile range) during each session.In the second phase , the reward probabilities were reversed for each rat .t) and the choice index (Ic) to track the relative sampling times and the local choice ratios respectively. We defined two behavioural indices, the time index  between the high and the low reward spouts. Thigh is the median sampling duration at the high reward spout and Tlow is the median sampling duration at the low reward spout during the window of interest after excluding the rewarded trials. A 50 trial window is chosen for the analysis reported in Ic reflects the normalised choice difference between the two spouts across the same window of trials. Chigh is number of times the high reward spout was chosen and Clow is number of times low reward spout was chosen during the window of interest. As before, the first lick at either drinking spout was considered as the behavioural choice.Similarly, ITo quantify the overlap between the sampling time distributions , we foun


Citrus spp investigated through HRM analysis. Among those, 10 showed more genotypes by HRM analysis than capillary electrophoresis owing to the presence of SNPs in the amplicons. For the SSR markers without SNPs present in the flanking region, HRM also gave distinct melting curves which detected same genotypes as were shown in capillary electrophoresis (CE) analysis. Moreover, HRM analysis allowed the discrimination of most of the 15 citrus genotypes and the resulting genetic distance analysis clustered them into three main branches. In conclusion, it has been approved that HRM is not only an efficient and cost-effective alternative of electrophoresis-based method for SSR markers, but also a method to uncover more polymorphisms contributed by SNPs present in SSRs. It was therefore suggested that the panel of SSR markers could be used in a variety of applications in the citrus biodiversity and breeding programs using HRM analysis. Furthermore, we speculate that the HRM analysis can be employed to analyse SSR markers in a wide range of applications in all other species.High resolution melting curve analysis (HRM) has been used as an efficient, accurate and cost-effective tool to detect single nucleotide polymorphisms (SNPs) or insertions or deletions (INDELs). However, its efficiency, accuracy and applicability to discriminate microsatellite polymorphism have not been extensively assessed. The traditional protocols used for SSR genotyping include PCR amplification of the DNA fragment and the separation of the fragments on electrophoresis-based platform. However, post-PCR handling processes are laborious and costly. Furthermore, SNPs present in the sequences flanking repeat motif cannot be detected by polyacrylamide-gel-electrophoresis based methods. In the present study, we compared the discriminating power of HRM with the traditional electrophoresis-based methods and provided a panel of primers for HRM genotyping in Citrus. The results showed that sixteen SSR markers produced distinct polymorphic melting curves among the  Citrus taxonomy is very complex mainly due to sexual compatibility between Citrus and related genera, the high incidence of nucellar polyembryonic reproduction, the high frequency of bud mutations, the long history of cultivation, and worldwide distribution Citrus has been evaluated by a number of studies using various molecular markers The citrus species are widespread crops in over 100 countries and their production has experienced continuous growth in the last decade with a total annual production over 120 million tons SSRs have been highly popular genetic markers for last two decades because of their hypervariability, codominance, multiallelic nature, high reproducibility, extensive genome coverage and the amenability to automation and high throughput genotyping Recently, high resolution melting analysis (HRM) has been identified as a powerful method that can be applied to analyze the genetic variations including SNPs, insertions or deletions (INDELs), and methylations of DNA in PCR amplicons We hypothesize that HRM can efficiently and accurately distinguish the variation of nucleotide repeats in microsatellite markers, and the concurrent nucleotide variations mainly SNPs in the sequence flanking repeat motif can also be identified. The aim of this study was to evaluate whether HRM can be an alternative to electrophoresis-based methods such as capillary electrophoresis, and whether it can distinguish more polymorphisms than just repeat variations in citrus species. The outcomes of the work will also provide a reliable and robust set of SSRs to assess genetic diversity and conduct fingerprinting through HRM platform.Twenty four primer pairs amplifying fragments less than 250 bp were chosen from the study of Luro Citrus spp investigated through HRM analysis. In total, 66 alleles were detected altogether . In the dendrograms shown in de novo SNPs present in the fragments. The results showed that HRM is not only a method suitable for discriminating SSR genotypes, but also a more accurate approach which can detect more alleles that contain SNPs in the sequences flanking SSR repeat motifs.HRM has been proved an efficient, and cost-effective approach to detect sequence variations such as SNPs in humans, plants and microorganisms. In plants, this approach has been applied to detect SNPs and SSRs which were used for genotype identifications and genetic mappingHRM has been applied to analyze SSR markers in a few studies The HRM analysis allowed discrimination of the genotypes at the species level for the 15 citrus varieties/hybrids by using the panel of 16 SSR markers adopted from Luro . The genetic distance analysis of HRM data clustered the 15 citrus genotypes under investigation into 3 major groups, each containing one of the ancestral true species . The only exception was that satsuma mandarin was unexpectedly clustered in the branch close to lemon and citron. It was noted, however, that haplotypes formed by a SNP and SSR in 3 markers were unique to this cultivar but not present in other mandarin oranges. We are uncertain whether this result is true representation of its genetic distance from other mandarins, oranges and close varieties, or analysis bias may be generated from the selection of markers by chance. Another possibility is that the limited representation of citrus diversity in the present study can be an origin of unexpected genetic distance between Satsuma and the mandarin and orange group. Nevertheless, our clustering result is supported in general by many studies that verified the hypothesis that only 3 Citrus types, namely the citron, the mandarin and the pummelo, constituted true or valid species The two dendrograms obtained by CE and HRM data gave consistent results. In both, genotypes with same parental origin were generally clustered in the same groups. However, some genotypes of the same parental origin clustered closer with HRM data than CE data . Dendrogram based on HRM data also showed a clearer separation of the genotypes than that based on CE data owing to the additional SNPs detected by the HRM analysisde novo, can be widely used in all the plant and animals species in the areas such as biodiversity analysis, genetic mapping and breeding programs.The present study produced the HRM profiles for a panel of 16 SSR markers selected from the study of Luro In spite of many advantages of the HRM analysis on SSR markers, we should not overlook the limitation of HRM analysis. When the genotype number of an SSR marker becomes high, the melting curves produced by some distinct genotypes tend to be similar. While the sensitivity of the assay can be increased by using HRM difference plotting, the variations of the melting curves posed among the same genotypes increase such that the genotypes of the individuals can be inconclusive. This can be expected when the population under investigation is large and involves many species which lead to a large number of alleles. To address this limitation, the ambiguous genotypes can be resolved by mixing them equally with a sample of known homozygous genotypes and then performing HRM analysis The study presented here has shown that HRM is not only an efficient and cost-effective alternative of traditional electrophoresis-based method for SSR markers, but also a method to uncover more polymorphisms caused by SNPs present in the regions flanking nucleotide repeats. Consequently, more polymorphism and/or polymorphic haplotypes can be discriminated using this approach. It is also suggested that the panel of SSR markers can be used in a variety of applications in the citrus biodiversity and breeding programs worldwide by using HRM analysis established in this study. Furthermore, we believe that the HRM analysis can be employed to analyse SSR markers in a wide range of applications in all other taxa including plant and animal species. However, some limitations of the HRM analysis cannot be overlooked despite its advantages.A group of 15 genotypes belonging to 11 citrus species and hybrids was used as a population for analysis . The leaTotal DNA was extracted from young leaves according to the protocol of Doyle and Doyle C. clementina Hort. ex Tan.) by Luro Primers were selected from EST-SSRs developed on ‘Nules’ clementine , approximately 30 ng of template DNA, 0.2 mM dNTPs, 1× PCR buffer II, 2 mM magnesium chloride, and 1 U of MyTaq DNA polymerase . PCR was performed at 95°C for 12 min; followed by 35cycles of 95°C for 30 sec, 53°C for 30 sec, and 72°C for 45 sec; and one final cycle of 72°C for 15 min, on GeneAmp 9700 and 2700 amplifiers . For HRM analysis, PCR amplifications were performed in a total volume of 10 µL on a Rotor-Gene 6500 realtime PCR Thermocycler  and PCR reaction preparation was automated by a CAS1200 liquid handling system (Corbett Research). The reaction mixture contained 20 ng of genomic DNA, 1x PCR buffer , 2.5 mM MgCl2, 0.2 mM dNTP, 300 nM forward and reverse primers, 1.5 µM Syto® 9 , and 0.5 U Biotaq DNA polymerase (Bioline). The amplification was achieved by a touchdown PCR protocol: first denaturation at 95°C for 2 min, then 50 cycles denaturation at 95°C for 5 s, annealing and extension for 10 s at 60°C for the first cycle and thereafter at 0.5°C decrease each for 10 cycles, and a final extension at 72°C for 2 min.For capillary electrophoresis, PCR reaction included the two specific primers (0.3 µM) plus a labeled M13F primer  was mixed with 10 µl of formamide and 0.35 µl of LIZ-500 size standard and denatured at 95°C for 5 min. Up to three PCR products labelled with FAM, PET, or NED were pooled before separation in the ABI 310 Genetic Analyzer (Applied Biosystems) and analysis was conducted using Genemapper 4.0 software.The HRM-SSRs showing different results from CE-SSR analysis were sequenced using an ABI310 genetic analyzer (Applied Biosystems). Fragments amplified from genomic DNA were bidirectional sequenced to eliminate sequencing errors. PCR products were purified using a PCR purification kit following the protocol provided by the manufacturer (Bioline). Seqman software  was used for contig assembly and SNP identification. Sequences with a mixture of alleles of unequal lengths were reconstructed manually or using Champuru 1.0 software Genetic distances were calculated based on the proportion on shared alleles Table S1SSR and SNP haplotypes in 15 citrus genotypes as shown by CE and HRM analyses performed with 16 selected EST-SSRs markers. Fragment sizes include the M13 tail (19 bp).(PDF)Click here for additional data file.


Numerous host factors were found to affect the profiles of viral RNA recombinants and significant differences in recombination frequency were observed among various RNA viruses. Comparative analyses of viral sequences allowed for the development of evolutionary models in order to explain adaptive phenotypic changes and co-evolving sites. Many questions remain to be answered by forthcoming RNA recombination research. (1) How various factors modulate the ability of viral replicase to switch templates, (2) What is the intracellular location of RNA–RNA template switchings, (3) Mechanisms and factors responsible for non-replicative RNA recombination, (4) Mechanisms of integration of RNA viral sequences with cellular genomic DNA, and (5) What is the role of RNA splicing and ribozyme activity. From an evolutionary stand point, it is not known how RNA viruses parasitize new host species via recombination, nor is it obvious what the contribution of RNA recombination is among other RNA modification pathways. We do not understand why the frequency of RNA recombination varies so much among RNA viruses and the status of RNA recombination as a form of sex is not well documented.RNA recombination is one of the driving forces of genetic variability in (+)-strand RNA viruses. Various types of RNA–RNA crossovers were described including crosses between the same or different viral RNAs or between viral and cellular RNAs. Likewise, a variety of molecular mechanisms are known to support RNA recombination, such as replicative events  along with non-replicative joining among RNA fragments of viral and/or cellular origin. Such mechanisms as RNA decay or RNA interference are responsible for RNA fragmentation and  Plus-stranded RNA viruses include some of the most dangerous pathogens for animals and humans. Moreover, a vast majority of plant viruses are (+) RNA viruses. RNA viruses demonstrate a large level of variability in their genetic information, due to either mutations, RNA–RNA crossovers (RNA recombination), or reassortment. RNA recombination was demonstrated for many RNA virus species, whether under natural or experimental conditions. Similar to genetic recombination in DNA-based organisms, viral RNA recombination is defined as the process of swapping RNA fragments among RNA molecules. If crossovers occur amongst the same RNA templates in a homologous fashion, the exchanges are functionally equivalent to DNA meiotic crossing-over. In some viruses, the frequency of homologous crossing-over is very high and practically every replicated viral RNA molecule can be considered as chimerical in nature, as we have demonstrated for brome mosaic virus (BMV) RNAs  complex in mRNA viruses [reverse transcriptase (RT) in retroviruses] changes templates during synthesis of the nascent strand , BMV, Cucumber mosaic virus (CMV), and Cowpea chlorotic mottle virus (CCMV)  and illegitimate (nonhomologous) recombinants, respectively  RNA viruses are replicating in membranous structures called spherules or replication factories  during BMV RNA recombination  RNA transcripts containing nonoverlapping deletions did restore the infectious virus  RNA virus systems are now being progressively elucidated. Comparison among three plant RNA virus replication systems  reveals general patterns within the stepwise process of viral replicase complex assembly which requires concerted involvement of protein–protein, RNA–protein, and protein–lipid interactions  RNA viruses operate in the cytoplasm, as the miRNAs do, there are likely either structural and/or functional constrains against such snatching events. Future studies will certainly bring further insight to this question.RNA recombination between viral and micro (mi)RNAs has not yet been reported. However, given the fact that this would be a useful source of already adapted elements to be acquired by the virus in order to secure the in-Recently, a reverse scenario was observed. Nonretroviral RNA sequences of Bornaviruses and other (−) strand RNA viruses were integrated into the host genome, including the human genome , many unanswered questions remain to be addressed. They include, but are not limited to, the sub-cellular location of recombination events, the role and availability of host RNA degradome for recombination, or the link between the elements of RNA degradation pathways and viral RNA recombination. The molecular mechanisms of such crossover events are not well understood, especially whether template-switching or re-ligation processes are involved. More data, especially from plant RNA virus systems are required to assess the general nature of these processes in plant vs. animal/human tissues.The above examples illustrate that the cytoplasmic RNA processing mechanisms are able to cross paths with viral replication pathways inside the cell. Despite diverse examples of viral RNAs recombining with host RNA sequences  and the mechanism of recombination . As regards to the evolutionary studies, various computer programs are used for massive comparisons of viral sequences in order to reveal the recombination footprints. The examples of such software include, but are not limited to, Topali, RECCO, GARD, RDP, GENECONV, Chimaera, MaxChi, BOOTSCAN, SISCAN, PHYLPRO, DIPLOMO, SImPlot, Lard, and 3SEQ. These programs can identify the recombination sites among different viral strains, different viral species, and even between the virus and the host  and other viral- and/or host-encoded proteins, or the mutual host–virus effects in short-term virus evolution. The main analytical effort in the recombination experiments is to identify RNA recombination products and to map the location of cross sites. Usually, viral RNAs are extracted and amplified by RT-PCR and the resulting cDNA products are cloned followed by sequencing and/or restriction digestion of a large number of clones. This way the crossovers are detected and mapped within the sequence markers, providing information about both frequency and distribution of recombination events. Proper controls are required to guard against RT-PCR generated recombinants.Genetic RNA recombination is a major driving force for RNA virus diversity. By understanding the factors and the mechanisms that affect recombination, one can ultimately develop better means for controlling RNA virus infections. In this review I have described the current status of RNA virus recombination research and its future directions. I have also noted its progress over the last several years emphasizing on some future research venues. Evidently, there is still much to be learned about the mechanistic details of RNA recombination. For example, it is not yet clear how various factors modulate the ability of viral replicase to switch templates, such as the role of RNA template structures, the molecular and structural features of replicase proteins, or the functions of other viral and host factors during cross-over events. Also, the intracellular location of the RNA–RNA template switching has not been confirmed. Besides copy-choice, RNA viruses can recombine with non-replicative RNAs. It is not exactly known what mechanism is responsible for ligation of viral RNA fragments, or where inside the cell this process occurs. RNA viruses were found to recombine with cellular RNAs, but again where in the cell and what factors enable such events, is not well known. And the opposite, the exact steps and the molecular mechanisms of the RNA viral sequence integration with the cellular DNA have not been untangled. Amongst other questions, not much is known about how splicing or active ribozymes can contribute to the RNA virus recombination (Edgell et al., via recombination (Domingo, From the evolutionary stand point, RNA recombination seems to play a key function during virus speciation and emergence, but its shared contribution that parallels other RNA modification pathways has not yet been assessed. We do not fully understand how RNA viruses achieve their high potential of parasitizing new host species Domingo, . The entDomingo, .The frequency of RNA crossing-over varies among RNA virus species and there is little evidence that recombination was favored by natural selection. Because of this and since recombination rates follow the patterns of RNA genome organization, Simon-Loriere and Holmes  postulatvia recombination can be reduced during construction of plant RNA viral vectors (Nagyová and Subr, Despite the above deficiencies, the so far accumulated knowledge about viral RNA recombination has already found some practical applications. For example, measures could be taken to reduce recombination while designing the antiviral resistance in transgenic plants with artificial micro RNAs (Fahim and Larkin, The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Use of constructs with a deleted cox2 intron, however, led to a loss of RNA editing at almost all editing sites, with only a few sites still partially edited. Likewise, one deletion in exon 1 and three in exon 2 abolish RNA editing at all cox2 sites analyzed. Furthermore, intron splicing is abolished using these deletions. Mutation of a cytosine residue, which is normally edited and localized directly adjacent to the intron, to thymidine did not result in restoration of splicing, indicating that the loss of splicing was not due to loss of RNA editing. One deletion in exon 2 did not lead to loss of splicing. Instead, most editing sites were found to be edited, only three were not edited. Unexpectedly, we observed additional RNA editing events at new sites. Thus it appears that deletions in the cox2 RNA sequence can have a strong effect on RNA processing, leading to loss of splicing, loss of editing at all sites, or even to a gain of new editing sites. As these effects are not limited to the vicinity of the respective deletions, but appear to be widespread or even affect all editing sites, they may not be explained by the loss of PPR binding sites. Instead, it appears that several parts of the cox2 transcript are required for proper RNA processing. This indicates the roles of the RNA sequence and structural elements in the recognition of the editing sites.As previously demonstrated, the maize  However, for efficient editing, an upstream sequence of 40 nucleotides was required [in vitro assay not only confirmed this result, but identified one nucleotide downstream of the atp9 editing site as essential [Mitochondrial RNA editing in higher plants has long been described –3, and ing sites ,10. Usinrequired . A caulissential . Arabidopsis thaliana. These proteins are characterized by 4-20 repeats of 34-36 amino acids each [ccb203, cox3, and nad4 transcripts [A. thaliana plastid editing sites, and hundreds of mitochondrial ones [In recent years nuclear encoded mitochondrial or plastid proteins have been identified, which are required for RNA editing of specific sites. Almost all belong to the pentatricopeptide repeat protein family, which has 450 members in ids each . At currids each . All of ids each . All PPRids each . Indeed,ids each . Howevernscripts –19. Receial ones . NeitherCurrent data suggest that the target RNA motif of PPR editing factors is rather little defined, resulting in a network of PPR trans-factors which bind to RNA sequences at a low level of specificity . Such a in organello systems for mono- and dicotyledonous plants. These systems were successfully employed to analyze mitochondrial mRNA editing and the ability of plant mitochondria to recognize and correctly process RNAs from different taxa [in organello editing and splicing of in vitro transcribed cox2 mRNA [We previously had established and described ent taxa –27. In aox2 mRNA .in organello system to analyze the effect of different sized deletions in the cox2 coding sequence on RNA splicing and editing. We show that deletions in the RNA sequence can have a strong effect on RNA processing, leading to loss of splicing, loss of editing at all sites, or even to a gain of new editing sites. Previous work did indicate a potential role of the secondary or tertiary structure of the RNA on editing site recognition . Hence, cox2 mRNA from mono- and dicot origin [A growing number of PPR proteins have been implicated in editing site recognition. However, their target motifs appear to be little defined . In prevt origin . One wayin organello incubation, mRNA was isolated and RT-PCR was performed. While the control vector pNB475 gave rise to an mRNA which was spliced  and pCH754 (intronless) are given in The other plasmids carrying deletions in exons and shown in cox2 mRNA. The data obtained for pHS571 (exon2∆99 bp)  hint to cox2 mRNA and all deletion constructs used. These data are shown in cox2 mRNA affects RNA editing. pNB475 and pHS571 (exon2∆99 bp) differ in 3’ deletion of one bulge, two hairpin and two interior loops and three stems , pCH765 (exon2∆52 bp), and pCH768 (exon2∆33 bp)  see , which hcox2 mRNA editing in cauliflower, which is not readily explained by the current knowledge on mitochondrial editing factors and their interaction. The first example of loss of editing involving all editing sites of a transcript was observed in atp6 mRNA in cytoplasmic male sterile Sorghum bicolor [in organello editing system and maize cox2 mRNA and vice versa hinted to a function of the mRNA structure in the editing process [cox2 mRNA even if the editing sites are absent in their own endogenous mRNA. Most intriguingly two monocot-specific editing sites, which are pre-edited on the DNA level in dicots, are properly edited in cauliflower mitochondria [atp6 mRNA from Sorghum bicolor in maize mitochondria. RNA editing depended on the presence of part of the 5´ UTR and the open reading frame from maize in a chimeric transcript [cox2 mRNA editing, which may be explained best by changes in the RNA secondary structure. Unfortunately, RNA secondary or tertiary structure predictions by means of bioinformatics are still rather crude and results obtained are hard to confirm or evaluate. The structural predictions presented in this study provide hints that both sequence and structural elements are essential for RNA editing. Similar results are reported for ADAR2 based RNA editing that converts adenosine to inosine (A I) in mammals [If indeed PPR editing factors recognize editing sites either on their own or acting in a network, one might argue that deletions should not have an effect on distant editing sites. Instead only local effects on neighboring sites might be expected, e.g. due to loss of the PPR binding sites. In this study all but one deletion had a global effect on maize  bicolor , althoug bicolor . While n process . Cauliflchondria . Additioanscript . These danscript . This mo mammals . Howeverin vitro mutagenesis. Oligonucleotides CH2294, CH2322, CH2328, CH2337, CH2338, CH2355, CH2356, CH2381, CH2382, CH2383, CH2385, CH2386, CH2387 were employed in order to introduce deletions into the different vectors. Oligonucleotides FK864 and FK865 were used for construction of vector pHS571. Oligonucleotides FK 770, FK789, NB852, IH977, CH2319 through CH2322, and CH2385 were used for specific amplification of RNA transcribed from the introduced vectors. All oligonucleotides are shown in Table S1 in cox2 gene under control of the Arabidopsis thaliana cox2 promoter and 3´ region. Plasmids pHS571, pCH737, pCH753, pCH754 and pCH765, pCH767, and pCH768 are derived from pNB475 by PCR based deletion using the respective oligonucleotides listed in the table. Plasmids pCH736, pCH755 and pCH756 were generated by in vitro mutagenesis of pHS571. All vectors are listed in Table S2 in All plasmids are derivatives of plasmid pNB475 , which cCauliflower heads were obtained from local grocery stores or grown in greenhouses in the Botanical Garden of the Christian-Albrechts-University (CAU) at Kiel.in organello incubation and 1200 µg mitochondrial protein was used. In organello incubation of mitochondria was carried out for three hours, as described previously [All mitochondrial procedures were performed as described ,39, howeeviously ,39.Mitochondrial RNA was isolated as described . QuantifThese procedures were performed as described previously  using anin vitro introduction of point mutations and deletions into plasmid DNA recombinant PCR-based site-directed mutagenesis was applied. In the case of in vitro mutagenesis, two complementary oligonucleotides containing the desired mutation served as primers for the amplification of the whole plasmid with proofreading polymerase Pwo . Deletions were achieved using two phosphorylated inverse primers directly flanking the desired deletion for the amplification of the remaining plasmid. As template for the PCR reaction 25-50 ng plasmid DNA was employed. The PCR product was digested with DpnI , which selectively hydrolyzed the methylated original template DNA. PCR product DNA carrying a point mutation was transformed directly into E. coli and recombined in vivo while the linear plasmid DNA of the in vitro deletion had to be ligated first via its phosphorylated blunt ends.The methodology is based on the QuikChange Site-Directed Mutagenesis Kit and the ExSite PCR-Based Site-Directed Mutagenesis Kit (Stratagene). For specific CLC Genomics Workbench (www.clcbio.com) was used for RNA structural analysis which uses a dynamic programming algorithm for free energy minimization which includes free energy increments for coaxial stacking of stems when they are either adjacent or separated by a single mismatch . The theSequence logo of sequence flanking RNA editing sites was created using WebLogo 3.3 .All other standard molecular biology techniques were performed according to published standard procedures .Figure S1Intron mRNA sequencing. (A) Oligonucleotide pairs CH2319/CH2320, and CH2321/CH2322 were used to amplify unspliced mRNA. Cox2 exons are shown in green and the intron in yellow color. (B) Editing site 11 (in exon 1) exhibits partial editing, which is expected in unspliced mRNA. (C) Evidence for partial editing of one position (+840) in the intron (reverse sequencing).(TIF)Click here for additional data file.Figure S2Sequencing of unspliced amplicon from  No evidence for RNA editing is observed.(TIF)Click here for additional data file.Figure S3Sequences adjacent to new editing sites observed for spliced mRNA from pCH767. Sequence logo of sequence flanking RNA editing sites was created using WebLogo 3.3 [Logo 3.3 . No obvi(TIF)Click here for additional data file.Figure S4Models for RNA secondary structure. (A-H) Editing status of RNA from different plasmids and comparison with RNA secondary predictions. (PDF)Click here for additional data file.File S1This file contains all supplementary tables.(DOCX)Click here for additional data file.


This mini-review summarizes the process of reverse-transcription, an obligatory step in retrovirus replication during which the retroviral RNA/DNA-dependent DNA polymerase (RT) copies the single-stranded genomic RNA to generate the double-stranded viral DNA while degrading the genomic RNA via its associated RNase H activity. The hybridization of complementary viral sequences by the nucleocapsid protein (NC) receives a special focus, since it acts to chaperone the strand transfers obligatory for synthesis of the complete viral DNA and flanking long terminal repeats (LTR). Since the physiological microenvironment can impact on reverse-transcription, this mini-review also focuses on factors present in the intra-cellular or extra-cellular milieu that can drastically influence both the timing and the activity of reverse-transcription and hence virus infectivity. A genAmong Retroelements, the LTR-containing retrotransposons such as yeast retrotransposons (Ty) and human endogenous retroviruses (HERV) resemble simple retroviruses such as the gammaretrovirus murine leukemia virus (MuLV). In fact, these mobile elements encode the virion structural Gag proteins and Pol enzymes and contain non-coding regulatory sequences essential for genome replication, integration and expression such as the LTR . The endogenous retrotransposons that are abundant genetic elements in the host genetic make-up are probably playing key roles in genome reshuffling and variability. Thus, the replication of mobile retrotransposons by a transcription/reverse-transcription/integration process, also called ‘copy-and-paste’, is thought to have fueled the evolution of eukaryotic genomes from yeast to human  and transmembrane (TM), in the form of trimeric ensembles for HIV-1. The shape and oligomeric organization of the structural proteins of the inner core seem to be characteristics for each genera of the and MuLV –21).trans within the same cell to ensure co-replication of these DLV’s , occurs during the early phase of virus replication, soon after virus infection of a target cell. The process of viral DNA synthesis by RT initially takes place in the virion core after its entry into the cytoplasm. The virion core is thought to undergo structural changes to become the reverse transcription complex  of the full-length viral RNA are highlighted in ynthesis .In most retroviruses the PBS is 18 nt in length and annealing of replication primer tRNA, - tRNALys,3 for Lentiviruses - via its complementary 3′ 18 nt is promoted by means of the RNA annealing activity of NC proteins. This RNA annealing activity is exhibited by both the polyprotein Gag precursor and the mature NC protein  by copying the genomic RNA (thick black line) . Other fin vitro analysis on HIV-1, it appears that both the TAR upper loop and the R ends are important for this annealing reaction  –41 40–41 in vivo .3.The full length viral RNA, in addition to being the retroviral genome, plays a key role early in retrovirus assembly since it represents a scaffolding platform onto which Gag polyprotein molecules readily assemble to ultimately form the inner core structure –47. In aa.in vitro, a prerequisite for RNA packaging [cis-acting packaging signals, notably those in the 5′ UTR, since both the genomic RNA and the spliced viral RNA species are packaged in a competitive manner [cis-acting signals recognized by RT, and reverse transcription of notably the fully spliced viral RNAs takes place in virions and in newly infected cells as efficiently as that of the unspliced viral RNAs [Significant amounts of subgenomic viral RNAs are packaged into virions alongside the full length viral RNA –51. Undeackaging –57. All ackaging . While gackaging . Howevere manner . The subral RNAs . In the ral RNAs .b.In the course of virion morphogenesis, abundant cellular RNAs are incorporated into viral particles, which, in the case of wild-type retroviral particles, reside in the core together with the genomic RNA. Interestingly, incorporation of cellular RNAs into viral particles is greatly enhanced when the genomic RNA lacks the Psi packaging signal. Under these conditions, cellular RNAs can constitute more than 50% of the total RNA mass per particle ,49. ThesFigures vary for the extent of cellular RNA incorporation into virions since RNA can be selectively excluded or enriched for in retroviral particles. Among abundant species, there are many small Pol-III-generated RNAs such as tRNA, 5S rRNA, U6 snRNA, mY RNAs, 7SL RNA and 7SL RNA-derived SINE RNAs (B1 RNA or Alu RNA) ,64–68. FThe 7SL RNA exemplifies specific host RNA encapsidation since it is enriched at a level similar to that of the genomic RNA in MuLV and HIV-1 virions –66. In aSimilarly, mY RNAs are among the most highly represented non-coding RNAs specifically packaged in MuLV. Interestingly, mY RNAs are recruited even in Ro60 knockout cells, where mY RNA is degraded in the cytoplasm while there is a residual pool in the nucleus –69. ThesTaken together, the mechanism by which non-genomic, viral and cellular RNAs are packaged into virions is as yet poorly understood, or controversial, with the exception of the primer tRNA. The roles of subgenomic RNA and some cellular RNAs in reverse transcription and in the biology of the virus remain a matter of speculation, with the notable exception of the replication primer, and of 7SL RNA, thought to recruit the APOBEC 3G restriction factor ,74.4.The overall process of Retrovirus assembly is considered to take place at the plasma membrane of infected cells where Gag and Gag-Pol molecules assemble via major interactions between two platforms, the N-terminal myristate and basic residues of the Gag-Matrix with plasma membrane phospholipids, and the zinc finger and the flanking basic residues of the Gag-NC with the genomic RNA (reviewed in –79). Oncin vitro conditions using mild detergents that preserve the core integrity [However, it has long been shown that small amounts of minus-strand viral DNA are present in viral particles of Rous sarcoma virus (RSV), Moloney murine leukemia virus (MoMuLV), and HIV-1 generated in cell cultures. This indicates that reverse transcription can already be ongoing during virus assembly or in the released particles, in a process which has been called natural endogenous reverse transcription (NERT) –89. Thisntegrity –93.More ntegrity –90. HIV-ntegrity ,90,91.in vitro as well as in HIV-1 virions , which suggests that SEVI might act as a multifunctional cofactor enhancing HIV-1 infection through numerous modes.Furthermore, SEVI seems to display a potent nucleic acid chaperoning activity and to greatly enhance the reverse transcription process Taken together these findings show that viral DNA synthesis can indeed start during virus formation and that the so-called NERT process is largely influenced by components of the physiological microenvironment such as polyamines, dNTP’s and SEVI. This in turn can have a significant impact on HIV-1 infectivity of primary target cells –91.Arguably, Retroviruses such as HIV should be viewed as RNA/DNA viruses rather than being strictly categorized as RNA viruses. Furthermore, the evidence highlights the need to identify microbicide compounds aimed at inhibiting components of the seminal fluid which facilitate HIV-1 infection .5.In the course of assembly, Gag structural precursor molecules are targeted to the plasma membrane by the N-myristate and basic residues of the Gag matrix (MA) on the one hand, and the genomic RNA, notably the Psi Packaging signal ), on the other. In fact, NC – consisting of either one or two highly conserved zinc fingers flanked by basic residues – directs the selection of the genomic RNA through multiple interactions between the RNA Psi sequences and both its zinc finger(s) and its basic residues ,21,75,95In the case of HIV-1 NC, the central globular domain corresponding to the two zinc fingers and the basic linker forms a hydrophobic platform which specifically binds the Psi stem-loops of the genomic RNA . Mutex vivo [However reverse transcription does take place in CCHC mutant virions as evidenced by the accumulation of newly made viral DNA .6.i.e. the Capsid protein (CA)), almost invariably display a defect in viral DNA accumulation [In cells undergoing HIV-1 infection, the process of reverse transcription occurs in a densely populated environment, the cytoplasm. There, it is intimately linked to at least two other steps, namely uncoating, the process through which the viral core is reorganized, and trafficking towards the nucleus . Given tmulation –103.cis-element [i.e. nuclear import. This model is seducing as it finds parallels in other viruses whose capsids undergo profound changes in the proximity of nuclear pores that are required for the nuclear import of their genome [However, the relationship between HIV-1 reverse transcription and viral core reorganization is poorly understood. Specifically, it is not known whether uncoating occurs subsequent to the completion of viral DNA or if a reciprocal co-stimulation is established between the two processes. The former hypothesis was raised following evidence indicating that viral cores gained stability in the absence of the central polypurine tract -central termination sequence (cPPT-CTS) -element . In thisr genome –106. Wher genome –110 and r genome –113.Why is the relationship between reverse transcription and uncoating important? We propose it is so to counteract in a timely fashion possible antiviral defenses that target viral cores. Viral cores are characteristic molecular signatures of Retroviruses and they are known to be targeted shortly after cell entry by antiviral factors. For example, the Tripartite motif protein 5 alpha (TRIM 5α) recognizes viral cores and destructures them, impacting on reverse transcription (108). If uncoating does not occur until the completion of viral DNA synthesis, then a structure identical to the one entering the cell will be present throughout the journey of the viral core across cytoplasm. If, instead, uncoating occurs progressively together with reverse transcription this characteristic complex will undergo change, as a consequence of which the viral structures sensed by cellular antiviral defenses will be markedly distinct.It remains to be seen if TRIM5α or the Friend susceptibility virus gene 1 (Fv1) are unique factors or if other proteins exert similar functions, but it would not be surprising to find these characteristic retroviral structures to be the target of multiple factors. In this respect, by providing a signal for the ordered reorganization of viral cores, reverse transcription may protect the virus from attack within the cytoplasm environment simply by promoting shedding of the components through which viral cores are recognized.The major difference between the two models outlined above on the relationship between core reorganization and reverse transcription is the length of time during which the initial viral core structure persists as such in the cytoplasm of infected cells. It is well appreciated that viral DNA completion varies greatly among target cells. For example, while the reverse transcription process takes only 4 to 8 hours in transformed cell lines such as HeLa cells, widely used to study viral infection, it can take up to 20–30 hrs in primary lymphocytes and macrophages or days in non-stimulated monocytes –116. ThuIndeed a correlation between the extent of reverse transcription and the permissivity of primary cells to infection is widely reported. In the past, this has been hypothesized to be a consequence of poor availability of dNTPs in cells resistant to infection. Yet, more recent data indicates that RT functions correctly even at low dNTP concentrations, such as those normally found in restrictive cells including macrophages or monocytes ,117. It One of the major problems in studying the early phases of infection is the long appreciated observation that the vast majority of viral particles entering cells are non-infectious, as they do not result in the establishment of an integrated proviral DNA and are progressively lost over time. Only 1 in 8 viruses is thought to be infectious in established cell lines where infection is efficient  but may 7.Studying reverse transcription in HIV-1 has shed some light on virus variability fueled by the error prone RT enzyme, which is, at least in part, counterbalanced by NC protein in order to control the process and to contribute to its completion and fidelity . At the To understand how HIV-1 genetic variability allows the virus to resist innate defenses, specific immunological responses and highly active antiretroviral therapies targeting the viral enzymes RT, PR and IN (HAART) , the cirSince the nucleocapsid protein of HIV-1 plays key roles in reverse transcription, mainly through its highly conserved zinc fingers, it represents a target of choice for compounds and approaches  that see