Therefore, we conclude that most current prokaryotes may still contain relics of the primeval RNA World and that both extended RNA codes may well represent two plausible evolutionary paths between the RNA code and the current SGC.Herein two genetic codes from which the primeval RNA code could have originated the standard genetic code (SGC) are derived. One of them, called extended RNA code type I, consists of all codons of the type RNY (purine-any base-pyrimidine) plus codons obtained by considering the RNA code but in the second (NYR type) and third (YRN type) reading frames. The extended RNA code type II, comprises all codons of the type RNY plus codons that arise from transversions of the RNA code in the first (YNY type) and third (RNR) nucleotide bases. In order to test if putative nucleotide sequences in the RNA World and in both extended RNA codes, share the same scaling and statistical properties to those encountered in current prokaryotes, we used the genomes of four Eubacteria and three Archaeas. For each prokaryote, we obtained their respective genomes obeying the RNA code or the extended RNA codes types I and II. In each case, we estimated the scaling properties of triplet sequences via a renormalization group approach, and we calculated the frequency distributions of distances for each codon. Remarkably, the scaling properties of the distance series of some codons from the RNA code and most codons from both extended RNA codes turned out to be identical or very close to the scaling properties of codons of the SGC. To test for the robustness of these results, we show, via computer simulation experiments, that random mutations of current genomes, at the rates of 10 Several properties have been unveiled using different methods for their analysis. In particular, the fact that DNA sequences display fractal scaling and the finding that some bacterial chromosomes possess an inverse bilateral symmetry (IBS) was demonstrated by means of a renormalization group (RG) approach The premise of RG is that in a critical phase transition, the equations describing the system are independent of scale. In this work we have applied the RG transformation in order to look for vestiges of the RNA World and of two putative intermediate states between the RNA World and the LCA.The notion that present genomes may still retain remnants of their ancestry for more than three billion years has been a subject of controversy In this work, we followed the concept that likely a large fraction of genes within a genome are ultimately related by descent to a small number of genes that arose early in our evolutionary history Borrelia burgdorferi, Bacillus subtilis, Mycoplasma genitalium, and Escherichia coli) and three Archaeas . From each of these organisms, we derived their respective genomes obeying the RNA code, the extended RNA code type I and II. Third, in order to assess if our in silico constructed genomes could be biologically meaningful in regard to the different proposed genetic codes, we performed a fractal scaling analysis based upon a renormalization group (RG) technique The article is organized as follows. First, following the criteria of relaxing the RNY code and maintaining the constraint of symmetry between complementary strands M. jannaschii and M. kandleri. In order to test the hypothesis of preservation of patterns, we performed computer simulation experiments in which we allowed for random mutations of current genomes during three billion years. Finally, we reconcile previous debates by concluding that genomes are systems that are constantly under a critical state and therefore, they may show universal properties of scale invariance.We show that several scaling and statistical features of the RNA and the extended RNA type I and II codes are still detectable in current prokaryote chromosomes since they show a critical state, with the interesting exception, to some extent, of Borrelia burgderfori, Bacillus subtilis, Escherichia coli, Mycoplasma genitalium, Nanoarcheum equitans, Methanopyrus kandleri, and Methanococcus jannaschii were retrieved from the NCBI, Genbank resource from the NIH (http:www.ncni.nlm.nih.gov) with the following corresponding accession numbers: NC_001318, NC_000964, NC_000913, NC_000908, NC_005213, NC_003551 and NC_000909. We used bacterial and archaeal genomes with only open reading frames (ORF), i.e. only the protein coding region of the chromosomes. Since our goal was to study statistical properties of whole chromosomes, the original structure of the strands had to be preserved, and therefore we assembled all reported ORF one after the other as originally ordered and oriented in the chromosome (OOO), as if we were reading all ORF in only the leading strand. For example, in the case of Borrelia burgderfori the first ORF is located in the leading strand, so it was included without any change to the sequence. However, the second ORF is located on the lagging strand, and if we joined it after the first ORF, we would be mingling both strands. From the flatfile downloaded we got the information about the location of the second ORF (c1796-768), and we added the nucleotides 768 to 1796 of the leader strand to the first ORF. So was done for each ORF. An ad hoc program was written in MATLAB in order to create the OOO sequences.The complete sequences of ad hoc program written in language Perl. This program permitted to generate the following three different genomes:In order to construct the different genomes, we discarded from the OOO genomes the triplets which did not correspond with the desired genetic code by means of an Genomes having only RNY triplets in the first reading frame only, i.e., those that are governed by the RNA code.Genomes with only RNY, NYR and YRN triplets, i.e., those that obey an extended RNA code type I.Genomes with only RNY, RNR, and YNY triplets, i.e. those that follow an extended RNA code type II.In order to simplify the description of the results, the first, second and, third types of genomes will be called, here and further, RNA genomes, extended RNA type I genomes, and extended RNA genomes type II, respectively.ATG ATA CTA ATG AAG TAT AGT GCT ATT TTA TTA ATA TGT AGC GTT AAT TTA TTT …For example, if the OOO sequence is: AGT GCT ATT AGC GTT AAT …Then, the RNA genome is: ATG ATA CTA ATG TAT AGT GCT ATT TTA TTA ATA TGT AGC GTT AAT TTA …The extended RNA type I genome is: ATG ATA ATG AAG TAT AGT GCT ATT ATA TGT AGC GTT AAT TTT …The extended RNA type II genome is: ATACTAATGAAGTATAGTGCTATTTTATTA ATATGTAGCGTTAATTTATTTTGTTTTCAAAATAAATTAACTACTTCATG… the triplet ATA lies in positions 4, 17, 34, 65, … This series is used for the CPP. The distance series will be 13, 17, 31, … In this way, although we start with a categorical series, we end up with a numerical one.Instead of using the classical stochastic random walk mapping rules of DNA, such as the purine-pyrimidine (RY) rule e. g. , the actMost scientists are familiar with the normal or Gaussian distribution, where the moments, such as the mean and variance, have well defined values. In a fractal process, as more data are analyzed, rather than converge to a definite value, the mean continues to increase toward ever larger values or decrease toward ever smaller values. For a fractal process, the moments, such as the arithmetic mean, may not exist. Self-similarity means that the small irregularities at small scales are reproduced as larger irregularities at larger scales. These increasingly larger fluctuations become apparent as additional data are collected. Hence, as additional data are analyzed from a fractal process, these ever larger irregularities increase the measured value of the variance. When the mean is infinite, no natural scale exists by which to gauge measurements. Self-similarity implies that no natural spatial or temporal scale exists, that is, there are events at all scales. Statistical self-similarity implies that a value measured for a property depends on the scale or resolution at which it is measured, and this is called scaling. It has been shown that in DNA sequence analysis, the critical concept is that of scale given the heterogeneity of DNA sequences Power-law distributions are known to have statistical self-similarity resulting in the probability density function obeying a functional scaling law which is a renormalization group property. A group is a collection of objects sharing some property in common. The renormalization group relation is the mathematical expression of the underlying process not having a fundamental scale. This lack of a single scale is similarly manifested in the divergence of the central moments.Classical scaling principles are based on the notion that the underlying process is uniform, filling an interval in a smooth, continuous fashion, and thereby giving rise to finite averages and variances. The new principle is one that can generate richly detailed, heterogeneous, but self-similar structures at all scaling. Thus, length, area, and volume are not absolute properties of the system but are functions of the units of measure Now we present the basic ideas of the RG approach in order to illustrate how this procedure can be applied for analyzing the genome of a prokaryote. Briefly, the renormalization group (RG) analysis, introduced in field theory and in critical phase transitions, is a very general mathematical and conceptual tool, which allows one to decompose the problem of finding the macroscopic behavior of a large number of interacting parts into a succession of simpler problems with a decreasing number of interacting parts, whose effective properties vary with the scale of observation decimation and rescaling. When we go from the fine scale to the coarse scale, the process is called decimation. The idea of RG is to decimate the degrees of freedom, while rescaling so as to keep the same scale by calculating, for example, the relative dispersion (the ratio of the standard deviation to the mean). The procedure can be repeated using groupings of two, three, four and more data points. In this way the fractal dimension that is independent of the degree of coarse-graining can be determined.The concept of RG is useful for systems that exhibit the properties of scale invariance and self-similarities of the observables at the critical point of the system All figures related to the RG operation were obtained by aggregating one by one the distance series and the length of the final distance series was dependent upon the length of the distance series. The whole collection of plots regarding the CPPs, the scaling analysis, and the frequency distributions of triplets in all types of genomes of the seven chosen prokaryotes, are available upon request.Methods for assessing the fractal characteristics of time-varying or distance-varying signals are useful for DNA sequences analysis. DNA sequences which vary, apparently irregularly, have been considered to be driven by external influences which are random. The Hurst exponent The exponent Mandelbrot In each case we determined the scaling properties of the distance series of triplets and their reverse complementary triplets via a renormalization group (RG) approach from which the Hurst exponent The RNA World code consists of only RNY codons that comprises 16 out of the 64 possible triplets and which codify for 8 amino acids. To wit, RNY codons are AAY (Asn), AGY (Ser), ACY (Thr), AUY (Ile), GAY (Asp), GGY (Gly), GCY (Ala), and GUY  see .The 3 sets of codons, RNY, NYR, and YRN are here defined as the codons of the extended RNA code type I  and 3 stop triplets, emerge. Konecny et al. Now, if we allow for transversions in the RNA code in the 1st or the 3rd nucleotide bases, codons of the type YNY and RNR arise. We define the extended RNA code type II as consisting of the 3 sets of codons RNY, RNR, and YNY . This coIt has been proved that in order to study the statistical properties of DNA sequences in bacterial chromosomes it may be necessary to consider both halves of the chromosome because they can display inverse bilateral symmetry (IBS) E. coli, M. genitalium, N. equitans and M. kandleri in E. coli, M. genitalium, N. equitans and M. kandleri are illustrated in E. coli for the triplets GGC and GCC, in the 1st half of the genomes, goes from M. genitalium for the triplets AAU and AUU, in the 1st half of the genomes, lies between B. burgdorderi and B. subtilis, slightly more than 50% of the scaling exponents of a given triplet in one half of the genome are statistically similar to its corresponding reverse complementary triplet in the other half of the genome, a fact which clearly indicates that their chromosomes display IBS (not shown). However, in the case of E. coli, M. genitalium, and the three Archaeas, the type of symmetry of each chromosome is in general difficult to discern. In the case of E. coli  half is similar to the estimate of the scaling exponent of AUU in the 2nd (or in the 1st) half. Note that the magnitude of the scaling exponents for the reverse complementary triplets AGC and GCU in M. kandleri, are not preserved. For M. kandleri and M. genitalium, the aggregation analysis deviates from straight lines, which means that there is an inverse power-law decorated by a dominant log-periodic modulation. Despite all these plethora of behaviors, it is noteworthy that the scaling exponent values obtained for most codons and its complementary in the original genomes of all organisms analysed turned out to be similar or very close to the ones obtained with their respective genomes governed by any of the extended RNA codes and even with the RNA code.We present results for coli see , it is acoli see . In the N. equitans. Similarly, there are only 2 out of the 16 codons in the 1st half of the RNA genome which display scale invariance in comparison with the current genome of M. kandleri. Note that the codons NYR and YRN that belong to the extended RNA code type I as well as the codons RNR and YNY that pertain to the extended RNA code type II share the RYR and YRY codons. In both extended codes there are more RNY codons exhibiting scale invariance than their corresponding RNA genomes of the RNA world. This is true for all prokaryotes except in the case of the 2nd half of the RNA genome of M. jannaschii in which there are 8 codons in comparison with its corresponding 2nd half of the extended RNA genome type I in which there are 7 codons. The number of RYR-YRY codons whose scaling exponents are statistically equal to those of the current genomes ranges from 8 to 15 out of the 16 codons that belong to any of the extended RNA codes. Note that scale invariance is less frequent in codons RRR-YYY, that comprise the complementary code of the extended RNA code type I, than the ones found in the extended RNA code type II. These types of codons do not show scale invariance in the extended RNA code type I in the genome of M. jannaschii, but all 16 codons of this type exhibit scale invariance in the extended RNA code type II. Conversely, the number of codons YYR-YRR, which constitute the complementary code of the extended RNA code type II, that display scale invariance, are in general less than the ones encountered in the extended RNA code type I. The exception to this rule is again M. jannaschii. Overall, the total number of codons that exhibit scale invariance of the extended RNA genomes ranges from 20 (M. jannaschii) to 54 (N. equitans) out of the 64 codons of the SGC.In B. subtillus, M. genitalium and E. coli, whereas the corresponding distributions in the extended RNA code type II are similar to the current genome of B. burgdorferi. The oscillatory decaying pattern with 3-base periodicity of the FDD can clearly be ascribed to the fact that the count of a given triplet is carried out by considering its three reading frames. But this condition is not necessary, since in the case of the FDD of RRR or YYY codons that appear only in the 2nd and 3rd reading frames of the extended RNA code type I, also exhibit an oscillatory decaying pattern every 3 bases (not shown). The same holds true for the YRR and YYR codons that pertain to the complementary code of the extended RNA code type II.It is well known that coding and non-coding sequences display different statistical and evolutionary properties e. g. . One of Herein, we examine the robustness of the estimates of the scaling exponents in order to see if not all information of ancestral organisms has been erased at least for the last 3 billions years of evolution. Additionally, the effect of this perturbation upon the CPP's and FDD's was also examined.−10 and 10−9 mutations per site per year. The number of point mutation occurring in one million years per chromosome 9 years. The simulated experiments were made blindly regardless of synonymous or non-synonymous mutations, transitions or transversions, or the specific codon usage of each organism. Results for N. equitans and B. burgdorferi are shown in −10 the CPPs, the FDD's are practically unaltered for both organisms whereas for a rate of 10−9 clear deviations from the current patterns start to emerge. Likewise, at 10−10 the estimates of the corresponding scaling exponents still reflect long-range correlations whereas for a rate of 10−9 the exponents tend to indicate random behavior , but considering the 2nd and 3rd reading frames there are 48 codons that specify 17 amino acids and the three stop codons. If transversions in the 1st or 3rd nucleotide bases of the RNY pattern are permitted, then there are also 48 codons that encode for 18 amino acids but no stop codons.An outstanding result regarding the scaling properties of the distance series of some codons from the RNA genomes and most codons of the extended RNA genomes type I and II, is that they turned out to be identical or very close to those of the present genomes. Since the magnitudes of the scaling exponent of the distance series of these codons are unaltered, both in evolutionary time and in actual distance series, we can say that they show critical scale invariance according to the renormalization group theory.In this work we have used a renormalization group approach to obtain an expression for the aggregated relative dispersion. The aggregated relative dispersion indicates that the process has a preferential scale length, M. kandleri and M. jannaschii depart from the RNA genomes of other prokaryotes rules out the possibility that the scale invariance is due to the self-similarity of the DNA sequences or that they are due to artifacts of the RG technique.The fact that the RNA genomes of counts of certain correlated base combinations are only given at separation intervals of three bases and are zero at all other separations ”. Shepherd estimated that the time of last use of the comma-less code appears to be of the order of 3 billion years ago The FDD of the RNA genomes never show an oscillatory pattern every 3 bases, as was proposed some years ago by Shepherd Our perturbation analysis suggest that critical scale invariance was selected since the RNA world and that an enrichment of RNY codons in the genes for proteins could be a primitive remnant due to this fact and is not necessarily due to codon usage In summary, the attractive and appealing idea of fossilized remnants of the primeval and the extended RNA codons in today existing genes is given credit in this work.Appendix S1Mathematical aspects of the scaling analysis.(0.10 MB DOC)Click here for additional data file.


Silence [Silence springs from the extraordinary, yet brief, history of RNA silencing. In just two decades, we have seen the anomalous properties of plant and fungal transgenes connect with a series of amazing experiments in which injected double-stranded RNA triggered silencing in worms. These diverse lines of research revealed the essence of RNA interference (RNAi), and the importance of these discoveries has been recognized through numerous awards and accolades including a Nobel Prize for Fire and Mello [Welcome to Silence , a new ond Mello ,3.Our current understanding of RNA silencing derives from experiments performed in organisms from three kingdoms, experiments that directly inspired billion-dollar investments by biotechnology and pharmaceutical companies to use RNA-silencing both to diagnose and to treat disease in humans. Both small interfering RNAs and microRNA-blocking antisense oligonucleotides are now in human clinical trials . Some ofRNA silencing has excited scientists and non-scientists alike: witness front-page headlines in the American and British press -10, evenThe study of RNA silencing has now travelled far from its posttranscriptional roots. The link between RNA and epigenetic silencing by chromatin modification, for example, is well established in many organisms . In otheSilence will enthusiastically publish papers on these and other RNA-based mechanisms in addition to studies of the canonical RNA silencing pathways.In parallel, other types of RNA-mediated mechanisms have been discovered, from CRISP RNAs  in bacteSilence. First, the history of silencing is one of extensive cross-fertilization among different research communities. Such inter-organism as well as inter-disciplinary collaboration and discussion explains the remarkable productivity of our field. Unfortunately, the expansion and diversification of RNA silencing research threatens to fragment our intellectual community. Increasingly, the opportunity for a plant researcher, for example, to read a paper on genomic rearrangements in protozoa or for a fly geneticist to learn of the discovery of a novel mechanism of transposon control in fungi is being lost. Of course, existing journals devoted to the study of RNA will always publish some RNA silencing papers, but it is unreasonable to think that these journals can allocate a high proportion of their pages to our field. As RNA silencing research diversifies, we risk losing the excitement generated by the common enthusiasm for RNA silencing that unites distinct research communities. Silence seeks to nurture that enthusiasm by sustaining the interdisciplinary flavor of our field.Papers with (RNA) AND (silence OR silencing) in their titles or abstracts first appeared in the mid 1990s; there are now more than 1,400 each year and the trend is increasing (source: Web of Science) . So why Silence is a response to the rise of genomics and high throughput sequencing. These developments challenge molecular biologists because they demand a new, computational outlook on biological data. There is an enormous opportunity for bioinformaticians, mathematicians and statisticians to work together with experimental biologists to meet this challenge. They will extract useful and interesting information from genome sequences and large datasets and integrate them with similarly large datasets dealing with various other "omic" analyses of experimental systems. Modeling as a basis for hypothesis generation and testing will become increasingly important.Second, Silence can help molecular biologists and geneticists communicate effectively with computational scientists. We would be pleased, for example, to publish computational tools and research papers that use "dry science" to investigate RNA silencing or non-coding RNAs. We welcome reviews and commentaries in which computationalists introduce novel ideas, approaches and concepts in a style that is accessible to experimentalists.Silence presents a selection of articles on different topics and an insightful review to illustrate the type of paper that we would like to include as the journal grows. Our renowned and diverse editorial advisory board [This inaugural issue of ry board  ensures ry board  and othery board  of many ry board -23.Silence is meant to be yours; your comments and submissions will ensure it succeeds as the hub of the RNA silencing field.We look forward to receiving your manuscripts for publication and your feedback about the journal.


PTGS  is used to counter pathogenic invasions, particularly viruses. In return, many plant viruses produce proteins which suppress silencing directed against their RNA. The diversity of silencing suppression at the species level in natural hosts is unknown.Rice yellow mottle virus) in rice. The RYMV-P1 protein is responsible for cell-to-cell movement and is a silencing suppressor. Transgenic gus-silencing rice lines were used to investigate intra-specific and serogroup silencing suppression diversity at two different levels: that of the virion and the P1 silencing suppressor protein. Our data provide evidence that silencing suppression is a universal phenomenon for RYMV species. However, we found considerable diversity in their ability to suppress silencing which was not linked to RYMV phylogeny, or pathogenicity. At the level of the silencing suppressor P1 alone, we found similar results to those previously found at the virion level. In addition, we showed that cell-to-cell movement of P1 was crucial for the efficiency of silencing suppression. Mutagenesis of P1 demonstrated a strong link between some amino acids and silencing suppression features with, one on the hand, the conserved amino acids C95 and C64 involved in cell-to-cell movement and the strength of suppression, respectively, and on the other hand, the non conserved F88 was involved in the strength of silencing suppression.We investigated the functional diversity of silencing suppression among isolates of the African RYMV (We demonstrated that intra-species diversity of silencing suppression is highly variable and by mutagenesis of P1 we established the first link between silencing suppression and genetic diversity. These results are potentially important for understanding virus-host interactions. PTGS  is conserved among eukaryote kingdoms and is a gene-regulatory mechanism involved in several control processes, including development, maintenance of genome stability, and defence against invasive pathogens. This molecular mechanism is initiated by double-stranded RNA (dsRNA) molecules, or RNA with secondary structures, and results in a reduced steady-state level of cognate cytoplasmic mRNA ,2, in whRice yellow mottle virus, belonging to the Sobemovirus genus, is endemic to Africa and is the major pathogen of irrigated rice [Nicotiana benthamiana [The ted rice ,16. Its ted rice ,18. P1 ited rice . With upted rice . This prted rice . It alsothamiana ,21.iudA), we investigated by GUS activity reversion the functional diversity of PTGS suppression upon virion inoculation or ectopic P1 gene expression. We thus were able to demonstrate for the first time the high diversity of silencing suppression at the intra-species level, suggesting a complex mechanism in silencing suppression at the virus scale. Using P1 mutants we found that cell-to-cell movement and the efficiency of silencing might be linked with variation in the efficiency of silencing suppression.It is well known that viral suppressors differ considerably in their structure and function . HoweverO. sativa L. (O. s.) spp. japonica cv. Nipponbare containing the uidA (gusA) transgene from pCAMBIA 1301 (GenBank accession No. AF234297) and carrying a single copy of T-DNA [uidA gene and the transgenic line L10 is gus-silenced  was used.Transgenic of T-DNA   ,23, thesTwo weeks after seeding, plants (2 leaves) were mechanically inoculated as described elsewhere  with dilAAGCTTGACAATTGAAGCTAGGAAAGGAGC, reverse GAATTCTCTAGACGCGGCC GCTATCAA, and cloned into the XbaI/HindIII (in bold) restriction site from the 35S cassette. These constructs were delivered twice onto pieces of L10 leaves (collected 2 weeks after seedling) by particle bombardment. For each P1 tested, 20 leaves were cut into 10 cm strips. In order to detect the specific effect of P1 and to avoid potentially unfavourable viral translation, we analysed leaves at 2 days post-delivery (dpd). Histological experiments were performed in the presence of ferro- and ferri-cyanide, so as to ensure wide range spread of coloration was not influenced by stain spreading and only reflected presence of GUS activity.P1 sequences with UTR (from 1 to 709 nt)  from fivgus-silenced line by biolistic delivery (Biorad Biolistic PDS 1000/He) after 3 h incubation in MES buffer containing 10% (w/v) sucrose. Leaves were bombarded at a pressure of 1100 psi and a flight distance of 6 cm with tungsten-coated particles. Each construct was delivered to 20 leaf pieces that were bulked for analysis at the end of the experiment. Leaves were then placed at 26°C for 2 days in 16 h light, 8 h dark at a relative humidity of 60%, on 1.5% phytagel and 0.5 w/v MES containing 3% (w/v) sucrose.Five micrograms of sP1s were inoculated onto pieces of leaves from the L10 ®-Gly (Ambion®) as recommended by the manufacturer. Radio-labelling of the probe and hybridization was performed as done in ref. [GusA was used for the specific probe preparation. Radio-labelled signals were detected either by autoradiography or by scanning with Typhoon (Amersham). RNA band intensities were quantified using Image Quant software (Molecular Dynamics).Total RNA was extracted from rice leaves with TRIzol reagent (Invitrogen™) according to the manufacturers' instructions. Analysis of low molecular weight RNA was performed on 15 μg total RNA as described previously . Analysi in ref. . A 808 b4, 10 mM Na2EDTA, 1 mg.ml-1 N-Laurylsarcosine, 0.1% v/v Triton ×100) by two successive centrifugations of 20000 g at 4°C. The Bradford colorimetric method  [® Transfer Medium, Biorad). A diluted rabbit polyclonal antibody directed against the P1 protein (1/500) was used and revealed by a second (1/40000), horseradish peroxidase-conjugate (Pierce) through Supersignal® West-Pico chemiluminescence substrate. Blots were exposed to CL-XPosure™ film (Pierce).Soluble proteins were extracted from rice leaves in a sodium phosphate buffer (50 mM NaHPO Pierce)  was usedThe virus concentration was evaluated by DAS-ELISA as described previously . DAS-ELIHistochemical staining for GUS activity and GUS assays were performed according to Jefferson  and invo®). Fluorescence was measured at 15 min intervals for 2 h with a Fluoroskan fluorimeter (Labsystem), with a 365 nm excitation filter and a 455 nm emission filter. Calibration was performed with quantification of fluorescence of soluble 4-methylumbelliferone .As described previously , proteingus-silenced rice line (L10). As seen in Figure gus transgene, leads to an absence of GUS activity, and is accompanied with a strong accumulation of gus specific siRNA. This accumulation is maintained during the life-cycle of rice plants  whereas in non-inoculated (NI) and mock-inoculated (BI) L10 leaves, GUS activity was undetectable    Figure . Moreove) Figure , and theFurthermore, as previously demonstrated at the virion scale, the efficiency of these silencing suppressors is not correlated with viral phylogeny. Indeed, according to RYMV evolutionary history , P1s froPotato potexvirus X (PVX) are linked [As we found a qualitative effect of the different P1s, and as it has been demonstrated that silencing suppression and cell-to cell movement of e linked , we invegus-siRNA and mRNA, in the delivery and distal areas for each sP1 protein previously selected for a range of variation in their efficiency of silencing suppression . For the sCI63 protein, we observed a weak accumulation in the both areas for gus-siRNA, and strong accumulation mainly in del area for gus-mRNA (lanes 8–9). For the sMg1 protein, we observed a contrasting situation between the del and dis areas; lower accumulation of gus-si-RNA and accumulation of mRNA in the del area, and an absence of gus-mRNA in the dis area , we attempted to assign amino acid diversity to functional effects with ClustalW alignments  Figure .2C67(x)24C92(x)2C95) which is conserved across the RYMV phylogeny y Figure . This kiy Figure . We usedgus-specific si- and mRNA detection), we demonstrated that none of the four mutations completely abolished the suppression effect of sTz3. Nevertheless, the single amino acid mutation C95S impaired the ability of P1 to suppress uidA silencing in the distal part of leaves (high level of gus-siRNA), suggesting that this mutation affects the cell-to-cell movement of P1  and Mg1 (lowest suppressor) P1 proteins. P1 mutants only showed partial alteration of the silencing suppression function. Cystein C95 has been shown to be essential for the cell-to-cell movement of protein P1, and C64, F88 essential for the strength of silencing. This contrasts with p19 from the  on PTGS . These r on PTGS . Based o on PTGS . MutatioIn its natural host we highlighted the functional diversity of RYMV isolates on silencing suppression. The functional diversity at the scale of virion is not correlated to genetic diversity of the P1 protein suppressor. In addition, we demonstrated that this functional diversity is not linked to the virus phylogeny or its pathogenicity. Our overall results suggest that the mechanism of silencing suppression due to the virus is more complex than previously thought. By studying the P1 silencing suppressor alone we demonstrated that the functional diversity of silencing suppression occurs at the P1 protein level and mutagenesis experiments demonstrated that cystein C95 is essential for cell-to-cell movement of P1, and that C64 and F88 are involved in the efficiency of silencing suppression. As both cysteins (C95 and C64) are conserved in the RYMV phylogeny, and as F88 is not, we could assume that this last mutation could be one of the amino acid involved in silencing suppression diversity of P1. This mutation opens the way to identify other amino acids involved in the functional diversity of silencing suppression.rice yellow mottle virus; dsRNA: double strand RNA; miRNA: microRNA; siRNA: small-interfering RNA; PTGS: post-transcriptional gene silencing; ORF: open reading frame; GUS: β-glucoronidase; uidA: GUS gene; cv: cultivar; O.s: Oryza sativa; TAI: Taipei; BF: Burkina Faso; CI: Ivory Coast; Mg: Madagascar; Ni: Nigeria; Tz: Tanzania; DAS-Elisa: double antibody sandwich-enzyme linked immunosorbent assay; UTR: untranslated region; CaMV: cauliflower mosaic virus; CP: coat protein; GFP: Green fluorescent protein; PVX: potato potexvirus ; EtBr: ethidium bromide; NI: non-inoculated; BI: buffer-inoculated, ND: non-biolistic delivery; BD: biolistic delivery; dpd: day post delivery; del: delivery; dis, distal.RYMV: The authors declare that they have no competing interests.CS carried out experiments and drafted the manuscript. MB-R participated in the design, cloning, mutagenesis and transitory expression assays; DF participated in the design of experiments in Figs


Human cells depend critically on the signal recognition particle (SRP) for the sorting and delivery of their proteins. The SRP is a ribonucleoprotein complex which binds to signal sequences of secretory polypeptides as they emerge from the ribosome. Among the six proteins of the eukaryotic SRP, the largest protein, SRP72, is essential for protein targeting and possesses a poorly characterized RNA binding domain.We delineated the minimal region of SRP72 capable of forming a stable complex with an SRP RNA fragment. The region encompassed residues 545 to 585 of the full-length human SRP72 and contained a lysine-rich cluster (KKKKKKKKGK) at postions 552 to 561 as well as a conserved Pfam motif with the sequence PDPXRWLPXXER at positions 572 to 583. We demonstrated by site-directed mutagenesis that both regions participated in the formation of a complex with the RNA. In agreement with biochemical data and results from chymotryptic digestion experiments, molecular modeling of SRP72 implied that the invariant W577 was located inside the predicted structure of an RNA binding domain. The 11-nucleotide 5e motif contained within the SRP RNA fragment was shown by comparative electrophoresis on native polyacrylamide gels to conform to an RNA kink-turn. The model of the complex suggested that the conserved A240 of the K-turn, previously identified as being essential for the binding to SRP72, could protrude into a groove of the SRP72 RNA binding domain, similar but not identical to how other K-turn recognizing proteins interact with RNA.The results from the presented experiments provided insights into the molecular details of a functionally important and structurally interesting RNA-protein interaction. A model for how a ligand binding pocket of SRP72 can accommodate a new RNA K-turn in the 5e region of the eukaryotic SRP RNA is proposed. The signal recognition particle (SRP) participates in the vital compartmentalization of every cell by guiding proteins towards their membrane translocation sites. Except in chloroplasts , SRP is The mammalian SRP is composed of an 303-nucleotide SRP RNA and proteins SRP9/14, SRP19, SRP54 and SRP68/72 . ProteinCryo-electron microscopy combined with site-directed mutagenesis and footprinting experiments demonstrated that SRP68 possesses numerous contacts with the SRP RNA of the large SRP domain ,14. In cSeveral high-resolution structures of substantial portions of the SRPs from a variety of taxa have been solved and this knowledge contributed significantly to our understanding of how the SRP assembles and fulfills a multitude of functions ,17-19. D7 M-1. The RNA-binding SRP72 fragments shared a conserved region with the consensus sequence PDPXRWLPXXER at positions 572 to 583 (X is for any amino acid residue) and a cluster of positively charged amino acid residues at positions 552 to 559 [The focus of the presented work is human protein SRP72, the largest of the SRP proteins (74.6 kDa), and its association with the SRP RNA. Full-length SRP72 as well as a fragment of SRP72  were shown to bind independently and specifically to human SRP RNA with a K'a of 2.9 × 102 to 559 . We usedHuman SRP72, composed of 671 amino acids, had a molecular mass of 74,602 dalton, a pKi of 10.01, and 98.8% identity to its canine homologue . The proPurified polypeptide 72e was used as the substrate for the initial site-directed mutagenesis experiments designed to identify the smallest polypeptide with undiminished RNA binding activity. The RNA ligand was a 46-nucleotide hairpin which contained the 5e motif Figure . RecombiA recently updated alignment of 113 eukaryotic SRP72 sequences  confirmeEscherichia coli, purified by adsorption on Ni-NTA beads, and the eluates were incubated with in vitro transcribed 5e RNA. Binding was measured by incubating fixed amounts of RNA with increasing concentrations of the purified wild-type or mutant polypeptides. The proteins and RNAs eluted from the Ni-NTA magnetic agarose beads were analyzed by SDS PAGE followed by staining with Coomassie blue and Ethidium bromide (see Methods). The results shown in Figure As substrate for mutagenesis we used the coding region of 72h which exhibited optimal binding to the 5e RNA  and 581-RE-582 (69 ± 14%). Interestingly, the mutation of the evolutionary conserved P579 within PDPXRWLPXXER to an alanine substantially reduced the binding activity (26 ± 1%). At the same time, the mutation at the variable M580 allowed the formation of complexes at wild-type levels (100 ± 4%).The alanine changes at 585-YY-586 caused a significant reduction 60 ± 3%) in the ability to form complexes. Similarly, the deletion of seven amino acid residues, including 585-YY-586 and S584, in 72l yielded inactive polypeptides. Together with the dissimilar RNA binding activities of 72f and 72g . Changing or removing six lysine residues abolished binding completely. These data suggested that the lysine-rich region contributed substantially to the RNA binding activity of SRP72.Chymotrypsin was shown to preferentially cleave peptides at trptophane, tyrosine and phenyalanine residues . This prWe investigated whether different chymotryptic digestion patterns could be observed when the RNA binding domain of SRP72 formed a complex. Increasing amounts of 72 j polypeptide was incubated with a fixed amount of the human Δ35  or the 5The 5e motif was discovered in an alignment of the SRP RNAs as one of four conserved elements ,24. It hTo investigate the possibility of a structural contortion of the 5e RNA motif we adapted a previously developed strategy for the study of DNA bending . The potUsing synthetic DNA and RNA oligonucleotides we generated three different assemblies with 10-nucleotide staggered complementary overhangs Figure , lane 3.Haloarcula marismortui ribosome [K-turns, a widespread feature of many medium size and large RNA molecules, have been characterized as being composed of a canonical stem, three unpaired nucleotides, and two adjacent non-canonical GA pairs . Within ribosome  yielded ribosome  protrudeThe three-dimensional structure of SRP72 has not been determined experimentally. We therefore used homology modeling of the whole protein and its RNA binding domain in order to gain insights into the mechanism of the binding of RNA by SRP72. The initial SRP72 model was created using the I-TASSER server  followedThe structure of the RNA binding region was predicted to contain two crossed alpha-helices separated by a large loop formed by residues R555 to P572  were constructed using as starting material NcoI and BstXI-restricted DNA fragments from a previous study . The N-tPlasmids encoding wild-type or mutant SRP72 fragments with a C-terminal his-tag were generated by PCR using as primers the oligonucleotide GGA CCA TGG TTC GGA AGA AGG GTG GAA AAG TTA CTGG and various other oligonucleotides depending on the desired construct, e.g CGC AAG CTT TCA GTG GTG GTG GTG GTG GTG CTT TCT TCC CCG GTA G. The PCR products were digested with NcoI and HindIII and ligated to the purified NcoI and HindIII-digested 72e vector DNA and clones were selected as described above. To delete or change residues within the lysine-rich cluster, a silent HindIII site was introduced into the coding region for 561-KLP-563 by PCR site-directed mutagenesis . This inEscherichia coli strain Rosetta (pLysS). Transformants were selected by an overnight incubation at 37°C on LB agar plates containing Ampicillin and Chloramphenicol [For the expression of the his-tagged SRP72 fragments plasmids were transformed into the competent cells of the phenicol . ColonieThe frozen pellet from 2.5 ml of cell culture was resuspended in 2.5 ml of ice-cold lysis buffer  supplemented with 20 ul of protease inhibitor cocktail equivalent to 1/75th of one EDTA-free complete mini protease tablet (Roche). The cell suspension was sonicated  6 times for 15 sec each at a setting of 35 percent with 1 minute interruptions during which the sample was placed on ice. The lysate was subjected to centrifugation in a Beckman ultracentrifuge for 1 hr at 50,000 rpm and 4 C (Beckman TLA-100.3 rotor) and the supernatant was added to a 200 ul suspension of Ni-NTA Superflow beads (Quiagen) which had been pre-equilibrated in lysis buffer. The sample was mixed by slow rotation for 1 hr at 4°C and transferred to 0.5 ml mini-columns (Evergreen Scientific). After the column had settled by gravity flow, the beads were washed with 1 ml of lysis buffer. The his-tagged bound proteins were collected by washing in two steps with 150 μl of lysis buffer containing 150 mM and then 300 mM Imidazole. Sample aliquots were subjected to electrophoresis on 12.5% polyacrylamide SDS Tricine gels followed by staining of the polypeptides with Coomassie blue with known amounts of Lysozyme for reference to calculate protein concentrations from a standard curve.Full-length human SRP RNA (hR) or its fragment from the large SRP domain (Δ35 RNA) were synthesized by run-off transcription of DraI- or BamHI-restricted plasmids with T7 RNA polymerase . The conBinding of the his-tagged polypeptides to the 5e or Δ35 SRP RNA was measured using Ni-NTA magnetic agarose beads (Quiagen) essentially as described . Bead-boPolypeptides at a concentration of 0.25 to 0.3 mg/ml were incubated in a volume of 10 μl lysis buffer containing 300 mM Imidazole (from the purification step) with 0.3 μg of alpha-Chymotrypsin  for up to 10 minutes at ambient temperature. The digestion was terminated by adding 6 μl of SDS-loading buffer  and the samples were analyzed by electrophoresis on 12.5% Tricine-SDS polyacrylamide gels followed by staining with Coomassie blue.For the identification of his-tagged chymotryptic fragments, the digested samples were adjusted to 100 μl and a final concentration of 50 mM Na-phosphate, pH 7.5, 500 mM NaCl and 0.1% Tween 20 (binding buffer). 15 μl of a Ni-NTA magnetic bead suspension (Quiagen) were added follwed by occasional mixing with a pipette at room-temperature for 1 hr. The beads were separated from the unbound polypeptides by placing the samples into a magnetic field, the beads were washed with 50 μl of binding buffer and the bound polypeptides were eluted with the same buffer but containing 250 mM Imidazole. Samples were analyzed by SDS PAGE and the gels stained with Coomassie blue.2, pH 7.9 with increasing amounts of in vitro transcribed Δ35 RNA. The samples were incubated for 10 minute at 37°C and then placed at room temperature. 0.3 μg of Chymotrypsin were added and complexes were digested for 5 minutes. The digestion was terminated by adding 4 μl of Tris-acetate EDTA buffer followed by SDS PAGE as described above.To measure the ability of the RNA to protect protein from the digestion by Chymotrypsin, the purified 72j polypeptide was mixed at room temperature in 12.5 μl of 50 mM Tris-HCl, 300 mM KOAc, 5 mM MgCl2 and 16% glycerol. Samples were incubated for 10 minutes at 37°C and separated by electrophoresis at room temperature on a 6% poyacrylamide gel (Acryl/Bisacryl 19:1) in 20 mM HEPES, 0.1 mM EDTA, pH 8.3 at 15 mA for 2 hrs. A picture was recorded after staining of the RNAs with buffer containing 0.1 mg/mL Ethidium bromide and placing the gel onto a UV-transilluminator. The number of pixels in each band was measured using NIH Image software [For the formation of complexes between the 72j polypeptide and the SRP RNA, 0.25 μg of 5e or Δ35 RNA were mixed with variable amounts of 72j protein in 10 μl of 50 mM Tris-HC, l pH 7.9, 300 mM KOAc, 5 mM MgCl400 μmole of each complementary nucleic acid were annealed in 400 μl of TE containing 100 mM NaCl. Samples were placed in a boiling water bath and allowed to gradually cool to room temperature during a time period of about 30 minutes. The annealed nucleic acids were extracted with an equal volume of phenol/chloroform and the aqueous supernatant of the centrifugation was mixed with 15 μl of 5 M NaCl and 3 volumes of ethanol. The nucleic acids were incubated at -70°C for 20 min and the precipitate was collected by centrifugation, washed with 300 μl 80 percent ethanol and dissolved in 100 μl of TE.2. Samples were allowed to gradually cool from 70°C to 4°C within approximately 30 minutes. 5 μl aliquots of the samples were subjected to electrophoresis at 4°C and 160 V using a 8% acrylamide gel (Acryl/Bisacryl 19:1) in TBE or in TB with 1 mM MgCl2 until the Bromophenol blue dye had migrated 10 cm. The gel was stained for 10 minutes with 0.1 mg/ml Ethidium bromide and the nucleic acids were visualized using a UV-transilluminator (Bio-Rad) to record a picture.4 μmole of the annealed DNA fragments were mixed with equivalent molar amounts of synthetic oligoribonucleotides in 20 μL of 50 mM Tris-HCl, pH 7.5, 25 mM NaCl, with or without 1 mM MgClThe sequences of the nucleic acids are provided in Additional file The three-dimensional model of the complete amino acid sequence of human SRP72 was created by the I-Tasser server . From thEI purified wild-type and mutant polypeptides, transcribed the RNAs, and carried out all binding assays. AI created the three-dimensional model of the human SRP72 and participated in the assessing the spatial relationship between RNA and its interaction with SRP72. CZ conceived the study, generated the mutant plasmids, carried out the RNA bending experiments, and drafted the manuscript. All authors read and approved the final manuscript.Sequences of the synthetic polynulceotides used to investigate kinking of the 5e motif in human SRP RNA. Oligonucleotides are numbered as in Figure Click here for fileAtomic coordinates of the predicted structure of human SRP72 in pdb format.Click here for filePredicted atomic coordinates of human SRP72's RNA binding domain.Click here for file


How much do we really understand about how +RNA viruses usurp and transform the intracellular architecture of host cells when they replicate? Viruses containing RNA genomes, or RNA viruses, constitute an extensive genetic group of plant, animal, and human viruses. RNA viruses include many viruses that are important human pathogens, and have been shown to have the ability to move from animals to humans, resulting in zoonotic-human epidemics. RNA viruses that contain single-stranded RNA genomes of positive polarity are known as positive-strand RNA (+RNA) viruses. +RNA virus families of animals and humans include the Coronaviridae (SARS-coronavirus), Picornaviridae , Flaviviridae  and Togaviridae . The objective of this Primer is to focus on established and new approaches to understand the interface of +RNA virus replication and cell biology.Positive-strand viruses replicate in the cytoplasm of the host cell in association with cytoplasmic membranes. To complete a productive infection, they must enter cells, institute cytoplasmic replication factories, replicate genome RNA, express their genomes, sort and traffic proteins and RNA, and assemble and release virus particles—in some cases within an 8–12 hour life cycle. Investigators who seek to dissect the replication strategies of +RNA viruses are drawn inexorably into the complexities of cellular biology. To determine the mechanisms by which +RNA viruses navigate these steps may necessitate an understanding of cell biology processes as diverse as cytoplasmic organelle structure and membrane biogenesis, endoplasmic reticulum (ER) formation, microtubule motor movement, actin polymerization, and trafficking in the secretory pathway. +RNA viruses also share a common requirement for translation of the input mRNA-sense +RNA genome to yield nonstructural proteins that are responsible for both induction of membrane modifications and RNA replication and transcription. These genomes range in size and complexity from the small, segmented 4.5 kb flock house virus (FHV) genome to the 27–32 kb nonsegmented coronavirus (CoV) genomes, which express an ~800 kDa replicase fusion polyprotein processed by multiple viral proteinases into 16 mature nonstructural proteins (nsp1 to 16) . Mor. Mor3]. An example of this is the nodavirus FHV, which forms discrete spherular invaginations in outer mitochondrial membrane . The sphPLoS Biology by Knoops et al. [The present study in s et al.  uses simThe study is important for the CoV and +RNA virus cell biology field for several reasons. First, it suggests more common mechanisms between coronaviruses and other +RNA viruses, such as the Flaviviridae, that also induce DMVs, CMs, and VPs. Second, it may allow the unification of seemingly disparate results of previous studies of CoV RC membrane origins and evolution. Third, it presents a model for testing questions that have been raised by other studies. For example, sites of CoV virion assembly appear to be distinct from RCs, but it is not known how progeny genome RNA is delivered from RCs to sites of assembly. Analysis of the RVN over time with multiple cellular and replicase markers will likely define the mechanism for RNA and protein movement from RCs to sites of assembly. Finally, the model raises exciting new questions. The results suggest that the majority of SARS-CoV double-stranded RNA, and possibly RNA synthesis, resides within the interior of the DMV vesicles, while virion budding occurs from the cytosol. However, the careful 3-D reconstructions did not identify any vesicle opening or pore in the DMVs that would allow movement of genome RNA from the inside to the cytosol. This suggests a novel mechanism both for access to the membrane-protected interior and for egress of genome RNA.The integration of novel approaches for high-resolution reconstruction of +RNA RCs is an exciting evolution of viral cell biology. Future studies to address the remaining and new questions of RC formation and function will require approaches to define the RCs by their viral and cellular proteomes, and to determine protein interactions, membrane changes, RNA synthesis, and virion assembly. Recent studies have tracked viral RNA in real time by tagging it with green fluorescent proteins (GFPs) or “molecular beacons.” Engineered viruses expressing GFP-fusions with viral replicase proteins have been used for direct fluorescent studies of replicase protein targeting, and will allow real-time analysis of protein expression, interactions, and movement . GFP-repIn summary, the level of resolution and understanding of +RNA RC formation and function is rapidly increasing, and new technologies are being developed and applied to understand how “simple” +RNA viruses can usurp and transform the intracellular architecture in a rapid and comprehensive manner. We will undoubtedly continue to be surprised by the approaches used by the viruses and the insights they provide into the fundamental biology of the cell.


M of the synthetic androgen methyltrienolone (R1881), whereas 0.01 nM of this compound induced the same effect in parental cells. At later passages (> 75), androgen exerted an inhibitory effect on growth of LNCaP-abl cells. The non-steroidal anti-androgen bicalutamide stimulated proliferation of LNCaP-abl cells. AR protein expression in LNCaP-abl cells increased approximately fourfold. The basal AR transcriptional activity was 30-fold higher in LNCaP-abl than in LNCaP cells. R1881 stimulated reporter gene activity in LNCaP-abl cells even at 0.01 nM, whereas 0.1 nM of R1881 was needed for induction of the same level of reporter gene activity in LNCaP cells. Bicalutamide that acts as a pure antagonist in parental LNCaP cells showed agonistic effects on AR transactivation activity in LNCaP-abl cells and was not able to block the effects of androgen in these cells. The non-steroidal AR blocker hydroxyflutamide exerted stimulatory effects on AR activity in both LNCaP and LNCaP-abl cells; however, the induction of reporter gene activity by hydroxyflutamide was 2.4- to 4-fold higher in the LNCaP-abl subline. The changes in AR activity were associated neither with a new alteration in AR cDNA sequence nor with amplification of the AR gene. Growth of LNCaP-abl xenografts in nude mice was stimulated by bicalutamide and repressed by testosterone. In conclusion, our results show for the first time that the non-steroidal anti-androgen bicalutamide acquires agonistic properties during long-term androgen ablation. These findings may have repercussions on the natural course of prostate cancer with androgen deprivation and on strategies of therapeutic intervention. © 1999 Cancer Research CampaignAdvanced prostate cancer is treated by androgen ablation and/or androgen receptor (AR) antagonists. In order to investigate the mechanisms relevant to the development of therapy-resistant tumours, we established a new tumour model which closely resembles the situation in patients who receive androgen ablation therapy. Androgen-sensitive LNCaP cells were kept in androgen-depleted medium for 87 passages. The new LNCaP cell subline established in this manner, LNCaP-abl, displayed a hypersensitive biphasic proliferative response to androgen until passage 75. Maximal proliferation of LNCaP-abl cells was achieved at 0.001 n


Mycobacterium tuberculosis. Post-transcriptional regulation of gene expression by sRNA molecules has been demonstrated in a wide range of pathogenic bacteria and has been shown to play a significant role in the control of virulence. By screening cDNA libraries prepared from low-molecular weight RNA from M. tuberculosis we have identified nine putative sRNA molecules, including cis-encoded antisense transcripts from within open reading frames and trans-encoded transcripts from intergenic regions. sRNAs displayed differential expression between exponential and stationary phase, and during a variety of stress conditions. Two of the cis-encoded sRNAs were associated with genes encoding enzymes involved in lipid metabolism, desA1 and pks12. These sRNAs showed complementarity to multiple M. tuberculosis genes, suggesting the potential to act as both cis-encoded and trans-encoded sRNAs. Overexpression of selected trans-encoded sRNAs had profound impact on growth of M. tuberculosis and M. smegmatis. This is the first experimental evidence of sRNAs in M. tuberculosis and it will be important to consider the potential influence of sRNA regulation when studying the transcriptome and the proteome of M. tuberculosis during infection.In spite of being one of our most prominent bacterial pathogens, the presence of small regulatory RNAs (sRNAs) has not previously been investigated in  Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the most successful human pathogens. It is estimated that a third of the world's population has been infected, with new infections occurring at a rate of approximately one per second. Less than 10% of infected individuals go on to develop active disease, resulting in around 9 million new cases and 1.6 million deaths annually. The remaining 90% will not develop any symptoms but the bacteria may persist in the form of an asymptomatic latent infection with the potential to reactivate at any time.M. tuberculosis during active or latent infection remains poorly understood, considerable efforts have been invested in exploring transcriptional changes associated with bacterial adaptation to changing environments in in vitro and cell culture models. The genome of M. tuberculosis encodes 13 sigma factors, 11 two-component sensory transduction systems, and over 100 annotated transcriptional regulators  molecules, which can act by base pairing or by interaction with proteins . Most of targets . The lat targets . sRNA bied sRNAs . RegulatM. tuberculosis cultures : 1: mRNA fragments within open reading frames (ORFs) (11 clones); 2: rRNA spacer fragments (5 clones); 3: tRNA fragments (1 clone); 4: unknown RNA encoded in intergenic regions , named according to the original library clones; 5: unknown RNA encoded antisense to annotated ORFs , named according to their antisense partner; 6: fragments that could not be assigned to a single region due to small size (< 17 base pairs) or chimeric sequence (149 clones). The intergenic regions containing B11 and F6 were represented 16 and three times respectively, and in both growth phases, indicating that transcripts from these regions were relatively abundant.As we had observed differential expression of Rv 2050 and therefore considered to be the 5′ UTR of this gene and thus not investigated further. The sizes and genome locations of the remaining sRNA candidates are shown in The genomic positions and context of the unknown RNAs were identified in order to determine the likelihood of each to be a genuine sRNA or part of the adjacent gene . If an itrans-encoded  and four cis-encoded . To further clarify if the identified candidates were genuine sRNAs, we performed Northern blotting with riboprobes complementary to the original cDNA clones. Before hybridization the membranes were stained with methylene blue to ensure uniform loading between exponential and stationary phase RNA (not shown). The results of the Northern blotting, shown in trans-encoded sRNAs were expressed at significantly higher levels than the cis-encoded ones. All transcripts  were larger than the cloned fragments, suggesting that the latter were probably degradation products. The majority of sRNAs were seen as more than one transcript, and most of the transcripts appeared to be differentially expressed and/or processed in the two growth phases. The ASdes and ASpks blots showed a significant smear representing larger species (> 150 nucleotides), specifically in exponential growth, suggesting the presence of larger transcripts/precursors in addition to the small transcripts. The C8 sRNA appeared as a large smear with the majority of the signal between 110 and 140 nucleotides in exponential phase, less so in stationary phase where a distinct band around 85 nucleotides was seen, suggesting extensive trimming of the primary transcript to the 85-nucleotide RNA. In addition there was a faint band around 55 nucleotides. A summary of the approximate sizes of transcripts found by Northern blotting is shown in Our cDNA libraries identified nine sRNA candidates; five Experimental procedures for details). The results, shown in cis-encoded sRNAs appeared to have several different 5′ ends, primary as well as processed, of varying intensity. However, some of the larger products from the F6 and G2 reactions turned out to be concatemers of transcripts with the same 5′ end as the library clones. As the concatemerization was TAP dependent we consider these 5′ ends to be transcription start sites. This means that G2 was the only trans-encoded sRNA with more than one 5′ end, while all of the cis-encoded RNAs had multiple 5′ ends.sRNA transcripts were further characterized by RNA ligase-mediated rapid amplification of 5′ and 3′ complementary DNA ends (RLM-RACE). The 5′ ends were mapped by comparing the RACE products obtained with and without prior treatment with tobacco acid pyrophosphatase (TAP), which facilitates the differentiation of transcription start sites from processed 5′ ends . In the case of B11 this would result in a 93-nucleotide transcript in reasonably good agreement with the Northern blot. The situation for C8 was not as straightforward and hence we performed 3′ RACE to verify the C8 3′ end (see below). In the case of F6, a 102-nucleotide transcript is in good accordance with the faint signal of approximately 100 nucleotides seen on the Northern blot. This transcript could then be processed to the shorter and more abundant ∼55-nucleotide transcript.Experimental procedures. In some cases an additional 3′ RACE was performed on poly-C-tailed RNA for comparison. The 3′ ends thus identified were compared with the 5′ ends and the transcript sizes on the Northern blots in order to identify accurate transcript boundaries. Results of 3′ RACE analysis are summarized in In order to map the remaining 3′ ends, we performed 3′ RACE. A poly-A tail was added to the RNA and 3′ RACE was carried out as described in trans encoded sRNA candidates were encoded on the same strand as one of the adjacent protein-encoding genes (Rv0609A (Rv0609A and B55 is 100% conserved (in M. tuberculosis complex only), this suggests the possibility that B55 is part of the 3′ UTR of the Rv0609A mRNA rather than being an sRNA. Although we had identified two 3′ ends for C8 by RACE, as well as a possible terminator between C8 and the downstream  Rv3722c, we found that C8 was co-transcribed with Rv3772c .Once we had identified the 5′ and 3′ termini of the transcripts we assigned the most probable boundaries for the transcripts seen on the Northern blots. Each of the resulting sequences was subsequently analysed using ructures . Strikinructures . In partM. tuberculosis sRNAs responded to stresses similar to those encountered during infection. Therefore, we subjected cultures of M. tuberculosis to oxidative stress (induced by H2O2), DNA damage (induced by Mitomycin C) and acid stress. Total RNA was isolated and sRNA expression detected by Northern blotting and subsequently normalized to 5S RNA levels to compensate for uneven loading. If an sRNA had multiple bands, we quantified all the bands. Each signal was compared with a control sample, i.e. unstressed, exponential growth for oxidative stress and DNA damage, and a pH control for the acid stress . A representative Northern blot for each sRNA upon stress is shown in Bacterial sRNAs are often stress induced. We wanted to investigate how the expression of trans-encoded sRNAs, B11, F6 and G2 were cloned in plasmid vectors under the control of the strong rrnB promoter of M. smegmatis , in spite of the absence of any obvious homologue of this sRNA in the rapid-growing strain. The expression of F6 did not result in any apparent growth defects in M. smegmatis, although the 5′ end and central, loop-containing part of this sRNA is conserved  as well as short antisense transcripts encoded within ORFs (cis-encoded sRNAs). One of the trans-encoded sRNAs was identified as the M. tuberculosis homologue of the 4.5S RNA molecule that forms part of the signal recognition particle involved in protein secretion .Although the percentage of putative sRNAs clones that we obtained (13%) was high in comparison with that reported using a similar approach with RNA from Streptomyces, a related actinomycete, predicted 32 sRNAs, of which 20 were confirmed experimentally  promoters as well as having a canonical terminator structure.A recent bioinformatic screen in Mycobacteria nor Streptomyces have identifiable homologues of the Hfq RNA chaperone that has been widely implicated in the mechanism of trans-encoded sRNAs in Gram-negative bacteria  is an essential gene for growth of M. tuberculosis (desA2/Rv1094)  12 is implicated in synthesis of mannosyl-β-1-phosphomycoketide molecules, which are recognized as antigens by CD1-restricted T cells  , and couthe sRNA .trans-encoded sRNAs, and the possibility remains that some of these could be structural rather than regulatory RNAs, as was the case of 4.5S RNA, meaning they would not have mRNA targets as such. However, genome and operon associations with genes of known function may give a clue. Thus, F6 is located within a region encoding enzymes involved in lipid metabolism as well as co-transcribed with fadA2, for which the transcript is downregulated in macrophages  to a final concentration of 10 mM for 1 h. To induce DNA damage, Mitomycin C was added to an exponentially growing culture (OD∼0.3) at a final concentration of 0.02 μg ml−1 for 24 h. Acid stress was induced by spinning down an exponentially growing culture at room temperature and resuspending in acidified medium (pH 5.0) at 37°C to OD 0.3–0.4 and subsequently grown for 24 h. In order to take into account the hypoxia and cold shock induced during centrifugation, a control culture was treated identically but resuspended in standard 7H9 medium (pH 6.5) and likewise grown for 24 h and used for normalizing the acid stressed samples.To induce oxidative stress, 30% HrrnB promoter from M. smegmatis  and RNA ligase (Ambion) according to instructions. Riboprobes were made with Ambion's mirVana miRNA Probe construction kit using the cDNA cloned sequence as template .Depleted RNA was end-labelled with template  and 32P-E. coli poly(A) polymerase (Ambion) and CTP. The RNA was treated with TAP and a 5′ linker  and RNA marker low from Abnova (20–500 nucleotides).5′ RACE was carried out essentially as described by E. coli poly-A-polymerase (Ambion), reverse transcribing with Superscript III (Invitrogen) using the GRdT oligo or GRdG oligo (3′ RACE was performed by adding a poly(A) or poly(C) tail to total RNA with dG oligo  and finadG oligo .cDNA was made with random primers and Superscript II (Invitrogen). A control reaction was carried out in parallel without enzyme. A standard PCR reaction was subsequently carried out with primers listed in


Laser capture microdissection enables the isolation of single cells or small cell groups from histological sections under direct microscopic observation. Combined with quantitative PCR or microarray, it is a very powerful approach for studying gene expression profiles in discrete cell populations. The major challenge for such studies is to obtain good quality RNA from small amounts of starting material.We have developed a simple, flexible, and low-cost method for simultaneously producing RNA from discrete cell groups in embryonic day 15 mouse brain. In particular, we have optimized the following key steps in the procedure: staining, cryosectioning, storage of sections and harvesting of microdissected cells. We obtained the best results when staining 20 μm-thick sections with 1% cresyl violet in 70% ethanol and harvesting the microdissected tissue in RNA stabilization solution. In addition, we introduced three stop-points in the protocol which makes the tedious process of laser capture microdissection more flexible, without compromising RNA quality.Using this optimized method, we have consistently obtained RNA of high quality from all four simultaneously microdissected cell groups. RNA integrity numbers were all above 8, and long cDNA fragments (> 1.2 kb) were successfully amplified by reverse transcription PCR from all four samples. We conclude that RNAs isolated by this method are well suited for downstream quantitative PCR or microarray studies. There has been an increasing interest in studying and comparing the gene expression profiles of small discrete cell populations. Such studies have been greatly facilitated by the recent development of laser capture microdissection (LCM) technology -3. This For most applications, LCM requires staining of the tissue prior to dissection, which exposes RNA to aqueous solutions and chemical components. In addition, LCM is a lengthy process and is performed at room temperature. These factors can cause RNA degradation and have to be carefully considered when carrying out LCM experiments.A large number of reported studies using LCM do not provide information about RNA integrity after the microdissection. Those that do assess the RNA quality have reported RNA integrity numbers (RINs) below 8, indicating that there had been at least some degree of degradation ,5. RIN iOriginally LCM was developed to analyze cancer specimens. Therefore, most protocols were optimized for the dissection of tumour cells in different organs e.g. prostate or colon ,7,8. LCMGene expression profiling of specific cell populations usually requires the isolation and comparison of at least two adjacent cell groups. A rigorous assessment demands that the different cell groups are collected from the same tissue sections. Obtaining equally high quality RNA from these individual populations simultaneously is an essential prerequisite to a meaningful expression profile comparison. This poses a particular challenge as the time necessary for collecting the different samples from each section increases many-fold.In addition to the technical challenges, many researchers using LCM also face organizational difficulties. Laser capture systems which are shared among different research groups only allow time-limited access, and separation of histology from LCM facilities further complicate the management of the experiment. LCM can also be very costly, as special membrane-covered slides have to be used.We have addressed these issues and developed a simple, convenient and low-cost method of producing high quality RNA from small cell groups, microdissected by UV laser. This method has been specifically adapted for comparing gene expression profiles in multiple adjacent cortical layers from the same embryonic brain tissue. We report here a full protocol that considerably improves RNA integrity and allows maximal flexibility in the experimental procedure.In order to optimize the protocol for gene expression analysis between multiple areas, we designed the following experimental approach Figure : anterioThe following four parameters were investigated and optimized:We compared four different staining protocols – hematoxylin, hematoxylin and eosin (H&E), 0.1% cresyl violet and 1% cresyl violet Figure . We founAs it had been previously reported that ethanol-based staining solutions improve RNA integrity , we alsoManufacturer's guidelines and many protocols recommend cutting 5–12 μm sections ,17. We tWe further verified whether efficient cutting and catapulting was also possible for other tissue types on the same 20 μm sections (data not shown). We found that the laser parameters used for cortex could be applied to other nervous tissues , whereas tongue muscle and cartilage could be successfully microdissected after some minor adjustments to the laser settings (UV energy of 78 vs. 76 points).We tested whether storage of sections at -80°C before LCM would noticeably impair RNA quality as previously suggested  by compaCollecting samples from multiple regions of the same brain section and pooling samples from several slides increases the time the tissue is in the harvesting tube. We compared collection of tissue in RNA stabilization solution and lysis buffer Figure . SamplesThe complete optimized protocol is summarized in Figure In addition to the RIN, we amplified long fragments from selected genes  by RT-PCR analysis Figure . The preRNA yields from LCM samples are difficult to evaluate as concentrations are usually below the detection limit of spectrophotometers (10 ng/μl), and bioanalyzers only give an inaccurate estimate of quantity at this concentration. In order to estimate RNA quantities more reliably, we microdissected and pooled 20 μm-thick strips from approximately 140 sections. RNA extracted from this large number of tissue samples was successfully quantified by spectrophotometry, and we obtained yields of total RNA between 120 ng (subplate) and 180 ng  per sample. Performing cell counts on cresyl violet stained material, we estimated that each microdissected strip (580 μm × 40 μm) contains approximately 600 cells in the cortical plate and 300 cells in the subplate at E15. This results in an average RNA yield of 2.5 pg per cell.One of the major difficulties when using LCM for gene expression profiling is to obtain RNA of good quality for subsequent microarray analysis. This is particularly challenging if multiple cell groups are dissected simultaneously from the same tissue sections as in this study. In addition, LCM can be very time-consuming and difficult to manage, especially if complex logistics are involved . Finally, LCM may be expensive if a large number of membrane-coated slides or special commercial kits are used. LCM requires a combination of critical procedures including tissue collection, cryosectioning, fixation/staining/dehydration, storage and microdissection. Each can have a serious impact on the subsequent RNA quality. To address these issues, we have analyzed and optimized four crucial steps in the LCM procedure: tissue staining and dehydration, section thickness, storage of sections and harvesting and storage of microdissected samples.The ability to accurately identify different cell groups is essential in most LCM studies. Optimal staining allows clear visualization of the histological structure and at the same time minimizes RNA degradation. For the latter, all staining protocols have to be very short to reduce the time the tissue is exposed to room temperature, aqueous solutions and chemical components. We evaluated four different staining methods – hematoxylin, H&E, 0.1% cresyl violet and 1% cresyl violet. Hematoxylin and H&E were tested because RNAse inhibitors can be added to the solutions which would potentially reduce RNA degradation . We founIn most studies, LCM is performed on very thin sections (5–12 μm) ,17. By iIt is important to note, however, that increased tissue thickness requires a lower numerical aperture and results in a broader, less precise laser beam. This effect is not noticeable when collecting relatively large areas as in our study but has to be considered when aiming to microdissect very small cell groups or single cells.Previous studies have reported that cryosections should be processed by LCM immediately after staining to avoid RNA degradation . This apIt took us 30–40 minutes to harvest four cell groups from the 4–6 sections on each slide. For tissue collection, one tube was placed on the laser microdissector at room temperature while the three tubes for the other three cell groups were kept on ice. In each LCM session, six to eight slides were processed, and samples were collected in the same four tubes. Under these circumstances, we found that RNA stabilization solution preserved RNA better and yielded slightly better RNA quality than when dissected tissue was collected directly in lysis buffer. In addition, the stabilization solution allowed us to visualize and to confirm the number of dissected strips after tissue harvesting and before RNA extraction Figure . We estiAlthough recently developed amplification methods make it possible to analyze degraded samples, we believe that part of the gene expression information will be lost, especially from rare transcripts . In addiCurrently, there is no reliable method for measuring low concentrations of RNA (< 10 ng/μl). In order to estimate the RNA yield, we microdissected a large number of strips and quantified the extracted RNA by spectrophotometry. We found an approximate yield of 1.3 ng of total RNA per cortical plate strip and 0.85 ng per subplate strip, which is consistent with the different cell densities of these two layers. Based on cell counts using cresyl-violet stained sections, we estimated an RNA recovery of 2.5 pg per cell. This is within the range of yields previously reported for brain cells collected by LCM ,21. It iWith the recent development of very efficient amplificiation methods, it has become possible to perform microarray analysis on very small quantities of total RNA, starting from as little as 500 picograms. In order to obtain more robust results and to avoid loss of rare transcripts, we believe it is desirable to use at least 10–20 ng of RNA as starting material, if time and material limitations allow for it. Based on our estimates of RNA recovery, this corresponds to 5000 to 10,000 brain cells, or 20–30 microdissected strips in our experiment.The optimized method described here allows a flexible workflow as not only tissue blocks but also sections and microdissected tissue can be stored at -80°C for at least several weeks. We believe that this is particularly important as many research groups only have access to shared LCM facilities which can be used sporadically. In addition, all the steps in this protocol except for the RNA extraction are performed with routine lab equipment and solutions, which makes this approach very cost-effective and easily attainable.We describe a protocol for UV-laser capture microdissection of multiple specific cell groups from the same tissue section. In particular, we have optimized four crucial procedures: 1) staining and dehydration of the tissue, 2) section thickness, 3) storage of sections, 4) harvesting and storage of microdissected cells with possible points of interruption. Using this protocol, we have consistently obtained RNA of very high quality (RINs > 8) from all simultaneously harvested cell groups and long PCR products were successfully amplified from the transcripts.All procedures were performed in an RNase-free environment. Working surfaces were treated with RNase decontamination solution  and rinsed with RNase-free water. Glassware was baked at 280°C for 4 h to inactivate RNase. Certified RNase-free plasticware was used in each process. All solutions were made from chemicals of molecular biology grade and RNase-free water. No additional RNase inhibitors were added into the individual solutions.Time-pregnant mice were killed by cervical dislocation in accordance with the UK Animals (Scientific Procedures) Act (1986). Whole heads of embryonic day 15 (E15) mice were flash-frozen directly in isopentane on dry-ice. Once hardened, the heads were embedded in O.C.T compound (Tissue-Tek) and stored at -80°C. The tissue was sectioned (10 μm or 20 μm) on a cryostat  and mounted on membrane-coated 1 mm PEN slides (Zeiss). Before use, the slides had been treated with RNase decontamination solution and UV irradiation at 320 nm for 30 min as recommended by the manufacturer. Once the first section had been mounted, the slide was kept inside the cryochamber (-18°C) while the next section was cut. Four to six sections were mounted in this way on each slide. Cutting and mounting was performed as quickly as possible to ensure that all sections adhered properly to the membrane. Two slides were prepared at a time and processed in parallel.Sections were allowed to air-dry for at least 2 min before fixation in 95% EtOH for 30 sec, both in the cryochamber.2O, stained with Mayer's hematoxylin (Sigma) for 15 sec, rinsed with H2O followed by 70% EtOH, stained with Accustain Eosin Y (Sigma) for 10 sec (for H&E only), dehydrated with 95% EtOH, 10 sec, 100% EtOH, 40 sec, and cleared with xylene, 60 sec.Hematoxylin and hematoxylin and eosin (H&E) staining were performed as previously reported for LCM samples . Briefly2O and 1% cresyl violet in H2O or 70% EtOH, filtered and stored at 4°C for up to one month.For cresyl violet staining, sections were rehydrated in 95% EtOH, 75% EtOH, 70% EtOH, stained in cresyl violet for 30–45 sec and dehydrated in 75% EtOH, 95% EtOH, 100% EtOH, 100% EtOH, 30 sec each. 0.1% cresyl violet was prepared in H100% EtOH was stored with desiccant beads  to prevent any rehydration.After dehydration, sections were air-dried and either processed immediately or stored in a box with desiccant at -80°C for up to several weeks.Frozen sections were postfixed in 4% paraformaldehyde in PBS for 20 min and quenched in 1.5% hydrogen peroxide for 30 min. Sections were then blocked for 2 h at RT with 5% donkey serum  in Tris-buffered saline (TBS) with 0.1% Triton-X100  and incubated with goat anti-Nurr1  1/200 in 1% donkey serum in TBS at 4°C overnight. Biotinylated donkey anti-goat (Abcam) 1/500 in 1% donkey serum in TBS was applied for 2 h at RT and reacted with avidin-biotinylated enzyme complex (ABC) using the Vectastain Elite kit  and diaminobenzidene (DAB) according to the manufacturer's instructions.LCM was performed using a PALM Microbeam IP 230V Z (Zeiss).Slides which had been stored at -80°C were transported and kept in a box with desiccant on dry ice. Slides were subsequently dried one at a time in a 50 ml Falcon tube with desiccant at room temperature. From each section, all four areas were cut and catapulted into separate caps (0.6 ml Eppendorf tube). Caps either contained 50 μl of RNA stabilization solution  or 50 μl lysis buffer . Tubes were kept on ice with the cap down until all areas were collected. The approximate time required to collect all four areas from one slide (4–6 sections) was 40 min. The tissue strips that were in RNA stabilization solution were directly stored at -80°C with the cap down. Strips in lysis buffer were vortexed for 45 sec, spun down and stored at -80°C.In order to harvest enough tissue for downstream microarray analysis, we collected the same cell groups from 3–4 brains. Sections from each brain were processed in a different LCM session and thus collected in a separate cap.Cell groups collected in lysis buffer were defrosted on ice and pooled with the same cell lysates from different brains. Tissues stored in RNA stabilization solution were defrosted on ice and stabilization solution was replaced with lysis buffer under a dissecting microscope using a syringe. After 5 min incubation, the samples were vortexed for 45 sec and spun down. Then the same cell lysates derived from different brains were pooled.Total RNA was extracted using the RNeasy micro kit (Qiagen) following the manufacturer's instruction. On-column DNase digestion step was performed. RNA quantities were determined using spectrophotometry . RNA integrity after LCM was analyzed both on RNA 6000 Pico Chips (Agilent Technologies) and by reverse-transcription PCR (RT-PCR).Nurr1 (subplate marker), Tbr1  and β-actin cDNAs were amplified using the following sets of forward (F) and reverse (R) primers:Approximately 20 ng of total RNA were used as a template for reverse transcription using Superscript III Reverse Transcriptase together with random hexamers  following the manufacturer's instructions. Long PCR fragments corresponding to the regions of the mouse Nurr1 (1244 bp):F 5'-CATGGACCTCACCAACACTG-3'; R 5'- CTGGGTTGGACCTGTATGCT-3'Tbr1 (1204 bp):F 5'- TCTCGACCACTGACAACCTG-3'; R 5'- GCGTAGTTGCTCACGAACTG-3'β-actin (677 bp):F 5'- AGCCATGTACGTAGCCATCC-3'; R 5'- ACATCTGCTGGAAGGTGGAC-3'PCR products were visualized on a 1.2% agarose gel. DNA molecular weight marker 1 kb  was used to determine product size.Images were taken on the PALM Microbeam IP 230V Z (Zeiss) or on a DMR transmission light microscope (Leica, Figure W-ZW and FO developed and optimized the described method, performed all the experiments and wrote the manuscript. SL analyzed the RNA qualities and quantities and provided useful suggestions. ZM conceived of the study and contributed to the planning of the experiments and to the writing of the manuscript. All authors read and approved the final manuscript.


Recent experimental and computational studies have provided overwhelming evidence for a plethora of diverse transcripts that are unrelated to protein-coding genes. One subclass consists of those RNAs that require distinctive secondary structure motifs to exert their biological function and hence exhibit distinctive patterns of sequence conservation characteristic for positive selection on RNA secondary structure.The deep-sequencing of 12 drosophilid species coordinated by the NHGRI provides an ideal data set of comparative computational approaches to determine those genomic loci that code for evolutionarily conserved RNA motifs. This class of loci includes the majority of the known small ncRNAs as well as structured RNA motifs in mRNAs. We report here on a genome-wide survey using RNAz.Drosophila genome show the hallmarks of stabilizing selection action of RNA structure, and hence are most likely functional at the RNA level. A subset of RNAz predictions overlapping with TRF1 and BRF binding sites , which are plausible candidates of Pol III transcripts, have been studied in more detail. Among these sequences we identify several "clusters" of ncRNA candidates with striking structural similarities.We obtain 16 000 high quality predictions among which we recover the majority of the known ncRNAs. Taking a pessimistically estimated false discovery rate of 40% into account, this implies that at least some ten thousand loci in the et al., Nat. Biotech. 23: 1383–1390 (2005)] shows that qualitatively similar fractions of structured RNAs are found in introns, UTRs, and intergenic regions. The intergenic RNA structures, however, are concentrated much more closely around known protein-coding loci, suggesting that flies have significantly smaller complement of independent structured ncRNAs compared to mammals.The statistical evaluation of the RNAz predictions in comparison with a similar analysis of vertebrate genomes [Washietl  High-throughput transcriptome data obtained in particular using tiling arrays -6 and cDEfficient computational methods ,18 have Computational screens for structured RNAs have been reported so far for mammalian ,23, urocDrosophila melanogaster: Isogai et al. . In . In DrosDR performed the RNAz screens and coordinated the analysis; SJP initiated this study. All authors closely collaborated in annotation, statistical analysis, and interpretation of the data, contributed to writing and approved the final manuscript.Machine readable annotation files and annotation tables for all RNAz predictions can be found at . SupplemD. melanogaster antennapedia complex. Figure 2: The D. melanogaster bithorax complex. Figure 3: Exemplary consensus secondary structures of two RNAz predictions. Figure 4: Comparison of obtained p-values. Figure 5: Complete WPGMA cluster tree of RNA candidates overlapping TRF and BRF binding regions. Table 1: Comparison of RNAz predicted ncRNAs using normal and randomized alignments. Table 2: Summary of RNAz predicted ncRNAs. Table 3: Number of predicted ncRNAs which overlap with Transfrags from [Supplemental figures and tables. Figure 1: The Click here for file