Francisella tularensis is a highly pathogenic Gram-negative bacterium that causes the disease tularaemia in humans and animals. Relatively little is known about the regulatory networks existing in this organism that allows it to survive in a wide array of environments and no sRNA regulators have been identified so far.Regulation of bacterial gene expression by small RNAs (sRNAs) have proved to be important for many biological processes. in silico prediction to identify sRNAs in F. tularensis strain LVS. Using a cDNA cloning and sequencing approach we have shown that F. tularensis expresses homologues of several sRNAs that are well-conserved among diverse bacteria. We have also discovered two abundant putative sRNAs that share no sequence similarity or conserved genomic context with any previously annotated regulatory transcripts. Deletion of either of these two loci led to significant changes in the expression of several mRNAs that likely include the cognate target(s) of these sRNAs. Deletion of these sRNAs did not, however, significantly alter F. tularensis growth under various stress conditions in vitro, its replication in murine cells, or its ability to induce disease in a mouse model of F. tularensis infection. We also conducted a genome-wide in silico search for intergenic loci that suggests F. tularensis encodes several other sRNAs in addition to the sRNAs found in our experimental screen.We have used a combination of experimental assays and F. tularensis encodes a significant number of non-coding regulatory RNAs, including members of well conserved families of structural and housekeeping RNAs and other poorly conserved transcripts that may have evolved more recently to help F. tularensis deal with the unique and diverse set of environments with which it must contend.Our findings suggest that  Trans-acting sRNAs regulate the translation and/or the stability of their target RNAs and each often regulate more than one target. For the most part, these sRNAs affect target genes in a negative fashion by binding to the region surrounding the start codon and ribosome binding site, but can act through base pairing in a region far upstream, and occasionally affect translation positively . Some rview see ). Many oStaphylococcus aureus [Salmonella typhimurium [Vibrio cholerae [Streptococcus [Pseudomonas aeruginosa [Clamydia trachomatis [Clostridium perfringens [RNA regulators furthermore control pathogenesis in bacteria such as s aureus -6, Salmohimurium -10, Vibrcholerae -13, groutococcus ,15, Pseuruginosa -19, Clamchomatis , and Clofringens ,22.Francisella tularensis is a Gram-negative bacterium that causes the disease tularaemia in humans and in a large number of animals and is one of the most virulent bacterial pathogens known. The ability of F. tularensis to invade and replicate in host cells, particularly in immune cells such as macrophages, is critical for its capacity to cause disease. Once inside host cells, F. tularensis resides transiently inside a phagosome that matures into a late endosomal stage and the bacterium then escapes to replicate in the cytoplasm. The mechanism by which the bacterium escapes the phagosome is not well understood, but genes encoded in a pathogenicity island (FPI) are required for this step in the infectious cycle. Several regulatory proteins regulate virulence gene expression in F. tularensis by activating transcription of the FPI. These comprise the proteins MglA [F. tularensis unlike many other bacteria encodes only one alternative sigma factor [ins MglA ,24, SspAins MglA , PrmA [2ins MglA , and Fevins MglA ,28. F. ta factor , no compa factor , and verF. tularensis [F. tularensis LVS. We have assessed and identified the sRNA species that are expressed at high levels and found the commonly known 4.5S RNA, 5S rRNA, and the transfer messenger RNA (tmRNA). Additionally, we identified two novel sRNA species by cDNA cloning and have characterized these. Studies of sRNA mutant strains suggested potential targets for regulation. As an alternative approach to identify sRNAs in F. tularensis, we performed a bioinformatic prediction of sRNAs. Interestingly, this analysis found both the experimentally determined sRNA loci and additionally identified a number of other putative sRNA-containing intergenic regions.We have recently studied the role of Hfq in the physiology and virulence of larensis . TranscrF. tularensis we first assessed the highly expressed sRNAs by polyacrylamide gel electrophoresis followed by ethidium bromide staining  or in stationary phase (S), after oxidative stress (10 mM H2O2) or after growth at high osmolarity (Schaedler broth + 2% NaCl). We chose to assess the RNA profile under different conditions because sRNA expression in other bacteria was found to be constitutive for some sRNAs (mostly cis-encoded sRNAs), but induced under specific conditions (such as exposure to stress) for most trans-encoded sRNAs [To identify sRNAs in g Figure . This ap. aureus . We isole stress 0 mM H2O2trans-translation, which is a reaction that transfers the translational complex to the tmRNA and ultimately leads to degradation of the polypeptide . This RNA is also designated SsrA and is 421 nt in size. tmRNAs are found in all bacteria and are highly expressed. The tmRNA has properties of both a tRNA and a mRNA. It participates in iew, see ).Based on the size of RNA #2, we presumed it was the 5S rRNA (114 nt). This was confirmed after cDNA sequencing. Next, RNA #1 was identified as the 4.5S RNA component of the signal recognition particle . FTL_R0044 was found in one of ten clones sequenced, whereas the other nine clones were the 5S rRNA. This is not very surprising considering the very similar size of these two transcripts and the high expression of the 5S RNA. Despite several attempts, we did not obtain any cDNA clones of RNA #3, the RNA with an apparent size of ~200 nt.As our analysis of highly expressed RNA did not result in the identification of novel small RNA transcripts, we proceeded to construct a limited cDNA library of low molecular weight RNAs. For this, we used the same approach as described for the highly expressed sRNAs. Total RNA from bacteria grown under the different conditions described above, but depleted of ribosomal RNAs (16S and 23S rRNA), was separated on a 8% denaturing polyacrylamide gel and RNAs of approximately 100-200 nt were eluted from the gel and processed the same way as for the highly expressed RNAs. The gel was divided into smaller areas before eluting the RNAs and ten clones from each area , showing that deleting either ftrA or ftrB does not affect the ability of F. tularensis to survive a number of stresses. Likewise, we did not observe any change in expression of either FtrA or FtrB after exposure to oxidative or osmolarity stress (data not shown).To study the role of the sRNAs, we proceeded by creating mutant strains carrying a chromosomal deletion of either the s Figure . NeitherF. tularensis intracellular multiplication. As shown in Figure in vitro.We next tested if either gene plays a role in F. tularensis, we assessed the ability of each mutant strain to induce disease in the mouse model of infection. No major difference in survival was observed between mice infected with wild-type LVS or with either of the mutant strains, showing that neither gene is required for virulence (data not shown). In addition, after infecting mice with a 1:1 mixture of mutant and wild-type bacteria we recovered equal numbers of wild-type and mutant bacteria from the spleen (data not shown). This further demonstrates that deleting ftrA or frtB does not affect F. tularensis pathogenicity in the mouse model.To examine if FtrA or FtrB is required for virulence of in vitro characterization of the mutant strains therefore do not rule out the possibility that FtrA or FtrB may indeed control functions related to growth, stress resistance or virulence.It is important to recall that most sRNAs affect gene expression negatively. Deleting a specific sRNA gene and thus alleviating the repressive effect may not be of major consequence for the bacterium. Our results from ftrA or the ftrB mutant. This way we identified several potential targets for FtrA and FtrB , FTL_0207 , FTL_0902 (encoding an oxidoreductase), and FTL_1922 (encoding a YggT family protein). Interestingly, pyrF transcripts were also found at higher levels in a hfq mutant [hfq or ftrA affects pyrF mRNA levels in the same manner, in agreement with a regulatory mechanism in which Hfq-mediated FtrA-pyrF mRNA base-pairing leads to degradation of the target mRNA. This, however, needs to be demonstrated experimentally. The mRNA levels of five genes were changed in the ftrB mutant compared to wild-type bacteria: two were found in higher levels (FTL_0836 and FTL_1754) and three at lower levels  in the ftrB mutant. Of these, FTL_0421 (encoding a lipoprotein) was also found to be down-regulated in the F. tularensis hfq mutant [Most characterized sRNAs control expression of their target genes by base-pairing with a target mRNA. In addition to affecting translation, base-pairing between a sRNA and its target frequently leads to changed stability of both sRNA and mRNA. Therefore, to experimentally identify potential targets for regulation by FtrA and FtrB, we compared the transcriptomes of LVS wild-type bacteria grown in regular broth to that of either the rB Table . None ofq mutant . Thus, dq mutant . This fiF. tularensis that may have been missed in our physical screen, we used the bioinformatic tool SIPHT [F. tularensis LVS. SIPHT identifies candidate intergenic loci based on the co-localization of intergenic conservation and Rho-independent terminators. It then annotates these candidates for several features, including the similarity of their primary sequence and predicted secondary structure to intergenic sequences in other bacterial replicons, their proximity to the putative binding sites of various transcription factors, and their homology to previously confirmed or predicted sRNAs and cis-regulatory RNA elements.While our cloning based screen proved effective in identifying previously unknown sRNAs, the relatively small number of clones sequenced meant that only abundant small transcripts could be found using this approach. In an effort to identify putative sRNAs in ol SIPHT  to conduStaphylococcus aureus [Pseudomonas aeruginosa. For fourteen of the loci identified, including FtrA and FtrB, conservation of secondary structure was predicted by QRNA [ftrA, one of which in a position consistent with the larger band seen by Northern analysis and the results of the 5'-RACE.A total of 24 candidate loci were identified by SIPHT, including three intergenic regions (IGRs) each containing two overlapping candidate loci, leading to a total of 21 IGRs  of the F. tularensis genome leads to spurious hits in BLAST searches that are more likely due to low sequence complexity than to actual sequence conservation. Second, as described above, it is also likely that existing terminator prediction programs, most of which had been trained on E. coli, are less effective in identifying terminators in AT-rich species such as Francisella. Indeed, in a kingdom-wide search for putative sRNAs in over 500 different strains, all Francisella sp. were among the 20 strains with the lowest density of putative intergenic Rho-independent terminators. However, while these factors likely contribute to both the decreased sensitivity and specificity of our sRNA predictions, the fact that both FtrA and FtrB were identified in our in silico screen suggests that the predictive algorithm used by SIPHT is effective in identifying novel sRNAs even in species such as Francisella that are AT-rich and have relatively few sequenced relatives.Several characteristics of F. tularensis contain ORFs encoding small peptides , previously non-annotated, have been identified in other bacterial species ,38. We tF. tularensis of the well-known non-coding RNAs tmRNA and 4.5S RNA after direct purification and cloning of the cDNAs. Two additional sRNAs expressed by F. tularensis were identified from a cDNA library prepared from low molecular weight RNA. The two putative sRNAs , which are encoded in intergenic regions, were confirmed to be genuine sRNAs by Northern blot analysis and the transcripts were characterized with respect to transcription endpoints. The sRNAs do not show sequence similarity to any known sRNAs and their DNA sequences are only found in Francisella species, indicating that these are novel sRNAs. Deletion of either the ftrA or ftrB gene had no effect on bacterial survival during normal growth or stress, and did not affect the capacity of Francisella to induce disease in mice. This suggests that these sRNAs affect functions that are not required under the conditions tested or that additional sRNAs with redundant functions may exist. Specifically, in silico analysis identified conserved sequence between FtrB and sRNA candidate #20 , on chocolate agar plates supplemented with IsoVitalex vitamin solution , or in defined medium [Escherichia coli was grown in LB. When needed, media was supplemented with kanamycin (50 μg ml-1 for E. coli and 10 μg ml-1 for F. tularensis). Oligonucleotides used are described in Table All strains and plasmids used in this study are listed in Table d medium . EscheriCandidate sRNAs were predicted using the SIPHT program, as described previously . Thresho600 = 0.3 were exposed to 10 mM H2O2 for 30 min before harvested. For experiments at high osmolarity, LVS was grown in complex medium containing 2% NaCl to OD600 = 0.3 before being harvested. Total RNA was extracted using Trizol reagent (Invitrogen) according to the protocol provided by the manufacturer and treated with Turbo DNase (Ambion). For cDNA library construction, total RNA was depleted of 16S and 23S rRNAs using the MICROBExpress kit (Ambion) according to the protocol provided by the manufacturer before loaded on gel.LVS was grown to mid-exponential phase or to stationary phase in Schaedler-K3 before harvesting the bacteria. For oxidative stress experiments, bacteria at OD20 μg of total RNA was heated and loaded onto an 8% polyacrylamide/8M urea gel with the RNA Century Plus (Ambion) as size marker. After separation, the gel was stained with ethidium bromide and a gel piece containing RNA of a certain size was cut out. RNA was eluted from the gel after crushing and incubation at 37°C in 0.5 M ammonium acetate and 1 mM EDTA (pH 8.0) for 6 hours under constant agitation. Supernatants were extracted once with chloroform before precipitation of the RNA with isopropanol in the presence of glycogen. Next, 3' adapter was ligated to the dissolved RNA with T4 RNA ligase in the presence of DMSO and RNase inhibitor for 90 min at 37°C. The RNA was purified from a 15% polyacrylamide/urea gel as described above to remove non-incorporated adapters. The eluted RNA was treated with tobacco acid phosphatase (TAP) before ligation of 5' adapter (using the same method as for 3' adapters) and subsequently non-incorporated adapters removed by passing and twice washing the RNA on a Microcon YM-30 (Millipore) column. The RNA was reverse transcribed using the 3'PRIMER and SuperScript II reverse transcriptase (Invitrogen), PCR amplified using 5'PRIMER and 3'PRIMER and ligated into pCR2.1-TOPO. All RNAs were identified using this protocol, except the tmRNA for which we used a protocol that ligates the 3'adapter before the 5'adapter and utilizes different adapters  and quantified on a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). After extraction, 10 μg of total RNA were loaded on a polyacrylamide gel (8% acrylamide/8M urea). After migration, the RNA was transferred to a Hybond-N+ membrane (Amersham) and crosslinked with UV light. The membrane was prehybridized in Rapid-Hyb Buffer (Amersham). Then, For 5'-RACE, 15 μg of total RNA was incubated with TAP for 2 hours at 37°C after which 5' adapter was ligated with RNA ligase. RNA was purified from gel and reverse transcribed with a gene specific primer and SuperScript II RT. cDNA was then used as a template in PCR reaction with the 5'PRIMER and the gene specific primer (GSP_5'RACE) and cloned into pCR2.1-TOPO before sequencing.3'-RACE was performed by ligating the 3' adapter to total RNA, followed by gel purification of adapter ligated RNA, reverse transcription with 3'PRIMER and finally conducting PCR with 3'PRIMER and GSP_3'RACE. The 3' end of each sRNA was determined by sequencing of cloned PCR products in pCR2.1-TOPO.ftrA and ftrB were amplified by PCR using primer pairs DelR/DelB and DelO/DelT. The upstream and downstream fragments were purified from gel, annealed and extended in 20 cycles of PCR without primers and the product further used as template in a PCR reaction with primers DelR and DelT. The ~2 kb PCR products were cloned into pCR2.1-TOPO (Invitrogen) and mobilized as a NotI-SacI fragment into the sacB based suicide vector pMP812 [ftrA and pMP-ΔftrB. pMP-ΔftrA and pMP-ΔftrB were introduced in LVS by electroporation and integration of the plasmid into the chromosome confirmed by PCR. Strains were then passed once in medium without selection, subsequently streaked on solid medium containing 6% sucrose and isolated colonies were tested for loss of the gene by PCR (using primer pairs DelCheck1/DelCheck4 and DelCheck2/DelCheck3). Deletion of the gene was confirmed by sequencing.Regions of approximately 1 kb upstream and downstream of r pMP812  creatingF. tularensis microarrays  contain 70-mer oligonucleotides, in five copies, representing all genes of strains SchuS4 and LVS. Microarrays were scanned with a Genepix 4000B scanner (Molecular Devices). To quantify signal fluorescence intensities, TIFF images were analyzed using the Genepix Pro 6.0 software. Statistical analyses were performed using publicly available software, the R/Bioconductor package LIMMA (available from http://www.bioconductor.org). A list of statistically significant differentially expressed genes was obtained using lowess normalization (after inspection of MA plots) and applying the empirical Bayes moderated t-test. Microarray data are available at ArrayExpress.RNA used in microarray experiments was extracted using Trizol reagent combined with purification of the aqueous phase on RNeasy columns (Qiagen). cDNA labeling and microarray hybridizations were performed as described . Three iTo validate the microarray results, two ORFs were selected (for each mutant strain) for real-time quantitative reverse transcription PCR (qRT-PCR) analysis with the same RNA samples used in the microarray hybridizations. For the analyses, one microgram of RNA was reverse transcribed using random hexamers and SuperScript II reverse transcriptase (Invitrogen) according to the protocol provided by the manufacturer. Real-time PCR was performed with gene-specific primers using an ABI PRISM 7700 and FastStart SYBR master mix (Roche Diagnostics). To calculate the amount of gene-specific transcript, a standard curve was plotted for each primer set using a series of diluted genomic DNA from LVS. To compare the transcript amounts in the different strains, the amounts of each gene transcript was normalized to DNA helicase (FTL_1656) as this gene has been shown to change little in expression during growth . The exp5 cells per well in 12-well tissue plates  and monolayers were used 24 hours after seeding. J774 macrophage monolayers were incubated for 90 min at 37°C with the bacterial suspensions (approximate multiplicities of infection 100) to allow the bacteria to enter. After washing , the cells were incubated in fresh culture medium containing gentamicin (10 μg ml-1) to kill extracellular bacteria. At several time-points, cells were washed three times in PBS and processed for counting of surviving intracellular bacteria. For this, bacteria were recovered by lysis of macrophages with distilled water and the titer of viable bacteria released from the cells was determined by spreading preparations on agar plates. For each strain and time in an experiment, the assay was performed in triplicate. Each experiment was independently repeated two times and the data presented are from one experiment.J774 cells were propagated in RPMI or Dulbecco's Modified Eagle's Medium (DMEM) medium containing 10% fetal calf serum. Cells were seeded at a concentration of ~2 × 101 to 104 bacteria) and the mortality was followed for 9 days. The actual number of viable bacteria in the inoculum was determined by plating dilutions of the bacterial suspension on chocolate plates. For competitive infections, LVS and mutant bacteria were mixed in 1:1 ratio and a total of approximately 400 bacteria were used for infection of five mice. After four days, mice were sacrificed. Homogenized spleen tissue from the five mice in one experiment were mixed, diluted and spread onto chocolate agar plates. PCR to distinguish wild-type and mutant bacteria were performed on 100 colonies. Animal experiments were performed according to the INSERM guidelines for laboratory animals' husbandry.LVS and mutant strains were grown in Schaedler-K3 to exponential growth phase and diluted to the appropriate concentrations. 6 to 8-week-old female BALB/c mice  were i.p. inoculated with 200 μl of bacterial suspension. Groups of five mice were inoculated with various doses of bacteria  and the fold difference (in mutant strain relative to wild-type strain) and standard deviations are shown for the FTL_0045 and FTL_1922 genes (ftrA mutant) and FTL_0421 and FTL_0836 genes (ftrB mutant).Click here for filein silicoPutative sRNAs predicted . Table contains data provided in Table Click here for file


High-throughput RNA sequencing (RNA-seq) is considered a powerful tool for novel gene discovery and fine-tuned transcriptional profiling. The digital nature of RNA-seq is also believed to simplify meta-analysis and to reduce background noise associated with hybridization-based approaches. The development of multiplex sequencing enables efficient and economic parallel analysis of gene expression. In addition, RNA-seq is of particular value when low RNA expression or modest changes between samples are monitored. However, recent data uncovered severe bias in the sequencing of small non-protein coding RNA , such that the expression levels of some RNAs appeared to be artificially enhanced and others diminished or even undetectable. The use of different adapters and barcodes during ligation as well as complex RNA structures and modifications drastically influence cDNA synthesis efficacies and exemplify sources of bias in deep sequencing. In addition, variable specific RNA G/C-content is associated with unequal polymerase chain reaction amplification efficiencies. Given the central importance of RNA-seq to molecular biology and personalized medicine, we review recent findings that challenge small non-protein coding RNA-seq data and suggest approaches and precautions to overcome or minimize bias. Based on the detection of open reading frames (ORFs), entire transcriptomes are differentiated into two main RNA classes. The first comprises all protein coding messenger RNAs (mRNAs), and various subclasses of non-protein coding RNAs constitute the second ,2. AlthoThe high capacity and comparably low cost of modern deep-sequencing analysis suggest that high-throughput, small non-protein coding RNA sequencing  might be a superior tool when applied to personalized medicine. The commonly used microarray-based analysis has to compensate for differences in hybridization efficacies in order to ensure true representation of relative expression levels. Hybridization-based assays must also be sensitive enough to permit isoform-specific miRNA detection. Such requirements are sometimes hard to attain since individual miRNAs often display very different melting temperatures . FurtherHere, we review recent data indicating various sources of bias in sRNA-seq inherent to the common multiple experimental steps involved in library preparation. Experimental strategies and methods to overcome or minimize such distortion in expression levels are suggested wherever possible. Application of sRNA-seq-related methods in mRNA deep sequencing and personalized medicine are also discussed.Supplementary Data and Supplementary Table S1). The template-independent addition of oligo(C) or (A) stretches is catalyzed by Escherichia coli poly(A) polymerase. Alternative strategies of RNA end-modification rely on RNA ligation to introduce adapter oligonucleotides. RNA ligation is also applied to modify RNA 5′-ends.The analysis of full-length non-protein coding RNAs in sequencing projects requires RNA end-modification or equivalent strategies to ensure identification of native RNA termini as a precondition for cDNA construction . IrrespeE. coli poly(A) polymerase is a well-established approach  polymerase accepts only ATP as a substrate; in vitro it is reported to utilize, albeit with unequal affinity, all NTPs as substrates for transfer - and oligo(C)-tailing reactions  or plant miRNAs, is achieved by periodate treatment. The reaction relies on vicinal hydroxyl groups to oxidize sugar moieties and is accompanied by pentose ring opening. The resulting vicinal dialdehyde reacts with water and forms a chemical equilibrium with the corresponding dioxane derivative upon ring closure. The 2′-O-methyl group-containing RNAs are insensitive to oxidation and remain accessible for adapter ligation or tailing . Irrespective of the actual reason, the advice for minimizing this bias is identical and relies on the application of adapter pools to increase the likelihood of productive interactions .The chemical properties of RNA 5′-ends display structural variations. Depending on the starting material and experimental design, it may be necessary to alter the chemistry of RNA 5′-termini prior to cDNA generation. For example, neither 5′-triphosphorylated RNAs in prokaryotes nor primary eukaryotic RNA polymerase III transcripts are accessible for direct 5-adapter ligation. Similarly, RNA polymerase II-specific trimethylguanosine cap structures render RNA 5′-ends inaccessible. Thus, enzymatic pretreatment is usually conducted to avoid bias in the starting material (Supplementary Data and Supplementary Table S1). RNAs that contain monophosphorylated 5′-ends and 3′-hydroxyl groups are directly accessible to cDNA generation procedures. Consequently, unaltered RNA starting material leads to its specific enrichment (Supplementary Table S1). In contrast, triphosphorylated or 5′-capped RNAs require TAP treatment to generate 5′-monophosphorylated RNAs and to permit 5′-end ligation.Although the various chemical properties of RNA 5′-ends establish limitations for unbiased representation, they also permit specific enrichments  . Therefore, different chemistries of RNA 5′-ends not only distort sRNA-seq, but also allow for specific enrichment protocols. Subsequent analysis permits the distinction among RNA subsets, such as primary transcripts, capped RNAs and other products of RNA 5′-end processing  molecules ,74. ReceTo investigate potential influences on 5′-end modification derived from RNA secondary structure, ligation yields were compared among test sets of fully randomized RNAs. A remarkable underrepresentation of double-strand folds at 5′-ligation sites indicated strong preferences for single-stranded RNA substrates in ligation reactions conducted with T4Rnl1 .DNA-based adapter oligonucleotides perform well in RNA 3′-end ligation reactions , whereas RNA-based adapters are preferred for RNA 5′-end modification . Kineticin vitro transcribed non-protein coding RNAs and MID-modified adapters, ligation product levels  ranged from 60–70% to undetectable  experimental goal and (ii) lane capacity. According to Illumina® TruSeq Small RNA Sample Prep Kit guidance, 5 million sRNA-seq reads are considered appropriate for novel miRNA discovery within human-sized genomes. For miRNA profiling usually about 2.5 million reads are recommended. These approximations permit recalculation geared toward specific needs and requirements.et al. . Therefore, techniques to reduce the amount of reads that represent empty adapter dimers are of central importance, as they permit an increase in sequencing depth, reducing costs and ultimately enabling novel small non-protein coding RNA gene discovery.Apart from novel third-generation sequencing technologies that permit direct sequence analysis of unamplified cDNA, all commonly used protocols implement PCR amplification . In addiRecent analysis also emphasized the impact of PCR buffer compositions on PCR amplification bias . Remarkahttp://biz.yahoo.com/e/121115/hlcs8-k.html)) system, avoid PCR amplification steps in cDNA preparation and permit direct sequencing of unamplified samples  . In partThe miRNAs lack structural features such as mRNA poly(A) tails that could serve as class-specific enrichment or priming of reverse transcription . AlternaThus far, two alternative approaches are commonly utilized to reverse transcribe miRNA templates ,106. miRBecause the relative RNA G/C-content dictates primer Tm, qRT-PCRs are also strongly influenced by forward primer design, thereby affecting both qRT-PCR specificity and sensitivity. Altering the length of primer-template complementarity permits adjustment of the actual primer Tm. Conversely, in cases where RNA nucleotide composition would dictate very low melting temperatures, LNA-modified nucleotides might be incorporated into the primer sequence to increase the corresponding primer Tm . HoweverDeep sequencing is not limited to expression profiling of miRNA or other small non-protein coding RNAs. Even entire transcriptomes, including all protein coding and long non-protein coding RNAs, are subjected to high-throughput sequencing methods. Currently, as the length of single sequence runs is limited , longer RNAs require fragmentation prior to cDNA synthesis . MillionBias in general describes systematic errors that reflect method-related distortion from the truth. Bias might be detected easily when data generated by different methods are subjected to analytical comparison. Because it is systematic in nature, bias is not subject to variation in repeated experiments. Here, based on biochemical and deep sequencing data analyses, we have presented various steps in sRNA-seq protocols likely to cause severe distortions in the relative expression levels of individual sRNAs.The efficacy of RNA end-modification to permit cDNA construction depends not only on differences in strategies and reagents but also on changes in, among others, buffer compositions and additives that could increase molecular crowding. Therefore, meta-analysis of data generated with non-identical methods, reagents or even buffers is likely to be error-prone ,39,63,78In addition to the more obvious quantitative aspects of bias, there are also qualitative aspects because RNA-seq methods are also employed for RNA discovery. Individual RNAs might ‘drop out’ from detection because they are not amenable to cDNA construction under chosen conditions. Certainly, to increase the likelihood of productive RNA end-modification and increase the complexity of sRNA-seq library content, the parallel application of different strategies and reagents is required ,39,63,79The source of expression level distortion and the failure to detect certain RNA species at all, appear not to be limited to reactions of RNA/cDNA-end modification, but also apply to cDNA amplification by PCR . DiffereThe sRNA-seq approach is also attractive to medical research. Deep sequencing might permit the screening of multiple patient samples to monitor the progression of disease or treatment. Therefore, it is urgently necessary to establish rigid international standardizations to minimize distortion and to avoid misleading conclusions during RNA-seq data interpretation. In summary, qualitative and quantitative RNomics remain more challenging than anticipated.Supplementary Data are available at NAR Online.National Genome Research Network [NGFNIII 01GS0808 to J.B. and T.S.R.]; the Deutsche Forschungsgemeinschaft [BR 754/4-1 to J.B.]; USM Research University Grant [1001/CIPPT/81305 to T.H.T.]. Funding for open access charge: The licence was funded by Dr. Brosius’s research budget.Conflict of interest statement. None declared.


Crataegus spp.) extract WS 1442, a herbal therapeutic with unknown mechanism of action, on the mechanics of the ESL of ex vivo murine aortae. Furthermore, we measured the impact of WS 1442 on the sodium permeability of endothelial EA.hy 926 cell monolayer. The data show that (i) the ESL contributes by about 11% to the total endothelial barrier resistance for sodium and (ii) WS 1442 strengthens the ESL resistance for sodium up to about 45%. This mechanism may explain some of the vasoprotective actions of this herbal therapeutic.The endothelial glycocalyx (eGC) plays a pivotal role in the physiology of the vasculature. By binding plasma proteins, the eGC forms the endothelial surface layer (ESL) which acts as an interface between bloodstream and endothelial cell surface. The functions of the eGC include mechanosensing of blood flow induced shear stress and thus flow dependent vasodilation. There are indications that levels of plasma sodium concentrations in the upper range of normal and beyond impair flow dependent regulation of blood pressure and may therefore increase the risk for hypertension. Substances, therefore, that prevent sodium induced endothelial dysfunction may be attractive for the treatment of cardiovascular disease. By means of combined atomic force - epifluorescence microscopy we studied the impact of the hawthorn ( The daily sodium intake in industrial countries greatly exceeds physiological needs. There is a strong correlation between the daily sodium intake and the prevalence of arterial hypertension, one of the main risk factors of cardiovascular disease and mortality worldwide +-influx by counter-ion migration along the polysaccharide. This process causes a vasodilatory response Fluid shear stress induces a conformational change of heparan sulfate proteoglycans of the eGC from a random coiled state to an unfolded state. The unfolded structure reveals sodium binding sites and promotes transmembrane NaHawthorn extracts have been used in Europe for many decades to treat cardiovascular diseases. It has been shown that hawthorn extract WS 1442 induces an endothelium dependent, NO and EDRF mediated vasorelaxation Na+P) of the endothelial cell monolayer under control conditions and under conditions where the eGC was enzymatically digested. Furthermore, we measured the Na+P of WS 1442 treated cells to analyze the link between the mechanical state of the ESL and Na+P.To test, whether WS 1442 modulates the functional properties of the ESL, we established a method using combined atomic force microscopy (AFM) - epifluorescence microscopy. This approach makes it possible to observe in real time eGC height and stiffness 2. For GM 7373, minimal essential medium  supplemented with 20% fetal calf serum , 1% MEM vitamins (Invitrogen), 1% MEM nonessential amino acids (Invitrogen) and 1% Pen-Strep (Invitrogen) was used. EA.hy 926 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 1% Pen-Strep solution and 10% FCS.Human endothelial EA.hy 926 cells  and bovine aortic endothelial GM 7373 cells  were cultured at 37°C and 5% COAll animal procedures were performed in accordance with the guidelines of the Charité, Universitätsmedizin Berlin, and were specifically approved by the Landesamt für Gesundheit und Soziales  for the use of laboratory animals (G0355/09) and followed the “Principles of Laboratory Animal Care” , as well as the current version of German Law on the Protection of Animals.2) of the whole aorta was removed and attached on Cell-Tak® coated glass, with the endothelial surface facing upwards. The artery preparation was bathed in PBS (PAA Clone). After preparation, the segments were stored in minimal essential medium supplemented with 20% FCS, 1% MEM vitamins, 1% MEM nonessential amino acids and 1% Pen-Strep.Mouse aortae were isolated and freed from surrounding tissue. A small patch ; pH 7.4), supplemented with 1% FCS, was added.As primary antibody, mouse anti-heparane sulfate (10E4 epitope) antibody  and as secondary antibody, Qdot 655 goat anti-mouse antibody conjugate (Invitrogen), were used. GM 7373 cells were seeded on Ø35 mm cell culture glass bottom dishes  for 72 h until a confluent cell monolayer was formed. The primary antibody (1∶500) was added to the living cells directly into the cell culture medium. After 30 min incubation at 37°C the cells were washed 3×5 min with PBS (37°C). Next, the secondary Qdot antibody conjugate was added (1∶800). The cells were again incubated for 30 min and washed 3×5 min in PBS at 37°C. PBS was removed and HEPES buffered solution  combined with a Leica DMI 6000 B inverted fluorescence microscope , the nanomechanical properties of the ESL were monitored in dependence of the amount of heparan sulfates. Simultaneous indentation measurements and immunofluorescence microscopy  were perK, the sample stiffness. K is the mechanical resistance of a sample against deformation along a defined degree of freedom:F is the applied force and x is the displacement caused by the applied force to the sample. In this work, K is measured as the slope of the obtained force-distance curve  was used with the same parameters as for the Catalyst AFM setup. ESL stiffness and ESL height of murine ex vivo aortae were obtained before and after application of 10 µg/ml WS 1442 for 30 min.Force-distance measurements on murine 2, the basolateral sodium concentration was determined. The sodium concentration of the medium was obtained by means of a Radiometer EML 105 electrolyte analyser . The sodium permeability was calculated as follows:BLC is the change of the basolateral Na+ concentration and AC is the apical sodium concentration (considered to remain constant through the experiment). BLV is the basolateral fluid volume, S is the surface area of the filter membrane and Δt the time of incubation. To test whether Na+P depends on the ESL, we applied heparinase I  directly into the medium for 30 min prior to the experiment. Again, the medium was replaced with high sodium medium and Na+P was obtained as described above.EA.hy 926 cells were grown on filter membranes  in 12-well plates  for 4 days. The control group was grown in standard cell culture medium, the WS 1442 group was grown in standard cell culture medium containing 10 µg/ml WS 1442. Prior to the experiment, medium with a sodium concentration of 155 mM  and 130 mM  was applied. Osmolarity was maintained constant by the addition of appropriate amounts of mannitol. After 2 hours of incubation at 37°C and 5% CO2R = 0.75, p<0.01; The extracellular matrix of human cervical epithelial cells can be characterised by using AFM methods ex vivo arteries were used, in order to emulate physiological conditions as closely as possible. That it is feasible to use ex vivo arteries for AFM measurements has been shown previously p<0.01, Mann-Whitney U-Test). Due to the lack of transparency of the aortic preparations, the simultaneous AFM and fluorescence microscopy could not be applied. To test, whether the changes of the mechanical properties of the ESL were due to conformational (e.g. collapse) or structural (e.g. loss of ESL constituents) alterations, we compared the amount of heparan sulfates between WS 1442-treated cells and untreated cells. Interestingly, the amount of heparan sulfates was not altered after 30 min of WS 1442 - treatment ) are as follows: Na+P = 11.7*10−6  cm/s for the control group and Na+P = 9.8*10−6  cm/s for the WS 1442-treated group. After heparinase treatment, the Na+P of the control group rises by 12% to 13.1*10−6  cm/s while the Na+P of the WS 1442-treated group rises by 24% to 12.2*10−6  cm/s . WS 1442-treated endothelial cells show significantly lower Na+P (p<0.017) in contrast to the cells of the control group. This WS 1442 induced reduction of the Na+P is most likely caused by modifications of the ESL since both the control group and the WS 1442-treated group show no difference in Na+P after heparinase treatment.In a permeability assay we studied the 12) cm/s . StatistThe results show that WS 1442 induces a change of the mechanical properties of the ESL within a few minutes. The ESL becomes 30% softer and 22% thicker. As WS 1442 does not influence the amount of heparan sulfate molecules at the surface of endothelial cells, it is assumed that the nature of the eGC-modulation is a result of conformational alterations. From our results it appears likely that the packing density of glycosaminoglycans in the ESL is a regulated process. Loss of ESL constituents like heparan sulfate leads to structural changes of the ESL and thus to decreased height and stiffness. In contrast, as observed after WS 1442 treatment, the amount of ESL constituents can remain also constant while height and stiffness change. A similar behaviour was observed by Sui et al. ex vivo arteries physiological conditions are mimicked as closely as possible in vivo situation may be different because of the absence of factors such as fluid shear stress or hormones ex vivo. Application of transmission electron microscopy shows that the ESL-height could be up to a few µm With the use of Na+P of control cells to those after heparinase digestion of the eGC does reveal that the ESL significantly contributes to the Na+P of the endothelium. In addition, the permeability depends on the physiological state of the ESL. This conclusion is derived from the observation that WS 1442 treated cells show a significantly lower Na+P in comparison to untreated cells. This difference completely disappeared after heparinase treatment, so it is assumed that the changes of Na+P are a result of modulation of the ESL. This conclusion is strengthened by the assumption that the total resistance Na+R - the reciprocal value of permeability (Na+ = 1/PNa+) -R of the endothelial cell monolayer is determined by the combined resistance of the ESL, of the endothelial cell layer itself and of the filter membrane of the inserts:As the results revealed that WS 1442 modulates the mechanical properties of the ESL, there was a need to test whether these modulations cause an alteration of the ESL-dependent endothelial barrier function. Comparison of the Na+(filter)P is very small and thus negligible for further calculations. If it is assumed that after the ESL digestion Na+P depends only on the permeability of the endothelial cell itself, Na+(ESL)P can be calculated as follows:Na+P, Na+(cell layer)P and the calculated Na+(ESL)P. In vivo, a lower endothelial Na+P would result in a lower rate of sodium transfer from the blood across the endothelium to the interstitium. This mechanism may help retain sodium in the circulation and thus accelerate renal sodium excretion.Because the sodium permeability of the filter membrane is extremely high, the term 1/Na+P of the ESL decreases as shown in the present study. This may explain mechanistically how hawthorn extracts exert their protective effects on the cardiovascular system.A bath sodium concentration in the high physiological range impairs endothelial cell function e.g. NO-synthesis Vascular effects of WS 1442 have been related to its high content of oligomeric procyanidins (OPCs) (16). Red wine and purple grape juice polyphenols also exert beneficial effects on the cardiovascular system by enhancing NO-release Na+(ESL)P  . This ma


Genomic concepts are based on the assumption that phenotypes arise from the expression of genetic variants. However, the presence of non-Mendelian inheritance patterns provides a direct challenge to this view and suggests an important role for alternative mechanisms of gene regulation and inheritance. Over the past few years, a highly complex and diverse network of noncoding RNAs has been discovered. Research in animal models has shown that RNAs can be inherited and that RNA methyltransferases can be important for the transmission and expression of modified phenotypes in the next generation. We discuss possible mechanisms of RNA-mediated inheritance and the role of these mechanisms for human health and disease. Our basic understanding of transgenerational inheritance relies on observations made by our ancestors generations ago. Gregor Mendel's 1860s pioneering studies of pea plant crosses and breeding represent the foundation of what is known about inheritance mechanisms to this day. By crossing pea plants with pure-breeding, i.e. homozygous, characteristics and later also heterozygotes in different combinations, Mendel was able to propose two basic principles, or laws, which apply to sexually reproducing, diploid organisms: The first is the law of segregation, which states that the two alleles of a given trait from each parent segregate into two gametes, which are then passed down to the offspring. This law was confirmed in later studies on meiosis and ensures genomic stability by maintaining ploidy while allowing genetic diversity through new and random combinations of alleles of the same gene. Mendel further observed that different alleles of a trait (such as pea colors) do not blend, but either dominate or recess, allowing only one defined trait to be expressed at a time. Mendel's second law, the law of independent assortment, states that separate traits  are inherited independently from one another. This principle allows a vast variety of combinations of traits and thus ensures genetic diversity among the offspring.As a result of decades of research in the framework of Mendelian genetics, we now know that DNA is the main carrier of genetic information from one generation to the next. Methods for genetic analysis have evolved considerably and now allow the deciphering of entire genomes. DNA microarrays and next-generation sequencing have made it possible to identify millions of genetic variants, such as single nucleotide polymorphisms and copy number variants, in thousands of individuals. Currently, large-scale genome-wide association studies (GWAS) are used to unravel highly complex genotype–phenotype relationships and represent a sophisticated conceptual development that is based on Mendelian genetics Genetic information is organized in higher order structures, which consist of DNA, proteins, and RNA Transgenerational epigenetic inheritance has been described in various systems and detailed reviews on this topic have been published recently Kit paramutation” which describes a stable modification of Kit gene expression. Kit encodes a tyrosine kinase receptor with roles in developmental processes including hematopoiesis, germ cell differentiation, and melanogenesis. A homozygous deletion of Kit is lethal, whereas heterozygotes carrying one allele inactivated by a LacZ insertion (tm1Alf/+Kit) show a white-tail phenotype +/+Kit progeny and in subsequent crosses with wild type partners  represents the most prominent modification of mammalian mRNAs. The availability of m6A-specific antibodies provided an excellent opportunity to identify methylated RNAs by immunoprecipitation and sequencing of mRNA fragments. Indeed, two recent studies have identified adenine methylation marks in several thousand mRNAs, with a distinct enrichment in the vicinity of stop codons and in internal exons Cytosine-5 methylation (m5C) represents another prominent modification of RNA, which can be detected at single-base resolution by bisulfite sequencing Additionally, the regulatory role of RNA methylation in gene expression has also been investigated through the functional characterization of the DNMT2 RNA methyltransferase. DNMT2 is closely related to the established DNA methyltransferases and key epigenetic regulatory enzymes DNMT1 and DNMT3. However, DNMT2 does not methylate DNA Dnmt2 gene Kit RNA fragments into wild-type fertilized eggs induced up-regulation and possibly methylation of the Kit transcript, these processes could not be observed in Dnmt2-deficient mice. Importantly, corresponding Kit genomic DNA sequences remained unmethylated in wild-type as well as in Dnmt2-deficient mice Finally, it has been shown that the inheritance of paramutant mouse phenotypes requires an intact Interestingly, several recent findings suggest that the mechanisms of RNA-mediated inheritance might be relevant for human health and disease . For exaFurthermore, RNA-mediated inheritance could also provide an explanation for the missing heritability problem of complex human diseases. The “missing heritability” phenomenon was defined after even extensive GWAS failed to identify major risk factors for complex diseases


Whereas many cases of neurodegenerative disease feature the abnormal accumulation of protein, an abundance of recent literature highlights loss of RNA homeostasis as a ubiquitous and central feature of pathological states. In some diseases expanded repeats have been identified in non-coding regions of disease-associated transcripts, calling into question the relevance of protein in the disease mechanism. We review the literature in support of a hypothesis that intrinsically disordered proteins  are particularly sensitive to an age-related decline in maintenance of protein homeostasis. The potential consequences for structurally disordered RNA-binding proteins are explored, including their aggregation into complexes that could be transmitted through a prion-like mechanism. We propose that the spread of ribonucleoprotein complexes through the nervous system could propagate a neuronal error catastrophe at the level of RNA metabolism. Being comprised largely of postmitotic neurons that must persist for a lifetime, the central nervous system is at risk for damage that would be less dire in tissues wherein stem cells can be effectively mobilized. The accumulation of damage has important implications for neurodegenerative disease pathogenesis and may be responsible for features of the aged brain below the level of overt pathology. A hallmark of aged and diseased neurons is the aggregation of damaged or misfolded proteins, and it has long been thought that loss of protein homeostasis plays an important role in neurodegenerative disease . Recent Over the preceding several years RNA dysregulation has taken center stage in the pathophysiology of neurodegenerative diseases, most notably amyotrophic lateral sclerosis ALS; . With thDMPK gene , fragile X-associated tremor ataxia syndrome (FXTAS), spinocerebellar ataxia types 3, 8, 10, and 12, Huntington’s disease like 2  to RNA-mediated toxicity remain to be defined. Indeed, the majority of RNA-mediated diseases affect predominantly or even exclusively the CNS in an age-dependent manner. For example, FXTAS is manifest clinically as late adult onset ataxia and cognitive decline . It is cR-1 gene . PatholoR-1 gene . The mutR-1 gene . InteresR-1 gene . The conR-1 gene .in vitro evidence from structural analytic methodologies such as circular dichroism, solution state NMR, and small angle X-ray scattering , though it is recognized that the metaphor is inadequate in terms of the flexibility of proteins and their propensity to undergo structural changes in response to substrate binding and/or post-translational modifications. This textbook notion of the structure/function relationship is challenged by the growing catalog of proteins that are intrinsically disordered, in whole or in part. If the definition of an intrinsically disordered protein (IDP) restricts inclusion to those proteins containing disordered segments of 30 or more residues, roughly a third of eukaryotic proteins would qualify . Importaattering . A recurWhereas some IDPs appear to execute their function in the unstructured state . Hubs increase the robustness of networks; random node failure is less deleterious to highly connected networks containing hubs, a property exploited in communications networks such as the internet. Hubs also serve to shorten the distance between any two nodes, increasing the efficiency of network operations . The protein is predicted to be almost entirely disordered by the available algorithms , with the exception of a small central domain. The central segment of FUS is thought to mediate binding to nucleic acids ; one of the known functions of FUS is as a DNA binding transcription factor . FUS is a conserved protein whose orthologs have been identified in the fruit fly  has confirmed that they are efficiently degraded in this fashion . There is evidence that pharmacological enhancement of autophagy reduces the number of inclusions and corresponding loss of motor function in a transgenic model of TDP-43 proteinopathy  found in the catecholaminergic neurons of the substantia nigra and locus coeruleus of the non-diseased primate brain . It may be that age-related decline in proteolytic capacity could trigger the initial aggregation of the IDP, even in the absence of somatic mutations.Age-related decline in protein degradation may contribute to an RNA homeostatic crisis by promoting the accumulation of RNA-binding IDPs as described above, but recent speculation on mechanisms by which the pathology of neurodegenerative diseases may spread within an individual has increased the potential for IDP-mediated devastation. It has long been known that pathological changes in the nervous system follow anatomical patterns of spread that are stereotypic for each disease. In the case of sporadic Parkinson’s disease, for example, in vitro . It remaBased on the current literature we suggest that RNA homeostasis is a weak link in the aging brain, and the loss of RNA homeostasis underlies much neurodegenerative pathology. The precipitating event for an ensuing catastrophe may be the well-documented decline in proteolytic efficiency which would have immediate and deleterious effects on substrates such as the RNA-binding IDPs. In such a scenario the initiation of an “error catastrophe” in which aggregation of RNA-binding proteins promotes dysregulation of RNA splicing, RNA stability, RNA export, transport and translation would have knock-on effects on protein structure and function. The added burden to the proteasome and compromised function of proteolytic components would result in further perturbation of RNA homeostasis in a self-amplifying cycle. This catastrophe may be exported to neighboring cells through the prion-like spread of ribonucleoprotein complexes. We believe that the hypothesized mechanism of age-related neurodegeneration is experimentally tractable, and critical aspects of the hypothesis can be tested using mouse models and cell culture systems.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Phytophthora species belong to a phylogenetically distinct group of economically important plant pathogens that cause billions of dollars in yield losses annually as well as ecologically devastating outbreaks. We analyzed the small RNA-generating components of the genomes of P. infestans, P. sojae and P. ramorum using bioinformatics, genetic, phylogenetic and high-throughput sequencing-based methods. Each species produces two distinct populations of small RNAs that are predominantly 21- or 25-nucleotides long. The 25-nucleotide small RNAs were primarily derived from loci encoding transposable elements and we propose that these small RNAs define a pathway of short-interfering RNAs that silence repetitive genetic elements. The 21-nucleotide small RNAs were primarily derived from inverted repeats, including a novel microRNA family that is conserved among the three species, and several gene families, including Crinkler effectors and type III fibronectins. The Phytophthora microRNA is predicted to target a family of amino acid/auxin permeases, and we propose that 21-nucleotide small RNAs function at the post-transcriptional level. The functional significance of microRNA-guided regulation of amino acid/auxin permeases and the association of 21-nucleotide small RNAs with Crinkler effectors remains unclear, but this work provides a framework for testing the role of small RNAs in Phytophthora biology and pathogenesis in future work.In eukaryotes, RNA silencing pathways utilize 20-30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focused on plant and animal RNA silencing systems.  Phytophthora species are a diverse group of filamentous, eukaryotic plant pathogens that are related to other stramenopile (heterokont) eukaryotes within the chromalveolate super-group Phytophthora) was shown to be evolutionarily ancient. The oomycota lineage is estimated to have split from the photosynthetic stramenopiles in the ochrophyta lineage near the Proterozoic-Phanerozoic boundary ca. 570 million years ago Phytophthora contains some of the most devastating plant pathogens and contains upwards of 100 formally described species Phytophthora species are capable of infecting nearly all eudicot plants and several monocot species, causing multibillion-dollar damage to crops, ornamental plants, and natural environments. P. infestans infection of potatoes and tomatoes causes late blight disease and is best known as the cause of the Irish potato famine P. sojae is an economically important pathogen of soybeans resulting in estimated losses of $1–2 billion annually worldwide P. ramorum causes sudden oak death and infects a large variety of ornamental nursery crops P. ramorum infections have afflicted trees in the western United States since the mid-1990s and more recently gained notoriety for causing massive landscape-scale dieback in plantations of Japanese larch in the United Kingdom Phytophthora species have been sequenced Eukaryotes use RNA-based pathways to regulate gene expression at the transcriptional and post-transcriptional levels. RNA silencing pathways utilize small RNAs (∼20–30 nucleotides) to program effector protein complexes to recognize specific target nucleic acids in a sequence-dependent manner In plants and animals, and several other eukaryotic lineages, microRNAs are ∼20-24-nucleotide RNAs that mediate silencing of target transcripts post-transcriptionally. Primary miRNA transcripts (pri-miRNA) are generally transcribed by RNA polymerase II and contain self-complementary regions that fold to form imperfect double-stranded stem-loop structures. In animals, pri-miRNAs are initially processed by the RNaseIII protein DROSHA, or by the spliceosome in the case of mirtrons, to form precursor-miRNA (pre-miRNA). The miRNA and duplex strand (miRNA*) are released from the stem-loop RNA by the RNaseIII protein DICER (DCR) Another diverse class of small RNAs, referred to broadly as siRNA, are common to animals, plants, and fungi Phytophthora species. Dcl, Rdr and Ago genes were described in P. infestansP. infestansPhytophthora genus. We used deep sequencing and comparative genomic approaches to analyze the endogenous small RNA populations of P. infestans, P. ramorum and P. sojae. A bimodal distribution of small RNA sizes was observed across all three species with peaks centered on 21- and 25-nucleotide long small RNAs. A bimodal distribution is suggestive of separate processing by the two DCL homologs in each species. The larger small RNA size class is predominantly associated with loci encoding transposable elements or other repetitive DNA while the shorter class was associated with several gene families, including Crinkler effectors and type III fibronectins, inverted repeats and a novel, conserved miRNA family. This work is a crucial step in elucidating the endogenous RNA silencing pathways in these important plant pathogens and provides novel insights into the silencing machinery in the stramenopiles.Small RNAs have been studied in several pathosystems Phytophthora species Phytophthora. In agreement with the observed activity of RNAi, homologs of the core eukaryotic RNA silencing machinery were identified in P. infestans, including one Dcl, one Rdr, and five Ago genes Phytophthora species, we used the genomic resources available for P. infestans, P. sojae, and P. ramorum to identify homologs in the three phylogenetically diverse species (see methods) Phytophthora genome encodes a single Rdr protein  Trace Archive. We were able to assemble a 3962-nucleotide contig that contained a Dcl gene that was distinct from PiDcl1 and shared significant amino acid similarity to PsDcl2 and PrDcl2, confirming that P. infestans has two Dcl genes  is an established tool for studying gene function in  protein . PhylogeRdr gene . Two Dcld PrDcl1 . We hypocl genes , Table 1vergence .Ago homologs and six probable Ago pseudogenes were identified from all three Phytophthora species  and similar to the putative product of the PsAgo5_ps pseudogene  with 140,022 genomic matches, P. ramorum had 31,821 aligned reads  with 160,953 genomic matches and P. infestans had 5,594,216 aligned reads  with 54,692,467 genomic matches. The differences in genomic matches per unique sequence between species are probably caused by differences in genome size The P. infestans, the majority (84%) of small RNA genome alignments (loci) that could be assigned to an annotated feature category overlapped retrotransposons , and less than 10% overlapping inverted repeats, helitron transposons and satellite repeats    . The proir (Mbp) . In cont repeats . TherefoP. infestans feature-specific small RNA size profiles were similar to the genome-wide profile: bimodal with peaks at 21- and 25-nucleotide RNAs . Small RNA generating-loci were defined as segments of the P. infestans genome that contained mapped reads with up to 50 nucleotides of space between consecutive small RNAs. Segments were kept for further analysis if they contained at least one distinct pair of small RNAs that overlapped on opposite strands with 2-nucleotide 3′ overhangs, as expected for Dicer products bona fide small RNA-generating loci. For each of the 2,328 P. infestans segments that contained a Dicer-duplex pair , a proportional small RNA size profile was constructed, to remove abundance differences between segments, by calculating the proportion of small RNA reads for each size (20–27 nucleotides) relative to the total segment reads  is more than twice as large as the P. sojae and P. ramorum genomes  due to recent proliferation of transposable elements P. infestans genome was not uniform and resulted in a distinct genome architecture that consists of blocks of gene-dense and gene-sparse regions P. infestans are flanked by intergenic regions between 20 bp and 3 Kbp long , while another set are flanked by at least one intergenic region that is 5 Kbp or larger   . GDRs anns, BRs) . Similarns, BRs) . A similns, BRs) . In contns, BRs) . Small Rns, BRs) . Unlike ns, BRs) . Genes gns, BRs) , with thP. infestans genome coincides with the location of transposable elements and low gene density. Additionally, we observed enrichment of transposable element loci at 25/26-nucleotide-generating segments of the P. infestans genome. Therefore, we hypothesize that 25/26-nucleotide small RNAs function to repress transposable elements in Phytophthora species. In agreement with our observations, P. sojae strains that contain naturally silenced alleles of the avirulence gene Avr3a were shown to have abundant 24-26-nucleotide small RNAs at the Avr3a locus Avr3a was demonstrated in crosses between silenced and non-silenced strains, suggesting an epigenetic mode of inheritance Phytophthora is mediated through chromatin modifications and not DNA methylation The distribution of 25/26-nucleotide small RNAs across the P. infestans are derived from transposable elements, repeats and other intergenic regions, but some genes were identified at small RNA-generating loci . Interestingly, five of the eight families  are enriched in gene-sparse regions As described above, the majority of small RNAs in ing loci . To examP. infestans genome, some genes encoding ANX, CRN, FN3 and P-ATPases overlapped small RNAs that were primarily 18-23-nucleotides long . In contrast to genes associated with 25/26-nucleotide small RNAs, 18-23-nucleotide small RNAs, and in particular 21-nucleotide small RNAs, at ANX, CRN, FN3 and P-ATPase loci were more abundant on average and were almost exclusively located in the gene body  on Supercontig 16 and 63, the DC-type that were found primarily (28 of 34) on Supercontig 6, and the D2 and DXZ types that were found together primarily (63 of 73) on Supercontig 31, 48, 82, 85, 69, 104 and 143  to eliminate Rdr-dependent small RNA-generating regions. Finally, for each remaining foldback, the most abundant sequence was considered the mature miRNA and was required to have a complementary sequence with two-nucleotide 3′ overhangs (miRNA*) from the opposite arm of the foldback. Two loci each from P. infestans, P. sojae and P. ramorum matched our criteria (P. infestans was a 714-nucleotide inverted repeat (pin-IR2758) that produced heterogeneous 21-nucleotide siRNA and was therefore not considered a MIRNA (MIRNA loci in P. ramorum and P. sojae were from the same family (MIR8788) and were related to one of the loci from P. infestans. The foldback structures of all MIR8788 homologs were strikingly similar, and multiple sequence alignment of the primary foldback sequences also showed that the foldbacks were highly conserved  were consistent while the 3′ ends were variable   . In addi species . We useddetected . Gm1.7889 and 10 . FurtherMIR8788 is a bona fide miRNA-generating locus is strong, but it is perhaps surprising that only one MIRNA family was found in our analysis. To assess possible biases in our computational pipeline, we independently analyzed the P. infestans small RNA libraries using the ShortStack pipeline MIRNA locus, MIR8788. These analyses may have missed miRNAs that were not represented in our libraries because of specific spatiotemporal or environmental-dependent patterns. Furthermore, our analysis relied on libraries from mycelium grown in broth and it is possible that additional MIRNA loci specific to certain life-stages, particularly during host-pathogen interaction, remain to be discovered. Alternatively, the concept of a distinct miRNA biogenesis pathway may not be relevant to Phytophthora species if their small RNA pathways produce ∼21-nucleotide miRNA-like sequences from hairpin RNAs with differing precision and processing efficiencies, producing sets of small RNAs that range from heterogeneous (pin-IR2758) to precise (MIR8788)  . Indeed,all RNAs . It may Phytophthora species have a conserved set of core RNA silencing machinery, including one Rdr, two Dcl and four Ago proteins, with some species-specific duplicates. Our detailed analysis of small RNA sizes and distribution across the P. infestans genome, as well as small RNA sizes from P. sojae and P. ramorum, supports the presence of two primary, distinct small RNA size classes: 21- and 25/26-nucleotides. The 21- and 25/26-nucleotide small RNAs are not just distinct in size but are also generated from distinct features, with 25/26-nucleotide small RNAs predominantly associated with transposable elements and 21-nucleotide small RNAs associated with inverted repeats and several gene families. These results are largely in agreement with Vetukuri et al. Phytophthora have at least two distinct small RNA pathways that may be partitioned by Rdr, Dcl and Ago diversification as well as small RNA size. In this model, we propose that 25/26-nucleotide small RNAs define a siRNA pathway that targets transposable elements and other repetitive features and silences target loci through an epigenetic mechanism. Besides a role for genome defense and evolution of genome structure, epigenetic regulation of pathogenicity factors has important implications for the evolution of isolates and host range In plants, animals and fungi, duplication and diversification of genes that encode DCR/DCL, RDR, AGO and other accessory proteins has resulted in the evolution of distinct RNA silencing pathways CRN and FN3, appear to be siRNA and likely are dependent on Rdr activity. In contrast, other 21-nucleotide small RNAs may be generated from hairpin RNA (e.g. MIR8788) without the need for Rdr activity. It is unclear what the function of 21-nucleotide small RNAs is, but given the large number of P. infestans CRN genes associated with 21-nucleotide small RNAs they may be important components of pathogenicity, host range or variability of effector repertoire between isolates.Additionally, we propose that 21-nucleotide small RNAs define at least a second pathway that regulates targets by post-transcriptional control. The presence of a second pathway for small RNA biogenesis is further supported by the observed ancient divergence of Dcl1 and Dcl2, and the deep split in the Ago tree separating Ago1/2 from the other Agos, reflecting independent evolution. Some 21-nucleotide-generating loci, such as Phytophthora species; Phytophthora ramorum Pr-102, Phytophthora sojae P6497 and Phytophthora infestans T30-4 Phytophthora isolates were maintained on cleared 10% V8 agar medium ; 15 g agar; 900 mL deionized water) in a 20°C incubator in the dark P. sojae and P. ramorum and after 4 days for P. infestans.Mycelium was grown from the sequenced strains of three Total RNA was isolated as described previously P. ramorum and P. sojae were prepared for sequencing on the 454 platform using 5′ adaptors 5′-ATCGTAGGCUGCUGAUA-3′ and 5′-ATCGTAGGCCACUGAUA-3′ for P. sojae and P. ramorum, respectively, with strain-specific bases bolded, as described previously P. infestans libraries were prepared for sequencing on the Illumina 1 G platform as described previously Small RNA libraries for P. sojae (v3.0) and P. ramorum (v1.1) were obtained from the Joint Genome Institute (JGI) P. infestans was provided by the Broad Institute Sequencing reads were parsed to remove adaptors and collapsed to a unique set with read counts. Parsed sequences from each species were aligned to their respective genome using BOWTIE v0.12.8 Rdr, Dcl, and Ago were located using tBLASTn P. sojae, P. ramorum, and P. infestansPiDcl2, MEGABLAST PsDcl2 to the P. infestans whole-genome shotgun reads at the NCBI Trace Archive  using EINVERTED (-gap 12 -threshold 10 -match 3 -mismatch -4) A computational pipeline was used to identify putative miRNA from among the P. infestans small RNA libraries using the ShortStack pipeline v0.4 To assess possible biases in our computational pipeline, we independently analyzed the P. sojae, P. ramorum and P. infestans transcripts using TargetFinder v1.6 (http://carringtonlab.org/resources/targetfinder) Targets for miR8788 were predicted for P. infestans mycelium tissue was resolved by denaturing 17% polyacrylamide-urea gel electrophoresis. For size standards, 21- and 24-nucleotide synthetic RNA markers  were also resolved. RNA was transferred to a positively charged nylon membrane  by semi-dry electrotransfer. RNA was UV-crosslinked to the membrane twice at 1200 Joules. DNA probes for pin-miR8788 (5′-GCGGCGCTGGTACGCTTGGTAG-3′) and serine-tRNA (5′-CTGTGAGATTCGAACTCACGC-3′) were end-labeled by phosphorylation with γ32P-ATP and Optikinase (Amersham). Membranes were incubated with hybridization solution  at 38°C for 1 hour and then incubated with probes at 38°C overnight.Total RNA (10 µg) from P. sojae candidate psj-miR8788 target transcripts Physo2.s_450095 and gm1.7889_g were analyzed for miRNA-guided cleavage by modified RLM-5′RACE using the GeneRacer kit (Life Technologies) as described psj-miR8788 binding site. Gene-specific primers were 5′-AGCAGCGAGCCGCCCAGCACGAGGAA-3′ and 5′-TGGGCACCAGGCACACGGGCAGCAC-3′ for Physo2.s_450095 and 5′-AGCGTGGGAATGAGGCACACCGGCA-3′ and 5′-AGGTCGGGGAACGGCACACTCGGGT-3′ for gm1.7889_g. PCR products were cloned and sequenced to detect RNA fragment 5′ ends.The P. tetraurelia sequences were extracted from ParameciumDB T. thermophilia sequences were extracted from the Tetrahymena Genome Database T. gondii sequences were extracted from the NCBI protein database: Rdr1 [XP_002371017]; Ago1 [XP_002364262]. A. thaliana sequences were extracted from The Arabidopsis Information Resource (TAIR v10) Homo sapiens AGO2 [Q9UKV8] and Drosophila melanogaster AGO1 [Q27IR0] were extracted from UniProtKB http://www.ncbi.nlm.nih.gov/geo/) database under the series accession GSE50033. MIR8788 was submitted to miRBase (http://www.mirbase.org/).Figure S1MIR8788 processing in P. infestans, P. ramorum and P. sojae.MIR8788 foldback diagrams for P. infestans (A), P. ramorum (B) and P. sojae (C). miR8788 guide strands and miR8788* strands are highlighted in each foldback. Proportion of small RNA reads for the entire foldback are plotted as stacked bar graphs. Small RNAs are color coded by size.(EPS)Click here for additional data file.File S1Supplemental tables for small RNA-generating segments, genes and inverted repeats.(XLSX)Click here for additional data file.File S2Manually annotated gene sequences.(DOCX)Click here for additional data file.


Key carbonate producers including hard coral, crustose coralline algae and Halimeda are mapped accurately (mean R2 = 0.81). Community net production estimates correspond closely to independent census-based estimates made in-situ (R2 = 0.86). Reef-scale outputs are compared with historic rates of production generated from (i) radiocarbon evidence of island deposition initiation around 3200 years ago, and (ii) island volume calculated from a high resolution island digital elevation model. Contemporary carbonate production rates appear to be remarkably similar to historical values of 573 m3 yr−1. Anticipated future seawater chemistry parameters associated with an RCP8.5 emissions scenario are employed to model rates of net community calcification for the period 2000–2100 on the basis of an inorganic aragonite precipitation law, under the assumption of constant benthic community character. Simulations indicate that carbonate production will decrease linearly to a level of 118 m3 yr−1 by 2100 and that by 2150 aragonite saturation levels may no longer support the positive budgetary status necessary to sustain island accretion. Novel aspects of this assessment include the development of rate law parameters to realistically represent the variable composition of coral reef benthic carbonate producers, incorporation of three dimensional rugosity of the entire reef platform and the coupling of model outputs with both historical radiocarbon dating evidence and forward hydrochemical projections to conduct an assessment of island evolution through time. By combining several lines of evidence in a deterministic manner, an assessment of changes in carbonate production is carried out that has tangible geomorphic implications for sediment availability and associated island evolution.A geomorphic assessment of reef system calcification is conducted for past (3200 Ka to present), present and future (2010–2100) time periods. Reef platform sediment production is estimated at 569 m Reef islands are low-lying accumulations of biogenically derived sediments Reef islands are constructed from unconsolidated sediments derived from benthic calcium carbonate producers such as scleractinian corals, coralline algae, green calcified algae, molluscs and benthic foraminifera Because reef island sediments derive entirely from surrounding coral reef and reef flat environments, they are highly sensitive to environmental conditions that may modify reef community composition and productivity, including sea surface temperature in-situ census based estimates of carbonate production. Contemporary estimates of carbonate production are then compared to historical levels of island accretion, ascertained through a combination of i. radiocarbon evidence to identify the point at which island deposition began, and ii. the development of a high resolution digital elevation model of the island, such that an approximation of of average island accretion rate can be derived by dividing island volume by age. Finally, hydrochemical measurements taken from the reef flat are used to simulate future levels of carbonate production for the complete reef system using anticipated values of aragonite saturation under a RCP8.5 emissions scenario. Simulations are undertaken for 4 seasonal periods  and calculated across a complete tidal cycle to account for natural chemical fluctuations. Such an assessment is of particular interest because Lady Elliot Island is the southernmost coral Cay of the Great Barrier Reef and is therefore representative of an island site at the margins of environmental conditions (e.g. sea surface temperatures) that are suitable for carbonate production.The objective of the present study is to conduct a geomorphic assessment of reef system calcification and associated island evolution for past (3200 Ka to present), present and future (2010–2100) time periods at Lady Elliot Island (southern Great Barrier Reef). This is achieved through a combination of geospatial analysis, hydrochemical measurements and radiocarbon evidence. Contemporary levels of carbonate production are estimated for the complete reef system by mapping the magnitude and distribution of key benthic community calcifiers across the reef platform. This information is then utilised alongside estimates of reef surface rugosity in a rate law calculation to estimate net community carbonate production from inorganic aragonite precipitation. Results are validated against independent −1 and 60 m Ka−1 for the windward and leeward sequences respectively At 24°07′S and 152°43′E, Lady Elliot island lies 84 km offshore from the Queensland coastline and is part of the Capricorn-Bunker group . Lady ElLady Elliot Island is subject to a diurnal mesotidal regime with a mean spring tidal range of 1.7 m and a neap tidal range of 0.9 m in-situ census based methods for estimation of reef carbonate production within a Pacific context . The only calcified macroalgae observed was Halimeda, which was observed in clusters of small patches (<1 m2), rather than dense beds.A detailed inventory of the carbonate budget components for Lady Elliot island was undertaken to develop an initial conceptual model in order to constrain the relationship between reef benthic carbonate production and sediment delivery to the island, and ascertain the applicability of the  context . The invFramework erosion by internal borers including endolithic organisms such as bivalves and worms and substrate grazers such as urchins was either entirely absent or observed in such low quantities that their influence was inconsequential. Parrotfish had a more localized influence, being absent from most surveys but with schools of up to 17 bullethead parrotfish observed at in the northwest site 2, . Bioeros2 that incorporated a side profile of the reef community, including the understorey. The video camera was mounted on a 50 m length of cable, permitting the deeper reef platform to be sampled after attaching weights to the base of the camera head. The geographical position of each video sample point was recorded with a dGPS (accuracy <1 m).This work was undertaken with permission from the Great Barrier Reef Marine Park Authority (permit ref. G36002.1). Fifty snapshots of oblique underwater video footage of benthic cover were collected from a boat around Lady Elliot Island . Fugawi was mapped using a predictive habitat mapping approach in-situ. The bathymetric echo sounder survey was undertaken with a Ceeducer Pro system  with an integrated GPS. Soundings were undertaken following a zig-zag pattern across the reef platform profile around the entire island. The tidal signal was removed from the depth readings using Navy Seafarer tides to convert all readings to a datum of mean sea level.The distribution of four major carbonate producing benthic components , crustose coralline algae and For each digital pixel of a raster grid covering the entire reef platform, a spatially explicit regression model predicted the distribution of each carbonate producer on the basis of the independent physical variables around Lady Elliot Island. This followed a methodology that has successfully modelled the distribution of major carbonate producers around Lizard Island on the northern GBR net values measured on reefs and rates of inorganic aragonite precipitation Gi were used as the basis for deriving a rate law with which net community calcification could be estimated on the basis of aragonite saturation state Strong correlations between GThe inorganic aragonite precipitation rate law  was paracA is a measure of the fraction of reef area actively depositing CaCO3 and ′rk is the ratio between the total area of the reef actively precipitating CaCO3 and its planar area (i.e. the reef rugosity). netG was estimated in a deterministic manner by applying a modified version of the rate law on a per pixel basis. Different rates of inorganic aragonite precipitation were applied across the two reef zones by generating a reef flat and a reef slope mask  and employing a conditional statement that inserted the correct Gi value depending on pixel location. In this way, the different aragonite conditions of the reef flat and slope could be accounted for. The term cA was adjusted to reflect the range of contributions made to netG by different community components. For each pixel, cA was estimated according to the relative contribution of the different carbonate producing community components and expressed as a proportion of the theoretical maximum calcification value. This was a pixel comprised completely of branching coral, the community component with the highest calcification rate (i.e. 10 Kg CacO3 m−2 yr−1):iP is the proportional cover of each community component and cK is the calcification rate of that community component for n community components. To convert values for overall carbonate production of the reef platform to island accretion, the volume of carbonate produced each year was reduced to account for sediment loss from the system.Where −1 yr−1, which was comparable with rates of off-reef sediment and rubble transport elsewhere To quantify the annual volume of sediment loss from the island, cylindrical sediment traps (35 cm length by 5 cm diameter) were deployed at the edge of the reef terrace (approximately 14 m water depth) adjacent to each of the in-situ census validation sites. In line with the model objective (to estimate sediment availability for island maintenance and growth), sediment loss was defined as any outward sediment movement beyond the boundary of the upper reef terrace and down the reef slope to a deeper position (>14 m), from which it is unlikely to be transported back up the reef flat and deposited on the island. A single sediment trap was deployed at each of the five sites (marked 1 to 5 on in-situ using the ReefBudget census-based methodology −2 yr−1). This methodology yields continuous records of all cover types and transitions between different covers along the transect, thereby internally adjusting the sampling frequency on the basis of the spatial variation of the benthic cover itself. A detailed description of the methods, along with supporting datasets, field survey and data entry sheets can be downloaded from the ReefBudget website (http:/geography.exeter.ac.uk/reefbudget).To validate the modelled values of community carbonate production, calcification for the Lady Elliot island reef slope was measured Halimeda. Substrate topographic complexity was incorporated to derive an accurate measure of the true surface area covered by each taxon by running a measuring tape over the substrate to record the length of transect conforming to the topography (d1) and simultaneously measuring the planar distance of transect covered. Rugosity (topographic complexity) of the survey site was then determined as d1/d2 Halimeda  and the substitution of calcification rates derived from the Pacific for branching and non-branching live coral  and calcified macroalgae netG for the complete reef platform, the iG term was adjusted to incorporate anticipated values of aragonite saturation for the summer, autumn, winter and spring periods at 10 year intervals from 2010 to 2100. iG term calculated for anticipated future values of aragonite saturation state , with individual cover correlations as follows: hard coral branching (R2 0.89), hard coral non-branching 0.89, crustose coralline algae (R2 0.8) and Halimeda (R2 0.76)  . In-situ3m-2yr−1 . These v3 yr−1 of sediment. Over the simulation period from 2000 until 2100, this decreased by 79% to 118 m3 yr−1 because of the reduced aragonite saturation states associated with the RCP8.5 emissions scenario. While the aragonite saturation states decreased in an exponential fashion, simulation results indicated a linear decrease in reef scale carbonate production . Furthermore, reef island development is a function of interrelationships between reef basement, sediment supply and hydrodynamic process regimes that drive geomorphic fluctuations across centennial timescales 2 fluxes, to generate a realistic chemical scenario for the projections. This is particularly important at Lady Elliot Island because the water ponds over the reef flat at low tide, leaving it effectively isolated from offshore conditions for approximately 2–3 hours each tidal cycle, when extreme carbonate chemistry conditions occur Many studies of the impact of ocean acidification on coral reefs employ carbonate chemical parameters from oceanic as opposed to coastal settings e.g. . HoweverThe present study uses rate laws to generate estimates of total reef system carbonate production by combining spatially extensive remotely sensed information on community composition and rugosity with local hydrochemical measures of seawater carbonate chemistry. Estimates of carbonate production are constrained by realistic values and model outputs are coupled with reconstructions of historical island evolution from radiocarbon dates. This approach draws on the increasing suitability of remote sensing images to accurately and consistently upscale estimates of carbonate production from observable features, such as live coral A linear decrease in carbonate production has been simulated at the scale of the complete reef system, with a 79% reduction on current levels by 2100. This is consistent with documented declines in carbonate production elsewhere. Perhaps the most valuable and novel aspect of this study is that it has demonstrated how several disparate techniques can be combined to provide a deterministic means of modelling changes in reef carbonate production and consequent implications for island evolution through time.