ASH1 mRNA in budding yeast. It is particularly well suited to highlight the interdependence between different steps of mRNA localization. The second example is Staufen-dependent localization of oskar mRNA in the Drosophila embryo, for which the importance of nuclear events for cytoplasmic localization and translational control has been clearly demonstrated. The third example summarizes Egalitarian/Bicaudal D-dependent mRNA transport events in the oocyte and embryo of Drosophila. We will highlight general themes and differences, point to similarities in other model systems, and raise open questions that might be answered in the coming years.At all steps from transcription to translation, RNA-binding proteins play important roles in determining mRNA function. Initially it was believed that for the vast majority of transcripts the role of RNA-binding proteins is limited to general functions such as splicing and translation. However, work from recent years showed that members of this class of proteins also recognize several mRNAs via cis-acting elements for their incorporation into large motor-containing particles. These particles are transported to distant subcellular sites, where they become subsequently translated. This process, called mRNA localization, occurs along microtubules or actin filaments, and involves kinesins, dyneins, as well as myosins. Although mRNA localization has been detected in a large number of organisms from fungi to humans, the underlying molecular machineries are not well understood. In this review we will outline general principles of mRNA localization and highlight three examples, for which a comparably large body of information is available. The first example is She2p/She3p-dependent localization of  Transcriptional control of gene expression was long considered as the main mechanism to regulate cellular functions. The identification of large networks of posttranscriptional modifications challenged this view. They also demonstrated the great advantage of rapid response to external stimuli. More recently, it emerged that also translational control contributes to a wide range of biological processes. It shares with posttranslational modifications the advantage of rapid response, but also offers the great benefit of a regulated amplification of the genetic information from a single transcript into dozens to hundreds of protein molecules. Drosophila melanogaster yielded the surprising insight that about 70 % of the expressed transcripts are subcellularly localized [Drosophila embryogenesis might be an extreme case, it demonstrates the importance of this mechanism of gene regulation. Indeed, data from the last 20 years showed that mRNA localization is very common in eukaryotes [Drosophila melanogaster and Caenorhabditis elegans [Xenopus laevis, mice and human cell lines [Translation of mRNAs can be regulated in time , but also by controlling the location of protein synthesis . Often these two properties are combined to achieve a tight control over protein synthesis. Subcellular localization of mRNAs is used to generate asymmetric accumulation of proteins and thus cellular asymmetry. A genome-wide assessment of the intracellular distribution of mRNAs in embryos of ocalized . Althougkaryotes , inverte elegans , and verll lines .1, left) [1, middle) [1, right). An asymmetric distribution of a subset of transcripts can be achieved by different mechanisms. One possibility is that transcripts simply diffuse through the cytoplasm or reach a certain subcellular site by cytoplasmic streaming, where they become anchored and subsequently translated  . Local a middle) , 7. The 2A) [In this last scenario, mRNAs are recognized by dedicated RNA-binding proteins and transported as part of motor-protein containing messenger ribonucleoprotein particles (mRNPs) to subcellular sites most often in the periphery of a cell , 8. Thes2A) , 10. RecASH1 mRNA in yeast. Studies on ASH1 have let to the yet best characterization of an mRNA-localization mechanism. It is particularly well suited to exemplify the dynamics of mRNP assembly from the nucleus to its cytoplasmic destination. Since mRNA localization also serves functions that are specific to multicellular organisms, we will discuss two localization events occurring during the early development of the fruit fly [. The first of these examples is the localization of oskar (osk) mRNA to the posterior pole of the Drosophila embryo. We chose this example because it demonstrates the requirements of nuclear splicing events as well as oligomerization of mRNAs for their localization. Such an involvement of RNA-centered steps may be important also in other organisms. The second example from Drosophila is the dynein-dependent localization of several mRNAs by a protein complex containing Egalitarian (Egl) and Bicaudal D (BicD). This example was chosen because it highlights the close collaboration of motor-associated components and RNA recognition factors during mRNA localization. It should be noted that mRNA localization is also important for many vertebrate oocytes and somatic cells, such as neurons and fibroblast. However, in this review we will not cover these localization processes because they have been subject of excellent recent reviews [In this review, we will focus on the assembly of localized RNPs and their active transport to subcellular sites. In order to highlight general principles of mRNA localization, we will discuss three representative examples, for which a sufficient body of mechanistic insights is available. We will begin with summarizing the molecular events leading to the transport of ruit fly , 12. The reviews , 13-15. Saccharomyces cerevisiae, a set of about 30 mRNAs are transported from the mother cell into the daughter cell at various stages of the cell cycle [ASH1 mRNA. It encodes a transcriptional repressor that inhibits mating-type switching in the daughter cell and therefore ensures different cell fates of mother and daughter cell [ASH1 mRNA localization were identified in a genetic screen [ASH1-mRNA localization has been obtained  [In ll cycle -19. Theyll cycle -22. The ter cell , 24. Aboc screen . Since tFig. 2B) -22, 26. ASH1 mRNA already begins in the nucleus with the binding of She2p, a key trans-acting factor for localization of ASH1. It has been reported that She2p interacts with transcriptionally active RNA polymerase II [ASH1 transcript. However, the specificity of this early aspect of pre-mRNP assembly is still being disputed [Loading of RNA-binding proteins onto erase II , suggestdisputed  and requASH1 mRNA pass through the nucleolus [ASH1 mRNA [2B) [ ASH1 mRNA localization [ASH1 mRNA localization has not yet been unraveled.She2p has an unusual RNA-binding domain that consists of two dimers forming an elongated tetramer , 30. Nucucleolus , where tSH1 mRNA , 33 and RNA [2B) . AlthougASH1 mRNA already in the nucleus  [ASH1 mRNA during its cytoplasmic transport [ASH1 mRNA during its transport. Two other nuclear shuttling factors, the RNA-binding proteins Puf6p and Khd1p, also interact with Fig. 2B) , 34-36. ransport -38. The ASH1 pre-mRNP is exported to the cytoplasm, where it associates with a cytoplasmic, motor-containing pre-complex [2B) [ASH1 mRNA as well as to She2p and therefore has a central function in assembling the mature mRNP [The nuclear -complex . It conslex [2B) -46. She3ure mRNP , 41, 45.ure mRNP . This sp2B). With its C-terminal half She3p interacts with She2p to form the described specific ternary complex , 41, 45,ASH1 mRNA is controlled by the RNA-binding proteins Puf6p [ASH1 mRNA [ASH1 mRNA and its subsequent translation. Khd1p binds to the first half of the open reading frame of ASH1 mRNA [ASH1 mRNA. The combined action of Puf6p and Khd1p prevents premature translation of ASH1 mRNA during transport. After reaching the site of destination the transport complex is anchored, disassembled and translation is activated. Whereas translation activation by release of the translational repressors is well understood [Translation of ns Puf6p  and Khd1ns Puf6p . Puf6p bSH1 mRNA  and simuSH1 mRNA . The latSH1 mRNA , 38. Morderstood , 21, ancASH1 mRNA accumulates in a crescent-like shape at the plasma membrane, which has been interpreted as anchoring [per se [ASH1 mRNA with mutated start-codon shows strongly impaired crescent-like localization [After transport, nchoring , 52, 53.lization . ASH1 mRNA. This allows assembly of the 60S subunit with the 40S on the AUG start codon and translational activation [At the bud tip, translation is activated by phosphorylation of Khd1p and Puf6p. Khd1p becomes phosphorylated by the membrane-associated kinase Yck1p , which rtivation . Such phtivation . Thus, pASH1 mRNA more than 30 additional transcripts are transported by the She2p/She3p/Myo4p complex [ASH1, they are recognized by the transport machinery via their cis-acting zip-code elements [Besides  complex -19. Likeelements , 55-58. In yeast, the zip-code elements of localized mRNAs show only limited sequence or structural conservation and thus the features defining specificity have not been fully understood , 55-58. ASH1 mRNPs might consist of larger structural assemblies that enable the co-transport of multiple transcripts to their destination.Since all localized mRNAs in yeast are transported by the same machinery, the question arose whether each zip-code containing mRNA is localized independently or whether multiple transcripts are transported together in a large complex. Live-cell imaging combined with a dual tagging strategy of the localized mRNAs in fact showed that localizing mRNPs contain at least two different mRNAs . This fiDrosophila oogenesis, mRNA localization presents an initial key step for the establishment of the body axes and embryonic patterning. Several mRNAs of maternal origin, including bicoid, gurken and osk, are transported from the nurse cells into the oocyte and localized to distinct positions within the oocyte. After fertilization, their locally translated protein products provide positional information and establish a tightly controlled transcriptional regulatory network for the segmentation of the embryo [bicoid or osk has been studied in great detail [osk, its localization process can be structured into several phases, including mRNA export from the nucleus, dynein-dependent transport of osk from the nurse cells into the oocyte, and kinesin-dependent trafficking to the posterior pole of the oocyte. A number of RNA-binding proteins have been described that act in trans to facilitate the transport from the nurse cells into and within the oocyte [During e embryo . The loct detail , 62 and t detail . Especiae oocyte . Some ofosk transcript is associated with a dynamic collection of proteins. These factors orchestrate the synthesis, processing, export, translational control, localization, and degradation of osk mRNA. More functionally relevant trans-acting factors are known for osk RNA than for any other localized transcript.Throughout its lifetime from synthesis in the nuclei of nurse cells to degradation at the posterior pole of the oocyte, the osk mRNA localization [osk reporter mRNAs derived from cDNA are incompetent of localization in the absence of endogenous osk mRNA. In addition to EJC components, several other RNA-binding proteins whose loss of function result in defects in osk localization, shuttle between nucleus and cytoplasm. These include proteins of the heterologous nuclear RNP (hnRNP) family such as Hrp48 and Squid/Hrp40. Whereas Hrp48 directly binds to osk 5’- and 3’-UTR [osk 3’-UTR where it also binds to Hrp48 [osk in the nucleus. Unlike other hnRNPs binding to osk, the polypyrimidine tract-binding protein (PTB)/hnRNP I does not need to bind its target mRNA inside the nucleus. This conclusion was drawn based on the observation that an exclusively cytoplasmic variant of PTB is able to associate with osk and functionally replace endogenous PTB [Drosophila protein, the nuclear association of the Xenopus laevis PTB homolog with its target Vg1 mRNA has been proposed to be a crucial step during localization [Drosophila PTB binds to multiple sites within the osk 3’-UTR and mediates the formation of large complexes containing multiple osk molecules [osk translation by masking the mRNA from the translation machinery. During splicing in multicellular eukaryotes, a large multisubunit complex called the exon junction complex (EJC) is deposited upstream the exon-exon junction. Whereas this complex generally serves as a hallmark for the nonsense-mediated decay of mRNAs with premature stop codons , its asslization . This red 3’-UTR , 66, Hrpto Hrp48 . Since mto Hrp48 , Hrp40 anous PTB . In contlization . Drosopholecules . This asosk RNA protein particles also involves a second translational repressor, Bruno [osk 3’-UTR  and appears to repress translation via two different mechanisms. On one hand, Bruno recruits Cup, an inhibitor of cap-dependent translation initiation that interferes with the interaction of the translation initiation factors eIF4E and eIF4G [in vitro observations suggest that by binding to its cognate sites within osk mRNA the protein incorporates the transcripts into large 50 - 80S translation silencing particles [osk RNP particles has recently revealed that Hrp48 is also required for formation of large osk particles [osk mRNA comes from observations that BRE elements can act in trans and establish translational control on co-expressed osk mRNA mutants that lack BREs [osk RNP particles contain multiple osk RNA molecules with their corresponding RNA-binding proteins. Besides protein-driven multimerization, new data also suggest that RNA-RNA interaction between individual osk molecules could contribute to the formation of these large particles. A stem-loop region within osk 3’-UTR that does not encompass any known binding site for the above mentioned proteins is sufficient to drive homodimerization of two osk messages [osk mRNA. Interestingly, formation of large r, Bruno . Bruno cnd eIF4G . On the articles . This paarticles . Life imarticles . Additioack BREs . This fimessages . Togetheosk particles described above contain additional RNA binding proteins like Exuperantia (Exu) and Staufen (Stau). Exu lacks canonical RNA binding motifs but associates with osk mRNA and with many proteins involved in translational repression of osk [osk mRNA localization and found in RNPs that display dynamic movements consistent with active transport. Staufen contains several double-stranded RNA binding domains (dsRBDs) and is involved in RNA localization in a number of organisms [osk mRNPs already early on during microtubule-dependent transport [Xenopus homolog that participates in localization of Vg1 mRNA in oocytes, assembles with the RNA in the cytoplasm after nuclear export [Drosophila Stau associates with the mature osk particle in the cytoplasm, presumably after the transport of osk particles from the nurse cell to the oocyte [osk-containing mRNPs are localized to the posterior pole via active transport by microtubule-dependent motor proteins [osk mRNPs in living oocytes has revealed that this transport corresponds to a random walk [The large n of osk . Exu is rganisms . On one ransport . Althougransport , it has r export . Similare oocyte . In the proteins , 81. Thedom walk . Transpodom walk  but alsoosk mRNA localization has revealed that also in multicellular organisms multiple RNA-binding proteins participate in the localization of an mRNA and that nuclear events are important to guide cytoplasmic localization of transcripts. Some of the described proteins like Stau, Hrp48, or EJC components such as Barentz might have a more direct role in mRNA transport by e.g. recruiting different motor proteins (dynein or kinesin I) at various stages of localization. The involved RNA-binding proteins can have diverse but also overlapping functions during localization, ranging from translational control to particle formation and anchoring at the target site.In summary, the detailed analysis of Drosophila development, directional transport of several mRNAs also occurs in a dynein-dependent manner towards the minus-ends of microtubules. Cytoplasmic dyneins are unrelated to myosins or kinesins and much larger [During h larger . The funh larger , of which larger . Of thesh larger . Drosophila oogenesis, dynein motors transport several transcripts from the nurse cells into the oocyte. Later in the blastoderm embryo this motor complex also localizes mRNAs to its apical periphery [bicoid, fushi tarazu, gurken, hairy, fs(1) K10 (K10), orb, wingless, and the I Factor retrotransposon RNA [During eriphery , 87. In oson RNA -94.osk mRNA as well as for ASH1 mRNA a nuclear history is important for their respective cytoplasmic localization. For dynein-dependent transport during Drosophila development it remains unclear whether specific nuclear events are also required. Microinjection of dynein-dependent transcripts into Drosophila embryos resulted in their efficient localization to the apical periphery [For eriphery . AlthougDrosophila embryo, trafficking mRNPs switch their movement frequently from the minus-end direction towards the apical periphery to plus-end direction and vice versa [During transport of mRNAs to apical sites of the ce versa , 96. In ce versa , the molce versa . A rathece versa . It indice versa .Drosophila embryos is the lack of conserved sequence motifs [DrosophilaK10 mRNA [K10 zip-code RNA into blastoderm embryos efficiently localized to apical sites, whereas mutant versions with a narrower major groove showed impaired apical localization [K10 mRNA is expressed and transported much earlier in development than the developmental time-point of the injection experiments [K10 zip-code element are likely to be general features of zip-code elements for minus-end directed dynein transport.One puzzling feature of zip-code elements that mediate minus-end directed transport in e motifs . RecentlK10 mRNA , which iK10 mRNA , was detK10 mRNA . The stulization . This exeriments , it can The similarity of these processes in oogenesis and embryogenesis also extends to the involved protein co-factors. During both developmental stages, the two proteins Egalitarian (Egl) and Bicaudal D (BicD) are expressed and required for minus-end localization of transcripts , 101. Egin vitro. This is consistent with the observed bidirectional, unbiased transport of non-localizing mRNAs in vivo [The RNA-binding properties of Egl suggested that it mediates the recognition of zip-code RNAs. Alone, Egl showed a modest preference for zip-code containing RNAs over non-localizing control RNAs . The pre in vivo . Howeverin vivo injection experiments [An obvious question is how the recognition of zip-code mRNAs by Egl induces a net bias of movement towards minus ends of microtubules, whereas non-localizing RNAs are transported bidirectionally. The RNA-Egl-BicD complex might stabilize the dynein complexes and thereby increases the total copy number of active dynein motors associated with localizing transcripts. This hypothesis receives support from eriments . It has eriments . AlternaSeveral different transcripts are recognized and localized to apical sites of the embryo. In principle, they could either be transported in distinct particles or be co-transported in joint assemblies. Co-injection experiments with different fluorescently labeled, zip-code containing RNAs demonstrated that they are indeed incorporated into the same particles and transported together to apical sites . No suchgurken mRNA in the oocyte after its dorso-anterior transport depends on dynein as a static tethering factor [gurken mRNA, they are dispensable for its anchoring. Together these findings suggest that dynein-dependent transport and anchoring of mRNAs follow similar principles in the oocyte and in the blastoderm embryo. It will be interesting to see whether this assumption holds true also for minus-end directed transport in adult tissues and in higher eukaryotes.At the minus-end destination of dynein transport, the mRNA becomes anchored. For the apical attachment of transcripts, the dynein motor becomes a static tethering factor . Antibodg factor . AlthougS. cerevisiae SHE-dependent RNA transport and Myo2p-dependent vesicle and organelle transport both involve only a single type V myosin motor [Ustilago maydis hyphal transport of dozens of mRNAs also depends on directional transport [S. cerevisiae than in corresponding bidirectional events in Drosophila. As switching of direction is also a common feature in neurons [Drosophila. A major drawback, however, is the enormous complexity of mRNA-localization particles for instance in neurons [Drosophila that can be genetically challenged will continue to guide the way to a mechanistic understanding of mRNA-localization events in general. The direct comparison of the presented mRNA localization events suggests that in multicellular organisms rapidly alternating, bidirectional transport events by antagonizing motors is a common feature. In contrast in lower species like yeast either only one type of motor is involved or the opposing motors do not seem to constantly change direction of the transported particles. Although too few examples have been studied in sufficient detail to fully support this hypothesis, the general trend seems to follow this rule. In in motor . In the ransport . Althougransport  and thus neurons -113, bid neurons . Thus, fS. cerevisiae such reconstitution experiments yielded high mRNA specificities that are sufficient to explain specific transport in vivo [Drosophila a preference has been reported for zip-code RNAs [Yet another open issue is how specific individual mRNA transport events really are. A major problem with many mRNA localization events is the lack of biochemical insights into the composition and assembly of their corresponding mRNPs by reconstitution experiments where binding affinities can be quantified under controlled conditions. To date only in  in vivo . In the ode RNAs . HoweverU. maydis. Here, a range of RNAs are transported back and forth along microtubules without anchoring at a target site [An example where high specificity for RNA binding may not be required is mRNA localization in the fungus get site . It seemin vitro specificities of RNA-binding proteins with their apparent function in vivo, an interesting discrepancy can be observed. On one hand, several isolated RNA-binding domains lack high specificity in vitro, whereas in their cellular context their full-length proteins are involved in highly specific recognition events [ASH1 mRNA binding by the two transacting factors She2p and She3p [When comparing the reported n events , 115. Thn events -118. Othnd She3p  and the nd She3p . Because


Acropora and Montipora have almost disappeared from the reef. Indeed, almost all hard coral species have declined in abundance, with the result that total hard coral cover in 2010 (17.7%) was less than half what it was in 1985 (47.5%). In addition, macro-algal cover has increased from 11.3% in 2003 to 28.5% in 2010. The frequency of disturbance combined with possible chronic influence of a growing human population mean that a diverse reef assemblage is unlikely to persist on this reef into the future.Recurrent disturbances can have a critical effect on the structure and function of coral reef communities. In this study, long-term changes were examined in the hard coral community at Wanlitung, in southern Taiwan, between 1985 and 2010. In this 26 year interval, the reef has experienced repeated disturbances that include six typhoons and two coral-bleaching events. The frequency of disturbance has meant that species susceptible to disturbance, such as those in the genus  Coral reefs are declining rapidly on a global scale due to natural and anthropogenic disturbances Acropora, now rare or absent despite coral cover remaining relatively constant sensu 17] from corals towards a degraded state often dominated by macroalgae Diadema, causing a dramatic expansion of the Diadema population Diadema population throughout its geographic range Coral assemblages respond in a number of ways to disturbance In this study, changes in hard coral cover and composition on a fringing reef at Wanlitung were analyzed based on surveys conducted from 1985 to 2010. Wanlitung Reef is located on the west coast of Hengchun Peninsula, Kenting National Park (KNP), southern Taiwan, and had a well-developed fringing coral assemblage. However, the reef has suffered from both natural and anthropogenic disturbances, such as typhoons, coral bleaching, overfishing, and sewage discharge, in the last few decades Wanlitung Reef  is part of a fringing reef system along the west coast of the Hengchun Peninsula, southern Taiwan. Facing the junction of the Pacific Ocean (Bashi Strait) and South China Sea, it is influenced by a branch of the Kuroshio Current. Over 280 species of corals have been recorded in the area Ecological monitoring of the benthic community was conducted at Wanlitung Reef using various techniques to varying degrees of taxonomic resolution in the following years: 1985, 1987, 1999, 2003–2005 and 2008–2010 . Chang and Jan Acropora from 23% cover in 1985 to be almost locally extirpated in 2010 and Montipora that declined from 15% to 1.28%  the community was on the way towards the degraded state  in which all taxa are low in abundance. The assemblages between 2003 and 2005 (green circle) are dominated by Montipora, other corals and Faviidae but are distinct from the original 1987 assemblage due to a lack of Acropora (Changes in the relative abundance of coral taxa at Wanlitung from 1985 to 2010 are illustrated in Acropora .Acropora, are less abundant than in 1987. A six year period between 1999 and 2005, during which there were no major disturbances, allowed coral cover to return to 1987 levels. However, the Acropora have not been abundant in the assemblage since bleaching in 1998 and the genus is now virtually locally extirpated (Recurrent disturbances to the coral reef at Wanlitung have resulted in a degraded coral community in which most taxa, in particular, the structurally important tirpated . FurtherDiadema, and a potential pulse of nutrients which all contributed to a dramatic increase in algal cover, from 4% in 1977 to 92% in 1993 DiademaSimilar declines in coral cover due to natural disturbance have occurred in many other reefs worldwide. For instance, there was a dramatic loss in coral cover as a result of typhoons, from 80% in 1987 to 10% in 1989, on the southern GBR Acropora spp. in Tiahura Reef between 1979 and 2006 Acropora recruits on the reefs, suggesting that recruitment failure may also be a factor in the lack of recovery in this taxon although the cause of recruitment failure over such a long period remains unknown.The recovery of coral cover between 1999 and 2003 was associated with a shift in assemblage structure, similar to Tiahura Reef, Moorea. Recurrent disturbancesat Tiahura, including outbreaks of crown-of-thorns starfish, typhoons, and coral bleaching, have prevented the recovery of many taxa, most notably Turf and macro-algae cover were fairly stable since these taxa were first recorded separately in 2003, however, since 2008 macro-algal cover has increased 3-fold . In geneNumerous disturbances over the last 23 years, in particular typhoons and bleaching, have caused long-term changes to coral reef at Wanlitung, however, local stressors such as overfishing, pollution, and coastal development are also likely to have contributed to these changes. Without major changes to current development practices in Kenting, these reefs are unlikely to recover in the near future.


Most whole blood RNA stabilization and isolation methods also require significant volumes of blood, limiting their effective use in small animal species, such as rodents. To overcome these challenges, a QIAzol-based RNA stabilization and isolation method (QSI) was developed to isolate sufficient amounts of high quality total RNA from 25 to 500 μL of rat whole blood. The method was compared to the standard PAXgene Blood RNA System using blood collected from rats exposed to saline or lipopolysaccharide (LPS). The QSI method yielded an average of 54 ng total RNA per μL of rat whole blood with an average RNA Integrity Number (RIN) of 9, a performance comparable with the standard PAXgene method. Total RNA samples were further processed using the NuGEN Ovation Whole Blood Solution system and cDNA was hybridized to Affymetrix Rat Genome 230 2.0 Arrays. The microarray QC parameters using RNA isolated with the QSI method were within the acceptable range for microarray analysis. The transcriptomic profiles were highly correlated with those using RNA isolated with the PAXgene method and were consistent with expected LPS-induced inflammatory responses. The present study demonstrated that the QSI method coupled with NuGEN Ovation Whole Blood Solution system is cost-effective and particularly suitable for transcriptomic profiling of minimal volumes of whole blood, typical of those obtained with small animal species.Blood is an ideal tissue for the identification of novel genomic biomarkers for toxicity or efficacy. However, using blood for transcriptomic profiling presents significant technical challenges due to the transcriptomic changes induced by  To daPeripheral blood has obvious advantages for biomarker discovery due to its non-invasive collection and availability. In addition, biomarkers identified in blood should be easily translated from preclinical species to humans. A number of studies have demonstrated that transcriptomic changes in peripheral blood can serve as biomarkers of exposure to xenobiotics or as biomarkers for pathological changes occurring in other tissues –8.Peripheral blood mononuclear cells (PMBCs) can be used for genomics biomarker studies since they are the most transcriptionally active cells in blood. However, the extra fractionation procedure required for PBMCs can have a significant impact on the transcriptome . IsolatiFirst, RNA degradation and transcriptomic changes can occur quickly after the blood is drawn from subjects ,11. TradAttempts to overcome these hurdles led to the development of a number of new approaches for blood RNA stabilization . One of The second major concern for whole blood transcriptomic analysis is the interference from globin mRNA. Whole blood contains abundant α- and β-globin mRNA transcripts that comprise up to 70% of total mRNA . Such a In order to overcome these challenges, we have developed a simple whole blood processing method that immediately stabilizes RNA upon blood collection and yields high quality RNA from as little as 25 μL of rat whole blood. To circumvent the need for a separate globin reduction procedure, the NuGEN Ovation Whole Blood Solution was employed for microarray sample preparation. The NuGEN approach hybridizes biotinylated cDNA onto the microarray and has been shown to have sufficient sensitivity and reproducibility with small amounts of input RNA . Using b2.2.1.2.1.1.Total RNA was isolated from 2.5 mL rat whole blood using the standard PAXgene method or from 500 μL rat whole blood using the QSI method. When normalized with blood volume, the total RNA yield from the PAXgene method was approximately twice the yield from the QSI method .Given the small amount of input RNA required for NuGEN Whole Blood Solution, both isolation methods yielded a sufficient amount of RNA for microarray analysis. RNA quality from both isolation methods was excellent with an average RIN score of 9.2.1.2.P) for the RNA samples isolated using the QSI method were generally lower than those produced from the PAXgene method . However, there was no significant difference in the GAPDH 3′/5′ ratios and β-actin 3′/5′ ratios between the PAXgene and the QSI methods.The array performance metrics are summarized in 2.1.3.p-value = 0.44 and 0.43, respectively).LPS induced the highest number of gene expression changes in rat blood at 2 hrs, followed by 6 hrs and 24 hrs. 2 was over 0.9 in any abundance category.To address whether blood RNA isolation methods could selectively affect transcripts of different abundance, the gene expression levels of a panel of nine transcripts representing various abundance levels were further evaluated using the 2-hr time point samples. The signal intensities of the low abundance transcripts were less than 3-fold of the microarray background signals. The intensity of the mid abundance transcripts were 14–26-fold above background levels, while the intensities of the high abundance transcripts were over 80-fold above background, representing the top 5th percentile of the rat genome. Each abundance category contained a transcript that was up-regulated, down-regulated, or unchanged (less than 2-fold relative to control treatments) by LPS treatment. The list of transcripts is provided in the supplemental table. As shown in r2 > 0.9.To evaluate the impact of the RNA isolation method on characterization of biological functions, blood transcriptomic profiles were also subjected to pathway analysis using Ingenuity Pathway Analysis Software. The analysis was focused on the samples from the 2-hr time point since they represented the peak transcriptomic changes induced by LPS. As expected, LPS induced gene expression changes consistent with perturbation of immune responses. Regardless of the RNA isolation method, the top five regulated physiological functions were: hematological system development and function, immune cell trafficking, cell-mediated immune response, and tissue morphology. In addition, the top five most impacted canonical signaling pathways were the same with the two RNA isolation methods . Figure 2.1.4.To confirm the microarray results, the expression levels of the same panel of 9 transcripts representing different abundance levels were measured by real-time RT-PCR using 2-hr blood RNA samples isolated with the PAXgene method. Consistent with the microarray results, the transcripts were shown to be regulated in the same direction for all abundance levels . The tra2.1.5.To assess if small blood volumes are adequate for transcriptomic profiling using the QSI method, various volumes of whole blood (25–200 μL) were collected from rats treated with saline or LPS for 2 hrs. Transcriptomic profiles were generated using the QSI method for RNA isolation followed by the NuGEN method for RNA amplification and target labeling. When normalized with blood volume, the smaller amount of blood input yielded the greatest amount of total RNA produced by volume . FurtherThere were no significant differences in microarray performance using RNA isolated from various volumes of blood . Figure r2 > 0.9 between the two sets of samples   weighing approximately 250 g were obtained from Charles River Laboratories, Inc, Portage, MI, USA. The animals were permitted non-certified Rodent Chow and water ad libitum. Rats were housed two or three per cage for two days after receipt to aid in acclimation.Male Sprague-Dawley rats [Crl:CDGuiding Principles in the Use of Animals in Toxicology [Thereafter, rats were single housed in ventilated, stainless steel, wire bottom hanging cages equipped with feeders and an automatic watering system. The animals were administered a single intravenous injection of vehicle  or LPS  and euthanized 2, 6, or 24 hrs post dosing (3 rats/time point/group). Blood samples were collected via the abdominal vaudal vena cava at necropsy for RNA isolation . For thexicology  and appr3.2.RNA isolation from samples collected in PAXgene Blood RNA Tubes was performed according to the manufacturer’s specifications (BD Biosciences). For the QSI method, total RNA was isolated from 750 μL of blood-QIAzol lysate following the Qiagen Automated BioRobot 3000 RNeasy-96 RNA isolation protocol (QIAGEN). After manual centrifugation of a 96-well plate containing QIAzol lysate/chloroform mixture, the Qiagen BioRobot 3000 automatically transfers the upper, aqueous phase to a 96-well RNeasy plate for automated RNA purification. Nucleic acid concentration was determined by O.D. 260 nm . The RNA integrity was evaluated and an RNA Integrity Number (RIN) was generated using an Agilent 2100 Bioanalyzer and its accompanying software .3.3.All blood RNA samples were processed to generate microarray hybridization targets using the Ovation Whole Blood Solution , a module containing three NuGEN products. Briefly, 50 ng of DNAase-treated RNA aliquots were amplified using the Ovation RNA Amplification System V2 coupled with the Ovation WB Reagent following the manufacturer’s instructions (NuGEN). Approximately 4.4 μg of each amplified single strand cDNA sample was then biotinylated and fragmented using the FL-Ovation cDNA Biotin Module V2. The resulting cDNA was hybridized to an Affymetrix Rat Genome 230 2.0 Array  using the NuGEN-modified hybridization protocol, with 2 min of heat denaturation of the hybridization cocktail at 99 °C and a minimum of 16 hrs of hybridization at 45 °C using an Affymetrix Hybridization Oven 640. The array was subsequently washed and stained with streptavidin-phycoerythrin (Molecular Probes) on the Affymetrix Gene-Chip Fluidics Workstation 400 following the NuGEN-recommended protocol EukGE-WS2v4_450. The array was then scanned using an Affymetrix GeneChip Scanner 3000.3.4.Tcf7 (Rn_00493446_m1), Map2k6 (Rn_00586764_m1), Nrp1 (Rn_00595457_m1), Alas2 (Rn_00566201_m1), Flad1 (Rn_01512487_m1), Canx (Rn_00596877_m1), S100a9 (Rn0585879_m1), Tnf (Rn_99999017_m1), Il1b (Rn_01514151_m1), and 18S (Hs_99999901_s1). Expression levels of each gene were quantified by real-time RT-PCR with an ABI Prism 7900 Sequence Detection System . Briefly, 200 ng of total RNA was reverse transcribed using the iScriptTM cDNA Synthesis Kit according to the manufacturer’s instructions (Bio-Rad Laboratories). Real-time PCR analyses were then performed in a total volume of 10 μL containing 1:15-diluted synthesized cDNA using Taqman® Gene Expression Assays (Applied Biosystems). The PCR cycling profile was as follows: 10 min at 95 °C, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The relative fold changes were calculated using the delta delta Ct method and normalized to 18S ribosomal RNA, the endogenous control gene.The following primers and probes were selected from the Applied Biosystems Assays-on-Demand gene expression products: 3.5.α = 0.05. For microarray analysis, the scanned image and intensity files were imported into Rosetta Resolver gene expression analysis software version 7.2 . Individual gene expression ratios were built for each treatment animal versus the averaged vehicle controls at the corresponding conditions using the Rosetta Resolver error model [in silico pool of treated animals versus the corresponding vehicle controls using the Rosetta Resolver error model. Genes with a fold change over 2 and a p-value < 0.01 were mapped to pathways and biological functions using Ingenuity Pathway Analysis .All non-microarray statistical analyses were conducted using JMP 7.0.1 statistical software from SAS . Comparisons were made using the two-tailed t-test with a significance value of or model . Genes a4.In the present study, we have demonstrated that the QSI method can produce sufficient quantities of high quality RNA from small volumes (25–500 μL) of rat whole blood. Furthermore, the QSI method can generate microarray results comparable to the standard PAXgene method. Given the simplicity of this blood collection procedure and the minimal volume of blood required, the method can be easily applied for blood genomics profiling in pre-clinical species.


RNA helicase plays an important role in host mRNA and viral mRNA transcription, transport, and translation. Many viruses utilize RNA helicases in their life cycle, while human immunodeficiency virus type 1 (HIV-1) does not encode an RNA helicase. Thus, host RNA helicase has been involved in HIV-1 replication. Indeed, DDX1 and DDX3 DEAD-box RNA helicases are known to be required for efficient HIV-1 Rev-dependent RNA export. However, it remains unclear whether distinct DDX RNA helicases cross-talk and cooperate to modulate the HIV-1 Rev and Tat function.To investigate the potential role of distinct DDX DEAD-box RNA helicases in HIV-1 Rev or Tat function, we used the Rev-dependent luciferase-based reporter plasmid pDM628 or HIV-1-LTR-Luc reporter plasmid. 293FT cells were cotransfected with several HA-tagged DDX, including DDX1, DDX3, DDX5, DDX6, DDX17, DDX21, DDX56, pDM628 or pHIV-1-Luc, and/or HIV-1 Rev or Tat expression plasmid. We also examined immunofluorescence and immunoprecipitation analysis to analyze the interaction of DDX with Rev or Tat protein.In this study, we noticed that distinct DDX RNA helicases, including DDX1, DDX3, DDX5, DDX17, DDX21, DDX56, except DDX6, bound to the Rev protein and they colocalized with Rev in nucleolus or nucleus. In this context, these DEAD-box RNA helicases except DDX6 markedly enhanced the HIV-1 Rev-dependent RNA export. Furthermore, DDX3 interacted with DDX5 and synergistically enhanced the Rev function. As well, combination of other distinct DDX RNA helicases cooperated to stimulate the Rev function. On the other hand, DDX3 also interacted with HIV-1 Tat and modulated Tat-induced transcription. Indeed, the ATPase-dependent RNA helicase activity of DDX3 seemed to be important for both Rev and Tat function. In contrast, other nuclear and nucleolar DDX RNA helicases, including DDX5, DDX17, DDX21, and DDX56, could interact with HIV-1 Tat, while these DEAD-box RNA helicases did not coactivate the Tat-induced transcription.Altogether, these results suggest that distinct DDX DEAD-box RNA helicases cooperate to modulate the HIV-1 Rev and Tat function.


Begomoviruses that have adapted to a wide host range and are able to cause major diseases in agronomic crops. We analyzed the efficacy of induced resistance to PepGMV in Nicotiana benthamiana plants with two constructs: one construct with homologous sequences derived from PepGMV, and the other construct with heterologous sequences derived from ToChLPV. Plants protected with the heterologous construct showed an efficacy to decrease the severity of symptoms of 45%, while plants protected with the homologous construct showed an efficacy of 80%. Plants protected with the heterologous construct showed a reduction of incidence of 42.86%, while the reduction of incidence in plants protected with the homologous construct was 57.15%. The efficacy to decrease viral load was 95.6% in plants protected with the heterologous construct, and 99.56% in plants protected with the homologous construct. We found, in both constructs, up-regulated key components of the RNAi pathway. This demonstrates that the efficacy of the constructs was due to the activation of the gene silencing mechanism, and is reflected in the decrease of viral genome copies, as well as in recovery phenotype. We present evidence that both constructs are functional and can efficiently induce transient resistance against PepGMV infections. This observation guarantees a further exploration as a strategy to control complex Begomovirus diseases in the field.ToChLPV and PepGMV are  Begomovirus is the largest genus in the Geminiviridae family and includes approximately 192 species that infect dicot plants; are transmitted by the Bemisia tabaci whitefly species complex, and can be either monopartite (containing a single DNA-A component) or bipartite (DNA-A and DNA-B) [Begomoviruses have become one of the most important groups of plant viruses in tropical and subtropical regions and are responsible for the emergence of new diseases that cause important yield losses [Pepper golden mosaic virus (PepGMV) is one of the most widely distributed Begomoviruses species affecting tomato (Solanum lycopersicum L.), and pepper (Capsicum annuum L.) and it is disseminated over a large geographic area spanning southwestern United States, Mexico and Central America [BegomovirusesPepper huasteco yellow vein virus (PHYVV) [Tomato yellow leaf curl virus (TYLCV) [Physalis ixocarpa Brot.) and tobacco [Tomato chino La Paz virus (ToChLPV), has been found infecting tomato plants in the Baja California Peninsula [Solanum rostratum Dunal), and pepper plants in Sinaloa, San Luis Potosi, and Baja California Sur, respectively [d losses . Pepper  America . PepGMV, (PHYVV) ,6 and To (TYLCV) . These c tobacco ,5. Anotheninsula . In addiectively ,11. The ectively ,12,13. Begomoviruses. The AC1 gene has been an excellent target to generate broad resistance because it: i) is highly conserved; ii) is involved in the modulation of geminiviral gene expression, and iii) would impact viral replication by reducing viral DNA accumulation [A biotechnological tool that has been used to induce pathogen-derived resistance against begomoviral diseases in plants is the RNA interference (RNAi) ,15,16. Rmulation ,27,28,29Effects and effectiveness to evaluate transgenes are commonly based on severity of symptoms and incidence of the disease, which are analyzed by PCR and dot blot hybridization . In addiNicotiana benthamiana plants by Agrobacterium-mediated transient assay. Also, we analyzed by microarray hybridization the altered mRNAs to detect key components related to silencing pathways.The present study reports the engineering of two RNAi constructs based on the AC1-IR-AV1 region of PepGMV and ToChLPV. We evaluated their efficacy to induce resistance to PepGMV infections in Pepper golden mosaic virus (PepGMV), we engineered two hairpin-type (hpRNA) constructs designed to express dsRNA of the AC-IR-AVI segment of PepGMV and ToChLPV. The constructs were designed to express an intron-hairpin comprising the ≈146 nts of the 5´ end of the AV1 gene, the entire (326 nts) intergenic region (IR) and 714 nts of the 5´ end of the AC1 gene, the AC1-IR-AV1 segment total comprising 1186 nts . The presence of transgenes was detected by PCR. To evaluate the resistance efficacy against N. benthamiana plants with an infectious clone derived from PepGMV. In the resistance analysis, all the non-infected plants (negative control) showed a normal phenotype (The resistance generated by CIRP (Construct of the Intergenic Region of PepGMV) and CIRT (Construct of the Intergenic Region of ToChLPV) was determined by challenging henotype A. All poN. benthamiana plants infiltrated with either CIRP or CIRT and inoculated with PepGMV, did not show severe symptoms or showed only attenuated symptoms  of the amount of starting quantities (SQ) to the amount of the endogenous 18S rRNA gene was y = –3.450x + 13.0, with R² = 0.999 (efficiency of 94.9%). The standard curve to PepGMV AV1 gene was y = –3.385x + 18.5, with R² = 0.989 (efficiency of 97.4%).-1 copies. With CIRT we observed 2.27 × 10-2 copies and 2.20 × 10-3 copies in CIRP . Components common to both CIRT and CIRP treatments were: RNA polymerase IV, DCL3, AGO4, DRD1, DDM1, CMT3, and MET1. Other components of RNA silencing were differentially expressed (greater 1.5 up) in either CIRT or CIRP treatments. Components particular to CIRT included AT2G35160.1, which encodes SU(var)3-9 homolog 5 (SUVH5); AT3G15390.1, encoding silencing defective 5 (SDE5); AT3G49500.1, encoding RNA dependent RNA polymerase (RDR6); AT4G14200.1, encoding pentatricopeptide repeat (PPR), and AT5G23570.1, which encodes SGS3. Components particular to CIRP included AT1G08060.1, encoding Morpheus molecule maintenance of methylation ; AT1G09700.1, encoding dsRNA-binding protein 1 (DRB1); AT3G22680.1, encoding RNA-directed DNA methylation 1 (RDM1); AT3G42670.1, encoding chromatin-remodeling 38 (CHR38), and AT4G11130.1, encoding RNA-dependent RNA polymerase 2 (RDR2).To determine whether the efficacy of CIRT and CIRP was due to RNAi, we analyzed the transcriptome of Our results provide evidence that both CIRT and CIRP constructs are highly efficient to suppress the multiplication of a viral genome and can be explored in the control against PepGMV infections and other related species involved in mixed infections. Our results agree with several study groups , indicatPrevious studies mentioned that to obtain optimal results with heterologous RNAi constructs, the nucleotide (nt) identity should be at least 85% . NucleotIn a partial region of the AC1 gene, we found three ≈24 nt-long  that havIt has been documented that resistance is broad and effective against strains of the virus from which the AV1 gene is derived, or even against closely related virus species. The use of the non-coding IR region is an interesting strategy to induce broad resistance against other heterologous geminiviruses. This is because the generation of 24 nt siRNAs derived from activity of DCL3 modifies methylation processes, which is considered the principal mechanism of antiviral defenses through transcription gene silencing .Begomovirus-complex diseases.In addition, it has been reported that dsRNA can be generated by single and specific siRNA that can be elongated and consequently generate more specific siRNAs, thereby imparting resistance not only against homologous but also against heterologous virus species . The anaXbal to confirm the presence of the sense and antisense arms and orientation of the inverted repeat sequences. CIRP and CIRT were transformed into Agrobacterium tumefaciens GV2260 by electroporation (Bio-Rad Gene Pulser II Electroporator). The construct designed with PepGMV is homologous in respect to the challenging virus (PepGMV) and it was named CIRP (Construct of the Intergenic Region of PepGMV). The second construct derived from ToChLPV is considered heterologous and it was named CIRT (Construct of the Intergenic Region of ToChLPV). The generated amplicons to be used in the constructs were based on the degenerate primers PAL1v1978-PAR1c496  with addN. benthamiana seeds were grown for 30 days in chambers at 28 °C with a photoperiod of 16 h light: 8 h dark. To evaluate the protective effect against PepGMV infection and the effectiveness in the activation of the RNAi response, we conducted a completely randomized study consisting of four treatments: (1) CIRP, plants agro-infiltrated with CIRP and then inoculated with PepGMV four days after infiltration; (2) CIRT, plants agro-infiltrated with CIRT and then inoculated with PepGMV four days after infiltration; (3) positive control, plants agro-infiltrated with the empty vector (pH7GWIWG2(II)) and then inoculated with PepGMV four days after infiltration; and (4) negative control, plants agro-infiltrated with the empty vector but not inoculated with PepGMV. Eight plants per treatment were agro-infiltrated in the four-leaf stage according to a previously published protocol [protocol . The inoprotocol . The datSymptom severity was evaluated based on the following adjusted scale: 0, no visible symptoms; 1, presence of visible mild symptoms; 2, some yellowing and minor curling of leaf ends; and 3, severe symptoms of curling throughout the plant . The perThe incidence was calculated based on the detection of viral transcripts by PCR using the degenerate primer pair AC1048-AV494 . The for™EvaGreen (BioRad), 0.1 µL of specific primers (250 µM) and 1 µL of the DNA sample and was conducted in 96-well optical plates in the CFX96 qPCR Detection System (BioRad). The cycling parameters were as follows: 1 cycle at 95 °C/30 s and 39 cycles each consisting of 95 °C/0.05 s and 60 °C/20 s. Following amplification, a melting curve analysis program was performed to verify the amplified products by their specific melting temperatures (Tm) from 65.0 °C to 95.0 °C at 0.5 °C/0.05 s increments. For each sample, three replicates were amplified in parallel. Two standard curves were prepared: the first from the target gene  and the second from the internal report (plasmid containing the18S rRNA gene). The plasmid was serially diluted 10-fold, from 10 ng DNA/µL. Standard curves were obtained by linear regression analyses of the threshold cycle (Ct) value of each of the three standard-dilution replicates over the log starting quantity of DNA. Results were expressed as the log of the ratio of the quantity of virus DNA [The quantification of the PepGMV load was determined by qPCR and it was carried out using PepGMVCPq5 and PepGMVCPq3 primers that amplify 104 bp of the AV1 gene . The endirus DNA ,50. DataThe efficacy (E) of the RNAi system was calculated as a percentage of reduction of the disease in protected plants with respect to unprotected plants and based on the ability to decrease the disease severity, incidence and viral load. The efficacy of variables was calculated with the formula: E (%) =  for each variable). Arabidopsis thaliana V.3.0.3. microarrays’ for 14 h at 42 ºC, using hybridization solution UniHyb (Arraylt). Data acquisition, processing and quantification of the images obtained from the microarray hybridization were performed in an Array-Pro Analizer 4000 with the corresponding software (Packard BioChips). To perform the analysis of microarray data and select genes that are significantly differentially expressed between classes of samples, we used the genArise package developed by the Computer Unit of the Institute of Cellular Physiology (UNAM). Data were normalized and the mean and standard deviation of the distribution of log2 (ratio) values were calculated, defining a global fold-change difference and confidence. Genes were considered to be differentially expressed with a q value ≤0.05 and a fold change cut off of 1.5 (Z-score). The databases Arabidopsis Information Resource (TAIR), Blast2GO, Database for Annotation Visualization and Integrate Discovery (DAVID) V6 were used to analyze Gene Ontology (GO). The microarrays data have been submitted to the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/).Per treatment, i.e. CIRT, CIRP and positive control, two independent biological replicates were used to monitor differences in gene expression between treatments. For each biological replicate, three leaves from each plant per treatment were harvested, pooled, flash-frozen, ground in liquid nitrogen, and stored at –80 ºC. Total RNA was extracted using TRIzol (Invitrogen). Of total RNA, 10 µg were used for cDNA synthesis with incorporation of fluorophores Alexa fluor 555 and Alexa fluor 647. An equal quantity of labeled cDNA from either CIRT or CIRP was hybridized, together with labeled cDNA from controls, to the collection of ‘N. benthamiana. We found several up-regulated key components of the RNAi pathway. This demonstrates that the protection efficacy is due to the activation of the gene silencing mechanism, inducing efficiently an RNA-mediated cross-protection. Heterologous RNAi constructs are an interesting model to study antiviral strategies, a step forward for the control of PepGMV, and to understand the complex interaction between Begomovirus, RNAi, and the host.This study shows that CIRP and CIRT constructs are efficient to induce resistance against infections caused by PepGMV in


Since the emergence of next generation sequencing platforms, unprecedented opportunities have arisen in the study of natural vertebrate populations. In particular, insights into the genetic and epigenetic mechanisms of adaptation can be revealed through study of the expression profiles of genes. However, as a pre-requisite to expression profiling, care must be taken in RNA preparation as factors like DNA contamination, RNA integrity or transcript abundance can affect downstream applications. Here, we evaluated five commonly used RNA extraction methods using whole blood sampled under varying conditions from 20 wild carnivores.Despite the use of minute starting volumes, all methods produced quantifiable RNA extracts (1.4 – 18.4 μg) with varying integrity (RIN 4.6 - 7.7), the latter being significantly affected by the storage and extraction method used. We observed a significant overall effect of the extraction method on DNA contamination. One particular extraction method, the LeukoLOCK™ filter system, yielded high RNA integrity along with low DNA contamination and efficient depletion of hemoglobin transcripts highly abundant in whole blood. In a proof of concept sequencing experiment, we found globin RNA transcripts to occupy up to ¼ of all sequencing reads if libraries were not depleted of hemoglobin prior to sequencing.By carefully choosing the appropriate RNA extraction method, whole blood can become a valuable source for high-throughput applications like expression arrays or transcriptome sequencing from natural populations. Additionally, candidate genes showing signs of selection could subsequently be genotyped in large population samples using whole blood as a source for RNA without harming individuals from rare or endangered species. Adapting to an ever-changing environment is one of the most crucial functions of living organisms. Although we have a profound understanding of how some adaptive mechanisms have shaped phenotypic traits i.e. -3), thei, thei3])Whole blood offers several advantages for transcriptome studies on non-model organisms but is surprisingly underrepresented in the recent literature. Blood has an important role in mediating between the environment and the organism and hence exploring gene expression profiles derived from blood may provide deeper insight into immune response or stress metabolism -22. FurtA second challenge of using whole blood for transcriptome profiling is the heterogeneity of blood cells and the high content of interfering hemoglobin mRNAs. Globin mRNA in mammalian whole blood is estimated to account for 70% of all mRNA transcripts (i.e. ), an excAs encouraging as these preservation methods appear, to our knowledge, their performance has never been evaluated by concurrent experiments on different species and using similar collection conditions. In the present study, we evaluated four commercially available RNA preservation buffers applied to whole blood samples in seven different carnivore species. Furthermore, we explored the performance and the suitability of a new whole blood filter system for application under field conditions. All five sampling methods were evaluated with regard to RNA quality including DNA contamination and RNA yield as well as the integrity of the isolated RNA. In a proof-of-concept-experiment, we modified and validated conditions of mRNA preparation from whole blood samples for NGS applications. Finally, we provide guidelines on how to preserve and process RNA extracts from whole blood sampled from wildlife populations.We evaluated different RNA extraction methods applied to whole blood sampled under field conditions that resemble situations researchers face when investigating non-model organisms in their natural habitat. We used samples collected from 20 carnivores and RNA from each individual was preserved using five methods  resulting in a total of 99 RNA extractions  that filters leukocytes and depletes the amount of reticulocytes prior to extraction. We found that among the 19 pre-filtered extracts only four samples yielded PCR amplifications. This corresponds to only 11% amplification rate .When testing if the extraction method, the species or individual factors of each animal affected the DNA contamination in the 99 RNA samples, only the extraction method revealed a significant correlation . The A260/280 ratio averaged between 1.9 and 2.1 for all extraction kits : 5.0-43.9 μg using RiboPure™; 0–6.2 μg for the RNeasy®; 0.1-10.2 μg for the PAXgene™ and 0.2-15.1 μg for samples extracted using TRIzol® LS ; 6.9 ± 2.6 RNeasy® (stored in RNAprotect®); 7.7 ± 1.2 for PAXgene™ (stored in PAXgene™ blood buffer), 6.2 ± 2.9 TRIzol® LS . The values for RNA integrity differed significantly between methods (ANOVA: p = 0.007). In comparison to RNeasy® and PAXgene™, whole blood samples extracted with TRIzol® LS or RiboPure™ showed a broad range of well-preserved RNA samples to RNA samples totally degraded. Due to low RNA concentrations in 12 samples, we lowered the threshold parameters in the Agilent Bioanalyzer software for these particular samples in order to obtain a RIN value. These samples included a total of seven study animals  and all extraction/ storage methods were affected.We further investigated the integrity of each RNA extract as indicated by the RIN factor and observed the following average values: 4.6 ± 2.3 for samples extracted using RiboPure™  but these two factors are not mutually exclusive  when compared to those collected in the field (5.8 ± 1.2). Furthermore, we observed a significant effect of the sample collector as well as the species (p = 0.002 and p = 5.872e-16). However, the species were sampled by different researchers and this could consequently also contribute this association.In order to evaluate a potential reduction of hemoglobin transcripts in the filtered extracts, we used a quantitative PCR assay to estimate the relative ratio between hemoglobin and three housekeeping genes and compared them to the values derived from the four other whole blood extraction methods. The average log transformed ratio for hemoglobin in filtered blood was significantly higher than that of whole blood  and B2 with 35.0% (109 out of 309 contigs). We extracted the number of reads per gene in order to evaluate a potential over-representation of particular genes in the sample. Additional file The principle aim of this study was to assess the performance of commercially available kits and protocols commonly used to preserve and extract RNA from blood samples. We used total RNA yield, RNA preservation (RIN) and the degree of DNA contamination as measures of performance.DNA contamination is particularly worrisome if expression profiles are generated with high-throughput methods because even a single contaminating molecule can be detected, making sequencing efforts less efficient and potentially leading to false biological conclusions. Our DNA contamination tests did reveal a substantial amount of DNA co-extracted with all RNA extraction protocols for whole blood. Between 80.0-95.0% of the whole blood RNA extracts resulted in at least one single DNA amplicon Table . We obseThe applied DNase depletion protocols recommended for each of the tested method should in theory have removed moderate amounts of DNA (<50 μg DNA/ ml RNA extract). Surprisingly, irrespective of the extraction method, an additional DNase treatment was required in order to ultimately deplete any trace of DNA, which indicates severely contaminated RNA extracts. This is somewhat alarming and in agreement with results reported for other RNA extraction methods . Our finAside from DNA, other contaminants of RNA extracts derive from incomplete removal of cellular components such as proteins, lipids and carbohydrates or traces of salt and organic solvents stemming from the extraction procedure itself. With regard to protein depletion in RNA extracts, determined by the A260/280 ratio, all methods yielded samples with a ratio averaging above 1.9, which is considered suitable for NGS ,37. In cAfter depletion of interfering DNA, an accurate quantification of extracted RNA was possible. Depending on the extraction kit, the RNA yield for 500 μl blood is supposed to range between 1.6 μg up to 55 μg. While the yield obtained for PAXgene™ samples is similar to that suggested by the manufacturers (0.1-10.2 μg/500 μl blood vs. 1.6- 3.2 μg/500 μl blood as tested by the manufacturer), the amount of RNA retrieved using the RNeasy® kit was much lower than stated (0–6.2 μg/500 μl blood vs. 25–35 μg/ 500 μl blood). Many factors, such as the individual hydration condition of an animal or blood density differences in certain parts of the body  can inflIntriguingly, our study showed that samples extracted using organic solvents like phenol/chloroform (e.g. RiboPure™ kit) or TRIzol® LS reagent resulted in the highest RNA yields  as well as the preservation of the RNA (ANOVA: p = 0.002). The reasons for this pattern might include individual differences in sample handling procedures. However, we need to stress the fact that the effect of collectors and species could not be differentiated since in most cases samples from one species were collected by a single person under unique conditions  produced the extracts with the lowest and most widely scattered RIN values rendering its suitability for high-throughput applications questionable. This was surprising since both storage buffers have extensively been used to preserve RNA from a broad range of animal tissues including brain, liver, testis, tissue culture cells or white blood cells in many different species . -41. latees . We fountegrity ,37. Howea priori filters leukocytes from whole blood and by depleting an excess of hemoglobin mRNA, it ultimately reduces the amount of downstream inhibitors in RNA extracts. However, this filtering procedure comes with a cost as the average yield of 1.2 μg/ 500 μl blood in the LeukoLOCK™ filter system was the lowest in our analysis. Although low in quantity, the RNA itself was well preserved as indicated by high RIN values , this nInsufficient elimination of reticulocytes from whole blood RNA extracts not only affects DNA contamination but might also change the ratio of highly abundant versus rare transcripts. Hemoglobin is highly expressed in blood and warrants special treatment ,32,44. AWhether finding the causative mutations or simply cataloguing the wealth of expressed genes is a priority, normalizing the RNA extract is beneficial ,47. In alater® soaked membrane of the filter system allows flexibility in handling time as the RNA is stable for 24 hours at 37 °C, one week at 25 °C and one month or more at 4 °C. Second, the LeukoLOCK™ system works reliably even with small amounts of starting material. The system was originally designed for 9 ml human blood but we have demonstrated that much smaller volumes (1 ml whole blood) are sufficient as well and might therefore become the method of choice for small species or individuals that are difficult to handle or suffering from stress. One caveat about the system is the complex sample processing, which requires a certain familiarity with the steps in the protocol. However with appropriate practice and precautions the benefits of this system outweigh the extra effort, particularly in remote areas with limited access to electricity and refrigeration.The ultimate decision about which RNA extraction method to apply may reflect a balance between performance, the anticipated laboratory effort, the project’s logistics and finances and the scope of the project. Considering all tests employed in this study, we conclude that the most promising RNA storage and extraction method is the LeukoLOCK™ filter system. Besides its performance, the method includes practical amenities well suited for field applications. First, the RNAThe second best performing method was the PAXgene™ kit. The preservation buffer is intended for stabilization of blood for as much as three days at 18- 25 °C or for 5 days at 2-8 °C. This method has been designed for medical applications and is therefore more expensive than other kits. We would like to stress that great care has to be taken to ensure a sufficient removal of DNA co-extracted with this RNA extraction kit.We show that whole blood can be a valuable resource for transcriptome studies and that the efficiency of NGS technologies can be maximized by employing appropriate library preparation methods . Consequently, whole blood is a promising and easy accessible RNA source that has great potential for NGS studies in ecological and conservation genetics.Canis lupus baileyi), four grey foxes (Urocyon cinereoargenteus), three bobcats (Lynx rufus), two coyotes (Canis latrans), four California sea lions , two harbor seals (Phoca vitulina) and one California sea otter (Enhydra lutris) in different facilities/ field settings , RNAprotect® (Qiagen), PAXgene™ blood buffer  and TRIzol® LS Reagent (Invitrogen)  using the PAXgene™ blood buffer [later®. Once the filter was saturated with RNAlater®, it was sealed until further use. All samples were shipped to the lab at UCLA immediately after collection in the field and kept at −20 °C until further processing. Each individual’s blood was sampled at the same time for all five approaches simultaneously, except for one coyote where we failed to obtain a LeukoLOCK™ sample . However, some modifications were incorporated when using the PAXgene™ buffer as suggested for low amounts of starting materials . Species2, 4% DMSO, 0.2 mM each dNTP, 0.5 μM forward and reverse primer mix and 1 μl of the non-transcribed RNA. The PCR was performed on an Eppendorf Mastercycler EP Thermal cycler (Eppendorf) using the following cycling steps: initial denaturation at 94 °C for 9 minutes, denaturation at 94 °C for 20 seconds, annealing temperature 56 °C for 20 seconds, extension at 72 °C for 20 seconds; final extension at 72 °C for 4 minutes; 40 cycles total. PCR amplification was evaluated on a 2% Agarose gel stained with SYBR Safe DNA gel stain (Invitrogen) and visualized under UV light. Samples that successfully amplified in at least two out of the four PCRs were subjected to an additional DNase treatment using 4 U of rDNaseI (Ambion) according to the manufacturer’s recommendation.Extracted RNA samples were subjected to kit specific or recommended DNase treatments. The efficiency of DNA removal was determined in “no-RT” control PCR using a regular polymerase. We targeted four loci (see section below) by using the following conditions. A mastermix was prepared with 0.2 U TaqGold (Applied Biosystems), 1x buffer Gold, 1.5 mM MgClIn order to evaluate the RNA yield, 10 μl of 1:10 diluted, DNA-free RNA was used in a Quant-iT™RiboGreen® RNA reagent assay (Invitrogen), which uses a dye that intercalates with nucleic acids and absorbance (260 nm) could be measured at with a multimode DTX880 plate reader (Beckman Coulter). RNA integrity was assessed at the UCLA Sequencing and Genotyping Core using the RIN values calculated on an Agilent 2100 Bioanalyzer (Agilent Technologies).In order to assess whether prior filtering (LeukoLOCK™ filter system) would efficiently reduce the amount of hemoglobin transcripts compared to the whole blood approaches, we performed a qPCR targeting ß- hemoglobin as well as three housekeeping genes for all extracted samples. A volume of 4 μl undiluted DNA-free RNA was reverse transcribed using SuperScript III First-Strand Synthesis Super Mix (Invitrogen). Since random hexamers have shown to result in an overestimation of mRNA copy number by up to 19-fold , we usedUbc), Phospholipase A2 (YWHAZ) and the human acidic ribosomal protein .st strand cDNAs were diluted 1:100. Additionally, samples that were filtered through the LeukoLOCK™ system were first diluted 1:2 in order to account for the 1 ml whole blood starting material. 1 μl of each dilution was used in a 10 μl reaction of LightCycler 480 SYBR Green I Master mix (Roche). All samples from one individual were run at the same time in duplicates and on the same plate to reduce nuisance parameters such as technical variations within individuals. We included two DNA samples as a positive control/ positive calibrator on each plate. All primers were used in a 0.5 μM end concentration. The LightCycler protocol included 5 minutes activation at 95 °C, 10 seconds denaturing at 95 °C, primer annealing for 15 seconds at 55 °C, elongation for 30 seconds at 72 °C and a melting curve . The last step was a cooling step at 40 °C for 10 sec. The samples were subjected to 50 cycles. In addition to the qPCR set up, we evaluated the PCR efficiency for each primer pair by including 12 diluted DNA samples .1Blood samples from two confined wolves (Mexican and Arctic wolf) were collected into PAXgene™ blood tubes and prepared for sequencing utilizing Roches’ 454 GS FLX Titanium platform following four different procedures. After the extraction of RNA, each extract was treated differently Table , allowinR (http://www.R-project.org).To first evaluate the extent of DNA contamination of each RNA extract, we used two different analytical approaches. Assuming that higher concentration of DNA in an extract is more likely to yield amplification success, we first counted the number of positive PCR’s for each extraction method and performed a Pearson’s Chi square test. We further calculated the ratio of positive PCRs for each sample and performed an ANOVA using the statistical software package Total RNA yield was estimated using the Quant-iT™RiboGreen® RNA reagent assay comparing the estimates of RNA concentrations against a provided standard and adjusted for a starting volume of 500 μl whole blood. To obtain a reliable and comparable measure for the integrity of all extracted RNA samples, we used the RNA integrity number (RIN), a measurement that uses the entire electrophoretic trace of each RNA sample. We used the Agilent 2100 Bioanalyzer and accompanied software which calculates the RIN values within a range of 0 and 10, where 0 represents a totally degraded and 10 a completely preserved RNA sample . An ANOVß- hemoglobin) and the reference genes (HuPoUbcYWHAZ) using the relative quantification software . This approach compensates for differences in target and reference gene amplification efficiency within an experiment and between experiments. The PCR efficiency for each primer pair was evaluated once and used as an external standard throughout the experiment. The target was paired with all three reference genes and the geometric mean of the resulting ratios was calculated. By equalizing the average ratios of the whole blood approaches with 100% of globin transcripts, we were able to determine the average reduction of ß- hemoglobin in the filtered samples. To evaluate the significance of ß- hemoglobin reduction in filtered compared to whole blood samples, we performed both an ANOVA as well as a Students t-test.To assess the amount of hemoglobin transcripts for the whole blood and the filtered approach, we processed the outputs from the LightCycler runs (using LightCycler 480 software) by first undertaking a melting curve analysis in order to determine that each PCR worked properly and no unspecific amplification occurred . SamplesAfter on-machine filtering of raw reads, all retained reads of each sample were assembled into contigs using Roche’s gsAssembler software with minimum overlap of 40 bp and minimum overlap identity of 90%. In order to evaluate each sample treatment Table  for usefIn order to assess the number of reads derived from hemoglobin transcripts, we extracted the genomic regions containing hemoglobin genes from the publicly available dog genome  (chr.21:The authors declare that they have no competing interest.DS and OT developed the idea, designed the study and performed the experiments and the analysis. LEKS provided the majority of the blood samples and RKW contributed to the study design as well as provided funding. DS and OT wrote the manuscript with editing contributions from all authors. All authors read and approved the final manuscript.Supplementary Information. The accompanied pdf-file provides in depth information regarding the sample preparation for 454 sequencing, two additional tables and a figure that depicts the sampling set up [g set up -58.Click here for file


Given their sessile condition, land plants need to integrate environmental cues rapidly and send signal throughout the organism to modify their metabolism accordingly. Small RNA (sRNA) molecules are among the messengers that plant cells use to carry such signals. These molecules originate from fold-back stem-loops transcribed from endogenous loci or from perfect double-stranded RNA produced through the action of RNA-dependent RNA polymerases. Once produced, sRNAs associate with Argonaute (AGO) and other proteins to form the RNA-induced silencing complex (RISC) that executes silencing of complementary RNA molecules. Depending on the nature of the RNA target and the AGO protein involved, RISC triggers either DNA methylation or chromatin modification  or RNA cleavage or translational inhibition . In some cases, sRNAs move to neighboring cells and/or to the vascular tissues for long-distance trafficking. Many genes are involved in the biogenesis of sRNAs and recent studies have shown that both their origin and their protein partners have great influence on their activity and range. Here we summarize the work done to uncover the mode of action of the different classes of sRNA with special emphasis on their movement and how plants can take advantage of their mobility. We also review the various genetic requirements needed for production, movement and perception of the silencing signal. Plants produce miRNAs and siRNAs, but no piRNAs. The biogenesis pathways responsible for the production of miRNA and siRNA molecules share a few similarities. They all derive from double-stranded RNA (dsRNA) molecules that are cleaved into one or many small RNA (sRNA) duplexes by one of the four dicer-like enzymes (DCL) found in plants   in petunia. Unexpectedly, cosuppression of endogenous and transgenic CHS-A was observed, resulting in a loss of flower pigmentation instead of an increase  -C-terminal LEUCINE-RICH REPEATS (LRR) class of disease-resistant proteins. It is therefore possible that the reported induction of the miRNA reflects an attempt from the pathogen to use an endogenous plant system to lower its defenses rather than a bona fide plant defense response.In addition to plant antiviral defense responses that rely on siRNAs derived directly from the genome of the pathogen, there are now indications that host-, as opposed to parasite-encoded sRNAs might also participate in antiviral defense. Indeed, two miRNAs, bra-miR158, and bra-miR1885, are significantly upregulated during TIR1, AFB2, and AFB3 controling auxin response   associated with AGO1, whereas capped and polyadenylated transcripts from the TAS3 locus are cleaved by miR390 associated with AGO7 , most of which are inactivated or silent because of epigenetic suppression  gene trigger trans-TGS on ectopic FWA copies introduced by transformation  coding sequence  is restricted to the nucleus while other factors (associated with PTGS) remain in the cytoplasm. This has important consequences for the silencing models; for instance, it was long thought that the PTGS machinery was localized exclusively in the cytoplasm, forbidding that sRNA could be generated against a pre-mRNA. However, a recent study has showed that an intron introduced in plants as an IR could cause the silencing of the corresponding gene  is still under debate. Obviously, it must contain nucleic acids, most certainly RNA, to ensure sequence-specificity. However, whether it is a long or short, single- or double-stranded molecule remains unclear. Moreover, it is possible that different forms of silencing signals exist   genes (Smith et al., AGO1 in the companion cells of an ago1 mutant does not rescue the vein photo-bleaching phenotype associated with SUL silencing, indicating that AGO1 plays an essential role in the receiving cells (Dunoyer et al., DCL4 for which expression in the companion cells of a dcl4 mutant is sufficient to rescue the vein photo-bleaching phenotype.In the hope of revealing the factors involved in sRNA silencing movement, two similar approaches have been used. They involved the companion cell-specific expression of an IR triggering the silencing of an endogenous gene of Arabidopsis, either the DCL3, AGO4, DRD1, and POLV genes release partial TGS of IR-PDS inducing transgenes, thus increasing the amount of PDS 21-nt siRNAs and the photo-bleaching phenotype (Smith et al., The screens described above also confirmed that the 21-nt sRNAs are essential for the short distance propagation of PTGS. Indeed, the intensity of the photo-bleaching phenotype is directly correlated to the abundance of the 21-nt. It is therefore expected that any mutation influencing the abundance of these sRNAs would influence the overall phenotype. This is probably why mutations in RDR6 did not affect local PDS PTGS caused by IR-PDS inducing transgenes expressed in companion cells (Himber et al., N. benthamiana was shown to contribute to the virus-induced cell-to-cell silencing signal (Qin et al., Lastly, it is interesting to note that silencing of endogenous genes does not propagate systemically in an RDR6-dependent manner, and remains localized to the 10–15 cells neighboring the veins. Indeed, mutations in Obviously, the movement of RNA silencing signals is crucial for plants. What appeared primarily as a simple and straightforward mean to counter-attack RNA viruses has turned into a complex battery of traveling molecules insuring the coordinated development of the organism as well as genome stability and perhaps even the capacity for fast adaption to the environment. With such important implications in the various biological functions of the plant, it is not surprising that RNA interference raises high interest in the fields of agriculture and biotechnology. Indeed, sRNAs now appear as a promising mean to be able to modify the plant metabolism in any number of ways. The potential practical applications of gene silencing emphasize the importance of answering the leftover questions regarding the sRNA molecules. For instance, it remains to be confirmed if sRNAs are actually the silencing effectors of these processes. Also, the carriers allowing movement through plasmodesmata and/or into the phloem remain to be identified. Increasing our knowledge in this field will also be of vital importance for the understanding of cell fate, defense mechanisms, stress adaptation and evolution of plants.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.


Annexin A2 (ANXA2) is a protein implicated in diverse cellular functions, including exocytosis, DNA synthesis and cell proliferation. It was recently proposed to be involved in RNA metabolism because it was shown to associate with some cellular mRNA. Here, we identified ANXA2 as a RNA binding protein (RBP) that binds IBV (Infectious Bronchitis Virus) pseudoknot RNA. We first confirmed the binding of ANXA2 to IBV pseudoknot RNA by ultraviolet crosslinking and showed its binding to RNA pseudoknot with ANXA2 protein in vitro and in the cells. Since the RNA pseudoknot located in the frameshifting region of IBV was used as bait for cellular RBPs, we tested whether ANXA2 could regulate the frameshfting of IBV pseudoknot RNA by dual luciferase assay. Overexpression of ANXA2 significantly reduced the frameshifting efficiency from IBV pseudoknot RNA and knockdown of the protein strikingly increased the frameshifting efficiency. The results suggest that ANXA2 is a cellular RBP that can modulate the frameshifting efficiency of viral RNA, enabling it to act as an anti-viral cellular protein, and hinting at roles in RNA metabolism for other cellular mRNAs. Ribosomal frameshifing is a recoding process of translation where a specific messenger RNA (mRNA)-mediated signal directs a ribosome to shift its reading frame and to continue in the new frame. Programmed-1 ribosomal frameshifting is the most widely used translational recoding mechanism of many viruses Viral frameshifting signals are present in coding regions of mRNA, and are generally composed of a “slippery” sequence, a stimulatory downstream RNA hairpin or pseudoknot structure and an intervening spacer region Annexin A2 (ANXA2) is a multi-functional protein that has been implicated in a number of cellular functions, including calcium dependent regulation of exocytosis, DNA synthesis and cell proliferation To identify the proteins that might recognize frameshifting signals, we presently utilized IBV pseudoknot RNA as a model transcript. We identified ANXA2 as a RBP that can recognize viral pseudoknot RNA and reduce viral frameshifting. We confirmed the RNA binding activity of ANXA2 by various methods and tested its role in the regulation of frameshifting. By overexpression and knockdown approaches, we showed it to be an efficient modulator of viral frameshifting. We speculate that cellular ANXA2 protein may act as an anti-viral protein that can reduce detrimental viral frameshifting and successful replication of the virus. The present results offer a clue for the role of ANXA2 as a RBP for other cellular RNA transcripts.http://bibiserv.techfak.uni-bielefeld.de/pknotsrg/submission.html) Since IBV pseudoknot RNA was shown to induce ribosomal frameshifting during RNA translation, we utilized IBV frameshifting RNA sequences as the model transcript To search for cellular proteins that directly interacted with IBV pseudoknot RNA, a RNA pull down assay was performed in the presence of cell extracts . Wild-tyThe previous experiment indicated the possible interaction of ANXA2 to IBV pseudoknot RNA. To confirm whether ANXA2 could bind wild-type IBV pseudoknot RNA but not mutant IBV RNA, we performed UV crosslinking assay to show that radiolabeled wild-type IBV pseudoknot RNA bound to ANXA2 protein . After RNext, we tested whether ANXA2 could bind directly to IBV pseudoknot RNA. To explore this, GST-fused recombinant ANXA2 protein was purified and used for GST pull-down assay. More radioactivity was recovered after precipitating with GST-ANXA2 in comparison to GST, suggesting that recombinant ANXA2 could associate with IBV pseudoknot RNA. Moreover, wild-type IBV pseudoknot RNA tended to bind more than mutant IBV RNA did . We testSince we found that ANXA2 bound IBV pseudoknot RNA, we next tested whether it could control the efficiency of ribosomal frameshifting on IBV pseudoknot RNA in the cells. We constructed dual luciferase reporters controlled by IBV frameshifting sequences with or without pseudoknot RNA . FrameshTranslation is the complex RNA metabolism regulated by diverse factors, such as ribosome, tRNA and many ribosome associated proteins. Here, we identify ANXA2 as the regulator of ribosomal frameshifting. Since it was previously reported that ANXA2 binds RNA and regulates RNA localization RNA pseudoknots are structural elements formed upon base-pairing of a single-stranded region to a stretch of complementary nucleotides elsewhere in the RNA chain. Since the first discovery of pseudoknot structure in 3′-UTR of Turnip Yellow Mosaic Virus (TYMV), numerous viruses have been reported to contain pseudoknot RNA The detailed mechanism of pseudoknot function on frameshifting is being intensively studied. Recent data suggests that the mechanical strength of the pseudoknot structure is correlated with ribosomal frameshifting, with pausing of ribosome itself is not sufficient for frameshifting Deregulated expression of ANXA2 is implicated in many human tumors and diseases Plasmids containing wild-type (pFS-cass5) and mutant (pFS-cass5.15) pseudoknot sequences from IBV genomic RNA were generous gifts form Dr. Ian Brierley (University of Cambridge) Human embryonic kidney cell line HEK293T were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum . Human prostate cancer cell line LNCaP were purchased from the ATCC and cultured in RPMI1640 with 10% FBS. Human lung fibroblast IMR90 and human lung adenocarcinoma SK-Lu I cells were purchased from ATCC and cultured in MEM containing 10% FBS using standard techniques.Cells were harvested and washed twice with ice-cold phosphate buffered saline (PBS). Cell pellets were resuspended in two volumes of buffer A  supplemented with protein inhibitor cocktail . After incubating 15 min on ice, cells were lysed with 0.5% Nonidet P-40 and centrifuged at 4,000 rpm at 4°C for 30 sec. Clarified cytosolic extract was quantitated for protein concentration and stored in aliquots at −80°C.32 was added by incubating with γ-S32 ATP (10 mM) with T4 Polynucleotide Kinase (New England Bio Lab). Biotin was added at the 5′-end of the prepared RNA by incubating one and half hours with 10 mM PEO-Iodoacetyl Biotin (PIERCE) in the dark. Cytosolic extracts were pre-cleared with Streptavidin-coated magnetic beads (New England Bio Lab) and beads were pre-blocked with tRNA. Biotin and 4-thio labeled RNA was incubated with pre-cleared cell extract in binding buffer  for one hour in room temperature. Ultraviolet radiation (365 nm) was performed to induce binding between 4-thio UTP and interacting proteins. Pre-blocked magnetic beads were added and incubated for one hour to pellet RNA-protein complexes. Two volumes of washing buffer  was added and washed seven times, followed by elution with boiling in 2× sample buffer for 5 min. Proteins were separated by 10% SDS-PAGE and visualized by silver staining .Wild-type and mutant IBV RNAs were transcribed in the presence of 4-thio UTP (Ambion) by in vitro transcription with T7 RNA polymerase. Phosphate was removed from 5′-end by Calf Intestinal Alkaline Phosphatase (New England Bio Lab) and γ-SAfter pulling-down interacting proteins by a biotin pull-down assay as described below, protein identification was performed in the Functional Proteomics Center, Korea Institute of Science and Technology. Silver stained bands were excised and an automated in-gel tryptic digestion was performed on a Mass Prep Station (Micromass). The gel pieces were destained, reduced with DTT, alkylated with iodoacetamide and digested with sequencing grade modified trypsin (Promega). Resulting peptides were extracted from the gel and analyzed via MALDI-TOF and MALDI-TOF-TOF. Proteins were identified from the mass spectrometry data using MASCOT (Matrix Science) and searching the NCBI data base.32P]CTP. 32P-labeled IBV pseudoknot RNA (1.1×105 c.p.m) was incubated with 30 µg of cell extract with 2 µg of tRNA, 0.2 µg of poly d(I-C) and 40 U of RNase inhibitor. The RNA-protein binding reaction was carried out in a 30 µl reaction mixture containing 20 mM HEPES pH 7.5, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA and 5% glycerol. The mixtures were incubated at room temperature for 30 min, after which they were UV-irradiated (120 mJ/cm2) on ice for 30 min with an UV cross linker (Fisher Scientific). RNAs were digested with 1 µg RNase A, 25 U RNase S1 and 0.1 U RNase V1 at 37°C for 10 min and analyzed by 10% SDS-PAGE.IBV pseudoknot RNA was transcribed and labeled in vitro with T7 RNA polymerase (Ambion) with α-ATP. 32P labeled IBV pseudoknot RNA (1.1×105 c.p.m) and purified GST-ANXA2 or GST protein were incubated at room temperature for 30 min in a binding buffer containing 25 mM HEPES pH 7.5, 100 mM NaCl, 1 mM MgCl2 and 4% glycerol. RNA-protein complexes were resolved on 5% native polyacrylamide gels. Gels were dried and analyzed by autoradiography.The basic procedure for EMSA was previously described 5′-GTCGACTTTAAACTGATACGGGGTATC-3′ and 5′-GAAGGATCCCAGCTGAAAGGC-3′). qRT-PCR was performed with StepOne real-time PCR system (Applied Biosystems). Reactions were amplified using selective primers as described above with Power SYBR master mix (Applied Biosystems) according to the manufacturer's instructions. Quantification was carried out with the StepOne™ software (Ver 2.2). Percentages of PK RNA binding to ANXA2 were expressed as the ratio of comparative threshold cycle (TC) to input level in PK mRNAThe basic procedure for RNA-IP was previously described 5′-AATGTCGACTTTAAACTGATACGGG-3′ and 5′-TAAGGATCCCAGCTGAAAGGCTC- 3′ from of pFS-cass 5 and pFS-cass 5.15, respectively. In frame sequence was amplified with primers 5′-AATGTCGACACGGGGTATCAGTC-3′ and 5′-TAAGGATCCCAGCTGAAAGGCTC-3′. No frame sequence was amplified with primers 5′-AATGTCGACCTGATACGGGGTAT-3′ and 5′-TAAGGATCCCAGCTGAAAGGCTC-3′.To make IBV dual luciferase reporters, a frameshifting sequence (72 bp) was amplified from pFS-cass 5 and pFS-cass 5.15. The sequence was inserted between Renilla and firefly luciferase genes of a dual reporter, p2Luc vector  Fe  =  Frameshifting efficiency, F =  Wild-type or mutant Luc activity, Fn =  No-frame Luc activity and Fp  =  In-frame Luc activity.Cells were cultured and transfected with frameshifting reporters using Lipofectamine™ (Invitrogen). For the luciferase assay, cells were scraped into 100 µl of passive lysis buffer (Promega). Luciferase activity in the lysate was determined with a dual-luciferase reporter assay system (Promega), according to the manufacturer's instruction and measured with a Turner Luminometer TD-20/20. Firefly luciferase activity was normalized to the activity of renilla luciferase. Frameshifiting efficiency was calculated by the following formula:


BMC Evolutionary Biology article, Huiquan Liu and colleagues report two new genomes of double-stranded RNA (dsRNA) viruses from fungi and use these as a springboard to perform an extensive phylogenomic analysis of dsRNA viruses. The results support the old scenario of polyphyletic origin of dsRNA viruses from different groups of positive-strand RNA viruses and additionally reveal extensive horizontal gene transfer between diverse viruses consistent with the network-like rather than tree-like mode of viral evolution. Together with the unexpected discoveries of the first putative archaeal RNA virus and a RNA-DNA virus hybrid, this work shows that RNA viral genomics has major surprises to deliver.In a recent http://www.biomedcentral.com/1471-2148/12/91See research article:  Viruses are the most abundant and genetically diverse biological entities on earth as demonstrated by recent metagenomic studies . Unlike Owing to their (relatively) small genomes and often rapid reproduction, diverse viral genomes were characterized in the early days of genomics, and many unexpected evolutionary relationships have been revealed between viruses with different genomic strategies that infect widely different hosts. A small set of virus hallmark genes encoding proteins essential for virus replication and morphogenesis form different combinations in diverse viruses but are absent from cellular genomes . Along wBMC Evolutionary Biology article, Huiquan Liu and colleagues . Th. Th11]. Leviviridae and Cystoviridae, might not even have a common origin with the majority of eukaryotic RNA viruses [Leviviridae and Narnaviridae notwithstanding. So where does the bulk of RNA viruses come from? A hypothesis has been proposed that the RdRp was derived from the reverse transcriptase of a bacterial retro-transcribing element and the other genes from different bacteriophage and bacterial sources [Beyond discovering strange chimeras, metagenomics has the potential to change the existing fundamental ideas on the evolution of RNA viruses. Almost all extremely diverse RNA viruses infect various eukaryotes. The only two known families of bacterial RNA viruses,  viruses , the evo sources . However sources . This noWe are probably only starting to scratch the surface of the virosphere. An unbiased sampling of the viral habitats (and these span the entire biosphere), which with modern technologies is becoming realistic, is expected to result in a major expansion of the networks of virus evolution.