The division of labor between template and catalyst is a fundamental property ofall living systems: DNA stores genetic information whereas proteins function ascatalysts. The RNA world hypothesis, however, posits that, at the earlier stagesof evolution, RNA acted as both template and catalyst. Why would such divisionof labor evolve in the RNA world? We investigated the evolution of DNA-likemolecules, i.e. molecules that can function only as template, in minimalcomputational models of RNA replicator systems. In the models, RNA can functionas both template-directed polymerase and template, whereas DNA can function onlyas template. Two classes of models were explored. In the surface models,replicators are attached to surfaces with finite diffusion. In the compartmentmodels, replicators are compartmentalized by vesicle-like boundaries. Bothmodels displayed the evolution of DNA and the ensuing division of labor betweentemplates and catalysts. In the surface model, DNA provides the advantage ofgreater resistance against parasitic templates. However, this advantage is atleast partially offset by the disadvantage of slower multiplication due to theincreased complexity of the replication cycle. In the compartment model, DNA cansignificantly delay the intra-compartment evolution of RNA towards catalyticdeterioration. These results are explained in terms of the trade-off betweentemplate and catalyst that is inherent in RNA-only replication cycles: DNAreleases RNA from this trade-off by making it unnecessary for RNA to serve astemplate and so rendering the system more resistant against evolving parasitism.Our analysis of these simple models suggests that the lack of catalytic activityin DNA by itself can generate a sufficient selective advantage for RNAreplicator systems to produce DNA. Given the widespread notion that DNA evolvedowing to its superior chemical properties as a template, this study offers anovel insight into the evolutionary origin of DNA. At the core of all biological systems lies the division of labor between thestorage of genetic information and its phenotypic implementation, in otherwords, the functional differentiation between templates (DNA) and catalysts(proteins). This fundamental property of life is believed to have been absent atthe earliest stages of evolution. The RNA world hypothesis, the most realisticcurrent scenario for the origin of life, posits that, in primordial replicatingsystems, RNA functioned both as template and as catalyst. How would suchdivision of labor emerge through Darwinian evolution? We investigated theevolution of DNA-like molecules in minimal computational models of RNAreplicator systems. Two models were considered: one where molecules are adsorbedon surfaces and another one where molecules are compartmentalized by dividingcellular boundaries. Both models exhibit the evolution of DNA and the ensuingdivision of labor, revealing the simple governing principle of these processes:DNA releases RNA from the trade-off between template and catalyst that isinevitable in the RNA world and thereby enhances the system's resistanceagainst parasitic templates. Hence, this study offers a novel insight into theevolutionary origin of the division of labor between templates and catalysts inthe RNA world. At the core of all biological systems lies the division of labor between the storageof genetic information and the manifestation of genetic information, i.e. thefunctional differentiation between DNA, which is the information storage medium(template), and RNA and proteins, which are responsible for different aspects ofoperation . This fundamental property of life, however, is believed tohave been absent at the earliest stages of evolution. The RNA world hypothesis,which is currently considered to be the most, if not the only, realistic scenariofor the origin of life, posits that, in the first, primitive replicating systems,both the storage of genetic information, and chemical catalysis were embodied in asingle type of molecules, namely, RNA What selective advantage could there be for the evolution of DNA and proteins in theRNA world? Proteins are obviously superior to RNA as chemical catalysts because oftheir greater repertoire of chemical moieties and structural flexibility.Conversely, proteins are vastly inferior to RNA for the storage of geneticinformation because of the absence of mechanisms for template-directed replication.These properties of proteins are compatible with the view that proteins evolved asentities specialized in chemical catalysis owing to their superiority to RNA in thatcapacity.The case of DNA appears less straightforward. On the one hand, it remains somewhatunclear what would be the principal driving forces behind the evolution of DNA inthe RNA world. DNA is generally a less reactive molecule than RNA thanks to theabsence of the 2′-hydroxyl at its sugar moiety. In particular, DNA is markedlymore resistant to hydrolysis than RNA invitro evolution experiments On the other hand, there is no clear experimental evidence demonstrating that DNA isinferior to RNA as a chemical catalyst functionally equivalent to DNA?Given these considerations, we ask: What selective advantage could there be for anRNA-based evolving system to evolve an entity that is solely dedicated to thestorage of genetic information, i.e., an entity that isAs a first attempt to answer this question, we consider the evolution of DNA-likemolecules in RNA replicator systems, the simplest form of the RNA world that canundergo Darwinian evolution. Our aim is to examine whether there could exist purelypopulation dynamical factors, independent of specific nucleic acid chemistry, whichwould support selection for DNA-like molecules, i.e., molecules solely dedicated tothe storage of information, in RNA replicator systems. To address this question, weconstruct and investigate minimal computational models of an RNA-like replicatorsystem with a built-in possibility to evolve DNA-like molecules.There are two types of molecules in the models developed here: “RNA-likemolecules” and “DNA-like molecules” . The only difference between the two types of molecules is the presenceor absence of the catalytic capacity—all other possible differences areignored for the sake of simplicity and focus. Thus, an RNA molecule can be both atemplate for replication and a catalyst that replicates other templates, whereas aDNA molecule can only be a template for replication . Moreover, DNA and RNA compete fora common resource (precursors) for replication (this direct competition between DNAand RNA is expected to make the models more conservative with respect to theevolution of DNA). The models do not include protein-like molecules because weintend to investigate the simplest possible scenarios under which the evolution ofDNA can be considered  and actual replication of thetemplate:A molecule produced by replication  and another in which replicators are compartmentalized byvesicle-like boundaries that are impermeable to replicators (the compartment model).In the compartment model, the size of a compartment grows (or shrinks) in proportionto the number of replicators inside the compartment, and a compartment divides whenits size reaches a threshold given by a parameter The two models were implemented as described previously To simulate the dynamics of compartment boundaries, we employed the Cellular PottsModel (CPM) Before presenting the results of the simulations, let us first consider replicatorsystems consisting of RNA and DNA in general terms. In such a replicator system,there are four replication reactions as listed in the RNA, RpDNA, DpRNAand DpDNA), regardless of whether replication proceeds via thetranscription cycle or via the reverse transcription cycle. Assuming that the totalconcentration of molecules is constant, this increase in the complexity of thereplication cycle leads to a reduction in the concentration of each type ofmolecules and to the corresponding reduction in the rate of multiplication comparedto the self-replication system.By comparing the three replicator systems, we can see two effects that can be broughtabout by the inclusion of DNA molecules into a replication cycle. First, the systemsthat include DNA are more complex and thus less efficient than the self-replicationsystem. The inclusion of DNA requires the joint action of four types of molecules tocomplete a replication cycle , can evolve DNAmolecules and the ensuing division of labor between the template and thecatalyst.RNA with arbitrary chosen values ofRNA, and it expands into anempty region as the population of RpRNA grows. The back of a waveconsists of parasites, and it contracts, leaving empty regions, in thedirection of wave propagation due to the extinction caused by parasitesThe surface model was initialized with a homogeneous population ofRpof and  , No. 1. of and  .The frorasites , Note 1 esystem  andquicalue of  . In otheue of   do not spend any part of theirlifetimes replicating others, which reduces the advantage of the parasiteover the catalyst . Hence, when a traveling wave consisting of RpRNA can parasitize on the self-replicationsystem given that its The above explanation implies that parasites are one of the key factorsbehind the coexistence of the self-replication and transcription systems. Toexamine whether this is indeed the case, we conducted the followingsimulation ,No. 3. Can the transcription system supersede the self-replication system? In otherwords, can a transcription system, assuming that it emerged, be maintainedthrough evolution in the absence of the self-replication system? The answerturns out to be negative. To address this question, we continued theprevious simulation, in which the self-replication system and thetranscription system coexisted, by removing the entire population of theself-replication system , No. 4. eplicase . After tHow and why does the presence of the RNA replicase cause the evolutionarystabilization of the transcriptase? The RNA replicase has two distincteffects on the transcription system:RNA and DNA replicase DpRNA) aredirectly replicated by the RNA replicase;The catalysts of the transcription system  The RNA replicase does not recognize thetranscriptase as a template; 2) the transcriptase does not recognize the RNAreplicase as a template ; 3) the RNA replicase and the transcriptase do notrecognize each other (a combination of 1 and 2). For each of these threecases, we continued the previous simulation that had reached theevolutionary equilibrium. The results showed that the evolutionarystabilization of the transcriptase was obtained only in case 1. In thiscase, the modification did not qualitatively change the behavior of thesystem although the transcriptase went extinct after a long time due tofluctuation. In case 2, the transcriptase evolved into a dual specificityRp, which out-competed the original RNA replicase, and which then underwentspeciation into the RNA replicase and the transcriptase, re-establishing theoriginal system as we saw before . In case 3, thetranscriptase evolved into a dual specificity Rp, which went extinct afterits value of As shown above, parasites mediate the coexistence between theself-replication system and the transcription system. Does this mean thatthe evolution of Dp was caused by parasites? To analyze the cause and effectrelationship between the parasite and the evolution of the transcriptionsystem, the model was modified to exclude parasites that are explicitlydefined as a class of non-catalytic replicators with an advantage stabilization and evolutionary (long-term) stabilization. Theresults showed that, on the one hand, parasites enabled the transcriptionsystem to coexist with the self-replication system. In theoretical ecology,this is known as predator-mediated coexistence Second, exploration of the model showed that removing any of the threecomponents of the system, namely, the self-replication system, thetranscription system and the parasite, at the evolutionary steady stateresulted in the restoration of the deleted component through the evolutionof the remaining components . MoreoveGiven the evolution of the transcription system, it is interesting toconsider the separation between template and catalyst in terms of the flowsof genetic information. In particular, is the line of descent continuedthrough the replication of DNA in the transcription system? To address thisquestion, we conducted the following experiment. The previous simulation, No. 1wThe Central Dogma of molecular biology states that the flow of geneticinformation is unidirectional from nucleic acids to proteins RNA . Secondly,however, compartments containing only RpRNA experience fasterevolutionary deterioration of the internal replicator system thancompartments containing the transcription-like system, an effect thatconfers a selective advantage onto the compartments containing thetranscription-like system under sufficiently strong mutation pressure.The above two results, the repeated cycle of invasion and extinction ofcompartments containing only RpRNA, RpDNA,DpRNA and DpDNA in equal proportion. The values ofRNAalone .This result is understandable because, as discussed above, the inclusion ofDNA in a replication cycle leads to a fourfold increase in the number of thetypes of molecules required for multiplication and so reduces the efficiencyof multiplication and increases the chance of stochastic loss of essentialmolecules. Therefore, compartments increase their fitness by losing the DNAreplicase.To investigate the first effect, we directly measured the fitness ofcompartments as follows. The model was modified to make the boundaries ofthe compartments completely unchangeable so the model contained compartmentsbut no compartment dynamics. The model was initialized in a configurationwhere the size of the compartments was equal to the threshold above whichcompartments divide in the original model .This qualitative difference in the rates of evolutionary deterioration ofcatalysts supports the argument that compartments containing thetranscription-like system experience slower evolutionary deterioration ofthe internal replicator system (the origin of this difference will beelucidated in the next section).The result was that the system consisting only of Rp extinct . The ofolecules . This raolecules . AlthougRNA. In fact, thishas already been seen in one of the simulations described above . If the rate of theevolutionary deterioration of compartments containing the self-replicationsystem is sufficiently high, the advantage of the transcription-like systemmore than compensates for the disadvantage, and the evolution of thetranscription-like system becomes possible.We next consider the reason why, in the compartment model, thetranscription-like system displays slower evolutionary deterioration thanthe self-replication system. As already mentioned, in the self-replicationsystem, catalysts gain a selective advantage by decreasing the time theyspend replicating templates which simulates the population dynamics of the internal replicator system ofcompartments in the compartment model . AlthougDNA to DpDNA wasneither too large nor too small .The ODE model was numerically solved for various initial conditions. Theresults showed that the transcription system could survive under well-mixedconditions if the initial ratio of Rpoo small , No. 1. RNAplay both the role of catalysts and templates for RNAreplication, whereas DpRNA plays only the role oftemplates. Of course, DpRNA also plays the role of catalysts forDNA replication, but so does RpRNA for transcription—thereis no asymmetry between Dp and Rp with respect to reactions involving DNAmolecules. Due to the above asymmetry, DpRNA will be replicatedby RpRNA more often (per molecule) than RpRNA isreplicated by RpRNA assuming that the initial condition issymmetric with respect to Dp and Rp. This can be seen from the fact thatthree times more RpRNA is required than DpRNA in orderto produce an equal amount of the two complex molecules (i.e. that betweenRpRNA and RpRNA and that between RpRNAand DpRNA) We next considered the effect of adding either RNA replication or reversetranscription to the transcription system. Before describing the results, itis worth noting that adding either of these processes introduces asymmetryinto the system. For instance, adding RNA replication makes RpDNA to DpDNA which itselfdetermines the ratio of RpRNA to DpRNA. Let us supposethat the system is initially symmetrical between Rp and Dp. Now, by addingreverse transcription, RpDNA is produced at a slightly higherrate than DpDNA due to the aforementioned asymmetry in reversetranscription, so that the ratio of RpRNA to DpRNAincreases through transcription, which, in turn, leads to the furtherincrease of the RpDNA to DpDNA ratio, hence anamplifying factor. Next, for the case of RNA replication, assuming asymmetric initial condition again, adding RNA replication slightly increasesthe ratio of DpRNA to RpRNA. However, the change ofthe DpRNA to RpRNA ratio has no effect on theDpDNA to RpDNA ratio because there is no reversetranscription. Thus, transcription tends to bring the DpRNA toRpRNA ratio back to the DpDNA to RpDNAratio, hence a dampening factor. To summarize, provided the absence ofreverse transcription, the transcription of DNA molecules tends to bring thepopulation composition of RNA molecules towards that of DNA molecules and sodampens the bias in the population composition of RNA molecules generated bythe asymmetry in RNA replication. In other words, the population of DNAmolecules serves as a buffer to the population of RNA molecules.The results of the numerical calculation indicated that adding RNAreplication did not adversely affect the survival of the system , No. 2. The above argument implies that in the absence of reverse transcription,transcription prevents the evolutionary deterioration of catalysts becausethe selective advantage a catalyst would obtain by increasing the time itspends being a template is effective only in the RNA population, and such aselective advantage would be counteracted by the transcription of DNAmolecules. To examine whether this is indeed the case, we extended the ODEmodel to include the population of an additional species of Rp, which werefer to as Rp′. The strategy of the analysis was to treat Rp′as a mutant of Rp and examine whether Rp′ could out-compete theoriginal Rp when the small amount of Rp′ was introduced into thesystem, which mimicked the mutation of Rp into Rp′. The value ofThe above argument shows that the release of catalysts from thetemplate-catalyst trade-off depends not only on the presence oftranscription but also on the absence of reverse transcription, i.e.blockage in the flow of information from catalysts (RNA) to templates (DNA).To investigate this issue, we conducted a simulation of the type shown inatalysts .DNA). Therefore, fromthe actual flows of genetic information, the division of labor between thetemplate and the catalyst was established for the dual specificity Rpdespite the fact that it maintained a high RNA replicase activity. However,the situation differed for the DNA replicase. The simulation showed that twopopulations of compartments quickly arose in the system: in one of thesepopulations, the Dp was descended from DpDNA, whereas in theother population the Dp was descended from DpRNA. Because of thefiniteness of the system, the entire population of Dp was eventuallydescended either from DpRNA or from DpDNA, with thechoice determined by chance.As shown above, breakage in the flow of information from RNA to DNA(elimination of reverse transcription) is an important factor underlying theadvantage of the transcription-like system in a compartmentalized replicatorsystem. However, although reverse transcription activity was much reduced inthe transcription-like system, it was not completely absent due to themutation pressure. Moreover, the transcription-like system maintained a highrate of RNA replication . Therefore we were interestedto find out in which direction the information was transmitted amongreplicators in the long run: from RNA to DNA, or from DNA to RNA, or both?To address this question, we conducted the same simulation as for thesurface model in order to trace the line of descent over generations. Thissimulation showed that the population of the dual specificity Rp was alwaysdescended from its DNA templates , which can explain why the line of descent for Dp was not alwayscontinued through DNA replication in the original compartment model and the catalyst (RNA), which mitigated the adverse effect of parasites arising fromthe trade-off between templates and catalysts. In the compartment model, DNA couldcause the retardation of the evolutionary deterioration of the internal replicatorsystem of compartments by eliminating the advantage of RNA molecules beingnon-catalytic, i.e. evolving into parasites, which originated from theaforementioned trade-off. This retardation required the presence of transcriptionand the absence of reverse transcription. In other words, the information must flowfrom DNA-like molecules (template) to RNA-like molecules  but not viceversa. This unidirectionality of the information flow is also a form of division oflabor between the template and the catalyst. Therefore, both models effectivelyyield the same conclusion: DNA can nullify the disadvantage of RNA functioning as acatalyst—and hence the advantage of parasites—through establishing thedivision of labor between the template (DNA) and the catalyst (RNA). This advantagecan more than compensate for the disadvantage due to the reduced efficiency ofmultiplication caused by the increased complexity of the replication cycle.Although the transcription system avoids the trade-off between template and catalystthrough establishing the division of labor between the template and the catalyst, itgenerates the trade-off between replication and transcription whereby a template(DNA) must spend a part of its lifetime being transcribed in order to producecatalysts (RNA), and during these times, the template cannot be replicated. Thelatter trade-off causes a selection pressure for templates to evolve towardsdecreasing the rate of transcription in exchange for increasing the rate ofreplication. In the present models, however, this selection pressure does not affectthe evolution because the models do not allow templates to evolve their affinitiestowards Rp and Dp so as to differentiate between replication and transcription. Toexamine the effect of the replication-transcription trade-off on the evolution ofDNA, we slightly modified the models to allow templates to distinguish between Rpand Dp  in the RNA-protein world. In thiscase, DNA can release RNA from the trade-off between replication and translation sothat RNA can be dedicated to translation. This effect might cause a substantialreduction in the selective advantage of parasitic templates because the suppressionof RNA replication due to translation would be more severe than the suppression ofDNA replication due to transcription assuming equal rates of protein production . Moreover, if a high rate of protein production is selectivelyadvantageous to the system, releasing RNA from the replication function and soallowing it to be dedicated to translation might be a substantial advantage to thesystem, causing strong selection pressure for the evolution of DNA.The present models assume that Dp can emerge from Rp through a one-step mutation.This simplification was made because the central question of the current study waswhether there could be any selective advantage for an RNA-based evolving system toproduce DNA-like molecules independent of specific nucleic acid chemistry. Itappears that our main conclusion on the existence of such a selective advantageshould be valid independent of specific assumptions on the mutation. To furtherassess the validity of this conclusion, we also investigated the models under twodifferent assumptions on the effects of mutations. Under the first assumption, thedistinction between Rp and Dp was continuous. Each replicase is assigned twoparameters that determine the product specificity: Kaneko and Yomo not templates for each other, so bothribozymes must function as templates to transmit information to subsequentgenerations. In the present models, DNA allows RNA (ribozyme) to not function astemplate at all and releases it from the template-catalyst trade-off. In addition,the division of labor between template and catalyst evolved without assuming reducedcompetition between replicators.Zintzaras et al We previously noted an interesting difference between the concepts of genotype asapplied to modern cells and protocells conceived as vesicle-like compartmentscontaining replicators Text S1The text contains notes to the main text, the implementation details of thesurface model and the compartment model, the ODE models and the descriptionof the models that take account of the trade-off between replication andtranscription.(PDF)Click here for additional data file.


C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [C9ORF72 transcript implicate a loss of protein function due to haploinsufficiency, intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic [The GGGGCC G4C2) intronic repeat expansion within ia (FTD) ,2. The m intronicbe toxic .in situ hybridisation probes for G4C2 to detect RNA foci in transfected cells and human brain tissue form C9ORF72 mutation carriers. We assessed the toxicity by cell counting, activated caspase, PARP cleavage and Annexin V expression transfected cells and by TUNEL assay in zebrafish embryos. We probed transfected cells with antibodies to 30 RNA binding proteins and then tested the best candidates by immunohistochemistry in human brain tissues. We used biotinylated G4C2 x72 probes to pull down repeat binding proteins and confirmed binding by western blot.In order to determine whether expanded G4C2 transcripts might be toxic and sequester RNA binding proteins we generated G4C2 repeats with 8x, 38x and 72x and expressed them in a range of neuronal and non-neuronal cell lines, primary neurons and zebrafish embryos. We used fluorescent Cellular toxicity was associated with the nuclear retention of transcripts containing 38x and 72x repeats and the appearance of RNA foci. By placing the repeats 3’ to EGFP we were able to monitor nuclear export and show that transfected neuroblastoma cells, but not HEK cells, declined in number over time due to apoptotic cell death. This was most marked in those showing the greatest nuclear retention and foci, indicating cell type and neuron-specific toxicity of longer G4C2 repeats.In order to explore the sequestration hypothesis we screened antibodies to 30 RNA binding proteins on 72x transfected neuroblastoma cells and demonstrated colocalization of G4C2 RNA foci with three proteins. Using biotinylated 72x RNA we were only able to pull down one of these indicating that it is able to bind G4C2 RNA directly. On probing human cerebellar ALS and FTD tissues we detected a striking colocalization of this protein with 70% of RNA foci.Our observation that longer stretches of G4C2 RNA form neurotoxic foci and bind specific RNA binding proteins is similar to other intronic microsatellite expansion disorders where CUG (DM1) and CCUG (DM2) intronic repeats generate RNA foci in cells, animal models and in patients with myotonic dystrophy.


In the last years, numerous studies have focused on understanding the metabolism of RNA and its implication in disease processes but abnormal RNA metabolism is still unknown. RNA plays a central role in translating genetic information into proteins and in many other catalytic and regulatory tasks. Recent advances in the study of RNA metabolism revealed complex pathways for the generation and maintenance of functional RNA in amyotrophic lateral sclerosis (ALS). Interestingly, perturbations in RNA processing have been described in ALS at various levels such as gene transcription, mRNA stabilization, transport, and translational regulations. In this paper, we will discuss the alteration of RNA profile in ALS disease, starting from transcription, the first step leading to gene expression, through the posttranscriptional regulation, including RNA/DNA binding proteins and aberrant exon splicing to protein noncoding RNAs, as lncRNA and microRNA. In last decade, many studies have focused on understanding the RNA metabolism and its implication in both translational and regulatory aspect of disease processes. Currently, it is proved that RNA plays a central role in many cellular processes from translating genetic information into proteins, to catalytic and regulatory aspects. Recent advances in the study of RNA metabolism revealed complex pathways in the generation and maintenance of functional RNA and in cells survival related to defects in RNA.Mainly we may distinguish two principal families of RNA, coding (mRNA) and noncoding (miRNA and lncRNA), that are implicated in RNA metabolism that can produce cellular defects and can be causes of diseases.Changes in gene expression and splicing patterns are described in an increasing number of complex diseases such as the neurodegeneratives . PerturbAs for coding RNA, recently changes in gene expression in ALS patients have been demonstrated  and the About noncoding RNA, recent genome-wide analysis of the human transcriptome has revealed a plethora of long non-protein-coding RNAs (lncRNAs) whose biogenesis, regulation, and cellular roles are only starting to be elucidated. Increasing evidence points to deregulation of lncRNAs as an important, yet unexplored layer of complexity in human diseases, including neurodegenerative disorders . MoreoveIn this paper, we focused on RNA metabolism, starting from transcription to posttranscriptional regulation by DNA/RNA-binding proteins, noncoding RNAs as lncRNA and microRNA, and their contributions in the ALS pathogenesis.Alterations in gene expression have been documented in many papers in different tissues from ALS patients using microarray analysis but the data are discordant.Two main works have been published between 2010 and 2011 that reported mRNA expression data in Peripheral Blood Mononuclear Cells (PBMCs) of ALS sporadic patients , 10.Zhang and collaborators  performeκB p65 was higher in ALS spinal cord and inhibition of NF-κB in neurons overexpressing TDP-43 reduces vulnerability to toxic injury [κB.Concerning TDP-43 mRNA expression, Mougeot and collaborators  discoverc injury  suggestiInterestingly, Zhang and collaborators defined by microarray experiments the main pathways related to the RNA metabolism and transcription complexes pathways, as splicing, mRNA transport, and transcription factors . Also inAbout mutated SOD1 cellular model, Kirby  showed bDifferent aspects define post-transcriptional RNA regulation; here we proposed to review some of the different steps as the role of RNA binding proteins and the splicing in altered RNA metabolism.The discovery of mutations in different DNA/RNA-binding proteins, as causes of both familial (FALS) and sporadic ALS (SALS), has opened enormous perspectives on the implication of post-transcriptional mechanism alterations in ALS pathogenesis.It is homologous to the heterogeneous nuclear ribonucleoproteins (hnRNPs) , which aMutations in TARDBP gene have been found in about 3 to 4% of FALS cases and in about 2% of SALS patients . Most TAThe finding that TDP-43 protein aberrantly cleaved, hyperphosphorylated, and ubiquitinated is present in the cytoplasm of SALS spinal motor neurons but not in controls or in patients with Alzheimer's disease which defines the involvement of this protein in ALS , 23. MorStrong and collaborators have shown that TDP43 stabilizes the low molecular weight neurofilament (hNFL) mRNA through a direct interaction with the 3′-untraslated region (3′UTR) . In anotAbout TDP-43 effects on other genes expression, it has been reported that TDP-43 interacts with subunits of the Mediator complex , which b It is a protein that in humans is encoded by the ATXN2 gene, which has a physiological role on mRNAs, possibly through direct RNA binding, translation, transport, and stability . In 2010Fused in sarcoma, and translocated in lipoSarcoma (FUS/TLS) is a heterogeneous ribonucleoprotein (hnRNP) that, as TDP-43, is involved in RNA splicing, transportation, and stabilization , 31. UndThe C terminus that contains RNA recognition motif (RRM) allows the binding to nucleic acids, Arg-Gly-Gly (RGG) repeat rich, and zinc finger domains involved in RNA processing , 33.Mutations in FUS/TLS gene have been found in 4% of FALS .Thus, mutations in the latter region lead to redistribution of FUS to the cytoplasm , 33, 35.Subcellular localization of FUS/TLS has been studied in cells transfected with wild-type and mutant FUS/TLS gene by compartmental fractionation showing a significant increase of FUS mutants in the cytoplasmic fraction .κB, higher in ALS patients than in controls, by direct binding to NF-κB subunit p65 [κB is also a fundamental transcriptional factor involved in SOD1 expression, which already demonstrated increased in ALS patients [In relation to Swarup and collaborators paper , FUS is unit p65 . As we munit p65 , NF-κB ipatients .In contrast to TDP-43, no associations of post-translational modifications of FUS such as phosphorylation, ubiquitination, or truncation have been detected in ALS or FTLD cases . SOD1 is a soluble protein acting as a 32 kDa homodimeric enzyme to convert naturally occurring, but harmful, superoxide radicals to molecular oxygen and hydrogen peroxide. Mutations in SOD1 gene are responsible for approximately 20% of familial ALS cases . With re It encodes a protein that has RNA helicase activity determined by a domain at the C-terminal end of the protein. In 2004, mutations in SETX gene have been identified in ALS patients  but so fRNA helicases are also involved in the modification of chromatin structure, essential to the initiation of transcription, either directly through modulation of the chromatin structure, or through interactions with the assembly of the transcription-initiation complex. RNA helicases are associated with the spliceosome where they are postulated to be essential in regulating the base pairing between snRNAs and pre-mRNA . The proIt is a 14 kDa angiogenic ribonuclease whose activity is related to its ability in regulating ribosomal RNA (rRNA) transcription.Human angiogenin has been shown to play a role in regulating gene expression by direct binding to DNA . ANG binMutations in ANG gene provoke reduction of cell proliferation, angiogenic activities, and nuclear localization in ALS patients ; moreoveIt is a gene located on chromosome 9, composed of 10 exons and coding for 481 amino acids protein. In September 2011, two publications by Renton et al.  and DeJeRNA-based mechanisms exert a vital role in governing neuronal function; consequently, alterations in RNA processing have been involved in numerous neurodegenerative conditions leading to the identification of novel pathogenic pathways. Among these, alternative splicing of pre-mRNAs represents an important step of post-transcriptional gene regulation by increasing the coding capacity of transcripts and generating proteomic diversification. Disruption of alternative splicing regulation, due to either mutations in splicing regulatory elements or aberrant activity of RNA-binding proteins, can affect the finely tuned program of gene expression and cause the abnormal production of protein isoforms. Numerous papers described abnormalities in the RNA splicing machinery in ALS. Aerbajinai and colleagues  demonstrOnly 2% of the human genes encode for proteins, while the rest of the transcribed gene produces a great number of non-protein-coding RNAs (ncRNAs) as tRNA, rRNA, miRNA, natural antisense transcripts, and long noncoding RNAs (lncRNAs) . ActuallMiRNAs are small RNAs of 21 nucleotides and act as post-transcriptional repressors. They are able to interact with specific sequences of target mRNA (mainly in the 3′UTRs) recruiting the RNA-induced silencing complex (RISC) and, on the basis of complete or incomplete miRNA-mRNA complementary, they direct mRNA to degradation or to translational repression, respectively . Two RNaIt has been demonstrated that the proteins TDP-43 and FUS/TLS are able to interact physically with Drosha, suggesting their involvement in miRNA biogenesis . RecentlIn ALS pathogenesis, mislocalization of TDP-43 due to accumulation in cytoplasmatic aggregates probably reduces the processing of TDP-43-regulated by Drosha and Dicer and generates different miRNA expression profiling.As regards neuron survival, crucial in ALS, it has been demonstrated that ablation of Dicer in mice affects miRNA synthesis during development and leads to the loss of the ability to make functional miRNA postnatally . These mAbout ALS, miRNA profiling study on the skeletal muscle of SOD1-G93A mouse model has demonstrated that the muscle-specific miRNA miR-206 is upregulated in lower limbs of SOD1-G93A mice among 320 miRNAs tested .Furthermore, in mouse model with the deletion of miR-206 it has been observed that the lack of this miRNA accelerates the disease progression, suggesting that the higher amount of miR-206, in SOD1-G93A mice may be a compensatory effect to reduce motor neurons degeneration in ALS.All these observations sustain the importance of miRNA microprocessor complex in ALS disease, future studies on ALS patients may elucidate miRNA roles in ALS human mechanism.Recent genome-wide analyses of the human transcriptome have revealed a plethora of lncRNAs whose biogenesis, regulation, and cellular roles are only starting to be elucidated. LncRNAs are 300 to thousands nucleotides long that are involved in many biological processes as transcription, translation, and splicing .According to the competing endogenous RNA (ceRNA) theory defined by Salmena et al., all types of RNA transcripts, coding and noncoding, could communicate with each others in a keen way supervising different cellular mechanisms . For insIn particular, lncRNA could act as competitor molecules able to sequester miRNA and thereby protecting target mRNA from traslational repression . An examIncreasing evidence points to deregulation of lncRNAs , which hConsidering the literature data about ALS, it appears evident that this pathology has been often studied in the eyes of mitochondial or oxidative protein defects disease.Recently, modifications of RNA metabolism have been described in ALS at various levels: splicing, editing, transport, stabilization, translation, and degradation.In this paper, we tried to summarize the main data about the different steps of RNA metabolism, from transcription to post-transcriptional regulation and the rilevance of protein-noncoding RNA. Different genes have been found altered in gene expression profiling and often the same genes are involved in post-transcriptional pathways. Moreover, the RNA cyclelife can be controlled by protein noncoding RNA, that are playing more and more important roles in RNAs regulation.In summary, we have tried to underline the importance of going deep to the heart of RNA metabolism since we think it may provide new insights into the complex regulation of genes expression, possibly disclosing new mechanisms involved in ALS aetiology and identifying innovative therapeutic strategies for this disease.


N-(diphenylmethylene)glycinamides across chiral α-chloro-N-p-toluenesulfinylaldimines was developed. The resulting -γ-chloro-α,β-diaminocarboxylamides were formed with the opposite enantiotopic face selectivity as compared to the -γ-chloro-α,β-diaminocarboxyl esters obtained via Mannich-type addition of analogous N-(diphenylmethylene)glycine esters across a chiral α-chloro-N-p-toluenesulfinylaldimine. Selective deprotection under different acidic reaction conditions and ring closure of the γ-chloro-α,β-diaminocarboxylamides was optimized, which resulted in Nα-deprotected syn-γ-chloro-α,β-diaminocarboxylamides, N-sulfinyl-β,γ-aziridino-α-aminocarboxylamide derivatives, a trans-imidazolidine, and an Nα,Nβ-deprotected syn-γ-chloro-α,β-diaminocarboxylamide.The asymmetric synthesis of new chiral γ-chloro-α,β-diaminocarboxylamide derivatives by highly diastereoselective Mannich-type reactions of  In recent years, non-proteinogenic diaminocarboxylic acids have gained a lot of attention among organic chemists and biochemists –3. This DPP IVs are proteases that specifically cleave off N-terminal dipeptides and are involved in the degradation of incretin hormones, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). GLP-1 is involved in the regulation of glucose homeostasis through stimulation of insulin secretion, inhibition of glucagon release, and delay of gastric emptying. It has been demonstrated that the presence of intravenous GLP-1 increases insulin secretion as a response to elevated glucose levels, and as such, GLP-1 can offer therapeutic benefits for patients with type 2 diabetes. Unfortunately, therapeutic application of GLP-1 is problematic due to the lack of oral activity and the rapid degradation by plasma DPP IV. Therefore, DPP IV inhibitors could offer a solution to this problem, as they can extend the duration of action of GLP-1 and prolong the beneficial effects –12.S)-2,4-diaminobutanoylpiperidine, is a lead compound in the development of a large series of highly potent and selective DPP II inhibitors ..N-sulfinN-(diphenylmethylene)glycinamides 4 was performed starting from N-Boc glycine, in accordance with literature procedures  in this transition state, TS-6a is highly disfavored. The formation of the -γ-chloro-α,β-diaminocarboxylamides syn-5 can be explained by a boatlike transition-state model TS-6b involving the (E)-N-p-toluenesulfinylaldimines 3 [TS-6b, in which the haloalkyl group (–CClR2) occupies the less hindered pseudoequatorial position, and the corresponding Li-adduct 7 are stabilized by the interaction between the Li-cation, the diphenylmethyleneamino group, and the sulfinylimine nitrogen.Reaction of the l esters . Howeverimines 3 –35. ThisN-sulfinylimines 3 with the glycinamides 4, as compared to the reaction with glycine esters, is attributed to the α-coordinating ability of the chlorine atom with the lithium of the incoming enolate as depicted in transition state TS-6b. The coordinating α-chloro atom in TS-6b overrides the chelation of the sulfinyl oxygen  and allows the sulfinylimine to react in the conformation wherein the S=O bond and the lone pair of electrons on the nitrogen atom are antiperiplanar [N-p-toluenesulfinylimines containing an oxygen atom as α-coordinating group [syn-addition products syn-5 were subsequently cyclized to the corresponding N-sulfinyl-β,γ-aziridino-α-aminocarboxylamides 8 upon treatment with K2CO3 in acetone under reflux in a moderate to very good yield  at rt for 15 min. After a basic workup with NH4OH, it was concluded that the conversion towards the N-deprotected syn-β,γ-aziridino-α-aminocarboxylamide 9b was complete based on 1H NMR and LC–MS analysis of the crude reaction mixture. Unfortunately, all attempted purification techniques  to remove benzophenone and some other minor impurities from the crude reaction mixture, failed to provide the pure N-deprotected syn-β,γ-aziridino-α-aminocarboxylamide 9b.In order to extend the potential applicability of the synthesized nditions . In analsyn-γ-chloro-α,β-diaminocarboxylamides syn-5 was investigated en route towards the development of potential DPP inhibitors [syn-γ-chloro-α,β-diaminocarboxylamides syn-5 were treated with 5 equiv of trifluoroacetic acid in acetone/water (2:1) for 15 min . The obtained result was in accordance with the earlier reported selective deprotection of a benzophenone imine functionality, in the presence of an N-p-toluenesulfinyl moiety, of diamino esters with H3PO4/H2O/THF [After a basic workup with NH/H2O/THF ,40.syn-γ-chloro-α,β-diaminocarboxylamide 10b was chemoselectively cyclized to the corresponding N-sulfinyl-β,γ-aziridino-α-aminocarboxylamide 11b upon treatment with K2CO3 in acetone under reflux in 86% yield. In order to provide access to the Nα,Nβ-deprotected syn-γ-chloro-α,β-diaminocarboxylamides, syn-γ-chloro-α,β-diaminocarboxylamides syn-5 were subjected to some alternative acidic deprotection reactions  group was not removed under these reaction conditions but was trapped by the deprotected β-amino group, as the deprotection of analogous anti-substrates under the same reaction conditions led to unprotected anti-α,β-diaminocarboxyl esters [syn-γ-chloro-α,β-diaminocarboxylamide could subsequently ring close further to trans-imidazolidine 12b, which will be less sterically congested than an analogous cis-imidazolidine. In the literature, comparable non-halogenated trans-imidazolidines were already synthesized by 1,3-dipolar cycloaddition of N-benzylidene glycine ester enolates across N-sulfinylaldimines in the presence of a Lewis acid [trans-stereochemistry of imidazolidine 12b was ensured by the vicinal coupling constant 3JH4-H5 = 7.43 Hz and the 1H NMR chemical shift of H4 (3.85 ppm), which were in the same range as for closely related trans-imidazolidines and trans-oxazolidines [trans-imidazolidine 12b is a potential building block for foldamers, as the corresponding trans-oxazolidin-2-ones are already applied as such [trans-Imidazolidine 12b could also be used as a precursor of the corresponding Nα,Nβ-deprotected α,β-diaminocarboxylamide, by hydrolysis under acidic conditions, in analogy with deprotection reactions of imidazolidines, imidazolines and oxazolines in the literature [syn-γ-chloro-α,β-diaminocarboxylamide syn-5a was directly converted into the dihydrochloride of the Nα,Nβ-deprotected syn-γ-chloro-α,β-diaminocarboxylamide 13a, by stirring in 0.5 M (aq) HCl/EtOAc (2:1) for 30 min at rt, in a yield of 83%. In this reaction, the acid-catalyzed hydrolysis of the benzophenone imine functionality proceeds readily and prevents the formation of the corresponding trans-imidazolidine.In an initial reaction, ol at rt . This rel esters . This isl esters –42. The wis acid . The traolidines . The tra as such . trans-Iterature ,47–48. Hsyn-γ-chloro-α,β-diaminocarboxylamides are formed in acceptable to good yields and with excellent diastereomeric ratios by stereoselective Mannich-type reactions of N-(diphenylmethylene)glycinamides across chiral α-chloro-N-p-toluenesulfinylaldimines. Notably, a very high syn-diastereoselectivity was obtained in the synthesis of the -γ-chloro-α,β-diaminocarboxylamides with the opposite enantiotopic face selectivity as compared to the Mannich-type additions of N-(diphenylmethylene)glycine esters across chiral α-chloro-N-p-toluenesulfinylaldimines. The synthesized γ-chloro-α,β-diaminocarboxylamides were selectively deprotected under acidic conditions, and the resulting α,β-diaminoacylpyrrolidines and -piperidines have a potential applicability as dipeptidyl peptidase inhibitors, which is currently under investigation.In conclusion, it was demonstrated that new chiral File 11H NMR and 13C NMR spectra for compounds 3, syn-5, 8, and 10–13.General experimental conditions, experimental procedures and data, copies of File 2syn-5b.CIF-file of compound


Flock House virus (FHV), the best studied of the animal nodaviruses, has been used as a model for positive-strand RNA virus research. As one approach to identify host genes that affect FHV RNA replication, we performed a genome-wide analysis using a yeast single gene deletion library and a modified, reporter gene-expressing FHV derivative. A total of 4,491 yeast deletion mutants were tested for their ability to support FHV replication. Candidates for host genes modulating FHV replication were selected based on the initial genome-wide reporter gene assay and validated in repeated Northern blot assays for their ability to support wild type FHV RNA1 replication. Overall, 65 deletion strains were confirmed to show significant changes in the replication of both FHV genomic RNA1 and sub-genomic RNA3 with a false discovery rate of 5%. Among them, eight genes support FHV replication, since their deletion significantly reduced viral RNA accumulation, while 57 genes limit FHV replication, since their deletion increased FHV RNA accumulation. Of the gene products implicated in affecting FHV replication, three are localized to mitochondria, where FHV RNA replication occurs, 16 normally reside in the nucleus and may have indirect roles in FHV replication, and the remaining 46 are in the cytoplasm, with functions enriched in translation, RNA processing and trafficking. All viruses depend on host cell functions for multiple replication steps, and modulate host pathways to make infected cells a better environment for virus replication. Accordingly, both targeted and genome-wide studies identifying cellular factors and virus-host interactions that support or inhibit virus replication offer great value for understanding infection and developing novel antiviral approachesNodaviridae family of animal viruses. FHV has a small, bipartite RNA genome. The smaller genomic RNA, RNA2 (1.4 kb), encodes the capsid protein precursor. The larger genomic RNA, RNA1 (3.1 kb), encodes multifunctional protein A, which has RNA-dependent RNA polymerase, self-interaction and membrane-interaction domains and is the sole viral protein required for FHV RNA replication Drosophila cells, a natural host for productive FHV infection, FHV RNA replication and transcription occur in virus-induced, ∼50 nm invaginations of outer mitochondrial membranes The largest genetic class of viruses are the positive-strand RNA viruses, which encapsidate messenger-sense single-stranded RNA and include many important human pathogens like hepatitis C virus, West Nile virus, many animal pathogens, and the majority of known plant viruses. One model that has been used to analyze the mechanisms of positive-strand RNA virus replication and virus-host interaction is Flock House virus (FHV), the best-studied member of the Saccharomyces cerevisiae, a widely used model for genetics, cellular and molecular biology S. cerevisiae, including an ordered, genome-wide, single gene deletion or yeast knockout (YKO) library that allows systematically studying the effects of each gene on a selected process, such as FHV replication. This YKO library was previously used to identify genes that facilitate or inhibit brome mosaic virus (BMV) and tombusvirus RNA replication Nodaviridae. Moreover, very limited overlap was found among the host genes that strongly modulated BMV and tombusvirus replication. Thus, analysis of host factors required for FHV replication should shed further light on the replication pathways and host interactions of positive-strand RNA viruses.To identify host genes that facilitate or inhibit FHV replication, we utilized FHV’s ability to replicate in an unusually wide range of cells. Although originally isolated from insects, FHV RNA also efficiently replicates and directs the production of infectious virions in mammalian cells, plant cells, and the yeast Renilla luciferase reporter gene, whose expression depends on viral RNA replication and sub-genomic mRNA synthesis. Implicated candidate genes were then validated by further testing of their effects on the replication of wt FHV RNA1, confirming 65 genes whose deletion produced significant effects on FHV genomic RNA1 replication and sub-genomic RNA3 production.Here we present a systematic functional genomics analysis of host genes that affect FHV replication using ∼4,500 yeast deletion mutant strains, each with a single known ORF knocked out, covering 80% of the yeast genome. Initial high throughput screening was conducted with an RNA1 derivative containing a GAL1 promoter to express an FHV genomic RNA1 derivative with two engineered changes , as required for RNA1 replication and subgenomic RNA3 production 692 to E amino acid change in a conserved polymerase motif The data in 600 = 0.075 in SD medium with galactose for two more passes to induce virus replication. Yeast growth was monitored by measuring culture OD600. For each strain, Renilla luciferase activity was measured using a whole cell assay as described Except for 301 yeast deletion strains with previously annotated significant growth defects, the remaining 4,491 strains of the BY4743 homozygous diploid yeast knock-out (YKO) non-essential library were tested for their ability to support FHV RNA replication as follows: yeasts were transformed with plasmid pFA and pF1R, and the resulting yeast transformants were grown in liquid synthetic defined (SD) medium with histidine and leucine omitted to maintain plasmid selection. Glucose was provided as the carbon source for the first passage in liquid medium. After one day of growth, the yeasts were sub-cultured to a starting density of ODTwo independent passes of the above screen were performed across the 4,491 YKO strains, which were arrayed in 48 96-well plates. The YKO library contains 84 duplicate strains, which leaves 4,407 unique yeast deletion mutant strains. In the first and second pass, 204 and 212 unique strains respectively could not be assayed as they either failed to be transformed with pFA + pF1R or to grow in galactose liquid medium. Of the affected strains, 147 were not assayed in either pass, and 122 were only assayed once. The remaining 4,138 unique strains were tested twice for FHV replication, with a small portion tested four times because they were duplicated in the library. The Pearson’s correlation coefficient between the two passes is 0.446.To stabilize the variance between samples with high and low luciferase activity readings, a power transformation  of ≤5%, 46 strains (25%) showed significant differences from wild type yeast . The remaining 5 genes encode cytoplasmic proteins, including the PRR1 and PSK2 kinases involved in signal transduction VIP1 that functions in actin cytoskeleton organization EGD1, a ribosome-associated chaperone involved in folding and targeting of newly synthesized peptides, including some destined for mitochondria WHI5, a cell cycle-regulated transcriptional repressor that accelerates G1/S transition Among the 65 confirmed genes, only eight showed decreased FHV replication when deleted, indicating that their functions support FHV replication . Three oLEA1, FHV RNA1 accumulation in individual experiments was always increased above wt yeast, but ranged from 2.6- to 23-fold higher than in wt yeast  and proteins involved in nucleoside metabolism , other metabolic processes , protein ubiquitination (DIA2), signal transduction (ASC1), and unknown functions .Among the 57 confirmed genes whose deletion increased FHV RNA replication, 41 encoded proteins that localized to the cytoplasm, while 16 encoded proteins that normally localized to the nucleus . Of the trans (pFA + pF1R) and cis (pF1) FHV RNA replication systems for primary genome-wide screening and validation testing, these genes might also affect plasmid-directed transcription or nucleo-cytoplasmic export of viral RNA.The 16 genes with products normally localized to the nucleus include eight genes that function in transcription, two in chromatin remodeling, one in DNA repair and five in the cell cycle . Since FRenilla luciferase expressed through viral RNA replication and sub-genomic mRNA synthesis. Interestingly, however, few host genes were identified in common between these studies. Only one gene, e.g., was identified to affect all three viruses: VPS24, a subunit of endosomal sorting complex required for transport III (ESCRT-III), whose deletion decreased TBSV and BMV replication, but increased FHV replication. The only additional common genes revealed by pairwise comparisons were ribosomal protein gene RPL7A  Ski2, Ski3/5, and Ski7, which repress the levels of endogenous dsRNA replicons in yeast BUD31, an RNA splicing factor VID22, implicated in membrane import and DNA repair Among five host genes with similar effects on BMV and FHV replication, three are related RNA degradation genes NEW1, involved in mRNA export and ribosome biogenesis MCM16, which functions in chromosome segregation BUD26, a gene of unknown function. In addition to these whole genome screening results, our recent targeted studies Lsm1, Lsm6, Lsm7, and Dhh1 are required to efficiently recruit BMV genomic RNAs into both translation and RNA replication Three genes that facilitate BMV replication were found here to suppress FHV replication  increase virus replication when deleted, in contrast to TBSV, for which 90 of 96 confirmed genes (94%) decreased virus replication when deleted In keeping with the distinct groups of host genes required by these three positive-strand RNA viruses, individual retroviruses like HIV-1 and Moloney murine leukemia virus (MuLV) also each require or are restricted by many different host factors HindIII/SpeI fragment of pBDL7fs-GFPN2Renilla luciferase ORF downstream of FHV B1 AUG codon.pFA was generated by cloning the Renilla luciferase assay were performed as previously described Yeast strain BY4743 (WT) and the homozygous diploid deletion series (BY4743 strain background) were obtained from Research Genetics . Standard yeast techniques were used. 96-well transformation and the Renilla luciferase reporter gene as in 600 = 0.075, grown for one day at 26°C, and sub-cultured again in galactose-SD-medium at OD600 = 0.075 and grown at 26°C overnight. This final overnight culture was assayed as previously described Renilla luciferase activity.To assay for FHV directed replication and expression of the Renilla luciferase activity of each deletion strain in relative light units (rlu) was transformed by taking the 600 numbers of all strains were measured to monitor the growth of yeasts, and were normalized to the plate median as well. 5% of the total strains that grew too fast and 5% that grew too slow were removed from the final analysis.The For validation testing, selected candidate yeast strains were grown in 96-deep-well plates and transformed as described above with pF1. Four colonies of each transformed strain were picked to check for FHV replication. The yeast were again grown in glucose-SD-medium for one day, then sub-cultured twice in galactose-SD-medium as described above for primary screening. Total yeast RNA was isolated http://www.r-project.org/), one sided T-tests were performed to identify mutant yeast strains with significantly altered FHV replication relative to wild type yeast. Q-values were calculated based on t-scores to control the false discovery rate under 5% Using the R statistical package (version R-2.11.1) (Table S1Whole screen data for FHV replication in yeast knockout library (YKO).(XLSX)Click here for additional data file.


In vivo imaging of subcellular RNA localization in Xenopus oocytes reveals domains of transport directionality mediated by distinct molecular motors, with dynein providing a directional cue for polarized transport. Xenopus oocytes. Using a novel approach to measure directionality of mRNA transport in live oocytes, we observe discrete domains of unidirectional and bidirectional transport that are required for vegetal RNA transport. While kinesin-1 appears to promote bidirectional transport along a microtubule array with mixed polarity, dynein acts first to direct unidirectional transport of RNA towards the vegetal cortex. Thus, vegetal RNA transport occurs through a multistep pathway with a dynein-dependent directional cue. This provides a new framework for understanding the mechanistic basis of cell and developmental polarity.Cytoplasmic RNA localization is a key biological strategy for establishing polarity in a variety of organisms and cell types. However, the mechanisms that control directionality during asymmetric RNA transport are not yet clear. To gain insight into this crucial process, we have analyzed the molecular machinery directing polarized transport of RNA to the vegetal cortex in  Like traffic on highways, molecular cargos are transported within cells on tracks that are collectively referred to as cytoskeletal networks. RNA molecules are one such cargo, and in many species, the localization of RNAs in egg cells or oocytes is essential for establishing the first asymmetries that are necessary for proper embryo development. RNAs can be actively transported by molecular motors that move cargos along the cytoskeletal tracks, but how such motors are capable of directing cargos to specific destinations within the cell is not yet known. Here we show that two motors, dynein and kinesin—known to carry out transport in opposite directions—are both directly involved in RNA localization in frog oocytes. To understand how these motors can promote directional cargo transport, we developed a system to monitor RNA transport in live oocytes. We find that the motor acting first in the pathway, dynein, is responsible for unidirectional transport. Bidirectional transport, mediated by kinesin, occurs subsequently on cytoskeletal tracks of opposing polarity near the RNA's final destination. Our results suggest a new model for directional transport comprising an initial directional cue that dominates over a later nondirectional step, acting to refine the ultimate cargo distribution. RNA transport underlies cell and developmental polarity in many organisms. Spatial regulation of gene expression mediated by subcellular RNA localization is required for embryonic axis formation, germ cell specification, and neuronal polarity Xenopus oocytes, localization of Vg1 mRNA to the vegetal cortex during oogenesis is essential for proper germ layer patterning during embryogenesis In Drosophila blastoderm embryo recruit more dynein motors than are recruited to nonlocalizing transcripts oskar RNA localization by the kinesin-1 motor relies on a slight bias in microtubule orientation within the Drosophila oocyte for directional transport Regulating the net direction of transport is crucial, both for RNAs and for other cargos such as vesicles and organelles. This important process is not well understood, although recent studies have suggested a range of possible mechanisms. Some models favor regulation at the level of the molecular motor, whereby control of motor number, motor activity, or both is responsible for directional transport Xenopus oocytes we have analyzed the functions of specific molecular motors in vegetal RNA transport. Through biochemical and in vivo interference experiments, we show that cytoplasmic dynein is required for an initial step in the vegetal RNA transport pathway. This is followed by a kinesin-dependent step that ultimately brings the RNA to the cortex. Using in vivo imaging approaches, we measured the rate and direction of RNA movement, revealing discrete regions of transport directionality in the oocyte cytoplasm. While transport near the vegetal cortex is bidirectional, dynein-dependent transport is strongly biased toward the vegetal pole and thus provides the initial directional cue for polarized RNA transport. Our results reveal an unexpectedly complex choreography, with multiple motors moving in several directions, to ensure delivery of RNA cargos to their precise destination.To uncover the basis for directional RNA transport in Xenopus oocyte is mediated in part by kinesin motors Glued  is significantly lower than that of a nonlocalizing βG-MS2 RNA (t1/2 = 16.6±1.92 s). Yet the mobility of VLE-MS2 RNA outside of the vegetal cytoplasm (1/2 = 16.4±0.97 s) to that of βG-MS2 RNA. The calculated diffusion coefficients outside of the vegetal cytoplasm (1/2 = 16.2±2.13 s). Since the FRAP experiments assess the mobility of the entire population of MS2-tagged RNAs in a given region, these results indicate that the majority of VLE-MS2 RNAs in the vegetal cytoplasm are not moving rapidly at any given time. Moreover, the reduced mobility of VLE RNA in the vegetal cytoplasm is likely due to interaction of the RNA with the microtubule cytoskeleton, presumably through an interaction with molecular motors.To measure RNA transport in vivo, we used Fluorescence Recovery After Photobleaching FRAP, . The larytoplasm  are consTo assess the effects of dynein disruption in live oocytes, we performed FRAP in the vegetal cytoplasm in oocytes overexpressing dynamitin and in control oocytes. We carried out FRAP in Regions 1 and 3, but not Region 2, due to enlargement of the cup region in oocytes expressing dynamitin . As showDirectionality is crucial to understanding the mechanisms controlling asymmetric RNA transport, and roles for motors that move in opposing directions on microtubules complicate this issue. We have previously described a subpopulation of microtubules, present at the vegetal pole during mid-oogenesis, which are oriented with plus ends at the cortex 1 and t2   at 18°C for 8–16 h as previously described For in vivo interference, kinesin-1 rigor g for 10 min, lysates were precleared with mouse IgG-agarose beads (Sigma) before overnight incubation with mouse IgG (Sigma) or mouse anti-dynein intermediate chain antibody  prebound to Protein A-Sepharose beads (Millipore). After washes, bound samples were reserved for protein blotting, or samples were sequentially treated with DNase and proteinase K before RT-PCR using primers for Vg1 and EF1α, as previously described Glued (Abcam), anti-Vera Oocyte cell lysates were made in 10 mM HEPES pH 7.4, 100 mM potassium acetate, 10 mM magnesium acetate, 5 mM EGTA, 0.1 M sucrose, 1 mM DTT, 0.4 mM Pefabloc SC (Sigma-Aldrich), 0.1% NP-40, 1 U/ml RNasin (Promega), 0.1 µg/ml leupeptin, 0.1 µg/ml antipain, and 0.1 µg/ml trypsin inhibitor. After centrifugation at 10,000× GluedOocytes were microinjected with purified function-blocking antibodies  or RNAs (at 250–500 nM) encoding kinesin-1 rigor 2b, DIC 74.1) and Alexa-594  fluorescent secondary antibodies (Invitrogen), washed with PBT, dehydrated, and stored in anhydrous methanol at −20°C. Oocytes were imaged on either a Zeiss LSM510 or LSM710 confocal microscope as previously described Oocytes were permeabilized with 50 µg/ml proteinase K for 3–6 min, fixed in 3.7% formaldehyde for an hour, blocked in 2% BSA and 2% goat serum, and incubated overnight with purified mouse anti-kinesin 1  and/or anti-dynein  antibodies, both at 1∶100 dilution. Oocytes were then washed with PBT  and incubated overnight with isotype-specific mouse Alexa-633 . Stage III albino oocytes were injected with 2 nl of either 250 nM mCh-MCP mRNA or 250 nM PA-mCh-MCP mRNA and incubated overnight in OCM to allow protein expression. Oocytes in some cases were incubated in 10 µg/ml nocodazole to depolymerize microtubules  were calculated using the previously determined rate constant (b) in the following equation: 1/2 values by fitting to a simple diffusion model r is the radius of the FRAP ROI.FRAP analyses were carried out using a Zeiss LSM 510 Meta Confocal Laser Scanning Microscope equipped with a 40× water correction C-Apochromat objective. Regions for FRAP were identified for each oocyte as follows: Region 1 was 15 µm into the vegetal cytoplasm from the germinal vesicle, Region 2 was 50 µm into the vegetal cytoplasm from the germinal vesicle, Region 3 was 25 µm into the vegetal cytoplasm from the vegetal cortex, and Region 4 was 25 µm into the animal cytoplasm from the animal cortex. Within each region, a 5 µm circular region of interest (ROI) was bleached using the 405, 488, 561, and 633 laser lines at 100%. Fluorescence recovery was monitored at 5-s intervals to track VLE-MS2 recovery and 1-s intervals for βG-MS2. FRAP data were fit as previously described A Zeiss LSM 710 Confocal Laser Scanning Microscope equipped with a 40× water correction C-Apochromat objective was used to acquire images. PA-mCh-MCP 1, t2). Change in fluorescence intensity within the collection windows was measured in order to quantify the population of moving RNA using the following formulas:RNA movement was calculated by defining 5 µm diameter collection windows at 5 µm and 15 µm distances left, right, animal, or vegetal from the site of activation. The fluorescence intensities from each collection window were plotted over time see  to identFigure S1Dynein antibody injection blocks vegetal RNA localization. Oocytes were injected with either (A) control IgG (Sigma) or (B) function-blocking (TIF)Click here for additional data file.Figure S2Time course of vegetal RNA localization. Oocytes were injected with fluorescently labeled VLE RNA and cultured for 4–24 h before fixation and imaging. (A) At 4 h, the injected RNA is found predominantly in the perinuclear cup region, with little accumulation at the oocyte cortex. (B) By 8–10 h, the injected RNA is evident throughout the vegetal cytoplasm, with significant accumulation at the oocyte cortex. (C) Twenty-four hours after injection, the RNA is predominantly cortical, with little RNA in the vegetal cytoplasm and none detected in the cup region. Representative confocal images are shown, with the vegetal pole towards the bottom. Scale bars, 50 µm.(TIF)Click here for additional data file.Figure S3glued of dynactin or (B) no exogenous protein (Control), and incubated overnight to allow exogenous protein expression. Microtubules were stained with anti-α-tubulin (Sigma) and fluorescent secondary antibodies before imaging by confocal fluorescence microscopy. Shown are representative confocal sections cropped to show the vegetal cytoplasm. Scale bars, 20 µm.Inhibition of dynein by dynactin disruption does not alter microtubule organization in the vegetal cytoplasm. Oocytes were injected with (A) the CC1 domain of p150(TIF)Click here for additional data file.Figure S4glued of dynactin (CC1) or (B) no exogenous protein (Control), and incubated overnight to allow expression of exogenous protein. Dynein was detected with anti-dynein  antibodies and fluorescent secondary antibodies before confocal microscopy. Shown are representative confocal sections cropped to show the vegetal cytoplasm. Scale bars, 50 µm.Inhibition of dynein by dynactin disruption does not alter dynein distribution in the vegetal cytoplasm. Oocytes were injected with (A) the CC1 domain of p150(TIF)Click here for additional data file.Figure S5Kinesin-1 Δmotor mutant disrupts vegetal RNA localization. (A) Uninjected oocytes or (B) oocytes injected with 250 nM kinesin-1 heavy chain lacking the motor domain XKHCΔm  were sub(TIF)Click here for additional data file.Figure S6Transport directionality is not evident for RNAs that are not undergoing localization. Oocytes expressing PA-mCh-MCP were microinjected with (A) βG-MS2 RNA or (B–C) VLE-MS2 RNA. Time courses after activation in (A–B) the lower vegetal cytoplasm (Region 2) in untreated (A) and nocodazole-treated (B) oocytes or (C) the animal hemisphere cytoplasm (Region 4) are shown. Fluorescence intensities in the V (black) and A (grey) quadrants are shown. After activation (A–C), fluorescence returned to background levels within 20–30 s.(TIF)Click here for additional data file.Figure S7n = 10 oocytes), (B) the lower vegetal cytoplasm (n = 10 oocytes), (C) the animal hemisphere (n = 19 oocytes), and (D) in nocodazole-treated oocytes activated in the vegetal cytoplasm (n = 13 oocytes). The value for a given quadrant was calculated as the percentage of total intensity (V+A or L+R), and all error bars indicate standard deviation. p values were generated using an unpaired Student's t test; *p = 0.00001, **p = 0.16.Averaged RNA transport directionality in specific regions. After activation of PA-mCh-MCP, as in (TIF)Click here for additional data file.Figure S8Dynein remains colocalized with VLE RNA after localization. Oocytes injected with VLE RNA were incubated for 24 h to allow the majority of the injected RNA to complete localization, then probed with anti-dynein  and fluorescent secondary antibodies before confocal microscopy. Shown is a confocal section: (A) VLE RNA and (B) dynein. A cropped and zoomed view (C) shows co-localization in the vegetal cytoplasm, with VLE RNA in red, dynein in green, and co-localization in yellow. Scale bars, 50 µm.(TIF)Click here for additional data file.


Mosquito-borne infectious diseases pose a severe threat to public health in many areas of the world. Current methods for pathogen detection and surveillance are usually dependent on prior knowledge of the etiologic agents involved. Hence, efficient approaches are required for screening wild mosquito populations to detect known and unknown pathogens.Culex pipiens molestus mosquitoes. The majority of the viral transcripts (.>80% of the region) were covered by the small viral RNAs, with a few peaks of very high coverage obtained. The +/− strand sequence ratio of the small RNAs was approximately 7∶1, indicating that the molecules were mainly derived from the viral RNA transcripts. The small viral RNAs overlapped, enabling contig assembly of the viral genome sequence. We identified some small RNAs in the reverse repeat regions of the viral 5′- and 3′ -untranslated regions where no transcripts were expected.In this study, we explored the use of Next Generation Sequencing to identify viral agents in wild-caught mosquitoes. We extracted total RNA from different mosquito species from South China. Small 18–30 bp length RNA molecules were purified, reverse-transcribed into cDNA and sequenced using Illumina GAIIx instrumentation. Bioinformatic analyses to identify putative viral agents were conducted and the results confirmed by PCR. We identified a non-enveloped single-stranded DNA densovirus in the wild-caught Our results demonstrate for the first time that high throughput sequencing of small RNA is feasible for identifying viral agents in wild-caught mosquitoes. Our results show that it is possible to detect DNA viruses by sequencing the small RNAs obtained from insects, although the underlying mechanism of small viral RNA biogenesis is unclear. Our data and those of other researchers show that high throughput small RNA sequencing can be used for pathogen surveillance in wild mosquito vectors. The sequencing technologies offer distinct advantages over traditional viral detection and surveillance methods that generally require prior knowledge of the etiologic agents, as well as depending on virus-specific primers, probes or antibodies. These traditional techniques are, therefore, unsuitable in situations where the causative agent of an outbreak is entirely novel, or is a pathogen variant with several mutations to key priming regions. Hence, high throughput sequencing techniques provide a powerful new opportunity for surveillance and discovery of novel pathogens. The techniques provide a cost-effective mechanism for massive parallel sequencing generating extreme sequencing depth, whilst providing multiplex analyses for etiologic agent identification.Mosquito-borne infectious diseases have been emerging and re-emerging in many areas of the world, especially in tropical and subtropical areas where agents such as West Nile virus (WNV), dengue virus (DENV), chikungunya virus (CHIKV) and yellow fever virus (YFV) are present. Surveillance of infectious agents carried by mosquitoes is important for predicting the risk of vector-borne infectious disease outbreaks. Recently, a new strategy based on small interfering RNA (siRNA) immunity to virus infection was proposed for detecting novel RNA viruses in laboratory reared drosophilae and mosquitoes, as well as RNA/DNA viruses in plants using high throughput sequencing techniques AAAAA…, GCGCGC…), or multiple unresolved nucleotides, which were resulted from sequencing procedures, mappable sequences were generated. By mapping to the miRNA database, we identified about 200 known miRNAs for each mosquito species. Using miRNA prediction software, one to two thousand miRNA candidates were predicted , Anopheles gambiae densonucleosis virus (GenBank Accession: EU233812), and Aedes aegypti densovirus strain 0814616 (GenBank Accession: FJ360744). Further analysis demonstrated that the matched A. albopictus Parvovirus sequences were also present in the A. gambiae densonucleosis virus genome and the A. aegypti densovirus strain 0814616 genome. The A. gambiae densonucleosis virus and A. aegypti densovirus strain 0814616 shared most of their matched sequences. Sequence alignment showed that these three viruses exhibited more than 80 percent sequence identity, indicating that a virus with homologous sequences to these three viruses was present within the C. pipiens molestus sample. For the other two samples (C. tritaeniorhynchus and A. sinensis), no significant amount of sequence was found that corresponded to any specific virus. To discover potential novel viruses which may be remotely related to known viruses, a BLAST strategy proposed in the literature We performed BLAST analysis (using the blastn program) to identify potential viral sequences in the cleaned unique sequences. Preliminary results revealed that a large number of unique sequences in the C. pipiens molestus, C. tritaeniorhynchus and A. sinensis) with CLC Bio  using the default parameters. The results showed that the small RNA reads overlapped which allowed contig assembly. Of the three viruses, A. gambiae densonucleosis had the most mapped reads (4481) and the longest assembled consensus sequence (3248 bp) which covered 78.5% of the whole genome (4139 bp) (A. gambiae densonucleosis virus was about 98% (3182/3248). The distribution of the lengths of the matched small viral RNAs showed that the majority (>60%) of them were 20–24 nt in length, with a peak distribution of 21 nt, while the total library small RNA (majority of them were endogenous siRNA) displayed a peak distribution of 22 nt  . The oveof 22 nt . This isAlthough most of the viral coding transcripts  were covered by the small viral RNA sequences, the small RNAs were not evenly distributed along the transcripts. There were 10–20 sites with relatively high coverage and 4 of these had very high coverage indeed (greater than 320× coverage compared with the average coverage of 24×) . The corThe densovirus genome contains two-pairs of inverted repeats, which constitute two stem-loop structures at the 5′ and 3′ untranslated regions of the genome termini . It alsoIt is notable that the high coverage small RNAs in the coding regions and the small RNAs mapping to the non-coding stem-loop regions are both highly conserved (0/385 nucleotide difference), compared to the genome as a whole which is roughly 2% different to the reference densovirus (EU233812). Such high evolutionary conservation suggests that these sequences are of functional importance.C. pipiens molestus mosquito sample, but not from C. tritaeniorhynchus or A. sinensis  sequence (and the siRNA mature forms generated from dsRNA), it is reasonable to expect that only RNA viruses which contain dsRNA as genomic RNA or replicate via a dsRNA intermediate can be identified using this strategy. This perception is consistent with previous reports where only RNA viruses were identified using small RNA sequencing techniques An alternative explanation for the small RNAs derived from the DNA virus may be degradation of virus transcripts. However, this hypothesis cannot explain at least two things: one is the very high incidence of some small RNAs that have 3–4 adenines at the 5′ terminus, the other is the biogenesis of the small RNAs that map to the inverted regions of the genomic termini. These are predicted to form a T- or Y-shaped structure that may participate in genome packaging signaling or replication initiation A. aegypti mosquitoes showed that the virus had significant efficiency, although most densoviruses take 2–20 days to kill their insect hosts Mosquitoes are the most important vector of WNV, DENV, CHIKV and YFV, and controlling mosquito populations is an important way of preventing epidemics of these life-threatening diseases. Among the many approaches to mosquito control Traditional generic methods for identifying and characterizing novel viral diseases have included electron microscopy, virus isolation in cell culture, immunological approaches and PCR. Recently technologies such as diagnostic microarrays and mass spectrometry have been proposed as generic tools for identifying viruses Although the densovirus identified here was not a significant etiologic agent, this discovery proves that the approach is applicable not only for discovery of RNA viruses, but also DNA viruses in mosquitoes. Currently all known human pathogenic viruses found in mosquitoes are RNA viruses, but this does not preclude DNA viruses from using mosquitoes as vectors for human, animal or plant diseases. Indeed the African swine fever virus is an arthropod-borne double-stranded DNA virus In conclusion, our study is the first to explore the application of convenient small RNA high throughput sequencing for virus discovery in wild-caught vectors. Our results suggest that small RNA sequencing is able to identify not only RNA viruses, but also DNA viruses in wild-caught mosquitoes, obviating the need for culture-based virus isolation or for prior knowledge of the etiologic agent. These results suggest that small RNA high throughput sequencing could be an ideal tool for surveillance of novel emerging viral disease or even non-viral infectious diseases.Culex tritaeniorhynchus, Culex pipiens molestus, and Anopheles sinensis were collected from Yunnan province, China, in 2009. The samples were stored in liquid nitrogen until RNA extraction. No specific permits were required for the described field studies; the samples collected were not privately owned or protected and did not involve endangered or protected species.The mosquitoes, including Prior to RNA extraction, mosquitoes were cleaned in sterilized water and dried with hygroscopic filter paper. Mosquitoes of the same species were pooled together. Total RNA was extracted separately from the different mosquito species using the Total RNA Purification Kit , according to the manufacturer's instructions. The quality of total RNA was analyzed on an Agilent 2100 Bioanalyzer system and by denaturing polyacrylamide gel electrophoresis. A small RNA library was generated according to the Illumina sample preparation instructions http://www.sanger.ac.uk/software/Rfam) and in the GenBank non-coding RNA database (http://www.ncbi.nlm.nih.gov/) to clarify degradation fragments of non-coding RNA. In addition, all sequences were mapped to miRNA sequences from the miRNA database, miRBase 16.0 (http://www.mirbase.org/).Clean-up of the raw data and subsequent small RNA mapping and prediction were performed with a proprietary software package, ACGT101-miR v3.5 . First, low-quality reads were removed from the raw reads. After removal of the adaptor sequences, and filtering of the low quality reads and simple artificial sequences, the mappable reads were extracted and the unique sequences generated by collapsing the identical sequences, with the occurrence count of each unique sequence as the unique sequence tag. These unique sequences were compared with the sequences of non-coding RNAs  available in Rfam (http://www.ebi.ac.uk/embl/) as a query, in order to expedite the BLAST process. BLAST results were then analyzed manually to screen for potential virus sequences.BLAST searches were conducted to identify the virus sequences in the cleaned unique reads using the blast-2.2.22 package 5′-ATA AAT TGA TCA GTC GTC CTC CAA C-3′; reverse primer: 5′-CTT GGG ATC ATT TCG GTC ATA T-3′) were selected from the viral sequence assembled with the mappable reads. The PCR was conducted in a 50 µl reaction mixture containing 1×Easy Taq PCR SuperMix , 1 µM each of the forward and reverse primers and 10 ng of template DNA. After pre-denaturation at 94°C for 3 minutes, 35 cycles of amplification  were performed, followed by a final incubation at 72°C for 5 min. PCR products were visualized on a 1% agarose gel stained with ethidium bromide.Total mosquito DNAs were extracted with TRIzol reagent  according to the manufacturer's instructions. A pair of primers  as the reference sequence. The read alignment is saved in the ace file format and can be viewed with common read alignment program like tablet (freely available on http://bioinf.scri.ac.uk/tablet/). All the read sequences can be retrieved from the ace file with any text editing program.(ACE)Click here for additional data file.