C. elegans. We find that SYD-1 mediates an UNC-6 (netrin) independent UNC-40 activity to promote ventral axon guidance. Genetic analysis suggests that SYD-1 function in axon guidance requires both UNC-40 and SAX-3 activity. Moreover, the cytoplasmic domains of UNC-40 and SAX-3 bind to SYD-1 and SYD-1 binds to and negatively regulates the MIG-2 (Rac) GTPase. We also find that the function of SYD-1 in axon guidance is mediated by its phylogenetically conserved C isoform, indicating that the role of SYD-1 in guidance is distinct from its previously described roles in synaptogenesis and axonal specification. Our observations reveal a molecular mechanism that can allow two guidance receptors to function interdependently to regulate a common downstream effector, providing a potential means for the integration of guidance signals.During development, axons must integrate directional information encoded by multiple guidance cues and their receptors. Axon guidance receptors, such as UNC-40 (DCC) and SAX-3 (Robo), can function individually or combinatorially with other guidance receptors to regulate downstream effectors. However, little is known about the molecular mechanisms that mediate combinatorial guidance receptor signaling. Here, we show that UNC-40, SAX-3 and the SYD-1 RhoGAP-like protein function interdependently to regulate the MIG-2 (Rac) GTPase in the HSN axon of  C. elegans, ventral axon guidance is mediated by the UNC-40 (DCC) and SAX-3 (Robo) guidance receptors. Here, we use genetic and biochemical analysis to identify a molecular mechanism that can mediate combinatorial signaling involving UNC-40, SAX-3 and the RhoGAP-like protein SYD-1.The nervous system is comprised of a complex network of axonal connections. This network is formed during development, when axons navigate to their target regions. Axon navigation requires multiple signaling pathways to detect and respond to extracellular guidance cues. Many of the guidance cues, receptors and signaling pathways have been identified. However, little is known about how the information encoded by different guidance cues is combined to arrive at a directional response. A key part of how this occurs is likely to involve combinatorial receptor signaling, when different guidance receptors work in combination with each other. However, the mechanisms that underlie combinatorial receptor signaling remain mostly unknown. In  Axonal migrations are guided through the developing nervous system by multiple extracellular guidance cues that activate receptors on the surface of the axonal growth cone. The axonal growth cone must integrate these multiple signals to arrive at a single directional response –7. ProgrIntegration of multiple guidance signals could be mediated by combinatorial receptor signaling . In factC. elegans, the AVM neuron is simultaneously exposed to both the UNC-6 (Netrin) and SLT-1 (Slit) guidance cues. Both of these cues guide the AVM axon towards the ventral nerve cord. UNC-40 (DCC) can function individually as a receptor for UNC-6, whereas SAX-3 (Robo) can function individually as a receptor for SLT-1. However, UNC-40 can also function independently of UNC-6, when it binds to the SAX-3 receptor to promote the response to SLT-1 [Guidance signaling pathways can also exhibit synergistic interactions. For example, in to SLT-1 . DespiteSYD-1 is an intracellular protein that contains a RhoGAP-like domain that has been implicated in synaptogenesis and axonal specification –21. HereC. elegans and extends an axon ventrally to the ventral nerve cord. To determine the relative contributions of guidance cues in HSN ventral axon guidance, we examined HSN axon guidance in slt-1(eh15) and unc-6(ev400) null mutants  temperature sensitive mutation. The single sax-3(ky200) mutant at 24°C has guidance defects with a penetrance of 19%, suggesting that this allele behaves as a strong loss of function at 24°C (unc-40(e1430); sax-3(ky200) double mutant. Although these animals were mostly embryonic lethal at 24°C, we were able to analyze axon guidance in the survivors and found that guidance defects in unc-40(e1430); sax-3(ky200) double mutants were significantly enhanced relative to either single mutant (unc-6(ev400) sax-3(ky200) double mutants were significantly enhanced relative to either single mutant. These results suggest that UNC-40 and SAX-3 can function in parallel to each other and that these two receptors mediate most, if not all, of the ventral guidance signals in the HSN axon.To investigate interactions between the signaling pathways that transduce the UNC-6 and SLT-1 cues, we examined guidance defects in mutants that disrupt their receptors, UNC-40 and SAX-3, respectively. We attempted to construct an  at 24°C . We conse mutant . Likewissax-3(ky123) null mutants had 20% defects, whereas slt-1(eh15) null mutants had only 2% defects ; unc-6(ev400) double null mutants relative to unc-40(e1430) and unc-6(ev400) single null mutants (unc-40(n324) null allele  null mutation could enhance guidance defects in unc-6(ev400) null mutants, but not in unc-40(e1430) null mutants  null allele and two different hypomorphic unc-40 alleles  null mutation, consistent with the idea that unc-40 and syd-1 function in a genetic pathway.To further test the idea that UNC-40 and SYD-1 function together, we analyzed double mutants between the  alleles . When twsyd-1(ju82) mutation that disrupts SYD-1A and SYD-1B, but spares SYD-1C  allele disrupts the SYD-1A and SYD-1B isoforms, but not the SYD-1C isoform. The syd-1(ju2) and syd-1(tm6234) alleles disrupt all three isoforms. We found that the syd-1(ju82) allele, did not enhance guidance defects in unc-6 null mutants. However, both syd-1(ju2) and syd-1(tm6234) did enhance guidance defects in unc-6 null mutants. These genetic interactions suggest that the role of the syd-1 gene in guidance may be mediated by the SYD-1C isoform. To further test the role of the SYD-1C isoform, we used the unc-86 promoter to drive expression of SYD-1C in the HSN neuron. We found that expression of SYD-1C in the HSN neuron can rescue guidance defects in syd-1(ju2); unc-6(ev400) double null mutants  null allele and mig-2 gain of function mutations. The mig-2(gm103) mutation encodes a fully activated MIG-2, whereas mig-2(gm38) encodes a partially activated MIG-2 [syd-1 can enhance the defects associated with the partially active mig-2(gm38) allele (mig-2(gm103) allele, consistent with the expectation that fully activated MIG-2 cannot be further activated. To further test the functional relationship between SYD-1 and MIG-2, we transgenically expressed SYD-1C in the mig-2(gm38) partially activated gain of function mutants (mig-2(gm38) gain of function mutation. To further test the interaction between SYD-1 and MIG-2, we constructed a syd-1(ju2); mig-2(ok2273) double null mutant. We found that penetrance of HSN guidance defects in these double null mutants was not enhanced relative to mig-2(ok2273) single mutants  allele , suggest mutants . Consist mutants , furthermig-2 in HSN axon guidance, we examined the HSN axon in mig-2(ok2273) null mutants  null mutants. To determine if mig-2 functions in a genetic pathway with sax-3, we examined sax-3 mig-2 double null mutants. If two genes function in a genetic pathway, a double null mutant should have defects similar to the greatest of the single mutants. We found that the sax-3 mig-2 double mutants had a penetrance of guidance defects similar to that of sax-3 single mutants, indicating that mig-2 and sax-3 do function in a genetic pathway. Consistent with a function for mig-2 in the sax-3 pathway, we also found that the mig-2 null mutation can enhance defects in the unc-40 null mutants. Together, these genetic interactions support the conclusion that MIG-2 functions in the SAX-3 pathway.To consider the role of  mutants . We founsyd-1 and sax-3. We found that the syd-1(ju2) null mutation fails to enhance axon guidance defects in sax-3(ky123) null mutants, suggesting that SYD-1 might function with SAX-3 (sax-3(ky200) hypomorphic allele and found that the syd-1(ju2) null mutation enhances guidance defects in sax-3(ky200) hypomorphic mutants  has been identified as a GAP that negatively regulates Rac activation to promote Robo signaling [Drosophila [We report that UNC-6 independent UNC-40 signaling is mediated by SYD-1 and that SYD-1 negatively regulates MIG-2. Thus, UNC-6 independent UNC-40 signaling can promote axon guidance by negatively regulating MIG-2. This idea is consistent with previous results showing that negative regulation of Rac promotes signaling downstream of Robo (SAX-3). In ignaling ,37. In mignaling –39. Posiignaling ,41. Likeosophila –37. TogeSYD-1C resembles its mammalian homolog mSYD1A, in that both are predicted to contain an Intrinsically Disordered Domain (ID domain) at their N-terminus, rather than a PDZ domain . ID domasax-3 null mutants relative to slt-1 null mutants. In fact, SLT-1 independent SAX-3 signaling has been observed in several different processes, including AVM and PVM axon guidance [sax-3 null mutants relative to slt-1 null mutants [slt-1 null mutants relative to sax-3 null mutants. The reason for the differences in slt-1 and sax-3 mutant defects in the AVM relative to the HSN and PVM are not known. However, these differences could be explained by a greater degree of redundant signaling in the HSN and PVM neurons relative to the AVM neuron.Although our study has focused on UNC-6 independent UNC-40 function, we have also observed evidence for SLT-1 independent SAX-3 function. This idea is supported by the higher penetrance of HSN guidance defects in guidance ,23,25,44 mutants . Genetic mutants . This in mutants ,44. Howeunc-40 null mutants to have a greater penetrance of guidance defects relative to unc-6 null mutants. However, we have found that unc-6 and unc-40 null mutants have a similar penetrance of HSN guidance defects. One possible explanation could be that UNC-6 can also function independently of UNC-40 in the HSN. Indeed, the existence of UNC-40 independent UNC-6 activity is apparent in AVM and PVM axon guidance, where unc-6 mutants have a higher penetrance of guidance defects relative to unc-40 mutants [Since we have found that UNC-40 can function independently of UNC-6, one might expect  mutants . Moreove mutants .unc-6(ev400) [slt-1(eh15) [sax-3(ky123) [unc-40(e1430) [syd-1(ju2) [syd-1(tm6234), syd-1(ju82) [mig-2(ok2273) [sax-3(ky200) [unc-40(ev546) [unc-40(tm5504) [mig-2(gm38) [mig-2(gm103) [unc-40(ev546) allele was obtained from Joseph Culotti. The syd-1(tm6234) allele was obtained from Shohei Mitani. All other alleles were obtained from the Caenorhabditis Genetics Center (CGC).Genetic manipulations were carried out using standard procedures. The N2 Bristol genetic background was used in all experiments and all animals were maintained at 20°C on NGM plates seeded with OP50 bacteria. The following alleles were used and are considered to be null: 6(ev400) , slt-1(e-1(eh15) , sax-3(k3(ky123) , unc-40(0(e1430) , syd-1(jd-1(ju2) , syd-1(t-1(ju82) , mig-2(o(ok2273) . The fol3(ky200) , unc-40(0(ev546) , unc-40((tm5504) . The fol-2(gm38) , mig-2(g2(gm103) . The uncThe pAGC5 plasmid includes Punc-86::syd1c::sl2::tagrfp and was created using Gibson Assembly and cloned into pCFJ910, provided by Erik Jorgensen via Addgene (Addgene Plasmid #44481). The DNA sequence encoding TAGRFP was obtained from Julie Plastino . pRSET-STransgenes expressing SYD-1C in the HSN neuron were created by injecting the pAGC5 plasmid at 4 ng/μl with 50 ng/μl of odr-1::RFP and 50 ng/μl Bluescript into wildtype worms. Two independent transgenic lines were established and crossed into the syd-1(ju2); unc-6(ev400) genetic background. HSN axon guidance was analyzed as described below.Axon guidance was analyzed as described previously ,52. The Escherichia coli DH5α with plasmid pGEX6P and pGEX4T-MIG-2 (obtained from Hiroshi Qadota [E. coli BL21(DE3) with pRSET-SYD-1C (pCZGY658) by incubating for 16 h at 22°C in the presence of 0.3 mM IPTG. GST- and His6- tagged proteins were extracted by sonication in PBS pH 7.4 with 0.5% Triton X-100 and protease inhibitors (Roche) and centrifuging. GST-MIG-2 was incubated with 0.1 mM of GTPγS or 1 mM of GDP (Sigma-Aldrich) in PBS pH 7.4 with 0.5% Triton X-100, 10 mM MgCl2, 10 mM EDTA, 2% glycerol and protease inhibitors for 30 min at 30°C, and then supplied additional MgCl2 to 75 mM. For pull-down analysis, His-SYD-1 was immobilized to HisPur Cobalt Resin (Thermo), incubated with GST or GST-MIG-2 proteins for 1 h at 4°C, washed with PBS pH 7.4 with 0.1% Triton X-100 and 20 mM imidazole for 4 times, eluted with 300 mM imidazole in PBS pH 7.4, 0.1% Triton X-100. The eluate and input were subjected to SDS-PAGE, and GST- and His- tagged proteins were detected by western blotting with mouse monoclonal Anti-GST antibody (Upstate Biotechnology) and Anti-His-tag mAb , respectively, using ECL Advance Western Blotting Detection Kit .Recombinant GST and GST-MIG-2 proteins were produced in i Qadota , respectin vitro transcription and translation system (Promega) and labeled with biotin. GST::UNC-40 was produced in bacteria and coupled to Glutathione-Sepharose. Binding assays were conducted for 16 hours at 4°C in PBS with 0.1% Triton X-100, 1% BSA and protease inhibitors. After binding, samples were washed 3 times with wash buffer (PBS and 0.1% Triton X-100). Bound material was detected by SDS-PAGE electrophoresis and western blotting with Strepavidin-Alkaline Phosphatase.SYD-1C protein was produced with the TNT SP6 quick coupled S1 FigHSN axon guidance was scored as defective if the axon failed to reach the ventral nerve cord. For all experiments, n≥200. Brackets indicate statistically significant difference, Z test for proportions (***p<0.0001).(TIF)Click here for additional data file.S2 Figsyd-1(tm6234) mutation is predicted to affect all three isoforms of SYD-1 and does not enhance guidance defects associated with the unc-40(e1430) null mutation. The syd-1(ju2) null mutation is expected to affect all three isoforms of SYD-1 and does not enhance guidance defects associated with the unc-40(n324) null mutation. HSN axon guidance was scored as defective if the axon failed to reach the ventral nerve cord. For all experiments, n≥200.The (TIF)Click here for additional data file.S3 Fig(A) In the heterodimer model, UNC-40 and SAX-3 form a heterodimer that interacts with SYD-1. In this model UNC-40, SAX-3 and SYD-1 function together to negatively regulate MIG-2. MIG-2 can also be activated by SAX-3. (B) In the cross talk model, UNC-40 and SAX-3 are not physically associated. UNC-40 associates with SYD-1 and negatively regulates MIG-2, which functions in the SAX-3 pathway.(TIF)Click here for additional data file.S4 FigUNC-40 can function individually by activating effectors such as CED-10, MIG-10, UNC-34 and UNC-115. SAX-3 can function individually by activating MIG-2. UNC-40 and SAX-3 can also function in a combined pathway, where they collaborate to regulate MIG-2. SYD-1 is specific to the combined pathway. This example is depicted with the heterodimer model see , however(TIF)Click here for additional data file.


Caenorhabditis elegans are enhanced by uptake of environmental RNA and amplification and systemic distribution of RNAi effectors. Therapeutic exploitation of RNAi in treating human disease is difficult because these accessory processes are absent or diminished in most animals. A recent report challenged multiple paradigms, suggesting that ingested microRNAs (miRNAs) are transferred to blood, accumulate in tissues, and exert canonical regulation of endogenous transcripts. Independent replication of these findings has been elusive, and multiple disconfirmatory findings have been published. In the face of mounting negative results, any additional positive reports must provide the proverbial “extraordinary proof” to support such claims. In this article, we review the evidence for and against a significant role for dietary miRNAs in influencing gene expression, and make recommendations for future studies.If validated, diet-derived foreign microRNA absorption and function in consuming vertebrates would drastically alter our understanding of nutrition and ecology. RNA interference (RNAi) mechanisms of  Eating is an engagement with the world  that traScience editorial [A recent experimental study reported that microRNAs (miRNAs) from plants may control target genes in the consumer , making ditorial . HoweverCaenorhabditis elegans, is largely absent in other organisms, including closely related nematodes [C. elegans [Realizing the tantalizing prospect of therapeutic RNA interference (RNAi) in humans has been a challenge in part because the confluence of three biological characteristics that facilitate RNA silencing in the nematode worm, ematodes . RNAi it elegans , and the elegans . Eukaryo elegans .C. elegans, long dsRNA injected into one part of the organism exert silencing effects in remote locations [C. elegans, dsRNA distribution may not apply equally to RNA molecules of different size or type, as dsRNA of 100 nucleotides or longer are the best silencing effectors [In ocations . A transocations –12. Consocations . Some foocations  and the ocations  to planaocations . Howeverocations –21. Evenffectors ,23. Thisffectors . TransfeA different sort of extracellular RNA (exRNA)-mediated communication may exist in some eukaryotes –30. RNA C. elegans SID-1, SIDT1 [into a recipient cell – with retention of function such as miRNA-mediated regulation – and stimulation of signaling from outside the cell by exRNA. In addition to recognition of dsRNA molecules by TLR3, toll-like receptors can sense short single-stranded RNA molecules [C. elegans.Nevertheless, the details of how exRNA is transferred in vertebrates between extracellular carrier and recipient cell remain largely unclear. One possible mediator is a human ortholog of 1, SIDT1 . This tr1, SIDT1  or conta1, SIDT1 . We expeolecules . Activatolecules ,43. In sC. elegans characteristic contributing to the remarkable implementation of RNAi is amplification of RNAi effectors [C. elegans is generally dependent on the products of the rde-1 and rde-4 genes [A second ffectors . Seconda-4 genes . Althoug-4 genes ,47. We aC. elegans. Whether soaked in a dsRNA solution [C. elegans assimilate dsRNA from their surroundings, often rendering experimental injection unnecessary. In contrast with SID-1-mediated spreading of dsRNA within the organism, a second protein, SID-2, allows entry of ingested RNA [C. elegans SID-2 gene in uptake-refractory organisms, including other nematodes, permitted uptake of ingested dsRNA [Efficient uptake of dsRNA molecules from environmental sources C also ensolution  or fed wsolution , C. elegsted RNA ,51. Expred dsRNA ,52. SID-C. elegans is manifestly not the only organism that can “sample” environmental RNA, although studies in organisms without amplification mechanisms necessarily involve low copy numbers and are thus fraught with the possibility of contamination artifacts [C. elegans [Apis (bee), was transferred to an ectoparasite with functional consequences [rtifacts ,53. The rtifacts . There i elegans , and it  elegans . For exa elegans ,57. (Mam elegans .) Uptake elegans . The lis elegans ,61, inse elegans –65, even elegans , and two elegans . dsRNA c elegans ,68–71. Aequences .Drosophila [Even so, simple feeding of RNA is insufficient for uptake by some insect species, including osophila , which losophila ,73; thisosophila . Formulaosophila  for stabosophila ,77 or inosophila  or otherosophila ,80. Althosophila . As a reC. elegans RNAi accessory mechanisms or to develop facile oral delivery methods for RNAi [Cell Research, a team led by Chen-Yu Zhang described a series of striking findings on “cross-kingdom” functional RNA transfer. In this study, Zhang et al. [Because of the failure – after more than a decade of intensive inquest – to find robust mammalian homologs of for RNAi , a reporg et al.  used senAt least four central findings of this work seemed poised to overturn prevailing RNAi paradigms: (i) Food-derived plant miRNAs entered mammalian circulation naturally and achieved levels comparable to those of abundant endogenous extracellular miRNAs. (ii) Plant miRNAs were detected not only in circulation, but also in all murine tissue types examined, and at copy numbers rivaling those of endogenous miRNAs. In liver, for example, some plant miRNAs were more abundant than let-7a. (iii) In the mouse, plant miRNAs were said to downregulate at least one endogenous target in liver, the low-density lipoprotein receptor adapter protein 1 (LDLRAP1), within hours of dietary intake. Interestingly, LDLRAP1 downregulation was also reported to increase circulating LDL. (iv) Perhaps most unexpectedly, uptake from the diet was independent of chemical modifications, engineered delivery vehicles, or even vesicles or other protecting carriers in the food, as chemically synthesized oligonucleotides with the same sequence as plant MIR168a were reportedly taken up with similar results.Dietary miRNA transfer thus appeared to be an attractive candidate to explain several unsolved mysteries in nutritional science. Observational studies have suggested that specific nutrients contribute to the observed benefits of plant food consumption, but randomized controlled trials have failed to support these conclusions . Could aCell Research publication ranged from enthusiastic optimism to incredulity. Coverage in journals and the popular press included a “you are what you eat” theme and explored implications of interspecies and interkingdom information exchange. Another leitmotif was dietary miRNA as a new type of nutrient [Response to the nutrient , one thanutrient . Debate nutrient . We noteAmidst intense specialist and lay interest in the Zhang et al. findings, questions have been raised about systemic distribution of xenomiRs following dietary intake and about the likelihood of post-transcriptional regulation. These questions have stemmed from critical review of the initial publication and examinations of related studies. Importantly, numerous negative experimental studies, including several initiated expressly to confirm the findings of Zhang et al. , have noSeveral features of the initial, positive publication have been critiqued ,94. OnlyPooling samples can be an effective strategy in certain circumstances but will also mask biological variability. In Zhang et al., though, plant miRNA reads in ten serum pools derived from 10 to 11 donors each varied strikingly ,53 Fig.. MIR168aA minority of individuals were responsible for all or most pooled reads. As a corollary, most donors may not have had detectable plant miRNAs in serum.Several donor pools represented distinct yet remarkably uniform populations. XenomiRs were detected at low frequency in donor pools 7 and 8, such that just one donor with detected miRNAs around the population average would have resulted in an upward revision of the pooled average by an order of magnitude or more. However, this presupposes non-random pooling; other than the male and female pools, there was no indication of pooling based on distinct population characteristics.Technical variability resulting from RNA extraction, sequencing, or library construction. Variability of RNA extraction, even in replicate purifications, can be large, especially with technically challenging non-kit-based methods [ methods . Technic methods , perhapsContamination and batch effects. Contamination by sources such as oligonucleotide standards and non-dietary environmental plant matter presents a risk of false positives. Pollen contains miRNAs [s miRNAs ,98,99 anThree hours after feeding, there was no significant difference in plasma or liver levels of MIR168a between mice fed regular chow or raw rice. By 6 hours, a 50% increase in serum and a twofold increase in liver were observed, accompanied by >50% decrease of putative target LDLRAP1. Such a striking, rapid downregulation requires a confluence of several factors. First, a fold change in serum MIR168a within the usual margin of error for qPCR assays would have to result, on average, in a doubling of copy numbers in liver cells in less than 3 hours. Second, baseline suppression of the LDLRAP1 transcript would have to reach near 100% suppression despite the modest fold-change in miRNA. (Reporter assays used to confirm miRNA-mediated regulation through the 3′ UTR may require thousands-fold changes in the levels of targeting miRNAs to obtain observable regulation over 24–48 hours or more.) Third, even in the unlikely event of immediate, 100% suppression of LDLRAP1 translation, the extant protein would have to have an extremely short half-life to experience >50% reduction in less than 3 hours. It is uncertain whether or not these prerequisites are fulfilled for MIR168a and LDLRAP1. With small fold changes derived from a semi-quantitative Western blot [To approximate the exposure of mice on a raw rice diet , a 55 kgOf 83 public HTS datasets analyzed for evidence of plant miRNAs in animal fluids and tissues , 53 yielIndependent studies have now presented data that are difficult to reconcile with general uptake of exogenous miRNAs at the levels reported by Zhang et al., levels that might be consistent with regulatory effects.RNA Biology that plant miRNAs were undetectable or indistinguishable from background signal in experiments conducted with three organisms: humans, mice, and bees. In plasma of human athletes with diets that included fresh fruits, plant miRNAs could not be detected. Bees also did not appear to take up plant miRNAs.In 2013, Snow et al.  reportedWild type mice with diets containing very different levels of specific miRNAs did not have above-background levels of plant miRNAs in tissues . AlthougRNA Biology, arrived at similar conclusions [A smaller study, also published in clusions . Blood wRNA Biology studies did not repeat the Zhang et al. mouse experiments or examine LDLRAP1, the putative target of miR168a, but a comprehensive attempt by Monsanto and Miragen researchers to replicate the findings was published several months later in Nature Biotechnology [The chnology . In thischnology .In late 2012, detection of numerous exogenous RNA sequences in human circulation was reported . HoweverBMC Genomics [In a second study by some of the same researchers, blood samples were taken from mice that did or did not receive liver-damaging doses of acetaminophen . ExogenoGenomics  or proviGenomics , but simGenomics , the datIt does not appear that transfer of miRNAs from the diet is a reliable occurrence in vertebrates, or that transferred molecules are normally sufficient in number to exert regulatory pressure. Nevertheless, it remains possible that transfer happens under certain circumstances. There may be room for additional research in this area Table  to addreGiven the instability of naked RNA, transcytosis – vesicular uptake of RNA carriers and their cargo A on one Degradation-resistant RNA molecules in dietary materials would be packaged into protein complexes and/or vesicles. Since transport mechanisms would recognize the carrier, not its cargo, it is difficult to envision how hypothetical sequence specificity could be achieved at the level of uptake. Specific sequences could be incorporated into protective carriers in the source material , but it is not clear that this occurs.Every biological barrier has some degree of leakiness. With sufficient dietary intake, some foreign RNA might survive the trip to the intestine. A small number of molecules could traverse the gut and enter the bloodstream. In the absence of sequence-specific uptake, which has not been demonstrated, the transfer of foreign RNA molecules should mirror their distribution and abundance in the source material.As we have stated, one explanation of the reported human donor pool variability would be uptake of plant xenomiRs by only a small number of individuals, perhaps as a result of altered intestinal permeability. Inflammatory states associated with infectious and non-infectious GI tract disease, stress, and malnutrition can lead to a leaky barrier, and the influences of obesity and alcohol use have also been described. Traditional folk remedies have not been well characterized and might include alteration of gut permeability. Admittedly, the work of Wang et al.  does notO-Methylation of plant miRNAs on the ribose of the 3′ nucleotide [2′ cleotide , a modifcleotide , has beecleotide ,106. BiaWhile optimization of plant- and animal-specific library preparation protocols is useful, protocol differences cannot explain the discrepancies between Zhang et al. and other studies. Plant HTS libraries have demonstrably been prepared successfully using standard protocols, and any plant-specific ligation issues would simply reduce the apparent plant/animal ratios; they would not explain the near or complete absence of plant miRNAs in most datasets. There is no indication in the Zhang article  or paperToo much sensitivity, rather than too little, is often a problem. The pervasiveness of nucleic acid contamination was elegantly illustrated by Charlson et al. . SearchiSince plant biology laboratories on the thoroughly pollen-dusted surface of our planet contain many potential contaminants – natural and artificial – special care must be taken to rule out such contaminants in the case of positive results. Sequencing and amplifying technical controls to assess sequencing instrument, reagent, supply, and sample contamination may seem wasteful, but it might help to answer the many questions about positive results. In this regard, investigators would do well to borrow a page from the experience of forensic scientists, who have developed particularly rigorous procedures to reduce, if never entirely eliminate, the chance of contamination.Finally, although the weight of the current evidence may convince many that plant RNA uptake by vertebrate animals is an infrequent event, a consortium approach may be advisable to answer the question definitively à la the XMRV Scientific Research Working Group . SamplesNon-physiologic expression of both target and putative effector RNA in cultured cells is a common approach to establishing canonical miRNA regulation through RISC. However, miRNA copy numbers in the hundreds or more per cell may be helpful but not always necessary to achieve some measure of canonical target silencing by a targeting miRNA. Mullokandov et al. , for exaNor is canonical RISC-mediated suppression the only conceivable or known mechanism of small RNA function. Nuclear effects of small RNA A have beHowever, although it is tempting to speculate that low copy number miRNA could exert influence through canonical or non-canonical means, homeopathic effects are implausible: at least a few copies of a targeting miRNA would need to be added to a cell to achieve a change in target amount. As Snow et al.  observedAs we previously mentioned, intestinal permeability may be altered by certain substances, allowing measurable uptake of dietary RNA. Disease states could also affect uptake and/or clearance. In these cases, the observed effects would likely be general. Detection should closely follow RNA distribution in the dietary material, and gut bacterial products should also be detected at higher levels. Permeability and clearance aside, the results of plant vesicle administration ,80 suggeSnow et al. astutely reviewed non-dietary means of systemic xenomiR transfer . Indeed,The scientific community has had two years to digest the first report that oral consumption of plant-derived miRNAs affects vertebrate metabolism. The popular and scientific press has accorded at least the requisite 15 minutes to this concept. However, as the poet Emily Dickinson wrote, “Fame is a fickle food – Upon a shifting plate.” This adage may be appropriate here, as several well-designed studies have been unable to confirm that gastrointestinal uptake is a general means of miRNA transfer and subsequent gene regulation. As the initial mixture of enthusiasm and skepticism has shifted, we must carefully consider alternative explanations for the original results. If xenomiRs as a field of inquiry are to endure, any future positive studies should rule out contamination, address rigorously the kinetics and mechanisms of dietary miRNA absorption, distribution, and clearance, and include sensitive assay systems to measure functionality in a reproducible and physiologically relevant manner for any putative diet-derived miRNA.


RNA-editing is an important post-transcriptional RNA sequence modification performed by two catalytic enzymes, "ADAR"(A-to-I) and "APOBEC"(C-to-U). By utilizing high-throughput sequencing technologies, the biological function of RNA-editing has been actively investigated. Currently, RNA-editing is considered to be a key regulator that controls various cellular functions, such as protein activity, alternative splicing pattern of mRNA, and substitution of miRNA targeting site. DARNED, a public RDD database, reported that there are more than 300-thousands RNA-editing sites detected in human genome(hg19). Moreover, multiple studies suggested that RNA-editing events occur in highly specific conditions. According to DARNED, 97.62 % of registered editing sites were detected in a single tissue or in a specific condition, which also supports that the RNA-editing events occur condition-specifically. Since RNA-seq can capture the whole landscape of transcriptome, RNA-seq is widely used for RDD prediction. However, significant amounts of false positives or artefacts can be generated when detecting RNA-editing from RNA-seq. Since it is difficult to perform experimental validation at the whole-transcriptome scale, there should be a powerful computational tool to distinguish true RNA-editing events from artefacts.We developed RDDpred, a Random Forest RDD classifier. RDDpred reports potentially true RNA-editing events from RNA-seq data. RDDpred was tested with two publicly available RNA-editing datasets and successfully reproduced RDDs reported in the two studies  while rejecting false-discoveries .http://biohealth.snu.ac.kr/software/RDDpred.RDDpred automatically compiles condition-specific training examples without experimental validations and then construct a RDD classifier. As far as we know, RDDpred is the very first machine-learning based automated pipeline for RDD prediction. We believe that RDDpred will be very useful and can contribute significantly to the study of condition-specific RNA-editing. RDDpred is available at  RNA-editing event is defined as a post-transcriptional RNA sequence modification . CurrentMoreover, there are multiple studies that showed direct relation of RNA-editing to biological phenotypes. For example, Galeano’s group showed that the editing events in glioblastoma by ADAR2 enzymes are crucial for pathogenesis and claimed that ADAR-class enzyme can be considered as a tumor-suppressor . And in DARNED, a well-curated public RNA-editing database have more than 300-thousands editing sites detected in the human genome hg19 . InteresRNA-seq, a high-throughput sequencing of transcriptome, is a powerful method for investigating whole-transcriptome status. Since the nature of the technology is taking a snapshot of cells with massive sequencing reads, it is suitable for detecting condition-specific events in whole-transcriptome scale. Therefore, it is also suited for detecting RNA-editing events that have such condition-specific characteristics. There have been a number of studies that used RNA-seq to reveal condition-specific editing patterns in whole-transcriptome scale –7.Even though RNA-seq is suitable for RNA-editing detection, it is also true that the current computational pipelines of RNA-editing detection with RNA-seq have considerable false-positive risks. In 2012 Nature Biotechnology journal, an article “The difficult calls in RNA editing”, reports interviews with eight prominent RNA-editing researchers. They pointed out that false-positive calling is one of the most challenging problems in RNA-editing detection with RNA-seq .The false-positives caused by mis-alignment of short-reads can be termed “Systematic Artefacts” due to their inherent and reproducible characteristics. Systematic artefacts can be caused by various reasons, (a) inherent duplications/repeats within genomic sequences, (b) ambiguity caused by splicing-junctions, (c) prevalent polymorphisms between individuals and (d) shortness of sequencing reads , 18. ThiTo assess the significance of confounding effects caused by systematic artefacts, we performed a simple simulation test in order to measure the false-detection rate of RNA-variants caused by mis-alignments. We used RNA-STAR, a state-of-art alignment tool in base-accuracy , 21, to We generated 10 simulated data and aligned them Fig. . In addiA priori knowledge based filtering [To handle systematic artefacts in RNA-seq, a number of computational approaches have been developed. These can be categorized into three groups in terms of features they used: (a) iltering , 27, (b)iltering , (c) Maciltering , 28.A priori knowledge based filtering used public genomic features, such as Alu repeats, genomic duplications, and pseudogenes, to assess the detected editing-sites directly. For instance, Li’s group used public annotation of genomic repeats to filter out potential artefacts within the detected RDD(RNA/DNA Difference) sites [e) sites . On the e) sites .Unlike the filter-based methods that directly assess RDD candidate sites with pre-defined filters, machine-learning based methods generates a predictor in advance. The predictor, or machine-learning classifier, is trained to learn the differences between true and false examples. As an example, Laurent’s group generated a Random Forest predictor that utilizes read-alignment patterns as attributes. With 77 attributes, Laurent’s group generated a predictor and demonstrated it has 87 % of estimated accuracy by experimental validation . As mentLaurent’s work  was the Here we introduce RDDpred, a software package that is generally usable and do not need an experimental validation to prepare condition-specific training examples. Hence, RDDpred prepares condition-specific training data directly from input sequencing data or raw RDD candidates. In order to collect positive examples without experimental validations, we utilized two well-organized RNA-editing databases, RADAR and DARNED , 29. SinWe tested RDDpred in Python (2.7.3), Samtools-Bcftools (1.2.1), WEKA (3.6.12) package, in linux environment.RDDpred takes alignment results as input data and gives the prediction results of each SNVs, or RDD candidates as outputs. The raw RDD candidates are detected with Samtools-Bcftools pipeline  while thRDDpred can take inputs from any kind of alignment methods providing BAM-format outputs. However, we recommended RNA-STAR for its high degree of overall accuracy and ultra-fast performance , 21.RADAR and DARNED databases include 2.5 million, 300-thousands of curated sites respectively , 29. TheTo build a predictor, we also need negative examples. To address this issue, we utilized the MES method, a computational simulation for predicting error-inducible regions within genomes  Fig. 5)5). With It is known that there are various types of artefacts from RNA-seq, such as amplification errors during library construction, sequencing errors, and errors by mis-alignments. Unlike former events, errors by mis-alignments shows different characteristics that they are more reproducible. Since the errors from library construction and sequencing procedures are transient in general, they can be excluded by replicating experiments. On the other hand, since the errors by mis-alignments, or systematic artefacts, are inherent to specific alignment method, they might not be excluded even after multiple replications. Therefore, RDDpred mainly focuses on detecting systematic artefacts with considering other artefacts as well.Read-alignment pattern is defined as local status of alignments in variant sites that has been utilized to distinguish artefacts . There aAs mentioned, the 15 attributes are categorized into six category, (a) “Read Depth” category represents read-count in editing sites. (b) “Allele Segregation” category includes four attributes, such as VAF, SGB, FQ, and CallQual, respectively. All of these attributes are calculated from edited read-ratio against total reads. (c) “Mapping Quality” category of attributes reflects how the alignments of reads are well-performed, which utilizes alignment scores that the aligner generates. Four attributes, such as PV3, MQB, MQ0F and MQ belongs to this category. (d) “Read Position” category includes three attributes, such as VDB, RPB, and PV4, which represent how the positions of variants are biased within sequencing reads. (e) “Base Quality” category uses base-quality information generated by sequencing machine to detect whether low-quality bases are significantly biased to editing-sites. Two attributes, PV2 and BQB belongs to this category. (f) Finally, “Read Strand” category includes single attribute PV1, that represents how the strands of edited reads are biased than non-edited reads Negative examples: Predicted as artefact sites by MES method The entries overlapped with test-data are excluded from training-dataTraining datasets Positive examples: Positively detected sites by experimental validation (Sanger-seq)Negative examples: False discovery sites proved by experimental validation (Sanger-seq)Test datasets We constructed condition-specific model independently from each datasets and evaluated RDDpred separately with the corresponding validation results as test-data. And the test-data was the results of Sanger-seq by each groups. In the process of constructing each models, we carefully arranged the training-data in order not to contain any of information related to test-data. In other words, we did not use test-data for the construction of the model. RDDpred predicted 105,564 sites from 6,856,440 initial RDDs candidates, showing that not only the model was able to reproduce (95.32 %) the results but also was able to reduce potential artefacts (98.46 %) from Bahn’s study. Moreover, RDDpred have successfully reject most of the false discoveries in Bahn’s prediction. Resulting 84.21 % NPV. The following results indicate that RDDpred showed relative robustness than Bahn’s by reducing potential artefacts and rejecting false-discoveries Figs. .Fig. 6OIn Peng’s study, RDDpred predicted 3,076,908 sites from 58,666,976 initial RDDs candidates, which also proves that the model was able to successfully reproduce (90.37 %) the results but also reduce potential artefacts (94.79 %) from Peng’s study. Moreover, RDDpred have successfully reject most of the false discoveries in Peng’s prediction. Resulting 75.86 % NPV . The following results indicate that RDDpred also showed relative robustness than Peng’s by reducing potential artefacts and rejecting false-discoveries  Xeon(R) CPU E5645 @ 2.40 GHz)To provide information about actual running-time and memory-usage of RDDpred, we monitored the resource-usage while processing the Peng’s dataset. With the machine specified below, RDDpred took 18.33 hours to process 583,640,030 reads of 101,787,059,720 bases. We believe that the resources used here are not too excessive for common research groups. We developed a software package for RNA-editing prediction from RNA-seq data. RDDpred utilizes current published database and methods such as RADAR, DARNED , 29 and In order to investigate working principle of RDDpred in terms of prediction power of each categories, we calculated information-gain values with WEKA . The ranDuring the high-throughput sequencing, the sequencers generate bases-qualities that represent the confidence of sequencing. Therefore, unlike other five metrics, the “Base Quality” reflects the molecular status of bases that are directly recorded by sequencer. Until now, we only knew bases modified by editing enzymes are somehow recognized as guanine (or thymine for APOBEC class), but did not know how these recognitions are observed in the perspective of sequencing machines. The base-quality issue indicates that there might be some distinctions between normal and edited bases at the molecular level. Thus, it implies that more detailed recording of molecular characteristics during sequencing process might be a key to improve the accuracy of RNA-editing detection.RNA-seq is one of the most powerful methods to investigate transcriptome and the amount of RNA-seq has recently increased nearly exponentially . In spithttp://biohealth.snu.ac.kr/software/RDDpred.RDDpred prepares training examples that are specific to the condition of input data without experimental validations. RDDpred proved good performances by reproducing the detection of two previous studies and correcting most of their false-discoveries. Moreover, as far as we know, RDDpred is the very first automated pipeline that utilizes machine-learning technique with a well-evaluated performance. Thus, we believe that RDDpred will be very useful and can contribute significantly to the study of RNA-editing. RDDpred is available at


There is a strong genetic component to many disease subtypes  and we are just beginning to understand the relevant genetic factors. Several studies have related RNA splicing to cardiovascular disease and circular RNAs (circRNAs) are an emerging player. circRNAs, which originate through back-splicing events from primary transcripts, are resistant to exonucleases and typically not polyadenylated. Initial functional studies show clear phenotypic outcomes for selected circRNAs. We provide, for the first time, a comprehensive catalogue of RNase R-resistant circRNA species for the adult murine heart. This work combines state-of-the-art circle sequencing with our novel DCC software to explore the circRNA landscape of heart tissue. Overall, we identified 575 circRNA species that pass a beta-binomial test for enrichment  in the exonuclease-treated sequencing sample. Several circRNAs can be directly attributed to host genes that have been previously described as associated with cardiovascular disease. Further studies of these candidate circRNAs may reveal disease-relevant properties or functions of specific circRNAs.For several decades,  In 2013, several ground-breaking manuscripts demonstrated a clear phenotypic outcome after circRNA perturbation and sparked enormous interest in this molecular species trans-splicing and tandem duplications as mentioned above, genomic alterations, or simply from read mapping artefacts Circular RNAs (circRNAs) are a recently-rediscovered RNA class, which were initially described as “scrambled” exons in the early 1990s The identification and quantification of circRNAs from next-generation sequencing (NGS) data is an area of ongoing research. The “circSeq” approach is the current gold standard for high-throughput circRNA identification Relative abundance changes in circRNA expression are typically approximated by comparing normalized back-splice junction read counts of specific circRNAs under different conditions and/or time points in vitro experiments It is widely assumed that circRNAs belong to the growing group of non-coding RNAs (ncRNAs) that encompasses microRNAs (miRNAs), long ncRNAs (lncRNA), or small-interfering RNAs (siRNA). Intriguingly, a recent study in endothelial cells showed that circRNAs are regulated in response to hypoxia and have a biological function as proven by In this manuscript, we aim to provide a catalogue of validated circRNA species in the adult murine heart. To this end, we employ the latest developments in bioinformatics and sequencing technology, and relate our catalogue to previously-identified cardiomyopathy-related genetic loci.+) and the other left untreated (RNase R−) (see Methods). No polyA-containing RNA removal step was carried out, in accordance with other recent reports on fragment-based circRNA detection Total RNA was extracted from hearts of adult mice which were 2, 3, 6, or 12 months old. After depletion of rRNA, each RNA sample was split into two fractions with one treated with RNase R . Due to the employed circle sequencing approach, RNase R− and RNase R+ samples should be clearly separated within the enrichment plot since the RNase R digestion step removes the bulk of linear transcripts and more sequencing reads actually cover circRNAs structures instead of linear transcripts in the untreated sample. Therefore, this graphical representation is a first quality measurement for the success of enrichment process. In an ideal experiment all linear fragments in the RNase R treated sample should be digested, leaving only circular constructs in the sample. A drastic increase of reads covering circRNAs would therefore be expected when comparing untreated and treated samples. Also this ideal situation does not translate exactly to practice. The enrichment plots in Ryr2, Hectd1, and Ppp2r3a that encode ryanodine receptor 2, HECT domain-containing E3 ubiquitin protein ligase 1, and protein phosphatase 2, regulatory subunit B, respectively, for further inspection.The vast majority of circRNAs were only found at only one location within the annotated gene regions . For the remaining 2.4% circRNAs, more than 3 reads were found at a secondary location within the annotated gene regions; however, hardly more than 10 reads were detected at the secondary locations for these circRNAs. Therefore, circRNAs seem to be predominantly emerging from single, specific gene regions. Among the 575 significant circRNAs identified above, we selected the top three most significant loci, Ryr2, throughout all experiments, showed the most significant enrichment in general  and is located within the last third of Hectd1 coding region  (Ryr2), or hypertrophic cardiomyopathy , and therefore are central parts of the respective KEGG pathways  These candidate genes are known to play key roles in different kinds of cardiomyopathy such as Ryr2 circRNAs regardless of the age of the murine heart examined (Ryr2 encodes ryanodine receptor 2 (RYR2), which is a sarcoplasmic reticulum membrane-embedded transport protein for Ca2+ and is responsible for the rhythmic muscle contractions generating the heartbeat Ryr2 circRNAs is relatively constant for all 4 time points, which peaks at month 6. Traces of Ryr2 circRNA are detectable even without the enrichment step, whereas after enrichment the abundance is 2–3 times higher when compared to the untreated sample.There is an overwhelming presence of examined A. PrimarTtn fits in the same picture, as it is responsible for the passive elasticity of muscles and therefore plays a critical role in heart muscle cells Ttn generally follows the pattern of Ryr2 albeit with relatively modest changes. In comparison, the enrichment process was not as successful as for Ryr2, as higher circRNA counts were detected for month 4 and month 6 in the untreated samples than the treated samples. For example, very long circRNA molecules may get nicked or sheared during the isolation process, making them sensitive to RNAse R treatment.The detection of Dmd-associated dilated cardiomyopathy is directly linked to mutations in the Dmd gene Dmd circRNA could be another valuable target for cardiomyopathy research. The expression pattern of Dmd circRNA differs from those of Ryr2 and Ttn, demonstrating maximal expression for month 12 instead of month 6. We achieved non-optimal enrichment for circRNAs from mice aged 3 or 6 months, possibly due to portions of the circRNAs lost during the RNase R digestion process.Ryr2 and Ttn may be more connected to young and active hearts, Dmd-encoded protein strengthens the cardiomyocyte sarcolemmal integrity, and its maximal expression may be important for optimal function (hemodynamic and stress responses) of the fully developed, mature adult heart. Although the described properties are related to the protein-coding linear transcripts, their corresponding circRNAs may be involved in those diseases directly or indirectly.The pattern of abundance of circRNAs for all three genes may be influenced by the age and activity of the mice. While the linear transcripts circTest. Many of these candidates coincide with disease-associated gene loci. For instance, significant candidates originate from the Ryr2, Hectd1, and Ppp2r3a gene loci that have been linked to cardiovascular disease.In this work we present circle sequencing as a highly efficient and sensitive tool for circRNA studies in cardiology. In summary, we compiled a catalogue of 575 candidate circRNAs that pass a test for enrichment in RNase R-treated samples in comparison to untreated samples using As a long term goal, how these circRNAs might influence cell processes in disease-related phenotypes and healthy tissue needs to be addressed. Furthermore, more efforts are also needed as to how this information may translate into new treatment approaches of cardiovascular disease.Total RNA extracted from heart tissue of CD1-strain mice of ages 2, 3, 6, and 12 months was commercially acquired from AMS Biotechnology . The rRNA depletion step was carried out with 20 μg of total RNA using the Ribo-Zero Magnetic Kit  and 5 μg input per reaction. Depletion was performed in parallel, followed by subsequent pooling. For digestion of linear RNA, 3 quarters of the depleted RNA was treated with RNase R, whereas the remaining one quarter of the depleted RNA only received water and acts as a negative control. Both samples were treated equally for all further steps. After heating to 70 °C and cooling to 35 °C, 10× RNase R reaction buffer and RNase R  were added to the 3-quarter fraction, followed by heating to 37 °C for 40 min. Samples were cleaned up using Agencourt RNAClean XP beads . Subsequent library preparation was performed with ScriptSeq-v2 RNA-Seq Library Preparation Kit (Epicentre). PCR amplification was performed with 15 cycles, without any further size selection. Sequencing was carried out on an Illumina HiSeq2500 system for 100 cycles using paired-end mode. Sequence data were deposited in the NCBI Sequence Read Archive (SRA) with the accession number SRP071584.Paired-end rRNA-depleted sequencing data of RNase R treated and untreated samples were analysed in detail. After initial quality assessment, low quality regions and adapter sequences were removed with Flexbar (version 2.5) -M -Nr 2 2 -fg -G -F -L 20”. Further statistical analyses were performed with the circTest R package provided with DCC and additional custom-tailored Perl scripts.To optimally exploit the advantage of the circSeq technique, we have developed the DCC software to efficiently detect and quantify circRNAs in sequencing data CD designed the study; LC and RR performed all RNA and sequencing experiments. TJ and CD analysed the data and wrote the manuscript. All authors read and approved the final manuscript.The authors declared that there are no competing interests.


Thrips tabaci. On the other hand, presence of “hidden break” was depicted in whiteflies' 28S rRNA despite their evolutionary grouping under same order with aphids. Divergence of 28S rRNA sequences confirms variation of both size and composition of gap region among insect species. However, phylogeny reconstruction does not support speciation as a possible source of the hidden break in insect's 28S rRNA. In conclusion, we show that RNA from a given insect order does not conform to a particular banding profile and therefore this approach cannot be reliably used to characterize newly discovered species.With an exception of aphids, insects' 28S rRNA is thought to harbor a “hidden break” which cleaves under denaturing conditions to comigrate with 18S rRNA band to exhibit a degraded appearance on native agarose gels. The degraded appearance confounds determination of RNA integrity in laboratories that rely on gel electrophoresis. To provide guidelines for RNA profiles, RNA from five major insect orders, namely, Diptera, Hemiptera, Thysanoptera, Hymenoptera, and Lepidoptera, was compared under denaturing and nondenaturing conditions. This study confirmed that although present in most of insect's RNA, the “hidden break” is absent in the 28S rRNA of onion thrips, Insects (Arthropoda: Insecta) constitute up to 55% of characterized species, playing different roles in the environment . For insα and β subunits [β segment of 28S rRNA molecule. In addition, the presence of “hidden break” has also been reported in the 5.8S rRNA of Drosophila and in higher plant chloroplasts [Success of downstream RNA application depends on the quality and integrity of the extracted RNA. Isolation of high quality RNA from biological samples is however challenged by the ubiquitous presence of ribonucleases (RNases) that rapidly degrade freshly prepared RNA . This masubunits , which csubunits . The UAAroplasts . In controplasts .Efforts to understand molecular mechanisms involved in the introduction of the gap have been hindered by unavailability of 28S rRNA secondary structures and high conservation of sequences reported in the regions that flank the “hidden break.” This makes it difficult to compare the “hidden breaks" . Neverth Thrips tabaci.In the present study, banding profiles of RNA from five major insect orders considered of economic importance including Diptera, Hemiptera, Thysanoptera, Hymenoptera, and Lepidoptera were compared with an aim of providing guidelines to the appearance of RNA for insects belonging to these orders. Importantly, the absence of a hidden break was observed in the onion thrips, icipe insectaries and screen houses. They included 150 aphids (Hemiptera); Brevicoryne brassicae, 60 mosquitoes (Diptera); Anopheles arabiensis, 20 tsetse flies (Diptera); Glossina fuscipes fuscipes, 16 honey bees (Hymenoptera); Apis mellifera, 200 whiteflies (Hemiptera); of Aleyrodidae family, six stem borers (Lepidoptera); Chilo partellus, four silkworms (Lepidoptera); Bombyx mori, 40 wasps (Hymenoptera); Fopius arisanus, 10 fruit flies (Diptera); Dacus bivittatus and 1000 thrips (Thysanoptera); Thrips tabaci. The sample size was informed by the size of the fly and experiments were done in replicates.Representative insects for the five orders were obtained from insect colonies maintained at theμL of TRI-reagent and centrifuged in an Eppendorf AG 5417R centrifuge  at 12,000 ×g for 10 min. The supernatant was transferred into a new tube followed by addition of one volume of absolute ethanol into the supernatant. After vortexing, 700 μL of the mixture was transferred into a Zymo-Spin IIC Column in a collection tube, centrifuged at 12,000 ×g for 1 min and the flow through discarded. The spin column was transferred into a new collection tube and 400 μL Direct-zol RNA prewash solution was added and centrifuged at 12,000 ×g for 1 min. The flow through was discarded and this step was repeated. Into the column, 700 μL RNA wash buffer was added and centrifuged at 12,000 ×g for 1 min. The flow through was discarded and the column spun again at 12,000 ×g for 2 min to completely remove any residual wash buffer. The column was then transferred into an RNase-free 1.5 mL Eppendorf tube and 25 μL of DNase/RNase-free water was added onto the column matrix, centrifuged at maximum speed for 1 min for RNA elution. The eluted RNA was immediately used for downstream processes.RNA from the insect samples was extracted using the Direct-zol RNA Mini Prep Kit , following the manufacturer's instructions. Fresh whole insect flies (≤50 mg) were homogenized in 500 μL of extracted RNA at A260 nm and A280 nm, respectively. Any sample with A260 nm/A280 nm ratio below 1.7 was discarded.The quantity and quality of the extracted RNA yield were determined using a Nanodrop (ND) 2000c Spectrophotometer,  by measuring optical density of 2 μL of 10 mg/mL ethidium bromide were added. The mixture was thoroughly but gently mixed, then poured onto a casting tray, and left to polymerize under a laminar flow chemical hood.A 1.2% formaldehyde agarose (FA) gel was prepared by mixing 1.2 g of agarose with 10 mL of 10X FA gel buffer . This was topped to 100 mL using RNase-free water. The mixture was heated to melt the agarose and cooled to approximately 65°C, after which 1.8 mL of 37% 12.3 M formaldehyde and 6 μL of saturated aqueous bromophenol blue solution was mixed with 80 μL of 500 mM EDTA of pH 8.0, 720 μL of 37% 12.3 M formaldehyde, 2 mL of 100% glycerol, 3.084 mL formamide, and 4 mL of 10X FA gel buffer and topped up 10 mL with RNase-free water. One volume of the dye was added to three volumes of RNA sample and mixed by pipetting. The sample-dye mixture was down spun and incubated at 65°C for 5 min, then chilled on ice, and immediately loaded onto the FA gel.The RNA samples were mixed with 1 volume of 5X RNA loading dye prepared as per the Qiagen protocol . SixteenThe gel was run at 70 V in the 1X FA gel running buffer  for 1 hour followed by visualization of the gel under UV illumination and analyzed using KODAK Gel Logic 200 Imaging System .μL of 10 mg/mL ethidium bromide was added to the mixture and the sample RNA then mixed with a non-denaturing 6X DNA loading dye and the samples loaded onto the gel, alongside a molecular weight DNA marker (O'GeneRuler 1 Kb plus DNA Ladder). Electrophoresis, visualization, and gel image analysis were carried out as outlined above.A 1.2% agarose gel was prepared by adding 1.2 g of agarose into 100 mL of 1X TAE buffer and heating to dissolve. Six  G. fuscipes fuscipes and Brevicoryne brassicae were substituted with G. morsitans morsitans and A. glycine, respectively. No relative sequences were recovered to represent Dacus bivittatus and Chilo partellus in phylogeny reconstruction. Multiple sequence alignment was performed using Muscle version 3.6 [Representative 28S rDNA sequences for the studied insect orders were obtained from GenBank  Table 2Table 2. sion 3.6  and a phsion 3.6 . Generalsion 3.6 . B. mori [ G. morsitans morsitans, T. knoxi, and A. glycine. The species were chosen as representatives for presence and absence of “hidden break” as observed in the denatured gel. T. knoxi sequence was used in place of T. tabaci based on its length. Secondary structures of these regions were determined using “The Predict a Secondary Structure server” at http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html which combines four prediction algorithms. Structures with maximum free energies were adopted for comparison and mapping of the hidden break regions.A sequence containing mapped hidden break and its flanking regions in B. mori  was aligμL which was high enough to be viewed under gel electrophoresis.Purity and quantity of extracted RNA were determined using a Nanodrop. Observed absorbance 260/280) ratio for the samples was above 2.0 , which i0 ratio f Brevicoryne brassicae and their close “cousins” Thrips tabaci, other insects' RNA exhibited single banding profiles under denaturing condition , T. knoxi variable region sequence recorded a higher GC composition (47%) compared to that of A. mellifera (39%), B. mori (35%), and G. m. morsitans (22%). Evolutionary relatedness of 28S rRNA molecules of the studied insects was assessed by constructing a phylogenetic tree of their 28S rRNA sequences or of their close relatives in case of missing sequences. The resulting tree with bootstrap values is shown in GC content of the region around the gap region was assessed using EMBOSS geecee tool  T. knoxi sequence and compared against that of B. mori, G. morsitans morsitans, and A. glycine  that are seen in case of whiteflies . In conclusion, we report absence of “hidden break” in one more insect, thrips, and suggest that the banding profile of all other insects may not always conform to a single band under denaturing conditions; thus RNA profiling should not be used as a means of characterizing newly discovered species.In addition to aphids' RNA (equences  did not


Caenorhabditis elegans has provided critical insights into our understanding of the breadth of small RNA-mediated gene regulatory activities. The first microRNA was identified in C. elegans in 1993, and the understanding that dsRNA was the driving force behind RNA-mediated gene silencing came from experiments performed in C. elegans in 1998. Likewise, early genetic screens in C. elegans for factors involved in RNA interference pointed to conserved mechanisms for small RNA-mediated gene silencing pathways, placing the worm squarely among the founding fathers of a now extensive field of molecular biology. Today, the worm continues to be at the forefront of ground-breaking insight into small RNA-mediated biology. Recent studies have revealed with increasing mechanistic clarity that C. elegans possesses an extensive nuclear small RNA regulatory network that encompasses not only gene silencing but also gene activating roles. Further, a portrait is emerging whereby small RNA pathways play key roles in integrating responses to environmental stimuli and transmitting epigenetic information about such responses from one generation to the next. Here we discuss endogenous small RNA pathways in C. elegans and the insight worm biology has provided into the mechanisms employed by these pathways. We touch on the increasingly spectacular diversity of small RNA biogenesis and function, and discuss the relevance of lessons learned in the worm for human biology.In the past 20 years, the tiny soil nematode  Caenorhabditis elegans is a simple organism in many ways, the worm has been a keystone of several conserved areas of biological exploration, including apoptosis, neurobiology, the establishment of developmental axes, and small RNA-mediated gene regulation  were successful in identifying the first known C. elegans Argonaute RDE-1 (RNAi DEficient) and other factors necessary for exo-RNAi , which synthesize particular classes of small RNAs in the worm using target transcripts as a template  and Dicer , are generated by two distinct mechanisms, and act as upstream triggers for synthesis by RdRPs of 22G-RNAs that silence target transcripts at both the transcriptional and post-transcriptional levels. One set, the canonical piRNAs, are transcribed by RNA Polymerase II from clusters of piRNA loci on chromosome IV (PIWI-interacting RNAs (piRNAs), bound by PIWI-clade Argonautes, are germline-expressed small RNAs that function in genome defense and germ cell development in diverse metazoans . In C. eosome IV . Anotherosome IV . Both tyosome IV . piRNAs osome IV . Owing tosome IV . This fadcr-1 mutants and bear some characteristics of Dicer processing  produce the 22G-RNAs and 26G-RNAs . The 26G-RNAs are synthesized by the RdRP RRF-3, and are thought to be processed in some manner by Dicer, as 26G-RNAs are depleted in de novo RdRP synthesis (and thus bear a 5′ tri-phosphate). Both EGO-1 and RRF-1 interact with the DEAD-box helicase DRH-3 and the dual Tudor domain protein EKL-1 to form functional RdRP modules . Targeting by the PRG-1/piRNA pathway and by the CSR-1 pathway are largely mutually exclusive, and these two pathways act in competition with one another to determine the ultimate pattern of gene expression in the germline. Together, silencing  and licensing (CSR-1 22G-RNAs) activities maintain a critical balance of germline gene expression. Furthermore, recent studies indicate roles for these same pathways in reinforcing patterns of gene expression over many generations, by transmitting an epigenetic memory  to progeny  complexes carry out diverse downstream functions? We focus here on recent progress toward understanding the questions of how both specificity and diversity of function are accomplished by these related pathways.While functional redundancy has been demonstrated among some of the 27 members of the Likewise, while a great deal of work remains to be done in the identification and characterization of all of the molecular players in these pathways, a picture is beginning to emerge of a set of strategies that cooperate to achieve both functional diversity and molecular specificity for small RNA pathways in the worm: (1) Spatial and temporal resolution of the expression of AGOs and other pathway factors allows tissue-specific differences in small RNA function; (2) Distinct biochemical properties of the AGOs, their interacting small RNAs, and an incompletely characterized catalog of interacting partners contribute both to the specificity of small RNA sorting and to the diversity of downstream molecular functions. (3) Compartmentalization of small RNA processes into subcellular structures, particularly in the germ lineage, allows for functional specialization among AGOs and 22G-RNAs with overlapping expression patterns.At the simplest level, some of the differences in the functional outcome in small RNA pathways can be explained by tissue-specific differences in gene expression. For instance, if any of the key components of a small RNA pathway are not expressed in a particular tissue or at a particular point in development, its function simply cannot be mediated. Likewise, due to the nature of RdRP-mediated pathways, which utilize transcripts both as template for small RNA synthesis and as target, the specific profiles of genes expressed in particular tissues may impact which genes are available to be targeted.While developmentally staged mRNA-seq studies are beginning to shine a light on differences in expression of small RNA pathway components, a comprehensive examination of expression of these factors has yet to be undertaken. Nonetheless, examples of differential AGO expression exist in the literature. For example, the expression profiles of the two 26G-RNA associated AGOs, ALG-3, -4 and ERGO-1, are distinct. While ALG-3, -4 are enriched in stages of development in which spermatogenesis occurs , ERGO-1 expression is enriched in mature adult hermaphrodites undergoing oogenesis and in embryos . HoweverPost-translational modification of AGOs and other pathway components could also contribute to differential pathway activity, as various post-translational modifications of AGOs in other organisms have been shown to regulate AGO function . FurtherO-Methylated oligonucleotides followed by immunoblotting to identify the associated AGO protein . Loading specificity has functional consequences, as RDE-1-bound miR-243 functions as a primary siRNA, triggering secondary siRNA production and silencing of a gene that contains a near-perfect complementary target site  is absent in many C. elegans WAGOs, where it is replaced by a histidine. Y529 is preserved in ALG-1, -2 and RDE-1, which bind to mono-phosphorylated small RNAs (as does HsAgo2). We speculate that this difference and other as-yet unknown differences in the 5′ small RNA binding pocket could contribute to preferences in loading of C. elegans AGOs. Clearly, in-depth structure-function studies will be necessary to dissect the features of C. elegans AGOs necessary for small RNA binding specificity.Although the specific structural features of worm AGOs that confer specificity to small RNA binding have not yet been identified (no structure for worm AGOs has been solved yet), it is likely that different features of the 5′ and 3′ small RNA binding pockets of the various cificity . NotablyIt is clear that small RNA properties are not sufficient to explain AGO binding specificity, since CSR-1 and a host of WAGO proteins bind (thus far) biochemically indistinguishable 22G-RNAs that target mostly non-overlapping transcripts . SpecifiC. elegans AGOs examined to date, including CSR-1, WAGO-1, and PRG-1, localize to the perinuclear germline granules of worms, called P granules . Although MUT-14 helicase activity is required for the production of 22G-RNAs in the germline, it is dispensable both for production of secondary siRNAs (22G-RNAs) in the soma and for the 22G-RNAs . Lendinggranules .Figure 2B). Although WAGO-1 accumulates in P granules, it is possible that loading occurs during transient occupancy elsewhere (that is not easily detectable by immunofluorescence studies). If this is the case, the compartmentalization of the EGO-1 versus RRF-1 RdRP modules in P granules vs. Mutator foci may play a central role in maintaining specificity of CSR-1 versus WAGO loading. Clarification of the role of subcellular compartmentalization in the specificity of and competition between these two functionally opposing pathways awaits further experiments.The model that emerges, then, is one where germline transcripts exported through the nuclear pore transit through P granules, where they are scanned for complementarity to CSR-1 and/or PRG-1-bound small RNAs. In this manner, transcripts targeted in P granules by CSR-1 might be distinguished and protected, while PRG-1 targeted transcripts could be identified and shuttled to the Mutator foci. The matter of where WAGO-1 loading ultimately occurs remains unresolved . The role of the (in some cases presumed) slicer activity of these Argonautes has been explored for only some of these proteins.In -pathway . Since tC. elegans RDE-1, which binds DCR-1-produced primary exo-siRNAs, appears to be required only for passenger strand removal, with little direct role in target silencing  strand of the siRNA duplex, which facilitates loading of the guide (targeting) strand . In contilencing . As prim elegans , it is p elegans . It appein vitro and in vivo (in vitro (in vivo (see below). Similarly, although PRG-1 has some slicer activity in vitro , as well as silencing of some endogenous genes, through a mechanism that involves inhibition of RNA polymerase II elongation and small RNA-dependent trimethylation of Histone H3 at Lysine 9 . HRDE-1 shows 22G-dependent localization to the nucleus, where it associates with the nascent pre-mRNAs of targets and directs H3K9me3 modification . GeneticLike NRDE-3 and HRDE-1, CSR-1 localizes to chromatin at its target loci in a 22G-RNA-dependent manner . BecauseIn support of this model, CSR-1 has been shown to positively regulate the expression of germline genes on a genome-wide scale, and is particularly important during sperm development for promoting the expression of genes involved in sperm differentiation downstream of the ALG-3, -4 26G-RNA pathway . AlthougS. pombe. The similarity between the C. elegans HRDE-1 and NRDE-3 pathways and the S. pombe centromeric small RNA pathway is evident: small RNAs guide AGO to nascent transcripts, where AGO then recruits chromatin modifying factors that induce heterochromatin formation to silence transcription at the locus. Likewise, it appears that CSR-1 and its associated euchromatin-promoting activities participate in a similar self-reinforcing loop, albeit one that promotes euchromatin domains, and for which a histone modifying component has not yet been identified.Each of these nuclear small RNA pathways in the worm seems to function in a manner with parallels to the role of AGO and chromatin modifiers in pericentromeric heterochromatin formation in C. elegans genome, there is some probability that their expression will be silenced in the germline in a PRG-1- and WAGO 22G-RNA-dependent manner. Transgenes that are not silenced are instead targeted by CSR-1 22G-RNAs  appear to be mechanistically distinct from those used in humans or other model animals, it is becoming increasingly clear that the regulatory themes accomplished by these pathways are recurrent.From the elegant simplicity of the discovery of RNAi in pathways . AlthougC. elegans, acts in a similar manner by silencing at both the transcriptional and post-transcriptional level to defend against the mutagenic effects of transposon mobilization. In addition, piRNA pathway components in insects and mammals are localized to germ granules similar to the P granules and Mutator foci of the worm, and most of the factors in these pathways are required for organismal fertility  that are not formally considered piRNAs are actually functionally equivalent to mammalian piRNAs. To reiterate, although the mechanisms of small RNA biogenesis may diverge between organisms, the functional downstream consequences are recurrent themes.Protection of germline genome integrity is an intrinsic goal of piRNA pathways in animals. Because the germ lineage is essential for preserving the integrity of a species, it is not surprising that many overlapping regulatory methods act to guard its integrity . Certainertility . In thisDrosophila and mammals that positive gene regulatory roles for Argonautes and small RNA pathways may be a more widespread phenomenon  interact with these mechanisms to influence phenotypic variation. In fact, such studies are already underway: a recent study revealed that small RNAs are generated in response to nutritional stresses, and that these small RNAs mediate inheritance of phenotypic responses to the stress for multiple generations . ClearlyThe 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.