Caenorhabditis elegans is guided towards a ventral source of secreted UNC-6 (netrin). The axon’s outgrowth response to UNC-6 is mediated by the UNC-40 (DCC) receptor. We have proposed that in response to the UNC-6 molecule the direction of UNC-40-mediated axon outgrowth is stochastically determined. The direction of guidance is controlled by asymmetric cues, including the gradient of UNC-6, that regulate the probability that UNC-40-mediated axon outgrowth is directed on average, over time, in a specific direction. Here we provide genetic evidence that a specialized extracellular matrix, which lies ventral to the HSN cell body, regulates the probability that UNC-40-mediated axon outgrowth will be directed ventrally towards the matrix. We show that mutations that disrupt the function of proteins associated with this matrix, UNC-52 (perlecan), UNC-112 (kindlin), VAB-19 (Kank), and UNC-97 (PINCH), decrease the probability of UNC-40-mediated axon outgrowth in the ventral direction, while increasing the probability of outgrowth in the anterior and posterior directions. Other results suggest that INA-1 (α integrin) and MIG-15 (NIK kinase) signaling mediate the response in HSN. Although the AVM axon also migrates through this matrix, the mutations have little effect on the direction of AVM axon outgrowth, indicating that responses to the matrix are cell-specific. Together, these results suggest that an extracellular matrix can regulate the direction of UNC-6 guidance by increasing the probability that UNC-40-mediated axon outgrowth activity will be oriented in a specific direction.How extracellular molecules influence the direction of axon guidance is poorly understood. The HSN axon of  Caenorhabditis elegans HSN axon is a model for studying axon guidance. Details of HSN axon development were described in Adler et al., 2006. Immediately after hatching, the neuron extends short neurites in random directions from the cell body. However, the neurites soon become restricted to the ventral side of the cell where a ventral leading edge forms. This leading edge is just dorsal of a band of longitudinal body-wall muscle cells  mutation, and by mutations that disrupt the function of UNC-53, a cytoskeletal binding protein unc-40(ur304) mutation encodes an UNC-40 variant, UNC-40 (A1056V), that is proposed to partly mimics the confirmation of UNC-40 when UNC-6-ligated unc-40 and unc-53 mutants, randomly directed UNC-40-mediated axon outgrowth activity is induced in the unc-6(−) background and the axon has a probability of protruding in each direction. In the unc-6(+) background, UNC-40-mediated axon outgrowth activity and axon protrusion is ventral. The interpretation is that in the mutants an UNC-40 signal induces stochastically directed axon outgrowth activity, which in the unc-6(−) background cannot be oriented towards the ventral UNC-6 source.The major extracellular cue that directs HSN axon formation and guidance is UNC-6 (netrin) The conclusion that a separate process orients the outgrowth activity raises the possibility that external asymmetric factors, besides the UNC-6 gradient, could help regulate the probability that the UNC-40-mediated outgrowth activity will be oriented ventrally. Although an extracellular molecule might not induce the UNC-40-mediated axon outgrowth activity itself, the asymmetric distribution of that molecule could provide directional information for UNC-6 guidance by altering the probability of UNC-40-mediated axon outgrowth at each side of the neuron. Previous evidence suggests that the Wnt EGL-20 functions in this manner egl-20, the probability that UNC-40 will asymmetrically localize to the ventral side of the neuron is comparatively lower, whereas the probability of localization to the anterior and posterior sides is higher. Yet, the HSN axon is still guided to the ventral midline by UNC-6. This observation is evidence that the direction of UNC-40 axon outgrowth activity, and the outward force it generates, fluctuates during axon outgrowth egl-20, the formation of a morphologically mature axon is delayed in an UNC-40-dependent manner Our conclusion that the direction is stochastically determined in response to UNC-6 raises the question of how the neuron decides the direction of UNC-6 guidance. In wild-type animals, the probability that the HSN axon will protrude ventrally is very high. However in some mutants, such as We reasoned that the extracellular matrix that is just ventral of the HSN cell body could, along with the gradients of UNC-6 and EGL-20 wnt, affect the probability that the UNC-40-mediated axon outgrowth activity directs the axon ventrally. In part because the basement membrane molecule UNC-52 (perlecan) is strongly associated with this specific matrix mig-15 mutants they abnormally spread and will sometimes elongate in the anterior and posterior directions, i.e., the invasion of this matrix is disrupted In the anchor cell, the integrin heterodimer INA-1/PAT-3 is required to localize UNC-40 (DCC) to the invasive membrane ina-1 and mig-15 mutants, and we confirm that mig-15 functions cell autonomously in HSN. Axon development is consistent with the mutations causing the direction of UNC-40-mediated axon outgrowth activity to fluctuate within the neuron. As predicted by the biased random walk model, the axon may protrude from the cell body in the anterior or posterior direction but it nearly always reaches the ventral midline. Furthermore, there is an UNC-40-dependent delay in axon development. We also note that the effect the muscle/epidermis extracellular matrix has on the direction of axon outgrowth is cell specific; similar outgrowth patterns are not observed for the AVM axon, which also is guided into and through this matrix by UNC-6 and UNC-40 signaling.Here we provide evidence that the muscle/epidermis extracellular matrix and integrin/MIG-15 signaling regulate UNC-6 axon guidance by helping create a directional bias for UNC-40-mediated outgrowth activity in HSN. We show that mutations that alter the muscle/epidermal extracellular matrix do not prevent UNC-6 from inducing an asymmetric localization of UNC-40 within the neuron. However, they do affect the probability of where UNC-40 asymmetrically localizes within the neuron. In comparison to wild-type animals, the probability of UNC-40 localizing to the ventral side of the neuron decreases, while the probability of UNC-40 localizing at the anterior or posterior sides of the neuron increases. This phenotype is also observed in LGI, unc-40(e1430), zdIs5[mec-4::GFP]; LGII, unc-52(e998), unc-52(e444), vab-19(e1036); LGIII, ina-1(gm39), ina-1(gm144);LGIV,unc-5(e53), unc-112(r367), kyIs262 , zdIs13[tph-1::GFP];LGX, unc-6(ev400), unc-97(su110), mig-15(rh80), mig-15(rh326).Strains were handled at 20°C unless stated otherwise by using standard methods . A Bristol strain N2 was used as wild type strain. The following strains were used in this paper: kyEx1212 , urEx386, gmEx593.Transgenes maintained as extrachromosomal arrays included: C. elegans cDNA library (Invitrogen) was performed to make genetic vectors containing mig-15 sequence using forward (AAAACCGCTAGCGGATCCATGTCGTCATCAGGACTCGAC) and reverse (ACCAGTGGTACCTTACCTAGGCCAATTTGTCAACCCTGGCTTA) primers. The PCR product was cut with NheI and KpnI and inserted into pSM [unc-86::unc-40::gfp] vector  for forward direction. The constructed unc-86::mig-15 (forward) vector was cut by either NheI and AgeI or XmaI and AvrII, and those two fragments were combined to make unc-86::mig-15 (reverse). Both vectors containing forward and reverse direction of mig-15 sequences are injected with myo-3::mCherry as a co-injection marker.For cell-specific mig-15 knock-down experiment, PCR from unc-86::myrGFP transgenic strain, kyIs262. Synchronized populations were obtained by allowing eggs to hatch overnight in M9 buffer without food. The resulting L1, L2, and L3 nematodes larvae were mounted on a 5% agarose pad. Larval staging was determined by the gonad cell number. An outgrowth was defined as a protrusion if it extended a distance greater than the length of two cell bodies. A precocious phenotype was scored in the L1 or L2 stage if either HSN in an animal showed a predominate axon. Only one HSN was counted as having an axon per animal. The ventral neurite was scored if it had any ventral filopodia or a leading edge formation during middle L3 stage, and the most prevalent direction of growth was noted. The standard error of proportion was calculated for this purpose. Images were taken using epifluorescent microscopy with a Zeiss 63× water immersion objective.HSN neurons were visualized using an integrated zdIs5[mec-4::GFP] transgene. Axons were scored as having an anterior protrusion if the axon traveled laterally more than three cell body lengths from the cell body. Axons were scored as having dorsal or posterior protrusion if the axon traveled dorsally or posteriorly for a distance greater than two cell body lengths from the cell body. The AVM was considered multipolar if more than one process, greater than one cell body length, was observed.For analysis of the AVM axon protrusion phenotype, L4 stage larvae were mounted on a 5% agarose pad. The AVM axon was visualized in L4 stage larvae expressing the unc-86::myrGFP transgenic strain, kyIs262. L4 stage larvae were mounted on a 5% agarose pad. An anterior protrusion was scored if the axon extended from the anterior side of the cell body for a distance greater than the length of three cell bodies. A dorsal or posterior protrusion was scored if the axon extended dorsally or posteriorly for a distance greater than two cell body lengths. The HSN was considered multipolar if more than one process extended a length greater than one cell body. Ventral parallel HSN axon growth was counted if two axons grow ventrally in parallel after crossing into the muscle/epidermis interface. Images were taken using epifluorescent microscopy with a Zeiss 40× objective.HSN neurons were visualized using an integrated kyEx1212 transgene were mounted in M9 buffer with 10 mM levamisole. Images were taken using epifluorescent microscopy with a Zeiss 63× water immersion objective. To determine the HSN position and the stage of the animal, all animals were imaged by differential interference contrast (DIC) microscopy as well. The UNC-40::GFP localization was determined by measuring the average intensity under lines drawn along the dorsal and ventral edges of each HSN cell body by using ImageJ software.Staging was determined by the gonad cell number and gonad size. For analysis of UNC-40::GFP localization, L2 stage larvae expressing the http://www.physics.csbsju.edu/stats/anova.html) test was used to determine if there is a significant difference between intensities of three segments. The dorsal distribution was considered uniform if p≥0.05 and was considered asymmetrical if p≤0.05. Within an asymmetric population, the highest percent intensity was considered to localize UNC-40::GFP to either anterior, posterior or central domain of the dorsal surface. In addition, UNC-40::GFP is regarded as anterior-posteriorly localized if both anterior and posterior regions show significantly higher intensities than the central region.For analysis of the anterior–posterior orientation of UNC-40::GFP along the dorsal surface, the dorsal segment was geometrically divided into three equal lengths . The line-scan intensity plots of each of these segments were recorded. ANOVA  allele has a cold-sensitive phenotype; at 22.5°C about 90% survive to the adult stage, whereas at 15°C most arrest at the twofold stage of embryonic development . The mutation disrupts epidermal elongation and muscle attachment to the epidermis. UNC-97 is expressed in touch neurons, sex muscles, and body wall muscles, where it colocalizes with integrin PAT-2/PAT-3 unc-97 function is embryonic lethal, whereas the unc-97(su110) allele is viable; it causes defective muscle attachments Several proteins are known to play prominent roles in the structure and function of this matrix. These include: UNC-52 (perlecan), an extracellular protein of the muscle/epidermal matrix; UNC-112 (kindlin), a protein required for muscle cell-matrix adhesion complexes; VAB-19, an ankyrin repeat protein that regulates epithelial cell–matrix attachment structures; and UNC-97 (PINCH), a LIM domain adaptor protein found at extracellular matrix attachment sites. Null alleles of unc-52(e998), unc-52(e444), unc-97(su110), unc-112(r367), and vab-19(e1036) mutations alter the pattern of UNC-40 asymmetric localization in HSN. In particular, the mutations increase the probability of UNC-40 asymmetrically localizing in the anterior and posterior direction, while decreasing the probability of UNC-40 asymmetrically localizing in the ventral direction ; unc-6(ev400), unc-112(r367); unc-6(ev400), and vab-19(e1036); unc-6(ev400), are similar to unc-6(ev400) (i.e. there is a more uniform distribution of UNC-40::GFP around the periphery of HSN (unc-52(e998), unc-52(e444), unc-112(r367), and vab-19(e1036) mutations have on the pattern of intracellular UNC-40 asymmetric localization depends on UNC-6 signaling.Despite any changes to the extracellular matrix caused by the mutations, HSN still responds to UNC-6. This is evident because UNC-6 signaling induces the UNC-40 asymmetric localization and in these mutants UNC-40 becomes asymmetrically localized. In fact, double mutants, 6(ev400) , i.e. thy of HSN . Therefounc-40(e1430); unc-52(e998), unc-52(e998); unc-6(ev400), unc-40(e1430); unc-52(e444), unc-52(e444); unc-6(ev400), unc-40(e1430); unc-112(r367), and vab-19(e1036); unc-6(ev400). In these cases, the probability of dorsally directed protrusion is greater than in any of the single mutants ;unc-5(e53) and unc-5(e53); unc-112(r367) mutants is similar to the pattern observed in unc-52(e998) and unc-112(r367) mutants, respectively (unc-52(e998) and unc-112(r367) mutations on the direction of protrusion do not require UNC-5 function. This is consistent with the previous finding that UNC-5 functions through a parallel pathway to induce UNC-40 asymmetric localization in HSN We previously found that the UNC-6 receptor UNC-5 also can play a role in the UNC-6 guidance of the HSN axon ectively . This suC. elegans has one beta (PAT-3) integrin chain and two alpha (INA-1 and PAT-2) integrin chains. INA-1 is expressed in migrating neurons, the pharynx, and the DTCs, but has not been detected in the body-wall muscles ina-1 and mig-15 mutations cause the same intracellular UNC-40 localization phenotypes as the mutations that perturb the muscle/epidermal matrix.We hypothesize that integrin signaling in HSN may regulate the response to the muscle/epidermal matrix. Integrins are heterodimeric transmembrane receptors and are one of the major cell surface receptors used to mediate cell-matrix interactions mig-15 and ina-1 mutations similarly alter the pattern of UNC-40 asymmetric localization in HSN. That is, the mutations decrease the probability of UNC-40 asymmetrically localizing ventrally, while increasing the probability of UNC-40 asymmetrically localizing in the anterior and posterior direction , the probability of anterior or posterior localization is reduced in comparison to mig-15(rh326) mutant  mutation and the UNC-40::GFP transgene. This may reflect a critical relationship between integrin and netrin signaling.To test whether  mutants . We alsohenotype . Further) mutant . A signi) mutant . Similarunc-6 and unc-40 mutations can alter the timing of HSN axon development unc-6 mutants UNC-40 remains more uniformly distributed across HSN and the initial neurites that form do not develop a strong bias for the ventral direction. Normally by the early L2 larval stage HSN is polarized ventrally with neurites primarily restricted to the ventral side where a leading edge forms. Around the L3–L4 transition a single ventral axon becomes evident. However in unc-6 and unc-40 mutants axon development is delayed and neurite extension is not confined to the ventral side during the L3 stage. A predominate axon, which protrudes anteriorly, eventually forms however not until the L4 stage egl-20, mig-1, or vang-1 mutations, the axon forms ventrally in response to UNC-6 but the development is delayed Previously it was noted that ina-1(gm39), ina-1(gm144), unc-52(e998), unc-52(e444), and mig-15(rh326) mutants. Similar to the Wnt/PCP mutants, we find that the neurons don’t clearly show ventrally oriented neurites until around the L3–L4 transition (ina-1(gm39);unc-6(ev400), unc-52(e444);unc-6(ev400) and unc-40(e1430);unc-52(e444), is similar to the unc-6(ev400) or unc-40(e1430) mutants. These results are consistent with the idea that INA-1, UNC-52 and MIG-15 function to promote the UNC-6 and UNC-40 activity responsible for directing axon formation in the ventral direction.We examined the duration of axon development in ansition . Furtherlin-4 microRNA and the POU homeodomain transcription factor UNC-86 ina-1(gm39), ina-1(gm144), unc-52(e998), and unc-52(e444) mutations also cause this precocious phenotype. Distinct axon extensions can be observed in the L1 stage (unc-52(e444);unc-6(ev400), is similar to that of the single unc-52(e444) mutant. This suggests that the precocious development caused by the loss of UNC-52 function is independent of UNC-6 signaling. However, we also observe that the precocious phenotype is suppressed in unc-40(e1430);unc-52(e444) double mutants, suggesting that the precocious axon in unc-52(e444) mutants is caused by UNC-40 activity that is independent of UNC-6.Whereas only immature neurites are observed at the L1 and L2 stage in wild-type animals, some mutations can cause early development of a distinct axon, which is defined as a process that extends greater than a distance equal to two times the anterior to posterior length of the cell body. This phenotype has been observed in mutants for EGL-20, MIG-1, and VANG-1 L1 stage  and the L1 stage . The pheunc-52(e998), ina-1(gm39), mig-15(rh80), unc-97(su110), and vab-19(e1036) mutations have little affect on the direction of axon protrusion (unc-6(ev400), unc-40(e1430), egl-20(n585), ina-1(gm39), unc-52(e444), or mig-15(rh326) mutants (data not shown).The AVM axon also migrates ventrally through the muscle/epidermal matrix and the guidance of AVM also requires UNC-6 and UNC-40. However, AVM is a mechanosensory neuron that develops a specialized extracellular matrix and forms attachments with the epidermis; in fact, the epidermis envelops the AVM process otrusion . Furtheri.e., there is a probability of UNC-40-mediated axon outgrowth activity occurring in any direction We previously showed that the directional response of a neuron to UNC-6 is stochastic; Axons respond to the surrounding environment and follow highly stereotyped and reproducible trajectories to their targets The results presented here indicate that the muscle/epidermis extracellular matrix is a directional cue for UNC-6 guidance. Matrix molecules, such as UNC-52 perlecan, may not themselves cause a directional response from the neuron. However, when UNC-6 induces the direction of UNC-40-mediated axon outgrowth activity to stochastically fluctuate within a neuron the interaction between the matrix and the neuron can cause directionality by increasing the probability that UNC-40-mediated axon outgrowth occurs at the surface of the neuron that is in contact with the matrix. We show that when the muscle/epidermis extracellular matrix is disrupted by different mutations, UNC-6 still induces the asymmetric localization of UNC-40 in HSN, but the probability of anterior and posterior UNC-40 localization and axon outgrowth increases, whereas the probability of ventrally directed localization and outgrowth decreases. Consistent with inducing fluctuations in the direction of UNC-40-mediated axon outgrowth activity and with movement described by a biased random walk model, the HSN axon is guided by UNC-6 to the ventral nerve cord in the mutants even though axon outgrowth from the HSN cell body can be directed anteriorly or posteriorly.unc-52 and ina-1 mutants there is a probability that the axon will also protrude from other sides ;ina-1(gm39), a substantial number of axons will initially protrude in the ventral direction. Nevertheless, the system does not create a ventral directional bias. The graphs also illustrate the strong bias for anteriorly directed axon outgrowth in unc-6 and unc-40 mutants and that the unc-52 and ina-1mutations also affect this UNC-40-independent mediated activity.i.e. there was always a 100% probability of outgrowth in just one direction, than a corresponding line in If a mutation did not create any fluctuations in the direction of axon outgrowth activity, The ability of the direction of axon outgrowth activity to fluctuate could also underlie precocious axon formation in mutants . Before unc-52 and ina-1 mutations alter the probability of UNC-40 localizing to each side of neuron (pre se. For example, contact with the matrix might alter some property of the neuron’s membrane that in turn leads to an event that influences the probability of UNC-40 complexes assembling or disassembling at that site.What does our analysis reveal about the molecular mechanisms of axon outgrowth? We observe that the f neuron  and 5. TThe probabilistic approach used to generate C. elegans plays a role in anchor cell invasion and in the formation of presynaptic specializations Besides guiding axons, UNC-6 and UNC-40 signaling in The model we propose could have broad implications. In general netrin/DCC signaling may be used to polarize cellular activities and orient them to the surrounding environment. That is, netrin causes the direction of DCC activity to stochastically fluctuate. By increasing or decreasing the probability that DCC activity will become oriented to one particular side of the cell, external asymmetric factors orient polarized DCC activity within the cell. The cellular activities regulated by DCC may depend on the cell type and the stage of development.


Circular RNAs (circRNAs) constitute a new class of noncoding RNAs in higher eukaryotes generated from pre-mRNAs by alternative splicing. Here we investigated in mammalian cells the association of circRNAs with proteins. Using glycerol gradient centrifugation, we characterized in cell lysates circRNA-protein complexes (circRNPs) of distinct sizes. By polysome-gradient fractionation we found no evidence for efficient translation of a set of abundant circRNAs in HeLa cells. To identify circRNPs with a specific protein component, we focused on IMP3 , a known tumor marker and RNA-binding protein. Combining RNA-seq analysis of IMP3-co-immunoprecipitated RNA and filtering for circular-junction reads identified a set of IMP3-associated circRNAs, which were validated and characterized. In sum, our data suggest that specific circRNP families exist defined by a common protein component. In addition, this provides a general approach to identify circRNPs with a given protein component. Circular RNAs (circRNAs) are a new and large class of noncoding RNAs comprised of hundreds to thousands of members, and present in all eukaryotes investigated so far, from protozoa, yeast to human145This type of circRNAs contains one or more adjacent exons, that are spliced out of the pre-mRNA to yield instead of the normal, linear protein-coding mRNA a circular RNA product. CircRNAs are cell-type specifically expressed, more stable than the corresponding linear mRNA, evolutionarily conserved, and mostly cytoplasmic in cellular localization5789Drosophila1in vitro conditions16Functionally, circRNAs are still largely uncharacterized, except for a miRNA sponge function, which has been experimentally validated for two cases, ciRS-7 and SRY circRNAs6Here we describe the initial characterization of circRNA-protein complexes (circRNPs). For a set of relatively abundant circRNAs we demonstrate that they exist in the form of discrete RNPs, stable in sedimentation analysis through glycerol gradient centrifugation. We then focus on IMP3 , a known oncofetal and tumor marker RNA-binding protein with multiple post-transcriptional roles181918To identify circRNA-protein complexes, we subjected cytoplasmic extract (S100) from HeLa cells to glycerol gradient fractionation, whereby RNAs and RNPs sediment according to their molecular size and shape. We focussed the analysis on S100 extract, since circRNAs predominantly localize in the cytoplasm. 22 fractions were collected, followed by RNA preparation from every second fraction and RT-PCR assays for 12 relatively abundant circRNAs, based on circular-junction-specific primers. In parallel, and for direct comparison, total RNA prepared from S100 extract was fractionated through glycerol gradients under the same conditions, followed by the RT-PCR analysis of the circRNA distribution across the gradient .The free circRNAs distribute in the glycerol gradient between fractions #7 and #11, depending on the size of the respective circRNA . In contrast, in the S100 extract the circRNAs peak at fractions #9 to #14, but in most cases with a clear peak, indicating that each circRNA exists in the form of a major distinct large complex in the 10–15 S region. To obtain direct evidence that this is due to protein components, we also analyzed the gradient distribution of circRNAs after proteinase K treatment: Clearly, the circRNAs shifted back to the position of free RNA , demonstg), as well as in nuclear extract , which allows mapping of RNA-protein contacts at single-nucleotide resolutionIntroduction). We mapped transcriptome-wide binding sites for IMP3 protein by iCLIP in HepG2 cells (human liver carcinoma cell line), followed by searching for iCLIP tags spanning the characteristic circular junctions .Second, IMP3-associated RNAs were obtained by anti-IMP3 immunoprecipitation, RNA-seq library construction, and sequencing . The major part of the total sequenced reads (~127.5 mio.) was from the IMP3-immunoprecipitated material , comparing in each case input (5%) and immunoprecipitate (90%); as a negative IP control, anti-FLAG IPs were analyzed in parallel . As anotAs a result, we were able to confirm all three IMP3 circRNP candidates as IMP3 targets ; both linear and circular isoforms of CAMSAP1, in contrast, exhibited only background or undetectable levels after IMP3 immunoprecipitation. These semiquantitative results were further confirmed by real-time PCR assays : The antAs the next step, we fractionated HeLa cytoplasmic extract (S100) by glycerol gradient centrifugation, in parallel with total RNA prepared from the same extract, and proteinase K-treated S100 extract . The twoFinally, we measured IMP3 association for two of these gradient-purified circRNPs, NFATC3 and ANKRD17, using anti-IMP3 immunoprecipitation from the respective peak fractions , followed by real-time RT-PCR for the circular splice junctions; as negative controls we used IP against eIF4E, an abundant cytoplasmic RNA-binding protein, which binds to the cap structure of linear mRNAs, and CAMSAP1 circRNA, which was not among the IMP3 targets identified . The IP in vitro SELEX procedure, using four rounds of selection with an N20 RNA pool and recombinant GST-IMP3, which contains the full-length IMP3 protein . Based on kernel density estimation and a p-value of 1.077e-07 (Welch two sample t-test), we conclude that C/A-rich motifs are significantly enriched in IMP3-associated circRNAs in vitro analysis (CA-rich motif)in vivo or act in a combinatorial manner. Therefore we have to consider that IMP3 may bind in 3′-UTR versus coding-exon regions through different ways and with different cofactors, reflected in different enriched motifs.In conclusion, the IMP3 protein by itself can recognize certain C/A-rich motifs. The specificity of IMP3 RNA recognition is most likely complex, due to its domain structure with two RNA-recognition (RRMs) and four KH motifs. This may contribute to why the motif from a PAR-CLIP-based S100), cytoplasmic extract , and nuclear extract (NE) from HeLa cells (IpraCell), as well as RNA isolated from S100 extract (RNA), proteinase K-treated S100 (S100 + PK), and proteinase K-treated cytoplasmic extract (CE + PK) were analyzed by glycerol gradient centrifugation. For the gradient with free RNA, RNA from 500 μl S100 was isolated by TRIzol (Ambion). Proteinase K (PK) treatment was done by incubating 500 μl extract (S100/CE) in 1x PK buffer with 100 μg/ml PK (Roth) and 0.5% SDS for one hour at 37 °C. Samples of 500 μl each were loaded onto 10–30% (v/v) glycerol gradients (10 ml) and subjected to ultracentrifugation for 16 hours at 4 °C . After centrifugation, the gradient was fractionated manually into 21 fractions of 500 μl each, whereas the resuspended pellet was labeled as fraction 22. RNA was isolated from 200 μl of every second fraction by TRIzol (Ambion), followed by RT-PCR (described below). PCR products were analyzed on 2% agarose gels and quantified . As size markers, the sedimentation of ribosomal RNAs was analyzed .Cytoplasmic S100 fraction , containing cycloheximide or puromycin. After centrifugation , the cytoplasmic lysates were loaded onto 10–50%  linear sucrose gradients . Gradients were centrifuged for 3 h at 35,000 rpm in a Beckman centrifuge (SW40Ti rotor), followed by fractionation (12 × 1ml), using the Brandel tube piercer (ISCO) in combination with an ÄKTA purifier . RNA was isolated from 500 μl of the fractions (#1–11) by phenol/chloroform extraction (Roth), followed by RT-PCR (described below).HeLa cells were treated with cycloheximide (100 μg/ml) for 10 min or puromycin (250 μg/ml) for 30 min, washed once with ice-cold PBS and lysed for 10 min on ice in gradient-lysis buffer  were used, for the negative control the antibody was omitted. For validation of immunoprecipitation by Western blotting, an IMP3-specific monoclonal antibody  was used. Sequencing was performed on an Illumina MiSeq instrument (75 bp single-end reads). For details on iCLIP experimental procedures and data analysis, see Rossbach Cell lysates were prepared in RIPA buffer  and subsequently cleared by centrifugation as well as by pre-incubation with protein-G dynabeads (Life technologies) without antibodies. Antibody binding was performed o/n at 4 °C, using a polyclonal IMP3 antibody (Millipore) and as mock control, FLAG-antibody (Sigma-Aldrich). Bead capturing was carried out for one hour at room temperature with protein-G dynabeads, and protein-RNA complexes were washed five times with 500 μl washing buffer , increasing the stringency during the washing steps. RNA from the input (5%) and from the immunoprecipitated (IP) fractions was extracted by TRIzol (Ambion), followed by RQ1 DNase (Promega) digestion and ethanol precipitation. cDNA synthesis and RT-PCR assays are described below. RNA from co-IP experiments (mock-IP and IMP3-IP) was used for cDNA library preparation, using the TruSeq stranded total RNA sample preparation kit (Illumina) according to the manufacturer’s protocol starting with RNA fragmentation. Libraries were sequenced on a HiSeq instrument . Immunoprecipitation from gradient fractions was done as described above, starting with 100 μl of glycerol gradient fractions. The IP was carried out with a monoclonal IMP3 antibody and a monoclonal eIF4E antibody as mock control (Santa Cruz Biotechnology).Sequence read map to the same chromosome and the same strand, with the two sequence segments mapping to the genomic region in reverse order.The overhang spanning the “back-spliced” junction is ≥12 nts.The alignment score of the chimeric mapped reads (using column 14:aS of Standard SAM attributes) must be greater (>2) than the linear alignment with genomic sequences and annotated transcripts (Comprehensive Gene Annotation Set from GENCODE Version 19).Both 5′ and 3′ splice sites are either annotated or conform to canonical splice sites. The circRNA abundance was predicted on the basis of circRNA specific junction read counts.Sequence reads were aligned to the human genome sequence (hg19) using STAR, an ultrafast universal RNA-seq aligner with chimeric alignment optionsHepG2 total RNA (2 μg) was treated with or without RNase R  for 20 min at 37 °C. RNA was phenolized, ethanol-precipitated, and 20% were used for RT-PCR as described below.RNA from gradient fractions or immunoprecipitated RNA was extracted by TRIzol (Ambion). cDNA was prepared by reverse transcription of 1 μg total RNA (RNase R assay), 10% of RNA from gradient fractions, or 10% of the coimmunoprecipitated RNAs (input/IP), using the qScript flex cDNA synthesis kit (Quanta) and random hexamer primers. Circular isoforms were PCR-amplified with divergent primers detecting the circRNA junction and linear isoforms with primers detecting the canonical splice junction downstream of the circRNA producing exons . The fraction of bound target RNAs in co-IP assays was calculated from technical triplicates by the ΔΔCt-method, with each target normalized to the corresponding input fraction (results represented as percent of the input). Biological replicates were used to calculate standard deviations.Real-time PCR was carried out, using PerfeCTa SYBR Green Fast-Mix (Quanta) and an Eppendorf realplexWestern blotting was performed with 1% of glycerol gradient fractions and a polyclonal IMP3 antibody (Millipore), which was secondarily detected with an anti-rabbit antibody.EcoRI, XhoI) into the pGEX-6P2 expression vector . The expression of the GST-IMP3-TEV-His fusion protein was induced by IPTG (1 mM) in E. coli BL21, followed by a two-step purification. Cells were lysed in His lysis and washing buffer  by sonication (three times 20 sec). The fusion protein was purified from cell lysate by incubation with Ni-NTA agarose (Qiagen) and subsequent elution . The His tag was cleaved off , and the remaining protein was purified in a second step via the GST tag . SELEX selections were carried out with the fusion protein bound to glutathione-Sepharose.The IMP3 (IGF2BP3) open reading frame was PCR-amplified with IMP3_fwd/IMP3_TEV_His_rev primers, including His-tag and TEV-cleavage site, and cloned  with 40 pmol GST-IMP3 or GST (as negative control) bound to pre-blocked glutathione-Sepharose beads  and 4 nmol SLX-N20 transcript. After a 20 min incubation at room temperature, the samples were washed three times with 1 ml of washing buffer , treated with proteinase K (Roth), phenol/chloroform extracted, and ethanol precipitated. The stringency of washing steps was increased during the rounds of selection .An RNA pool with a degenerated sequence of 20 nucleotides was prepared by T7 transcription, using as a template annealed SLX-N20 and T7-fw oligonucleotides . The selection was performed in a total volume of 200 μl  and SLX_R18 (GST) reverse primers. cDNA libraries were amplified by PCR . All libraries were pooled in equal amounts and purified by Caliper . The final library pool was subjected to high-throughput sequencing on a MiSeq instrument . For primer sequences, see Selected RNAs were reverse-transcribed , using the SLX_RT reverse primer, followed by PCR amplification with SLX_RT and SLX_T7-fw primers (16 cycles). Transcripts for the next round of selection were produced by Sequence reads were first sample-barcode sorted, trimmed by PCR primer sequences on both ends, and further random-barcode filtered to obtain 18- to 20-nt sequence tags of the enriched RNA pools minus selection; generated by ENCODE Consortium Long RNA-seq] were analyzed. The expression of circRNAs was determined by circRNA-specific junction counts. Based on three criteria , 117 circRNAs were selected as “IMP3 non-target group”.How to cite this article: Schneider, T. et al. CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs. Sci. Rep.6, 31313; doi: 10.1038/srep31313 (2016).


Two genomic fragments  of novel, positive-strand RNA viruses that infect archaea were first discovered in an acidic hot spring in Yellowstone National Park . To investigate the diversity of these newly identified putative archaeal RNA viruses, global metagenomic datasets were searched for sequences that were significantly similar to those of the viruses. A total of 3,757 associated reads were retrieved solely from the Yellowstone datasets and were used to assemble the genomes of the putative archaeal RNA viruses. Nine contigs with lengths ranging from 417 to 5,866 nt were obtained, 4 of which were longer than 2,200 nt; one contig was 204 nt longer than JQ756122, representing the longest genomic sequence of the putative archaeal RNA viruses. These contigs revealed more than 50% sequence similarity to JQ756122 or JQ756123 and may be partial or nearly complete genomes of novel genogroups or genotypes of the putative archaeal RNA viruses. Sequence and phylogenetic analyses indicated that the archaeal RNA viruses are genetically diverse, with at least 3 related viral lineages in the Yellowstone acidic hot spring environment.The online version of this article (doi:10.1186/s40064-015-0973-z) contains supplementary material, which is available to authorized users. Almost all life forms can be infected by viruses. To date, thousands of viruses have been identified  against the NCBI non-redundant nucleotide database. Hits with a significant level  included those two nucleotide sequences of JQ756122 and JQ756123, which were identified as nucleotide sequences of putative archaeal RNA viruses, suggesting that JQ756122 was archaeal RNA virus-specific and was well conserved, making it easy to map reads in metagenomic databases.The nucleotide sequences of the putative archaeal RNA viruses (GenBank accession no. JQ756122) was downloaded from GenBank and was searched  all of the databases on the CAMERA 2.0 portal . Hits were obtained from four databases  against the broad phage metagenome database in the CAMERA 2.0 portal. Reads that were significantly similar to the contig were downloaded from the CAMERA 2.0 portal and checked for RNA origin. The contig then served as a reference sequence to assemble these retrieved reads. Once an extended contig with a relatively longer size and higher coverage was obtained after reference assembly, it was used to search the broad phage metagenome database again. This procedure was repeated until the assembled sequence stopped extending. All of the sequence assemblies were generated using the Geneious Pro . A schematic presentation of the sequence assembly procedure is shown in Figure Subsequently, JQ756122 was used to search  and against the NCBI non-redundant protein database using BLASTX  for the potential homologous sequences in the databases. The REPuter program  and used to reconstruct the phylogenetic trees. Maximum likelihood analyses were performed using phyML  and BLASTX  analyses showed that all 9 contigs were significantly similar to the sequences of the putative archaeal RNA viruses (JQ756122 or JQ756123)  collected by Bolduc et al. (Bolduc et al. Sulfolobus islandicus and Sulfolobus acidocaldarius. These findings suggest that the RNA viral genomes replicate in an archaeal host belonging to the Sulfolobales, a cell type commonly found in NL10 and acidic hot springs worldwide, and elicit a CRISPR-mediated immune response.” These 4 types of direct repeats were searched here against nine contigs. However, no identical matches were observed. These 4 types of direct repeats were also absent in the two contigs that were assembled by Bolduc et al. Therefore, we could not determine whether the potential host of the nine contigs here is Sulfolobus. However, Bolduc et al. demonstrated that the potential host of their two contigs was archaea. Stedman et al. argued that the host of the putative archaeal RNA viruses that were identified by Bolduc et al. is not archaea and may be a novel phylogenetic lineage based on the fact that the codon usage frequencies of the two contigs from Bolduc et al. are very different from that of the claimed host (Stedman et al. Bolduc et al. identified CRISPRs from cellular metagenomes (Bolduc et al. Bolduc et al. identify two genomic fragments of the putative archaeal RNA viruses (Bolduc et al. Thus far, little is known about the biological features of archaeal RNA viruses. Whether such viruses exist in the environment requires further study via isolation and identification. However, based on these available sequences, specific primers can be designed to survey the distribution, diversity and dynamics of these putative archaeal RNA viruses in various interesting environments. In addition, additional metagenomic sequencing work needs to be performed, which would contribute greatly to the discovery of novel archaeal RNA viruses, which in turn would provide additional insight into the diversity, evolution and ecology of archaeal RNA viruses and their hosts.


Reversible chemistry allowing for assembly and disassembly of molecular entities is important for biological self-organization. Thus, ribozymes that support both cleavage and formation of phosphodiester bonds may have contributed to the emergence of functional diversity and increasing complexity of regulatory RNAs in early life. We have previously engineered a variant of the hairpin ribozyme that shows how ribozymes may have circularized or extended their own length by forming concatemers. Using the Vienna RNA package, we now optimized this hairpin ribozyme variant and selected four different RNA sequences that were expected to circularize more efficiently or form longer concatemers upon transcription.  PAGE analysis confirms that (i) all four selected ribozymes are catalytically active and (ii) high yields of cyclic species are obtained. AFM imaging in combination with RNA structure prediction enabled us to calculate the distributions of monomers and self-concatenated dimers and trimers. Our results show that computationally optimized molecules do form reasonable amounts of trimers, which has not been observed for the original system so far, and we demonstrate that the combination of theoretical prediction, biochemical and physical analysis is a promising approach toward accurate prediction of ribozyme behavior and design of ribozymes with predefined functions. RNA processing plays a fundamental role in the cellular life cycle. RNA molecules are permanently synthesized, modified, edited, truncated, or abolished. In viruses, viroids, and satellite RNAs with circular RNA genomes, replication follows a rolling circle mechanism, thus initially producing linear concatemeric versions of the RNA genome . FurtherHairpin ribozyme catalyzed RNA cleavage and ligation reactions follow a transesterification mechanism . CleavagHerein we address the question whether it is possible to design entirely self-reactive RNAs to efficiently circularize OR polymerize to large RNA entities. In contrast to previous work, our purpose was to optimize RNA sequences for particular conformations favoring monomeric variants or multimerization, rather than tuning the cleavage and ligation rate itself. Self-reactive RNA molecules changing their properties by circularization or increasing their length by polymerization provide a good case-study to exploit the repertoire of state of the art computational design algorithms and to improve them by experimental verification. Good heuristics to embed catalytic activity into RNA molecules with desired functions are highly amendable for synthetic biology, since RNA cleaving or ligating ribozymes constitute an additional layer of gene regulation. Additionally, successful designs would have direct implications on the RNA world theory to explain the emergence of RNA genomes in an early RNA world.Using the ribozyme CRZ-2 , which wvia consistent mutations guided by a dependency graph. The mutations are meant to increase the probability of forming catalytically active structures and influence the conformations formed upon dimerization of the individual species. Using switch.pl, ∼10,000 bistable RNA molecules conforming to the above design objective were designed and ranked according to two scoring functions κ1 and κ2  and those that do not (higher κ2). Detailed explanation and formulas can be found in Materials and Methods and Supplemental Figure S2a,b. Compared with manual design that we had applied in previous work , compute1) compared with PBD3 and PBD4.Compared with the reference RNA CRZ-2, PBD1–4 differ by various base replacements in the nonconserved regions . HoweverThe five RNAs, CRZ-2 and PBD1 to PBD4 were prepared by in vitro transcription with T7 RNA polymerase  and incubated at conditions favoring self-cleavage followed by ligation . First, b and c  or 105mers (PBD3 and PBD4) are typically located below the 100 nt size standard mainly due to the triphosphate at the 5′-end resulting from in vitro transcription of the ribozymes  the linear 83mer is the only RNA that may perform cyclization due to the chemical constitution at its 3′- and 5′-end. However, the migration behavior of an unknown cyclic species in a PAA gel is impossible to predict by common size markers, since the overall shape and the migration behavior of the cyclic RNA strongly depend on the sequence . PreviouIntermolecular backbone ligation can only occur upon dimerization of the 83mer and/or the intermediate cleavage products, carrying the required termini . A summary of these species can be seen in c in lane 1) and 92/94mer (b in lane 1). The bands further up are hard to interpret and might show a little of c83mer and 186mer (92 + 94), as well as a 258/260mer (83 + 83 + 92/94) next to the 300 bases ladder.Identification of monomeric cleavage products was straight forward since they move roughly according to their size. Identification of higher ligation products is challenging, because their movement can be irregular . Howevera) and the c83mer (d). Since we can see a clear band for 92/94mers (b) we suggest that these species took part in intermolecular ligation reactions with 83mers resulting in a diverse set of dimers (e). However, we cannot exclude that a low running trimer is present as well. PBD3 and PBD4  show mostly the same species with different intensities. Analogous to PBD1 they show noncircular species that most likely represent the homo-dimer (83 + 83) and possibly hetero dimers and/or a trimer.Assignment of bands becomes more difficult for PBD1 to PBD4. PBD1 (lane 3), our most efficient ribozyme concerning 5′- and 3′-end cleavage, shows two bands in addition to the c83mer, which most likely correspond to the linear 166mer (83 + 83) and 249mer (83 + 83 + 83), respectively. PBD2 (lane 4) shows four species between the 105mer (With the purpose of identifying cyclic dimers in the reaction mixture we designed and synthesized an inactive dimer (CRZ*) which should mimic the behavior of its CRZ-2 equivalent . To obtain deeper insight into the self-processing behavior of the designed RNAs and in particular into chain lengths distribution, we supplemented the biochemical analysis by Atomic Force Microscopy (AFM). AFM imaging is able to visualize RNA chains on single molecule level , allowinThe contour-length histogram of the linear 83mer shows three contour-length peaks at 13.4, 24.5, and 36.5 nm, as well as very few molecules with even higher lengths . These peaks closely map to expected values for regularly folded monomers, dimers and trimers, respectively, making an interpretation straightforward. The segment lengths, representing individual helical regions, showed peaks at 5.9, 8.5, and 13.8 nm , which most likely correspond to the monomer helices and dimer-variants of these helices. AFM imaging reveals that the monomer of the l-83mer typically consists of one “short” and one “long” segment, which enclose an angle of ∼110° B. The diZ-like conformation with a total contour length of 25.3 ± 1.8 nm, matching well the expected value for a regularly folded dimer (24.9 nm). Using the same reasoning, the peaks ∼6.4 ± 0.3 and 9.0 ± 0.6 nm may be interpreted as “partially back folded” monomer and the one ∼30.7 ± 1.5 nm as a “partially back folded” trimer.Full-length CRZ-2, as well as PBD1 and PBD4 show an even wider spectrum of contour-length peaks and chain conformations , which is expected from the computational design and the biochemical analysis. In the AFM measurements, full-length CRZ-2 creates predominantly species being shorter than 24 nm . This “cut-off” shifts to 36 nm for PBD1, while much longer RNA chains (up to ∼80 nm) are observed for PBD4. Hence, the population shifts progressively to longer RNA products from CRZ-2 over PBD1 to PBD4, which is in agreement with the gel electrophoretic analysis. All structures show contour-length peaks at expected values close to those from the l-83mer, which makes an identification of monomers, dimers, and trimers straightforward. Monomers appear mostly in a rod-like conformation, and a kink (similar to the l-83mer monomers) is rarely resolvable . HoweverTo compare the AFM findings with the computational design and the results of the biochemical analysis, we calculated the number frequency of each species from the contour-length histograms. The ratio between observed monomers, dimers, and trimers are given in the last two lines of Taken together, the results of the biochemical analysis in combination with AFM imaging confirm the predicted behavior of the self-processing RNAs CRZ-2, PBD1, PBD2, PBD3, and PBD4. All RNAs undergo two initial cleavage events that truncate the full length transcript at the 5′- and 3′-end to a linear 83mer with 5′-hydroxyl group and 2′,3′-cyclic phosphate required for ligation. The subsequent intramolecular ligation delivers exclusively cyclic versions of the 83mer, whereas intermolecular ligation produces dimers and longer concatemers, which apart from PBD4, have no or rather low tendency toward cyclization. This implies that formation of cyclic dimers is extremely unfavored, since it requires the ligation at two sites simultaneously. The same applies to longer concatemers that are rather rare anyway.1). Thermodynamic optimization, however, resulted in less efficient cleavage reactions compared to CRZ-2, since the cleaved ends remain tightly bound and equilibrium is shifted toward ligation. (ii) PBD1–PBD4 vary in the formation of monomers and multimers (as intended by scoring function κ2), but interestingly, molecule optimization for stable dimers reduced the variety of dimer species and did not lead to a higher yield of trimers. Comparing experimental results with theoretical predictions, we observed two major points, as discussed in detail below. (i) All designed species are highly efficient in circularization or ligation of cleavage products  has to overcome a higher energy barrier than in the case of manually designed CRZ-2 . This isb in the CRZ-2 lane in The cleavage cascade can start with either of two reactive conformations resulting in cleavage of the 5′- or 3′-end. In the case of CRZ-2, none of these conformations correspond to the ground state of the molecule, rather they are 4.2 and 8.5 kcal/mol above the ensemble free energy. Cleavage of the 5′-end is the favored reaction, but results in a structure that has to overcome a high barrier to fold into the reactive 92mer conformation for 3′-end cleavage. In equilibrium, the reactive structure is sparsely populated, since it is 7.2 kcal/mol above the ensemble free energy. We therefore expect that the prominent band e in lane 3). CRZ-2 and PBD2 show the greatest variety of concatemeric species. In the case of CRZ-2, we see stable 92-/94- and 83mer cleavage products (b and c in lane 1). The probabilities to ligate their reactive ends are higher than the probabilities to cleave off the remaining terminal sequence patches, which corresponds to the fact that we observe a variety of concatemeric species. PBD2 shows only the 92-/94mer band b and no band for the 83mer c . For PBD4 also the 97mer is detectable , different monomeric cleavage products from the full length transcript (contour lengths differ by <2 nm), or to distinguish, which type of dimer, trimer, etc. is observed in the AFM image. However, AFM resolved structural features  and observed segment and contour lengths that match with secondary structure prediction for monomers, dimers, and trimers. In the case of the linear 83mer of CRZ-2 (which can only form multiples of itself) the typical pitch of 0.30 ± 0.02 nm per base pair in a helix  matches d , making a combination of these techniques promising and very powerful. Our study revealed that (i) self-processing activity can be programmed into RNA structures, (ii) self-processing activity can be predicted and optimized by computer-aided design, and (iii) AFM turned out to be a powerful technique to image the reaction products at the single molecule level, even for short RNAs (<100mer). Dynamic processes like self-induced topology changes and oligomerization and their sensitivity upon sequence variation are essential for biological self-organization and evolution. Moreover, a large number of publications over the past two years have shown that biological processing of RNA into circular species with often still unknown function is widespread in nature. Thus, nowadays the emergence of circular RNAs and their cellular functionalities are actively investigated , making L (left) and R (right), respectively, and the linear core as C (center). An initial ribozyme species therefore consists of three parts and can be abbreviated as “LCR” molecule. Additionally we introduce the term O for the circular version of C. Despite the ability of C to form a circular O, multiple copies of C can ligate to one long strand that itself can form a maxi-cycle .To have a consistent, length-independent annotation for all possible RNA species that can emerge from a starting (full-length) ribozyme, we introduce the following notation: We denote the 5′- and 3′-ends of the full-length molecule as 1 and κ2) were used to select for ribozymes which are able to process themselves efficiently into cyclic monomers (κ1) and to differentiate between those, which predominantly form catalytically active or inactive dimers (κ2). Both functions estimate rates for cleavage reactions by computing the probabilities of catalytic secondary structures, hence following two hypotheses: First, a cleavage/ligation rate is proportional to the equilibrium probability of a catalytically active secondary structure; second, the cleavage reaction leads to dissociation of the shorter fragment and is therefore irreversible. Equilibrium probabilities of RNA molecules can be calculated from the equilibrium partition function (Z); Z can be calculated using the McCaskill algorithm (Z(LCR) be the equilibrium partition function over all feasible secondary structures compatible with the molecule LCR, and Z(LCRL) be the constraint partition function over all reactive secondary structures in which L can be cleaved off, then the probability P(LCRL) can be computed asThe following two scoring functions (κlgorithm  implemenlgorithm . Let Z(LLCR) that can process itself into the linear catalytic core in two parallel ways. Either the 5′-end (L) of the sequence is cleaved first and the resulting truncated version (CR) cleaves the 3′-end (R), or vice versa. For both of these parallel, two-step reaction pathways we are interested in the rate limiting step which determines the speed of the cascade. Since we approximate cleavage rates from probabilities of catalytic secondary structures, the rate limiting cleavage reaction is the minimum of both probabilities, and the total rate is the sum of both parallel cleavage pathways. The yield of circular reaction products is computed as the fraction of circular molecules in equilibrium (Z(O)/(Z(O) + Z(C))) resulting in the following scoring function:All computations were done using the Vienna RNA package Version 2.1.6 with standard energy parameters at 37°C. Supplemental Figure S2a shows our model of the cleavage cascade yielding cyclic monomers. It starts with a full-length molecule  from a LCR dimer Our model of the cleavage/ligation cascade which forms circular dimers is shown in Supplemental Figure S2b. It follows the assumption that dimerization between full-length molecules happens first, then an intramolecular cleavage cascade is triggered, and finally the system equilibrates between all dimeric cleavage products. While monomers have one reactive ground state with two conserved interior loops to cleave one of their ends, dimers can form up to two reactive centers in three different ways to cleave one end . The two interior loops needed for a reaction are commonly called loop A (harboring the reactive site) and loop B. Our computations to score the dimer-cleavage cascade require at least one of these loop regions to be formed intermolecularly, since κ1, the yield of circular dimers is computed as the fraction of circular dimers in equilibrium (Z(O2)/(Z(CC) + Z(C2) + Z(O2))) with CC and C2 denoting noncovalently and covalently bound dimers, respectively. The second scoring function κ2 is therefore computed asLCRd]θ/[LCR]θ computes the equilibrium ratio between dimers and monomers at a given concentration θ (here 100 nM) for the LCR molecule following Bernhart et al.  and EDTA (50 mM) for the following PAGE analysis.RNAs (11.25 pmol) were solved in Tris–HCl buffer . After denaturation for one minute at 90°C, RNA folding was allowed for 10 min at room temperature. To initiate the cleavage reaction, MgClFermentas) being composed exclusively of linear RNAs was added. Each individual mixture was separated in the first dimension gel. Then, the gel piece corresponding to the entire lane was cut out and used for electrophoresis in the second dimension, upon which linear RNAs are supposed to form a diagonal. Covalently closed cyclic RNAs possess reduced degrees of freedom, thus migrating in nonlinear dependence on the linear species and occurring beyond the diagonal  were analyzed using 2D PAGE . First dimension: denaturing conditions (7 M urea) 15% polyacrylamide; second dimension: 17.5% denaturing polyacrylamide or 15% native polyacrylamide.Ribozyme reactions were carried out as described above using 400 nM RNA. After reaction, the product mixture was diluted 1:10, and 5 µL of this solution were lyophilized. The pellet was taken up in 50 µL of semidenaturing buffer  to a final RNA concentration of 4 nM for imaging. Resolved RNA samples were frozen in liquid nitrogen until use.2 steps on silicon wafer; MicroMasch) and PG .AFM imaging was performed in air using a Multimode atomic force microscope  equipped with a Nanoscope IIIa controller. The AFM piezo scanner was calibrated using calibration gratings TGZ01 . For the investigated RNA constructs, adsorption times of 30 sec to 2 min were sufficient to obtain a suitable RNA surface coverage on the mica substrate. After adsorption, the RNA samples were rinsed in Milli-Q water (Millipore) and dried in a laminar flow hood, followed by AFM imaging.ABCR (Karlsruhe) one day prior usage.The images were recorded with conventional Tapping Mode in air using standard tapping mode cantilevers . Before usage the cantilevers were tested with a Nioprobe self-imaging sample (Aurora Nanodevices) and only cantilevers with tip radius <5 nm were used for imaging. To reduce tip contamination by RNA uptake during imaging process, cantilevers were functionalized with 3-aminopropyldimethyl-ethoxysilane (APDES) from In contrast to DNA samples, whose structure often remains unchanged even after storage periods of several months , samples had to be imaged within few days after preparation. The highest resolutions were always obtained directly after preparation, while storing in air often led to post-preparational RNA chain degradation already after few weeks.Images were analyzed using the software supplemented with the AFM. The shape of an RNA chain was “retraced” in terms of a sequence of connected straight segments, which allowed to calculate the contour length as sum of Euclidean distances . As showSupplemental material is available for this article.


Much research on coral reefs has documented differential declines in coral and associated organisms. In order to contextualise this general degradation, research on community composition is necessary in the context of varied disturbance histories and the biological processes and physical features thought to retard or promote recovery. We conducted a spatial assessment of coral reef communities across five reefs of the central Great Barrier Reef, Australia, with known disturbance histories, and assessed patterns of coral cover and community composition related to a range of other variables thought to be important for reef dynamics. Two of the reefs had not been extensively disturbed for at least 15 years prior to the surveys. Three of the reefs had been severely impacted by crown-of-thorns starfish outbreaks and coral bleaching approximately a decade before the surveys, from which only one of them was showing signs of recovery based on independent surveys. We incorporated wave exposure (sheltered and exposed) and reef zone  into our design, providing a comprehensive assessment of the spatial patterns in community composition on these reefs. Categorising corals into life history groupings, we document major coral community differences in the unrecovered reefs, compared to the composition and covers found on the undisturbed reefs. The recovered reef, despite having similar coral cover, had a different community composition from the undisturbed reefs, which may indicate slow successional processes, or a different natural community dominance pattern due to hydrology and other oceanographic factors. The variables that best correlated with patterns in the coral community among sites included the density of juvenile corals, herbivore fish biomass, fish species richness and the cover of macroalgae. Given increasing impacts to the Great Barrier Reef, efforts to mitigate local stressors will be imperative to encouraging coral communities to persist into the future. The last two decades of coral reef research have seen a proliferation of publications charting the declining condition of coral reefs. These include reports of reductions in coral cover across entire regions or oceans Coral cover, when evaluated in isolation, is a fairly crude measure of coral reef condition, and may mask other changes in ecological processes or species composition The Great Barrier Reef (GBR), Australia, is not immune to the general degradation of reef condition reported for many other reef regions of the world. Indeed, a series of large scale and influential publications have documented the declining coral cover and condition of the GBR, particularly in the mid to southern sections and in-shore habitats Here we assessed the spatial community composition of five mid-shelf reefs of the Great Barrier Reef with known recent disturbance histories. Two of the reefs had relatively undisturbed communities for at least 15 years prior to the study, whereas coral cover on three of the reefs had been severely impacted by COTS and coral bleaching approximately 7–10 years prior to our surveys. Of these, only one of the reefs appeared to be recovering well, based on independent surveys. We assessed reef condition on these five reefs including data on coral cover, coral composition, coral recruit density, reef structural complexity and fish assemblages. For each reef we surveyed three reef zones , at each of three sites on the wave exposed side of the reefs and at three sites on the sheltered side of the reefs. The extensive data we collected allowed a spatial assessment of the community composition of these reefs, in the context of known disturbance histories. Specifically we aimed to answer the following research questions: 1) How does coral cover, composition and structural complexity vary among reef zones and exposure levels on degraded or recently recovered reefs compared to reefs subject to little recent disturbance? 2) Which ecological predictor variables best correlate with coral compositional differences among these reefs?We surveyed five mid-shelf reefs on the central section of the Great Barrier Reef near the city of Townsville . These rWheeler reef is in a no-take ‘green’ zone of the GBR, whereas the other four reefs are in ‘yellow’ and ‘blue’ zones where fishing activities are allowed. The effects of the zoning on fish assemblages of the GBR are generally related to a small suite of target predatory species We examined the influence of wave exposure and reef zone, surveying six sites at each of the five reefs between November 2010 and January 2011. The predominant wave energy influencing reefs of the GBR comes from the south-east, particularly during the southern hemisphere winter trade wind months. Therefore, to capture any influence of exposure, we surveyed three sites on the south-east facing, wave exposed side of the reefs, and three sites on the north-east facing, wave sheltered side of the reefs. At each site, we collected data from 4 replicate transects in three different zones . Surveys on the reef slope were between 7–9 m depth, parallel to the reef crest. The reef crest was well defined on all sites and survey depth was generally between 2–3 m depth. The reef flat communities were surveyed approximately 100 m back from, and parallel to, the reef crest.All fieldwork was observational, non-extractive, data collection, and was conducted under research permit number G10/33239.1, issued by the Great Barrier Reef Marine Park Authority.We surveyed the benthic and fish communities along four 50 m replicate transects within each zone of each site. This resulted in a rich dataset of 72 transects per reef and 360 across all five reefs. The benthic composition was surveyed using the point intercept method, where the substrate directly below the transect tape was surveyed every 50 cm along each transect tape. Categories included sand, rubble, pavement, algae, hard and soft coral, and other benthic invertebrates (e.g. sponges). Hard corals were surveyed to the genus level and growth form noted, whereas algae were surveyed to broad functional classifications, such as turf, crustose coralline algae (CCA) and fleshy macroalgae. The density of coral recruits (colonies <1 cm diameter) and juvenile corals (colonies between 1 and 5 cm diameter) was quantified in a 10 m by 1 m belt transect at the start of each 50 m transect tape. The structural complexity of the reef at each transect was estimated visually on a six point scale, following Polunin & Roberts All diurnally active, non-cryptic, reef associated fish species >8 cm total length (TL) were surveyed. Larger more mobile species were counted as transects were laid in a 5 m wide belt, and small territorial species (mostly pomacentrids and some labrids) were surveyed on the return pass along the transect in a 2 m wide belt. Individual fish were identified to species, their abundance counted and their size estimated to the nearest centimetre (TL). Length estimation was calibrated at the start of each day's diving by estimating the length of a random selection of PVC pipes. A total of 261 species of fish from 27 families were recorded in the surveys. Fish survey data were converted to biomass using published length-weight relationships Broad differences in the percent cover of live coral, structural complexity and the coral genus richness per reef were initially assessed using one-way ANOVAs and Tukey's post-hoc tests to identify which reefs were driving any differences. A more detailed assessment of the variation in these three response variables was assessed using a hierarchical model, with wave exposure (exposed and sheltered) and zone  as fixed factors that also include their interaction, and reef as a random effect. Normality of the residuals and homogeneity of variances were assessed by reviewing plots of residuals against fitted values and Q-Q plots, and all assumptions of the test were met.We classified our coral genera/growth form data into the four life history strategies defined by Darling et al. We assessed differences in square root transformed coral composition (based on the above life history categorisation) among the reefs, zones and exposure levels using non-metric multidimensional scaling (MDS) on a Bray-Curtis similarity matrix. We used a three-way PERMANOVA, with interactions (maximum permutations  = 9999), to test whether the coral group composition differed significantly among sites experiencing different levels of 3 factors: reef, zone  and exposure . We ran the same analyses using the original coral genera/growth form data as a comparison to the results of the life history categorisation of the corals.We assessed broad differences among the reefs in a range of key variables that are known to have the potential to influence coral community dynamics using one-way ANOVAs. These variables included total fish biomass (kg/ha), herbivore biomass (kg/ha), number of coral juveniles (1–5 cm), number of coral recruits (<1 cm), macroalgae cover (%), fish species richness per sites, and CCA cover (%). We used Tukey's post-hoc tests to identify which reefs drove any identified differences. Assumptions of the test were assessed by reviewing plots of residuals against fitted values and Q-Q plots. To assess which combination of these predictor variables was best correlated with the site level patterns in the coral communities, we first normalised the predictor variables listed above to put them on the same scale, and log transformed them to improve the spread of the data. We then used the rank-correlation BEST BIO-ENV routine to assess which combination of the predictor variables best correlated with the patterns in the coral life history community data. The technique computes rank-correlations for all possible combinations of predictor variables, and converges on the combination with the strongest relationship with the dependent community composition dataset 4,85 = 32.89, p<0.001), structural complexity , and coral genus richness   . Specifip<0.001) . In compAssessing coral cover, structural complexity and coral genus richness among zones and wave exposure treatments exposed more of the variation in the data . The ranFor structural complexity there were also significant effects of reef zone and the interactions between exposure and zone . OverallThere were significant differences for coral genus richness values among sites, driven by wave exposure and reef zone . Coral gAcropora corals accounting for of the cover in the recovered reef category , but positively correlated with total hard coral cover .Assessing variables that are thought to influence reef dynamics at a broad reef scale highlighted significant differences in fish biomass, herbivore biomass, the number of juvenile corals and the cover of macroalgae . Fish biWe found substantial differences among reefs, zones and exposure for coral cover, structural complexity and the richness of coral genera. Although the recovered reef appeared to be reaching coral cover levels of the undisturbed reefs, the coral composition differed substantially, particularly in reef slope and crest habitats. This may support previous studies showing long-term shifts in coral composition following disturbances and recovery Acropora) and low coral recruit mortality Rib reef was the only one of our three disturbed reefs that had demonstrated substantial recovery of live coral cover . The recovery time period (∼1 decade) is similar to other published examples of rapid reef recovery following severe disturbance Acropora corals Acropora corals was also apparent for John Brewer reef in the 1990's Acropora in the current spatial surveys. Succession theory suggests that while biomass or cover may recover rapidly, it can take a lot longer for species diversity and community composition metrics to reach similar levels as undisturbed communities The substantial compositional differences observed in our data beg the question as to whether these are natural differences, slow successional processes that will converge through time, or if these differences in composition are likely to be permanent. The composition of corals at Rib reef reflect recovery back to compositions seen on that reef in the past. Data from Rib reef in the 1980's and 1990's indicate a dominance of (plating) Acropora species. This genus is known to be fast growing, and has been shown to underpin rapid recovery trajectories Favites and Montipora) are in the stress-tolerant group Assigning coral genera and growth forms into life history groupings provided a mechanism to simplify the information presented, and highlights some interesting patterns in the data. Most of the cover recorded for Rib reef was associated with competitive corals, which are principally Our BEST BIO-ENV and RDA results highlighted strong associations between sub-sets of our predictor variables and patterns in coral community structure in our study. While the predictor variables included were selected as they are known to be potential drivers of reef community dynamics, it should be noted that our predictor variables could be both causes and consequences of the observed differences in coral communities. When all the reefs were included, the most important variables were herbivore biomass and fish species richness, which were higher on undisturbed and recovered reefs, and macroalgae cover, which was generally higher on unrecovered reefs. Higher herbivore biomass is known to negatively influence macroalgae cover The potential for an interaction between herbivore biomass and juvenile corals could be important for degraded reefs. Similar to Trapon et al. Reef zonation is well known to influence both coral cover and the composition of species present Shortly after the surveys reported here were conducted, cyclone Yasi, a very large category 5 storm passed over the study reefs and caused extensive damage to both coral cover and the structural complexity of the reefs Figure S1Cover of four coral life history categorisations at reef sites of different disturbance, zone and exposure. a) Stress-tolerant, b) weedy, c) generalist, d) other coral cover (%) among disturbance category, reef zone and wave exposure. Bars represent means per site ± standard error.(DOCX)Click here for additional data file.Figure S2Differences in coral community composition by genera with disturbance, zone and exposure. Non-metric multidimensional scaling analysis of coral genera/growth form cover (%). Colour and shape of symbols represent disturbance category, reef zone and wave exposure. Vectors represent the relative contribution of selected coral genera/growth form to the observed variation among sites. Vectors represented have a minimum correlation of 0.6 with MDS1 or MDS2.(DOCX)Click here for additional data file.Figure S3Relationships between predictor variables and coral community composition patterns. Redundancy analysis of coral life history composition patterns on reef sites of different disturbance history, zone and wave exposure level, with the biological predictor data overlaid to demonstrate the direction of influence of the various variables.(DOCX)Click here for additional data file.Table S1Classification of coral genera/growth forms into the coral life history groups defined in Darling et al. (2012).(DOCX)Click here for additional data file.Table S2Random factor “reef” results of the hierarchical models for a) hard coral cover, b) structural complexity and c) coral genus richness. The intercepts and 95% confidence intervals (CI) of each reef and their corresponding disturbance are presented.(DOCX)Click here for additional data file.Table S3Pairwise tests among reefs for PERMANOVA results on coral community composition data.(DOCX)Click here for additional data file.Table S4Hierarchical model results assessing exposure and zone and their interaction with reef as a random factor for: a) competitive, b) non competitive, c) stress-tolerant, d) weedy, e) generalist, f) other coral cover (%).(DOCX)Click here for additional data file.Table S5Random factor “reef” results of the hierarchical models for a) competitive, b) non competitive, c) stress tolerant, d) weedy, e) generalist, f) other coral cover (%). The intercepts and 95% confidence intervals (CI) of each reef and their corresponding disturbance are presented.(DOCX)Click here for additional data file.


Leptomonas seymouri Narna-like virus 1 (LepseyNLV1). A 2.9-kb segment encodes an RNA-dependent RNA polymerase (RdRp), while a smaller 1.5-kb segment showed no database search matches. This is the first report of bisegmented Narnaviridae from insect trypanosomatids.Genome sequences were determined for a novel RNA virus,  From a total of 300 million reads, 0.22% were assembled into a contig of 2,914 nucleotides (nt), predicting a 932-amino acid (aa) protein that bore motifs typical of narnaviral RNA-dependent RNA polymerase (RdRps) (1–L. seymouri strain ATCC 30220 using TRIzol reagent (Thermo Fisher), followed by digestion with DNase I (Thermo Fisher), purification with RCC-25 kit (Zymo Research), and digestion by S1 nuclease (Thermo Fisher).From aviridae –3, whichTwo cytoplasmic double-stranded RNAs (dsRNAs) of ~3 and ~1.5 kb were visualized after agarose gel electrophoresis , 5, whicL. seymouri Narna-like virus 1 (LepseyNLV1) L and S segments; however, their functional association remains to be proven. Some data suggest that both segments are unstable during culture, as sensitive RT-PCR tests of this strain acquired independently from another laboratory did not reveal them . As in other multisegmented RNA viruses, loss of the RdRp would be expected to result in the loss of the remaining segments.Provisionally, we term these elements the Narnaviridae (“naked RNA”) lack capsids or envelopes and do not form infectious viral particles. They reside in the cytosol as an RNA-protein complex containing one single-stranded RNA segment of about 3 kb, with a single open reading frame (ORF) encoding the RdRp (–Narnaviridae place it firmly within Narnavirus as a new species, since the overall amino acid divergence exceeds 80%. Interestingly, the trisegmented Ourmiavirus spp. show a closer relationship to Narnavirus than to Mitovirus (Narnavirus and Ourmiavirus.Viruses in the family the RdRp –3. Two ghe RdRp –. Phylogeitovirus , 7, suggLeptomonas is a monoxenous kinetoplastid parasite of insects, and related parasites are widespread in insects around the world (L. seymouri has been repeatedly isolated from patients infected by Leishmania donovani (in vitro, it is unable to survive in mammalian macrophages (Leishmania guyanensis have been associated with elevated pathogenicity in animal models (Leptomonas-Leishmania coinfections (he world . Interesdonovani , althougrophages , 10. Becl models , LepseyNfections .KU935604 and KU935605.The genome sequences of the LepseyNLV1 L and S segments have been deposited in GenBank under accession numbers


Candida albicans adaptation to the host requires a profound reprogramming of the fungal transcriptome as compared to in vitro laboratory conditions. A detailed knowledge of the C. albicans transcriptome during the infection process is necessary in order to understand which of the fungal genes are important for host adaptation. Such genes could be thought of as potential targets for antifungal therapy. The acquisition of the C. albicans transcriptome is, however, technically challenging due to the low proportion of fungal RNA in host tissues. Two emerging technologies were used recently to circumvent this problem. One consists of the detection of low abundance fungal RNA using capture and reporter gene probes which is followed by emission and quantification of resulting fluorescent signals (nanoString). The other is based first on the capture of fungal RNA by short biotinylated oligonucleotide baits covering the C. albicans ORFome permitting fungal RNA purification. Next, the enriched fungal RNA is amplified and subjected to RNA sequencing (RNA-seq). Here we detail these two transcriptome approaches and discuss their advantages and limitations and future perspectives in microbial transcriptomics from host material. Fungal pathogens of mammals are able to live and proliferate in a wide range of host body sites including skin surfaces and mucosa, but also internal organs. In order to successfully colonize or infect tissues offering such different conditions, pathogenic fungi need effective adaptation mechanisms. Adaptive processes are controlled by transcriptional programs and their understanding can provide critical clues in fungal pathogenesis .Cryptococcus neoformans and Aspergillus fumigatus  from two AIDS patients with cryptococcal meningitis prior to antifungal therapy. The RNA was extracted from fungal cells obtained after CSF centrifugation that corresponded to 106–108 cells. Due to this enrichment step, the RNA extraction yielded almost only fungal material which could be further processed for direct RNA-seq analysis. The authors could analyze the profile of about 97% of all C. neoformans genes . Some genes were identified as significantly upregulated in vivo as compared to in vitro conditions and were genes previously recognized as contributing to pathogenicity. For example, genes with known stress response functions, such as RIM101 (a pH-dependent regulator), ENA1 (an ATPase transporter gene) and CFO1 (a ferroxidase) as well as several transporters were upregulated in host samples  samples from which RNA was directly extracted. Microarray hybridizations were carried out after amplification of A. fumigatus RNA. This approach allowed the resolution of 95% of the A. fumigatus genes . Alternatively, other studies have co-cultured C. albicans with mammalian cells or tissue cultures to obtain transcript profiles reflecting the adaptation of this fungal pathogen to different host cell types (see Table 1). In these conditions, the recovery and enrichment of C. albicans cells is not technically difficult. These data may partially reflect the real gene expression landscape of C. albicans in the host. It is now understood that transcriptional networks can be shaped by different profiles between in vitro and in vivo experiments, thus highlighting the value of conducting such studies directly during infection  between the in vitro and in vivo response to caspofungin, although 18 and 13 transcription factors genes are each upregulated by caspofungin in vivo and in vitro, respectively . The different attempts to resolve the C. albicans transcriptome in vivo have turned out to be a great challenge for researchers since fungal RNA ratios in recovered infected organs were very low as compared to host RNA. Low fungal RNA ratios in transcript profile experiments that use microarray compromise data quality due the low signal/noise ratio. Likewise, when using RNA-seq approaches, the number of fungal reads may be too low for a comprehensive coverage of the fungal ORFome.Even though it is technically challenging, several transcriptional studies have been performed on mice organs after systemic infection with albicans , or micealbicans , as wellTable 2). Enrichment of cells before RNA extraction exposed them to environmental changes before stopping transcription and RNA degradation, thus potentially modifying the observed transcriptional response . RNA-seq has been used in several genome-wide C. albicans transcriptional studies in vitro . Briefly, two probes are specifically designed for each target gene. One probe, called the capture-probe, is linked to biotin and helps to immobilize the molecules of interest onto a counting stand. The second probe is target-specific and is called the reporter probe. This probe is made of six fluorochromes of four different colors, defining a fluorochrome code specific to each target molecule. This color code confers the technique a very high sensitivity and enables the analysis of quantity-limited biological samples. The mRNAs of selected genes can hybridize to both their corresponding capture-probe and the reporter probes, which are both in excess in the reaction liquid. The main interest of this detection method is that it is direct and does not require linear (array) or exponential (PCR) amplifications. The method provides simultaneous digital readouts of the relative abundance of the selected mRNA species within a sample. The restricted number of sample manipulation steps together with the absence of enzymatic reaction (in most cases) allows precise and physiologically correct quantifications from starting material.Novel approaches to enable transcript profiling directly from host samples have emerged recently. The first technology, also called “nanoString” , consistC. albicans and allowed the fungal transcription profiling on mice samples containing less than 0,1% of C. albicans RNA  from host samples originating from systemic and intra-peritoneal candidiasis in animal models , the design of biotinylated oligonucleotides can be undertaken and customized. A total of 55,342 bait probes were designed to cover 6,094 C. albicans ORFs. Due to cost limitations, the first 250 nucleotides of each gene were not covered in the bait design, resulting in an average of nine probes for each ORF. The use of this capture system on RNA obtained from infected host tissues resulted in enrichments in the proportion of C. albicans transcripts of more than 500-fold and in RNA-seq libraries containing more than 50% of fungal transcripts. To verify that neither this enrichment nor the predominating background of host material introduced a bias in the results, a simple validation was carried out. RNA from C. albicans was mixed with host RNA . This spiked RNA was next subjected to enrichment with the biotinylated bait system. The results obtained from sequencing these samples were compared to those of sequencing the same C. albicans RNA without the presence of host RNA and without enrichment. Using this approach, we were able to verify that there was no bias for 97% of the genes. Therefore, the relative amount of each gene was the same whether the samples were subjected to the enrichment procedures or not. Moreover, a machine learning approach helped to delimitate the necessary features for the baits in order to efficiently capture their target genes. Such information will be useful for future designs of bait libraries, either targeting C. albicans or other organisms.Another technology that enables specific enrichment of a microbial transcriptome in a host is the bait capture method , whose principle is outlined in an exome  and conss fungus . Using aC. albicans from infected mouse kidneys, after 16 and 48 h of infection, and from infected Galleria mellonella larvae, 2 and 24 h post-infection. These samples revealed a high resolution transcriptome, showing the expression levels of over 80% of all C. albicans genes, constituting a huge improvement in resolution relatively to previous in vivo transcriptional analyses of this microorganism. Over 1000 genes were found to be statistically up- or down-regulated in vivo relatively to in vitro when performing a meta-analysis that identified genes commonly regulated in the four different conditions. Several functions were enriched among these genes, including some typically associated with virulence, such as adhesion, iron homeostasis, stress response, response to starvation, and biofilm formation. On the other hand, such detailed landscape of the C. albicans gene expression profile allowed the identification of a large number of genes that were so far ignored to participate in the process of host invasion and infection, and these alone will be targets of investigation for years to come.This enrichment method allowed RNA-sequencing of C. albicans, highlighting the adequation of this insect model to study C. albicans virulence. These data are consistent with studies that revealed a good correlation between C. albicans virulence in mice invasive models and the insect larvae  between observed gene expression levels . This high correlation in gene expression profile is remarkable, given the different experimental conditions between both studies  and the different analytical approaches taken. Among the genes that were commonly upregulated in both data sets, genes relevant for C. albicans pathogenesis can be identified including: (i) HWP1  and ALS3 , (ii) ZRT1 and PRA1  and (iii) FTR1 (a high-affinity iron permease) and SIT1 (a transporter of ferrichrome siderophores). These results suggest that the enrichment/RNA-seq method can be an alternative to nanoString when whole genome data are required.Taking datasets produced from both technologies analyzing C. albicans mutants with reduced virulence. So far, this task was even more challenging than for wild-type strains due to the reduced fungal burdens often found for such mutants in host organs, thus further reducing the proportion of fungal versus host biomass. With the potential of these transcriptome technologies, it will be also possible to investigate C. albicans gene expression in other contexts, such as from different organs during systemic infection or during other types of infections, like oropharyngeal candidiasis, mucosal infections or gastrointestinal colonization. Importantly, since these methods allow the selective sequencing of fungal transcripts, it will be now possible to perform such studies directly on human biopsies from solid tissues, even if containing low amounts of fungal cells. Such data will be useful to estimate to which extent the C. albicans transcriptome from clinical samples differs from animal models. Moreover, the possible applications of transcriptome technologies are obviously not limited to C. albicans. To apply them to other pathogens, one needs simply to design a set of probes matching the ORFome of the organism of interest. Both approaches currently implicate significant costs given their technical specificities and the need to perform experiments in duplicates or triplicates . The significant costs of these technologies are originating from capture probe biosynthesis, which might be reduced in the future by novel chemistry. On the other hand, the expansion of their future use will probably contribute to decrease their price.The two novel transcriptional approaches, nanoString and RNA-seq following enrichment, represent powerful tools to analyze the behavior of pathogens during infection. With RNA samples directly taken from within its host, the technical challenges of quantification of fungal mRNA in a complex host mixture have now been overcome, with the difference that nanoString targets still a limited number of genes of interest while enrichment/RNA-seq is genome-wide. Future applications of enrichment/RNA-seq may include the transcriptome analysis of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.