Arabidopsis thaliana. The absorption, elution, and the long-term stability of the RNA were monitored by using quantitative real-time RT-PCR. The results indicate that adsorbed RNA can be eluted from the sorbents using simple buffers and employed directly for downstream molecular diagnostic assays without any further processing. RNA integrity can be maintained for extended time periods under refrigeration temperatures in the presence of covalently immobilized stabilizing compounds. This study provides initial evidence of the potential for application of MSNs in transportation and storage. They may also have utility in sample collection and processing in restrictive environments.The potential for encapsulating RNA within tunable, semi-permeable structures for storage and transportation purposes offers an interesting approach to the reduction of stringent storage requirements that often hamper the field application of genetic analysis methods. In this study, we assessed the potential for application of functionalized, porous silicate sorbents in maintaining nucleic acid integrity. Mesoporous silica nanoparticles (MSNs) with and without incorporated stabilizing reagents were used to encapsulate triosephosphate isomerase mRNA of  Alternatively, RNA can be protected within a physical barrier employing materials similar to those used in DNA encapsulation: liposomes, micelles, or polymers i.e. RNases, can provide ideal environments to prevent RNA degradation resulting from exposure to chemicals or enzymes which in turn provide the potential for storing and transporting nucleic acids at room temperature in a cost-effective, environmentally friendly manner.With the advancement of molecular diagnostic technologies, detection of nucleic acid targets has been increasingly applied in medical, biological, environmental, and food-related diagnostic assays 2/g and ordered pore systems with narrow pore size distributions. Reaction conditions can be chosen to yield relatively monodisperse particle sizes (50–200 nm). Small particle sizes allow for capping of mesopores or other modifications that may prevent undesired release of encapsulated cargo. The nanoparticle morphology also offers advantages in adsorption rates and saturation loading levels One example of these types of structures is mesoporous silica nanoparticles (MSNs). These materials offer high surface areas and ordered or semi-ordered pore structures. Similar to the MCM-41 mesoporous silicate, MSNs are often synthesized with surfactant templates to provide surface areas of up to 1,000 mIn this study, we demonstrated successful adsorption of RNA using silicate nanoparticles. The adsorbed RNA can be eluted from the sorbents using simple buffers and employed directly for downstream molecular diagnostic assays without any further processing. We also evaluated the long term stability of the encapsulated RNA. The results indicate that RNA integrity can be maintained for extended time periods under refrigeration temperatures when adsorbed in the presence of covalently immobilized stabilizing compounds. This preliminary study assesses the potential for application of tailored, functionalized, porous silicate sorbents in maintaining nucleic acid integrity. The goal is to extend the functional lifetime of reagents and samples under less than ideal conditions. Possible applications range from sample collection, transportation, and processing to extended storage or storage in restrictive environments.i.e. cetyltrimethylammonium) bromide (CTAB) were obtained from Sigma-Aldrich Corporation . 3-Aminopropyltrimethoxysilane (APS) and 3-isocyanatopropyltriethoxysilane (ICS) was obtained from Gelest, Inc. . 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) was purchased from Pierce Chemical Company . NaOH (1N solution) and hydrochloric acid 37.6% were obtained from Fisher Scientific , and 200 proof ethanol was from the Warner-Graham Company . Chemicals were used as received. Deionized water (18.2 MΩ-cm) was obtained using Milli Q UV-Plus water purification system .Bovine serum albumin (BSA), trehalose, glucosamine, tetraethyl orthosilicate 98% (TEOS), mesitylene 98% (TMB), and hexadecyltrimethylammonium  method; average pore size (50 Å) was calculated by the Barrett-Joyner-Halenda (BJH) method from the adsorption branch of the isotherm; and total pore volume (0.750 cm3/g) was determined by the single point method at relative pressure (P/P0) 0.97. Scanning electron microscopy (SEM) samples were mounted on SEM stubs using conducting carbon tape. Sputter coating with gold under argon was accomplished using an auto sputter coater (Cressington 108) for a duration of 60 s. SEMs were collected using a LEO 1455 SEM .Nitrogen adsorption experiments were performed on a Micromeritics ASAP 2010 porosimeter at 77 K ethanesulfonic acid; pH 5.5). The solution was incubated with agitation overnight, rinsed thoroughly with water, and dried at 50°C for 24 h. From this point, all solutions coming into contact with the materials were prepared using nuclease free water.Modification of the silicate structure by stabilizing compounds was accomplished by first providing functional groups on the silicate surface . MateriaArabidopsis thaliana was chosen as control RNA. A 1061 nucleotides segment of the gene was PCR out of cDNAs of A. thaliana and cloned into TOPO4.0 vector . The plasmid containing the TIM gene was used as template for PCR with M13 primers, then digested with restriction enzymes PstI and XbaI , then cloned into pSP64 polyA Vector  digested with the same enzymes to generate pSP64poly(A)-TIM. TIM RNA transcripts were generated from pSP64poly(A)-TIM linearized with EcoRI and in vitro transcribed from the SP6 promoter using the MEGAscript high-yield transciption kit (Life Technologies) according to the manufacture's recommended protocol. RNA labeling reactions were carried out in 30 µL reaction volume containing 2 µg of TIM RNA using ULS™ fluorescent Labeling kit for Agilent arrays with Cy3  according to the manufacture's recommended protocol. Cy3-labeled RNA was stored at −20°C freezer until ready to be used. A NanoDrop 2000 spectrophotometer  was used for analysis of Cy3-labeled RNA. Microarray mode was used with analysis correction at 340 nm and automated parameters for RNA and cy3. Label analysis indicated a labeling ratio of 15 cy3 to RNA copy.Triosephosphate isomerase  of Adsorption of RNA by the porous sorbents was evaluated by placing a fixed quantity of sorbent in aqueous solution  with a known concentration of TIM RNA in a 0.5 mL Eppendorf tube. Samples were briefly vortexed and placed on an agitator. Initial samples were incubated for varying time periods from 10 min. to 3 h. with agitation. Increased incubation time did not result in an increase in the RNA adsorbed. All data presented here are the result of a 10 min incubation. Following incubation, samples were centrifuged at 2,500 rpm for 5 min., and supernatants were separated from the precipitated sorbents. This protocol was followed for samples used in PCR analysis as well as in fluorescence analysis.RNA elution was performed using 20 µL of EB buffer  at room temperature. NS with encapsulated RNA were mixed with EB buffer and vortexed briefly to mix, then incubated at room temperature for 10 minutes. After incubation, the samples were centrifuged at 2,000 rpm for 10 min.; the supernatants were used for quantitative real-time reverse transcription-PCR (qRT-PCR). Other elution buffers, such as nuclease free water and NEB RNA elution buffer , were also used. For NEB RNA elution buffer, the sorbents were incubated at 50°C for 10 min.. For nuclease free water, sorbents were incubated at either room temperature or 65°C for 10 min. Alternatively, the nuclease free water was heated to 95°C then added to sorbents, and incubated at room temperature for 10 min.5′-CCGCCGTCTCCTCCCACCAA-3′) and TIM252R (5′-TCCGGATCCAGCCATGGCAAC-3′). One µL of eluted RNA was used as template for 25 µL RT-PCR reactions using MyiQ Thermal Cycler (Bio-Rad Laboratories). 10× serial diluted TIM RNA (1–105 fg/µL) were used as standard curve. The qRT-PCR reactions were performed using the following conditions: 50°C, 10 min, and 95°C for 3 min 30 sec, followed by 30–35 cycles of 10 sec of denaturing at 95°C; and 20 sec of annealing/extension at 64°C.qRT-PCR was performed using iScript™ one-step RT-PCR kit  with primers TIM134F (2/g and a pore volume of 0.75 cm3/g. Nitrogen sorption characterization yielded a type IV isotherm with a steep increase in adsorption in the relative pressure range (ca. 0.2–0.45), corresponding to capillary condensation in channel-type mesopores . The pore size distributions also showed changes with a loss in definition of the peak at 92 Å for glucosamine and trehalose functionalization  were functionalized with trehalose (NS-T), glucosamine (NS-G), and BSA (NS-B). Functionalization of sorbents resulted in a loss in surface area for the NS-G and NS-T materials to 320 mlization Fig. 2B.k), the saturated loading capacity of the sorbent (sq), and the site heterogeneity (n) within the sorbent based on the free  and bound target  RNA adsorption isotherms were determined for the four materials variants Fig. 3A.−4 pg−1; standard deviation 1.8×10−5) were greater than those determined for NS-B  and NS-G . Fits of the data indicated homogeneous interaction sites within the sorbents .We defined criteria requiring a difference of greater than three times the standard deviation in the measurements over a range of no less than five consecutive data points as an indicator of uniqueness in the data sets. Differences in adsorption of RNA by the NS and NS-T materials were within the noise of the measurements and failed to meet these criteria. The data sets for NS-B and NS-G sorbents met the criteria for uniqueness. Fitting of the data using the LF binding isotherm indicated a saturation capacity of 205 pg/µg for the NS and NS-T materials. The saturation capacity for NS-B was similar at 219 pg/µg and slightly less for NS-G at 102 pg/µg. Affinity constants for NS and NS-T . We first evaluated the recovery of bound RNA using EB buffer  at room temperature. Recovery of RNA from NS-B (∼1%) was lower than that for any of the other sorbents 5–18%; Fig. 4A.Adsorption of RNA to the silicate sorbents described here is intended to provide interactions that improve the stability of the RNA. Retained RNA from that initially adsorbed to the sorbents was evaluated following storage at 4°C over a period of 16 months. Because these studies were initiated prior to completion of studies on varied elution conditions, extraction of the RNA into EB buffer at room temperature was utilized. Multiple samples for each sorbent were prepared as described for the adsorption studies. The supernatant was removed from the samples, but they were not dried; adsorbed RNA on the sorbents was stored in the presence of residual water. RNA remaining in the supernatant was evaluated using the PCR protocol described above. On day one of the trial, elution of RNA from samples of each material was evaluated. The amount recovered in this experiment was used to normalize recovered amounts on subsequent days in the experiment. All recovered values are for individual sacrificial samples taken from those prepared at the initiation of the experiment Fig. 5. This preliminary study assessed the utility of functionalized porous silicate sorbents in maintaining RNA integrity with the idea of facilitating storage and transportation of the materials under less stringent conditions. The simple sorbents described here offer a first step in providing this capability. Silicate materials were functionalized with compounds expected to offer interactions with nucleic acids that would further stabilize the absorbed nucleic acids. Sugars, such as sucrose, dextran, and trehalose, are known to effectively protect nucleic acids during lyophilization and freeze-drying processes Immobilization of stabilizing reagents, with the exception of trehalose, resulted in a decrease in sorbent affinity for RNA. Glucosamine immobilization also resulted in a decreased saturation loading level while all materials were described as having homogeneous binding sites. The homogeneity of binding sites is not surprising since binding of RNA by the sorbents is through nonspecific interactions that will vary based on the orientation and concentration of the functional groups on the surface as well as on the orientation of the RNA oligomers and their proximity to one another. The affinity coefficient and homogeneity factors determined based on the LF isotherm capture averages across all of the possible interactions. The significantly lower affinity and saturation capacity of NS-G are likely due to unfavorable interactions between the sugar and RNA. Glucosamine may also interact with the surface of the sorbent in a way that prevents the interaction of RNA with that surface.Fluorescence analysis of RNA binding and elution would have provided a method for evaluation of sorbents during continued development requiring less time and offering reduced error as compared to PCR analysis. Unfortunately, the attempt to evaluate adsorption using fluorescently labeled RNA was not successful. The binding isotherm observed based on the supernatant component of the assay tends to indicate the formation of multiple layers of target on the sorbent surface in contrast to the monolayer binding isotherm observed based on PCR analysis. It is likely that this change in behavior results from the interactions between the fluorescent labels on different RNA copies or from the interaction of fluorescent labels with the functionalized sorbents. When the adsorbed component of the fluorescently labeled RNA was analyzed as well, deviation was noted between the total applied and recovered RNA. It appears that adsorbed RNA does not obey the concentration curve established for RNA in solution. The likely causes of this variation are similar to those cited for the change in supernatant binding isotherm: fluorophore-fluorophore and fluorophore-sorbent interactions. The problems identified here eliminated the potential usefulness of fluorescence based evaluations utilizing this particular labeling technique. The technique employed resulted in a labeling ratio of approximately 15 Cy3 per RNA copy. It is possible that a reduced ratio of Cy3 to RNA achieved using an alternative labeling approach would facilitate fluorescence analysis while minimizing the impact on adsorption behavior.A critical consideration in development of sorbents intended to stabilize RNA is the ability to recover the material from the sorbent. The intention is for these materials to provide stabilization of nucleic acids without complicating follow-on processing steps through the addition of compounds which may interfere with PCR analysis, use of gene arrays, or other genetic techniques. It may be possible to find a range of harsh conditions under which RNA can be eluted from the sorbents. Treatment with highly basic solutions, for example, will result in hydrolysis of the sorbent scaffold. The scaffold components in this case would be present in any following processes. In order to avoid this type of down-stream contamination, studies of these sorbents have been limited to simple considerations such as variations on temperature, pH, and salt concentrations. As a result the evaluated elution conditions provided less than perfect recovery of the RNA. The results do provide a demonstration of the potential for use of these mild techniques provided properly designed conditions and sorbents.The recovery rate of RNA from materials functionalized with BSA was lower than that for the other sorbents though the affinity coefficients calculated indicate that the NS-T material offers a higher affinity for RNA. It is important to consider these materials as heterogeneous systems. An equivalent mass of the NS-T and NS-B sorbents (as used in the experiment) does not produce an equivalent concentration of interaction sites. The surface of the silicate, the immobilized protein (BSA), and the bound RNA form a complex system of interactions. As a result, the specific interactions and the impact of the elution buffers on these interactions can only be broadly considered. The NEB buffer has a higher salt concentration (20 mM) than the EB buffer (10 mM) and includes ethylenediaminetetraacetic acid (EDTA). Interaction of the RNA with the sorbent is through nonspecific mechanisms including electrostatic interactions. The surface of the silicate sorbent will have a negative charge under aqueous conditions, while the protein will provide sites of both negative and positive charge. The salt in the two buffers considered here (Tris-Cl) provides positive charge through primary amine groups. The lower pH of the NEB buffer (7.5 versus 8.5 for EB) provides a higher concentration of these charged groups (pKa∼8). This is one possible contributing factor to the increased effectiveness of the NEB buffer. The possible role for EDTA in the elution buffer is less clear. EDTA is negatively charged and is typically used for chelation of metals. Future studies will seek to optimize elution conditions to improve the RNA recovery rate.Assessment of RNA stability indicated that the selected functionalization reagents offer improved RNA stability over the bare NS sorbent for at least three months under refrigeration conditions. No significant improvement over long term storage was observed with all sorbents showing similar RNA retention after 1 year. It is interesting to note, however, that the NS-G sorbent consistently retained more RNA than other sorbents over a 9 month period. These results indicate that this approach does have the potential to improve RNA stability. Other stabilizing reagents could provide greater benefits than those evaluated here. Furthermore, as a preliminary study, only one storage condition was tested. It is possible that variations in performance would be observed under different circumstances. Further study is needed to address these points.Encapsulating RNA within porous sorbents offers the potential to design an ideal environment for stabilizing the molecules. These materials offer the potential for storing and transporting nucleic acids under less restrictive conditions in a cost-effective, environmentally friendly manner. This effort ultimately seeks to control detrimental interactions such as enzymatic and microbial degradation, the action of oxidizing and alkylating agents, base-catalyzed hydrolysis, and inter/intramolecular nucleophilic activity. Some of these mechanisms are controlled through the porous nature of the sorbents described, for example, the pores do not allow movement of bacteria through the sorbent structure. Other actions are limited due to the interaction of the RNA with a solid support which limits the mobility of the RNA. Overall, for stabilization of RNA it is desirable to disrupt 3D structures, control solvent interactions, provide reducing sites and chelating groups, and inhibit nuclease activity. The simple sorbents described here offer a first step in providing the types of materials needed. We have demonstrated the potential of the sorbents to adsorb RNA which can then be eluted at a later time. We have also shown that even these simple sorbents can provide enhanced RNA stability during storage under less than optimal conditions. Our ongoing effort will evaluate additional sorbent modifications as well as combinations of modifications. We will also continue to seek a method for extraction of higher percentages of the bound RNA without the need for harsh processing steps.


Gyrodactylus salaris, an important but small (500 μm in length) monogenean pathogen of Norwegian Atlantic salmon .Describing and evaluating miRNA inventories with Next Generation Sequencing is a goal of scientists from a wide range of fields. It requires high purity, high quality, and high yield RNA extractions that do not only contain abundant ribosomal RNAs but are also enriched in miRNAs. Here we compare 6 disparate and commercially available totalRNA extraction kits for their suitability for miRNA-preparations from Gyrodactylus salaris with the RNA Tissue Kit (GeneMole) or the Trizol Plus Kit (Invitrogen).We evaluated 1 salt precipitation method , 2 Phenol based extraction methods , 1 paramagnetic bead extraction method  and 2 purification methods based on spin column chromatography using a proprietary resin as separation matrix . The quality of the extractions from 1, 10 and 100 individuals, respectively, was assessed in terms of totalRNA yield, RNA integrity, and smallRNA and miRNA yield. The 6 RNA extraction methods yielded considerably different total RNA extracts, with striking differences in low molecular weight RNA yield. The Phenol-free Total RNA Purification Kit (Amresco) showed the highest totalRNA yield, but the best miRNA/totalRNA ratio was obtained with the ZR MicroPrep Kit (Zymo Research). It was not possible to extract electrophoretically detectable miRNAs from Gyrodactylus salaris from infected Atlantic salmon that delivers a totalRNA yield suitable for downstream next generation sequencing analyses of miRNA. Two of the six tested totalRNA kits/methods were not suitable for the extraction of miRNAs from Gyrodactylus salaris.We present an optimized extraction protocol for single and small numbers of  MicroRNAs (miRNAs) are key regulators of many biological processes in eukaryotes . BesidesCaenorhabditis elegans [MiRNAs are single-stranded, 22 nucleotide long, noncoding transcripts derived from different genome-encoded hairpin precursors, and regulate gene expression by various mechanisms . First d elegans  they rep elegans , develop elegans , cell de elegans , haemato elegans , neuron  elegans , tumorig elegans , DNA met elegans , and as  elegans . In evol elegans ,14. Clea elegans -17.Several methods for isolation of totalRNA have been developed, with the focus primarily on high molecular weight RNAs . CommercGyrodactylus salaris . This parasite is responsible for a major epidemic disease of wild salmon in Norway and Russia [Gyrodactylus salaris individuals respectively. Particular emphasis was placed on assessing (i) total RNA yield (ii) RNA integrity (iii) smallRNA yield, and (iv) miRNA yield.The current study focuses on the ectoparasitic platyhelminth d Russia , but gyrd Russia ,20. AparGyrodactylus salaris were maintained on Atlantic salmon  parr in 500 liter tanks in charcoal-filtered and dechlorinated, continuously running Oslo tap water at 5-6°C. Fish were fed daily on pellet feed (Ewos) and maintained under continuous dim illumination [mination .Gyrodactylus were removed from fins using a mounted needle, taking care to avoid contamination with fish tissue. Individual parasites were rinsed with ethanol at -20°C to further remove contaminating fish mucus or epithelial cells. For RNA extraction groups of 1, 10, and 100 G. salaris individuals were pooled in 10 μl of -20°C ethanol and immediately processed.Single infected fish were anaesthetised using 0.1% chlorbutanol and killed by pithing, taking care to avoid contamination with blood. Fins were cut and stored in -20°C ethanol in 2 ml RNAse-free DNA loBind tubes (Eppendorf) until further processing. Using a binocular microscope Six commercially available kits were compared: MasterPure™ Complete RNA Purification Kit , Molestrips totalRNA basic for the GeneMole extraction robot , Phenol-free Total RNA Purification Kit , ZR RNA MicroPrep kit , mirVana Kit , and the Trizol Plus Kit .The selected kits represent four different extraction strategies for total RNA: rapid desalting and precipitation (EPI), solid phase extraction on silica-coated magnetic beads (GM), solid phase extraction on filter membranes  and sequential organic and solid phase extractions (AMB and INV). EPI utilises gentle lysis in a SDS-containing buffer with proteinase K whereas GM, AMR, ZR, and AMB kits all employ guanidinium thiocyanate-(GTC) containing chaotropic buffers for the lysis of the tissue/cells and the simultaneous inactivation of RNAses. The INV kit uses Trizol, a GTC-containing chaotropic lysis-buffer premixed with phenol. The EPI kit consecutively precipitates proteins and nucleic acids from the sample, and uses RNAse free DNAse for the subsequent RNA purification. Following the protocols of the GM, AMR, and ZR kits the samples in the lysis buffer (LB) are directly loaded onto the respective binding matrix, whereas the AMB and the INV kits include an acid-phenol-chloroform extraction step prior to the binding of the RNA to a silica-based glass-fiber filter membrane in a spin cartridge. Under high concentrations of salt the RNA binds to the silica-matrices whereas DNA and Proteins flow through the column. The bound RNA is eventually washed from the filters/beads and collected Table .For all extractions we followed kit protocols with the minor modification that all extracted RNA was eluted or resuspended in 100 μl RNAse-free Water (Ambion). The AMB kit protocol offers also a microRNAs only option but this was not followed as the totalRNA was also in the focus of this study and would have been lost. For the ZR Kit the quantitative smallRNA recovery option was used which should deliver smallRNA enriched total RNA with reasonable high mass RNA as well.G. salaris individuals, respectively. The obtained RNA extracts were heat-denatured for 2 min at 70°C and immediately stored in aliquots in 0.5 μl RNAse-free DNA loBind tubes (Eppendorf) at -80°C until further use.All kits were assessed for extractions from 1, 10, and 100 http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1647). RNA Quality Indicators (RQI) and RNA integrity numbers , were derived using the system software. All kits were used following the manufacturers protocols, and quantifications were done in duplicate.Quantification of totalRNA yield and assessment of integrity was done with the Experion (Bio-Rad laboratories) and 2100 Bioanalyzer  was used to analyse smallRNA and miRNA content with the 2100 Bioanalyzer (Agilent). The Agilent Small RNA kit offers a fast detection of small RNA with a sensitivity higher than agarose or polyacrylamide gels  and a temperature profile of 25°C for 5 min, 42°C for 30 min, and 85° for 5 min. The cDNA was probed for 5S, 18S, and 28S rRNA Gyrodactylus salaris using the specific primer pairs 5Sforward: 5'-TCACTCGGCTCACGTGACGA-3', 5Sreverse: 5'-GCCCTTAGCCGCCATTTGCG-3'; 18Sforward: 5'-TGGTTAAACCGCAAACGGCT-3', 18Sreverse: 5'-GTCGTCTGGCAACGGTCCAT-3'; 28Sforward: 5'-CCCAGCACCGAAGCCTACGC-3', 28Sreverse: 5'-AAACCGCTTCGGCCTCCACC-3'. PCR primers were designed from GenBank entries Z72477, Z26942, and AJ542394. The PCR amplification was done using the AmpliTaq Gold® Fast PCR Master Mix (Applied Biosystems) and a protocol consisting of 30 cycles with 94°C for 30s, 60°C for 40s, and 72°C for 60s. Only Gyrodactylus salaris preparations yielded positive PCR products that were verified by DNA sequencing, for control preparations from uninfected fish no PCR products were obtained (not shown).To verify that totalRNA preparations contained Gyrodactylus salaris individuals with a yield of 15.4 ng  from single g Figure . The oveThe yield differences between the kits were highest for extractions from just 1 individual. This may reflect different developmental stages of the respective animals, but may also be due to uncertain estimates due to the sensitivity limitations of the electrophoretic systems. RNA concentrations of less than 50 pg/μl and 100 pg/μl (corresponding to 5 ng and 10 ng in 100 μl) are below the sensitivity limit of the 2100 Bioanalyzer and Experion systems, respectively. Such effects are expected to be less important for the extractions of 10 and 100 individuals.In general, the quality of totalRNA extractions from single worms was low (RQI/RIN 5.3 ± 3.1) but was much better for 10 (7.5 ± 3.4) and best for 100 (8.1 ± 2.4) individuals. All preparations with the ZR and the EPI kits had relatively low quality indices (2.3-6.2), an indication of increased levels of degradation. The rather poor baseline in the electropherogram shows that, particularly in the EPI preparation, the RNA integrity had suffered from a relatively long extraction time including DNAse treatment. For the ZR kit, however, the low RQI/RIN values seem to have a different explanation. The protocol efficiently extracts small RNAs and the high molecular weight RNAs contribute comparatively less to the totalRNA. Although there is no noise indicating RNA degradation . However the highest amount of miRNAs with a yield of 9.6 ng for one individual could be extracted using the ZR RNA MicroPrep kit (Zymo Research), which is also the only kit that allows elution with a volume as little as 6 μl, one order of magnitude smaller than for the other kits and recommended as input for some downstream NGS applications .We have demonstrated successful extraction of high quality RNA from a single Gyrodactylus salaris individuals is sufficient for a microRNAs sequencing run with an Illumina Genome Analyzer 2 , making such genomic and transcriptomic analyses feasible with manageable numbers of these pathogenic fish parasites.The amount of totalRNA extracted in this study suggests that a sample size of 100 Other totalRNA extractions kits such as the Molestrips totalRNA basic for the GeneMole extraction robot (GeneMole) and the Trizol Plus Kit (Invitrogen) were found to be inappropriate for extracting small RNA and microRNA from rather small samples. This may be of particular interest as kits from the Trizol (Invitrogen) family have frequently been used as the standard extraction method in recent microRNAs studies .The successful extraction of suitable amounts of RNA, especially miRNAs of reasonable quality offers an interesting novel tool for assessing the miRNA complement of single small sized parasites including those of different developmental stages.The authors declare that they have no competing interests.BF conceived the study, designed and carried out all the experiments and drafted the manuscript. LB and PDH participated in the design of the study and contributed to drafting the manuscript. All authors have read and approved the final version of the manuscript.


We have shown that Dicer processes 7SL RNA into different fragments ranging from ∼20 to more than 200 nucleotides. Here we addressed the molecular functions of these 7SL RNA fragments and found that some of them functioned as dominant-negative regulators of the full-length 7SL RNA, interfering with signal recognition particle (SRP) complex formation. Transfection of these 7SL RNA fragments inhibited the expression of cell surface glycoproteins, the targeting of a reporter protein to the endoplasmic reticulum, and the secretion of secreted alkaline phosphatase. These results suggest that some Dicer-processed 7SL RNA fragments interfered with SRP-mediated protein targeting. Moreover, we showed that Dicer knockdown enhanced SRP-mediated protein targeting and that transfection of a mixture of the 7SL RNA fragments partially restored this effect. Our data indicate that Dicer can fine-tune the efficiency of SRP-mediated protein targeting via processing a proportion of 7SL RNA into fragments of different lengths. The signal recognition particle (SRP) is a key component of the cellular machinery that couples the ongoing synthesis of proteins to their proper subcellular compartments and is essential for cotranslational insertion of membrane and secretory proteins into the lumen of the endoplasmic reticulum (ER) The eukaryotic SRP complex comprises six distinct polypeptides  bound to an RNA molecule (the 7SL RNA) 7SL RNA was first detected in Rous sarcoma virus particles In the present study, we investigated the cellular functions of the Dicer-processed 7SL RNA fragments and found that some function as dominant-negative regulators of the full-length 7SL RNA by interfering with the formation of the SRP complex and inhibiting SRP-mediated protein targeting.AAGGCTTACCTTCTCCAGGCT), Control (AATTCTCCGAACGTGTCACGT). RNA transfection was performed as described HepG2.2.15 and HEK293T cells were cultured in RPMI 1640 medium , RKO and T29 cells were cultured in DMEM (Hyclone), and HCT116 cells were cultured in McCoy's 5A medium . All media were supplemented with 10% fetal bovine serum. We obtained siRNAs from Invitrogen  and constructed plasmids expressing shRNAs (shDCR and shCon) by inserting the corresponding hairpin oligonucleotides into the pSUPER.neo+GFP vector . The target sequences of siRNAs and shRNAs were as follows: Dicer , 5′-AAGCTTAATTATAATACGACTCACTATAG-3′ and 5′-GACCTGCTCCGTTTCCGACCTGG-3′; 7SL(97-299), 5′-AATTATAATACGACTCACTATAGGGAGACCGCGCCCGGCCTGATGAGTCCGTGAGGACGAAACGGTACCCGGTACCGTCTGCCGATCGGGTGTCCGCAC-3′ and 5′-AGAGACGGGGTCTCGCTATGTTG-3′.The control RNA (a fragment of LacZ) was previously described Cells were lysed in RIPA buffer and equal amounts of denatured protein were subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes . Membranes were then incubated with primary antibody, followed by horseradish peroxidase-linked secondary antibody, and immune complexes were detected using ECL plus reagent (Millipore). The primary antibodies were as follows: Dicer and SRP19 , SRP9 and SRP14 , SRP54 and SRP68 , SRP72 , γ-tubulin and β-actin .GAPDH mRNA served as an internal control. Primer sequences used to detect Dicer and GAPDH mRNAs were as described 5′-GGCTTCCTTGGCTACCATAA-3′ and GGCATTCTGATGGGAACTTG; SRP14, 5′-CAGCCCATCCATGTTAGCTC-3′ and AGCTACCGATGGGAAGAAGA-3′; SRP19, 5′-AGACTTTGGTCAGCACCTCC-3′ and AAACAGGAAGATGGGAGCCT-3′; SRP54, 5′-TTCCAAGGTCTGCTAGAACCA-3′ and CTCCCAGCTTTCTTGGGTTT-3′; SRP68, 5′-CGCTCCTTAGTTTCTTCGCTT-3′ and AGAGGGAGAACGGTTCCAGT-3′; SRP72, 5′-AGCCATCTTTCTGGATCTGG-3′ and TACATTCGGAAGAAGGGTGG-3′.Total RNA was prepared using TRIzol® reagent (Invitrogen) and incubated with RNase-free DNase I  for 30 min. The DNA-free RNA was reverse transcribed using a M-MLV reverse transcription kit  according to the manufacturer's instructions. Samples prepared in the absence of reverse transcriptase served as negative controls. SYBR green real-time PCR was performed using the ABI PRISM 7300 Sequence Detection system (Applied Biosystems). The level of 2, 0.01% octaethylene glycol dodecyl ether , 1 mM dithiothreitol, 30 U/ml RNase inhibitors (Promega), 0.25% Triton X-100, and a cocktail of protease inhibitors). The homogenized cells were incubated at 0°C for 10 min, potassium acetate was added to 500 mM, and nuclei were isolated by centrifuging the homogenate at 800×g for 10 min. The postnuclear fraction (1 mg) was incubated with 30 pmol 7SL RNA fragments or control LacZ RNA at 0°C for 10 min, 37°C for 60 min, layered onto a 10–40% glycerol gradient, and centrifuged at 5°C for 8 h at 40,000 rpm in a Beckman SW41Ti rotor. Ten equal fractions were collected from the top to the bottom of the gradient. One-third of each fraction was used for RNA extraction using TRIzol LS (Invitrogen), and the level of 7SL RNA was measured by real-time RT-PCR using the following primers, 5′-GGAGTTCTGGGCTGTAGTGC-3′ and 5′-ATCAGCACGGGAGTTTTGAC-3′ to specifically detect the full-length 7SL RNA. The remainder of each fraction was treated with trichloroacetic acid to precipitate proteins for western blot analysis.Fractionation of postnuclear extracts was carried out as described with modifications http://facs.scripps.edu/software.html).Cells were washed three-times with phosphate-buffered saline (PBS) and stained with Alexa Fluor® 488 conjugate-lectin GS-II  at room temperature for 30 min. The cells were then washed three-times and resuspended in PBS. Fluorescence was detected using FACSAria (BD Biosciences) and analyzed using WinMDI software (HEK293T cells were transfected with the plasmid pECFP-ER (Invitrogen). This plasmid encodes enhanced cyan fluorescent protein (ECFP) fused at its 5′ and 3′ ends to the endoplasmic reticulum (ER) targeting sequence of calreticulin and the ER retrieval sequence, KDEL, respectively. A cell line stably expressing pECFP-ER was transfected with specific RNAs as indicated, and images of fluorescent cells were obtained using a Leica DMI3000 B microscope. When compared with the corresponding control, the images were taken with the same light intensity. However, to present the results more clearly, different light intensities were applied in different experiments.Cells were co-transfected with a plasmid encoding SEAP  and different RNAs or shRNAs as indicated. The growth medium was changed every 24 h and collected at indicated time points. SEAP activity was determined using the Great EscAPe SEAP kit (BD Biosciences) following the manufacturer's instructions.Small RNAs less than 200 nt were extracted from HepG2.2.15 cells using the E.Z.N.A. Micro RNA Kit  and polyadenylated using the Poly(A) Polymerase Tailing Kit . Reverse transcription was then performed using the SMARTScribe M-MLV Reverse Transcriptase System  with CDS III/3′ PCR Primer and SMART IV Oligonucleotide. Next, the 7SL RNA derived-cDNA was annealed with a biotin-labeled 7SL sRNA5cd oligonucleotide (biotin-GAGTTCTGGGCTGTAGTGCGCTA) and purified using Dynabeads® M-280 Streptavidin magnetic beads (Invitrogen) according to the manufacturer's instructions. The purified DNA was PCR-amplified using CDS III/3′ PCR and 5′ PCR primers and cloned into the pGEM-T Easy Vector (Promega). Primer and oligonucleotide sequences are as follows:ATTCTAGAGGCCGAGGCGGCCGACATGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3′); SMART IV Oligonucleotide (5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3′); and 5′ PCR Primer (5′-AAGCAGTGGTATCAACGCAGAGT-3′).CDS III/3′ PCR Primer , 7SL RNA is also processed by Dicer into longer fragments We then tested whether these 7SL RNA fragments interfere with the formation of the SRP complex. Postnuclear extracts incubated with the control RNA or 7SL RNA fragments were fractionated on glycerol gradients. As shown in We reported that Dicer-processed 7SL RNA fragments are reduced in Dicer-knockdown cells To further investigate whether enhanced SRP function in Dicer knockdown cells is due to reduction of Dicer-processed 7SL RNAs, we transfected Dicer-knockdown cells with a mixture of the 7SL RNA fragments, including 7SL(1-96), 7SL(1-212), and 7SL(97-299). The 7SL RNA fragment mixture not only partially inhibited the secretion of SEAP , but alsDicer is essential for the biogenesis of microRNAs that may target SRP subunits, thus decreased Dicer expression may result in elevated levels of SRP proteins. To exclude the possibility that the increased efficiency of ER-mediated protein targeting in Dicer knockdown cells was mediated by increased levels of SRP proteins, the expression of SRP subunits was monitored using real-time RT-PCR and western blotting analyses. We found that knockdown of Dicer did not alter the expression of SRP proteins .We previously demonstrated that Dicer processes 7SL RNA into fragments of different lengths Of note, 7SL RNA fragments longer than 200 nt or lacking the 7SL sRNA5cd sequence cannot be detected by our cloning method. Another limitation of this method is that any RNA shorter than 200 nt that contains a sequence similar to that of 7SL sRNA5cd can be cloned. Therefore, further effort is necessary to develop a more specific and efficient method to clone all of the 7SL RNA fragments.Our data indicate that Dicer can fine-tune the efficiency of SRP-mediated protein targeting via processing a proportion of 7SL RNA into fragments of different lengths that interfere with the function of the full-length 7SL RNA.Figure S17SL sRNA5cd does not modulate SRP-mediated protein targeting. (A) HEK293T cells were co-transfected with the pSEAP2-control plasmid and different concentrations of 7SL sRNA5cd or the control small RNA. SEAP activity was determined 48 h post-transfection. (B) HEK293T cells stably expressing ECFR-ER were transfected with 7SL sRNA5cd or the control small RNA. Fluorescence was measured 48 h after transfection. (C) HepG2.2.15 cells were transfected with 7SL sRNA5cd or the control small RNA. Cell surface glycoproteins were measured 48 h later. Horizontal and vertical axes denote intensity of fluorescence and number of events, respectively. The filled histogram represents unstained cells, the black line represents cells transfected with the control small RNA, and the red line represents cells transfected with 7SL sRNA5cd.(TIF)Click here for additional data file.Figure S2Flow chart for cloning long 7SL RNA fragments. Small RNAs shorter than 200 nt were polyadenylated and reverse transcribed. The cDNAs derived from 7SL RNA fragments were purified using a biotin-labeled 7SL sRNA5cd oligonucleotide and PCR-amplified. The purified PCR products were cloned into the pGEM-T easy vector and sequenced.(TIF)Click here for additional data file.Figure S3Dicer-processed 7SL RNA fragments. (A) 7SL RNA Dicer cleavage sites  were predicated according to the sequences of 7SL sRNA5cd and 7SL sRNA8b. (B) Diagram of the synthetic Dicer-processed 7SL RNA fragments. Red arrows indicate the PCR primers that can only amplify the full-length 7SL RNA but not any of the synthetic 7SL RNA fragments(TIF)Click here for additional data file.Figure S47SL(1-96) inhibits secretion of SEAP in a concentration-dependent manner. HEK293T cells were co-transfected with the pSEAP2-control plasmid and different amounts of 7SL(1-96) or LacZ RNA. SEAP activity was determined 48 h post-transfection, *p<0.05, **p<0.01 as compared with LacZ RNA.(TIF)Click here for additional data file.Figure S57SL(1-212) inhibits the secretion of SEAP in different human cell lines. The pSEAP2-control plasmid was co-transfected with 7SL(1-212) or LacZ RNA, and SEAP activity was determined 48 h post transfection. **p<0.01 compared with LacZ RNA.(TIF)Click here for additional data file.Figure S6Knockdown of Dicer in different human cell lines. Representative western blot of Dicer. Detection of β-actin was used as the loading control. Con: control cells, K/D: Dicer knockdown cells.(TIF)Click here for additional data file.Figure S7Dicer knockdown enhances the expression of cell surface glycoproteins. Cells were transfected twice with siDCR or siCon, and cell surface glycoproteins were measured 96 h later. Horizontal and vertical axes denote intensity of fluorescence and number of events, respectively. The filled histogram represents unstained cells, the thick line represents Dicer knockdown cells, and the dashed line represents the control cells.(TIF)Click here for additional data file.Figure S8Dicer knockdown does not alter the expression of SRP proteins. HepG2.2.15 cells were transfected twice with siDCR or siCon, the expression of SRP proteins were measured by real-time RT-PCR (upper panel) and western blotting (lower panel) 96 h after transfection. Detection of β-actin was used as loading control. Con: control cells, K/D: Dicer knockdown cells.(TIF)Click here for additional data file.


Mammalian spermatogenesis is a complex hormone-dependent developmental program where interactions between different cell types are finely regulated. Mouse models in which any of the sperm maturation steps are perturbed provide major insights into the molecular control of spermatogenesis. The Twitcher mouse is a model for the Krabbe disease, characterized by the deficiency of galactosylceramidase (GALC), a lysosomal enzyme that hydrolyzes the terminal galactose from galactosylceramide, a typical component of the myelin membrane. In addition, GALC catalyzes the hydrolysis of the terminal galactose from galactosyl-alkyl-acyl-glycerol, precursor of seminolipids, specifically expressed on the membrane of germ cells. Previous data reported by our group demonstrated that glycolipids play an important role in sperm maturation and differentiation. Moreover, we hypothesized that the severe impairment of the central nervous system that affects the Twitcher mouse could interfere with the hypothalamus-pituitary-gonadal axis function, contributing to infertility. To highlight this hypothesis we have determined, at molecular level, the potential variation in expression pattern of brain hormones involved in spermatogenesis regulation. Infertility is a major medical problem worldwide. Male infertility affects 1 in 25 men in the Western world and is the cause of considerable social and financial burden , genetic disorders caused by lysosomal enzyme deficiencies, demonstrate that lysosomal enzymes can elicit pleiotropic effects specifically on spermiogenesis , 16. In New insights come also from the Twitcher mouse, a naturally occurring model of Krabbe disease, characterized by deficiency of galactosylceramidase (GALC) , 21. GALGALC gene suggesting its importance in the development and function of the male reproductive system and indicating in its deficiency the cause of infertility of the Twitcher males.At ultrastructural level, the most severe alterations are detected in the acrosomal complex Figure D: the inIt is known that hormones play a key role in controlling spermatogenesis and, moreover, that neurological impairment is often associated to infertility as demonstrated in several neurological mouse mutants. We have, therefore, hypothesized that an unbalanced hormonal profile, owing to severe brain degeneration, could contribute to male infertility in the Twitcher mouse.At testicular level, the Leydig and Sertoli cells are the target of pituitary hormones, such as LH and FSH. The close interaction between germ cells and somatic cells, present in testis, was demonstrated to be essential for correct spermatozoa differentiation. Any alteration in their morphology/metabolism would result in the impairment of this relationship.Among the testicular interstitial cells, Leydig cells are very important in testis development since they produce testosterone, a steroid hormone with a pivotal role in the regulation of spermatogenesis. To evaluate potential Leydig cells dysfunction, a careful morphological investigation of the tubular tissue of Twitcher mouse was performed in 35 days old mice, when the spermatogenetic process is already completed.We observed, at light microscopy level, that Twitcher mouse tubules compared to age matched wild type were smaller in size and that the interstitial space was reduced allowing the tubular membranes to become adjacent Figures A,B. ThesAt the ultrastructural level the Leydig cells of wild type mice were found in small clusters and most of them showed a normal ultrastructural pattern, with cell cytoplasm containing many lipid droplets Figure C. Leydign = 6) and Twitcher mice (n = 6) at PNDs 35, were normalized to the validated housekeeping gene eEF-2 (Eukaryotic elongation factor 2 kinase)  expressed by Leydig and Sertoli cells, respectively. Thus, GnRH, FSH, and LH are the brain hormones that regulate testicular function and spermatogenesis. To establish if the hypothalamic-pituitary-gonadal axis is deregulated in the Twitcher mice, we have investigated by qRT-PCR the mRNA expression levels of genes encoding these hormones. The expression levels of the analyzed genes in wild type ( kinase)  and refep < 0.01). LH and FSH expression, were also significantly decreased  in the Twitcher brain compared to aged matched wild type mouse.Our results indicated that, at PND 35, GnRH expression is reduced by 70% in the Twitcher brain compared to wild type (Thus, gene expression analysis performed at brain level proved that hypothalamus and pituitary functions were affected.Since mammalian spermatogenesis is a complex hormone-dependent developmental program that ultimately give rise to spermatozoa, mouse models in which any of this step is perturbed have provided major insights into the molecular control of spermatogenesis.The studies presented are the follow up of previous observations published by our group providing clues to the pleiotropic effect of the GALC gene and its importance in the development and function of the male reproductive system . The datIn conclusion, the data presented demonstrate that, in this mutant, the infertility may not be exclusively caused by the metabolic abnormalities in the sphingolipid pathway due to the GALC defect but, rather, to the severe involvement of the CNS that causes disruption of the hypothalamus-pituitary-gonadal axis.Although further work is needed to fully clarify the complex interaction between brain and testis hormones, our data offer a new approach to study the spermatogenesis defects associated to CNS pathologies. Furthermore, the Twitcher mouse can be considered a model system for the study of hormone signaling orchestration between brain hormones with their testicular receptors.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.


Post-transcriptional 3′ end processing is a key component of RNA regulation. The abundant and essential RNA subunit of RNase MRP has been proposed to function in three distinct cellular compartments and therefore may utilize this mode of regulation. Here we employ 3′ RACE coupled with high-throughput sequencing to characterize the 3′ terminal sequences of human MRP RNA and other noncoding RNAs that form RNP complexes.The 3′ terminal sequence of MRP RNA from HEK293T cells has a distinctive distribution of genomically encoded termini (including an assortment of U residues) with a portion of these selectively tagged by oligo(A) tails. This profile contrasts with the relatively homogenous 3′ terminus of an in vitro transcribed MRP RNA control and the differing 3′ terminal profiles of U3 snoRNA, RNase P RNA, and telomerase RNA (hTR).3′ RACE coupled with deep sequencing provides a valuable framework for the functional characterization of 3′ terminal sequences of noncoding RNAs. The addition of non-templated nucleotides to the 3′ ends of RNA molecules is a widespread mechanism for their regulation. Beyond the familiar long poly(A) tails of messenger RNAs, short 3′ tailing of uridine -4 and adRNase MRP is a ribonucleoprotein complex with a single RNA component (MRP RNA) transcribed by RNA polymerase III . MutatioBecause 3′ end processing is a prevalent and potent means of regulation for other noncoding RNAs and because one instance of dramatic 3′ extension of MRP RNA was previously reported , we sougTo comprehensively define the 3′-hydroxyl ends of endogenous RNA molecules in HEK293T cells, we adapted an RNA ligase-mediated 3′ RACE strategy coupled to deep sequencing Figure . While sWe obtained an average of 955,000 trimmed reads  per experiment for endogenous MRP RNA. Unlike typical pipelines, alignment of sequences to the reference genome was not performed, since this would have eliminated detection of extensions not mapping to the genomic template. Instead, raw sequencing reads were filtered to obtain a rigorous set where each read contained both a 3′ region of MRP RNA , spiked it into our cellular RNA, and prepared a single library for deep sequencing. The template for ivt-MRP was designed to have the commonly annotated …CU 3′ end produced by run-off transcription. As expected for this template, and in contrast to endogenous MRP RNA, ivt-MRP showed no appreciable abundance of the …CUGU ending nor a penchant for additional U’s or A’s Figure b. RatherAbout 1% of ivt-MRP endings were strikingly complex Figure a. These One key advantage to this 3′ RACE sequencing method is the transcriptome-wide scope of the ligated cDNA library. Thus, with this library in hand, any RNA of interest can be amplified and subjected to deep 3′ terminal profiling Figure . We founTo assess whether similar 3′ termini are captured in other published datasets, we used our bioinformatic pipeline to reanalyze raw poly(A)-depleted RNA sequencing data ,40,41 anThe information content of the eukaryotic genome is greatly expanded in the transcriptome through post-transcriptional processing events. Well-studied examples include alternative splicing, RNA editing, and modifications including methylation and pseudouridylation. Recently, Li et al. characterized extensive differences between RNA and DNA sequences within protein coding genes that produce corresponding peptides with sequences that deviate from the genomic template . The RNAHigh-throughput sequencing is a powerful technology with continually emerging and illuminating applications. The coupling of deep sequencing to the classic RACE technique has provided unprecedented insights into low abundance functional 3′ termini heretofore underappreciated . GeneralOur 3′ RACE sequencing analysis of the steady-state populations of four noncoding RNAs indicates that each RNA has a distinct repertoire of 3′ termini. Like MRP RNA, RNase P RNA is transcribed by RNA polymerase III and also has additional uridines beyond the annotated 3′ end. However, oligoadenylation is much less prominent with RNase P than with MRP RNA. Turning to the RNA polymerase II transcripts, we find U3 snoRNA to have a very homogeneous end. While processing intermediates with additional U’s have been observed in reporter expression systems of U3 snoRNA , endogenThe oligo(A) additions found in this study are consistent with those added by the TRAMP complex to nuclear RNAs targeted by the RNA exosome (for review see ). Along Much of the initial incentive for this work came from the finding of 3′ extensions on MRP RNA by Maida et al. , who repWhile evidence for 3′ heterogeneity on MRP RNA was presented as many as 30 years ago ,53, to o2, 5 mM NTPs, 10 mM DTT, 4 mg/mL yeast pyrophosphatase (Sigma) and T7 RNA polymerase. In vitro transcription proceeded for 30 min at 37C and was stopped by phenol extraction with ethanol precipitation. Precipitated RNA was treated with 50U RQ1 DNase (Promega) and stopped by phenol extraction with ethanol precipitation according to the manufacturer’s instructions. DNase-treated RNA was then PAGE purified and stored in water at -80C.MRP RNA 265 nt) was amplified from human genomic DNA using forward primer mrpF1  according to the manufacturer’s instructions, and quantified for total RNA by absorbance at 260 nm (via Nanodrop) and MRP RNA by northern blot comparison with ivt-MRP. 50 μg whole cell RNA containing approximately 15 ng endogenous MRP with or without 15 ng ivt-MRP was subjected to 50U of RQ1 DNase (Promega) and phenol extracted with ethanol precipitation according to the manufacturer’s protocol. Four 5 μg aliquots of each sample were then depleted of ribosomal RNA using the RiboZero rRNA Removal Kit (Epicentre), and 800 ng rRNA-depleted sample were subsequently treated with 2U alkaline phosphatase (Roche) and phenol extracted with ethanol precipitation as per the manufacterer’s instructions.2, 10 mM DTT and 200 U of Superscript III (Invitrogen). Reverse transcription lasted 50 min followed by heat inactivation at 85C for 5 min. 5 μL RT reaction was used as template for RACE and addition of Illumina adapters with 1X GC Buffer, 2 mM MgCl2, 10% DMSO, 400 nM primers X_RACEF/3’universal_R1, 400 μM dNTPs and 1.25U Phusion polymerase in 25 μL final volume with the following program: 98C for 2 min, 18 cycles of 98C for 30 s, 65C for 30 s, 72C for 1 min, and final extension at 72C for 10 min. The entire RACE reaction was loaded on 2.5% agarose gels and amplicons of ~150-600 bp were excised for purification (Qiagen). Purified RACE products (30 μL) were used as template for library amplification in 100 μL reactions containing 1X HF Buffer, 2 mM MgCl2, 400 nM primers Gen_primer-F2/Gen_primer-R2, 400 μM dNTPs and 5U Phusion polymerase with the following program: 98C for 2 min, 14 cycles of 98C for 30 s, 58C for 30 s, 72C for 1 min, and final extension at 72C for 10 min. These nested amplicons were subjected to PCR cleanup (Qiagen), quantified by Qubit and Bioanalyzer, diluted to 2 nM in 10 mM Tris-HCl, pH8.5, and combined for sequencing. 3–3.2 pM pooled libraries containing 30% phiX control V3 (Illumina) were run on the Illumina MiSeq following the manufacturer’s instructions.Library preparation was performed with a protocol similar to methods previously reported  with mod300 basepair reads were assessed for quality using FASTX software and trimmed from adapters to isolate relevant insert content Figure f using aThe authors declare they have no competing interests.KCG and TRC conceived the study and wrote the manuscript. KCG carried out the experiments and bioinformatics analysis. All authors read and approved the final manuscript for publication.Search primers for bioinformatics assessment of 3′ ends.Click here for file3′ termini of MRP RNA from human K562 cells.Click here for fileNorthern analysis of ivt-MRP and total RNA from HEK293T cells.Click here for fileSummary of 3′ termini from publicly available datasets ,40,41.Click here for fileExamples of complex “extensions” found in publically available datasets.Click here for filePrimers used in this study.Click here for fileAppendix RNA oligonucleotide sequences.Click here for file


Xenopus laevis oocytes by injecting fluorescent RNAs, followed by confocal microscopy of the oocyte cortex. Concentrating on RNA encoding the Xenopus Nanos homologue, nanos1 (formerly Xcat2), we find that it consistently localised into aggregated germ plasm ribonucleoprotein (RNP) particles, independently of cytoskeletal integrity. This implies that a diffusion/entrapment-mediated mechanism is active, as previously reported for previtellogenic oocytes. Sometimes this was accompanied by localisation into scattered particles of the “late”, Vg1/VegT pathway; occasionally only late pathway localisation was seen. The Xpat RNA behaved in an identical fashion and for neither RNA was the localisation changed by any culture conditions tested. The identity of the labelled RNP aggregates as definitive germ plasm was confirmed by their inclusion of abundant mitochondria and co-localisation with the germ plasm protein Hermes. Further, the nanos1/Hermes RNP particles are interspersed with those containing the germ plasm protein Xpat. These aggregates may be followed into the germ plasm of unfertilized eggs, but with a notable reduction in its quantity, both in terms of injected molecules and endogenous structures. Our results conflict with previous reports that there is no RNA localisation in large oocytes, and that during mid-oogenesis even germ plasm RNAs localise exclusively by the late pathway. We find that in mid oogenesis nanos1 RNA also localises to germ plasm but also by the late pathway. Late pathway RNAs, Vg1 and VegT, also may localise into germ plasm. Our results support the view that mechanistically the two modes of localisation are extremely similar, and that in an injection experiment RNAs might utilise either pathway, the distinction in fates being very subtle and subject to variation. We discuss these results in relation to their biological significance and the results of others.We have studied the localisation of mRNAs in full-grown  Drosophila and Caenorhabditis elegans, the Xenopus oocyte has also proved a productive model for studying RNA localisation because the cells are large and readily cultured in vitro. Tagged or labelled RNA molecules may readily be injected into the cell and their localisation followed to positions that mirror endogenous molecules The oocytes of Metazoa are usually highly polarised cells in which mRNAs are localised and stored as part of a mechanism to enable rapid early diversification of embryonic cells. Since the mechanisms of polarisation are not unique to oogenesis, but later operate in somatic cells, the tractability of embryonic systems has made the oocytes of many model species paradigms for the study of general polarisation and RNA localization mechanisms Xenopus oocytes; localisation to the animal pole is relatively weak and is non-cortical, whereas the concentration of vegetal RNAs is much stronger and these RNAs are anchored in the cortex (1) The germ plasm, a field of structures consisting of tightly bound cortical aggregates of RNPs and other components. It directs the development of the future germline. Classically germ plasm has been identified with cytological stains or by in situ hybridisation using chromogenic detection methods. These approaches identify large structures that have been called germinal granules. Electron microscopy shows that these consist of various components, including RNPs, mitochondria and cytoskeleton (2) The Vg1/VegT compartment, widely scattered RNP particles that are dependent on at least two RNAs, VegT and Xlsirts, for their structure Endogenously expressed RNAs may be localised either to the animal or vegetal pole of Drosophila and mouse oocytes, even though the latter do not form germ plasm Wnt11 RNA initially localises to the Balbiani body, but subsequently it is present in late pathway granules; in contrast other RNAs only localise to the Balbiani body in late previtellogenesis  Xenopus the germ plasm is continuous with the Balbiani body, even if its composition changes. While RNA localisation (at least of injected RNAs) into the Balbiani body is mediated by diffusion/entrapment nanos mRNA to germ plasm in DrosophilaThe existence of these two compartments has been established by using in situ hybridisation to analyze the distribution of endogenously stored RNAs, and complemented by conducting RNA localisation experiments with injected tagged or fluorescently labelled RNAs, often testing the localisation ability of regions within the 3′UTR. Many endogenous germ plasm RNAs are initially localised into the single large Balbiani body, or mitochondrial cloud, of early previtellogenic oocytes. The Balbiani body is a dense structure composed of messenger RNPs, cytoskeleton, endoplasmic reticulum and mitochondria. While it has been identified in only a proportion of animals, this number is steadily growing and it may be universal. For example it was recently described in Vg1 compartment RNAs are generally distributed throughout the cytoplasm, but are excluded from the Balbiani body. At the onset of yolk accumulation (vitellogenesis), they become localised into particles dispersed throughout the vegetal cortex; this form of localisation was therefore called the “late pathway” Vg1, localise by this pathway in mid stage oocytes, using microtubule transport Initially RNAs localised to the Hermes, Fatvg, Grip2), while others localise both to the Balbiani body and throughout the oocyte, then later are found in the germ plasm (Germes). Wnt11 RNA initially localises by the early pathway, but subsequently it is present in late pathway particles. Lastly, the 3′UTRs of early RNAs have been reported to localise like late ones when injected into vitellogenic oocytes Recent data suggest that the proposed dichotomy into two pathways is an over-simplification. As already mentioned, some RNAs localise to the Balbiani body only in late pre-vitellogenesis . The Xenopus protein Nanos1 is a homologue of Nanos, a protein encoded by a widely distributed germline RNA, first identified in DrosophilaXenopus, acting as a translational repressor Xpat and Xvelo1 RNAs. Xpat is a germ plasm RNA whose protein is also present in germ plasm, in particles which we show here are distinct from those containing Hermes protein and the various germ plasm RNAs tested, including that encoding Xpat itself Xvelo1, is a vegetally localized RNA whose function is unknown, but it encodes the homologue of Bucky ball, which is essential for germ plasm formation and oocyte polarity in zebrafish We have studied the localisation of several germ plasm RNAs injected into mid-stage and full-grown In this study we find that germ plasm RNAs usually localise into the RNP particles of germ plasm islands in large oocytes. Here they are co-localised with the RNA-binding protein Hermes, a known germ plasm component nanos1 RNA injected into stage V–VI oocytes. In the experiments described here, unless otherwise stated, oocytes were maintained within intact follicles, including both follicle and surrounding thecal cells, to simulate natural conditions as closely as possible Oocytes were cultured in the same OCM medium originally used by Yisraeli and Melton We initially investigated the localisation of Cy5-labelled nanos1, but not YFP-Hermes (see below) (Pattern II). An example is shown later in Figure Four. Occasionally, we observed an exclusively late pathway mode of localisation (Pattern III). The frequencies of these patterns is summarised in nanos1 3′ UTR alone was injected, except that the proportions showing Patterns II and III were higher.Three distinct modes of localisation were evident in injected oocytes ; the firnanos1 RNA, specifically containing the ORF, and nanos1-3′Δ4, fail to show any localisation at all. These sequences were cloned in the vector pSPJC2L GFP-Xpat-ORF fusion, failed to localise (data not shown). Separately we showed that mRNA from this construct translates into large amounts of protein, so it must be stable It is important to emphasise however that not all injected RNAs were localised. We show later in Figure seven that mutants of Routinely we injected 3 ng of Cy5-labelled RNA into each stage VI oocyte. In proportion to volume we injected about the same amount as did Messitt et al. into stage III oocytes Xenopus and zebrafish germ plasm particles Hermes RNA is localised in germ plasm particles in the vegetal cortex of vitellogenic oocytes nanos1 mRNA has been detected as a component of immunoprecipitated Hermes RNP complexes from early stage oocytes Xenopus oocytes both fusion proteins were expressed at high levels and they were localised to the vegetal cortex in a field of islands typical of germ plasm. The depth of focus in the low power image shown in nanos1 mRNA  os1 mRNA . Thus Finanos1 RNA was coincident with YFP-Hermes this RNA must also have been incorporated into existing RNPs, within existing germ plasm islands.The ability of YFP-Hermes fusion proteins to behave in a manner similar to the endogenous protein was confirmed by staining injected oocytes with Hermes anti-serum and an Alexa 633 secondary antibody. The fluorescent signals corresponding to endogenous and exogenous protein were coincident (not shown), indicating that YFP-Hermes is incorporated into all of the endogenous Hermes particles. Since Cy5-nanos1 and YFP-Hermes protein.In this report, when it is stated that molecules were co-localised within specific particles, we are obviously limited by the resolution of the light microscope. However the particles were large enough to be resolved (∼1 µm), the fluorescent colour coincidence was homogeneous and when the particles were observed to move around by Brownian motion or perhaps microtubule transport, they behaved as unitary structures. There is, therefore no reason to believe that the particles were not single RNPs, like those seen in electron microscope studies, containing both Cy5-nanos1 exactly co-localise in aggregated particles, they must also both be associated with the mitochondrial aggregates and hence must be localised in germ plasm islands. Other experiments confirmed that the Cy5-nanos1 co-localised with endogenous Hermes, stained with antiserum (data not shown), and we show later that Xpat, another germ plasm protein was concentrated in these islands. We have found that nanos1 RNA localised in the same way whether labelled separately with Cy5-UTP, Cy3-UTP or Aminoallyl-UTP-ATTO390.A diagnostic feature of germ plasm in diverse species is that it contains dense mitochondrial aggregates. We therefore stained oocytes with the dye TMRE, which is sequestered by mitochondria. Xenopus serum, although other workers often add this. Serum containing vitellogenin was originally reported to be important for Vg1 RNA localisation nanos1 RNA and/or Hermes protein. There was no noticeable effect of changing any of these conditions, either individually or in combination, in any given experiment.Previously early pathway RNAs had been reported to follow the late pathway when injected into vitellogenic oocytes. In the experiments just described there were some differences in procedures adopted by us and those usually followed by others. We have routinely used OCM lacking high concentrations of vitellogenin within Xpat RNA was localised in the germ plasm of full-grown oocytes, just like its protein Xpat RNA or its 3′UTR alone, localised to the vegetal cortex of mid-stage oocytes Xpat RNA was localised to the germ plasm of stage VI oocytes, just like nanos1 RNA . Thus intact microtubules and microfilaments are not necessary for localisation to vegetal RNP particles, at least within the experimental time scale required for injected nanos1 RNA localisation . The existence of microtubules resistant to disruption by agents like colcemid has been suggested, however the tubulin staining in nanos1 RNA and YFP-Hermes protein to germ plasm, since colcemid and cytochalasin D incidentally disrupted these. It will be noticed that colcemid caused the germ plasm islands to run together into a reticular structure. In these oocytes there were a few scattered Cy5-nanos1 particles lacking Hermes, as will be seen below in some stage IV oocytes (Pattern II). It is likely that these were late pathway particles and their frequency was greatly increased by cytochalasin D treatment. While the cytochalasin effect was not observed in experiments in which no late pathway granules were labelled in controls untreated with cytochalasin D, the possibility that actin filaments have a role in discriminating the two pathways might be worth investigating further.In nanos1 failed to show recovery, indicating that this RNA was more stably incorporated into germ plasm. Given that this RNA seems to localise by diffusion, either the RNA exchanges much more slowly than Hermes protein throughout the cytoplasm, or the germ plasm is the final target of this RNA and therefore there is little exchange only within cortical particles. Perhaps a mixture of the two possibilities is most likely.If cytoskeletal transport is not essential for the localisation of injected RNAs and newly expressed Hermes protein, it suggests that diffusion and exchange into pre-existing particles occurs. We have tested this by FRAP analysis of YFP-Hermes. Thus nanos1 RNA. We were able to confirm that this is indeed the case, as exemplified by the low power image shown in nanos1 RNA and RNA encoding YFP-Hermes into the equator a large area of the vegetal pole was imaged. The YFP-Hermes is evenly distributed over the whole vegetal pole, whereas the Cy5-nanos1 RNA is concentrated over only part. This result is consistent with the idea that nanos1 RNA and Hermes protein spread by diffusing separately. The slower movement of Cy5-nanos1 could also relate to its more stable incorporation into germ plasm. It is also possible that most of the diffusion of Cy5-RNA is through internal cytoplasm and the RNPs might be less stable there, but we cannot image this in FRAP experiments , whereas others use early to mid-vitellogenic oocytes (stage III–IV). This is probably a consequence of the original report that nanos1 and Xpat mRNAs, we saw patterns of localisation similar to those at stage VI  directed localisation to the Balbiani body, but not to the particles. Thus they identified an RNP targeting element in the distal 3′UTR, but only by the effect of its deficiency. We have directly tested an equivalent fragment, nanos1-3′Δ6, for its localisation ability at stage VI and found that it produced localisation into germ plasm particles at vitellogenic stages, although the overall frequency and strength of localisation was very poor (nanos1-3′Δ3 & Δ4 ), directed efficient localisation into particles. Thus the overall function of fragments appears to be similar in our experiments and in those conducted by others at earlier stages, although the details of particle localisation are different.Kloc et al. ery poor , Table 2nanos1-3′Δ6) contains only a single VM1 motif. Thus it is possible that quite different sequences are involved in localisation of this region of the RNA to germ plasm.Several studies have shown that vegetal localisation in mid oogenesis depends on multiple E2 and VM1 elements nanos1 3′UTR was co-injected with Cy3-nanos1-3′Δ6 RNA. Localisation of the Cy3-nanos1-3′Δ6 mutant transcript (containing the last 163 nucleotides of the 3′UTR) was normally very weak, but was enhanced by co-injected nanos1 3′UTR  was localised to germ plasm, but in some instances there were also isolated particles . This was true whether vitellogenin was present or absent and it was also true when microtubules were dissociated with colcemid . Thus in our experiments, the late pathway RNAs localise wholly or substantially into germ plasm particles in large oocytes.colcemid ;. The Cyarticles  and Cy5-nanos1 and Xpat RNAs to germ plasm (Pattern I). nanos1, VegT and Vg1 RNAs localised substantially into germ plasm in these stage IV oocytes. The latter two RNAs co-localised with the germ plasm RNA nanos1  also localises into germ plasm In the experiments described here we find that within the cortex labelled germ plasm RNAs injected into large Xenopus Balbiani body by diffusion and entrapment nanos mRNA to localise into the germ granules of DrosophilaXenopus oocytes has not gone unchallenged. Heinrich et al. Drosophila oocyte, or in C. elegans, where it is actin dependent By elimination, the mechanism by which the RNA and protein is localised appears to be diffusion and entrapment, since it is not dependent on the integrity of microtubules, microfilaments or cytokeratins, within the time scale of the experiments here. The mechanism is thus identical to that previously identified in previtellogenic oocytes, where it was concluded that the RNAs are localised into RNP particles of the Our data make it likely that Hermes and the injected RNAs do not co-migrate during localisation because, when labelled RNA and mRNA encoding Hermes are co-injected into the equator, the translated protein diffuses faster across the oocyte than the labelled RNA. This contrasts with Xpat, which forms fields of ectopic particles in the absence of vitellogenin in the external medium and requires microtubules for transport to the vegetal pole Vg1 mRNA localises into late pathway granules in larger oocytes. Since most localisation studies have focused on early and mid oogenesis, we also looked at the localisation of nanos1 and Xpat RNAs in stage IV oocytes. In most of our oocyte batches the RNAs were localised into germ plasm granules, as seen at stage VI. However, oocytes from several other females of a different cohort in our colony displayed isolated Cy5-labelled RNPs that lacked Hermes protein, presumably late pathway structures, in addition to a typical germ plasm RNA localisation pattern. Because exclusive late pathway localisation was expected from the literature, we looked for differences in our methods from those used by others, who have reported localisation of early RNAs by the late pathway. However, the behaviour of labelled RNA and protein was not affected by changes in the experimental conditions employed .Our observation of RNA localisation into cortical particles at late stages of oogenesis is at odds with the original report of Yisraeli and Melton nanos1-3′UTR construct was inefficient and highly variable in both stage VI and stage IV oocytes. Again this appeared to be an oocyte batch-sensitive issue and the UTR-only data are skewed by the fact that in the initial part of this work the UTR was localising to late pathway particles, while full-length nanos1 was consistently and robustly localising to germ plasm. The more ambiguous localisation of the 3′UTR-only construct emphasises that the final fate of the RNA is finely balanced in injection experiments. It seems that the coding region (ORF) has a substantial influence on localisation to germ plasm, but the ORF itself is not localised within the oocyte.The localisation of the Although our data support the contention, discussed in the Introduction, that the distinction between the early and late pathways is less absolute than originally thought, we clearly find that germ plasm RNAs can localise into germ plasm robustly at late stages . The seqHowever it may be that oocytes from frogs in other labs differ from ours, and we ourselves have found differences between animals, particularly with regard to the nanos1-3′UTR construct. One possibility that may explain this variation is that some oocytes contain spare capacity to form ectopic RNP particles. These would require cytoskeletal transport, just like Xpat protein particles Xvelo1 is a Xenopus RNA that is excluded from the Balbiani body at early stages, and localised to the vegetal cortex at late stages, both classic features of late pathway RNAs Xenopus Xvelo1 protein is a constituent of the Balbiani body and germ plasm at all stages (in preparation). Xvelo1 mRNA is unusual because it does not have conventional late pathway sequences, although it and the Vg1 localisation element bind similar proteins Xvelo1 RNA localises into germ plasm or jointly into germ plasm and late pathway granules. The differences in the localisation patterns observed may be because the in situ methods originally used to identify endogenous Xvelo1 transcripts When we examined the behaviour of RNAs naturally localised by the late pathway the results were also surprising. VegT and Vg1 were more surprising, since we found them to be substantially localised into germ plasm structures. However, it must be noted that this was in oocytes that exclusively localised nanos1 RNA into germ plasm. Unfortunately, we cannot predict how oocytes behave, so those with the mixed localisation pattern were not tested. Overall our experiments fit with the view that the localisation pathways are only subtly different.Our results with the injected definitive late pathway RNAs nanos RNA in DrosophilaWhat, one might ask, is the biological significance of a process which localises RNAs into germ plasm, or sometimes late pathway RNPs, by a cytoskeleton-independent mechanism, as previously seen in earlier Balbiani body stages? Drosophila it has been shown that oskar mRNA derived from intronless genes localises to germ plasm only if accompanied by oskar RNA that has been through a splicing process Xenopus RNA localisation is unclear. However in Drosophila nanos mRNA is localised by diffusion/entrapment nanos gene was also involved was not excluded. Nevertheless, in Xenopus nucleus-independent processes are likely to be important because many late pathway RNAs are dispersed throughout the oocyte, prior to later localisation. This property is not restricted to late RNAs, since this is also a feature of some RNAs which ultimately localise to germ plasm.One might debate how far one is looking at an artificial situation by injecting large numbers of molecules at one time, when they are normally made over a prolonged period, and in the case of RNAs they are transcribed and processed within the nucleus, then transported to the cytoplasm. While it is quite possible that RNAs exported from the nucleus wholly, or in part, localise by diffusion, in C. elegans P-granules are very dynamic and appear to dissolve and reform continuously. It seems that their localisation depends more on localised assembly/disassembly than on transport, with assembly being favoured in the germline lineage Xenopus.Since components apparently diffuse in and out of germ plasm particles, as we show for Hermes, one could argue that at the very least diffusion plays a role in particle maintenance. In nanos1 RNA does not do so, at least on the short timescale of a FRAP experiment. This model would produce net targeted diffusion of RNA, but proteins would be maintained in internal particles, as well as at the cortex. Two predictions of this model are that internal particles bind RNAs less stably and that they would have a different composition from those in cortical germ plasm islands. The latter is more easily testable. It should be mentioned that, even in Drosophila, the transport of Oscar-containing particles via micro-tubules has only a very small bias towards the posterior pole of otherwise random, kinesin-based movement, yet the result is tight localisation nanos1 are also labelled by YFP-Hermes; not shown). It is therefore possible that the distinction between the germ plasm and late pathway compartments occurs only at the cortex.What is the significance of the internal particles? One can envisage a facilitated diffusion process from nucleus to vegetal cortex in which proteins and RNAs move by a sort of “pass the parcel” mechanism. If the RNA is incorporated more stably into structures at the vegetal cortex than to those found internally, there would be a net diffusion towards the vegetal pole – in the analogy the “parcels” would end up with selfish individuals that do not hand them on. This fits our observation that Hermes protein diffuses in and out of the cortical particles, but C. elegans mislocalised P granules and their components are destroyed Xenopus to refine the distribution of germ plasm, and like its counterpart in C. elegans, germ plasm in Xenopus is much more labile than previously supposed One striking incidental result of our study is the demonstration that the amount of germ plasm decreases radically in the progress from oocyte to egg via maturation. The significance of this is unclear. In Capped mRNA was synthesised in vitro using linearised plasmid templates and the Message Machine in vitro transcription kit (Ambion). To prepare fluorescently labelled transcripts for microinjection Cy5 or Cy3-UTP  or Aminoallyl-UTP - ATTO-390 (JENA Bioscience) was included in the transcription reaction.nanos1 was synthesised, using T7 RNA polymerase from the plasmid pSPORT1 linearised with BamHI Vg1 RNA was transcribed using SP6 RNA polymerase from the plasmid pSP64TVg1 linearised with XbaI Xpat RNA was transcribed with T3 RNA polymerase from the plasmid pBSK linearised with XhoI VegT transcripts were synthesised using SP6 RNA polymerase from pBratCS2 linearised with NotI.Full length sense mRNA for Xvelo1 mRNA was transcribed using T3 RNA polymerase from the plasmid Xvelo1-MC118 (in pBK-CMV) linearised with XhoI (provided by T. Pieler).Sense nanos1 (pSPORT1) plasmid was mutated to TTG to prepare the mutant nanos1 (mut-nanos1) construct using the Stratagene site directed mutagenesis kit and the primers 5′-GAACAATTCCAACTTGGATGGCGGTC-3′ and 5′-GACCGCCATCCAAGTTGGAATTGTTC-3′. RNA was transcribed using T7 RNA polymerase following linearisation with BamHI.The ATG codon in the nanos1-ORF construct was prepared by amplifying the coding region in nanos1 (pSPORT1), with the following primers 5′-AAAAGATCTATGGATGGCGGTCTCTGCTTTGAC-3′and 5′-AGAATTCTCAGTGTCTCAGCTTTGGGTTATTAC-3′. The product was digested with BglII and EcoRI and ligated into the corresponding sites in pSPJC2L. RNA was transcribed using SP6 RNA polymerase following linearisation with XhoI. YFP-Hermes in pCS2+ was transcribed with SP6 RNA polymerase following linearisation with NotI.The Xenopus nanos1 5′ deletion mutants were constructed by PCR using the nanos1 (pSPORT1) plasmid as a template. For nanos1-5′Δ1 the primers 5′-AAAGAATTCACTCATGGAGCGACTACTTG-3′ and 5′-CACGCGTACGTAAGCTTGGATCCTCTAG-3′ were used to amplify nucleotides 38–793 of the nanos1 cDNA; nanos1-5′Δ2 primers 5′-AAAGAATTCCGTTACCCAGTAATGAGTC-3′ and 5′-CACGCGTACGTAAGCTTGGATCCTCTAG-3′ amplified nucleotides 148–793; nanos1-5′Δ3 primers 5′-AAAGAATTCCAGTACTAAGAGGCTACAC-3′ and 5′-CACGCGTACGTAAGCTTGGATCCTCTAG-3′ amplified nucleotides 267–793 and for nanos1-5′Δ4, primers 5′-AAAGAATTCCGTCGGCTCCTCAGAGAT-3′ and 5′-CACGCGTACGTAAGCTTGGATCCTCTAG-3′ were used to amplify nucleotides 344–793.nanos1-5′Δ3 where T7 RNA polymerase was used.Amplified products were then ligated into pGEM-T Easy. All constructs were linearised with BamHI, and RNA transcribed with SP6 RNA polymerase, except for nanos1-3′Δ1, nanos1-3′Δ3, nanos1-3′Δ5 and nanos1-3′Δ6 were constructed by PCR using the nanos1 pSPORT1 plasmid as a template. For nanos1-3′Δ1 the primers 5′-AAAAGATCTCAGAACAATTCCAACATGGATG-3′-3′ and 5′-AAAGAATTCTCTGGAGAGCTGGAATGTCCGG-3′ were used to amplify nucleotides 1–682 of the nanos1 cDNA; nanos1-3′Δ3 primers 5′-AAAAGATCTCTGAACTGGAAGGAACCCTGGG-3′ and 5′-AAAGAATTCTCTGGAGAGCTGGAATGTCCGG-3′ amplified nucleotides 399–682; nanos1-3′Δ5 primers 5′-AAAAGATCTGATGTCTCACTCGGCACCCACG-3′ and 5′-AAAGAATTCTGACGTCGCATGCACGCGTACG-3′ amplified nucleotides 574–793; nanos1-3′Δ6 primers 5′-AAAAGATCTGGGGCCAAGCGGTGCCCATCCC-3′ and 5′-AAAGAATTCTGACGTCGCATGCACGCGTACG-3′ amplified nucleotides 631–793.Deletion mutants nanos1-3′Δ2 was prepared by digesting the nanos1 pSPORT1 plasmid with SmaI to release the first 630 bp of the nanos1 cDNA. The SmaI fragment was ligated into EcoRI digested/blunted pSPJC2L. nanos1-3′Δ4 was prepared by digesting the nanos1-3′UTR construct in pCS2+ with BamHI and SmaI, releasing an insert corresponding to nucleotides 402–630 of nanos1. The insert was blunted and ligated into EcoRI-digested/blunted pSPJC2L. For sense RNA all constructs were linearized with XhoI and RNA transcribed with SP6 RNA polymerase.Amplified products were digested with BglII and EcoRI and ligated into the corresponding sites in pSPJC2L. Xenopus laevis (Daudin) were obtained by manual dissection from ovaries if they were to be cultured within intact follicles, or by collagenase treatment for isolation without follicle cells Oocytes of To disrupt microtubules, oocytes were cultured in OCM containing 5 µg/ml colcemid (Sigma). Actin polymerisation was disrupted by incubating oocytes in 1 µg/ml cytochalasin D (Sigma). Incubation of injected/uninjected oocytes in inhibitors was for 24–48 hours at 21°C.Oocytes were matured in vitro by addition of 125 µg/ml progesterone. Maturation was scored by the appearance of the germinal vesicle (white spot formation) at the animal pole of the oocyte. Eggs were obtained and handled as described previously Oocytes were stained and analyzed with antibodies as described previously


Dicer, Toll, and autophagy pathways have distinct roles in this Drosophila dsRNA pathology. Dicer dependence is accompanied by cleavage of rCAG.rCUG100 repeat dsRNA down to r(CAG)7 21-mers. Among the “molecular hallmarks” of this pathway that have been identified in Drosophila, some  correlate with observations in affected people  or in related animal models . The Toll pathway is activated in the presence of repeat-containing dsRNA and toxicity is also dependent on this pathway. How might the endogenously expressed dsRNA mediate Toll-dependent toxicity in neuronal cells? Endogenous RNAs are normally shielded from Toll pathway activation as part of the mechanism to distinguish “self” from “non-self” RNAs. This typically involves post-transcriptional modification of the RNA. Therefore, it is likely that rCAG.rCUG100 repeat dsRNA has a characteristic property that interferes with or evades this normal mechanism of shielding. We predict that repeat expansion leads to an alteration in RNA structure and/or form that perturbs RNA modification, causing the unshielded repeat RNA (in the form of its Dicer-cleaved products) to be recognized by Toll-like receptors (TLRs), with consequent activation of the Toll pathway leading to loss of cell function and then ultimately cell death. We hypothesize that the proximal cause of expanded repeat neurodegenerative diseases is the TLR recognition (and resultant innate inflammatory response) of repeat RNA as “non-self” due to their paucity of “self” modification.Previously, we hypothesized that an RNA-based pathogenic pathway has a causal role in the dominantly inherited unstable expanded repeat neurodegenerative diseases. In support of this hypothesis we, and others, have characterized  PubMed search “trinucleotide repeat disorders” gives >3,700 results). Much of this research is aimed at identifying the mechanism of pathogenesis underlying diseases caused by this form of mutation. Individual diseases can follow either dominant or recessive mode of inheritance indicating distinct pathogenic pathways. Repeat sequences that are expanded in copy number are the basis for ~20 dominantly inherited neurodegenerative diseases, including Huntington’s disease (HD). Despite some of the responsible genes being identified as long as 20 years ago, the identity and nature of the disease-causing pathogenic pathway remains a gap in knowledge for these diseases, i.e., no definitive molecular pathway from the mutation to the clinical symptoms has yet been identified. For at-risk individuals in families affected with dominantly inherited late-onset neurodegenerative diseases due to expanded repeats, the majority opt not to have the definitive pre-symptomatic diagnostic test. Their preference is to live with the uncertainty of not knowing, than the certainty of getting the disease, as no treatments are yet available. Therefore, determining the pathogenic pathway and identifying therapeutic targets for intervention is an urgent priority for reducing the impact of these devastating diseases. This understanding is essential for rational approaches to delay onset, slow progression, or ultimately effect cure.Since the first discovery of trinucleotide repeat expansion as the basis for many important human genetic diseases , there hThere are common properties exhibited by the various repeat expansions that give rise to human disease. The vast majority of these diseases originate from an existing repeat sequence that exhibits copy number variation in the human population. In each case, the disease alleles arise when copy number increases beyond a critical threshold. The repeat composition varies, but most are trinucleotide repeats. In some diseases, the repeat expands to the point where gene expression at the expanded repeat locus is either substantially reduced or lost altogether, resulting in loss-of-function of the repeat-harboring gene. Typically such diseases are inherited in a recessive manner. Many repeat loci, however, give rise to dominantly inherited diseases in a manner that is not gene-dose dependent  typically manifest as neurodegenerative and/or muscular diseases, some with a high degree of clinical overlap, despite affecting distinct proteins and unrelated loci. Where expanded repeats are translated, they generally code for polyglutamine; however, the proteins in which they are located are all unrelated in the remainder of their amino-acid sequence. Therefore, much attention has been focused on expanded polyglutamine as the common basis of pathology . This suggests that there may be a common pathogenic agent or agents in the translated and untranslated repeat diseases.The unstable expanded repeat diseases  is more controversial . Yet evesis ALS; . Further of SCA3 .SCA7 both appear to involve two cell types  contribute to progression of expanded repeat neurodegenerative diseases. In support of this possibility, ALS and Figure 2.What precedents and potential mechanisms are there for RNA to initiate pathogenesis in human diseases? See DM1 and DM2 repeat expansions that both give rise to myotonic dystrophy (DMPK and ZNF9). In muscle cells, RNAs from expanded alleles of either repeat are able to bind and sequester alternative splicing factors (muscleblind and CUG-BP) and in so doing, perturb the splicing pathways of proteins for which alternative splicing is a necessary step for their complete range of functions . Upon detailed characterization, this degenerative phenotype line was found to have the repeat transgene inserted into an endogenous gene (cheerio) in the opposite orientation to normal transcription. Transcripts containing expanded repeats would, therefore, originate from both strands via bi-directional transcription. This finding coincided with numerous reports in the literature that expanded repeat disease loci are typically transcribed from both DNA strands ] to produce repeat-containing dsRNA 7 also implicated specific Dicer processing of the rCAG.rCUG~100 dsRNA as a pathogenic pathway in this model  models employ copy numbers well in excess of those that cause pathology  in some of these diseases. This is thought to be due to an inverse relationship between repeat copy number and age-at-onset, the basis of which could be somatic repeat instability over time 7 cleavage product seen in the Drosophila models, providing evidence in support of the activity of this pathway in HD pathogenesis.equences . In one equences , a singlands see . Therefong dsRNA . Flies eis model . Similarla model . There aDrosophila, microarray analyses of Drosophila expressing rCAG.rCUG~100 dsRNA have been undertaken  function in normal biology to protect an organism from infection by viruses and bacteria. They recognize foreign pathogen molecules including DNA and RNA through specific receptors  and can distinguish these nucleic acids (as “non-self”) from the endogenous nucleic acids (“self”). Therefore, while the rCAG.rCUG~100 dsRNA is being expressed endogenously in this Drosophila model, it is being recognized by the Toll signaling pathway as foreign or “non-self” – a recognition that then activates innate inflammatory regulatory pathways, ultimately leading to cell death.The requirement for While it can be difficult to ascribe specific functions to RNA in pathogenic pathways, there are some noteworthy instances of disease-causing mutations in proteins that functionally interact with RNAs. By implication, the RNAs that these proteins normally act upon are, therefore, likely contributors to and/or mediators of the relevant pathogenic process.Recent discoveries regarding the importance of RNA–protein recognition in disease pathogenesis have led to a renewed interest in the role that these interactions play in biological processes. While they have long been recognized as key regulators of gene expression, only a small fraction have been functionally characterized. A recent compendium of RNA-binding motifs  highlighFRAXA rare, folate-sensitive chromosomal fragile site . In its milder forms, these neurological symptoms are diminished or even absent, but peripheral symptoms outside the nervous system are common to the phenotypic spectrum and include thrombocytopenia, hepatosplenomegaly, and elevated hepatic transaminases along with intermittent fever. Chilblains are also a typical feature. Together these symptoms demonstrate phenotypic overlap both with systemic lupus erythematosus and with the sequelae of congenital infection (Drosophila model of expanded repeat neurodegenerative diseases.Aicardi–Goutières syndrome (AGS) is a genetically heterogeneous disorder that is due  to the mutation of various nucleic acid-metabolizing enzymes, including various subunits of ribonuclease H2 or the RNA-editing enzyme ADAR1 .Repeat RNA sequences represent a pivotal point of potential weakness in processes that utilize RNA–protein recognition, as the repeat RNA sequence will harbor either a paucity or excess of sequence-binding motifs. Expansion of repeat RNA sequences, therefore, clearly has the potential to give rise to too much or too little of an interaction that is a rate-limiting factor in a crucial biological process. RNA modification is one process that is sequence motif-dependent and known to be key to the distinction between “self” and “non-self” by components of the innate immune pathways. Indeed, it has been shown that exogenous “non-self” RNAs require  in vivo . The expDrosophila. What evidence is there that this pathway of TLR recognition of expanded repeat RNA and subsequent activation of the innate inflammatory cascade is active in the human dominantly inherited neurodegenerative diseases due to expansion of repeat sequences?Double strand expanded repeat RNA pathology has been modeled in r(CAG)7 21mers from the much greater copy number double strand repeat RNA by Dicer. This r(CAG)7 21mer has been identified in the brain RNA of individuals affected with HD 7 21mers in HD brain is therefore an important “molecular hallmark” of this pathway and a key indicator of its activity in the human disease. Another, albeit less direct, indicator of this pathway is seen in the increased activity of components of the innate inflammatory response mechanism in human diseases associated with expanded repeats. Elevated TNF is seen in the Drosophila model as one read-out of innate immune activation (One of the key steps in dsRNA pathology is the generation of  with HD . The actla model  and, thetivation  and bothtivation . Anothertivation . Varioustivation  – a disetivation .Toll receptor pathway of expanded repeat RNA as “non-self” and consequent activation of the innate immune inflammatory cascade provides a mechanism and a common pathogenic pathway for the neurodegenerative diseases due to expanded repeats. This new understanding, once proven in the relevant human diseases, will provide new targets for intervention and ultimately, we hope, therapeutic targets for drugs to delay onset and/or alleviate disease progression.A growing body of literature indicates a consistent association between innate immunity, neuroinflammation and neurodegeneration . Where tRobert I. Richards drafted the initial version of the manuscript, including the hypothesis, then edited in the additions and changes made by the other co-authors. Saumya E. Samaraweera and Clare L. van Eyk provided original unpublished data on which the manuscript and its hypothesis is based, as well as contributions to the development of the hypothesis, the text and figures. Louise V. O’Keefe contributed to the development of the hypothesis and additional text and revision of the manuscript. Catherine M. Suter contributed to information on RNA-binding proteins, the development of the hypothesis and content of the text and figures.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.