C. elegans zygote, germline RNA granules, called P granules, assemble preferentially in the posterior cytoplasm. We present evidence that P granule asymmetry depends on RNA-induced phase separation of the granule scaffold MEG-3. MEG-3 is an intrinsically disordered protein that binds and phase separates with RNA in vitro. In vivo, MEG-3 forms a posterior-rich concentration gradient that is anti-correlated with a gradient in the RNA-binding protein MEX-5. MEX-5 is necessary and sufficient to suppress MEG-3 granule formation in vivo, and suppresses RNA-induced MEG-3 phase separation in vitro. Our findings suggest that MEX-5 interferes with MEG-3’s access to RNA, thus locally suppressing MEG-3 phase separation to drive P granule asymmetry. Regulated access to RNA, combined with RNA-induced phase separation of key scaffolding proteins, may be a general mechanism for controlling the formation of RNA granules in space and time.RNA granules are non-membrane bound cellular compartments that contain RNA and RNA binding proteins. The molecular mechanisms that regulate the spatial distribution of RNA granules in cells are poorly understood. During polarization of the DOI:http://dx.doi.org/10.7554/eLife.21337.001 Animal cells contain many smaller compartments known as organelles that perform particular roles. For example, a compartment called the nucleus stores most of the cell’s genetic information. The nucleus and many other organelles form inside layers of membrane that physically separate them from the rest of the cell. However, some organelles, such as the germ granule, do not have a membrane. It is thought that these organelles may form in the same way that oil droplets tend to come together when mixed with water. However, oil droplets form in water spontaneously and do not fall apart, so it is not clear how cells could control the assembly and destruction of such organelles.C. elegans are destroyed and reassembled in cycles. Smith et al. investigated how the worm cells control these cycles. The experiments show that a protein called MEG-3 is required to allow the components of granules to transition from behaving like individual molecules dissolved in water (similar to being dissolved in cell fluid) to assembling into droplets. When MEG-3 is mixed with molecules of ribonucleic acid (RNA) it can bind very tightly to the RNA and then separate out from the rest of the fluid to form distinct droplets.The germ granules inside the cells of a worm called Smith et al. also show that another protein called MEX-5 can destroy these droplets by attaching itself to RNA in place of MEG-3, which causes MEG-3 to dissolve back into the rest of the fluid. The physical properties of the MEG-3 droplets are still not known and so the next step following on from this work will be to find out whether germ granules behave like liquids, gels or hard solids.DOI:http://dx.doi.org/10.7554/eLife.21337.002 RNA granules are concentrated assemblies of RNA and RNA-binding proteins that form without a limiting membrane in the cytoplasm or nucleoplasm of cells . RNA graIn cells, RNA granule assembly is regulated in space and time. For example, stress granules assemble within seconds of exposure to toxic stimulants that require the temporary removal of mRNAs from the translational pool . In eggsC. elegans are an excellent model to study the mechanisms that regulate granule assembly (1 and excluded from the anterior somatic blastomere AB.The germ (P) granules of assembly . For mosassembly . As the assembly . By the mex-5 mex-6 double mutants, P granule still undergo cycles of assembly and disassembly but these are no longer patterned along the anterior-posterior axis, and small granules remain throughout the cytoplasm  and the intrinsically-disordered protein MEG-3  . PGL-1 ar stages . Phosphor stages . In thisTo determine the genetic hierarchy that controls granule asymmetry, we first compared the distributions of MEX-5, MEG-3, PGL-1 and GLH-1 during the earliest stages of zygote polarization. We monitored MEX-5, MEG-3 and GLH-1 localization using tagged alleles generated by genome editing  foci . During mex-5 and mex-6 (mex-5/6(RNAi) zygotes), the MEG-3 gradient did not form and MEG-3 and PGL-1 granules remained uniformly distributed throughout the cytoplasm ; pgl-3(bn104) zygotes, the MEG-3 and MEG-4 gradients were unaffected and MEG-3/MEG-4 granules formed in the posterior as in wild-type, except that the granules appeared smaller especially in zygotes  or genetic mutants . In zygytoplasm . In meg-properly , Figure properly . In pgl- zygotes , Figure  zygotes . These a zygotes .mex-5 transgenes, mex-5 locus using CRISPR/Cas9-mediated genome editing  (mex-5(S404A) zygotes), MEG-3 did not form a gradient or granules. Instead, MEG-3 remained uniformly distributed in the cytoplasm throughout the 1-cell stage , MEX-5(ZF-) did not form a gradient and was uniformly distributed in zygotes (mex-5(S404A) zygotes, however, mex-5(ZF-) zygotes assembled posterior MEG-3 granules as in wild-type (data not shown). mex-5 is partially redundant with its paralog, mex-6 (mex-6 by RNAi in mex-5(ZF-) zygotes caused MEG-3 granules to assemble throughout the cytoplasm, as in mex-5/6(RNAi) zygotes (mex-5(ZF-) is a loss-of-function allele. The loss of mex-5 activity was not due to reduced expression as MEX-5(ZF-) was expressed at the same level as wild-type MEX-5  hermaphrodites. We found that mex-5 zygotes assembled posterior MEG-3::GFP granules, as is observed in mex-5(ZF-) and wild-type zygotes. Depletion of mex-6 by RNAi in this background yielded zygotes with uniform MEG-3::GFP granules, as expected for a mex-5/6 loss-of-function phenotype  ability to suppress MEG-3::GFP granule assembly throughout the cytoplasm, we introduced the S404A mutation by genome editing into henotype . We concIDR) and MEG-3(aa545-862) (hereafter referred to MEG-3Cterm)  of MEG-3 for poly-U30 to be ~32 nM, similar to that of MEX-5  . MEG-3Cterm did not bind RNA significantly by EMSA or in the FP assay   . We test-3Cterm) . EMSA exphoresis . Using F~ 29 nM) . MEG-3ID3000 nM) . We concTo begin to explore the specificity of MEG-3 RNA binding, we challenged MEG-3/poly-U30 complexes with increasing concentrations of competitor RNAs and examined their behavior by EMSA. We found that poly-U, and to a lesser extent poly-A, were effective competitors, but not poly-C or poly-G . These oIDR in solution in the presence of high salt . Therefore to examine the phase separation properties of MEG-3, we used concentrated (100–320 μM) preparations maintained in 6M urea, diluted these into aqueous buffer (150 mM NaCl) and used light microscopy to immediately observe the mixture (Method). We found that MEG-3 and MEG-3IDR readily formed phase-separated condensates within 10 min at room temperature. Control proteins (BSA and MBP) subjected to the same treatment did not phase separate (data not shown). MEG-3 condensates were observed across a range of protein concentrations (0.5 μM to 5 μM) and became larger and more abundant with increasing protein concentration , MEG-3 formed more condensates than MEG-3IDR, and in the high concentration range (>5 μM) MEG-3IDR tended to form larger condensates (Concentrated (<1 μM) solutions of RNA-binding proteins containing IDRs spontaneously phase separate when switched from high to low salt (150 mM NaCl) . We werentration . MEG-3 adensates .IDR  as a His fusion . We pre-incubated recombinant MEX-5 with poly-U in buffer for 30 min before adding MEG-3. We found that MEX-5 strongly inhibited MEG-3 phase separation induced by RNA. Addition of excess RNA (3-fold increase) restored robust phase separation in the presence of MEX-5 . AdditioIDR is sufficient to promote RNA binding and phase separation, but does so less efficiently than full length MEG-3 at low protein concentrations. To examine the behavior of MEG-3IDRin vivo, we deleted the C-terminus of MEG-3 by CRISPR/Cas9 genome editing to generate a meg-3 allele that only expresses MEG-3IDR  zygotes, MEG-3IDR did not form a gradient and did not form granules  zygotes. The MEG-3 IDR granules and cytoplasmic gradient extended further towards the anterior compared to wild-type . The MEG-3IDR is essential for binding, but on its own binds with lower affinity . One possibility is that the MEG-3 IDR extends beyond the region predicted by IUPRED  with nanomolar affinity in vitro . These observations suggest that the IDR confers on MEG-3 an intrinsic tendency for phase separation that is tunable by RNA. RNA-induced phase separation has also been observed for Whi3, a fungal RNA-binding protein, and for hnRNPA1, a stress granule protein  along the anterior-posterior axis of the zygote . Our obsHow MEX-5 regulates MEG-3’s access to RNA in vivo remains to be determined. As suggested by the in vitro observations, MEX-5 could compete directly with MEG-3 for RNA binding. Alternatively, MEX-5 could function indirectly by recruiting factors to MEG-3/RNA complexes that reduce MEG-3’s affinity for RNA. The MEX-5 gradient arises as a consequence of phosphorylation by the posteriorly-enriched kinase PAR-1, which decreases the size of MEX-5 complexes and increases MEX-5’s diffusion rate . One posWhile MEX-5 was sufficient to inhibit the RNA-induced phase separation of MEG-3 in vitro, it was not able to reverse it. Addition of MEX-5 to preformed MEG-3 condensates was not sufficient to dissolve them (data not shown). In contrast, in vivo, MEG-3 granules dissolve in the anterior cytoplasm as MEX-5 concentration increases . We showC. elegans 3’ UTRs .IDR (aa1-544) fused to an N-terminal 6XHis tag in pET28a were expressed in Rosetta (DE3) cells at 16°C in LB + ampicillin (100 µg/mL) to an OD600 of ~0.4 and induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside at 16° C for 16 hr. Cells were resuspended in Buffer A  glycerol, 1% Triton-X100, 6M Urea, 6 mM βME, pH7.4) with added protease inhibitors and TCEP, lysed by sonication, spun at 13,000 rpm for 25 min, and incubated overnight at 4C. Lysate was passed over a His Prep FF 16/10 column . Bound protein was washed with Buffer B  glycerol, 6M urea, 6 mM βME) and eluted in Buffer C  Glycerol, 6M Urea, 6 mM βME). After each purification, aliquots of the peak elution fraction were run on 4–12% Bis Tris gels, and stained with Simply Blue Safe Stain . Proteins were concentrated to a final concentration of 100–320 µM in elution Buffer C. For use in RNA binding assays, proteins were dialyzed into storage buffer B  glycerol) and stored at −80° C.MEG-3 (aa1-862), MEG-3Cterm purified under native conditions was soluble in aqueous buffer even at high concentrations (>1 uM) and was used for RNA-binding assays.MEG-3 (545–862) was purified as above and also natively using the same protocol without urea. MEG-3MEG-3 that was utilized in RNA binding experiments was step dialyzed out of urea, into 4.5M Urea, 3M Urea, 1.5M Urea, and 0M Urea in MEG-3 Storage Buffer . The protein was aliquoted and flash frozen in MEG-3 Storage Buffer and stored at −80C.MEX-5(aa236-468) was purified as an N-terminal 6xHis:MBP fusion expressed in Rosetta (DE3) competent cells. Cells were grown at 37° C in LB + ampicillin (100 µg/mL) to an OD600 of ~0.4, before induction with 0.2 mM isopropyl β-D-1-thiogalactopyranoside and 100 μM zinc acetate at 16° C for 16 hr. Cells were resuspended in lysis buffer  glycerol, 1 mM TCEP, 100 μM zinc acetate, Roche complete EDTA-free protease inhibitor tablet), lysed by sonication and pelleted at 10,000 rcf for 15 min. The supernatant was passed over a HisTrap HP column  and washed with wash buffer  glycerol, 100 μM zinc acetate,1 mM TCEP). Column was eluted using elution buffer  glycerol, 100 μM zinc acetate). Elution fractions were pooled and run over a HiTrap Heparin HP column . Column was then washed in wash buffer B  and eluted using a gradient of wash buffer B and elution buffer B . Elutions were pooled and dialyzed into storage buffer as in Adult worms were placed into M9 salt solution on epoxy autoclavable slides  and squashed with a coverslip to extrude embryos. Slides were frozen by laying on pre-chilled aluminum blocks for 20 min (chilled using dry ice). Embryos were permeabilized by freeze-cracking  followed by incubation in methanol at −20°C for >15 min, and in acetone (pre-chilled at −20°C) at room temperature for 10 min. Slides were blocked in PBS-Tween (0.1%) BSA (0.5%) for 15 min x 2, and incubated with 50 ul primary antibody overnight at 4°C in a humid chamber. Antibody dilutions (in PBST/BSA): K76 , Rat α OLLAS-L2 , mouse α FLAG . Secondary antibodies were applied for 2 hr at room temperature.Fluorescence microscopy was performed using a Zeiss Axio Imager with a Yokogawa spinning-disc confocal scanner. Images were taken and stored using Slidebook v 6.0 software (Intelligent Imaging Innovations) using a 63x objective. For live imaging, embryos were dissected from adult hermaphrodites in M9 salt solution and mounted onto 3% agarose pads. All embryo images are z stack maximum projections using a z step size of 1 μm, spanning the entire width of the embryo.0 (14 min prior to mitosis). Final values represent average of three embryos. Error bars show standard deviation of the mean.Equally normalized time-lapse images were quantified using Slidebook v 6.0. Average fluorescence intensity relative to area of anterior (60%) and posterior (40%) of zygote were quantified and average fluorescence intensity relative to area of background (outside of zygote) was subtracted from each of these values. For each time-point, anterior and posterior fluorescence were expressed as fractions of total fluorescence and then normalized to TEMSA were carried out as described in Equilibration reactions were performed using same protocol as for EMSAs. Reactions were transferred to 384 well microplates . The apparent fluorescence polarization was determined using a Clariostar monochromator microplate reader with fluorescein-sensitive filters and polarizers. Polarization values were normalized relative to saturation polarization value. For each experiment, values of three reads were averaged. Average values and standard errors from at least three technical replicates were calculated and plotted against each protein concentration. These data were fit to a quadratic equation , to calc2 were quantified. Quantifications were manually verified for each image used. At least three technical replicates were quantified for each condition. Average number of condensates and S.E.M were calculated using all technical replicates.His::tagged MEG-3 fusions were quickly diluted out of urea into condensation Buffer  in the presence and absence of poly-U30 RNA. Dilutions were performed by adding buffer to protein in low-binding siliconized Eppendorf tubes and mixing briefly by pipetting. The reaction was either spun at 3000 rpm for 2 min or transferred directly into 35 mm glass bottom dish  for imaging. Phase separation assays with MEX-5 were performed by pre-incubating 3.5 μM 6xHis::MBP::MEX-5 protein  with condensation buffer and poly-U30 RNA for 30 min before diluting in MEG-3. Differential interference contrast (DIC) images were obtained on an Olympus inverted microscope, using a 100X objective. Images were taken and stored using Slidebook v 4.0 and 5.0. All images are a single focal plane focused on the slide surface. For each phase separation experiment, we took three separate images of an 80×80 micron field and counted the condensates using Image J64. To recognize condensates, background was subtracted from each image using a rolling ball radius of 10 pixels, a pixel brightness threshold was set to 15–255. Remaining pixels were smoothed three times and size and number of objects greater than 0.032 μmWestern blots were performed by running worm lysates on 7% Tris Acetate SDS PAGE precast gels . Protein was transferred to nitrocellulose membrane which was pre-blocked in 5% Milk diluted in PBS-Tween (0.1%) for 5 min (three times). The membrane was then incubated with Primary antibody for at least 18 hr at 4° C or 2 hr at room temperature. Membranes were washed and blocked in 5% milk for 5 min (three times) and incubated with secondary HRP conjugated antibody for 45 min at room temperature. Membranes were washed in 5% milk for 5 min (two times) and PBST for 5 min (one time). The membranes were then exposed to ECL substrate for 1 min and then exposed to film. Primary antibody dilutions (in 5% Milk PBST): Rat α OLLAS-L2 , Mouse α Tubulin .All in vivo biological replicates refer to experiments performed on independently isolated hermaphrodites  or independently treated hermaphrodites . In vitro biological replicates refer to experiments performed with independently purified protein preps. All in vivo technical replicates refer to observations in the same strain from separate zygotes. In vitro technical replicates refer to separate experiments performed using the same purified protein preps. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Julie Ahringer as the Reviewing Editor and Vivek Malhotra as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. A compiled list of essential revisions is given below. Full reviews are appended at the end of this letter for your information.Summary:C. elegans. The authors show P granule assembly occurs stepwise and critically depends on the function of Mex 5/6 and Meg3/4. They further demonstrate that Mex-5 needs to be phosphorylated and have intact Zn fingers that can bind to poly U stretches to generate meg-3/4 gradient. The most interesting aspect of the study is the analysis of the ability of Meg3 (4) to bind RNA primarily through its N-terminal IDR domain. Interestingly in vitro the affinity for polyU is similar between Mex5 and Meg3, suggesting a competition between these two proteins for RNA. The authors demonstrate in vitro phase transitions of purified IDR and full length Meg3, that this is increased by polyU RNA and can be competed by Mex5 binding to RNA. in vivo the Meg3 IDR is able to form granules however at a later stage or under increased RNA levels. Overall this is a very interesting study that outlines how non-sequence specific RNA interactions with IDR domains can facilitate phase transitions.The article by Smith, Seydoux and colleagues provides new insight into the organizational principles of P-granule assembly in Essential revisions:pgl mutants on Meg 3 granules appear quite different. In mex 5/6 mutants for Pgl or Meg granules, again is this just the images presented or a consistent difference.1) Quantification of granule number and size to back up qualitative claims. —The images in 2) This careful study is in striking conflict to a study published by Hyman and colleagues jut last month in Cell. In this study a role for Pgl proteins akin to the function of Meg3/4 proposed here is populated. The authors briefly discuss this in the present manuscript, but a more complete discussion would be helpful. In particular, in vitro Pgl proteins can phase separate and recruit other granule components but apparently in vivo granule formation depends on Meg3/4. Can this be related to a functional significance for germ line development?eLife showing that MEG-3 is a substrate of the kinase MBK-2 and the phosphatase PPTR-1/2 . In the Wang study, phosphorylation of MEG-3 by MBK-2 in the anterior of the C. elegans zygote promoted P-granule disassembly, while dephosphorylation by PPTR-1/2 promoted P-granule assembly. Attempts to reconcile these findings with the new discovery that asymmetric MEG-3 distribution and assembly now depends on an mRNA-binding competition with MEX-5 need to be included.3) The findings in this paper are a significant departure from the conclusions this same group published two years ago in pgl-1/3 RNAi embryos; however, the degree of knock down is not confirmed so PGL-1/3 may still be present. Co-stains of MEG-3/4 (anti-FLAG/OLLAS) with anti-PGL-1/3 in pgl-1/3(RNAi) embryos should be straight forward with existing antibodies.4) A central argument that distinguishes this paper from Saha et al. is that MEX-5 drives P-granule disassembly through MEG-3 instead of PGLs. Two findings support this. The first is that posterior enrichment of MEG-3::OLLAS is observed before posterior enrichment of PGL-1. PGL-3 is not examined and should be as the Saha et al. paper showed a specific PGL-3 response. There are available PGL-3 antibodies to test this. The second finding is that MEG-3 is still enriched in the posterior of 5) Findings presented in 6) It is not obvious as to why the ZF- mutated MEX-5 (R247E and K318E) also does not form a gradient, even though it has the necessary serine at position 404.7) Reviewer #1:C. elegans. The authors show P granule assembly occurs stepwise and critically depends on the function of Mex 5/6 and Meg3/4. They further demonstrate that Mex-5 needs to be phosphorylated and have intact Zn fingers that can bind to poly U stretches to generate mg3/4 gradient. The most interesting aspect of the study is the analysis of the ability of Meg3 (4) to bind RNA primarily through its N-terminal IDR domain. Interestingly in vitro the affinity for polyU is similar between Mex5 and Meg3, suggesting a competition between these two proteins for RNA. The authors demonstrate in vitro phase transitions of purified IDR and full length Meg3, that this is increased by polyU RNA and can be competed by Mex5 binding to RNA. in vivo the Meg3 IDR is able to form granules however at a later stage or under increased RNA levels.The article by Smith, Seydoux and colleagues provides new insight into the organizational principles of P-granule assembly in mex5/6 and Meg3/4, are not new, but in this study the use of crispr mutations and tagging of the endogenous locus makes the analysis more rigorous and less likely to be conflicted by trans gene expression. Overall this is a very interesting study that outlines how non-sequence specific RNA interactions with IDR domains can facilitate phase transitions. in vivo, granules are not composed of single molecules and the authors have shown in previous studies that P granules are structurally organized. How this organization is affected by the non-sequence specific RNA interactions is not clear.Many of the results with regard to reciprocal gradient formation of eLife.Given the interest in phase transitions and granule formation including plenty of controversies, this is a refreshingly solid and technically particularly careful study which in my mind seems perfect for pgl mutants on Meg 3 granules appear quite different. In mex 5/6 mutants for Pgl or Meg granules, again is this just the images presented or a consistent difference.1) Quantification of granule number and size. The images in meg4 mutant? Meg 3 and 4 are redundant is this at the level of function or phase transition. For example, can Meg3 and Meg4 form hetero-polymers in vivo?2) Biological role of Meg protein phase transitions. What is the phenotype of Meg3 IDR with regard to germ line development in a 3) This careful study is in striking conflict to a study published by Hyman and colleagues jut last month in Cell. In this study a role for Pgl proteins akin to the function of Meg3/4 proposed here is populated. The authors briefly discuss this in the present manuscript, but a more complete discussion would be helpful. In particular, in vitro Pgl proteins can phase separate and recruit other granule components but apparently in vivo granule formation depends on Meg3/4. Can this be related to a functional significance for germ line development?Reviewer #2:C. elegans zygote prior to mitosis. MEX-5 is necessary to block phase separation of P granules, but posterior MEX-5 is destabilized so P granules phase separate and segregate with the nascent germline. P granules consist of a heterogeneous mix of MEG, PGL, and GLH proteins (among other things), but the link between MEX-5 and P-granule proteins was unknown. Recently, a separate group  reconstituted P granule-like droplets in vitro, demonstrated that competition between PGL-3 and MEX-5 for mRNA can regulate droplet formation, and used theory to show that a MEX-5 gradient can drive PGL-3 and P-granule assembly in vivo. Smith et al. complement the findings of Saha et al., confirming that MEX-5 does act as an RNA sink to prevent phase separation, but in contrast they also demonstrate that the mRNA binding competition in the zygote is between MEX-5 and MEG-3 instead of MEX-5 and PGL-3. These findings make a significant contribution to what drives phase separation and P-granule assembly in the cytoplasm.In this paper, Smith, et al., address the poorly understood process of cytoplasmic RNA-granule phase separation during development. Previous work has shown that a series of asymmetric protein gradients are established after fertilization, culminating in the anterior enrichment of an RNA-binding protein called MEX-5 in the eLife showing that MEG-3 is a substrate of the kinase MBK-2 and the phosphatase PPTR-1/2 . In the Wang study, phosphorylation of MEG-3 by MBK-2 in the anterior of the C. elegans zygote promoted P-granule disassembly, while dephosphorylation by PPTR-1/2 promoted P-granule assembly. Attempts to reconcile these findings with the new discovery that asymmetric MEG-3 distribution and assembly now depends on an mRNA-binding competition with MEX-5 need to be included.1) The findings in this paper are a significant departure from the conclusions this same group published two years ago in 2) A central argument that distinguishes this paper from Saha et al. is that MEX-5 drives P-granule disassembly through MEG-3 instead of PGLs. Two findings support this. The first is that posterior enrichment of MEG-3::OLLAS is observed before posterior enrichment of PGL-1. PGL-3 is not examined and should be as the Saha et al. paper showed a specific PGL-3 response. There are available PGL-3 antibodies to test this. The second finding is that MEG-3 is still enriched in the posterior of pgl-1/3 RNAi embryos; however, the degree of knock down is not confirmed so PGL-1/3 may still be present. Costains of MEG-3/4 (anti-FLAG/OLLAS) with anti-PGL-1/3 in pgl-1/3(RNAi) embryos should be straight forward with existing antibodies.3) Findings presented in Reviewer #3:C. elegans embryo is studied. Recent work by these authors demonstrated that a group of intrinsically disordered, serine-rich proteins  regulates the dynamics of P granule assembly and disassembly. Here, with a combination of in vivo and in vitro work, the authors describe a model in which RNA binding competition between MEX-5 and MEG-3 results in P granule asymmetry. in vivo, the authors first show the opposing localization patterns of MEX-5 and MEG-3, and through mutational studies show that MEX-5 gradients drive MEG-3 granule asymmetry. in vitro, the authors show that purified MEG-3 (and its disordered region) can phase separate, and that this phase separation is greatly enhanced by addition of RNA through MEG-3 RNA binding. Finally, it is shown that addition of MEX-5 in vitro inhibits phase separation of MEG-3. These findings dovetail nicely with recent work showing a similar phenomenon with MEX-5 and PGL-3 . Overall, this study provides a clear and logical presentation of experimental work to support the majority of its conclusions, and I fully support its publication in eLife.In this manuscript by Smith et al. 2016, the nature of RNA dependent phase separation in relation to P granules in the early eLife. Your revised article has been favorably evaluated by Vivek Malhotra (Senior Editor), and Julie Ahringer, (Reviewing Editor).Thank you for resubmitting your work entitled "Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3" for further consideration at We are satisfied with your revisions except for your response to point 3, which needs to be addressed before acceptance, as outlined below:eLife). Please explain (at least briefly) in the Discussion how the new results in this paper fit with your results in Wang et al. on MBK-2 and PPTR-1/2, for example, including your ideas above that MBK-2/PPTR-1/2 may act permissively. It will be confusing to readers that you have only in passing mentioned your previous results on MEG-3 in this paper.The single sentence included in the Introduction is not sufficient to address point 3, requesting that you reconcile the findings in this paper with your study of MEG-3 in Wang et al.  Quantification of granule number and size to back up qualitative claims. —The images in pgl-1/3 zygotes, and this difference is most striking in the 1-cell stage and becomes less striking by the 4-cell stage. We previously reported this finding in Wang et al., 2014. The point of the experiment here, however, is that pgl-1/3 are not required to localize the MEGs to the posterior in the 1-cell stage or to the P blastomere in the 4-cell stage, which is clearly illustrated The embryos shown in 2) This careful study is in striking conflict to a study published by Hyman and colleagues jut last month in Cell. In this study a role for Pgl proteins akin to the function of Meg3/4 proposed here is populated. The authors briefly discuss this in the present manuscript, but a more complete discussion would be helpful. In particular, in vitro Pgl proteins can phase separate and recruit other granule components but apparently in vivo granule formation depends on Meg3/4. Can this be related to a functional significance for germ line development?meg-3/4 and does not correlate temporally with the MEX-5 gradient. Finally, the Hyman model assumes that PGL-3 is the driver of P granule asymmetry. In fact, neither PGL-3  are required for P granule asymmetry: zygotes lacking PGL-1 and PGL-3 still assemble MEG granules in the posterior. Future genetic studies will be needed to compare the different contributions of MEG and PGL proteins to germline development, but with respect to P granule asymmetry in zygotes, the MEG proteins are the drivers.We have now included a more complete discussion of the Hyman paper in the Discussion. The Hyman paper is a theoretical study that makes several predictions that are not consistent with in vivo observations. For example, the Hyman model predicts that the RNA binding domain of PGL-3 is essential for PGL-3 to form granules in the posterior cytoplasm in response to MEX-5. In fact, Hanazawa et al., showed in 2011 that PGL-3 lacking its RNA binding domain still forms posterior granules in vivo. The Hyman model also predicts that PGL-3 responds directly to the MEX-5 gradient. In fact, we find that PGL-3 asymmetry requires 3) The findings in this paper are a significant departure from the conclusions this same group published two years ago in eLife showing that MEG-3 is a substrate of the kinase MBK-2 and the phosphatase PPTR-1/2 . In the Wang study, phosphorylation of MEG-3 by MBK-2 in the anterior of the C. elegans zygote promoted P-granule disassembly, while dephosphorylation by PPTR-1/2 promoted P-granule assembly. Attempts to reconcile these findings with the new discovery that asymmetric MEG-3 distribution and assembly now depends on an mRNA-binding competition with MEX-5 need to be included.permissively to stimulate granule assembly/disassembly throughout the cytoplasm and that MEX-5 is responsible for the patterning. We now clarify this point in the Introduction.The Wang et al. paper showed that assembly/disassembly of P granules requires the activities of MBK-2 and PPTR-1/2. That study, however, did not claim that these regulators function in a spatially-restricted manner. In fact, in the Discussion section of Wang et al., we discussed the possibility that MBK-2/PPTR-1/2 act only 4) A central argument that distinguishes this paper from Saha et al. is that MEX-5 drives P-granule disassembly through MEG-3 instead of PGLs. Two findings support this. The first is that posterior enrichment of MEG-3::OLLAS is observed before posterior enrichment of PGL-1. PGL-3 is not examined and should be as the Saha et al. paper showed a specific PGL-3 response. There are available PGL-3 antibodies to test this. The second finding is that MEG-3 is still enriched in the posterior of pgl-1/3 RNAi embryos; however, the degree of knock down is not confirmed so PGL-1/3 may still be present. Co-stains of MEG-3/4 (anti-FLAG/OLLAS) with anti-PGL-1/3 in pgl-1/3(RNAi) embryos should be straight forward with existing antibodies.meg-3/4 zygotes. Second, we clarify that the pgl-1/3 zygotes shown in pgl-3(bn104) hermaphrodites treated with pgl-1(RNAi) and we include the control staining with anti-PGL-1 to demonstrate loss of PGL-1 protein  Findings presented in We now include an additional control figure  that sho6) It is not obvious as to why the ZF- mutated MEX-5 (R247E and K318E) also does not form a gradient, even though it has the necessary serine at position 404.Griffin et al., 2011 reported that the MEX-5 RNA binding domain is required for gradient formation. We have added an explanation of this to the text.7) Done.We are satisfied with your revisions except for your response to point 3, which needs to be addressed before acceptance, as outlined below:The single sentence included in the Introduction is not sufficient to address point 3, requesting that you reconcile the findings in this paper with your study of MEG-3 in Wang et al. (eLife). Please explain (at least briefly) in the Discussion how the new results in this paper fit with your results in Wang et al. on MBK-2 and PPTR-1/2, for example, including your ideas above that MBK-2/PPTR-1/2 may act permissively. It will be confusing to readers that you have only in passing mentioned your previous results on MEG-3 in this paper.We now have incorporated a discussion of our earlier findings to explain how phosphorylation and RNA-induced phase separation cooperate to regulate P granule dynamics.We have also expanded the last paragraph of the Discussion to outline remaining questions for future investigations.


The lncRNA sequences and data regarding their predicted functions in controlling the RNA processing and stability are available for browse, search and download in KTCNlncDB (http://rhesus.amu.edu.pl/KTCNlncDB/), the first online platform devoted to KTCN transcriptome.Keratoconus  is a degenerative eye disorder characterized by progressive stromal thinning that leads to a conical shape of the cornea, resulting in optical aberrations and even loss of visual function. The biochemical background of the disease is poorly understood, which motivated us to perform RNA-Seq experiment, aimed at better characterizing the KTCN transcriptome and identification of long non-coding RNAs (lncRNAs) that might be involved in KTCN etiology. The Database URL: http://rhesus.amu.edu.pl/KTCNlncDB/ Keratoconus (KTCN) is a degenerative disorder associated with stromal thinning and conically shaped protrusion of the cornea, resulting in loss of visual function . It is cab initio transcriptome assembly, expression estimation, and differential expression analysis and put much emphasis on bioinformatics study of lncRNAs. In order to investigate their potential molecular functions in KTCN, we compiled a list of known and newly identified lncRNAs based on RNA-Seq data from corneas and tried to characterize them functionally, using a previously published methodology (http://rhesus.amu.edu.pl/KTCNlncDB/). We believe that the data obtained from the first ever use of large-scale RNA-Seq approach of human KTCN cornea samples , followed by innovative bioinformatics study make KTCNlncDB a unique resource that will be of interest for researchers working in the fields of molecular biology, medicine and biotechnology.Here, we performed bioinformatics analysis of RNA-Seq data from KTCN and non-KTCN corneas, including hodology . In thishodology . There ahodology , while ahodology . Here, wet al., under review). A 75- or 100-bp paired-end run was performed on a HiSeq1500 platform (Illumina). Adapter and trimming of poor quality regions in short reads were performed using Trimmomatic  according to the manufacturer’s protocol, with slight modifications. The performed experimental and in silico procedures have been previously described ; (iv) transcripts with Ensembl biotypes belonging to ‘Protein coding’ or ‘Short non-coding’ groups were excluded, and in the case of newly discovered transcripts, only those predicted as non-coding by TransDecoder with default settings  were used as a starting point. In the search procedure, we followed the commonly accepted criteria , so thatsettings  were keplastal program from the LAST package was used to identify potential lncRNA–RNA interactions . Briefly, the ractions . A custoractions  using Biractions  and the The lncRNAs, by base-pairing with other RNA molecules, could affect their processing, stability, and expression levels in a number of ways. Therefore, the identified lncRNA–RNA interactions were scanned with the aim of classifying them into the following regulatory mechanisms: modulating alternative splicing through masking splicing signals, abrogation of miRNA functions, triggering RNA editing events, and guiding protein-coding transcripts to degradation in an SMD pathway.In terms of splicing regulation, interactions between lncRNAs and pre-mRNAs were filtered to keep only those that span exon–intron borders. Then, alternative splice sites were identified based on exon coordinates from Ensembl using in-house Python scripts, and only alignments that overlapped at least one alternative splicing event were kept for further consideration.in silico prediction of mature miRNA target sites was performed with miRanda . They were required to be located entirely within the lncRNA–mRNA alignment region and in a 3′ UTR of protein-coding transcripts. Finally, to study lncRNAs in the context of mRNA editing events, known mRNA editing sites were obtained from the RADAR database , MySQL 5.5 (http://www.mysql.com/), and Bootstrap 3 framework (http://getbootstrap.com/). The layout was enhanced with JavaScript Highcharts library (http://www.highcharts.com/) for interactive data plotting.The database was constructed using Hypertext Markup Language, cascading style sheets, PHP 5.4  and Mac OS X (Mavericks and Yosemite) operating systems using Internet Explorer , Mozilla Firefox (versions 3.6 and 30), Chrome (version 42), Opera (version 12.15) and Safari (version 8) web browsers. In case of Internet Explorer versions older than 9 the web interface was visualized improperly.Supplementary Table S1).Using our search criteria, we identified 16 331 lncRNA transcripts, making up 6.38% of the transcriptome. While most of them represent alternative splice forms of known genes, 735 novel lncRNA transcripts belonging to 160 genes were discovered as well. Their functions and possible links to KTCN pathogenesis were investigated, focusing on the putative roles of lncRNAs in the context of RNA–RNA interactions. A total of 56 395 potentially functional base pairings between lncRNAs and differentially expressed genes have been identified, most of which occurring in UTRs of protein-coding genes (91.18%), followed by non-coding transcripts (6.66%) and coding regions (0.99%), and overlapping untranslated and translated regions of protein-coding genes (1.17%). Due to data redundancy, resulting mainly from the fact that a particular lncRNA often base-pairs with multiple splice isoforms of a single gene, these interactions involve 870 unique lncRNAs and 996 transcripts predicted to be under their control. The highest number of lncRNAs has been associated with splicing regulation (789), followed by triggering mRNA editing events (663), masking miRNA target sites (534) and guiding protein-coding transcripts to the SMD pathway (514). The potential functions of 405 (55.10%) novel lncRNAs were also identified, with splicing regulation (403 lncRNAs) being the most prevalent association (http://rhesus.amu.edu.pl/KTCNlncDB/. It contains three main components: (i) ‘lncRNAs page’ with general information concerning all discovered lncRNAs, (ii) ‘lncRNA-RNA interactions page’ with details on functional interactions between lncRNAs and transcripts coming from differentially expressed genes and (iii) ‘BLAST page’, which enables sequence-based search of lncRNAs catalogued in KTCNlncDB.To enable access to the vast lncRNA-associated data generated in this study, we developed KTCNlncDB, a dedicated online database, freely available at lncRNA–RNA interactions page. This page grants access to lncRNA-RNA interactions data and is divided into three sections, ‘search options’, ‘search summary’ and ‘search results’.Search options. Here, the user can select criteria for data search and filtering: lncRNA id, interacting transcript id, potential function , gene name, and gene description.Search summary. In this section, user-selected search parameters are provided and the number of found records is reported. In order to download the filtered data into a tab-delimited text file, one needs to press the ‘Download current results’ button. Additionally, a pie chart summarizes lncRNA functions associated with the shown data.Search results. The search results are displayed in a table with one row per lncRNA–RNA interaction. The presented data include IDs of a lncRNA and its mate transcript, predicted regulatory role(s) of the lncRNA, gene name and description, transcript biotype (both referring to a transcript being under lncRNA control) as well as transcript region involved in the interaction . There is also a ‘Details’ button—upon clicking the user gets access to more information on the selected lncRNA–RNA interaction, including functional predictions, expression data and links to other interactions that involve the two RNAs. From this ‘details page’ the user can return to a ‘search page’ without losing search parameters by clicking a ‘Back to search’ button on top of the page.lncRNAs page. This page provides access to all predicted lncRNAs, including those with no functions assigned. Similarly to ‘lncRNA–RNA interactions page’, it is divided into three sections: ‘search options’, ‘search summary’ and ‘search results’ in a form of a table with the following fields: lncRNA transcript and gene ids, genomic coordinates, gene name, transcript biotype and gene description from ENSEMBL. Also, upon clicking ‘Details’ button, more detailed information is displayed: lncRNA sequence in a FASTA format, expression data, best BLAST hits to NONCODE v4 and Swiss-Prot databases as well as in silico found short peptides and Ribo-seq data analysis results. The Ribo-seq results come from HEK293 cell line and were provided with a recently published software, RiboTaper  and maximal number of found hits, the search parameters are left default and they are provided for user reference in the same page.Download page. Besides customized downloads available at ‘lncRNA–RNA interactions’ and ‘lncRNAs’ pages, also bulk download is enabled for the lncRNA sequences and their annotations, identified lncRNA–RNA interactions, BLAST search results and peptides found in the lncRNAs.et al., under review). We focused on a scenario wherein lncRNAs play their roles through base pairing with transcripts from differentially expressed genes, based on comparison between KTCN and non-KTCN patients. We found that lncRNAs could regulate processing and expression of at least 996 of them (Supplementary Table S1). This includes a number of genes from the TGF-β, Hippo and Wnt pathways, such as ‘SMAD9’, ‘SMAD6’, ‘TGFB3’ and ‘TGFBR1’, which have already been associated with ocular health , as determined with ConsensusPathDB . To the best of our knowledge, KTCNlncDB is the first catalogue of lncRNAs and their putative functions in KTCN. As it provides an integrative and easy-to-use platform to investigate lncRNAs expressed in human corneas, we believe KTCNlncDB will significantly contribute to KTCN-related research and also other tasks aimed at deciphering the biology of lncRNAs.The most relevant lncRNA-related results, including predicted lncRNAs and their putative functions in KTCN and non-KTCN transcriptomes, are stored in KTCNlncDB .This work was supported by the Conflict of interest. None declared.


Intercellular RNA trafficking has been shown as a widely-existing phenomenon that has significant functions in many aspects of biology. Viroids, circular noncoding RNAs that cause plant diseases, have been a model to dissect the role of RNA structural motifs in regulating intercellular RNA trafficking in plants. Recent studies on potato spindle tuber viroid (PSTVd) showed that the RNA motif loop 19 is important for PSTVd to spread from palisade to spongy mesophyll in infected leaves. Here, we performed saturated mutational analysis to uncover all possible functional variants of loop 19 and exploit this data to pinpoint to a three-dimensional structural model of this motif. Interestingly, we found that two distinct structural motifs can replace loop 19 and retain the systemic trafficking capacity. One of the alternative structures rapidly emerged from the inoculation using a loop 19 abolished mutant that is not capable of systemic trafficking. Our observation indicates the flexibility of multiple structural arrangements interchangeably exerting similar function at a particular RNA locus. Taken together, this study deepens the understanding of RNA structural motifs-regulated viroid RNA trafficking, which has broad implications for studying RNA intercellular trafficking as well. Potato spindle tuber viroid, family Pospiviroidae) is composed of 359 nt folding in a rod-like secondary structure comprising 27 RNA loop motifs flanked by short double helices   UTP-labeled riboprobes, was used for Northern blotting.To prepare RNA inoculum from wild-type (WT) and PSTVd variants, HindIII-linearized pRZ6-2 plasmids harboring correct PSTVd sequences served as templates for T7 polymerase transcription (Thermo Fisher Scientific) following manufacturer’s instructions. The generated RNA was purified using a MEGAClear kit (Thermo Fisher Scientific) after DNase I treatment. SpeI-linearized pInter-template was subject to in vitro transcription using a T7 MAXIscript kit (Thermo Fisher Scientific). The product, [α-Int in N. benthamiana [Loop19 is known as a critical loop for the intercellular trafficking of PSTVdthamiana , likely thamiana . Mutatiothamiana . To bettthamiana . We alsohttp://ndbserver.rutgers.edu/ndbmodule/services/BPCatalog/bpCatalog.html). The RNA Basepair Catalog utilized a heat map (with scores) to show the structural similarities among all possible base pairings within a given geometric group. The darker the blue color, the more similar in 3D structures (isosteric) the two base-pairings are. Whereas the darker the red color, the more dissimilar 3D structures (non-isosteric) the two base-pairings are. In practice, we first assumed that the base-pairing in loop 19 forms one of 12 geometry, here taking cis Watson-Crick/Watson-Crick base-pairing pattern as an example, then we checked the structural similarities among the six functional variants when they form cis Watson-Crick/Watson-Crick base-pairing. In this case, we found that the WT “A●C” is only weakly similar to “U●U” (light blue) while there are four functional variants not isosteric to WT (dark red)  . After l pattern . TherefoIntC227U mutant, which is trafficking-deficient. We infected 320 plants with PSTVdIntC227U RNA and examined infection 28 days post inoculation. The 320 plants were divided into 10 groups with each group containing 32 plants. To check infection, we pooled leaves from all 32 plants in each group as one sample and found the presence of PSTVd in systemic leaves indicated by strong signals of four pools and a weak signal from one pool, based on Northern blotting , which we designated as loop 19′.We found the PSTVdhe mFOLD  program IntG134A/C227U and PSTVdIntG134U/C227U may be functional variants or generated after their emergence through replication errors from other variants. To sort out the possibilities, we further checked the systemic trafficking function of each of these mutants with the infection assay. We inoculated plants with PSTVdIntG134A/C227U or PSTVdIntG134U/C227U transcripts and detected their trafficking to systemic leaves 28 days post inoculation using Northern blotting. Both variants successfully trafficked to systemic leaves in most of the test plants , comparable to WT  [In 156 registered PSTVd sequences from the sub-viral RNA database , we founlustalw) . The exclustalw) . HoweverIntG134A using site-directed mutagenesis and tested its trafficking. We observed trafficking in nine out of ten plants , which may help better understand the molecular basis underlying host-viroid/virus interactions.Here, we provided evidence for the rapid


Pseudouridine is the most abundant internal RNA modification in stable noncoding RNAs (ncRNAs). It can be catalyzed by both RNA-dependent and RNA-independent mechanisms. Pseudouridylation impacts both the biochemical and biophysical properties of RNAs and thus influences RNA-mediated cellular processes. The investigation of nuclear-ncRNA pseudouridylation has demonstrated that it is critical for the proper control of multiple stages of gene expression regulation. Here, we review how nuclear-ncRNA pseudouridylation contributes to transcriptional regulation and pre-mRNA splicing. The proper control of gene expression in the nucleus is achieved by the actions of a diverse set of factors. In addition to proteins, noncoding RNAs (ncRNAs) participate in most, if not all, stages of nuclear gene expression, including both RNA polymerase II (Pol II) transcription and pre-mRNA splicing  through two distinct mechanisms, namely RNA-dependent pseudouridylation and RNA-independent pseudouridylation.via complementary base-pairing interactions between the box H/ACA RNA (the loop sequence) and the substrate RNA. Both hairpins of H/ACA RNAs can carry functional pseudouridylation pockets and can thus independently direct pseudouridylation of uridines in separate RNAs or separate uridines located within the same substrate RNA. In the guide RNA–target RNA interaction, the guide sequences hybridize 5' and one nt 3' of the target uridine to the substrate RNA immediately, thereby framing it. The distance between the target uridine and the H or ACA box of the guide RNA is usually 14–15 nts , eukaryotic box H/ACA RNAs adopt a hairpin-hinge–hairpin-tail secondary structure Figure A. WithinThe four core proteins associated with box H/ACA RNAs are Cbf5 (dyskerin in human and NAP57 in rodents), Nhp2 , Gar1, and Nop10  in mouse S91 melanoma cells, mPUS1p was unexpectedly identified. In addition, SRA turned out to be a substrate of mPUS1p . It is caused by an inactivating mutation in human PUS1p  is the major factor required to overcome this restriction. P-TEFb, composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1, phosphorylates and thereby antagonizes the inhibitory actions of NELF and DSIF, triggering the release of Pol II from promoter-proximal pausing. In addition to this, P-TEFb also phosphorylates the C-terminal domain of the largest subunit of Pol II, which then serves as a platform for assembling key transcription and RNA-processing factors that promote transcriptional elongation, cotranscriptional processing of pre-mRNA, and lastly termination  infection is to “shock,” or reactivate, the latent HIV reservoirs for their subsequent “kill” by Highly Active Anti-Retroviral Therapy  by the U1 snRNP Interestingly, all of the major spliceosomal snRNAs are posttranscriptionally pseudouridylated Figure A. U2 snRin vitro-transcribed U2 snRNA, which lacks modification, was unable to rescue a splicing defect in U2 snRNA-depleted oocytes, following prolonged reconstitution periods, U2 snRNA was pseudouridylated and able to reconstitute splicing activity. In addition, anti-snRNP immunoprecipitation coupled with glycerol-gradient sedimentation demonstrated that U2 snRNA lacking pseudouridine was unable to form functional 17 S U2 snRNP. Thus, a good correlation between modification status, U2 snRNP biogenesis, and pre-mRNA splicing was established. In addition, by creating chimeric U2 snRNAs between cellular-derived and in vitro-transcribed U2, Yu et al. demonstrated that the functionally important modifications primarily resided within the first 27 nts of U2 snRNA , which is involved in the epigenetic modulation of gene expression, as well as alternative splicing, is pseudouridylated at two distinct sites (Carlile et al., Although the mechanism and function of snRNA pseudouridylation in splicing is considerably well studied, there are still many unanswered questions. For example, in contrast to U2 snRNA, the functional significance of pseudouridylation within U1, U4, U5, and U6 is not clear. In addition, within higher eukaryotes, there exists a second spliceosome, the minor spliceosome, which consists of U5, in addition to four distinct U snRNAs, U11, U12, U4atac, and U6atac, and all of them are pseudouridylated Figure . DetaileAll authors contributed to writing the manuscript.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.


Alphaviruses are arthropod-borne viruses that represent a significant threat to public health at a global level. While the formation of alphaviral nucleocapsid cores, consisting of cargo nucleic acid and the viral capsid protein, is an essential molecular process of infection, the precise interactions between the two partners are ill-defined. A CLIP-seq approach was used to screen for candidate sites of interaction between the viral Capsid protein and genomic RNA of Sindbis virus (SINV), a model alphavirus. The data presented in this report indicates that the SINV capsid protein binds to specific viral RNA sequences in the cytoplasm of infected cells, but its interaction with genomic RNA in mature extracellular viral particles is largely non-specific in terms of nucleotide sequence. Mutational analyses of the cytoplasmic viral RNA-capsid interaction sites revealed a functional role for capsid binding early in infection. Interaction site mutants exhibited decreased viral growth kinetics; however, this defect was not a function of decreased particle production. Rather mutation of the cytoplasmic capsid-RNA interaction sites negatively affected the functional capacity of the incoming viral genomic RNAs leading to decreased infectivity. Furthermore, cytoplasmic capsid interaction site mutants are attenuated in a murine model of neurotropic alphavirus infection. Collectively, the findings of this study indicate that the identified cytoplasmic interactions of the viral capsid protein and genomic RNA, while not essential for particle formation, are necessary for genomic RNA function early during infection. This previously unappreciated role of capsid protein during the alphaviral replication cycle also constitutes a novel virulence determinant. Alphaviruses can cause significant disease in infected individuals; however, our understanding of the molecular interactions that enable infection and contribute to the development of disease is limited. The work detailed in this manuscript characterizes the interaction of a viral RNA-binding protein, Capsid, with the viral genomic RNA. Importantly, these interactions were found to be at specific sites on the genome but not essential for virus assembly. Mutation of the capsid / RNA interaction sites decreased the replication of the virus and the severity of disease in a mouse model of infection. Taken together, these findings identify a previously undiscovered determinant of disease severity, and provide a potential basis for the development of new vaccines. Alphaviruses are positive-sense RNA viruses that exhibit a broad host range; and as evidenced by the emergence of chikungunya virus (CHIKV), represent a significant burden on the public health systems of developed and underdeveloped communities –9. Alphain vitro assembly systems have largely indicated that the assembly of Capsid:nucleic acid complexes is a nonspecific process  / 150mM NaCl / 5mM EDTA) by centrifugation at 185,000xg for 1.5 hours in a 60Ti rotor. Second, the pelleted viral particles were then resuspended in HNE buffer and applied to a 30% / 60% (m/v) sucrose step gradient prepared in HNE. The SINV particles were then banded via centrifugation at 250,000xg for 2.5 hours in an SW41 rotor at 4°C. The purified SINV particles were collected via needle aspiration, aliquoted and stored at -80°C for later use.SINV particles were purified via a two-step process . First, 11 SINV Toto1101 particles in a volume of 500μl, or 2x107 HEK293 cells infected with SINV Toto1101 at an MOI of 5 PFU/cell at 18hpi, were irradiated with 5700x100 μjoules per square centimeter in a Stratalinker. After cross-linking the RNA-protein complexes were solubilized in RIPA buffer (50mM Tris [pH 7.6] / 150mM NaCl / 1.0% NP-40 / 0.5% Sodium Deoxycholate / 0.1% SDS) and frozen at -80°C for later use. Prior to immunoprecipitation the lysates were thawed on ice, vortexed and clarified via centrifugation at 16,000xg for 5 minutes at 4°C. The clarified supernatants (~500μl) were transferred to a fresh tube. The cross-linked lysates were then incubated with 50ul packed volume of Protein G sepharose beads for 15 minutes at 4°C prior to being clarified via centrifugation at 5,000xg for five minutes. The resulting pre-blocked lysates were then immunoprecipitated using a polyclonal anti-capsid antibody, or control rabbit IgG sera. The cross-linked RNA:protein complexes were incubated in the presence of antibody at 4°C for a period of 2 hours under constant agitation. After antibody binding, 100ul packed volume of Protein G sepharose was added to each sample and further incubated for another 2 hours. After resin binding the RNAs were fragmented via the addition of RNAse T1 (ThermoScientific) to each immunoprecipitation. The fragmentation reactions were allowed to incubate for 15 minutes at room temperature. After fragmentation, the immunoprecipitated complexes were purified via centrifugation at 5,000xg for 2 minutes and washed three times with RIPA buffer and twice more with sterile 1xPBS. The resulting purified, fragmented RNA:protein complexes were treated with Proteinase K for 30 minutes at 37°C to release the RNA fragments from the immunoprecipitated complexes. The purified RNA fragments were then extracted using TRIzol and resuspended in a minimal volume. The RNA fragments were then used as the input materials for cDNA library generation via the NEBNext sequencing kit, as according to the manufacturer’s instructions. The resulting cDNA libraries were sequenced using the MiSeq platform.Per CLIP-seq library, either purified SINV particles or infected tissue culture cells were cross-linked via exposure to shortwave (254nm) UV irradiation. Briefly, approximately 10The specificity of the immunoprecipitation protocol described above was confirmed independently using small scale analytical replicates and metabolic labeling. HEK293 cells were either mock treated, or infected with SINV at an MOI of 10 PFU/cell, and allowed to incubate for 16 hours under normal conditions. Thirty minutes prior to the labeling period the infected monolayers were washed twice with 1xPBS and the media was replaced with methionine / cysteine free DMEM supplemented with 5% dialyzed FBS. After the depletion period the media was removed and replaced with methionine/cysteine free DMEM supplemented with L-AzidohomoAlanine (L-AHA) at a concentration of 50um. The cells were allowed to incubate under normal conditions for a period of two hours prior to removal of the culture media. Prior to harvesting the monolayers were washed three times with 1xPBS. Whole cell lysates were generated via the addition of RIPA buffer. Protein concentration was quantified via Bradford Assay and equivalent amounts of protein were precipitated via methanol / chloroform treatment. The precipitated proteins were resuspended in 1xPBS containing 5uM DIBO-AlexaFluor 64 and incubated for 1 hour at room temperature while shielded from light.After the labeling period, the samples were diluted in 2x volumes of RIPA buffer and immunoprecipitated identically to that described for the CLIP-Seq experiments. These treatments only differed on the basis of scale- as the total materials used were reduced 100-fold relative to those used for library generation. After immunoprecipitation 6x Laemmli buffer was added to a final concentration of 2x and the samples analyzed via SDS-PAGE. Protein species were detected via a Pharos Molecular Imager using the appropriate excitation and emission detection settings. As shown in Supporting Information , the ant−25. The sequencing data directly relevant to the studies described herein are available accompanying this document as https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ncbi.nlm.nih.gov_geo_query_acc.cgi-3Facc-3DGSE99879&d=AwIEAg&c=SgMrq23dbjbGX6e0ZsSHgEZX6A4IAf1SO3AJ2bNrHlk&r=OEUL-Bhg_4jju0S0FtlcvxycNjpESQ60pJohUAWMntY&m=S87220zYVRm6qbr6ME7XEYiR1DQZkoyOpa4rz4m8xmQ&s=M1W8fvnSVBtk9BsSVmlJIUA0pXouRfnBZuKIY4xy5jg&e=The cDNA libraries were trimmed to remove indices and adaptors prior to alignment to a reference genome consisting of the parental Toto1101 strain of SINV. Alignments were performed using LAstZ with standard parameters. Only reads corresponding to the positive-sense genomic RNA were mapped to the reference genome sequence. Sequence coverage was clustered at the nucleotide level of resolution from the aligned sequence reads. Analysis of the clustered sequence data relied on a subtractive method whereby the sequence coverage of capsid-specific libraries was compared to nonspecific control libraries. For these analyses the coverage of both the Capsid and Nonspecific control libraries were normalized internally by dividing each base by the total number of represented nucleotides to gain a percentage which was then multiplied by an arbitrary amount to generate a standardized measure of each nucleotides relative representation amongst independent library sets. The resulting values from the control libraries were averaged and subtracted from the Capsid-specific libraries to generate a difference map . Briefly, PCR amplification of the parental Toto1101, or AR86, infectious cDNA clones were performed using the sequences indicated in The quantitative assessment of the capsid:RNA interactions of the individual C:R interaction sites for parental and C:R interaction site mutants utilized small scale extracts generated identically to that described above for the CLIP-seq process. Briefly, HEK293 cells were infected at an MOI of 5 prior to cross-linking via UV irradiation. After the preparation of whole cell lysates, the capsid:RNA complexes were immunoprecipitated and the bound RNAs fragmented with RNAse T1. The immunoprecipitates were washed extensively and the retained RNA fragments were release via proteinase K digestion. The RNA fragments were extracted via TRIzol according to the manufacturer’s instructions.The precipitated RNAs and paired input controls, were used as the input materials for reverse transcription using random hexamer. The resulting cDNAs were assessed quantitatively via qRT-PCR as described below using the following primer pairs:SINV.nt9300F 5’-GCACCGCCATCAAGCAATGTGTGGC-3’; SINV.nt9300R 5’-CAATTTCCCTTGGGCCGTGTGGTCG-3’;SINV.nt10100F 5’-TGTTCCAAATGTGCCACAGATACCG-3’; SINV.nt10100R 5’-AATGTACTCTTGGTTGGTGGAAGGC-3’;SINV.nt10400F 5’-CAGCAGATTGCGCGTCTGACCATGC-3’; SINV.nt10400R 5’-GACTCCGTTCACGTACACATCTAGG-3’.The viral growth kinetic assays performed in these studies were essentially as previously described . BrieflyAt the indicated times post infection, infected HEK293 cells were washed twice with 1xPBS and harvested into TRIzol. Total RNA was extracted according to the manufacturer’s directions, with carrier glycogen being added to the precipitations. Equal amounts of total RNA (0.5μg) were used as template for the synthesis of cDNA and assessed quantitatively via qRT-PCR using the method and oligonucleotide primers previously described , 69, 70.To determine the relative infectivity of each SINV C:R interaction site mutant the number of total viral particles per unit volume, as measured by the genome equivalents per ml, was determined using qRT-PCR, as previously described . BrieflyThe quantitative analyses of viral gene expression described in these studies was performed via one of two methods-Gross viral and cellular gene expression was determined via metabolic labeling of infected cells , 72. BriQuantitative analysis of genomic RNA gene expression early during infection was performed using a process previously described, with one notable exception , 73. SpeViral RNA half-lives were assessed as previously described . HEK293 The production of soluble type-I IFN was assayed as previously described . BrieflyFour-week old WT C57BL/6J mice were obtained from the Jackson Laboratory. Mice were inoculated in the left rear footpad with virus in diluent  in a volume of 10 μl. On the termination day of each experiment, mice were sedated with isoflurane and euthanized by thoracotomy, blood was collected, and mice were perfused extensively by intracardiac injection of PBS. Serum was obtained by collecting blood in serum separator tubes (BD). PBS-perfused tissues were removed by dissection, placed into PBS-1% FBS and homogenized using a MagNA Lyser (Roche). The amounts of infectious virus in tissues were quantified by standard plaque assays using BHK-21 cells.This study was conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals and the AVMA Guidelines for the Euthanasia of Animals. All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee at the University of Colorado School of Medicine (Assurance Number: A3269-01) under protocol B-86514(10)1E. Experimental animals were humanely euthanized at defined endpoints by exposure to isoflurane vapors followed by thoracotomy.Unless otherwise noted, the quantitative data reported in this study are the means of a minimum of three independent biological replicates. Where appropriate, the statistical analysis of comparative samples was performed using variable bootstrapping, as previously described . The errS1 Table(PDF)Click here for additional data file.S1 DatasetStatistical summary of sequencing data. SINV-specific sequence reads. Read size analysis across the identified capsid interaction sites and a control region in the nsP4 coding sequence. QC was performed using Trimmomatic. Quality scores were averaged over a sliding window of 4 nucleotides and nucleotides with values less than 20 were removed.(XLSX)Click here for additional data file.S1 FigA) Metabolically labeled mock control and infected HEK293 cells cell lysates were immunoprecipitated with either non-specific control sera or anti-capsid sera using conditions identical to those used for the preparation of the cDNA libraries used in CLIP-seq. The input lane represents 1/10th of the starting material for the IP. Data shown is representative of several biological replicates. B) SINV infected HEK293 cells were either mock- or UV-crosslinked via shortwave UV irradiation at 18hpi. The cell monolayers were harvested via gentle scraping and solubilized in RIPA buffer to form whole cell lysates. The cell lysates were then precipitated with antibodies specific for either SINV Capsid, or control rabbit IgG, as indicated on the figure. All purification conditions were identical to those described for the development of the CLIP-Seq cDNA libraries utilized in this study, with the only exception being that RNA fragmentation was omitted. After purification, cDNA was generated from the immunoprecipitated materials, and the presence of the nsP1 coding region was detected via RT-PCR and agarose gel electrophoresis. Data shown is representative of three biological replicates.(TIF)Click here for additional data file.S2 FigIn vitro transcribed genomic RNAs for parental SINV, and each of the individual C:R interaction site mutants were assessed for translation using Rabbit Reticulocyte Extracts according to the manufacturer’s directions. The amount of translation was detected using nanoluciferase detection. Data shown is the mean of three independent biological replicates, with the error bar representing the standard deviation of the mean.(TIF)Click here for additional data file.S3 FigThe one-step growth kinetics of parental and a non C:R interaction site mutant as observed in HEK293 cells. Briefly, a region of the SINV genomic RNA detected, but identified as statistically insignificant, by CLIP-seq analysis was mutated using identical parameters to the bona fide C:R interaction site mutants. This region corresponds to nt41-98 of the viral genomic RNA, which includes the genuine start site of nsP1. The quantitative data in this figure represents the mean of three independent biological replicates, the error bar representing the standard deviation of the mean.(TIF)Click here for additional data file.


As ribonucleic acid (RNA) nanotechnology has advanced, it has been applied widely in RNA‐based therapeutics. Among the range of approaches, enzymatically synthesized RNA structures for inducing RNA interference in cancer cells have potential for silencing genes in a target‐specific manner. On the other hand, the efficiency of gene silencing needs to be improved to utilize the RNA‐based system for RNAi therapeutics. This paper introduces a new approach for efficient generation of siRNA from bubbled RNA‐based cargo (BRC). The presence of bubbles in between to avoid nonfunctional short dsRNAs allows the RNA‐based cargoes to contain multiple Dicer‐cleavage sites to release the functional siRNAs when introduced to cells. BRCs can be synthesized easily in a one‐pot process and be purified by simple centrifugation. Furthermore, efficient target gene silencing by the bubbled structure is confirmed both in vitro and in vivo. Therefore, this bubbled RNA cargo system can be utilized for target‐specific RNAi therapeutics with high efficiency in the generation of functional siRNAs in the target cells. Ribonucleic acid (RNA) has attracted considerable attention for its potential applications in nanomedicine for the treatment of cancer or infectious disease as well as for immunization.Rolling circle transcription (RCT)‐based enzymatic self‐assembly,Among the abovementioned RNA structures, the efficiency of Dicer cleavage plays a critical role in inducing RNA interference by RNA‐based materials. Although the Dicer‐mediated generation of siRNA from a RNAi microsponge was ≈21% in a previous study,FigureTo synthesize BRCs, two types of closed circular DNAs were prepared, as reported previously.One of the advantages of the synthetic approach of RCT‐induced self‐assembly is that the sizes and sequence‐dependent biological functions of the resulting materials can be manipulated easily. As observed in previous research,2+) plays an important role in packing the RNA strands due to its negative charge.Optical and electron microscopes, transmission electron microscopy‐based energy dispersive X‐ray scattering (TEM‐based EDX), and dynamic light scattering (DLS) were used to characterize the BRCs in a range of aspects. Scanning electron microscopy (SEM) of the BRCs revealed their spherical structures with highly porous surface Figure C. To exaz‐axial resolution, was used to analyze the longitudinal dimension of the BRCs , and nanoparticle tracking analysis (NTA) . The low PDI also indicates that the RNA structures are synthesized uniformly . Furthermore, Brownian motion of each nanoparticle was also tracked by NTA , which has a good s Figure F. Cross‐s Figure G and theFigureBefore evaluating its therapeutic efficacy, the in vitro siRNA generation was assessed by treating the BRCs with the Dicer enzyme. The Dicer enzyme is part of the RNase III family, and it cleaves dsRNA to short dsRNA fragments with lengths of 20–25 bp with a two‐base overhang on the 3′ end.To examine the cellular uptake of the BRCs covered with the transfection reagent, the BRCs were first labeled fluorescently with cyanine5 (cy5). During the RCT process, cy5‐labeled uridine triphosphate (cy5‐UTP) was added to the label, resulting in RNA molecules labeled with cy5. The fluorescently labeled BRCs were then analyzed by cytometry Figure C. The inCytometry analysis Figure D and fluFigureTo examine the in vivo RNAi ability of BRCs, noninvasive real‐time fluorescence imaging was performed from the subcutaneous implantation of nude mouse models of GFP‐expressing HeLa after the single intratumoral administration of GFP‐targeting BRCs (0.8 fmole of BRCs per mouse) Figure3A,B. TIn summary, bubbled RNA‐based cargoes were synthesized by an enzymatic approach with a preprogrammed sequence for the specific cleavage sites for Dicer to process. The overall size of the nanoparticles was controlled rationally by adjusting the ratio of template DNAs to the T7 RNA polymerase in the RCT reaction. The BRCs were synthesized by a one‐pot process, which avoided the labor‐intensive synthetic process, as is the case for many synthetic processes of other types of nanoparticles. Moreover, the resulting nanoparticles have a narrow size distribution, and the efficient Dicer‐mediated release of siRNAs from the BRCs was confirmed in vitro. The gene‐specific knockdown was also confirmed in vivo by an analysis of the gene silencing activity of GFP‐targeting BRCs, compared to the nontargeting BRCs. Overall, this study showed that the BRCs can be synthesized and purified easily, and this system can be utilized for target‐specific RNAi therapeutics with high efficiency in the generation of functional siRNAs in the target cells.The methods detailing the BRC synthesis, characterization, Dicer‐mediated siRNA generation, and in vitro and in vivo gene knockdown analysis are included in the Supporting Information.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.


The World Health Organization (WHO) international standard for hepatitis A virus (HAV) RNA nucleic acid assays was characterized by complete genome sequencing. The entire coding sequence and noncoding regions were assigned HAV genotype IB. This information will aid the design, development, and evaluation of HAV RNA amplification assays. Hepatovirus genus within the Picornaviridae family that causes acute liver infection, potentially resulting in HAV disease (Hepatitis A virus (HAV) is a nonenveloped single-stranded RNA virus with a genome of 7.5 kb and is a member of the  disease . World H disease . ElucidaThe first WHO IS developed for HAV RNA genome amplification techniques (HAV IS 00/562) represents a cornerstone of HAV diagnostic algorithms (Taq HiFi (Invitrogen) using primer pairs described previously  of an HAV-positive plasma donation sourced from the National Plasma Repository, South African National Blood Service, in 2011. To elucidate sequence identity, viral RNA was extracted using total nucleic acid isolation (TNAi) extraction methodologies on an Ampliprep instrument (Roche Ltd.). Rescued RNA was reverse transcribed and directly amplified with the SuperScript III one-step reverse transcription-PCR (RT-PCR) system with Platinum eviously . In combHAV-specific amplicons were sequenced  using a cycling profile of 25 cycles  on a 3130XL sequencer (ABI Ltd.) in forward and reverse directions and assembled and analyzed using Geneious v7 software. A contiguous sequence was represented by 7,386 nt, with a total GC content of 38.2%. Phylogenetic analyses indicated that the 3rd HAV IS clusters among HAV genotype IB sequences, with no subtype recombination events identified. The complete genome sequence reported here, derived from the 3rd WHO HAV RNA IS, will support genome amplification assay development for HAV RNA detection and quantification in environmental samples and clinical studies.KY003229.The genome sequence of the 3rd WHO IS for HAV RNA reported here has been deposited in GenBank under accession number


A bimolecular ribozyme consisting of a core ribozyme (ΔP5 RNA) and an activator module (P5abc RNA) has been used as a platform to design assembled RNA nanostructures. The tight and specific assembly between the P5abc and ΔP5 modules depends on two sets of intermodule interactions. The interface between P5abc and ΔP5 must be controlled when designing RNA nanostructures. To expand the repertoire of molecular recognition in the P5abc/ΔP5 interface, we modified the interface by replacing the parent tertiary interactions in the interface with artificial interactions. The engineered P5abc/ΔP5 interfaces were characterized biochemically to identify those suitable for nanostructure design. The new interfaces were used to construct 2D-square and 1D-array RNA nanostructures. Nanometer-sized biomolecules and their assemblies with precisely controlled shapes are attractive materials for the expanding field of nanobioscience, which includes nanomedicine and nanobiotechnology. Nucleic acids  are among the most promising biomaterials for nanobioscience purposes ,2,3,4. NTetrahymena group I ribozyme and served as a structural platform.RNA is synthesized enzymatically in single-stranded form in the cellular environment, and several classes of RNAs, such as ribozymes and riboswitches, exhibit their functions after folding into defined 3D structures. These properties suggest that artificial RNA nanostructures designed by RNA origami  could be constructed in vivo . To expaThe bimolecular group I ribozyme consists of a core ribozyme module (ΔP5 RNA) and an activator RNA unit (P5abc RNA), which form a complex expressing the full catalytic activity of the ΔP5 core ribozyme . P5abc RTo increase the structural variety of the assembled RNA nanostructures, the repertoire of RNA–RNA interacting motifs is important because it governs the scope of RNA nanostructure design and assembly. In this study, we exploited RNA–RNA interacting motifs that can be used for RNA nanostructure assembly. We examined artificial RNA–RNA interacting motifs structurally unrelated to the GNRA/receptor family but that act as modular parts to functionally substitute for GNRA/receptor motifs in unimolecular ribozymes.2+ (15 mM). The DNA template in the reaction mixture was removed by DNase treatment. The transcribed RNA was purified in a 6% or 9% denaturing polyacrylamide gel. A published protocol was used for 3′-end labeling of RNAs with the BODIPY-fluorophore synthesized in our laboratory [Plasmids encoding mutants of ΔP5 RNA and P5abc RNA were generated by PCR-based mutagenesis of pTZ-ΔP5 and pP5abc , respectboratory .Electrophoretic mobility shift assays (EMSA) for bimolecular P5abc/ΔP5 complexes were performed according to the procedure reported by Tanaka et al. . EMSAs w2) containing 2 mM guanosine triphosphate  was added to the RNA solutions, which were further incubated at 37 °C for 30 min. Ribozyme reactions were initiated by adding 5′-FAM-labeled substrates  and allowed to proceed at 37 °C. Aliquots were taken at given time points and treated with 1.5 volumes of stop solution containing 75% formamide, 0.1% xylene cyanol, and 100 mM EDTA. The mixtures were electrophoresed in 15% denaturing polyacrylamide gels. The gels were then analyzed with a Pharos FX fluoroimager . All assays were repeated at least twice. The mean values are plotted in the figures, with the minimum and maximum values indicated by error bars.Aqueous solutions containing ΔP5 RNA  and P5abc RNA  were heated separately at 80 °C for 5 min and then cooled to 37 °C. The 10× concentrated reaction buffer  pair  pair formed a bimolecular complex. However, neither the P5b-GAAC/P6-R(GAAC) pair nor the P5b-C-loop/P6-R(C-loop) pair showed a retarded band  interaction belonging to the GNRA/receptor family.EMSA with 10 mM MgAA) pair . We then to 5 mM B. In EMSded band , indicat2+, three biomolecular ribozymes possessing matched P5b–P6 interactions exhibited higher catalytic activity than the remaining six pairs possessing mismatched P5b–P6 interactions , since the remaining two P5abc RNAs could not activate the core ribozyme (−1), among the three matched complexes. On the other hand, the P6-R(GGAA) variant was also moderately activated by a mismatched P5abc with a GAAC loop  pair weakly supported the P5abc/ΔP5 complex  was added to activate the ΔP5 core ribozyme in the presence of equimolar amounts of the substrate RNA (0.5 μM) and the ΔP5 RNA (0.5 μM). In the presence of 3 mM Mgractions . On the ions −1, B was the −1, −1, D. The coribozyme C. A ΔP5 AAC loop D. This w complex . The C-lIt has been reported that complex formation of P5abc RNA and ΔP5 RNA is supported not only by the RNA–RNA interaction between P5b and P6, but also by four Watson–Crick base pairs between P5c in P5abc RNA and P2 in ΔP5 RNA ,31. The 2+  and its M5 variant (kobs = 0.31 min−1) showed closely similar activity, and were 2-fold more active than the M4 variant (kobs = 0.16 min−1). The M3 variant (kobs = 0.03 min−1) was distinctly less active than the three complexes. The rate constant of the M3 variant (0.03 min−1) was even smaller than those of two mismatched complexes having the P5c-M5/P2-M4 mismatched pair (kobs = 0.069 min−1) and the P5c-M4/P2-M5 pair (kobs = 0.076 min−1). These observations are qualitatively consistent with those from structure prediction and EMSA.We then investigated whether the binding affinity and the specificity of base pairs, which were predicted in er P5abc . The parTo design artificial RNA nanostructures with new P5b/P6 and P5c/P2 interacting pairs through modular engineering of the group I ribozyme, we chose the GAAC-R(GAAC) pair and the M5 pair as new modular parts for a closed RNA tetramer with square shape. Monomer units of this square tetramer are engineered variants of the group I ribozyme A that weWe have previously characterized the closed tetramer consisting of L2 RNA and L3 RNA. L2 RNA possesses P2-M3, P6-R(GAAA), P5c-WT, and P5b-GGAA, whereas L3 RNA possesses P2-WT, P6-R(GGAA), P5c-M3, and P5b-GAAA  interaction in the context of the bimolecular ribozyme demonstrated that this interacting motif, which can be used as a possible alternative to the naturally occurring GNRA–receptor interactions , showed 2+, the mobility of F-RNA was retarded gradually in an RNA concentration-dependent manner (In the presence of 50 mM Mgt manner A in P5c,t manner . The molWe have characterized artificial RNA–RNA interactions to determine their abilities to support the assembly of the two module RNAs in the P5abc/∆P5 bimolecular ribozyme. Some of the new RNA–RNA interactions characterized in this study were used as alternative modular parts to produce new P5abc/∆P5 interfaces. These new interfaces can be applied to design ribozyme-based RNA nanostructures.