Caenorhabditis elegans, we found that the highly conserved UNC-119 protein provides a link between the membrane-associated Ankyrin (UNC-44) and the microtubule-associated CRMP (UNC-33). Together they form a periodic membrane-associated complex that anchors axonal and dendritic microtubule bundles to the cortex. This anchoring is critical to maintain microtubule organization by opposing kinesin-1 powered microtubule sliding. Disturbing this molecular complex alters neuronal polarity and causes strong developmental defects of the nervous system leading to severely paralyzed animals.The development of a polarized neuron relies on the selective transport of proteins to axons and dendrites. Although it is well known that the microtubule cytoskeleton has a central role in establishing neuronal polarity, how its specific organization is established and maintained is poorly understood. Using the in vivo model system  The structural organization and dynamic remodeling of the cytoskeleton is essential for neuronal development and survival. Axons and dendrites are filled with bundles of non-centrosomal microtubules, which are important for maintaining cellular structure as well as intracellular transport. Notably, the microtubule cytoskeleton plays a central role in neuronal development and polarization: axons have microtubules with their plus-end projecting away from the soma, while dendrites have either a mixed (vertebrates), or uniform minus-end out (invertebrates), microtubule organization . This diDrosophila and Caenorhabditis elegans indicate that the motor kinesin-1 has a role in setting up the characteristic axon-dendrite microtubule organization, by sliding microtubules against each other  and UNC-33 (CRMP) in vitro and in vivo. We show that this complex forms a similar periodic arrangement as the spectrin cytoskeleton and that it is critical to anchor the microtubule cytoskeleton to the neuronal cortex. In the absence of cortical anchoring, UNC-116 (kinesin-1) induces massive microtubule cytoskeleton sliding in axons and dendrites, leading to loss of axon-dendrite microtubule polarity. Thus, we propose that the balance between kinesin-1 dependent microtubule sliding and cortical microtubule anchoring is essential for axon-dendrite microtubule polarity and thus for neuron development and functioning.In this study, we use C. elegans identified many unc-genes, which lead to impaired (‘uncoordinated’) locomotion  and unc-44 (Ankyrin) mutants , medium (UNC-33M) and large (UNC-33L), but only the large isoform is able to rescue the neuronal polarity defects of the unc-33 mutant  and that the unc-44 mutant has very similar phenotypes as the unc-33 mutant  acts downstream of 3 mutant . However3 mutant . In a yer UNC-44 . This, tr UNC-44 . Using tr UNC-44 , which ir UNC-44 . To testr UNC-44 , showingr UNC-44 . Since munc-44 mutant, we found that the UNC-119 recovers rapidly and seems to diffuse freely in the absence of UNC-44 , it is not possible to endogenously tag specific isoforms. However, inserting an internal GFP resulted in a functional UNC-33L protein . Therefo3 mutant . We did 3 mutant . However3 mutant , whereas mutants , which ikgrounds . In concAnkyrin proteins are well-characterized component of the cortical actin-spectrin cytoskeleton , which fCRMPs are described as cytosolic phosphoproteins that can bind microtubules via their C-terminus . To incrunc-119 mutant  in the PVD neuron . With FR9 mutant , which sell body . Togetheunc-mutants we observed neuronal development defects and disorganization of the microtubule cytoskeleton; microtubules were no longer restricted to the primary dendrite, but penetrated the side branches as well (unc-44 (Ankyrin), the unc-119 and the unc-33 (CRMP) mutants  to the cortex, suggests that the complex anchors microtubules in neurons. To visualize the microtubule cytoskeleton we expressed the microtubule binding protein MAPH-1 in the PVD neuron  and foun as well . Additio as well . To exam as well . Upon ac as well , which i mutants  and we o mutants . These runc-mutants suggests that forces are exerted on the microtubule cytoskeleton, potentially by microtubule motors. Previously, unc-116 (kinesin-1) was shown to be important for dendritic microtubule organization, possibly by transporting microtubules  completely suppressed the microtubule mobility in the unc-33 (CRMP) mutant , also suppressed the microtubule mobility of the unc-33 mutant (unc-104 (kinesin-3) did not suppress the microtubule mobility in the unc-33 mutant (The displacement of the microtubules in the otubules . If the  lineage . As anti) mutant , but not) mutant  nor the 3 mutant . This sh3 mutant .Altogether we have found that UNC-119 bridges the membrane-associated UNC-44 (Ankyrin) to the microtubule binding UNC-33L (CRMP) to immobilize the microtubule cytoskeleton . While iunc-44::gfp knock-in animals  in neurons.To confirm this model, we examined whether artificial anchoring of UNC-33S (CRMP) is sufficient to balance kinesin-1 activity in the absence of the cortical anchoring complex. To do this, we fused UNC-33S, which cannot bind to UNC-119, to the GFP-nanobody (vhhGFP) and expressed it in the PVD neuron of  animals . Artific mutants  as well 3 mutant . Remarka9 mutant , showingC. elegans unc-119 mutant is severely paralyzed, a phenotype that can easily be rescued by reintroduction of the gene and which has extensively been exploited as a transgenesis background in C. elegans for many year  mutant. Future work will have to address the precise role of microtubule sliding by kinesin-1 in neuronal development.A proper balance of microtubule sliding and bundle immobilization was also shown to be important for neuron development in other organisms. In utgrowth . To accoutgrowth . Mutatin-end out , arguingadaptors . Howeverunc-119 were found to be important for neuronal development in other species as well; depletion of zebrafish UNC-119a led to various neuronal defects that are reminiscent of the C. elegans phenotypes . Moreover, we were able to rescue the unc-119 mutant microtubule organization and locomotion by directly attaching UNC-33 to UNC-44, demonstrating that UNC-119 functions as a structural scaffold to bring these proteins together for proper neuronal development and functioning.UNC-119 is a highly conserved protein both in sequence and in function . Interesenotypes  and deplenotypes . Thus faenotypes . As suchenotypes , where tenotypes . Similar defects . HoweverDrosophila, the kinesin-1 sliding is counteracted by the ‘mitotic’ kinesin MKLP1  isoform interacts with microtubules via UNC-119 and UNC-33 (CRMP) in both axons and dendrites. Also in mammalian neurons, CRMPs are undeniably important for microtubule organization, neuronal development, and neuronal polarity , or whether their binding is regulated, for example by phosphorylation , unc-44(hrt8) were generated by injection of the guide + Cas9 plasmid + Pmyo-2::tdTomato into STR318 and screening for Unc phenotype. Similarly, the deletion of cb-unc-119 form mosSCI lines was performed by injecting cb-unc-119 sgRNA with Cas9 protein and scoring for the Unc phenotype. To generate unc-44(hrt5[GFP]) we used pha-1 co-conversion approach (unc-119(hrt13[GFP]) we used the self-excising cassette-approach . The collected eggs were washed three times with fresh egg buffer. Then the eggs were separated using 30% final concentration of sucrose by centrifuging at 1200 rpm for 20 min. The separated top layers of eggs were collected in a new tube and washed 2–3 times with egg buffer. Then, the eggs were treated with chitinase (1 U/ml)) for 40 min to digest the egg shell. The chitinase reaction was stopped using L15 medium. After chitinase treatment the embryos were passed through 25 G needle for dissociating into single cells. The dissociated cell suspension was filtered through 5 µm Durapore filter (Millipore). Then the single cell solution was centrifuged for 3 min at 3200 rpm and the pellet was resuspended in a lower volume of L15 medium and seeded on to #1.5 glass bottom petri dish coated with peanut lectin and incubated at 25 C. The cell culture medium was changed every day and at day three post plating we proceeded with the immunocytochemistry.Embryonic cell isolation was performed using a previously described method with some modifications . BrieflyCells were washed 2x gently with media, followed by a PBS++ (Ca2+ and Mg2+) wash. Then the cells fixed cells with 4% (v/v) PFA in PBS++ (Ca2+ and Mg2+) for 10–20 min at RT. Then cells were washed 3x with PBS. Cells were permeabilized using 0.01% Triton in PBS for 20 min at RT. Then the cells were washed 3x with PBS, and then blocked in 2% BSA for 20–30 min at RT. The primary antibody  was directly added the blocking buffer at 1:1000 dilution and incubated at 4 C overnight. The next day the cells were washed 3x with PBS, 10 min each. Then, the secondary antibody  was used at a dilution of 1:1000 and incubate for 2 hr at RT. The cells were washed 5x with PBS, 10 min each. Then the samples were mounted in Mowiol and stored at 4°C until imaging.L4 or young adult worms were anesthetized with 10 mM tetramisole or 5 mM Levamisole on a 5% agarose pad and all live imaging was performed within 60 min. All live fluorescent imaging was performed on a Nikon Eclipse-Ti microscope and with a Plan Apo VC, 60×, 1.40 NA oil or a Plan Apo VC 100 × N.A. 1.40 oil objectives (Nikon). The microscope is equipped with a motorized stage , a Perfect Focus System (Nikon), ILas system  and uses MetaMorph 7.8.0.0 software (Molecular Devices) to control the camera and all motorized parts. Confocal excitation and detection is achieved using a 100 mW Vortran Stradus 405 nm, 100 mW Cobolt Calypso 491 nm and 100 mW Cobolt Jive 561 nm lasers and a Yokogawa spinning disk confocal scanning unit  equipped with a triple-band dichroic mirror  and a filter wheel  containing BFP (ET-DAPI (49000), GFP (ET-GFP (49002)) and mCherry (ET-mCherry(49008)) emission filters . Confocal images were acquired with a QuantEM:512 SCEMCCD camera (Photometrics) at a final magnification of 110 nm (60x objective), 67 nm (100x objective) per pixel, including the additional 2.0x magnification introduced by an additional lens mounted between scanning unit and camera (Edmund Optics). All EBP-2::GFP imaging was performed at one frame per second (fps) and when necessary Z stacks were taken with 1 µm step size and maximum intensity projections and further processing was performed using ImageJ software (NIH). For Photoactivation of PA-GFP-TBA-1, the red mKate2 fill was used to find the neuron and select the ROI. Photoactivation was performed with the 405 nm laser set at 8–12% laser power and imaged one fps for at least 200 s on the Nikon spinning disk setup. The SRRF optimized image of UNC-44::GFP; MAPH-1, an acquisition of 150 frames at one fps was taken and processed with the nanoJ plugin  in ImageThe PVD neuron morphology, RAB-3::mCherry and MAPH-1 localization were imaged on a LSM 880 confocal Laser Scanning Microscope with Plan Apo 63×/1.2 Imm Corr objective (Zeiss). All other images were acquired using LSM700 (Zeiss) with a 63x/1.40 Oil or 40x/1.30 Oil DIC objective using 488 nm and 555 nm laser lines.Animal locomotion was imaged on a ZEISS Axio Zoom.V16.To analyze the recovery of fluorescence, 20 pixel round selections were made in regions of bleached and non-bleached GFP fluorescence as well as in the background. The average fluorescence intensity was measured using ImageJ for each frame, the background intensity was subtracted from the bleached and non-bleached areas and normalized to the five frame average prior to the bleach. The bleached area was then divided by the non-bleached area, to correct for bleaching due to imaging and the first frame after bleach was set to 0. To calculate the immobile fraction was calculated as 1-the average recover from 50 to 100 s.Template Matching or StackReg. Images and time-lapse acquisitions were rotated and if necessary flipped to have the anterior to the left and ventral side down.To correct for drift time lapse acquisitions were corrected with the ImageJ plugins Z-stacks were acquired with stepsize of 1 µm and sum-projections were made of 6 planes for the axon, the cell body or the proximal anterior dendrite. Selections of 120 × 5 µm (axon), 10 × 10 µm (cell body) and 25 × 2 µm (dendrite) were made in ImageJ and the integrated density was measured.TIRF-SIM (structured illumination microscopy) is based on exciting the sample with evanescent sinusoidal illumination patterns and detecting the fluorescence response on a camera. Our custom-built TIRF-SIM setup is shown in Each super-resolved image was reconstructed from nine raw widefield images by adapted code of fairSIM in Java as described in E. colibacteria obtained from a 0.5 l overnight culture in lysogeny broth (LB) was added as food source. Animals were incubated at 20°C shaking at 200 rpm for 6 days. To harvest the animals, the culture was transferred to 50 ml conical tubes and cooled on ice for 20 min. Animals were then pelleted by centrifugation . After aspirating the supernatant, animals were pooled in a single 50 ml tube, and washed twice in ice-cold M9. After the second wash step, animals were resuspended in 20 ml of ice-cold M9, followed by the addition of 20 ml of ice-cold 60% sucrose in H2O. After vigorous mixing of the sucrose/worm mixture, 4 ml of ice-cold M9 was gently layered on top, and the worms were centrifuged at 400 g for 2 min at 4°C. A layer of animals was now visible on top of the sucrose, while contaminants sedimented at the bottom. The sucrose float steps were performed as quickly as possible, as otherwise the layer of animals failed to form properly. To maximize recovery, 30 ml of supernatant was aspirated from the sucrose float, and distributed into four 50 ml tubes that were subsequently filled by addition of room-temperature M9. The room temperature M9 allowed the animals to digest OP50 bacteria in their intestine. The four tubes were placed on ice to cool down for 30 min, after which the animals were washed twice in lysis buffer . During the first wash the animals were again pooled into one tube. After a final wash, C. elegans was divided in four 15 ml TPX hard plastic tubes and as much lysis buffer as possible was removed, and the C. elegans pellet were frozen in liquid nitrogen and stored at −80°C.Liquid cultures were started with semi-synchronized L1 animals obtained by starvation. Grow 12 worms on 60 mm nematode growth media (NGM) plates with OP50 bacteria until there are no bacteria were left on the plates and the plates were covered with starved L1 animals. All animals were washed off the plates with M9 and transferred to a two liter Erlenmeyer flask containing 500 ml S-medium supplemented with penicillin-streptomycin  diluted 1:100, and nystatin suspension  diluted 1:1000. A pellet of OP50C. elegans pellets were lysed by sonication using a Bioruptor (diagenode) fitted with a 15 ml tube holder. For this 1 ml of worm pellet was mixed with 1 ml of 2x lysis buffer ). Pellets were gently swirled until thawed. Tubes containing the lysates were placed in the Bioruptor in a bucket filled with ice-water. Samples were lysed 15 times for 30 s, with 30 s intervals. After the third and sixth lysis, lysates were mixed by gently swirling the tube. To remove cellular debris, lysates were distributed to 2 ml Eppendorf tubes, centrifuged at 13,000 rpm for 30 min at 4°C. The supernatant was incubated with GFP-trap magnetic beads (ChromoTek) for 2 hr at 4°C. The beads were then washed three times with the lysis buffer and frozen in liquid nitrogen and stored at −80°C for mass spectrometry analysis.The C. elegans). Carbamidomethylation of cysteines was set as a fixed modification and oxidation of methionine was set as a variable modification. Trypsin was specified as enzyme and up to two miss cleavages were allowed. Data filtering was performed using percolator, resulting in 1% false discovery rate (FDR). Additional filters were search engine rank one and mascot ion score >20.Beads were resuspended in 20 µL of Laemmli Sample buffer (Biorad) and supernatants were loaded on a 4–12% gradient Criterion XT Bis-Tris precast gel (Biorad). The gel was fixed with 40% methanol/10% acetic acid and then stained for 1 hr using colloidal coomassie dye G-250 . After in-gel digestion, samples were resuspended in 10% formic acid (FA)/5% DMSO and analyzed with an Agilent 1290 Infinity  LC, operating in reverse-phase (C18) mode, coupled to an Orbitrap Q-Exactive mass spectrometer . Peptides were loaded onto a trap column  with solvent A (0.1% formic acid in water) at a maximum pressure of 800 bar and chromatographically separated over the analytical column  using 90 min linear gradient from 7–30% solvent B (0.1% formic acid in acetonitrile) at a flow rate of 150 nl/min. The mass spectrometer was used in a data-dependent mode, which automatically switched between MS and MS/MS. After a survey scan from 350 to 1500 m/z the 10 most abundant peptides were subjected to HCD fragmentation. MS spectra were acquired in high-resolution mode , whereas MS2 was in high-sensitivity mode . Raw files were processed using Proteome Discoverer 1.4 . The database search was performed using Mascot  against a UniProt database (taxonomy Hek293T cells (authenticated and tested negative for mycoplasma) were co-transfected with BirA together with the indicated bio-mCherry and gfp constructs and incubated for 24–36 hr. Cells were harvested in ice-cold PBS and lysed with lysis buffer . Cell lysates were centrifuged at 13,000 rpm for 10 min and the supernatants were incubated with Dynabeads M-280 (Invitrogen) which were pre-blocked in buffer containing 20 mM Tris, pH 7.5, 20% glycerol, 150 mM NaCl, and 10 µg chicken egg albumin for 30 min followed by two washes of 2 min with wash buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Triton X-100. After incubating for 1 hr at 4°C, beads were washed five times with washing buffer. Samples were eluted with SDS/DTT sample buffer and boiled for subsequent western blot assay. For western blot, samples were loaded onto 10% SDS-PAGE gels and transferred to nitrocellulose membrane. Membranes were blocked with 2% BSA  in PBS/0.05% Tween-20. Primary antibodies  and anti-mCherry ) were diluted in blocking buffer and incubated with the membranes overnight at 4°C, washed three times with PBS/0.05% Tween 20 and incubated with secondary IRDye 680LT or IRDye 800LT antibodies for 45 min at room temperature. Membranes were then washed three times with PBS/0.05% Tween 20 and scanned on Odyssey Infrared Imaging system (LI-COR Biosciences).For all quantifications, n = the number of individual animals analyzed. Statistical analyses were performed using GraphPad Prism software version 8.0. All bar diagrams represent mean SD statistics was performed using a Kruskal-Wallis test with Dunn’s correction, except for the analysis of Data in  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:C. elegans as a model organism, this study demonstrates that microtubules need to be anchored at the cell cortex of axons and dentrites to prevent kinesin-1 driven microtubule sliding that would otherwise perturb microtubule polarity in neurons. Moreover, the authors have worked out the molecular details of this cortex attachment, providing new interesting insight into the molecular mechanism of the establishment of neuron polarity.Using Decision letter after peer review:eLife. Your article has been reviewed by Vivek Malhotra as the Senior Editor, a Reviewing Editor, and three reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Peter W Baas (Reviewer #1); Vann Bennett (Reviewer #3).Thank you for submitting your article "Cortical anchoring of the microtubule cytoskeleton is essential for neuron polarity and functioning" 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.Summary:C. elegans. This would cause them to slide if not for membrane-associated attachments of the microtubule bundles. The authors work out in meticulous detail the molecular components of the attachments. They demonstrate when the relevant molecular links are disrupted, the sliding forces of kinesin-1 cause massive disorder of microtubules, which results in morphological defects with functional consequences. The experiments overall are well designed and the results are striking. The observations bring novel insights into the architecture of the microtubule cytoskeleton in neurons. The main areas for improvement concern the presentation. More detail should be added explaining how experiments were done so that the work is more accessible to the non-specialist reader and some claims require modifications to accurately reflect the data as presented.This manuscript describes very interesting work that demonstrates that kinesin-1 generates substantial forces on the microtubules in the axons and dendrites of Essential revisions:The presentation of the work requires editorial changes. The authors should please describe experiments in sufficient detail, so that they can be reproduced and are also accessible to the non-specialist, should pay attention to the correct use of terms, and make sure that claims are fully supported by their data.1) It would be helpful for readers unfamiliar with the various polypeptides used here to include their domain maps.2) Particularly the experiments in Figure 2 are difficult to follow. Please detail how proteins were expressed and isolated, provide conditions for reassembly, and include relevant references. Also, please label relevant polypeptides on the gel panels. The goal should be that someone else can reproduce these experiments. Please ensure that it is always clear whether experiments are performed under conditions of overexpression or whether native protein levels are investigated.3) Please always state clearly at which stage the animals are when used in experiments .4) The wording about the periodicity of the membrane attachments shown in Figure 4 should be tempered a bit. The effect doesn't seem as dramatic as actin rings, for example. Moreover, finding the same periodicity as β-spectrin does not demonstrate that the two are connected . For this conclusion to be drawn, colocalisation between β-spectrin and the components of the complex should be demonstrated. Please also explain how the neurite was chosen for analysis and in which part of the neurite the periodicity is analysed? Is the neurite fragment close to the cell body, where is the AIS located?5) With regard to Figure 6 and related wording, the authors should avoid using the term dynamics if they mean sliding. That will confuse some readers.6) Figure 6: The authors do not discuss that the photoactivated microtubules are visibly weaker in the mutant strains than in the wild type animals. Is there an effect on microtubule dynamics in those mutants? Given that CRMP-2 has been reported to have microtubule assembly-promoting properties , the potential impact UNC-44-119-33 complex disruption on microtubule dynamics should be discussed.7) Figure 7: Does the rescue of microtubule sliding rescue the physiological phenotypes of the UNC-44-119-33 complex mutants, for instance neuronal architecture and worm movements? Summary:C. elegans. This would cause them to slide if not for membrane-associated attachments of the microtubule bundles. The authors work out in meticulous detail the molecular components of the attachments. They demonstrate when the relevant molecular links are disrupted, the sliding forces of kinesin-1 cause massive disorder of microtubules, which results in morphological defects with functional consequences. The experiments overall are well designed and the results are striking. The observations bring novel insights into the architecture of the microtubule cytoskeleton in neurons. The main areas for improvement concern the presentation. More detail should be added explaining how experiments were done so that the work is more accessible to the non-specialist reader and some claims require modifications to accurately reflect the data as presented.This manuscript describes very interesting work that demonstrates that kinesin-1 generates substantial forces on the microtubules in the axons and dendrites of Essential revisions:The presentation of the work requires editorial changes. The authors should please describe experiments in sufficient detail, so that they can be reproduced and are also accessible to the non-specialist, should pay attention to the correct use of terms, and make sure that claims are fully supported by their data.C. elegans names.As discussed below we have now better described the experiments and modified the wording for some of the conclusions to better reflect the data. Moreover, we changed the color use in Figure 3 and Figure 5 to be consistent to the model and added the model to the figures to help the reader through the data. In addition, in Figure 4, Figure 6 and Figure 7 we added the mammalian names next to the 1) It would be helpful for readers unfamiliar with the various polypeptides used here to include their domain maps.We added the proteins domain maps to Figure 2.2) Particularly the experiments in Figure 2 are difficult to follow. Please detail how proteins were expressed and isolated, provide conditions for reassembly, and include relevant references. Also, please label relevant polypeptides on the gel panels. The goal should be that someone else can reproduce these experiments. Please ensure that it is always clear whether experiments are performed under conditions of overexpression or whether native protein levels are investigated.We clarified the pull-down experiments in the legend and better labeled the relevant proteins and immunoblot conditions in the figure.3) Please always state clearly at which stage the animals are when used in experiments .We indicated the age of the animals in each figure.4) The wording about the periodicity of the membrane attachments shown in Figure 4 should be tempered a bit. The effect doesn't seem as dramatic as actin rings, for example. Moreover, finding the same periodicity as β-spectrin does not demonstrate that the two are connected . For this conclusion to be drawn, colocalisation between β-spectrin and the components of the complex should be demonstrated.We agree with the comment and have toned down the language to reflect the uncertainty about the physical interaction from these superresolved images.Please also explain how the neurite was chosen for analysis and in which part of the neurite the periodicity is analysed? Is the neurite fragment close to the cell body, where is the AIS located?C. elegans does not have an axon initial segment and thus precludes any analysis accordingly. For all conditions tested, we choose ROIs at random locations along the neurite with a minimum length of 2 μm. However, the labeling structure was variable along the neurite, thus only sections containing a visible periodicity were analyzed for the quantification. We clarified this in the figure legend.5) With regard to Figure 6 and related wording, the authors should avoid using the term dynamics if they mean sliding. That will confuse some readers.We changed “dynamics” to “mobility”.6) Figure 6: The authors do not discuss that the photoactivated microtubules are visibly weaker in the mutant strains than in the wild type animals. Is there an effect on microtubule dynamics in those mutants? Given that CRMP-2 has been reported to have microtubule assembly-promoting properties , the potential impact UNC-44-119-33 complex disruption on microtubule dynamics should be discussed.unc-33, unc-119 and unc-44 mutants and observed small changes compared to wild type .Indeed, the PA-GFP-tubulin signal was generally weaker in the mutants, which may suggest that there are fewer microtubules. We commented on this in the manuscript. Moreover, we quantified the microtubule dynamics in the 7) Figure 7: Does the rescue of microtubule sliding rescue the physiological phenotypes of the UNC-44-119-33 complex mutants, for instance neuronal architecture and worm movements?kinesin-1[floxed];unc-33 double mutant, which is not be rescued. We added this to the manuscript .As the full loss of function mutant for kinesin-1 is not viable, we here depleted kinesin-1 in a cell specific manner (the skin) using lox-sites approach. Therefore, we cannot assess the rescue of the worm locomotion. However we did analyze the microtubule polarity in the


We also found that the expression level of most PPR genes decreased with the temperature rises, which may contribute to the decline of RNA editing efficiency at high temperature. Our findings suggested that the RNA editing events were very sensitive to heat stress; the changes of amino acid in RNA editing genes may contribute to the stress adaption for grape.RNA editing is a post-transcriptional process of modifying genetic information on RNA molecules, which provides cells an additional level of gene expression regulation. Unlike mammals, in land plants, RNA editing converts C-to-U residues in organelles. However, its potential roles in response to different stressors  remains unclear. Grape is one of the most popular and economically important fruits in the world, and its production, like other crops, must deal with abiotic and biotic stresses, which cause reductions in yield and fruit quality. In our study, we tested the influence of the environmental factor temperature on RNA editing process in the whole mRNA from grape organelle. In total, we identified 122 and 627 RNA editing sites in chloroplast and mitochondria respectively with the average editing efficiency nearly ~ 60%. The analyses revealed that number of non-synonymous editing sites were higher than that of synonymous editing sites, and the amino acid substitution type tends to be hydrophobic. Additionally, the overall editing level decreased with the temperature rises, especially for several gene transcripts in chloroplast and mitochondria (The online version of this article (10.1007/s10142-019-00727-7) contains supplementary material, which is available to authorized users. RNA editing is a post-transcriptional process that could alter the nucleotide sequence of gene transcripts, potentially diversifies the transcriptome and proteomes beyond the genomic blueprint  proteins with several additional non-PPR protein factors that are both encoded in the nuclear genome (Ichinose and Sugita Vitis vinifera L.) is one of the most popular and economically important fruits in the world. Grape production, like other crops, however, must deal with various abiotic and biotic stresses, which cause its reductions in yield and fruit quality. It remains unclear that whether the RNA editing events could response to various stresses, such as temperature and water. In this study, we tested the influence of the environmental factor temperature on RNA processing based on whole mRNA deep-sequencing data. With the temperature increasing, reduced RNA editing efficiency was detected significantly, which may resulted from temperature-sensitive expression or stability of the RNA editing factors. Environmental cues, in this case temperature, rapidly reprogram the grape transcriptome through RNA editing, presumably resulting in altered proteomic ratios of edited and unedited proteins. Our findings also suggest that the changes of amino acid in these genes may contribute to the stress adaption for grape.Grape . The whole genome and annotation file of grape were also downloaded from the NCBI data repository for expression analysis  were downloaded from We performed the RNA editing sites identification for grape mitochondria and chloroplast separately, and the identification process was split into two steps. For each organelle genome, firstly, we aligned the transcriptome data against the reference and called SNPs; secondly, the RNA editing sites were identified based on the called SNPs-calling results. In order to increase the sequencing depth, we merged the three duplicates under one condition into one sample. Take mitochondria as an example, the quality control of paired-end Illumina sequencing data were firstly evaluated by NGSQCToolkit and low-quality sequence data were filtered out  (Patel and Jain www.ncbi.nlm.nih.gov/projects/Sequin/table.html) as inputs. Afterwards, the raw variants are filtered by ten rule-dependent filters and statistical filters to reduce the false positives as the following steps: (1) quality control filter, (2) depth filter (DP > 4), (3) alt proportion filter , (4) multiple alt filter, (5) distance filter, (6) spliced junction filter, (7) indel filter, likelihood ratio (LLR) test filter (LLR <10), (8) Fisher’s exact test filter , and (9) complicated filter model. Finally, all filtered RNA editing sites were identified and their corresponding annotation information files were also generated at the same time.Based on the SNP-calling results (in “VCF” format) and genome annotation files (in “tbl” format), we utilized the REDO tool to identify the RNA editing sites under default parameter values . In order to decipher the tendency of RNA editing efficiency for each organelle, cluster analysis and heatmap plotting were also provided based on the RNA editing efficiency matrix. Values of editing efficiency matrix were normalized by subtracting the row-wise mean from the values in each row of data and multiplying all values in each row of data by standard deviation value. A heatmap was plotted in all samples using “pheatmap” function in R, the distance matrix of different samples was calculated using “dist” function with the default Euclidean method, and the hierarchical clustering was computed using “hclust” function. Genes with significantly changes in editing sites were picked out for further functional analysis.For each organelle, the resulted editing sites were used for further statistics and feature analysis, including statistics of editing number, editing type, codon position, amino acid changes, and involved genes. The value of RNA editing efficiency at one site was expressed as the proportion between edited transcripts and total transcripts. If one site was edited, the C/G base (wild type) should be altered to the T/A base (edited type), since one editing site could be detected hundreds of times via sequencing, the number of wild type (C/G) or edited type (T/A) of bases could then be counted at this particular site, then the editing efficiency at one site could then be calculated by the formula: depth of edited bases (T and A)/total read depth of bases. Furthermore, we compared the RNA editing efficiency between each two conditions and identified the editing sites with statistical significance  of Two-tailed Wilcoxon rank-sum test was used to perform the pairwise comparison of RNA editing efficiency between each two neighboring conditions. As for the pairwise comparison for each editing site, Fisher’s exact tests were used.There were a total of 12 RNA-seq samples that treated with different temperatures  in our study; each condition has three replicates. We aligned the transcriptome data to the organelle reference genome respectively. The size of mitochondrion genome is about 773,279 bp, encoding 158 genes, for each sample; there were about 80,000 reads mapped to reference with mapping rate about 0.45% (std = 0.185). For chloroplast, the size of its genome is about 160,928 bp, encoding 120 genes. There were about 400,000 reads mapped to reference with mapping rate about 2.13% (std = 0.7681). Generally speaking, more genes are located in mitochondrion compared with chloroplast, and then, the mapping reads of mitochondrion should be more than that of chloroplast; however, it turned out, an opposite result beyond our expectation, chloroplast has a higher mapping rate significantly, which may be due to sources of samples; more chloroplast mRNA in leaves were extracted and sequenced in this study. The statics of reference-guided mapping rate was shown in Fig. In order to increase the sequencing depth and reliability of editing sites, the resulting bam files of three replicates under one condition were merged for subsequent identification of RNA editing sites. Based on the SNP-calling results and organelle genome annotation file, a total of 749 RNA editing sites were identified in both organelles; however, a few editing sites only appeared under certain condition. Take samples at 25 °C temperatures in chloroplast as illustration, the attributes of RNA editing sites were shown in Fig. SWe also found that RNA editing occurred in second codon position was mainly the largest in both organelles, followed by first codon position except three conditions  of chloroplast, as shown in Fig. p < 0.05) was detected in the comparison of RNA editing efficiency between each two neighboring conditions except for T40-T45 of chloroplast, as shown in Fig. We also performed statistics and cluster analysis for the RNA editing efficiency. On the whole, the average efficiency of RNA editing sites was about 0.56. For chloroplast and mitochondrion, the average RNA editing efficiency was 0.59, 0.58, 0.48, and 0.42 and 0.64, 0.61, 0.58, and 0.57 respectively under four conditions . With the increase of temperature, the average editing efficiency both declined gradually, as shown in Fig. maturase K (matK) and NADH dehydrogenase subunit 2 (ndhB) genes; both genes have four changed editing sites. All the sites of ndhB gene  were C-to-U editing type and demonstrated the trend of decreasing; the corresponding amino acid changes were His-to-Tyr, Pro-to-Leu, Ser-to-Phe, and Pro-to-Leu, the four amino acids all changed to be hydrophobic. Whereas three sites of matK gene demonstrated the trend of decreasing, one site showed a rising trend. For mitochondrion, more genes have editing efficiency changed sites, such as NADH dehydrogenase gene family (nad4/5/7), ATPase gene family (atp6/9), heme trafficking system membrane gene family (ccmB/C/FC/FC/FN), mitochondrial Cytochrome c oxidase gene family (cox1/2/3), and ribosomal gene family (rps4/7).We annotated the involved genes with editing sites of “step-up” and “step-down” RNA editing efficiency. Hence, a total of 68 genes were annotated , as shown in Fig. ndhB gene encodes components of the thylakoid ndh complex which purportedly acts as an electron feeding valve to adjust the redox level of the cyclic photosynthetic electron transporters. ndhB gene contains by far the higher number of editing sites, 10 in Arabidopsis, probably because the proofreading mechanism that ensures identical sequences of the two inverted repeated regions of plastid DNA makes improbable the fixation of C-to-U back mutations . Compared with 26 downregulated PPRs, there were still 5 upregulated PPR proteins, such as PPR proteins: GSVIVG01031345001 (PP284_ARATH), GSVIVG01012156001 (PP327_ARATH), GSVIVG01008664001 (PP425_ARATH), revealing different functions of PPR proteins. Hence, on a whole level, there is a positive correlation between the PPR proteins expression and RNA editing efficiency; it is reasoned that the reduced RNA editing efficiency may result from the dropped expression of most PPR proteins.Transcriptome analysis of RNA-seq data was also performed for measuring and comparing the levels of gene expression of RNA editing genes and PPR genes. However, for RNA editing genes, no expression difference was detected under different temperatures, suggesting that temperature stress only affect the RNA editing events and has no influence on expression level for those genes. In order to investigate the reason for the reduced RNA editing efficiency with the increasing of temperature, we also evaluated the expression of RNA editing genes and PPR proteins. After blast searching, a total of 419 proteins were identified as PPRs, and 414 PPR proteins were expressed. Interestingly, the expression level of most PPR proteins demonstrated a downregulated tendency along with the increasing of temperature, as shown in Fig. kinetoplastid protozoa followed by its reporting in a wide range of organisms  deficiency in Drosophila melanogaster indicated that RNA editing plays an integral role in temperature adaptation by sensing and acting globally on RNA secondary structure  mRNAs. Most of the C-to-U changes in the protein coding regions tend to locate at first, second positions, and the physicochemical property of amino acids was mostly modified. In addition, consistent with previous studies, we also found that amino acid changes tend to be hydrophobic; therefore, plant RNA editing is believed to act as an additional proofreading mechanism to generate fully functional proteins . Environmental cues, in this case temperature, rapidly reprogram the grape organelles transcriptome through RNA editing, presumably resulting in altered structure or function of edited proteins. However, the underlying molecular mechanisms of stress adaptation for RNA editing still require further investigation.C-to-U RNA editing is a highly conserved process that post-transcriptionally modifies mRNA, generating proteomic diversity. However, its potential role in response to different stressors  and growth development remains unclear. Our study suggested that RNA editing was responsive to environmental inputs in the form of temperature alterations. Using the angiosperms grape, we identified 122 and 627 RNA editing sites in chloroplast and mitochondria respectively with the average editing efficiency nearly ~ 60% and detected that acute temperature alterations within a normal physiological range result in substantial changes in RNA editing levels. Additionally, the analyses also revealed that number of non-synonymous editing sites were higher than that of synonymous editing sites, and the amino acid substitution type tends to be hydrophobic. The response of reduced RNA editing efficiency to temperature alterations further confirmed the relationship between environmental factors and RNA editing, which might be through intrinsic thermo-sensitivity of the RNA structures that direct editing or due to temperature-sensitive expression of the RNA editing enzyme Additional file 2:Table S2. A matrix for editing allele proportion of RNA editing sites in all chloroplast samples. (XLSX 16 kb)Additional file 3:Table S3. Pairwise comparison of editing allele proportion in all chloroplast samples. (XLSX 76 kb)Additional file 4:Table S4. Information of RNA editing sites in all mitochondria samples. (XLSX 140 kb)Additional file 5:Table S5. A matrix for editing allele proportion of RNA editing sites in all mitochondria samples. (XLSX 40 kb)Additional file 6:Table S6. Pairwise comparison of editing allele proportion in all mitochondria samples. (XLSX 359 kb)Additional file 7:Table S7. Gene differential expression analysis between samples under two conditions . (XLSX 12 kb)Additional file 8:Fig. S1. The attributes of RNA editing sites in chloroplast illustrated by samples at 25°C temperatures. Fig. S2. Reduced efficiency patterns of RNA editing sites in mitochondria. (DOCX 852 kb)


These findings assign a novel function for RNase 1, and position it as a strong candidate for generation of tRNA halves and Y RNA fragments in biofluids.Extracellular RNAs participate in intercellular communication, and are being studied as promising minimally invasive diagnostic markers. Several studies in recent years showed that tRNA halves and distinct Y RNA fragments are abundant in the extracellular space, including in biofluids. While their regulatory and diagnostic potential has gained a substantial amount of attention, the biogenesis of these extracellular RNA fragments remains largely unexplored. Here, we demonstrate that these fragments are produced by RNase 1, a highly active secreted nuclease. We use RNA sequencing to investigate the effect of a null mutation of RNase 1 on the levels of tRNA halves and Y RNA fragments in the extracellular environment of cultured human cells. We complement and extend our RNA sequencing results with northern blots, showing that tRNAs and Y RNAs in the non-vesicular extracellular compartment are released from cells as full-length precursors and are subsequently cleaved to distinct fragments. In support of these results, formation of tRNA halves is recapitulated by recombinant human RNase 1 in our  Cells secrete a variety of RNA molecules to the extracellular environment. These RNAs can be present outside cells in extracellular vesicles (EVs) , in lipoc, resulting in protection of cells from apoptosis following osmotic stress  noncoding RNAs. They were initially identified as components of ribonucleoprotein particles that form autoantigens in systemic lupus erythematosus . Intraceet al. , who shoet al.  and squaet al. . Furtheret al. .In spite of their regulatory and diagnostic potential, there is little knowledge about the biogenesis of extracellular tRNA and Y RNA fragments. Here, we show that tRNA halves and distinct Y RNA fragments in the extracellular environment are produced through the action of RNase 1, a broad specificity ribonuclease with proposed role as scavenger of extracellular RNA . Our res2. Cells were cultured in RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), penicillin (100 units/ml), streptomycin (100 μg/ml) and glutamine (4 mM) (Thermo Fisher Scientific), or in QBSF-55 serum-free medium . K562 cell line identity was verified by short tandem repeat profiling, and cells tested negative for the presence of Mycoplasma. Cell viability during culturing and medium conditioning was >95%, as monitored by trypan blue staining.Cells were grown in a humidified incubator at 37°C with 5% COStable Cas9 expression was established in the K562 human chronic myelogenous leukemia cell line (ATCC CCL-243) by lentiviral transduction of the pLentiV-Cas9-puro transfer vector. Lentiviral particles were produced by transfection of pLentiV-Cas9-puro , psPAX2 and pCMV-VSV-G plasmids into the 293FT cell line (Thermo Fisher Scientific). Upon 48 h post-infection (high multiplicity of infection), cells were subjected to puromycin selection to obtain an antibiotic-resistant bulk population.RNASE1 mutant cell lines, six sgRNA constructs  (RNASE1 null clone that was chosen for use in the present work was generated with sgRNA RNASE1_471.58 (RNASE1 that were used in this work represent a bulk population of GFP-positive K562-Cas9 cells infected with sgRNA Rosa26_(Mm). All cell lines were characterized by Sanger sequencing, PCR and RT-qPCR (To generate nstructs  were desa26_(Mm) . This sg1_471.58 . Cells c RT-qPCR .5 cells/ml. Cultures were spun 10 min at 300 g, the medium was removed, and the cells were gently resuspended in 10 ml of serum-free RPMI. The cell suspension was spun again as above, supernatant was aspirated, and cells were resuspended gently in serum-free RPMI to a density of ∼2.75 × 105 cells/ml. Cells were then returned to the incubator for 24 h, followed by collection of conditioned medium.For conditioning of RPMI medium, cells were grown in complete RPMI to a density of ∼2.75 × 105 cells/ml. Cells were then diluted 15-fold by addition of fresh medium, grown to a density of ∼4 × 105 cells/ml, followed by collection of conditioned medium.Cells grown in QBSF-55 serum-free medium were cultured for at least seven passages before medium conditioning experiments, to ensure full adaptation and normal growth. For medium conditioning in QBSF-55, cells were cultured to a density of ∼2 × 10Extracellular vesicles were collected essentially as previously described . Briefly2 and kept at −80°C until use. RNA was isolated with a mirVana miRNA isolation kit (Thermo Fisher Scientific). The manufacturer's protocol for total RNA isolation was used, with the following exceptions. For EV suspensions and non-EV conditioned medium, RNA isolation was started by addition of two volumes of mirVana Lysis Buffer, and volumes in subsequent steps were scaled up accordingly; organic extraction was spun at 9400 g instead of 10 000 g. RNA intended for sequencing was treated with DNase by using a TURBO DNA-free kit (Thermo Fisher Scientific). Up to 10 μg of RNA was incubated with 2 units of TURBO DNase in 1× DNase buffer (kit-supplied) for 25 min at 37°C. Reactions were inactivated by using the kit-supplied inactivation reagent. RNA was ethanol-precipitated, resuspended in diethyl pyrocarbonate (DEPC)-treated water (Thermo Fisher Scientific) and quantitated using a Qubit fluorometer (Thermo Fisher Scientific). RNA size profiles were analyzed on a Bioanalyzer instrument (Agilent Technologies), using RNA 6000 Pico chips for extracellular RNA samples and RNA 6000 Nano chips for cell RNA samples.Before RNA isolation, EV suspensions in PBS (250 μl) were treated with a combination of RNase A and RNase T1 before RNA isolation by addition of 10 μl RNase Cocktail (Thermo Fisher Scientific) and incubation at 37°C for 30 min. Non-EV conditioned medium was concentrated at 4°C from 15 ml to 1 ml on 3 kDa cutoff Amicon filters (Millipore-Sigma), as per manufacturer's instructions. Cells for RNA isolation were washed once in 1 ml ice-cold PBS and pelleted by centrifuging 7 min at 300 g at 4°C. Pellets were flash frozen in liquid NAll RNA samples for sequencing were decapped with Cap-Clip acid pyrophosphatase (Cellscript). RNA (up to 1 μg in 21 μl DEPC-treated water) was incubated 2 min at 85°C and transferred to ice for 1 min. 2.5 μl of manufacturer supplied Cap-Clip buffer, 20 units of RNase inhibitor  and 2.5 units of Cap-Clip enzyme were added and the reaction  was incubated for 1 h at 37°C. Following incubation, RNA was phenol-chloroform extracted and ethanol-precipitated. Unless otherwise noted, RNA samples were also treated with T4 polynucleotide kinase . RNA (up to 1 μg in 37.5 μl DEPC-treated water) was combined with 5 μl of manufacturer supplied PNK buffer and 2.5 μl (25 units) PNK. The reaction was incubated 10 min at 37°C, then 5 μl 10 mM ATP was added, followed by 30 min incubation at 37°C. RNA was phenol-chloroform extracted and ethanol-precipitated. Where applicable, the aforementioned PNK treatment was replaced by treatment with the rtStar tRF&tiRNA Pretreatment Kit (Arraystar) as per the manufacturer's protocol. The kit workflow includes 3′ deacylation, followed by end-healing with PNK in the presence of ATP and demethylation by AlkB.Starting material for RNA sequencing library preparations was 100 ng (before pretreatments as described above). Libraries were prepared using the NEXTFLEX Small RNA-Seq Kit v3 (Bioo Scientific) following the manufacturer's protocol. Reverse transcription reaction bead cleanup was performed in accordance with the manufacturer's alternative protocol without size selection. Libraries were amplified using 16 PCR cycles. Products were size selected on 2% agarose gels to include products larger than the adapter dimer and up to 350 bp (corresponding to RNA inserts up to ∼225 nt). The resulting libraries were sequenced on the Illumina NextSeq 500 platform.https://www.gencodegenes.org/) and used for all analyses in this study. A customized transcript annotation file was constructed as described below. The GENCODE comprehensive gene annotation for the primary assembly of the human genome GRCh38.p12 Release 28 was used as the main source of gene annotations. This annotation does not contain tRNA gene annotations or annotations of RNY5 and its pseudogenes. Therefore, it was complemented with the annotations of tRNA genes from GtRNAdb . Coordinates of RNY5 and its pseudogenes were converted from the gene annotation for the previous genome assembly (GRCh37.p13 Release 19) with the LiftOver tool in UCSC Genome Browser (https://genome.ucsc.edu/cgi-bin/hgLiftOver). The resulting customized transcript annotation file is available upon request. Genome indices were generated with STAR v.2.6.1d , 7.8 M urea gels. After electroblotting and UV-crosslinking to nylon membranes , RNA was detected using end-labeled DNA probes . HybridiVesicle-depleted conditioned medium was pre-incubated for 5 min at 37°C, followed by addition of recombinant human RNase 1 (Bon Opus Biosciences) to the indicated concentration. After 30 min incubation at 37°C, RNA was isolated as described above. RNA was also isolated from vesicle-depleted conditioned medium that was left on ice with no added enzyme. Isolated RNA was analyzed by northern blot.Pancreatic ribonucleases (RNases) are a family of secreted enzymes. Among human members of this family, RNase 1 has a unique combination of high catalytic activity and robust expression in a wide variety of tissues  and Supp. In addiRNASE1 gene in the human leukemia cell line K562, which expresses RNASE1  and non-EV conditioned media  showed a clear shift toward larger sizes in the absence of active RNase 1  and tRNA-Glu-CTC 17.3%), and 3′ halves derived from tRNA-Asp-GTC 25.8%) .The data reported in this paper were deposited in the Gene Expression Omnibus (GEO) database, gkaa526_Supplemental_FilesClick here for additional data file.


This paper describes a web based tool that uses a combination of sonification and an animated display to inquire into the SARS-CoV-2 genome. The audio data is generated in real time from a variety of RNA motifs that are known to be important in the functioning of RNA. Additionally, metadata relating to RNA translation and transcription has been used to shape the auditory and visual displays. Together these tools provide a unique approach to further understand the metabolism of the viral RNA genome. This audio provides a further means to represent the function of the RNA in addition to traditional written and visual approaches.Sonification of the SARS-CoV-2 genomic RNA sequence results in a complex auditory stream composed of up to 12 individual audio tracks. Each auditory motive is derived from the actual RNA sequence or from metadata. This approach has been used to represent transcription or translation of the viral RNA genome. The display highlights the real-time interaction of functional RNA elements. The sonification of codons derived from all three reading frames of the viral RNA sequence in combination with sonified metadata provide the framework for this display. Functional RNA motifs such as transcription regulatory sequences and stem loop regions have also been sonified. Using the tool, audio can be generated in real-time from either genomic or sub-genomic representations of the RNA. Given the large size of the viral genome, a collection of interactive buttons has been provided to navigate to regions of interest, such as cleavage regions in the polyprotein, untranslated regions or each gene. These tools are available through an internet browser and the user can interact with the data display in real time.The auditory display in combination with real-time animation of the process of translation and transcription provide a unique insight into the large body of evidence describing the metabolism of the RNA genome. Furthermore, the tool has been used as an algorithmic based audio generator. These audio tracks can be listened to by the general community without reference to the visual display to encourage further inquiry into the science. Modern computers have had a great impact on biological experimentation and data analyses to reveal otherwise hidden patterns in complex data. This is apparent in the field of genomic data analyses. The viral genome of the first patient suffering from COVID-19 was submitted to GenBank . Within This paper demonstrates that sonification of RNA sequence data may also be useful to understand how the genome functions. The audio is generated using two approaches. The rules governing gene expression have been applied to the process of generating a linear audio stream similar to the expression of a linear sequence of amino acids. These methods are based on our previous approach to sonify DNA sequences . These mThe systematic and reproducible representation of data as sound is increasingly becoming a adjunct to the traditional visualization techniques of data inspection and analysis , 10. In Genomic data has also provided a good candidate for sonification. These studies include sonification of the spectral properties of DNA, molecular analyses , 16 and Previous studies into DNA sonification for sequence analyses  demonstrThe web tool described in this paper  operatesThe most fundamental building block of RNA is an individual nucleotide and these were sonified as one of four individual notes whereas di-nucleotides were sonified as one of 16 notes and together these were panned left and right in the auditory display. Another characteristic of nucleic acid sequences which is often used as a metric of genome status is the GC content which is often represented as a ratio. Typically in Coronavirus the count of U is above average and C is below average whereas A is preferred over G  leading In the translation mode, codons represent an important feature of RNA and these were sonified as 20 notes when representing translation into amino acids. No distinction was made between the start methionine or that which occurs in the body of the peptide sequence. Additionally, stop codons were sonified as an additional note since these are highly significant in the function of the genome. Overlapping codons in each of the three reading frames were sonified during translation to detect ORF’s in either frame. An important consideration in the modelling of translation was to use the start and stop codons in each reading frame to trigger or halt the audio derived from other codons. Additionally, in the visual display the audible codons were shown using the one letter amino acid representation. Using this simple method all gene sequences reported in the GenBank metadata were accurately represented in both the audio and visual displays. Additionally, all open reading frames throughout the RNA genome are shown and sonified. However, only open reading frames that correlate with the known metadata (gene sequences) were labelled in the visual display. This is consistent with prior approaches of mapping either individual bases , codons In the display representing transcription, codons per se were not considered. Instead tri-nucleotide features were considered for sonification, however, these were considered to be positioned adjacent in the sequence rather than overlapping. Given that there are 64 different tri-nucleotides it is not possible to use a traditional scale. A traditional piano consists of 7 octaves plus a minor third (88 notes). Given that there are 7 scale notes in an octave it would require over 9 octaves to accommodate 64 trinucleotides. Using synthesised notes could overcome this limitation but this would entail playing shrill high pitched notes that would be grating to the ear. Therefore, linear mapping of 64 codons to individual notes was avoided. In the transcription display, tri-nucleotides were mapped to 16 individual notes since only the first and third position in each was considered. Since trinucleotides play no functional role in the process of transcription there was no loss of information content using this approach and the audio could be designed to complement the single nucleotides and di-nucleotides in the audio stream and avoid the mapping to shrill notes. Additionally, tri-nucleotides were not mapped to start or stop functionality and these are audible throughout the entire genome. Their occurrence had no further effect in the auditory display.Metadata specific to the Coronavirus SARS-CoV-2 sequence was used to supplement the audio generated from the intrinsic characteristics of the RNA sequence. Audio from un-translated sequences between the open reading frames were mapped to an audio stream at a reduced tempo so that they were more clearly distinguished from the coding regions. Additionally, the viral genome is known to contain 10 transcriptional regulatory sequences (TRS) and five known stem loop (SL) structures known to play a role in the function of the genome  and theiThe genome codes for a large polyprotein from a large open reading frame. This polyprotein is thought to be cleaved into 16 individual polypeptides (often referred to as NSP proteins) and the occurrence of the known cleavage sites was sonified. In addition to generating a short burst of notes, cleavage regions were also used to pause the progression of the play-head for a second or so by slowing the tempo to one tenth of the coding tempo. This effectively highlights the transition from one NSP sequence to the next. The occurrence of three or more identical nucleotides was also sonified since these are easy to detect by eye and their sound may help the user to keep track of where they are in the display.Audio generated from each of these sequence motifs and metadata were combined to create a complex auditory display to represent either transcription or translation. As the audio is played a sliding window of 60 nucleotides is shown on the screen. At any point in time the first nucleotide in the visual play-head can be heard in the auditory display. Other sequence features are determined relative to the position of this nucleotide.To play the entire genome takes approximately 96 min in translation mode which corresponds to approximately five nucleotides per second. This is slower than cellular translation which is thought to proceed at approximately 30 nucleotides per second , howeverThree sets of interactive buttons  was also used to reinforce the foundation of the basic scale harmony. The G or C bases, as nucleotide, di-nucleotide or trinucleotides were each matched to higher octaves and A and U were mapped to lower octaves. This was done consistently between these audio streams in an attempt to harmonise the otherwise random note selection based on sequence information. An exception to this principle was made for start and stop codons which were mapped to higher pitches than GC rich codons so that their occurrence was easily perceived in the auditory display (since higher pitched notes are perceived to be louder). Given that these codons are used to trigger and halt individual audio streams this approach further emphasises the occurrence of an open reading frame.The wider note range of the codons (20 notes) were used to introduce leading tones that often sound more dissonant and add complexity to the harmonic spectrum. This allows them to be easily discerned above the landscape tones of the simpler motifs. Lastly, less frequent audio from dispersed regions of the genome e.g. TRS or stem-loop (SL) motifs were pitched at the highest octave ranges or more dissonant notes within the diatonic scale to highlight their occurrence. All of this was done within a mode of the diatonic major scale. Translation was played in Bb Aeolian  whereas transcription was played in C Lydian . The parameters for mapping of each RNA feature into an audio stream is summarised in Table Each nucleotide generates a note on every beat whereas each di-nucleotide generates a note every second beat. Each codon (in an ORF) generates a not every third beat. Together these notes are syncopated to create a characteristic sound during peptide translation that is distinct from the surrounding untranslated region. Audio from the GC tracks are only triggered when the GC ratio changes by an increment of 0.1. If a note sequence has identical adjacent notes then the length of the note is extended rather than being repeated. This creates space and clarity for other notes layered in the auditory display.Translation of the genomic RNA leads to expression of a large polyprotein following ribosome binding to the 5′ prime untranslated region. However, from this genomic template the subsequent genes downstream from the polyprotein cannot be directly expressed presumably due to the stop codon at the end of the gene. In the display the sonification also stops at this point, however, play can be resumed to inspect the downstream sequence. Additionally, the tempo of the untranslated regions are slower than that of the coding regions so that the tempo increases as the play-head (in place of the ribosome) reads into a gene sequence. This was implemented to help the user distinguish between different sequence types during the display of translation.One of the more interesting characteristics of the viral genome is the phenomena of discontinuous transcription whereby a template switch occurs during the synthesis of sub-genomic negative-strand RNA’s . VariousThe website does not rely on a server and instead the entire RNA sequence is downloaded into the client browser when the page is loaded. All code is written in JavaScript and runs within the client browser. The React framework was used to create the environment state whereby each iteration of state represents a sliding window to the next base. Redux was also used to help manage state. Audio is generated in real time within the client browser using Tone.js. The Reactronica framework  was usedTranslation of the viral polyprotein is known to be subject to a frameshift mutation and since this does not follow the normal rules of gene expression a conditional expression was used to change the display for that instance so that the translated protein in frame 2 shifts to frame 1 in both the visual and auditory display.To understand the function of the viral plus RNA strand the information needs to be processed in the 5′ to 3′ direction during translation and in the reverse 3′ to 5′ direction for transcription (whereby nucleotides are extended to the newly synthesised minus strand at the 3′ end). In this study an auditory display of the sequence was generated with a sliding window moving in either direction. Processing of information within the sliding window was used to generate a synchronised auditory and visual display. This is advantageous since it mimics the behaviour of biological processes within the cell. To further emulate translation the generation of audio was triggered by start codons and silenced by stop codons. Furthermore, the visual display shows all possible peptide sequences and these are aligned with the RNA sequence being processed. From the sequence data alone the tool was able to detect and display all known open reading frames and metadata was used solely to label these in the display. Other open reading frames were detected throughout the genome in the displays, however, since these are not downstream of an in-frame ribosome binding site no claim is made that these are actually translated.High resolution analysis of gene expression in Coronavirus genomes has detected ribosome protected fragments which map to non-canonical ORF’s, these may be novel protein-coding ORFs and short regulatory uORFs. The tool highlights the occurrence of one such uORF of 30 nucleotides (including the stop codon) in the 5′ untranslated region downstream of TRS1  that is This uORF is clearly represented in Additiaonal file The Additional file The Additional file In the Additional file The Additional files In addition to using the tool to navigate and inspect the function of the genome, the tool has been used to generate isolated audio in both translation and transcription modes. A playlist of four tracks has been uploaded to SoundCloud. These audio tracks are to be listened to without reference to the visual display. The intention of this is to engage the non-specialised community with the concept of ‘the sound of the Coronavirus genome’ and hopefully encourage people to delve a little deeper into the ideas behind the concept. Without the context of the display and without a clear understanding of the molecular biology of RNA virus the audio has to engage purely on its own sonic qualities—as an example of algorithmic music. In translation mode two auditory displays were prepared, the first (Covid-19 Translation polyprotein) plays through to the end of the polyprotein lasting 1 h and 8 min, covering approximately 21,500 nucleotides. The second audio track from a sub-genomic RNA (Covid-19 Translation discontinuous) skips the polyprotein entirely to the untranslated region prior to the Surface glycoprotein and then plays through to the 3′ poly-A tail. This piece covers approximately 8500 nucleotides and lasts 27 min. In addition, two audio tracks were generated representing transcription/ RNA synthesis. The track representing genome replication (Covid-19 Transcription) last 2 h. The track representing discontinuous transcription (Covid-19 Transcription discontinuous) skips between TRS10 and TRS1 lasts only 1 min and 47 s.This paper extends prior work whereby DNA was sonified using the rules of gene expression to generate an auditory display. Previously an individual algorithm was used to produce an individual stream of audio from either a nucleotide, a di-nucleotide or codons and it was concluded that the sonification of codons was the most useful to identify mutations in repetitive DNA or to distinguish coding regions from non-coding regions . Here weMetadata was also used to affect the behaviour of the display to mimic what is known to occur during the Coronavirus life cycle. The polyprotein is the only product to be translated from the genomic RNA since this is thought to be the only ORF that has access to the ribosomal binding site in the 5′ untranslated region. To mimic this the default behaviour of the tool is to stop at the in-frame stop codon at the end of the polyprotein. The tool can be restarted at the adjacent untranslated region or elsewhere using the navigation buttons. The default behaviour in transcription mode is to read through the entire genome sequence from end to end to mimic genome replication to produce the complementary minus strand. A toggle switch has been implemented to mimic discontinuous transcription and in the first instance the polymerase will jump from TRS10 to TRS1 in the 5′ leader region. This can be overridden using the navigation buttons but if another TRS region is encountered by the play-head it will also jump to the TRS1 in the leader region. In translation mode the same toggle causes the ribosome play-head to skip the polyprotein and skip from TRS1 to TRS2 and into the Surface Glycoprotein sequence. From this point the play-head will continue to the 3′ end reading through the remainder of the genome. All other stop codons will be sonified but they will not influence the progression of the play-head. The auditory display in combination with real-time animation provide a unique insight into the large body of evidence describing the metabolism of the RNA genome. This provides another useful tool in the domain of genome browsers to further understand the complex function of the viral genome.Project name Real-time audio and visual display of the COVID-19 genome.Project home pagehttps://coronavirus.dnasonification.org/.Operating system: Platform independent.Programming language JavaScript (ECMAScript 6).Other requirements None.License GNU GPLv3.Any restrictions to use by non-academics: No restrictions.Additional file 1.Example 1 Sonification Untranslated ends.Additional file 2.Example 2 Sonification UTR to Surface Glycoprotein.Additional file 3.Example 3 Sonification of the Nucleocapsid Phosphoprotein.Additional file 4.Example 4 Sonification Sub-genomic RNA.Additional file 5.Example 5 Remix UTR through to Polyprotein.Additional file 6.Example 6 Remix ORF10 to the poly-A tail.Additional file 7.Example 7 Remix Membrane Glycoprotein.


RNA granules are ubiquitous. Composed of RNA-binding proteins and RNAs, they provide functional compartmentalization within cells. They are inextricably linked with RNA biology and as such are often referred to as the hubs for post-transcriptional regulation. Much of the attention has been given to the proteins that form these condensates and thus many fundamental questions about the biology of RNA granules remain poorly understood: How and which RNAs enrich in RNA granules, how are transcripts regulated in them, and how do granule-enriched mRNAs shape the biology of a cell? In this review, we discuss the imaging, genetic, and biochemical data, which have revealed that some aspects of the RNA biology within granules are carried out by the RNA itself rather than the granule proteins. Interestingly, the RNA structure has emerged as an important feature in the post-transcriptional control of granule transcripts. This review is part of the Special Issue in the Frontiers in RNA structure in the journal Molecules. The self-assembly of RNA-binding proteins (RBPs) and RNAs by phase separation into predominantly spherical, non-membrane bound condensates called RNA granules is a phenomenon observed across species. These granules form in distinct cellular locations and compartmentalize biomolecules. This compartmentalization creates cellular asymmetries, may enhance biological reactions, and promote molecular interactions required for cell growth and development. Phase separation imparts liquid-like properties to these assemblies, allowing granules to rapidly condense and dissolve depending on the environment in which they form and enables the exchange of granule components with the granule environment .Drosophila melanogaster, mutations in germ granule components induce sterility of the progeny  and stress granules A,B are wiewed in ) whereasrference . SeveralP-bodies have been first described in yeast cells in 2003  and haveP-bodies have long been seen as one of the cornerstones of RNA biology capable of organizing reactions fundamental to the physiology of a cell. However, a closer look challenges this view. Live cell imaging of single, fluorescently-labeled mRNAs revealed that in human epithelial (HeLa) cells mRNAs that enter P-bodies do not necessarily degrade in them . FurtherP-bodies could instead simply store transcripts and direct their flow through a translational cycle. For example, mRNAs appear to move between the polysome-depleted, P-body-enriched state and polysome-bound, cytoplasmic state ,24,25,26Stress granules form when messenger ribonucleoproteins (mRNPs) become stalled in translation initiation and can be induced by various cellular stresses such as heat shock, hypoxia, endoplasmic reticulum (ER) stress, arsenite poisoning, and viral infections ,29,30,31ATF4 , translates abundantly and equally efficiently in stress granules as outside [Purification and single molecule imaging techniques have also been applied to investigate the regulation of mRNAs in stress granules. These techniques revealed that while 185 mRNAs strongly accumulate in stress granules, most cellular transcripts enrich only at low levels (up to 30% of a given mRNA) ,29,37. F outside . These d outside . What fuCalligrapha punctate by Robert Hegner over 100 years ago [C. elegans) and nanos (nos) mRNA are essential germ granule components and conserved across species  or P granules (C. elegans), enrich mRNAs whose protein products are required for the specification of the germ cell fate and thus temporally and spatially instruct the formation of the germ cell linage in these two organisms  whilspecific ,43,44,45specific . Nuage ispecific ,47,48. Dy zygote Di,ii,F , nos, polar granule component (pgc), and germ-cell-less (gcl) enriched in these granules [gcl), specification (nos), transcriptional silencing (pgc), and division (CycB) of the primordial germ cells . Whiiewed in ,59. The  in [nos . Upon lo in [nos , preventby Smaug . A posteby Smaug ,63. In tethality ,63.Drosophila, the protein components of P granules of the early C. elegans embryo are structured. Here, the gel-like shell formed by two intrinsically disordered proteins MEG-3 and its redundant partner MEG-4 envelopes the liquid-like core composed of PGL-1 and -3 proteins [nos-1 and 2 [Drosophila polar granules, mRNAs in P granules in C. elegans are translationally repressed and only translate once they become released from P granules [C. elegans and their removal results in only approximately 30% reduction in the fecundity of the offspring [In contrast to polar granules of proteins ,65,66. P-1 and 2 . These t-1 and 2 . Howevergranules . In addiffspring .Drosophila, mediate transposon silencing  that, in iewed in ) and, iniewed in ). Duringiewed in . In addiiewed in . Therefoiewed in .C. elegans, P granules, Z granules, and mutator foci associate into PZM assemblages at the nuclei of germ cells. These tri-condensate assemblages are believed to be involved in RNAi inheritance, with localized functions of mRNA surveillance, RNA marking for memory storage, and transposon surveillance in P granules, Z granules, and mutator foci, respectively [In addition, different nuage functions appear to be segregated to different granules. For instance, in ectively .Despite the different functions of P-bodies, stress granules and germ granules in post-transcriptional regulation, fundamental features are nevertheless shared among these RNA granules. Perhaps most surprising is the fact that many granule properties are instructed by the transcripts themselves rather than the granule proteins, as discussed below.RNA granules form when the concentration of a protein which nucleates them reaches a critical concentration that triggers condensation . IntrinsLong and translationally-repressed (exposed) transcripts tend to enrich in RNA granules  17,29,3,67,87. TN6-methyladenosine (m6A), the most common mRNA modification [6A impacts the RNA structure by inducing the transition from paired to unpaired RNA strands [6A enhances phase separation of proteins that bind m6A [6A-modified mRNAs [6A modification in stress granules are approximately 50% higher than outside of granules [Drosophila of many polar granule-enriched mRNAs are similarly m6A modified [6A modifications enrich in mammalian stress granules and promote their formation [Drosophila germ granules demonstrated that RNAs are more stably associated within granules than the granule proteins themselves [Interestingly, the RNA structure is emerging as one of the principal regulators of phase separation . Highly fication . In mous strands  and expo strands . In addibind m6A  indicatied mRNAs ,100 and granules . Similarmodified . Thus, iormation . The divemselves ,85. RNAsnos in Drosophila oocytes demonstrated that this mRNA localizes to germ granules by a diffusion-entrapment mechanism and does not involve mRNA transport via molecular motors [C. elegans  assays combined with in vitro RNA-binding analysis identified the germ granule nucleator Oskar as a non-canonical RNA binding protein capable of interacting with a few germ granule mRNAs such as nos, pgc, and gcl with a low affinity and weak sequence specificity [C. elegans is also an RBP that interacts with mRNAs without sequence specificity and “prefers” longer transcripts with low ribosome occupancy [Drosophila germ granules and mammalian stress granules could in part occur through mRNA self-recruitment, where mRNAs anchored within granules could recruit additional mRNAs independently of granule proteins [Despite seeming permissiveness, mRNA enrichment to RNA granules is nevertheless highly selective. For example, in granules ,105,111,a embryo  only up a embryo . Similary embryo , fewer ty embryo . In humay embryo . Interesy embryo ,54,115 acificity ,117. Simccupancy . To ensuproteins ,118. Theproteins . RNA selDrosophila polar granules [Characteristics of RNAs enriched in P-bodies, stress granules, and germ granules  pertain granules ,105,111.Drosophila bicoid (bcd) and oskar (osk) mRNAs, the RNA genome of the human immunodeficiency virus (HIV) base-pair with each other in vitro and in vivo in RNA sequence-dependent manner [bcd and osk into multi-mRNP transport particles drives their enrichment to the opposite poles of the embryo and helps establish the body plan of the developing organism. However, genetic mutations, which drive inappropriate RNA-RNA interactions cause neurodegeneration. For instance, over-expanded CUG repeats in the 3′ untranslated region (UTR) of the myotonic dystrophy protein kinase (DMPK) gene are thought to engage in multivalent base-pairing, thus driving the formation of nuclear RNA inclusions [RNAs can spatially-organize independently of RBPs using intermolecular RNA-RNA interactions. t manner ,128,129.clusions . These iclusions ,131.via RNA base stacking, which can happen both with and without the Watson-Crick base pairing [bicoid mRNA dimers [RNA assemblies can also form  pairing . Within A dimers ,133,134.A dimers ,136 and A dimers ,138,139.Increasing evidence suggests that intermolecular RNA-RNA interactions also play a major role in the formation of RNA granules. Non Watson-Crick, non-specific intermolecular RNA:RNA, as well as sequence-specific intermolecular RNA-RNA interactions can promote homotypic and heterotypic RNA assemblies ,90,91,92Drosophila germ granules also self-assemble into homotypic clusters [Drosophila germ granules self-assemble in an RNA sequence-independent manner without detectable sequence-specific intermolecular RNA-RNA interactions , allowiocalized ,105. Whiocalized . For insocalized , a proceocalized . Thus, mDrosophila [Drosophila oocytes [Drosophila germ granule-enriched mRNAs are subjected to rapid degradation outside of germ granules and stabilized inside granules [trans RNA–RNA interactions (mRNA assemblies have been observed in diverse RNA condensates, including germ granules in osophila ,125 and osophila ,144, P-b oocytes , in mamm oocytes , and P-b oocytes . Thus, h oocytes , they magranules ,146 and ractions  [27,85,1It is unclear why some RNA assemblies, such as those that form in RNA granules appear benign or perhaps beneficial while others, such as those manifested in DM1, cause disease. The mRNA assemblies observed in RNA granules form within a proteinaceous granule environment, which could prevent possible harmful RNA aggregation observed for toxic RNA inclusions in DM1. Indeed, RNA helicases are core components of diverse RNA granules, which could dissolve potentially harmful RNA-RNA interactions that may form within highly compacted RNA assemblies. Alternatively, the location within a cell where mRNAs assemble could also be relevant. For instance, if the granule RNAs self-assembled in the nucleus rather than in the cytoplasm, as is the case with RNA inclusions in DM1, perhaps they too could become pathogenic. Future experimentation is needed to elucidate the role of RNA self-assemblies in normal cell growth as well as in disease.Enrichment of RNAs in RNA granules provides compartmentalization of post-transcriptional processes within a cell. The role of many RNA granules in post-transcriptional regulation remains unclear and requires further investigation. Surprisingly, RNA itself regulates many aspects of its own biology within RNA granules, including its recruitment to and organization within granules as well as stabilization of granules. From this perspective, RNA granules remind us of the primordial world in which the RNA was the “working” molecule that organized fundamental reactions. Thus, intriguingly, RNA granules could be seen as the remnants of the early world, in which the RNA takes center stage and carries out important functions, while the granule proteins provide the supporting role, enabling the RNAs to do their work. As for the past few decades, these mysterious condensates continue to surprise us and undoubtedly, future discoveries will reveal fascinating and fundamental new roles of granule RNAs in post-transcriptional regulation.