Caenorhabditis elegans. Proteomic analysis of the PIWI protein PRG-1 reveals an interaction with the constitutive P granule protein DEPS-1. DEPS-1 is not required for piRNA biogenesis but piRNA-dependent silencing: deps-1 mutants fail to produce the secondary endo-siRNAs required for the silencing of piRNA targets. We identify a motif on DEPS-1 which mediates a direct interaction with PRG-1. DEPS-1 and PRG-1 form intertwining clusters to build elongated condensates in vivo which are dependent on the Piwi-interacting motif of DEPS-1. Additionally, we identify EDG-1 as an interactor of DEPS-1 and PRG-1. Our study reveals how specific protein-protein interactions drive the spatial organisation and piRNA-dependent silencing within membraneless organelles.Membraneless organelles are sites for RNA biology including small non-coding RNA (ncRNA) mediated gene silencing. How small ncRNAs utilise phase separated environments for their function is unclear. We investigated how the PIWI-interacting RNA (piRNA) pathway engages with the membraneless organelle P granule in  C. elegans PIWI protein PRG-1 resides in the membraneless organelle P granule. Here, the authors identify the constitutive P granule protein DEPS-1 as an interactor of PRG-1 and show its function in piRNA-dependent silencing. DEPS-1 and PRG-1 form elongated condensates in vivo.The  These phase separated organelles, formed by proteins and nucleic acids, are sites for different aspects of eukaryotic RNA biology: the nucleolus is required for the assembly of ribosomes4, stress granules allow for translational stalling of mRNAs during stress-response5 and processing bodies (P-bodies) organise small RNA-mediated regulation of mRNA6. However, the molecular mechanisms of how RNAs and proteins assemble into phase separated organelles remain largely unexplored.The correct spatial organisation of molecules into organelles is essential for biological function. Recent studies reveal that membraneless organelles can be formed by proteins and nucleic acids condensing out of the bulk intracellular milieu, giving rise to liquid- or gel-like environments7. Various Ago proteins associate with membraneless organelles such as the P-bodies and germ granules9. Indeed, recently it has been shown that human Ago2 together with its binding partner TNRC6B can form biomolecular condensates entirely on their own10. A recently discovered condensate, the Z granule, contains the Ago protein WAGO-4 to establish transgenerational inheritance (TEI) of RNAi in C. elegans12. Hence, some small RNAs are routed through membraneless organelles. To further our understanding of how small RNAs operate within membraneless organelles, we turn to the piRNA pathway.Small non-coding RNAs execute diverse biological functions. mRNAs are targeted for silencing by small RNAs based on Watson-Crick base-pair complementarity in complex with members of the Argonaute (Ago) protein family15. Mutations in the piRNA pathway lead to varying degrees of infertility, indicating it plays an essential role in the survival of a species. For example, null mutations in each of the three piwi-coding genes lead to sterility in male mice18; depletion of the single functional PIWI protein in C. elegans leads to reduced fecundity19; in humans, a mutation blocking the ubiquitination of the PIWI protein HIWI has been implicated in azoospermia20.piRNAs associate with the PIWI clade proteins in the Argonaute family to repress transposable elements (TEs)C. elegans, piRNAs are 21 nt long with a 5′ preference for U21 and contain 2-O-methylation at the 3′ end23. piRNAs associate with the PIWI protein PRG-1 to scan for target mRNAs(21Us)25. These target mRNAs then serve as templates for the production of endo-siRNAs that are 22 nt long with a 5′ preference for G (22Gs) by the RNA-dependent RNA polymerases (RdRPs) EGO-1 and RRF-128. The C. elegans piRNA pathway offers a unique model for understanding how membraneless organelles engage with small RNA pathways as it requires proteins that can localise to two distinct and juxtaposed biomolecular condensates to achieve gene repression: the perinuclear P granules where PRG-1 resides24 and the secondary endo-siRNAs are entirely dependent on the mutator foci31.Mature piRNAs mediate transcriptional and post-transcriptional gene silencing. In C. elegans. First, we determine the protein interactome of the piRNA-binding protein PRG-1 and show a direct interaction with DEPS-1, a protein that associates with the P granule. We also identify the PRG-1-binding domain on DEPS-1 and show that this domain is required for silencing by piRNA, and for the typical morphology of PRG-1 condensates. We show that functionally, deps-1 and the interaction between DEPS-1 and PRG1 are required for the steady state levels of secondary endo-siRNAs. Hence, DEPS-1 functions as a bridge between piRNAs and secondary endo-siRNAs. Our study reveals that small RNA pathways and membraneless organelles are interdependent and that an essential P granule factor actively participates in small RNA regulation by directly binding to an Argonaute protein.In this work, we investigate how the piRNA pathway engages with the membraneless organelles for its function in 32 as well as for transgenerational inheritance (TEI) of exogenous RNAi33. It is not predicted to contain any known domain folds but consists of a poly-serine C-terminal end. Prediction for secondary structures suggests the protein is composed mainly of beta-sheets and loops  of PRG-1 followed by mass spectrometry . DEPS-1 and PRG-1 interact with  nM Fig. . So far PIWI) to be responsible for binding to full-length DEPS-1 using MST  only with a Kdapp of 151 ± 28 nM , one at the N-terminal and the other the C-terminal of DEPS-1. While the C-terminal region with similarity to dmGW182 is the poly-serine tail, the N-terminal region of DEPS-1 shares similarity with dmGW192’s Ago-binding motif II40  with a piRNA target site at its 3′ end, rendering its expression dependent on the piRNA pathway25. We analysed the effect of the PRG-1/DEPS-1 binding by removing the PBS from endogenous deps-1 and replacing it with a 5x glycine residue-linker via CRISPR-Cas9 gene editing (henceforth referred to as deps-1∆PBS) as well as two deps-1 null alleles (bn121 and bn124)32. Crossing deps-1 mutants with piRNA sensor animals, we found that the piRNA sensor is de-silenced in both deps-1null mutants, as well as the deps-1∆PBS mutant, as in the prg-1(n4357) mutant, indicating that deps-1 and specifically its PBS is required for piRNA-dependent silencing . We imaged the condensates at a high resolution to inspect how the DEPS-1 and PRG-1 cluster organisation is affected. As shown before wild-type DEPS-1 and PRG-1 clusters intertwine each other to form elongated condensates. While DEPS-1∆PBS localises to PRG-1 condensates when DEPS-1 is able to associate with the peri-nuclear region, PRG-1 condensates contain either very little or no DEPS-1∆PBS  become more compacted compared with the PRG-1/DEPS-1WT elongated condensates . This suggests the formation of these sub-organelle clusters are intrinsic to the protein sequences of DEPS-1 and PRG-1. We incubated the two proteins together in the presence of PEG2000 to see if they form intertwining clusters similar to those observed in vivo. Indeed, the DEPS-1 and PRG-1 clusters associate with each other  screen using full length DEPS-1 as a bait. We obtained one high-confidence candidate - the putative protein encoded by B0035.6. Y2H data indicated that B0035.6 interacts with DEPS-1 via its C-terminal region . Given the dependence of the piRNA pathway on the mutator foci, we tested if MUT-16 condensation was affected and found that they are not  mutant on piRNA abundance. We sequenced the small RNA population from 5’- independent libraries and show that the deps-1 mutant has a comparable level of 21U population as in wild-type animals  and prg-1 mutants  in deps-1 mutants  and mut-16 mutants  which also show greater than twofold reduction in mut-16 mutants , deps-1 has limited effects on ergo-1 targets45  and genes with >twofold reduction in secondary siRNA levels in deps-1 mutants  germline small RNA pathways which suggests the possibility that deps-1 might interact with other Ago proteins.We next asked if other small RNA populations are affected in deps-1 mutants, we asked whether changes in small RNA in our data correlate with previously published changes in mRNA expression32. Spike et al. show that in a deps-1 mutant, mRNAs of 13 genes are significantly downregulated and 32 genes are significantly upregulated, respectively32. We identified that 14 of these genes with different mRNA expression also have altered abundance of targeting small RNA in deps-1 mutants , suggesting the previously described effects on mRNA are mediated by the perturbations in small RNA populations. It remains to be determined which Argonuates are responsible for the changes in mRNAs and 22Gs given deps-1 is involved in multiple small RNA pathways.Having observed changes in secondary endo-siRNAs in 49 . We obtained a list of P granule factors from AmiGO under the GO term ‘P granule’ and manually curated the list to remove protein isoforms of the same gene. Comparing the P granule list with known endo-siRNA targets of various small RNA pathways, we observed that 55 out of 63P granule factors are endo-siRNA targets  or affected in deps-1 mutant (Hypergeometric Test: p < 0.1). Spike et al. show that RDE-4, a protein essential for RNAi, is downregulated in deps-1 null mutant which is likely the cause of deps-1 mutants being RNAi resistant32. Hence, some of the effect of deps-1 mutations on small RNA functions could be indirect and that deps-1 has a regulatory role on proteins with a direct role in these small RNA pathways.As most germline-expressed genes are targeted by endo-siRNA, it follows that P granule proteins may also be targeted by endo-siRNAsets Fig.  and thatant Fig. . As expeSmall RNA pathways associate with membraneless organelles. Here we reveal a role for the P granule factor DEPS-1 in the piRNA pathway, functioning as a link between piRNAs and secondary endo-siRNAs. Specifically, DEPS-1 directly binds PRG-1 through a conserved PIWI binding site (PBS) regulating 22Gs homoeostasis. Furthermore, the direct interaction with DEPS-1 is important for the organisation of PRG-1 sub-organelle clusters within the P granule Fig. .C. elegans primordial germ cells, these studies elegantly show that P granules are required for transgenerational inheritance of RNAi51. The direct interaction between PRG-1 and DEPS-1 provides an example for how small RNAs and P granules can be biochemically coupled.It has long been known that various small RNA machineries, such as Argonaute proteins, localise to P granules. However, it is only recently that a role for the P granule in ensuring appropriate small RNA-mediated silencing has been shown. Using mutant animals that lack P granules in the deps-1 null mutant, the piRNA population is normal in these animals suggesting that 21Us are still able to associate with PRG-126. In contrast, deps-1 mutations reduce endo-siRNAs of piRNAs origin and lead to brighter and fewer MUT-16 foci. The dependence on deps-1 for the normal level of 22Gs, but not 21Us, against piRNA targets places deps-1 downstream of prg-1. Therefore, DEPS-1 acts as a functional bridge between P granules and mutator foci. The C.elegans germline expresses several small RNA-associated Argonaute proteins that are not localised to the mutator foci, even though the function of these small RNA pathways depend on intact mutator foci30. The factors that bridge these Argonautes and mutator foci have yet to be fully characterised. ZNFX-1 is one such protein as it has been shown to associate with Argonaute proteins and a RdRP12.The piRNA pathway is dependent on protein factors reside both in P granules and mutator foci. Despite PRG-1 forming aberrant condensates in the deps-1 affects secondary siRNAs of many small RNA pathways, arguing for its function as a facilitator of multiple Argonautes. The PBS motif on DEPS-1 may be capable of recognising the universally conserved PIWI domain of several Argonautes52. Proteins capable of associating with multiple Argonaute proteins, as in the case of ZNFX-1, lead to balanced epigenetic signals. The possibility that DEPS-1 is critical in fine-tuning multiple piRNA and endo-siRNA pathways merits further study.Here we observe edg-1 specifically affects DEPS-1 condensates and not PRG-1 or the constitutive P granule protein PGL-1. Whether and how edg-1 regulate small RNA pathways remain to be determined. Indeed, whether EDG-1 is localised to P granules requires further investigation.We have identified EDG-1 as an interacting partner of DEPS-1 and PRG-1. Interestingly, knockdown of 53. The liquid-like property of membraneless organelles is thought to facilitate dynamic internal rearrangements as well as exchange of materials with their surroundings. Recently, a non-dynamic, gel-like scaffold has been found to envelope the liquid-core of P granules54; under electron microscopy, the crest and the base of P granules show distinct staining intensity55; differences in translational activity between the periphery and the core of the P-bodies have been observed in Drosophila oocytes56. These suggest subdomains exist within membraneless organelles to support or as a result of their functional complexities. We observe here that PRG-1 and DEPS-1 condensates are formed from smaller clusters of proteins that intertwine. Whether this organisation of PRG-1 and DEPS-1 substructures within the P granule is the result or reflective of the piRNA pathway activity is unclear. In this respect, Wan et al. show that ZNFX-1 forms a condensate that areas appose to both the P granules and Mutator foci11, while Ishidate et al. demonstrated that ZNFX-1 interacts with Ago proteins and promotes the Mutator foci machinery to position to the 3’ end of mRNAs12. Together, their studies provide evidence that there is a correlation between zones of intense molecular activities and protein localisation.Perinuclear germ granules are conserved features throughout the animal kingdom, and are sites of RNA metabolism and RNA-mediated gene regulation57. Furthermore, RNA:protein ratios determine phase-transition events in proteins prone to solid aggregation58. Given that a myriad of small RNA pathways are routed through the P granules and mutator foci in C. elegans59, it will be important to decipher how the various RNA species contribute to the formation of these organelles.While much focus has been placed on how proteins drive phase-transition in RNP foci formation, a flurry of recent studies investigated the importance of RNAs in the formation of RNP foci. Langdon et al. show that the secondary structure of mRNAs plays essential roles in specifying distinct Whi3-containing RNPsprg-1(n4357) animals were grown to 1 day-old adults at 20 °C on HB101. After washing thoroughly to remove bacteria, animals were resuspended in lysis buffer  and snap frozen in liquid nitrogen. The samples were then lysed by bead-beating, followed by centrifugation at 4 °C to remove insoluble debris. Anti-PRG-1 antibody (Custom) or rabbit IgG was pre-coupled to protein A/G matrix  and incubated with the supernatant of worm lysates for 4 h  lysates and three biological replicates of anti-IgG with N2 lysates). The immunoprecipitants were then washed with 3 × 1 ml of lysis buffer and eluted in elution buffer  with shaking at room temperature for 30 min.Synchronised wild-type N2 and 60. Peptides were desalted, dried and redissovled in 5% formic acid. RPLC was performed using a Dionex RSLC nano HPLC (Thermo Scientific). Peptides were injected onto a 75 μm × 2 cm PepMap-C18 pre-column and resolved on a 75 μm × 50 cm RP-C18 EASY-Spray temperature-controlled integrated column-emitter (Thermo Scientific) using a 4-h multistep gradient from 5% B to 35% B with a constant flow of 200 nl min−1 as described previously. The mobile phases were: 2% acetonitrile (ACN) incorporating 0.1% formic acid (FA) (solvent A) and 80% ACN incorporating 0.1% FA (solvent B). The spray was initiated by applying 2.5 kV to the EASY-Spray emitter and the data were acquired on a Q-Exactive Orbitrap plus (Thermo Scientific) under the control of Xcalibur software in a data-dependent mode selecting the 15 most intense ions for HCD–MS/MS. The survey scan was acquired over an m/z range 350–1600 with a 70,000 resolution, AGC target of 1e6 ions and a maximum IT of 20 ms. The subsequent MS2 scans were acquired over an m/z range of 200–200 m/z at 17,500 resolution, an AGC target of 1e5 and 60 ms maximum IT. Peptide ions were isolated with 1.4 Th precursor ion isolation window and fragmented using HCD with normalised collision energy (NCE) of 27. Data have been deposited in PRIDE (project accession code: PXD016838).Briefly, proteins eluted from immunoprecipitations were reduced and alkylated. Quantified proteins were then digested consecutively in solution using Lys-C and trypsin (both at 1:50 enzyme:substrate ratio)61. iBAQ values were divided by the total sum of intensity of each sample62. These normalised values were then log10 transformed to obtain normality and the resulting values were used for student’s t-test. To identify proteins enriched in immunoprecipitated PRG-1 from wild-type animals, the medians of the transformed values were used for fold-change calculations.Raw MS data were processed by MaxQuant600 ~0.8 and 1 mM IPTG was added to induce protein expression for 4 h. To purify the proteins, bacterial cells were lysed by sonication in PBS supplemented with protease inhibitors. 20 µg/ml RNaseA was added to the cleared lysates and incubated with gentle rotation overnight. Lysates were then applied to amylose resins  and washed with 20 column volumes of binding buffer supplemented with 14 mM beta-mercaptoethanol. Proteins were eluted with binding buffer supplemented with 20 mM maltose and 14 mM beta-mercaptoethanol. Proteins eluted from affinity column were subjected to size-exclusion chromatography equilibrated in assay buffer . Proteins used for MST assays were >85% in purity.All PRG-1 and DEPS-1 constructs were cloned using restriction enzymes into the pMAL-C5X vector. Recombinant proteins were expressed in BL21 (DE3) at 37 °C. Briefly, 10 ml of overnight pre-cultures were inoculated to 1 L of LB. Cells were grown to OD100 µM of purified proteins were incubated with 1 mM Atto fluor dye for 2 h in the dark. Free dye was subsequently separated from labelled proteins using a G-25 desalting column.DPES-1 peptide: IPLKFGEVILWNESDCDHDKFGFR2 peptide: PDFSSQPAVHKLTKRIPKdapp) was calculated by the law of mass action using data from either thermophoresis or thermophoresis with temperature jump.Proteins/peptides were labelled with atto-488 NHS ester . Free dye was separated from labelled protein using G25 desalting columns. Unlabelled proteins were serial diluted 1:2 and incubated with a constant amount of labelled protein. MST assays were carried in assay buffer supplemented with 0.01% NP-40. Fluorescence was monitored throughout the assay . The apparent dissociation constant . Animals were lysed with 5× freeze-thaw cycles in liquid nitrogen. Total RNA was isolated by chloroform extraction. For 5′ -independent libraries, 5 µg of total RNA was treated with 5′ polyphosphatase . Small RNAs were indexed using the TruSeq small RNA sample kit (Illumina) and size selected by gel separation in 6% TBE gels (Life Tech) and subsequently purified.https://basespace.illumina.com/ as fastq files after demultiplexing. Sequencing data is available in the European Nucleotide Archive under study accession number PRJEB31348 (Table Data 4). 3′ Adapter, reads below 18 nt length and reads with a length above 32 were removed using cutadapt. Remaining reads were aligned using STAR against the C. elegans genome WS235 allowing multimapping reads. To detect piRNAs, reads of 5′ dependent libraries were mapped against piRNA annotation WS235. Next, piRNA reads were counted using featurecount and abundance was calculated by correcting for library size using unique mapping reads. To compare piRNA sensor read distribution, reads of 5′ independent libraries were mapped against the piRNA sensor using STAR. Small RNA abundance was calculated by correcting for library size using H2B mapping reads. To compare small RNA changes in between worm strains of different gene set, small RNA reads per gene of a specific gene set were counted and abundance calculated by correcting for library size using unique mapping reads (cutoff > 50 reads per million). The mean small RNA abundance per gene was calculated, next the fold-change was calculated by divided the mean abundance in mutant animals by the mean abundance in wild-type animals. Gene sets were obtained from Bagijn et al. for piRNA targets25, Gu et al. for soma, germline and wago63, Claycomb et al. for csr-147, Conine et al. for alg-3/446, Vasale et al. for ergo-145, Buckley et al. for hrde-164 and repetitive element genes were detected using RepeatMasker.Small RNA sequencing results were obtained from 1 day-old adult animals were dissected for germline and freeze cracked on poly-lysine coated microscope slides. Dissected germlines were fixed in −20 °C methanol for 20 min. Fixed samples were washed with PBS-T (PBS supplemented with 1% tween-20) prior to primary antibody addition. Primary antibodies were incubated with the samples at 4 °C for overnight. Secondary antibodies were incubated at 37 °C for 1 h in the dark. Antibodies used: anti-PRG-1 ; anti-mouse GFP ; OIC1D4 for PGL-1 staining ; anti-RFP . All fluorescence secondary antibodies were from Invitrogen and used at 1:500. Dissected and stained germlines were mounted with Vectorshield antifading agent supplemented with DAPI.Images were taken on Leica SP8 confocal microscope. Images taken for granule quantification were of single slices, with pinhole set at 1 AU. HyVolution images were taken with pinhole narrowed to 0.5 AU to result in higher resolution. HyVolution images were deconvoluted using the HyVolution software. Some images were taken on the Zeiss LSM880 using the Airyscan mode.https://github.com/gurdon-institute/HKM-Segment) for ImageJ65, called by a macro (https://github.com/gurdon-institute/HKM-Segment/blob/master/Kin_granules.ijm) to detect granules and measure intensity, area and circularity in our piRNA-condensate paradigm . For fold change calculations, average intensity of all controls of one experiment was obtained and used to calculate the fold change of the individual mutant germline within the same experiment.Comparisons between different strains were usually obtained from three biological replicates, 2–4 germlines per replicate from 15 dissected germlines. Control and test strains were dissected and imaged in one setting. To calculate significant differences, mean values of each germline were obtained and tested for normality using the Shapiro–Wilk test. Two-sided Student’s 69, 1:1000); anti-tubulin ; anti-DEPS-1 .Proteins from 75–150 µg of worm lysates were resolved by SDS-PAGE and transferred onto PVDF membrane. Antibodies used: Antibodies used: anti-PRG-1  on NGM plates. Strains used in this study are listed in Supplementary Data Further information on research design is available in the Supplementary InformationReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4


Digestive system cancers are associated with high morbidity and mortality. Chemotherapy and radiotherapy are the main treatment modalities for these cancers. However, the development of therapy resistance leads to high rates of tumor recurrence and metastasis, resulting in dismal prognosis. Long non-coding RNA (LncRNA) H19, one of the most intriguing non-coding RNAs, has been shown to play a key role in the development and therapy resistance of various digestive system cancers  by regulating the abnormal expression of genes. In this review, we discuss the potential mechanisms of LncRNA H19 related therapy resistance in the context of digestive system cancers. LncRNA H19 is a potential novel therapeutic target for amelioration of cancer therapy resistance. Recent advances in the exploration of the human genome have shown that only 2% of all human genes are protein-coding genes, while the remaining genes are transcribed into non-coding RNAs. Depending on their length, non-coding RNAs are classified as long non-coding RNA (LncRNA), small nuclear RNA, small nucleolar RNA, micro RNA (miRNA), piwi interacting RNA, and small interfering RNA. The non-coding RNAs exhibit remarkable biological functions that involve a wide range of metabolic processes , interference with apoptosis, regulation of the expression of MDR genes, and transfer of exosomes  are only 9–18% and 16.4%, respectively . At present, LncRNA H19 in combination with chemotherapy has been shown to improve in vitro treatment efficacy against non-small-cell lung cancer (Zhou and Zhang


C. elegans the netrin receptor UNC-40/DCC controls the growth of dendritic-like muscle cell extensions towards motoneurons and is required to recruit type A GABA receptors (GABAARs) at inhibitory neuromuscular junctions. Here we show that activation of UNC-40 assembles an intracellular synaptic scaffold by physically interacting with FRM-3, a FERM protein orthologous to FARP1/2. FRM-3 then recruits LIN-2, the ortholog of CASK, that binds the synaptic adhesion molecule NLG-1/Neuroligin and physically connects GABAARs to prepositioned NLG-1 clusters. These processes are orchestrated by the synaptic organizer CePunctin/MADD-4, which controls the localization of GABAARs by positioning NLG-1/neuroligin at synapses and regulates the synaptic content of GABAARs through the UNC-40-dependent intracellular scaffold. Since DCC is detected at GABA synapses in mammals, DCC might also tune inhibitory neurotransmission in the mammalian brain.Increasing evidence indicates that guidance molecules used during development for cellular and axonal navigation also play roles in synapse maturation and homeostasis. In  AR at neuromuscular junctions in C. elegans. Here, the authors show that UNC-40/DCC assembles an intracellular synaptic scaffold, regulating the content of GABAAR and inhibitory neurotransmission.The netrin receptor UNC-40/DCC is required to recruit GABA Yet, it remains unclear for a given receptor what dictates the switch between a role in neurite guidance and a function in synaptogenesis or synaptic plasticity. Here, we identified the molecular mechanisms implementing the role of the netrin receptor DCC  in the control of synaptic GABAA receptor content in C. elegans.The precise patterning of synapses arises from a series of spatially and temporally regulated developmental events that include axon navigation, contact recognition of pre- and postsynaptic partners and synaptic differentiation. Axonal navigation is guided by multiple extracellular guidance cues that activate signaling receptors on the surface of the growth cone. Similarly, synapse formation relies on tightly regulated structural and molecular changes that are instructed upon interaction of synaptic adhesion molecules. Intriguingly, numerous evidences also point to a role of axon guidance molecules in synaptic differentiation and plasticityC. elegans initially identified three genes, unc-5, unc-6, and unc-40, that control the directionality of circumferential cell migrations and axon guidance5 . Subsequently, netrins were identified as diffusible chemotropic factors of the laminin superfamily homologous to UNC-68. The vertebrate netrin receptors DCC and UNC5 were recognized according to their homology to C. elegans UNC-40 and UNC-5, respectively10. DCC is a type I transmembrane receptor that belongs to the immunoglobulin superfamily (IgSF). Netrin binding to the DCC ectodomain causes receptor dimerization, which brings in close proximity the cytosolic regions of the receptors and enables their dimerization. This provides a docking platform to recruit activators of multiple signaling pathways, usually leading to an attractive response towards high netrin concentrations (reviewed by Boyer and Gupton11). In the presence of UNC5, netrin would trigger the formation of DCC/UNC5 heterodimers that mediate repulsive behaviors12. This long-range chemotatic gradient model has recently been revisited after analysis of axonal growth in mice where netrin expression was inactivated in specific subregions of the developing nervous system15. In these studies, the phenotypes were more consistent with a short-range haptotactic guidance model involving the interaction of growing axons with netrin present in the local environment. Detailed analysis of single axon outgrowth in C. elegans, together with in silico modeling, suggest that UNC-6/netrin biases the distribution of UNC-40/DCC at the membrane surface and stimulates an UNC-40-dependent protrusive activity towards growth direction17.Pioneer work in 1. Consistently, deletion of DCC in the adult forebrain results in shorter dendritic spines, behavioral defects, and loss of long-term potentiation. Activity-dependent secretion of netrin was recently involved in DCC-dependent potentiation of excitatory glutamatergic transmission in the hippocampus via Ca2+-dependent recruitment of GluA1-containing AMPA receptors18.Scarce reports also demonstrated a role of netrin/DCC signaling at synapses in the mature brain. In cortical pyramidal neurons, DCC is thought to be involved in LTP (long-term potentiation) upon activation of the Src kinase and phosphorylation of the NMDA receptorC. elegans, the UNC-6 netrin is secreted by ventral cells, and is thought to form a ventral-to-dorsal gradient19. C. elegans cells and axons utilize the polarized distribution of UNC-6 to orient circumferential migrations using the UNC-40 and UNC-5 receptors20. A well-documented UNC-40-dependent migration process is the outgrowth of body-wall muscle cell expansions towards motoneurons21. During post-embryonic development the muscle cells, which are located in four lateral quadrants along the animal’s body, extend projections called “muscle arms” to contact motoneurons at the medial ventral and dorsal cords and form en passant neuromuscular junctions (NMJs). UNC-40 drives muscle arm extension in response to the Punctin/MADD-4 guidance cue that is secreted by developing motoneuron axons22. On the ventral side, MADD-4 functions redundantly with UNC-6. UNC-40 activation triggers the remodeling of the actin network and involves multiple actors including the Rho guanine-nucleotide exchange factor (GEF) Trio-homolog UNC-73, members of the WAVE actin-polymerization complex and UNC-95, a component of focal adhesion complex23. The role of UNC-40/DCC was also carefully analyzed in the migration of several neurons .The dual role of DCC in both development and synaptic homeostasis is evolutionarily conserved. In C. elegans synaptogenesis. Netrin signaling was shown to control synaptic connectivity between the two interneurons AIY and RIA24. In this system, UNC-40 guides the migration of postsynaptic neuron RIA and drives presynaptic differentiation in the AIY neuron in response to local secretion of UNC-6 by a glial cell. Downstream signaling modulates actin assembly and recruits presynaptic components25. Recently, UNC-6 was involved in the male-specific maintenance of synapses between the sensory neuron PHB and the AVG interneuron26.Besides its canonical functions in guidance, UNC-40 plays direct roles in C. elegans27. In C. elegans, each body-wall muscle cell receives excitatory and inhibitory innervation from cholinergic and GABAergic motoneurons. The evolutionarily conserved CePunctin/MADD-4 protein is identified as an anterograde synaptic organizer that specifies the cholinergic versus GABAergic identity of postsynaptic domains28. CePunctin belongs to a family of poorly characterized extracellular matrix proteins, the ADAMTS-like proteins that contain multiple thrombospondin type-1 repeat (TSR) and immunoglobulin (Ig) domains as well as structurally unsolved domains in common with the ADAMTS family29. The functions of its vertebrate orthologs Punctin1/ADAMTSL1 and Punctin2/ADAMTSL3 are ill-defined, but Punctin2 is expressed in the brain and was identified as a susceptibility gene for schizophrenia30. Ce-punctin generates long (L) and short (S) isoforms by use of alternative promoters. MADD-4B/Punctin S and MADD-4L/Punctin L are differentially secreted by GABAergic and cholinergic neurons and trigger postsynaptic clustering of type A GABA receptors (GABAARs) and acetylcholine receptors (AChRs), respectively. At the inhibitory NMJ, MADD-4B-dependent clustering of GABAARs involves at least two molecular pathways 31. First, MADD-4B binds and clusters the synaptic adhesion molecule NLG-1/neuroligin in front of GABAergic boutons32. Second, it binds, recruits, and likely activates the netrin receptor UNC-40/DCC, which in turn promotes the interaction of GABAARs with neuroligin through a non-characterized mechanism27. Since UNC-40 also controls the growth of muscle arms, the inhibitory NMJ represents an interesting paradigm to compare the molecular pathways required for the guidance and the synaptic functions of UNC-40.We recently demonstrated that UNC-40 plays a role in the postsynaptic organization of inhibitory NMJs of ARs at inhibitory NMJs. We show that UNC-40 recruits FRM-3, a FERM  protein orthologous to FARP1/2, by a physical interaction between the intracellular P3 domain of UNC-40 and the FERM-FA tandem of FRM-3. In turn, FRM-3 recruits LIN-2, the ortholog of CASK , which might provide a hub to physically connect the GABAARs, FRM-3/FARP and NLG-1/neuroligin. All these processes are orchestrated by the main synaptic organizer MADD-4B/Punctin S, which both controls the synaptic localization of GABAARs through NLG-1/neuroligin and the synaptic content of GABAARs through an UNC-40-dependent intracellular scaffold.Using a genetic strategy, we identify here two proteins, FRM-3 and LIN-2, that implement the function of UNC-40 for the recruitment of GABAARs clustering, we mutagenized a C. elegans knock-in strain expressing fluorescently tagged GABAARs and performed a visual screen for abnormal fluorescence pattern. In this strain, the tagRFP-coding sequence was inserted in the unc-49 locus, fusing tagRFP to the extracellular N-terminus shared by the three GABAAR subunits UNC-49A, B and C. Among a wide range of phenotypes, we identified two strains with very strong decrease of synaptic fluorescence. Causative mutations were identified by whole genome sequencing, genetic mapping and rescue experiments.To identify synaptic proteins that control GABAfrm-3 gene, which codes for cytosolic proteins orthologous to the mammalian neuronal proteins FARP1 and FARP2 , further referred to as frm-3(0), which deletes most of the FERM coding region and introduces an early STOP  orthologous to CASK. The lin-2 locus generates two isoforms by the use of two different promoters. LIN-2A contains a CaM-kinase domain, two LIN-27 domains, a PDZ domain, a SH3 domain and a guanylate kinase domain. LIN-2B lacks the N-terminal CaM-kinase domain, and one of the LIN-27 domain  retrieved in our screen contained a deletion spanning the 1218-1331 nt, which caused a frameshift from valine 122 with subsequent introduction of an early stop codon. Interestingly, frm-3 and lin-2 were formerly shown to impact UNC-49 content at synapses, but the phenotypes that we observed seemed much more dramatic than previously reported33.One strain contained a mutation in the RP2 Fig. . Two FRMTOP Fig. . The secain Fig. . The linARs by immunostaining34 and detected a strong loss of UNC-49 staining at synapses in both frm-3(0) and lin-2(n397) mutants as compared with wild-type (WT) animals  mutant and found that the number and the morphology of muscle arms were normal  mutant  mutants33. To evaluate the functional consequence of frm-3 and lin-2 inactivation on GABAergic transmission, we recorded GABA-evoked currents in muscle upon optogenetic stimulation of GABA motoneurons. As compared with the WT, we observed a 75% and 90% decrease of GABA-evoked currents in frm-3 and lin-2 null mutants, respectively , where amino acids 137-463 of LIN-2A as well as part of LIN-2B N-terminus were deleted, the synaptic fluorescence level of RFP-UNC-49 was reduced by 75%, similar to frm-3(0) mutants , where the last 22 amino acids of the C-terminus common to LIN-2A and LIN-2B were deleted, GABAARs were also decreased by 70% at either 20 °C or 25 °C  mutant, which has been used as a reference allele in previous studies33, only caused a 40% reduction in RFP-UNC-49 fluorescence. Partial information on the lin-2(e1309) allele suggests that LIN-2A coding sequence is altered but that LIN-2B might still be expressed, since LIN-2B coding sequence and part of the LIN-2B promoter region might be intact  mutants and monitored RFP-UNC-49 fluorescence levels. Expressing either LIN-2A or LIN-2B completely rescued the lin-2(n397) mutant phenotype, indicating that the domains within the short LIN-2B isoform are sufficient for GABAAR clustering  null mutant animals, the overall fluorescence level of pHluorin-UNC-49 was decreased by 50% and small GABAAR clusters were fragmented and diffused away from GABA synapses  mutants, FRM-3B-GFP, and GFP-LIN-2A were present at excitatory and inhibitory synapses. The fluorescence level of FRM-3B-GFP was unchanged in nlg-1(0) mutants while the RFP-LIN-2 fluorescence level was slightly reduced  or frm-3(0) mutants   null mutants, RFP-LIN-2A and FRM-3B-GFP were reduced by approximately 50% and 70%, respectively  mutants  Fig. . We hypoely Fig. , whereasely Fig. . This defrm-3(0) mutants  mutant animals  mutants, FRM-3B-GFP was weakly detected in non-synaptic regions. Co-expression of myr-UNC-40 caused strong accumulation of FRM-3 at the membrane in the budding regions where myr-UNC-40 accumulated  mutants rescued the synaptic content of GABAARs  mutants, while the FERM domain only or the C-terminus of FRM-3B containing the RhoGEF and the two PH domains failed to rescue  null mutants that are small and have severe locomotion and egg-laying defects, unc-40::∆P3 mutants had normal body length and exhibited much less defects in locomotion and egg-laying  mutants while it completely disappeared in unc-40(0) mutants  mutants, FRM-3 is still detected at synapses while UNC-49 is decreased to the same extent as in frm-3(0) mutants, suggesting that FRM-3 is not sufficient to recruit UNC-49 receptors. Since LIN-2 is also decreased in frm-3(0) mutants, we wondered if LIN-2 and UNC-49 might directly interact. First, we performed a GST pull-down assay and demonstrated that HA-tagged LIN-2B interacts with the TM3-TM4 intracellular loop of UNC-49 fused to GST  or lin-2(0) mutant animals ; lin-2(0) or frm-3(0) double mutants than in nlg-1(0) single mutants, similar to lin-2 or frm-3 single mutants ; frm-3(0) double mutant is also comparable to lin-2 or frm-3 single mutants, suggesting that they indeed function in the same pathway.This model has at least three predictions. First, the interaction of NLG-1 and UNC-49 should depend on UNC-40 because UNC-40 recruits FRM-3 and LIN-2 at synapses. We thus compared the efficiency of retrieving RFP-UNC-49 after immuno-precipitation of NLG-1-GFP expressed either in wild-type or G-1 Fig. . Second,als Fig. . Third, 28. The MADD-4L (long) and MADD-4B (short) isoforms govern specific clustering of GABA and AChRs at inhibitory and excitatory NMJs. At GABA synapses, MADD-4B recruits NLG-1 on one hand and activates UNC-40 signaling on the other hand27. Whether MADD-4 would control the formation of the postsynaptic FRM-3-LIN-2 scaffold became an appealing hypothesis.Previous work in our laboratory identified MADD-4/Punctin as a master organizer of NMJsmadd-4(0) (null), madd-4B(0)  or madd-4L(0) (specific mutation of the madd-4L isoform) mutant animals. In madd-4(0) the synaptic localization of FRM-3 and LIN-2 was severely compromised, with a fluorescence level decreased by approximately 75 %  animals, FRM-3A-GFP and GFP-LIN-2A fluorescence levels were normal but FRM-3 and LIN-2 puncta were relocalized outside of GABAergic synaptic regions  mutation did not modify the level and localization of FRM-3 or LIN-2  mutant, suggesting that other pathways can drive synaptic targeting and stabilization of FRM-3.How Punctin triggers the recruitment of FRM-3 at synapses through UNC-40 remains to be further explored. Punctin was demonstrated to bind and localize UNC-40 at the tip of the muscle arms during development. Together with UNC-6/Netrin, it activates the GEF UNC-73/ trio and actin remodelers including the WAVE complexAR clustering. FRM-3 was initially related to the band 4.1 (EPB4.1) protein33, but the more recently identified isoform FRM-3B is the unambiguous ortholog of the mammalian FARP1 and FARP2. In neurons, the FARP proteins have been mostly implicated in the regulation of actin dynamics through GEF activity in response to guidance cues such as Sem6A/PlexA447 or Sema3A49. FARP1 directly interacts with the synaptogenic adhesion molecule SynCAM and regulates synapse number and dendritic spine morphology by activating Rac1 and regulating F-actin assembly50. In this study we demonstrated that the FERM-FA domain of FRM-3 is necessary and sufficient to rescue the synaptic defects of frm-3(0) mutants. Consistently, actin concentration is still detected in postsynaptic regions of frm-3(0) mutant, pointing to a specific function of the FERM-FA domain in the formation of a postsynaptic scaffold. This versatility might reflect an evolutionary diversification of FRM-3 functions, although a similar role of FARP proteins might be relevant at some synapses in mammals.Our study has identified a critical role of FRM-3 in promoting GABADrosophila, the FERM protein Yurt, an orthologue of mammalian EPB41L5, controls epithelial cell polarity38. Oligomerization of Yurt is crucial for its function in epithelial cell polarity and is supported by the FERM-FA domains. In Yurt, the oligomerization interface relies on a hydrophobic FxW motif which is conserved in FRM-3. In addition, the crystal structure of the zebrafish FARP1 and FARP2 FERM domains identified a positively charged patch that supports phospholipid binding and targeting to the plasma membrane51. This KRKK motif is also present in FRM-3  receptor subunit GluN2B56. In the present study we propose that LIN-2 is recruited to synaptic regions by FRM-3 and interacts with both neuroligin via its PDZ domain and with the TM3-TM4 cytoplasmic loop of the GABAAR via its SH3 and guanylate-kinase domains.LIN-2 is the ortholog of CASK, a member of the membrane-associated guanylate kinase family. Most studies have focused mainly on presynaptic roles for CASK. Interestingly, CASK interacts with the synaptic adhesion molecule neurexin via its PDZ domain and supports local actin assembly through the FERM protein P4.133 that proposed the existence of two GABAAR pools at the C. elegans NMJs, one synaptic pool depending on NLG-1/neuroligin, and a second synaptic pool depending on FRM-3/LIN-2. In this model, GABAARs were proposed to interact with FRM-3 based on a yeast-2-hybrid experiment. Although we could not detect this interaction by in vitro pull-down or yeast-2-hybrid (unpublished data), this interaction might exist. Yet, genetic data point to a central role of LIN-2 because we observe the same dramatic loss of GABAARs in lin-2(0) and in frm-3(0) mutants while FRM-3 remains enriched at synapses in lin-2(0). This is strong contrast with nlg-1(0) mutants where receptors are no longer clustered at GABA synapses but remain enriched along the cord, most probably by interacting with the FRM-3/LIN-2 complex. Removing LIN-2 in the presence or absence of NLG-1 causes the same loss of receptors, in contrast with previous studies which suggested that lin-2 and nlg-1 were synergistic. Few hypotheses might be proposed to reconcile these data. First, the previously used allele lin-2(e1309) might retain some expression of the LIN-2B isoform, which we showed to be sufficient for GABAAR clustering. Second, GABAAR localization and dynamics analysis was performed using transgenic expression of the UNC-49B subunit tagged in its TM3-TM4 loop, while we used CRISPR/Cas9 to modify the unc-49 locus and fuse RFP at the shared N-terminal part of the UNC-49 subunits. Tagged-UNC-49B will form a homomeric pentamer containing five fluorescent proteins in its cytoplasmic region while in our N-terminally tagged knock-in it will likely form a heteromeric B/C heteropentamer as thought for the endogenous receptor57. Third, since LIN-2 interacts with the cytoplasmic loop of UNC-49, we wondered if the presence of a fluorescent protein could modify the interactions with the FRM-3/LIN-2 scaffold. We therefore introduced by CRISPR/Cas9 the wormScarlet protein into the UNC-49B loop. This receptor localizes at GABA synapses but we observed only a 50% decrease of fluorescence in lin-2(0) mutants as compared with more than 80% when using the extracellularly tagged reporter  and lin-2(0) mutants unambiguously demonstrates that the FRM-3/LIN-2 scaffold is critical for endogenous GABAAR localization at the synapse.Our model differs from a previous studyAR clustering at C. elegans NMJs diverged from mammalian GABA synapses. In mammals, the self-oligomerizing protein gephyrin (GPN) is the core component of inhibitory synapses that binds the intracellular region of GABAARs to anchor them at the synapse59. Collybistin (CB), a Rho-guanine nucleotide exchange factor, binds GPN and GABAARs, which promotes the interaction of each protein with the intracellular region of Neuroligin-2. In addition, the transmembrane tetraspanins LHFPL3 and 4, also named GARLH (GABAAR regulatory LHFPL), form a tripartite complex with GABAARs and Neuroligin 2 and control synaptic clustering, of GABAARs61. In addition, GABAAR GPN-independent clustering mechanisms exist because GABAARs still form functional synaptic clusters in some neurons of GPN knockout mice63 59. In extrasynaptic regions, the targeting of GABAARs containing the α4 or α5 subunit is ensured by Radixin, a FERM protein that bridges GABAAR to the actin cytoskeleton66. GPN and CB are not present in the C. elegans genome, and the single C. elegans GARLH ortholog has not been involved in GABAergic transmission so far. However, it is remarkable to see that NLG-1, LIN-2/CASK and the intracellular domain of UNC-49 GABAAR form a tripartite complex promoted by FRM-3/FARP upon UNC-40/DCC activation. Despite using different elementary components, this configuration is functionally reminiscent of the arrangement of postsynaptic inhibitory scaffolds. Interestingly, DCC was recently identified in a proteomic analysis of the synaptic cleft components at inhibitory synapses67. Whether DCC might control the content of GABAARs at inhibitory synapses in the mammalian brain remains to be investigated.The evolutionary strategy for GABAC. elegans strains were derived originally from the wild-type Bristol N2 strain. Worm cultures, genetic crosses, and other C. elegans manipulation were performed according to standard protocols68. All strains were maintained on nematode growth medium (NGM) agar plates with Escherichia coli OP50 as a food source at 20 °C. A complete list of strains used in this study can be found in Supplementary data All frm-3  allele is a genomic rearrangement at the 5′ of lin-2 locus. The lin-2(n397) and lin-2(n105) alleles are deletions affecting both lin-2a and b isoforms. lin-2(kr357) is a deletion spanning nucleotides 1218-1331 at the lin-2a locus (position 1 is the ATG of lin-2a), resulting in an early stop codon.Molecular information on The constructs used and created in this study were described in Supplementary data tagRFP-T-unc-49(kr296) or pHluorin-unc-49(kr378) knock-in were generated using a MosTIC based technique where the fluorescence tags were inserted between Asp26 and Ser27 residues.The final kr392 allele, the wrmscarlet70 sequence was inserted between Met392 and Glu393 amino acid in UNC-49 genome encoding TM3-4 loop using CRISPR method based on 71. CrRNA sequence targeting insertion site of UNC-49 loop was designed and synthesized from IDT (Integrated DNA Technologies Inc.). CrRNA and tracerRNA (trRNA) were mixed as 1:1 ratio to form a duplex. Cr/trRNA duplex was incubated at 95 °C for 5 minutes and then placed at room temperature for 5 min. Injection mix contains Cas9 nuclease (10 μg) (IDT Inc.), cr/trRNA duplex 3 μL (100 μM), pOR2.2 repair template 50 ng/μL, pCFJ90 [Pmyo-2::mCherry] co-injection marker 2.5 ng/μL, RNAase/DNAase free water up to 10 μL. N2 young-adult animals were grown under 25 °C after injection. Hygromycin was added to plates 48 h later at concentration of 0.2 μg/μL for selection of resistant worms. Initial knock-in animals with roller phenotype were isolated and heat-shocked 2 h under 34 °C water bath to remove hygromycin selection cassette from the genome. Non-roller worms were then isolated and the PCR products were then sent for sequencing. Candidates were outcrossed with N2 twice to remove unspecific background mutations and used for the further studies.To generate kr457 allele, the coding sequence of P3 region (Ala1383-Lys1415) within the intracellular domain of UNC-40 was replaced by HA (Human influenza hemagglutinin) epitope tag sequence. CrRNA was designed and synthesized from IDT (Integrated DNA Technologies Inc.). An ultramer DNA fragment containing HA tag sequence, homology arms, NheI restriction enzyme site and a stop codon was produced by IDT Inc. Injection mix contains Cas9 nuclease (10 μg) (IDT Inc.), cr/trRNA duplex 3 μL (100 μM), ultramer repair template 1.25 μL (100 μM), pCFJ90 [Pmyo-2::mCherry] co-injection marker 2.5 ng/μL, RNAase/DNAase free water up to 10 μL. F1 progenies were checked by PCR and NheI restriction enzyme digestion (NheI site was introduced by ultramer repair template). Homozygotes were isolated and the PCR product were then sent for sequencing. Candidates were outcrossed with N2 twice to remove unspecific background mutations and used for the further studies.To generate krSi10 and krSi28 allele encoding NLG-1-GFP or NLG-1_∆PDZB-GFP (a deletion of the last 13 amino acid containing the PDZ binding motif) driven by myo-3 body wall muscle specific promoter were generated by MosSCI72. The single-copy insertion alleles generated by the miniMos method73 are listed in Supplementary Table. The fluorescent protein tagged genes are driven by myo-3 body wall muscle promoter or by him-4 distal muscle specific promoter. N2 worms were injected with 15 ng/μL of plasmid of interest containing the promoter and open reading frame, 50 ng/μL pCFJ601 (Mos1 transposase), 10 ng/μL pMA122 (negative selective marker Phsp16.2::peel-1), 2.5 ng/μL pCFJ90 (Pmyo-2::mCherry). Neomycin (G418) was added to plates 24 h after injection at 1.5 μg/μL final concentration. Candidate plates were heat-shocked for 2 h at 34 °C to remove extrachromosomal arrays. Worms with an insertion were isolated and homozygosed.The Freely moving worms were observed on nematode growth media (NGM) plates using an AZ100 macroscope (Nikon) equipped with a Flash 4.0 CMOS camera (Hamamatsu Photonics).Z-axis.For confocal imaging, young adult hermaphrodites  were used for imaging. Live worms were mounted on 2% agarose dry pads with 1% poly-lysine beads in M9 buffer. Fluorescence images were captured using an Andor spinning disk system (Oxford Instruments) installed on a Nikon-IX86 microscope (Olympus) equipped with a 60/1.2 oil immersion objective and an Evolve EMCCD camera. Images were acquired by IQ 3.4.1 software from Andor. Each animal was imaged as a stack of optical sections (0.2 μm apart) containing 33–36 slices and projected along P values for all the tests are provided in the source data file. For fluorescence intensity measurement, 30 μm (wide) × 5 μm (high) regions along ventral (between VD6 and VD7 neuron) or dorsal cord near the mid body were cropped and analyzed. Acquisition settings were the same across genotypes for quantitative analysis. For examination of each genetic background, each genotype of animals was imaged on at least 2 different days, and the data were pooled together. Data are presented as a percentage of the average fluorescence relative to that of the wild type. For co-localization quantification, images were captured along 40 μm in dorsal nerve cord anterior to the center of body. The plot file of either GFP-FRM-3/LIN-2/NLG-1 or SNB-1-BFP fluorescence signals were measured and processed by Mander’s overlapping correlation analysis modified from previous studies75. The co-localization between FRM-3, LIN-2, GABAR and NLG-1 were analyzed by Pearson’s coefficient correlation. Data are presented as min to max value for animals of each genotypes. For muscle arms growth quantification, number of arms from two body wall muscle cells in the quadrant connecting ventral nerve cord was counted. Average numbers of muscle arms per cell were compared between the wild type, unc-40(e1430), unc-40(kr457) or frm-3(gk585) mutants. For AVM neuron guidance quantification, pathfinding of AVM axon was characterized under spinning disk microscopy with a 60× optic lens. Ratio of abnormal/total number of animals counted from three independent days were compared between the wild type, unc-40(e1430) or unc-40(kr457) mutants. For the analysis of GABAR cluster size, the ROI of images were first thresholded by unique method provided by ImageJ. Then images were converted to 8-bit binary mask. The area of particles in each binary image was calculated. Average sizes were normalized to the mean of the wild type.Quantification of images was performed using ImageJ (v1.48 by NIH) with Fiji plugin add-ons. Statistic tests were performed by Prism6 from Graphpad and OriginLab software. The exact 76. Microdissection of C. elegans was adapted from previous work:77 the dissected region was always restrained to a small region (around 100 μm) before the vulva. Membrane currents were recorded in the whole-cell configuration using a MultiClamp 700B amplifier (Molecular Devices). Acquisition and command voltage were done using the Clampex 10 software driving an Axon Digidata 1550 (Molecular Devices). The bath solution contained (in mM) 150 NaCl, 5 KCl, 5 CaCl2, 1 MgCl2, 10 glucose, 15 HEPES, and sucrose to 320 mOsm/L (pH 7.2), and the pipette solution (in mM) 120 KCl, 4 NaCl, 5 EGTA, 10 TES, 4 MgATP and sucrose to 310 mOsm/L (pH 7.2). The slit worm preparations were exposed to 10 ms light stimulation performed with the pE2 system (CoolLED) at a wavelength of 460 nm drived by Clampex 10 software. The resistance of recording pipettes ranged between 3 and 4 MΩ. All chemicals were obtained from Sigma-Aldrich. All experiments were done at 20 °C. Data were analyzed with Clampfit 10 (Molecular Devices) and graphed with Origin software (OriginLab).The strains used for electrophysiology were derived from ZX426 straing for 20 min at 4 °C. Supernatant was collected and diluted to a final concentration of 0.2% Triton-X100 by WLB. Pre-cleaned 100 μL Trap-A bead coupled with anti-GFP or RFP nanobody  was added to the supernatant dilution and incubated at 4 °C overnight with gentle rotation. The beads were collected by centrifuge 1500 × g at 4 °C and washed three times with washing buffer  containing protease inhibitor and PMSF without triton-X100. Beta-mercaptoethanol containing Laemmeli buffer was added as 1/4 volume to the washed beads. Precipitated proteins were eluted by heating at 95 °C for 10 min and centrifuge 1500 × g at 4 °C for 10 min. Forty microliters of supernatant was loaded to 4–20% gradient precast Tris-HEPES gel  per lane for each group. Proteins were transferred to Nitrocellulose membrane and incubated with mouse monoclonal anti-GFP (Roche #11814460001) or RFP  antibody at 1:2000 dilution for 12 h at 4 °C. The membranes were then incubated with HRP conjugated secondary anti mouse antibody  at 1:2000 dilution. Blot pictures were taken by Bio-rad ChemiDoc XRS + imaging system.Mixed stage worms of each genetic background were collected from 40 of 10 cm dishes with 0.1 mM NaCl. After washing three times at 4 °C, worm slurry was frozen in liquid nitrogen to form worm beads around 15 mL in volume. Five miililiters of worm beads was grinded in liquid nitrogen and thawed in 7.5 mL ice cold worm lysis buffer  in 25 mL). Worm lysis was incubated at 4 °C for 2 h with rotation and then centrifuged at 15,000 × 78. To obtain GST-fusion proteins, overnight cultures (5 mL) of ArcticExpress E. coli strain  transfected with pGEX-3X vectors were further cultured in 100 mL LB medium with 100 mg/mL ampicillin, grown for 2 h at 37 °C with shaking until OD600 reached 0.6–0.8. Protein expression was induced with 1 mM IPTG and shaking at 11 °C for 24 h. The culture was centrifuged at 5000 × g for 10 min at 4 °C, the pellet was re-suspended in 10 mL ice-cold bacto-lysis buffer ). Cells were lysed on ice with 30 min of sonication. The lysed samples were then centrifuged at 15,000 × g for 30 min at 4 °C. Supernatants were incubated with glutathione-Sepharose 4B beads  at 4 °C for 12 h. Coding sequences of FRM-3B, FERM + FA, FERM, LIN-2B or UNC-49B_loops were cloned into pcDNA3.1(+) vector with a HA epitope tag to the N-terminus for in vitro expression. HA tagged proteins were transcribed and translated using TnT® Quick Coupled system , then incubated with GST-fusion coated beads for 12 h at 4 °C. The beads were then washed five times with 10 mM STE  buffer. Beads were boiled 10 min after the addition of an equal volume of 2×SDS sample buffer . Eluted proteins were separated on 4–20% gradient SDS/PAGE  and blotted to NC membrane. After blocking by 5% non-fatty milk, the blot was probed with anti-HA antibodies  at 1:1000 dilution, anti-GST  at 1:2000 dilution or stained immediately by ponceau S red.GST-pull down assay was modified from previously published protocolfrm-3(gk585) or lin-2(n397) animals were grown and collected from NGM rich medium plates. A freeze/cracking step was performed before acetone/methanol fixation at −20 °C. Primary antibody rabbit anti-UNC-49 was diluted at 1:250; mouse anti-UNC-17 (VAChT) was diluted at 1:50079. Secondary antibodies Cy3-labeled goat anti-rabbit IgG was diluted at 1:500 , Alexa 488-labeled goat anti-mouse IgG  was diluted at 1:500.The N2, Caenorhabditis elegans NLG-1 fragment [aa.710-798] was cloned as a bait. The prey fragments for LIN-2 were: LIN-2B full length [aa. 1-620] (GenBank accession number X92565.1). The constructs were checked by sequencing the entire inserts. These assays are based on the HIS3 reporter gene (growth assay without histidine). Controls and interactions were tested in the form of streaks of three independent yeast clones on DO-2 and DO-3 selective media. The DO-2 selective medium lacking tryptophan and leucine was used as a growth control and to verify the presence of the bait and prey plasmids. The DO-3 selective medium without tryptophan, leucine, and histidine selects for the interaction between bait and prey.Yeast-two-hybrid assays were performed by the Hybrigenics company. The coding sequence of the Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting Summary


ADAR1. Loss-of-function analysis showed that ADAR1 suppression profoundly repressed proliferation, invasion, and migration in thyroid tumor cell models. These observations were validated in an in vivo xenograft model, which showed that ADAR1-silenced cells had a diminished ability to form tumors. RNA editing of miRNAs has the potential to markedly alter target recognition. According to TCGA data, the tumor suppressor miR-200b is overedited in thyroid tumors, and its levels of editing correlate with a worse progression-free survival and disease stage. We confirmed miR-200b overediting in thyroid tumors and we showed that edited miR-200b has weakened activity against its target gene ZEB1 in thyroid cancer cells, likely explaining the reduced aggressiveness of ADAR1-silenced cells. We also found that RAS, but not BRAF, modulates ADAR1 levels, an effect mediated predominantly by PI3K and in part by MAPK. Lastly, pharmacological inhibition of ADAR1 activity with the editing inhibitor 8-azaadenosine reduced cancer cell aggressiveness. Overall, our data implicate ADAR1-mediated A-to-I editing as an important pathway in thyroid cancer progression, and highlight RNA editing as a potential therapeutic target in thyroid cancer.Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA. A-to-I editing of RNA is a widespread posttranscriptional process that has recently emerged as an important mechanism in cancer biology. A-to-I editing levels are high in several human cancers, including thyroid cancer, but ADAR1 editase-dependent mechanisms governing thyroid cancer progression are unexplored. To address the importance of RNA A-to-I editing in thyroid cancer, we examined the role of  Mutations in DNA are not the only source of modifications to the genomic content of human cells. RNA editing is a widespread posttranscriptional mechanism to engender genomic diversity by specifically and reproducibly changing selected RNA sequences without altering the genomic blueprint , 2. The As a result of RNA editing, inosines are interpreted by the cellular machinery as guanosines, and are base-paired with cytosine, making the effect of A-to-I editing similar to an A-to-G substitution . AccordiSome microRNA (miRNA) precursors undergo A-to-I editing, which regulates the expression and/or function of mature miRNAs. miRNAs are short noncoding RNAs that function posttranscriptionally to suppress gene expression through interactions of their seed region with complementary sequences in the 3′-untranslated regions (UTRs) of target messenger RNAs , and theThus, A-to-I alterations result in dynamic RNA mutations, which could ultimately lead to cancer outcomes similar to those caused by genomic mutations, but with higher flexibility as they affect only a fraction of the transcripts. Aberrant RNA editing is an underexplored mechanism that can reproducibly alter protein and regulatory RNA sequences, acting as a driver of carcinogenesis, and therefore, a potential therapeutic target . Indeed,ADAR1 may not directly reflect the editing activity  siADAR1 #1 [4390824] and siADAR1 #2 [AM5133]). miR-200b-3p mimics for the WT (C-300582-07-0002 miRIDIAN miRNA) and the edited form (mature sequence: UAAUGCUGCCUGGUAAUGAUGA) and the negative control (miRIDIAN miRNA Mimic Negative Control #1) were purchased from Cultek. In addition, the following vectors were obtained from Addgene: pCI-neo-RL-ZEB1 (Addgene plasmid #35535) and pCI-neo-RL-ZEB1 200bmut × 5 (Addgene plasmid #35537)  from Corning, and were seeded on the top of a 24-well plate containing 500 μL of Matrigel, which was added 30 min before seeding. The medium was replaced every 3–4 days.A total of 5000 Cal62 or TPC1 cells were resuspended in DMEM containing 5% Matrigel  as described . Cells wescribed , 48 h af6 Cal62-luc cells previously transfected with the corresponding siRNA and suspended in 50 μl of PBS mixed with 50 μl of Matrigel. Mice were randomly divided in three groups and xenografts were established in 22 animals. Tumor bioluminescent signals were determined in vivo at the indicated time points to calculate tumor growth, as described [Animal experimentation was performed in compliance with the European Community Law (86/609/EEC) and the Spanish law (R.D. 1201/2005), with the approval of the ethics committee of the Consejo Superior de Investigaciones Científicas . Xenotransplants were established in 6-week-old female BALB/c nude (nu/nu) mice by subcutaneous injection in the left flank of 1 × 10Renilla luciferase, activity of the ZEB1 3′UTR, the pCI-neo-RL-ZEB1 vector (ZEB1 3′UTR) or the pCI-neo-RL-ZEB1 200bmutx5 vector, containing mutations in the miR-200b-3p binding sites (ZEB1 3′UTR miR-200b-mut) was transfected together with a control Firefly luciferase plasmid and the corresponding siRNA or mimic. At 48 h after transfection, cells were harvested and assayed with the Dual-Luciferase Reporter Assay Kit (Promega Corporation). Renilla luciferase activity was normalized to firefly luciferase activity.To measure the reporter, Cells were treated with AKTi (Tocris), selumetinib (LC Laboratories), or a combination of both drugs for 24 h to final concentrations of 10 and 50 µM, respectively. Both drugs were dissolved in DMSO. 8-azaadenosine (8-aza)  was used to inhibit RNA editing. 8-aza dissolved in water was added to the cell culture medium at the indicated final concentrations.t test analysis (two tailed) and differences were considered significant at a P value < 0.05.Results are expressed as the mean ± SD of at least three different experiments performed in triplicate. Results from the in vivo studies and patient analysis are expressed as the mean ± SEM. Statistical significance was determined by Student’s Suppl_Table 1Suppl_Table2Figure Suppl-1Figure Suppl-2Suppl_ Figure Legends


Gastric cancer is associated with chronic inflammation, but there is still much to understand about the tumor microenvironment and the underlying tumor-promoting mechanisms. The Map kinase-activated protein kinase 2 (MK2) pathway is a regulator of inflammatory cytokine production that we have been studying in gastrointestinal cancers. Here, we set out to determine the significance of this gene in gastric cancer along with its downstream mediators and if there were differences in the primary tumors with and without metastasis.Human gastric cancer tissues with and without metastasis were examined for MK2 expression and cytokine profile in organ culture supernatants. Advanced statistical methods including a lower triangular correlation matrix, novel rooted correlation network, linear and logistic regression modeling along with Kruskal–Wallis testing with Sidak correction for multiple testing were applied to gain understanding of cytokines/chemokines linked to metastasis.The MK2 pathway is strongly linked with metastasis and a panel of cytokines. Gene expression was able to classify gastric cancer metastasis 85.7% of the time. A significant association with a panel of cytokines was found, including G-CSF, GM-CSF, Mip-1β, IFN-α, MCP-1, IL-1β, IL-6, and TNF-α. Mip-1β was found to have the strongest association with MK2 and metastasis after Sidak correction for multiple testing.MK2 gene expression and a novel associated cytokine panel are linked to gastric cancer metastasis. G-CSF is the strongest cytokine to differentiate between metastasis and non-metastasis patients and had the lowest P value, while Mip-1β showed the strongest association with MK2 and metastasis after Sidak correction. MK2 and associated cytokines are potential biomarkers for gastric cancer metastasis. The novel intercorrelation analysis approach is a promising method for understanding the complex nature of cytokine/chemokine regulation and links to disease outcome. Helicobacter pylori infection. Chronic inflammation has long been accepted as a risk factor for cancer development and progression, yet new treatment options targeting inflammation in cancer still remain limited. Thus, it is critical to understand the inflammatory pathways linked with more severe disease and poor outcome in order to identify inflammatory biomarkers in order to develop more effective treatment approaches.Gastric cancer is often diagnosed in later stages with high mortality rates associated with progression . Thus, tH. pylori infection may promote gastric cancer [We have been studying the Map kinase-activated protein kinase 2 (MK2) pathway as a potential target for inflammation and tumor growth in gastrointestinal cancers in mouse models , and herc cancer , 11 alonc cancer . Thus, MAlthough IL-1β, IL-6, and TNF-α have been shown to be regulated by MK2 signaling, cytokines often act in autocrine or paracrine manners to regulate production of other cytokines in the tumor microenvironment. We also have recently shown that MK2 regulates chemokine production in mouse models of gastrointestinal cancers , suggestHuman tissue samples were collected under an IRB approved human protocol at University of New Mexico Health Sciences Center with the assistance of the UNM Cancer Center Human Tissue Repository. Fresh samples were collected as matched tumor and normal tissues as determined by surgical pathology and transferred to the Beswick lab for processing. Tissue samples were divided into pieces for RNA extraction and cytokine assays.2, MultiScribe Reverse Transcriptase (3.125 U/μL) and its buffer, and 1 μg of cellular RNA. The RT step was performed according to the following protocol: 10 min at 25 °C, 60 min at 37 °C, 5 min at 95 °C. Obtained cDNA samples were stored at − 80 °C and used for the PCR reaction step. The PCR reaction mix was prepared using the Assays-on-Demand™ gene expression assay mix (Applied Biosystems) for human 18S and MK2. cDNA (2 µl) was added to a 20 × mix of PCR primers and TaqMan® FAM dye-labeled probe. The reaction was carried out according to the following protocol: 2 min at 50 °C, 10 min at 95 °C (1 cycle), and 15 s at 95 °C and one min at 60 °C (45 cycles) on Applied Biosystem’s StepOnePlus instrument. The endpoint used in real-time PCR quantification, CT, was defined as the PCR cycle number that crossed the signal threshold. Quantification of cytokine gene expression was performed using the comparative CT method and reported as the fold difference relative to 18S mRNA.RNA was extracted from tissue pieces using a trizol (ThermoFisher Scientific) method according to manufacturer’s instructions. RNA concentrations were measured using a Nanodrop instrument (ThermoFisher Scientific). Real-time PCR was performed according to Applied Biosystems’ two-step protocol. The RT reaction mixture includes random 2.5 μM hexamers, 500 μM dNTPs, 0.4 U/μL of the RNase inhibitors, 5.5 mM MgClNormal and tumor tissue pieces were divided into 8 mg (± 0.5 mg) sections and incubated in RPMI complete media for 16 h. Supernatants were analyzed by multiplex bead array for 26 cytokines/growth factors  according to manufacturer’s instructions and analyzed on a Luminex 200 machine.A/1—A from the A measure of stochastic superiority, which gives the odds that an individual in one group will score higher than an individual in the other group. Simple logistic regression was used to assess the odds of developing metastasis associated with an increase in MK2 utilizing the odds ratio (OR) and its associated 95% confidence interval. The logistic regression model was assessed using the Hosmer and Lemeshow goodness of fit test and model discrimination using the area under a receiver operator characteristic (ROC) curve (AUC) [MK2 and cytokines were described numerically using the median and interquartile range, from the first to the third quartile, for both being much less sensitive to outliers and non-normality. Graphically, we used (i) raw data (with jitter) overlaid with Box-plots, and (ii) Histograms with density curves overlay. The Kruskal–Wallis test was used to test for an overall difference in median levels of MK2 or cytokines between groups while using the Sidak adjustment for multiple testing . To asseve (AUC) . Simple ve (AUC) . A lowerp value was 0.001  and recruitment to promote colon tumor growth ; howeverIndependent of cytokines and chemokines, MK2 alone was able to correctly classify gastric cancer patients by metastasis status 85.7% of the time. In fact an optimal cut-off of 4.84 for MK2 was found to give a good accuracy as a diagnostic test for metastasis (AUC = 0.83) with both high sensitivity and specificity , while noting that the median of MK2 among metastasized subjects was 12.81 relative to only 3.36 among non-metastasized subjects . Data in mice have shown that MK2 does contribute to tumor progression by promoting M2 macrophage polarization and tumor angiogenesis which in turns promotes tissue renovation that governs cell invasion and metastasis . FurtherOur analysis with Sidak correction for multiple testing, showed significant elevation of G-CSF, IL-6, IL-8, MCP-1, IL-10, TNF-α, GM-CSF, and Mip-1α in metastasized subjects compared to non-metastasized ones with GCSF being the most significant biomarker. Of a great revelation is the strength of association between G-CSF and IL-10, which was found to be striking among those who metastasized compared to those who did not as illustrates in Additional file We are the first to show the significance of the MK2 pathway in gastric cancer. Of particular interest is that increased gene expression is strongly associated with metastasis. Gene expression was able to classify gastric cancer metastasis 85.7% of the time. Thus, MK2 is a potential biomarker for gastric cancer metastasis. Furthermore, MK2 expression was associated with a panel of cytokines, some of which are known downstream mediators, such as MCP-1, IL-1β, IL-6, and TNF-α. Others, such as G-CSF, GM-CSF, Mip-1β and IFN-α are previously unrecognized in associated with MK2. We found G-CSF to have the strongest association with metastasis with the lowest p-value. Furthermore, Mip-1β showed significant correlation with MK2 and metastasis after Sidak correction. Thus, MK2 and associated cytokines/chemokines are potential biomarkers for gastric cancer metastasis. The intercorrelation analysis approaches are a promising tool to more clearly understand the complexities of cytokine/chemokine regulation and association with disease outcome.Additional file 1: Figure S1. The association between GCSF and IL-10 stratified by metastasis status.


Mounting experimental evidence shows that non-coding RNAs (ncRNAs) serve a wide variety of biological functions. Recent studies suggest that a part of ncRNAs are critically important for supporting the structure of subcellular architectures. Here, we summarize the current literature demonstrating the role of ncRNAs and RNA-binding proteins in regulating the assembly of mitotic apparatus, especially focusing on centrosomes, kinetochores, and mitotic spindles. Non-coding RNAs (ncRNAs) are defined as a class of RNA molecules that are transcribed from genomic DNA, but not translated into proteins. They are mainly classified into the following two categories according to their length—small RNA <200 nt) and long non-coding RNA (lncRNA) (>200 nt). Small RNAs include traditional RNA molecules, such as transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), PIWI-interacting RNA (piRNA), and micro RNA (miRNA), and they have been studied extensively . Researc00 nt andChlamydomonas and Tetrahymena to nucleate microtubules in vitro is affected by RNase treatment; this suggests that centrosomes contain RNA molecules that perform essential functions as major microtubule-organizing centers . Al. Al14].  centers . Three y centers . However centers ,20, the  centers ,22 Centr centers . At pres centers . Compare centers , the und centers . Experim centers .This review focuses on recent literature demonstrating the functional implications of ncRNA molecules and RBPs in regulating centrosomes, kinetochores, and mitotic spindles.The centrosome, which comprises centrioles and PCM, is a non-membrane-bound organelle. Centrosomes serve as the major microtubule-organizing centers (MTOCs) in most animal cells. Therefore, centrosomes contribute to diverse biological processes, including cell division and cell polarity .Centrioles are small barrel-shaped structures characterized by the nine-fold radial symmetry of triplet-microtubules. A mature centriole is ~250 nm in diameter and ~450 nm in height in human cells . The cenXenopus egg extract was conducted in vitro. Centrioles  purified from Chlamydomonas or Tetrahymena were treated with several enzymes and subsequently added into the Xenopus egg extract. As a result, treatment with protease, RNase A, T1, or S1 nuclease significantly attenuated aster formation activity of centrioles. While protease disrupted the centriole, RNase treatment had no effect on its structure, suggesting that RNAs were not critical components of the centriole structure itself, but were somehow required for the MTOC activity of centrioles. Regarding PCM, RNase was reported to decrease the amount of PCM in mammalian cells [This chapter describes centrosomal RNA and RNA-binding proteins , Table 1an cells . Howeveran cells .Spisula) oocytes and named them centrosomal RNA (cnRNA) [Spisula, but whether these RNAs are required for cell division or organelle organization in this and other species remains unclear.Recent technological advances have made it possible to identify RNAs localized in the centrosome. In 2006, Alliegro et al. cloned five RNAs enriched in the centrosome of surf clam ( (cnRNA) . Since n (cnRNA) . A subse (cnRNA) . The int (cnRNA) . Nucleol (cnRNA) . This sa (cnRNA) . In summRegarding vertebrates, genomic-instability-inducing RNA (Ginir), a mammalian lncRNA conserved in rodents potentially involved in centrosome function, was identified as an oncogene in mouse melanoma cells . The Ginhttp://microkit.biocuckoo.org/) [In addition to RNAs, a large number of RNA-binding proteins reside in the centrosome. These proteins, such as EWS and NDH2, can be found in a database of protein localization  . Here, woo.org/) ,41.RNA-binding motif 14 (RBM14) was identified as a novel regulator for centriole biogenesis . RBM14 pGle1 regulates DEAD box RNA helicase and is responsible for mRNA export. Depleting Gle1 generates ectopic MTOCs in the cytoplasm, resulting in chromosome mis-segregation in human cells . Gle1 isHuR, an RNA-binding protein, may contribute to stabilizing mRNAs by binding to an ARE (AU-rich element) sequence in the 3’UTR region of the target mRNA. In U251 cells, HuR is transferred to the centrosome only when cells are stimulated by growth factors . SimultaThe heterodimer of RBM8A and MAGOH is a part of exon junction complexes formed on target mRNA. Intriguingly, this heterodimer is localized in the centrosome ; depletiC. elegans, undergoes condensation in vitro and in vivo [Recent work suggests that the pericentriolar matrix (PCM) is a phase-separated condensate . SPD-5,  in vivo . The SPD in vivo ,51. PCM  in vivo ,52. TherThe kinetochore is the macromolecular protein complex associated with sister chromatids and links them to the microtubule plus-end of mitotic spindles . AccordiCentromeres, the DNA sequences where kinetochore components assemble , are embThe constitutive inner-kinetochore proteins are called the “constitutive centromere-associated network” (CCAN) . CCAN prWhen the connections between the kinetochore and the spindle microtubules are incorrect, aurora-kinase B weakens the connections by phosphorylating some proteins in the outer layer ,62. AuroThe surveillance mechanism of the kinetochore-microtubule connections is called the “spindle assembly checkpoint” (SAC) . The impProper chromatid separation requires degradation of the link between sister chromatids, called cohesin . During Drosophila [The centromeric DNA sequence where the kinetochore structure is formed does not encode proteins, but is transcribed by RNA polymerase 2 (RNA polII) in human cells ,70. RNA osophila . Whereasosophila , those oosophila . Severalosophila ,73,74,75osophila . The imposophila ,76, buddosophila , fissionosophila , and fliosophila .The relationship between centromeric RNAs and kinetochore assembly was first investigated by experiments with maize in 2004 . This stIn 2007, experiments using human cells also revealed that transcripts of alpha-satellites in the centromere region bind to CENP-C. This interaction is necessary for accumulating CENP-C, INCENP, and survivin to the kinetochore. Furthermore, these three proteins were diminished from the kinetochore by RNase treatment; adding alpha satellite RNAs consistently recovered their accumulation at the kinetochore . Unless In addition to CENP-C, centromeric RNAs also promote centromeric deposition of CENPA . On the The neocentromere is a DNA region where the kinetochore is formed only when the normal centromere is dysfunctional. Similar to a normal centromere, the L1 retrotransposon on the 10q25 neocentromere is transcribed in human cells . The traXenopus egg extracts also showed that centromeric RNAs are associated with aurora-B and INCENP and promote their loading to the centromere [A study using murine erythroleukemia cells reported that binding of chromosome passenger complex (CPC) to CENP-A and aurora-B kinase activity were attenuated by RNase treatment, while adding minor satellite RNA, which is a centromeric RNA, rescued this phenotype . Furtherntromere ,81. Reduntromere ,81,82. FSgo1 protects the cohesin complex at centromeres. Sgo1 recognizes Histone H2A-T120 phosphorylation by Bub1  and therOverall, the function and significance of centromeric RNAs during assembling of major kinetochore and centromere proteins, such as CENP-A, CENP-C, aurora-B, and Sgo1 were introduced . Since cAlthough this review mainly focused on the role of centromeric RNAs on human kinetochore assembly, but the significance of centromeric transcripts are evident in animals, plants, and fungi. Since the sequence of centromeric DNA is little conserved among species, physiochemical properties, such as single-strand negatively charged RNA molecules, may be more important than the sequence itself, such as in the formation of open chromatin structures . InteresEukaryotic cells have three types of cytoskeletons—microtubules, actin fibers, and intermediate filaments. Here, we focus on microtubules, which contribute to cell morphology maintenance, intracellular transport, cilia formation, and cell division. Microtubules possess a hollow structure with 25 nm diameter made up of thirteen identical polymers (protofilament) of the α-βtubulin heterodimer. Tubulin polymers are highly dynamic and frequently grow and shrink, a characteristic called “dynamic instability”. Microtubule stability is regulated by post-transcription modification (PTM) and microtubule-associated proteins (MAPs) ,86,87. AIn mitosis and meiosis, microtubules form a rugby-ball-like mitotic/meiotic spindle critical for properly distributing sister chromatids to daughter cells. Spindle microtubules mainly nucleate at the centrosome, but chromosome-dependent and augmin-dependent pathways also exist . The chrXenopus egg extract attenuated kinase activity and thereby increased MCAK density on mitotic spindles, which lead to microtubule depolymerization. In contrast, transcription inhibitors or RNase treatment reduced the density of TPX2, a spindle assembly factor implicated in chromosome-dependent MTOC activity on mitotic spindles. Interestingly, inhibiting MCAK or adding purified TPX2 proteins relieved spindle destabilization caused by treatment with transcription inhibitor or RNase. These studies imply a non-coding function of RNA molecules as an activator for their interacting partner proteins [In this chapter, the role of RNA and RNA-binding proteins in microtubules and mitotic spindles are introduced . Given tproteins .Xenopus egg extract and identified enriched mRNAs and rRNAs [In 2007, Blower et al. performed a microarray analysis on purified mitotic spindles from nd rRNAs . The enrnd rRNAs . Althougnd rRNAs . 18S rRNnd rRNAs , the metXenopus egg extract, Rae1 is necessary for activating Ran-GTP to form microtubule asters, as well as for spindle assembly following sperm nuclei injection. RNase treatment can reduce aster nucleation from Rae1-beads in Xenopus egg extract [The microtubule-binding protein Rae1 is also essential for bipolar spindle formation in HeLa cells . Rae1 is extract , demonstIn addition to the aforementioned proteins, several RNA-binding proteins, such as spermatid perinuclear RNA-binding protein (Strbp) ,96, StauIn addition, ncRNAs can function as a seed for spindle matrix assembly through phase-separation. A spindle matrix is a protein structure observed around mitotic spindles that promotes spindle assembly . Since sPericentriolar material (PCM) is a mass of proteins surrounding centrioles. Considering that many proteins with an intrinsic disordered region exist in PCM, PCM may be formed by phase separation of its components. In 2017, a study using nematodes showed that the PCM protein SPD-5 has a property of phase separation in vitro and in vivo, and that the SPD-5 condensates promote microtubule nucleation in vitro when mixed with tubulin, microtubule polymerase ZYG-9 and microtubule-stabilizing protein TPXL-1 . RNAs maAs for mitotic spindle formation, a large number of RNA-binding proteins are present on mitotic spindles and have been shown to be required for their formation. Although the reported binding partners of these RNA-binding proteins are mostly mRNA, it is possible that these RNA-binding proteins also bind to a specific ncRNA which structurally functions in mitotic spindle assembly. The function of ncRNA may be to induce phase separation of proteins such as BuGZ, or to assemble several components or regulators that cooperatively promote mitotic spindle assembly.Several groups previously performed a knockdown screening using RNAi  or CRISP


Porifera, the marine sponge is one of the well-studied holobiont systems. In the present study, we elucidated the putative complete genome sequences of five novel RNA viruses from Hymeniacidon sponge using a combination of double-stranded RNA sequencing, called fragmented and primer ligated dsRNA sequencing, and a conventional transcriptome method targeting single-stranded RNA. We identified highly diverged RNA-dependent RNA polymerase sequences, including a potential novel RNA viral lineage, in the sponge and three viruses presumed to infect sponge cells.Invertebrates are a source of previously unknown RNA viruses that fill gaps in the viral phylogenetic tree. Although limited information is currently available on RNA viral diversity in the marine sponge, a primordial multicellular animal that belongs to the phylum  Porifera. Sponges are recognized as hosts of diverse symbiotic lives .The marine sponge is a primordial multicellular animal that belongs to the phylum ic lives , and theic lives . Sponge-To access RNA viromes, conventional total RNA-seq (single-stranded RNA [ssRNA]-seq) is the most commonly used method to detect RNA viral sequences in diverse environments and organisms, such as oceanic waters . FragmenSponges on tidal rocks in Tokyo Bay  were sampled in April 2014 and 2015. After washing with distilled water, samples were stored at –80°C until further analyses.Sponge samples were disrupted in liquid nitrogen in a mortar. Total nucleic acids were manually extracted from approximately 1 g of sponge powder. dsRNA was purified using a combination of the cellulose column method (–1) (Covaris) (Nuclease-treated dsRNA was fragmented by ultrasound at 4°C in Snap-Cap microTUBEs (Covaris) using Covaris S220  using random primers (9-mers). The resultant cDNA was quantified using the Qubit dsDNA HS Kit (Thermo Fisher Scientific) and Agilent 2100 Bioanalyzer (Agilent Technologies).–1), and fragmented cDNAs were used as templates for KAPA Hyper Prep Kit Illumina platforms (Kapa Biosystems). The resultant Illumina libraries were evaluated using the KAPA library quantification kit (Kapa Biosystems) and applied to the Illumina MiSeq platform (Illumina) according to the manufacturer’s protocol (600-cycle kit to perform 300-bp paired-end sequencing).Double-stranded cDNAs were fragmented using Covaris S220  with the following parameters: a minimum contig length of 500, word value and bubble size set to auto, and assemblies that were manually examined and extended using the Tablet viewer (–5. Sequences that matched a known RNA-dependent RNA polymerase (RdRp) gene by BLASTX with an e-value≤1×10–5 were collected from RNA virus contigs and segments. The presence of the RdRp domain  was confirmed using PfamScan  to an RNA viral polyprotein or hypothetical protein. Nucleotide sequences encoding the RdRp gene were clustered at 90% identity using VSEARCH (–5) to sequences in the nr database. Quality-based variant detection was performed using CLC Genomics Workbench ver. 11.0 (CLC Bio).Raw sequencing reads for dsRNA-seq were processed as described previously  and Short Read Archive database (accession nos. DDBJ: DRA008491).The sequences obtained in the present study are available in the GenBank database repository  mussel, and the abundance of other eukaryotic SSU rRNA sequences was lower than 0.2% of all SSU rRNA reads. No archaeal or bacterial SSU rRNA sequence was found from reads that dominated >2% of all SSU rRNA reads.To identify potentially active biomes in sponges obtained in 2014 and 2015, SSU rRNA sequences were reconstructed from total ssRNA-seq reads, and compositions were estimated based on mapped read numbers on SSU rRNA sequences  were identified and defined as operational taxonomic units (OTUs). Based on the taxonomic lineage of BLASTX top hit sequences, the richness of OTUs was analyzed and many viral OTUs were suggested to be closely related with (or belonging to) Totiviridae, Reoviridae, and Partitivirdae (Bryopsis cinicola (Chlorophyta)  succeeded in enriching viral reads and revealed RNA viromes associated with sponges collected in 2014 and 2015, while RNA virus reads were rare in the ssRNA-seq analysis, except for reads related to  samples . We obta samples . These s samples , and 7.7tivirdae . The majtivirdae . All of rophyta) .–5) with known viral sequences after the names of the most closely related virus family  with sequences in the nr database. However, an ORF of SRV RNA2 showed insignificant similarity  to the RT_like super family (cl02808) in the CDD search. Thus, we manually identified the signature amino acid residues conserved in the RdRp domain  showed lower read abundance than the ssRNA virus (SdRV) in ssRNA-seq data.To estimate the potential host of these RNA viruses identified in the dsRNA-seq analysis, we evaluated the abundance of these viral sequences in ssRNA-seq according to the previously proposed definition that viral reads sharing >0.1% in non-rRNA reads in conventional RNA sequencing may be infected with the sampled organism C. The fuCnidaria, not Porifera. The SdRV genome consists of one segment with two ORFs. The BLASTP search also revealed that the most similar sequence to SdRV RdRp was putative ORF1 of Caledonia beadlet anemone dicistro-like virus 2 isolate I38 (accession no. ASM93984) . A phylogenetic analysis of the RdRp sequence indicated that SdRV belonged to the positive-stranded ssRNA virus family Dicistroviridae   in the BLASTN analysis; its potential host Caledonia belongs to the phylum oviridae . The arroviridae . These rPartitiviridae)  found in the ssRNA-seq of invertebrates (–5) with sequences in Partitiviridae, SpaRV was not classified into the established genus of Partitiviridae by the phylogenetic analysis  ; however, a phylogenetic analysis of RdRp for Reoviridae revealed that SrRV was not positioned into the genus Orbivirus (Orbivirus) , while other segments showed no significant similarity  with known viral sequences.The sequence that was the most similar to RdRp in SrRV in the BLASTP analysis was Chuzan virus  was found in the nr database, but belongs to the β clade.The most significantly similar sequence in the BLASTP analysis for RdRp in SpiRV was Hubei picobirna-like virus 2. A phylogenetic analysis of RdRp suggested that SpiRV belongs to an undefined and tentatively named α clade of picobirnavirus-like viruses derived from marine microorganisms, diatoms, and invertebrates . In Pico–5) with sequences in the nr database in BLASTN and BLASTX analyses. The RdRp sequence was too distant from all known RdRp sequences to identify its phylogenic position. However, as discussed above, the genome structure of SRV resembled that of Reoviridae and the ratio of SRV reads in non-rRNA ssRNA-seq was similar to those of dsRNA viruses  to known, but unclassified RdRp identified from the chloroplasts of B. cinicola. Other RdRp-encoding segments and contigs were lower than 1,000× average coverage.Although we focused on numerically abundant viruses with putative complete genome sequences in the dsRNA-seq libraries, there was a RdRp-encoding contig with similar average coverage  to these five genomes. The contig (ID: Sponge_RNAV429) was identified in 2014 samples, and showed significant similarity  with polyA tails were retrieved by FLDS  dsRNA-seq Reveals Novel RNA Virus and Virus-Like Putative Complete Genome Sequences from https://doi.org/10.1264/jsme2.ME19132Supplementary Material 1Supplementary Material 2