Transport of messenger RNA (mRNA) from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. In the cell nucleus, a precursor mRNA undergoes a series of processing steps, including capping at the 5' ends, splicing and cleavage/polyadenylation at the 3' ends. During this process, the mRNA associates with a wide variety of proteins, forming a messenger ribonucleoprotein (mRNP) particle. Association with factors involved in nuclear export also occurs during transcription and processing, and thus nuclear export is fully integrated into mRNA maturation. The coupling between mRNA maturation and nuclear export is an important mechanism for providing only fully functional and competent mRNA to the cytoplasmic translational machinery, thereby ensuring accuracy and swiftness of gene expression. This review describes the molecular mechanism of nuclear mRNA export mediated by the principal transport factors, including Tap-p15 and the TREX complex. Eukaryotic cells consist of various organelles that execute different activities to sustain a range of cellular functions. The largest among them is the cell nucleus, which is surrounded by the nuclear envelope (NE) and stores genetic information in the form of chromatin. Transcription of genes, processing of various RNAs and replication and repair of DNA occur in the nucleus, whereas translation of proteins exclusively takes place on the ribosomes in the cytoplasm. Due to this physical separation, messenger RNAs (mRNAs) must be exported to the cytoplasm where they direct protein synthesis, whereas proteins participate in the nuclear activities are imported into the nucleus. In addition, some types of RNAs reenter to the nucleus after being exported to the cytoplasm . TherefoAmong the various RNA species, mRNA is the most divergent in sequence, length, and structure. In addition, as recently demonstrated by proteomic analysis ,3,4, mRNNuclear pore complexes (NPCs), which perforate the NE, are the main gateways through which RNAs and proteins are delivered to their proper destinations. The NPC is composed of approximately 30 distinct proteins that are collectively known as nucleoporins ,8,9,10. The importin/karyopherin-β family of proteins comprise the prototypical transport receptor family that mediates nucleo-cytoplasmic movement of most proteins and small non-coding RNAs, such as tRNA, uridine-rich small nuclear RNA (UsnRNA), and miRNA ,13,14,15mex67 accumulates poly (A)+ RNA in the nucleus under the non-permissive temperature .,17.12,17perature . Human Trganisms ,20,21,22rganisms ,28,29,30in vitro and are not able to distinguish different RNAs on their own [Both Tap-p15 and Mex67-Mtr2 are RNA binding proteins, but they bind nonspecifically to RNA heir own ,31,32. Their own ,35,36,37heir own ,39. In aheir own , and theheir own ,42,43,44heir own , as wellheir own ,47,48,49Recruitment of adaptor proteins to mRNPs is coupled with transcription and processing, causing mRNPs to be licensed to the mRNA-specific export pathway upon the completion of nuclear processing. Thus, transcription by RNA polymerase II (RNAPII) is a key determinant allocating mRNA to the appropriate export pathway. In addition, length is another important determinant that distinguishes mRNAs from UsnRNAs, both of which are transcribed by RNAPII in metazoans ,53. The It has been proposed that gene positioning to NPC-proximal site (known as “gene gating” ) is coupSeveral lines of evidence suggest that the mRNA export receptor directly interacts with an mRNA after its initial recognition via protein-protein interactions. This step has been shown to be regulated by post-translational modifications of the adaptor proteins ,69. SubsTap and Mex67 share a modular domain organization that comprises an RNA recognition motif (RRM) followed by leucine-rich repeat (LRR), NTF2-like (NTF2L) and ubiquitin-associated (UBA) domains  that areThe NTF2L and UBA domains each contain a single FG-repeat-binding site. Although structurally unrelated, these domains bind to FG-repeat sequences in a manner similar to that of the importin/karyopherin-β family transport receptors . These tin vivo and in vitro assays, Tap-p15 and Mex67-Mtr2 directly interact with RNA. Although the intrinsic RNA-binding activity of the mRNA export receptors is weak, D-type retroviruses, such as Mason-Pfizer Monkey Virus (MPMV) and Simian Retrovirus type 1 (SRV-1), have evolved an effective way to specifically exploit Tap-p15 to export unspliced viral mRNA [cis-acting RNA sequence that binds Tap-p15 with high affinity [i.e., the RRM, LRR and NTF2L domains, participate in RNA recognition  of the largest subunit of RNAPII ,114,115,1S2P3T4S5P6S7, the numbers of which vary across species, ranging from 26 in yeast to 52 in humans. The repetitive sequence functions as a binding platform for a wide variety of nuclear factors involved in capping, transcription elongation, splicing and cleavage/polyadenylation. Transcriptional stage specific modification of the CTD determines the types of factors that associate with it. For example, S5 of the CTD is phosphorylated during transcription initiation and is rapidly dephosphorylated upon the transition to transcription elongation. Thus, S5 phosphorylation is high at the promoter region and persists at low levels throughout the gene body. The capping enzyme is specifically attracted to the S5 phosphorylated CTD, and, thus, the cap is added to the 5' end of mRNA co-transcriptionally during transcription initiation [2 phosphorylation occurs later, increases during transcription elongation and decreases shortly after RNAPII reaches the transcription termination site. Factors that act later in the transcription cycle, such as splicing, cleavage and polyadenylation, selectively interact with an S2 or S2/S5 phosphorylated CTD. Earlier studies have revealed that the localization pattern of the yeast THO and TREX components on active loci, which increases from the 5' to the 3' region, resembles to that of S2-phosphorylated RNAPII [2/S5-diphosphorylated CTD. Thus, the 5' to 3' increase in S2 phosphorylation directly mediates the recruitment of the TREX complex to the active genes [The CTD consists of a highly conserved heptapeptide repeat: Yitiation . In contd RNAPII ,118. Howve genes . The elove genes . This trve genes .5 phosphorylation, S2 phosphorylation occurs always approximately 600 bps away from the transcription start site, irrespective of the gene length. Therefore, it is conceivable that short and long genes can have different CTD phosphorylation levels at the transcription termination site; short genes tend to have higher levels of S5 and lower levels of S2 phosphorylated RNAPII [It has recently been reported by different groups that the phosphorylation cycle of RNAPII CTD is very similar at all transcribed yeast genes ,56,57. Tin vitro. The binding of RNA and Aly/REF then stimulates the intrinsic ATPase activity of Uap56, inducing the dissociation of Uap56 from the complex. Since Uap56 and Tap-p15 competitively bind to Aly/REF, the release of Uap56 from the complex allows Aly/REF to recruit Tap-p15 [After being recruited to transcriptionally active genes, the mRNA-binding adaptor proteins Yra1 and Aly/REF in the TREX complex are transferred to the mRNA, where they then recruit the export receptors Mex67-Mtr2 and Tap-p15. This final step is proposed to be regulated by yeast Sub2 and metazoan Uap56, both of which serve as DExD/H-box-type RNA helicases in the TREX complex ,122. The Tap-p15 .The TREX components are also recruited to the mRNA during the transcription termination via interactions with the cleavage/polyadenylation factors ,125. SynIt has been known for years that splicing stimulates gene expression, but the step at which splicing acts has been elusive , indicating that properly processed mRNP is prone to translocation [In addition to the recruitment of the nuclear mRNA export factors, processing drastically alters the structure and composition of mRNPs. Indeed, it has been reported that the lengths of purified mature mRNPs from yeast were considerably shorter than the expected lengths of the mRNAs within them, indicating that processed mRNPs are highly compacted . The com tentans . In addilocation ,143.Recent evidence has indicated a role of splicing in the structural alteration of mRNPs at the molecular level. A comprehensive analysis of the endogenous EJC protein and mRNA interactomes in human cells has revealed that the EJCs are present on the vast majority of exon-exon junctions and form high-molecular-weight multimers with their associated SR proteins  Figure . The mulAny failure at each step during the biogenesis of mRNA results in production of faulty mRNP, which, in principle, threatens genome integrity and the normal proteome in the cell. To eliminate the functionally defective mRNP and ensure translational fidelity, cells have evolved multi-layered surveillance mechanisms. The translation-dependent quality control, which takes place in the cytoplasm, is the best-studied mechanism to degrade defective mRNAs and proteins deposited in mRNP in the nucleus play important roles . Al


Dnmt2/Trdmt1 is a methyltransferase, which has been shown to methylate tRNAs. Deficient mutants were reported to exhibit various, seemingly unrelated, defects in development and RNA-mediated epigenetic heredity. Here we report a role in a distinct developmental regulation effected by a noncoding RNA. We show that Dnmt2-deficiency in mice results in cardiac hypertrophy. Echocardiographic measurements revealed that cardiac function is preserved notwithstanding the increased dimensions of the organ due to cardiomyocyte enlargement. Mechanistically, activation of the P-TEFb complex, a critical step for cardiac growth, results from increased dissociation of the negatively regulating Rn7sk non-coding RNA component in Dnmt2-deficient cells. Our data suggest that Dnmt2 plays an unexpected role for regulation of cardiac growth by modulating activity of the P-TEFb complex. In Zebrin vivo . Phalke in vivo . Furtherin vivo . Recent in vivo  and thatin vivo . Interesin vivo . HoweverStudies in mouse myocardium have shown that induction of Cdk9/cyclin T1 or Cdk7/cyclin H activity is linked to cardiac hypertrophy . Cdk9/cyIn vitro studies suggested that the presence of B2 RNA at a promoter prevents phosphorylation of Ser5 residues on the CTD by TFIIH. Sequence analyses of B2 families showed that they are closely related to tRNA genes [Mouse B2 RNA inhibits RNA polymerase II (Pol II) CTD phosphorylation by TFIIH via interaction with the polymerase ,13. In vNA genes ,15.Rn7sk is a small nuclear RNA that inhibits Cdk9 activity through physical association with the Cdk9/cyclin T complex ,17. Rn7sWe have shown previously that transcriptional induction of Cdk9 following small non-coding RNAs (sncRNAs) injection into one-cell embryos results in cardiac hypertrophy in mice . To inveThe experiments described here were carried out in compliance with the relevant institutional and French animal welfare laws, guidelines and policies. They have been approved by the French ethics committee . Dnmt2 -/- homozygote knockout mice [-/- and Dnmt2+/+ animals. Histology and measurement of cardiomyocyte diameters was performed according to established protocols [n = 5 animals each), as well as cardiomyocyte diameters at the level of the nucleus in longitudinal sectioned cells of the interventricular septum for the HE staining  or cardiomyocyte diameters at the level of the nucleus in transversal sectioned cells of the interventricular septum for the WGA staining . Investigators were blinded for the genotype of the mice.Three months old male and female mice were used. Xylazine/Ketamine-anaesthetized mice  were examined by echocardiography using the iE33 xMATRIX system with a 12MHz transducer . Anesthesia lowered the heart rate compared to conscious mice without significant differences for Dnmt2rotocols ,21. ThreMouse embryonic fibroblasts (MEFs) prepared from 12.5 days embryos were inactivated by 10μg/ml mitomycin (Sigma). Dnmt2 knock out, kindly provided by T. Bestor, and control ES cells were grown on inactivated feeder cells in standard ES culture medium. According to well-established procedures , embryoin = 2) were quantified using ImageJ with the Gel Analysis tool.Total lysates from cell cultures and hearts were prepared, electrophoresed, and blotted. The following antibodies were used for immunodetection: polyclonal anti-CDK9 antibody from rabbit  in a 1:500 dilution in PBS, 2.5% Blotto, 0.05% Tween-20, polyclonal anti-RNA Pol II from rabbit  1:500, mouse monoclonal anti ß-actin (Santa Cruz Biotechnology), peroxidase-coupled goat anti-rabbit secondary antibody (Santa Cruz Biotechnology) 1:10000, and peroxidase-coupled rabbit anti-mouse secondary antibody (Santa Cruz Biotechnology) 1:10000. Western Blots (n = 6 each) using Trizol Reagent (Invitrogen). Ten μg total RNA were incubated with 2 μg of 5-mC monoclonal antibody  overnight at 4°C in 500 μl RIP buffer ) complemented with protease inhibitor . Afterwards, protein G beads  were added and after 1 hour incubation, 3 washes of pelleted beads performed in RIP buffer. 5-mC antibody precipitated RNAs were isolated using Trizol Reagent. cDNA synthesis was performed using the Maxima First strand cDNA Synthesis kit (Thermo Scientific) and quantitative PCR for Rn7sk using SYBR Select Master Mix (Applied Biosystems). Traces of non-specific bound GAPDH served for normalization.Total RNA was prepared from hearts of Dnmt2-deficienct mice and wild-type littermates , 2 mM vanadate (Sigma-Aldrich). One tablet of protease inhibitor mixture  was added just prior to use. Whole cell lysates were incubated on ice for 30 min and the protein concentration was determined using a BCA protein assay kit . 500 μg proteins were incubated with 10μg polyclonal anti-Cdk9 antibody from rabbit  for 4h. Alternatively, 10 μg of a polyclonal anti-Cyp2 antibody from rabbit  were used as a negative control. Protein A–agarose beads were added and rotated at 4°C for 4h. RNA protein complexes were washed 4 times with polysome lysis buffer , 2mM vanadyl ribonucleoside complexes solution (Sigma-Aldrich), 25μl protease inhibitor cocktail (pH 7.0). RNA protein complexes were eluted from beads with polysome lysis buffer including 0.1% SDS and 30μg proteinase K and reverse cross-linked at 50°C for 30 min, followed by TRIzol (Invitrogen) extraction and ethanol precipitation.Immunoprecipitation assays were performed as described . ES cellData are expressed as means ± SEM. ANOVA with the Bonferroni test as post hoc test was used versus controls. Differences between two groups were tested using the Mann-Whitney test for nonparametric samples. Differentiation of 200 wild-type and Dnmt2-deficient ES cell clones each was estimated for each time point using a contingency table with Fisher’s test and Yate’s correction. A p-value less than 0.05 is considered statistically significant.-/- mice compared to age-matched controls revealed increased left ventricular mass relative to body weight and higher left ventricular end-diastolic and end-systolic volumes whereas the ejection fraction as measure of cardiac contractility was preserved . Interestingly the proportion of phosphorylated versus total pol II, the form required for productive transcript elongation, increased in Dnmt2-deficient mice . Similarin vitro. Culture of the Dnmt2-deficient and control ES cells in hanging drops led to their aggregation in embryoid bodies (EB) [The morphology and growth characteristics of Dnmt2-deficient ES cells were comparable to wild-type ES cells; and they did not exhibit any sign of differentiation, neither into cardiac muscle nor otherwise. As it is established that activated RNA pol II plays a key role in cardiac growth and differentiation and we could detect more active form of RNA polymerase II in Dnmt2-deficient ES cells, we tested the differentiation potency of Dnmt-deficient ES cells ies (EB) . After pies (EB) , to be iin vivo and in vitro. Cdk9 positively and Rn7sk negatively regulates the P-TEFb complex, CTIP2 represses the Cdk9 kinase activity of P-TEFb [We thought that activation or over-expression of the P-TEFb complex may have a role in the observed phenotype in growth and differentiation of cardiac cells f P-TEFb . Cdk9 exf P-TEFb . Based oIn vitro methylation studies failed to show other substrates for Dnmt2 other than tRNA, which was achieved by incubation of Dnmt2 protein with purified total RNA [in vitro reconstitution studies rely on purified protein components and a controlled environment may not accurately reflect the complex intracellular atmosphere. Besides these in vitro studies, and the recent report on the role of Dnmt2 for bone formation in mice [It has been shown that Dnmt2 is part of the RNA processing machinery during cellular stress, by which Dnmt2-mediated tRNA methylation protects tRNA from endonucleolytic cleavage . In vitr in mice , little -/- than in Dnmt2+/+ hearts. This might explain the lack of inhibition of the P-TEFb complex, as RNA methylation protects them from cleavage [in vitro and in vivo results from increased occupancy of the promoters of these genes by the P-TEFb complex or is simply reflecting cardiac hypertrophy remains an open question.We have previously shown that small non-coding RNAs can induce hereditary epigenetic variations and act as the transgenerational signalling molecules , 28, 29.cleavage ; and unmcleavage . Whetherin vitro and in vivo. Taken together, we suggest that Dnmt2 is required to limit cardiac growth and differentiation by regulating RNA pol II phosphorylation, which involves the non-coding RNA Rn7sk.Although we cannot exclude that other yet unidentified mechanisms might contribute to the observed phenotype, the ablation of the Dnmt2 gene is changing the levels of several genes involved in cardiac hypertrophy


L appear to form another layer of the whole gene regulatory network. The degraded RNA such as a 5′-half-tRNA and an rRNA fragment function as small guide RNA (sgRNA) to guide the enzyme to target RNA. We were curious whether there exist RNAs in plasma that can function as sgRNAs for tRNase ZL, whether these RNAs are working as signaling molecules between cells to fulfill physiological roles, and whether there are any differences in plasma sgRNA species and levels between normal and pathological conditions. Here, we analyzed small plasma RNAs from three healthy persons and three multiple myeloma patients for potential sgRNAs by deep sequencing. We also examined small RNAs from peripheral blood mononuclear cells (PBMC) of three healthy persons and three myeloma patients and from various cultured human cell lines for sgRNAs. We found that read-number distribution patterns of plasma and PBMC RNAs differ between persons in the range of 5–40 nt and that there are many RNA species that exist significantly more or less abundantly in the plasma or PBMC of the myeloma patients than those of the healthy persons. Furthermore, we found that there are many potential sgRNAs in the 5–40-nt RNAs and that, among them, a 31-nt RNA fragment derived from 94-nt Y4-RNA, which can function as a 5′-half-tRNA-type sgRNA, is overwhelmingly abundant in the plasma of 2/3 of the examinees. These observations suggest that the gene regulatory network via tRNase ZL and sgRNA may be extended intercellularly.Several pieces of evidence suggest that small RNA degradation products together with tRNase Z L and guide the enzyme to artificial target RNA molecules via four base-pairings  × 1/6C3/2) and the probability of that happening is 2.4 × 10-43 in the binomial test, suggesting that the myeloma patients are significantly segregated from the healthy persons with respect to small plasma RNA abundance.Next we listed top 5 abundant RNA sequences for each of 5 to 40 nucleotides in each person and compared frequencies of each RNA sequence among the 6 persons in order to find small RNA species whose frequencies distinguish the myeloma patients from the healthy persons. There were 753 comparing sequences in total after excluding sequences overlapping between persons, and 79 sequences of them existed significantly  of the myeloma patients we examined was older than that (27 years) of the healthy persons , those dP = 0.0020 and 0.0013) more abundant in the myeloma patients  and three multiple myeloma patients (MM4–MM6) for their frequencies and sequences. Although, on the whole, frequency distribution patterns were similar among the six persons, each person showed his/her own distribution pattern . The disP = 0.013). Thus, using this value, we might be able to distinguish myeloma persons from healthy persons.Each read RNA sequence from the PBMC was assigned to its potential original transcript(s), and relative frequencies of the 10 RNA categories are shown for each person . The 6 pP = 0.00011–0.0498) more or less abundant in PBMC from the myeloma patients than in those from the healthy persons. Representative 6 comparisons and the others are shown in Val fragment, a 34-nt tRNAGly fragment, and a 35-nt tRNAGlu fragment were significantly (P = 0.0015–0.020) more abundant in the myeloma patients and a 31-nt snoRNA fragment was significantly (P = 0.00078) less abundant (P = 0.00014–0.0496) more or less abundant in groups of three persons containing one or two myeloma patients, while 109 were so in the group of the three myeloma patients. Given random distributions, an expected value of the numbers of the sequences that were significantly more or less abundant in the myeloma patients is 25.0 (= [109 + 141] × 1/6C3/2) and the probability of that happening is 4.2 × 10-43 in the binomial test, suggesting that the myeloma patients are significantly segregated from the healthy persons with respect to abundance of small RNAs from PBMC.We extracted top 10 abundant RNA sequences for each of 5 to 40 nucleotides in each person and compared frequencies of each RNA sequence among the 6 persons. Among 747 comparing sequences, 109 sequences were significantly  of the category tRNA in the myeloma patients  more abundantly in the PBMC from the myeloma patients than in those from the healthy persons  more or less abundant in PBMC from the myeloma patients than in those from the healthy persons, their frequencies in the 11 cell lines are presented in Relative frequencies of the 10 RNA categories for each cell line are shown in Although the patients MM1 and MM6 are the identical person, the frequency distribution pattern, the relative frequencies of the 10 RNA categories, and the abundant RNA sequences for each of 5 to 40 nucleotides differed clearly between the plasma and PBMC RNAs. This suggests that small RNAs in plasma are not secreted in proportion to those in PBMC. It is not clear which tissues secrete the plasma small RNAs and whether primary secreted RNA molecules are further degraded in plasma. We also do not know precise mechanisms of how the small degraded RNA products are generated in the cells.L and sgRNA may be extended intercellularly  In vitro tRNase ZL cleavage assays. 5′-fluorescein-labeled RNA-Y was incubated for the indicated time periods with recombinant human Δ30 tRNase ZL in the absence or presence of the Y4-RNA fragment. The cleavage products were analyzed on a denaturing 10% polyacrylamide gel.(A) A structure of a complex of the Y4-RNA fragment with the model target RNA-Y. An arrow indicates the expected tRNase Z(PDF)Click here for additional data file.S2 Fig(A) Frequencies of 5–40-nt RNAs. Read numbers of 5–40-nt RNAs in the human cultured cell lines A549, HepG2, HeLa, Jurkat, KMM-1, HL60, RPMI-8226, DAUDI, HEK293, Huh-7, and IMR-90 are graphed. (B) Relative frequencies of the 10 RNA categories. Each read RNA sequence was assigned to the categories mRNA, rRNA, scRNA, snRNA, srpRNA, miRNA, lncRNA, piRNA, snoRNA, and/or tRNA, and their relative frequencies are presented for the eleven cell lines.(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


C9orf72 causes familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but the underlying mechanism is unclear. Using RNA pulldown and immunohistochemistry in ALS biosamples, Cooper-Knock et al. identify proteins that bind to the repeat expansions. Disrupted RNA splicing and/or nuclear export may underlie C9orf72-ALS pathogenesis.Expansion of GGGGCC repeats in  C9orf72 represent the most common genetic variant of amyotrophic lateral sclerosis and frontotemporal degeneration, but the mechanism of pathogenesis is unclear. Recent reports have suggested that the transcribed repeat might form toxic RNA foci that sequester various RNA processing proteins. Consensus as to the identity of the binding partners is missing and whole neuronal proteome investigation is needed. Using RNA fluorescence in situ hybridization we first identified nuclear and cytoplasmic RNA foci in peripheral and central nervous system biosamples from patients with amyotrophic lateral sclerosis with a repeat expansion of C9orf72 (C9orf72+), but not from those patients without a repeat expansion of C9orf72 (C9orf72−) or control subjects. Moreover, in the cases examined, the distribution of foci-positive neurons correlated with the clinical phenotype (t-test P < 0.05). As expected, RNA foci are ablated by RNase treatment. Interestingly, we identified foci in fibroblasts from an asymptomatic C9orf72+ carrier. We next performed pulldown assays, with GGGGCC5, in conjunction with mass spectrometry analysis, to identify candidate binding partners of the GGGGCC repeat expansion. Proteins containing RNA recognition motifs and involved in splicing, messenger RNA nuclear export and/or translation were significantly enriched. Immunohistochemistry in central nervous system tissue from C9orf72+ patients with amyotrophic lateral sclerosis demonstrated co-localization of RNA foci with SRSF2, hnRNP H1/F, ALYREF and hnRNP A1 in cerebellar granule cells and with SRSF2, hnRNP H1/F and ALYREF in motor neurons, the primary target of pathology in amyotrophic lateral sclerosis. Direct binding of proteins to GGGGCC repeat RNA was confirmed in vitro by ultraviolet-crosslinking assays. Co-localization was only detected in a small proportion of RNA foci, suggesting dynamic sequestration rather than irreversible binding. Additional immunohistochemistry demonstrated that neurons with and without RNA foci were equally likely to show nuclear depletion of TDP-43 (χ2P = 0.75) or poly-GA dipeptide repeat protein inclusions (χ2P = 0.46). Our findings suggest two non-exclusive pathogenic mechanisms: (i) functional depletion of RNA-processing proteins resulting in disruption of messenger RNA splicing; and (ii) licensing of expanded C9orf72 pre-messenger RNA for nuclear export by inappropriate association with messenger RNA export adaptor protein(s) leading to cytoplasmic repeat associated non-ATG translation and formation of potentially toxic dipeptide repeat protein.GGGGCC repeat expansions of  C9orf72 gene represent the most common cause of familial amyotrophic lateral sclerosis (ALS) and familial frontotemporal degeneration  to examine the abundance and location of RNA foci in cerebellum, where p62-positive protein inclusion pathology is characteristic of C9orf72+ disease -incorporated DNA probe was used against the sense RNA hexanucleotide repeat . Slides with tissue, lymphoblastoid cells or fibroblasts were fixed in 4% paraformaldehyde for 10 min. Before use, formalin fixed paraffin-embedded tissue sections were deparaffinized. Slides were blocked with hybridization solution  for 3 h at 66°C and then incubated with 400 ng/ml of denatured probe in hybridization solution overnight at 66°C. After hybridization, slides were washed once in 2×SSC/0.1% Tween-20 at room temperature and three times in 0.1× SSC at 65°C. Slides were mounted with mounting medium containing DAPI . All solutions were made with DEPC-treated water.2 per image, 511 × 511 pixels) z-stack made up of images at 0.13-µm intervals through the entire nuclear volume of the cell under consideration.Primary visualization of foci was performed using a Leica SP5 confocal microscope system with a ×63/1.4 oil immersion objective lens. The presence of foci was assessed within a high resolution  lysis buffer . Lysates were cleared by centrifugation and supernatants taken for experiments. Nuclear extracts from SH-SY5Y cells were prepared using the Dignam method .5 and GGGGCC5 RNA molecules with 3’ biotin modifications were used to identify protein binding partners in pulldown assays. 60 μl aliquots of streptavidin sepharose  were blocked overnight on a spinning wheel at 4°C with RPD lysis buffer containing 2% bovine serum albumin. Total extracts were lysed in RPD lysis buffer whereas cerebellum homogenates and SH-SY5Y whole cell or nuclear extracts were mixed 1:1 with RPD lysis buffer (2×) supplemented with protease and RNase inhibitors. 1-2 mg of the appropriate total cellular or nuclear lysate was mixed with 15 µg biotin-labelled RNA, incubated at room temperature for 30 min and then on ice for 30 min. Mixtures were then transferred to a 6-cm petri dish and UV irradiated on ice at 0.3 J/cm2 in a UV crosslinker (Fisher). Mixtures were then applied to blocked streptavidin sepharose and incubated at 4°C for 2 h with agitation. Following binding, beads were washed three times with RPD lysis buffer and then twice with RPD wash buffer . Complexes were eluted by addition of RPD elution buffer  and 10 µg RNase A followed by agitation at room temperature for 30 min. Eluates were analysed by SDS-PAGE and proteins identified by mass spectrometry or western immunoblotting.AAAAUUIn solution tryptic digestions were performed on the eluted fractions by the addition of 100 mM final concentration ammonium bicarbonate and 0.1% ProteaseMAX™ surfactant. Trypsin was added to a mass ratio of (1:50) and incubated at 37°C overnight. Digestions were stopped with the addition of 1–2 µl glacial acetic acid and subsequently dried under vacuum. Tryptic digests were resuspended in 0.1% final concentration of trifluoroacetic acid. Five microlitres was used for liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) analysis. Peptides were separated using an UltiMate™ 3000 RSLC nano liquid chromatography system (Dionex), using a 150 mm × 75 µm I.D. PepMap™ reversed phase column (Dionex). Linear gradient elution was performed from 95% buffer A (0.1% formic acid) to 50 % buffer B  at a flow rate of 300 nl/min in 60 mins. MS/MS analysis was performed using a maXis UHR TOF mass spectrometer  using an automated acquisition approach. MS and MS/MS scans (m/z 50–2000) were acquired in positive ion mode. Lock mass calibration was performed using HP 1221.990364. Line spectra data were then processed into peak list by data analysis using the following settings. The sum peak finder algorithm was used for peak detection using a signal to noise ratio of 10, a relative to base peak intensity of 0.1% and an absolute intensity threshold of 100. Spectra were deconvoluted and the peak lists exported as Mascot Generic Files (MGF) and searched using Mascot 2.2 server (Matrix Science). The Swiss-Prot database  was searched using the following parameters . Search parameters were as follows: enzyme; trypsin; fixed modifications: carbamidomethyl (C); variable modifications: deamidation (NQ), oxidation (M); maximum missed cleavages: 1. Deamidation (NQ) were chosen as variable modifications. Additionally, we also used a peptide MOWSE score of <25 as a cut-off as calculated by Mascot. The false discovery rate was estimated to be 1% for peptide IDs after searching reverse databases. Protein identifications were based on a minimum of two unique peptides.5 RNA was 5’ end labelled with γ32P-ATP using T4 polynucleotide kinase (Fermentas). Reaction mixes were made up in RNA binding buffer  with 50 ng radiolabelled RNA and 5 µg purified recombinant protein. Mixes were incubated for 20 min at room temperature and 20 min on ice before being UV-irradiated on ice at full power. Complexes were analysed by SDS-PAGE and stained with Coomassie blue before being vacuum-dried and exposed on a phosphoimage screen.RNA-binding assays were carried out as described previously . GGGGCC5The following antibodies were used for immunohistochemistry: anti-TDP-43 (Proteintech 10782-2-AP) anti-FUS (Novus NB100-2599), anti-hnRNP H1/F (Abcam ab10689), anti-hnRNP A1 , anti-hnRNP D (Proteintech 12770-1-AP), anti-SRSF1 , anti-SRSF2 (Abcam ab30817), anti-ALYREF  and anti-hnRNP C1/C2 (Abcam ab10294). Poly-(Gly-Ala) dipeptide repeat protein was detected using anti-GA antibodies  as previously described ; in three C9orf72− cases with ALS the average proportion of neurons containing foci-like staining was 1.6% (range 1.1–2.5%); in normal controls the average proportion of neurons containing foci-like staining was 1.4% (range 1.3–1.6%). Only seven foci-like objects were observed in 300 neurons from the six non-C9orf72+ cases and never was more than one focus-like object was observed in a single cell; in contrast the average rate in C9orf72+ tissue was two foci per cell. RNase treatment in fibroblasts ablated foci, illustrating that the labelled product is RNA and in agreement with previous studies ; in these patients the average proportion of foci+ neurons was significantly higher in the ventral horn . In the fourth case, who presented with frontotemporal degeneration and later developed ALS, the pattern was reversed (40% versus 63%). Foci were primarily nuclear, however, some cytoplasmic foci were also observed in fibroblasts, cerebellar granule cells and in motor neurons .We generated 3’ biotinylated RNAs with the following sequences: 5’-[AAAAUU] repeats A–C. Cont repeats D. Eluted5-derived list of bound proteins was carried out using the Database for Annotation, Visualization and Integrated Discovery (DAVID) . This was particularly striking in the list of GGGGCC RNA-binders isolated from nuclear extracts of the SH-SY5Y human neuronal cell line (Gene ontology (GO) enrichment analysis of each GGGGCCin vivo some of the hits identified by mass spectrometry. For this purpose, eight well-described RNA recognition motif-containing proteins including splicing factors and messenger RNA nuclear export adaptors were prioritized and selected depending on available and efficacious commercial antibodies. The distribution of each protein relative to RNA foci was examined in approximately 200 cerebellar granule neurons and 50 motor neurons from a minimum of three C9orf72+ cases with ALS. Simultaneous co-staining was carried out in parallel in C9orf72− cases with ALS and neurologically normal control subjects. For all tested candidates, overall cellular protein distribution was not grossly different between C9orf72+ cases, C9orf72− cases and controls except for areas where co-localization was demonstrated. In cerebellar granule cells we demonstrated co-localization of hnRNP A1, hnRNP H1/F, ALYREF and SRSF2 with 27%, 30%, 26% and 33% of RNA foci, respectively .We used confocal microscopy to validate ectively A–D. In mectively E–G. In cin vivo, specificity of interaction with the (GGGGCC)5 RNA was assessed using RNA pull down assays from whole neuronal SH-SY5Y cell extract and western immunoblotting  .We also examined the co-incidence of RNA foci with depletion of TDP-43 from the nuclei of motor neurons of C9orf72+ patients with ALS (2P = 0.46) .In view of our prediction that the repeat sequence might sequester several proteins important for messenger RNA export, we wanted to explore the relationship between repeat associated non-ATG translated protein and RNA foci in specific neuronal populations. As expression of dipeptide repeat proteins is reported to be rare in the ventral horn of C9orf72+ disease. This genetic variant is the most common identified cause of ALS and frontotemporal degeneration. We and others  through disruption of the normal function of factors involved in nuclear speckles and thus messenger RNA splicing; and (ii) through inappropriate licensing of the transcribed


For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be) stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa) prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications.A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image ( The absence of mirror-image l-nucleic acids (l-RNAs and l-DNAs) from living organisms makes them interesting tools for applications in diagnostics, sensoring or therapeutics since they are resistant against nucleolytic degradation and are immunologically passive [Stereospecificity of chemical and enzymatic reactions is a basic principle of the (bio)chemistry in our homochiral world . The traynthesis . Likewis passive ,4.The current use of oligonucleotides for therapeutic and other purposes is based on either of two general mechanisms: hybridization between nucleic acids through Watson-Crick base pairing or binding of oligonucleotides to other molecules through their three-dimensional shape. Watson-Crick base pairing is exploited in technologies such as antisense, RNA interference, exon skipping or ribozymes in which an oligonucleotide is hybridizing to a messenger RNA either to arrest its translation, to eliminate or to correct it. The ability of oligonucleotides to adopt complex three-dimensional structures and to bind to a target  is the basis of aptamer technologies ,6. The qd-proteins or l-nucleic acids) requires some careful thoughts and also needs appropriate capabilities in synthetic chemistry since current molecular biology or biotechnology methods cannot be applied. A first breakthrough in this regard was published by Kent and co-workers in 1992 [l- as well as the non-natural d-configuration the authors established what is called “reciprocal chiral substrate specificity”: The l-enzyme was able to cleave an l- but not a d-substrate whereas the corresponding d-enzyme cleaved only the d- but not the l-substrate. As expected, an achiral inhibitor blocked both the l- and the d-enzyme. The analyzed interactions, however, were kept in their “own homochiral world”, i.e. the l-polypeptide acted on the l-substrate whereas the exact mirror-image d-polypeptide acted on the d-substrate. The next logical question was how to implement new, artificial heterochiral interactions. This was first achieved with l-peptide binders that were selected to bind to a d-peptide target  using phage display. When the selected l-peptide binders were converted to their d-configuration, the resulting d-peptide binders recognized the natural l-peptide target [d-nucleic acid libraries are screened against a non-natural mirror-image target (often a d-polypeptide). When the resulting d-aptamers are converted to l-aptamers, they will recognize the intended natural target (l-polypeptide) [l-RNA aptamer was identified to bind to a structured d-RNA (TAR RNA) by selecting d-aptamers against the mirror-image l-TAR-RNA [d- and l-configuration of an aptamer will bind with equal affinity [To make use of functional macromolecules of non-natural chirality ( in 1992 . By synte target . The same target . For thipeptide) . Recentl-TAR-RNA . Importa-TAR-RNA ,12. Howeaffinity .l-oligonucleotides to technologies that rely on the hybridization of complementary RNA or DNA strands. In the early 1990s there have been reports suggesting that l-oligonucleotides could hybridize to natural d-RNA or d-DNA and may therefore potentially be used for antisense applications. In these studies hybridization of l-homo-oligomers (oligo-l-U or -l-dA) to poly-d-T or poly-d-U was investigated and the formation of triple-stranded complexes was reported [l-DNAs containing all four bases with complementary d-DNA or d-RNA molecules they did not find any evidence for heterochiral hybridization [l-DNAs have no future as wide scope antimessenger compounds (…)”. Thereafter, no further progress was reported on the use of l-oligonucleotides for antisense or related strategies.In contrast to aptamers, it has not been possible to apply reported –17. Howedization . The autTwo decades later in early 2014, a recent publication now re-challenges the principle of stereospecific RNA and DNA hybridization by claiming that ribozymes and DNAzymes can hydrolyze RNA molecules irrespective of their chirality . As the l- and d-RNA phosphoramidites), Thermo Fisher Scientific , ChemGenes  and Metkinen phosphoramidite). Crude oligonucleotides were purified either by PAGE or ion exchange HPLC (IEX) followed by salt removal and subsequent size exclusion chromatography . Purity and identity was confirmed by IEX- and RP-HPLC and LC-MS (ESI-). PNA-oligonucleotides were synthesized by solid phase peptide synthesis following Fmoc/Bhoc strategy [DNA/RNA-oligonucleotides were synthesized on an ABI-394 synthesizer using standard solid-phase phosphoramidite chemistry and workup procedures . Phosphostrategy . Fmoc/Bhstrategy . Purity d- or l-RNA, d- or l-DNA and PNA strands was analyzed by measuring temperature-dependent hyperchromicity. Oligonucleotides (3 μM each) in 10 mM phosphate buffer (pH 7.4), 100 mM NaCl were heated to 95°C, cooled down to 25°C with 1°C/min, and heated up again to 95°C with 1°C/min. Absorption at 260 nm was measured in 0.5°C intervals. The melting temperature (Tm) was calculated from the 1st derivative of three 25°C to 95°C heating ramps. Experiments were performed in a Cary 100 spectrophotometer . Data were analyzed using Prism 5.00 software . The following oligonucleotides were used:Hybridization of anti-parallel complementary and parallel complementary d- or l-RNA template 5′-CUU CAA GUC CGC CA-3′d- or l-DNA template 5′-CTT CAA GTC CGC CA-3′d- or l-RNA 5′-UGG CGG ACU UGA AG-3′Anti-parallel complementary d- or l-DNA 5′-TGG CGG ACT TGA AG-3′Anti-parallel complementary d- or l-RNA 5′-GAA GUU CAG GCG GU-3′Parallel complementary d- or l-DNA 5′-GAA GTT CAG GCG GT-3′Parallel complementary PNA Ac-tgg cgg act tga ag-Gly-NH2Complementary et al. [d- or l-RNA substrate (200 nM) by d- or l-hammerhead ribozyme or d- or l-DNAzyme  was performed in 50 mM Tris (pH 7.5), 10 mM MgCl2. For control experiments, d- or l-RNA antisense oligonucleotides were added at 2 nM. Samples were heated to 95°C and cooled down to 25°C at 1°C/min. Hydrolysis was analyzed after 5 h incubation at 37°C using a denaturating 20% polyacrylamide, 7 M urea gel in TBE buffer. Gels were first stained with ethidium bromide staining and then documented under UV light. The following oligonucleotides were used:Experiments were performed according to the protocols provided by Wyszko et al. . Brieflyd- or l-RNA substrate 5′-fluorescein-CUUCA AGUCC GCCA-3′d- or l-ribozyme 5′-UGGCG CUGAU GAGGC CGAAA GGCCG AAACU UGA-3′d- or l-DNAzyme 5′-GGCGG AGGCT AGCTA CAACG ATTGA AG-3′d- or l-RNA antisense 5′-UCAAG UUUCG GCCUU UCGGCC UCAUC AGCG CCA-3′d-RNA/d-RNA (l-RNA/l-RNA (m = 70.9 ± 0.1°C and Tm = 70.8 ± 0.1°C for (mean ± sem of n = 3), respectively. In contrast, no sharp hyperchromicity transition was observed when heterochiral, anti-parallel, complementary RNA strands were tested (d-RNA/d-DNA (l-RNA/l-DNA (m = 57.8 ± 0.1°C and Tm = 56.9 ± 0.1°C (mean ± sem of n = 3), respectively, and d-DNA/d-DNA (l-DNA/l-DNA (m = 60.4 ± 0.1°C and Tm = 60.0 ± 0.2°C (mean ± sem of n = 3), respectively. Significant hyperchromicity indicating hybridization of heterochiral strands could not be detected for any of the tested examples , respectively. A similar picture was observed for d-DNA (l-DNA (m = 80.1 ± 0.3°C and Tm = 79.6 ± 0.1°C (mean ± sem of n = 3), respectively.To finally complete the hybridization experiments, we analyzed the interaction of achiral peptide nucleic acid (PNA) with RNA and DNA oligonucleotides of either chirality. As expected, PNA hybridization was independent of the chirality: the hybridization of d-RNA  and l-RNA  contaminating an l-ribozyme solution (2 μM) creates a false positive “heterochiral” cleavage product that is indistinguishable from that generated by real homochiral RNA hydrolysis  is largely tolerated  and, what is most important, to include appropriate controls. An example for such a control experiment is the use of antisense probes that may help to discriminate between a de facto cleavage and an artificial cleavage product derived from contaminations  hybridization does not occur between oligonucleotides of different chirality. Consequently, heterochiral ribozyme/DNAzyme cleavage of RNA molecules is not possible as long as the mechanisms of the catalytically active nucleic acids rely on hybridization to their respective substrates. However, the powerful techniques of in vitro selection and in vitro evolution are able to deliver mirror-image oligonucleotide candidates [cross-chiral RNA polymerase ribozyme has recently been reported [We have systematically investigated the hybridization behavior of oligonucleotides displaying the ndidates  that canndidates  but may reported .S1 Figd-RNA and (B) l-RNA with parallel complementary d-RNA (red) and l-RNA (blue). (C) d-RNA and (D) l-RNA with parallel complementary d-DNA (red) and l-DNA (blue). (E) d-DNA and (F) l-DNA with parallel complementary d-DNA (red) and l-DNA (blue). Mean of three melting ramps (25°C to 95°C) is given as normalized absorption A / Amax at 260 nm. First derivative is shown as dotted line. Data is representative of two independent experiments.Hybridization of enantiomeric, parallel complementary oligonucleotides  was followed by measuring temperature-dependent hyperchromicity at 260 nm. (A) (PDF)Click here for additional data file.S2 Figl-hammerhead ribozyme (50 μM), (B) l-DNAzyme (2 μM), (C) d-hammerhead ribozyme (50 μM) or (D) d-DNAzyme (50 μM) in 50 mM Tris (pH 7.5)  or in 50 mM Tris (pH 7.5), 10 mM MgCl2  for 5 h at 37°C. Samples were analyzed by PAGE and LC-MS (ESI-).Homochiral hydrolysis of 5′-fluorescein-labeled RNA substrate (5 μM) by (A) was incubated with (A) (PDF)Click here for additional data file.


Chronic bee paralysis virus (CBPV) is a serious viral disease affecting adult bees. We report here the complete genome sequence of CBPV, which was isolated from a honey bee colony with the symptom of severe crawling. The genome of CBPV consists of two segments, RNA 1 and RNA 2, containing respective overlapping fragments. Chronic bee paralysis virus (CBPV) is the causal agent of adult bee paralysis, which results in severe losses of worker bees , 2. AlthThis is the first full-length sequence of CBPV reported in China, and the genome analysis of this Chinese isolate of CBPV showed that it consists of two segments, RNA 1 and RNA 2. RNA 1 is 3,657 nucleotides (nt) in length and contains a large open reading frame (ORF), which forms three small overlapping ORFs, including a putative viral RNA-dependent RNA polymerase (RdRp). RNA 2 contains 2,267 nt and shares 95% and 97% homology at the nucleotide level with French (accession no. EU122232) and Japanese (accession no. AB682803) strains. RNA 1 and RNA 2 of the CBPV genome differ significantly from those of other regions of the world at the amino acid level (KX168412 and KX168413.The genomic sequence of the chronic bee paralysis virus has been deposited in the GenBank under accession numbers


Massively parallel cDNA sequencing (RNA-seq) experiments are gradually superseding microarrays in quantitative gene expression profiling. However, many biologists are uncertain about the choice of differentially expressed gene (DEG) analysis methods and the validity of cost-saving sample pooling strategies for their RNA-seq experiments. Hence, we performed experimental validation of DEGs identified by Cuffdiff2, edgeR, DESeq2 and Two-stage Poisson Model (TSPM) in a RNA-seq experiment involving mice amygdalae micro-punches, using high-throughput qPCR on independent biological replicate samples. Moreover, we sequenced RNA-pools and compared their results with sequencing corresponding individual RNA samples.False-positivity rate of Cuffdiff2 and false-negativity rates of DESeq2 and TSPM were high. Among the four investigated DEG analysis methods, sensitivity and specificity of edgeR was relatively high. We documented the pooling bias and that the DEGs identified in pooled samples suffered low positive predictive values.Our results highlighted the need for combined use of more sensitive DEG analysis methods and high-throughput validation of identified DEGs in future RNA-seq experiments. They indicated limited utility of sample pooling strategies for RNA-seq in similar setups and supported increasing the number of biological replicate samples.The online version of this article (doi:10.1186/s12864-015-1767-y) contains supplementary material, which is available to authorized users. Massively parallel cDNA sequencing (RNA-seq) is gradually superseding microarrays in quantitative gene expression profiling . Apart fBrd1+/−) and of their wild-type (WT) littermates , with Cuffdiff2, edgeR, DESeq2 and TSPM. Validation using independent biological replicates is preferred over in silico analyses, using online databases or simulated datasets, as well as technical validation, using the same RNA samples, to confirm true-positive DEGs between two or more biological conditions [Diverse methods are available to align RNA-seq reads , summarinditions , 20. HenPooling biological replicate RNA samples, such as those derived from a number of experimentally similar animals, may retain the biological information, while reducing the cost of sequencing. Validity and utility of sample pooling for gene expression analyses using microarrays have been evaluated extensively –23. BiolBrd1 as a DEG. None of the DEGs was identified by any three of these methods  on a congenic C57BL/6NTac background, and from their WT littermates. 8–10 weeks old Brd1+/− (n = 8) and WT mice (n = 8) were sacrificed. Their brains were snap frozen in 2-methylbutane and sectioned (1 mm) coronally using a slicer matrix  at −20 °C. Amygdalae were identified [RNA samples were obtained from female mice, heterozygous for a targeted deletion of exon 3–5 of the entified  and punc16 individual RNA samples  7.53 (SD 0.31) ), 4 poolReads that passed quality control  were aligned to the mouse genome (Mus_musculus.GRCm.38.72) with corresponding gene model annotation (Mus_musculus.GRCm38.72.gtf) by TopHat 2.0.6 . OverallBrd1+/− and WT mice (8/group). 180 ng total RNA was reverse transcribed by iScript Select cDNA Synthesis Kit . All eight DEGs, detected by DESeq2 and TSPM, were selected for validation. 107 more genes were randomly selected for validation from the list of remaining 191 DEGs, detected by Cuffdiff2 and edgeR. After 10–20 cycles of specific target amplification with PreAmp master mix , high-throughput qPCR was performed on the BioMark HD , using 48.48 dynamic arrays  and SsoFast EvaGreen Low ROX kit  [Additional files 16 RNA samples were obtained by the procedures, described above, from another batch of female Digital expression count matrix of our RNA-seq data is available as the Additional date file


Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor. We show that developing axons misorient in the absence of LON-2/glypican when the SLT-1/slit guidance pathway is compromised and that LON-2/glypican functions in both the attractive and repulsive UNC-6/netrin pathways. We find that the core LON-2/glypican protein, lacking its heparan sulfate chains, and secreted forms of LON-2/glypican are functional in axon guidance. We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor–expressing cells. We propose that LON-2/glypican acts as a modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration.Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we demonstrate that in  Work in nematodes shows that the proteoglycan glypican acts as an extracellular modulator of netrin signals during nervous system development, interacting with cells that express the netrin receptor. Caenorhabditis elegans. We identify that the response of migrating neurons to a key guidance cue, UNC-6/netrin, is modulated by a specific proteoglycan, LON-2/glypican. We show that LON-2/glypican may carry out this regulation by interacting with the UNC-40/DCC netrin receptor on the cell surface. We propose that LON-2/glypican acts as an extracellular modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration.During the development of the nervous system, migrating axons are guided as they navigate through complex environments to reach their target destinations. These directed migrations are essential to ensure the proper wiring and function of the nervous system and are instructed by guidance cues and receptors. There is a remarkably small set of guidance cues and receptors relative to the large number of neuronal migrations, suggesting that the actions of these guidance cues might be diversified by regulatory mechanisms. We have addressed this question in the genetically tractable nematode  Directed migrations of developing axons are essential for the proper wiring of the nervous system. A host of guidance cues and their receptors instruct axon guidance decisions. However, how these cues and the growth cone’s responses to them are spatially and temporally regulated in vivo remains largely unknown. Answering this question is central to our understanding of how growing axons navigate in complex environments to reach their targets during development and regeneration.Caenorhabditis elegans [UNC-6/netrin is a highly conserved secreted guidance cue with structural similarity to the extracellular matrix protein laminin –3. UNC-6 elegans  and bioc elegans . A secon elegans ,8, and a elegans ,10. WhilC. elegans, which has been instrumental for discovering major conserved axon guidance pathways. During larval development, the axon of the mechanosensory neuron AVM migrates ventrally as its growth cone integrates signals from two complementary guidance cues , the two glypicans (lon-2 and gpn-1), perlecan (unc-52), and agrin (agr-1). We found that the mild AVM axon guidance defects of sdn-1/syndecan mutants, including a null, [lon-2/glypican in double mutants lon-2 sdn-1  and misguides the axon of AVM [lon-2/glypican enhanced the defects caused by slt-1/Slit misexpression  and of the GABAergic motorneuron axons [lon-2/glypican single null mutants are defective in dorsal DTC migrations . In wild-type animals, PVM, like AVM, expresses the receptor unc-40/DCC, and its axon grows ventrally towards unc-6/netrin  in PVM ependent . In contgrations , highliglon-2/glypican functions in the same genetic pathway as known downstream mediators of unc-6/netrin signaling, we tested for genetic interactions between lon-2/glypican and unc-34/enabled. unc-34/enabled is a regulator of actin polymerization for axonal filopodia outgrowth [unc-6/netrin and slt-1/Slit guidance pathways renders the analysis of genetic interactions in the context of normal AVM axon guidance challenging. Therefore, we used the unc-6/netrin-specific gain-of-function approach as above, in which the dorsal migration of the PVM axon upon ectopic expression of unc-5/UNC5 is unc-34/enabled dependent  transgenes expressed under the heterologous epidermal promoters Pdpy-7 and Pelt-3  rescued lon-2 slt-1 double mutants back to slt-1 single mutant levels, as efficiently as when expressed under the endogenous promoter Plon-2 , in the migrating neuron itself (Pmec-7), in the intestine (Pelt-2), or in body wall muscles (Pmyo-3)  . Our ressdn-1(+) in the AVM neuron (using the heterologous promoter Pmec-7) rescued axon defects of lon-2 sdn-1 double mutants  cannot replace the function of lon-2/glypican; expressing sdn-1/syndecan in either the cells that normally express lon-2/glypican (using Plon-2::sdn-1) or the migrating neuron itself (Pmec-7::sdn-1) did not rescue the loss of lon-2/glypican (lon-2/glypican and sdn-1/syndecan have specific roles in axon guidance.We found that expressing wild-type copies of  mutants . Accordin-1::gfp ) revealeM neuron , at the glypican , supportGlypicans are composed of a core protein moiety with covalently linked HS chains attached via a tetrasaccharide linker at specific Serine residues , superfolder-GFP (SfGFP), and FLAG, respectively , sax-3/Robo, and sqv-5, suggesting that lon-2/glypican functions specifically in the unc-6/netrin pathway. Third, sdn-1/syndecan, cannot replace lon-2/glypican function, highlighting a requirement for lon-2/glypican that cannot be achieved by any HSPG. Given that the core protein of LON-2/glypican, devoid of its HS chains, is fully functional in guidance, the specificity of action of LON-2/glypican in netrin-mediated guidance appears to reside in the core protein itself. As a note, whereas lon-2/glypican mutants are defective in DTC migration, the lon-2/glypican mutant by itself does not show drastic alterations in AVM axon guidance as is observed with other modulators [lon-2/glypican, another HSPG may provide compensation or that our scoring of strong alterations in pathfinding did not include more subtle phenotypes, as could be expected from a modulator of the signal [The impact of dulators . It is pe signal .unc-6/netrin-mediated guidance. Thus, the core protein is the critical region of LON-2/glypican for netrin-mediated axon guidance. This is in line with previous studies showing a contextual dependence of HS chains for glypican function. For instance, the core protein of C. elegans LON-2/glypican and of Drosophila glypican Dally do not require HS chains to function in the transforming growth factor beta (TGFβ) pathway [Drosophila glypican Dally-like interacts with Wg and Hh through their protein core in a HS-independent manner [We show that the LON-2/glypican core protein, devoid of HS attachment sites, is able to associate with UNC-40-expressing cells and is functional in  pathway ,39. Simit manner ,40,41, at manner –45. Whilt manner ,39,41,42LON-2/glypican is predicted to localize at the cell surface via its GPI anchor . HoweverDrosophila glypican Dally-like protein is cleaved at the GPI anchor by the lipase Notum, to negatively regulate Wnts [Xenopus embryo [Glypican cleavage by lipases and proteases has been demonstrated to occur and to be functionally important in other contexts, such as in regulating fibroblast growth factor (FGF) and Wnt signaling during morphogenesis ,46. For ate Wnts . Similarate Wnts . The funate Wnts . In addis embryo  and incrs embryo , respectWe demonstrate that LON-2/glypican is secreted into the extracellular medium and decorates the outline of UNC-40/DCC-expressing cells. Deleting the extracellular domain of UNC-40 (UNC-40ΔNt) abrogated the association of LON-2/glypican with UNC-40/DCC-expressing cells, indicating that LON-2/glypican may interact with UNC-40/DCC at the cell surface. The association of LON-2/glypican with UNC-40/DCC may be direct or indirect through interactions with other molecules . Our explon-2/glypican functions in the same signaling pathway as the UNC-40/DCC downstream mediator unc-34/enabled during axon guidance.We found that LON-2/glypican leads to increased irregular morphology of UNC-40/DCC-expressing cells. Ectopic expression of DCC in mammalian cells activates downstream signaling via Cdc42 and Rac1, producing cytoskeletal rearrangements that lead to filopodia outgrowth and cell surface extensions . Our finOur results suggest a possible regulatory mechanism in the extracellular space in which secreted LON-2/glypican modulates the activity of the receptor UNC-40/DCC. LON-2/glypican may directly bind UNC-40/DCC, or alternatively, LON-2/glypican may instead interact with other molecules to impact UNC-40/DCC to modulate its stability, distribution, or activity. Alternatively, LON-2/glypican could potentially function as a co-receptor for UNC-6/netrin, where it may facilitate the formation of UNC-6/netrin-UNC-40/DCC-LON-2/glypican signaling complexes, similar to the situation in FGF signaling . It is aIn summary, our studies uncover a novel mechanism by which UNC-6/netrin signaling through its UNC-40/DCC receptor is modulated by the HSPG LON-2/glypican during axon pathfinding. Given the evolutionary conservation of the UNC-6/netrin pathway components (UNC-6/netrin and its receptors UNC-40/DCC and UNC-5/UNC5) and of glypicans  and that synthesis of HS chains is required for mammalian axons to respond to netrin-1 in vitro ,10, glypNematode cultures were maintained at 20°C on NGM plates seeded with OP50 bacteria as described . Strainsgpn-1 cDNA amplification, and primers oCB992 (TCGCTTCAAATCAGTTCAGC) and oCB993 (GCGAGCATTGAACAGTGAAG) were used for the control gene Y45F10D.4 [Total RNA was extracted from worm samples using Trizol (Invitrogen) according to the manufacturer’s instructions. 500 ng RNA was used to reverse transcribe using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and random primers. PCR reactions were carried out with first-strand cDNA template, primers oCB834 (ATCAAGACCGAGTGATAGTG) and oCB1321 (TGGCGAGTATTCCCGTTTAG) were used for 45F10D.4  cDNA ampAnimals were mounted on agarose pads, anaesthetized with 100 mM sodium azide, and examined under a Zeiss Axio Scope.A1 or a Zeiss Axioskop 2 Plus.zdIs5, an integrated Pmec-4::gfp reporter [Axons of neurons AVM and PVM were examined in L4 larvae and adult animals using reporter . AnimalsWorms were counted as mutant for AVM ventral axon guidance if (a) AVM failed to send an axon ventrally and instead projected laterally to the anterior or (b) the AVM axon projected laterally, in the anterior or the posterior direction, for at least ~15 μm (>3 AVM cell body lengths) before projecting to the ventral side. The angle between the anterior/posterior axon projection and the ventral axon projection had to be >45° to be counted as mutant, therefore excluding animals with a slight curve in the ventral axon of AVM from the mutant count.zdIs5 labeled neurons were too misplaced to be identified were excluded from analysis.Worms were counted as having their PVM axon misoriented dorsally if the axon of PVM was projecting to the dorsal side of the animal. The vulva was used as a reference for the ventral side. Worms whose The path of migration of the DTCs brings about the shape of the mature gonad arms  and oCB1124 (ATCATGGGGCCCTAAGCTGAATTCCCATAAC) were used to amplify a PCR product out of N2 genomic DNA containing bases 13,104 of cosmid C39E6 to 26,408 of cosmid F55D10 of the dpy-7 was cut with XmaI and ApaI and ligated with insert of lon-2 cDNA digested out of pCB251 (Pmec-7::lon-2) with XmaI and ApaI.Vector Pdpy-7::lon-2 (pCB268) was digested with HindIII and XmaI to release Pdpy-7 and ligated with insert of Pelt-3 2-kb promoter  amplified out of N2 genomic DNA (modeled after [HindIII and XmaI sites.Vector Ped after ) using pgrd-10::GFP (in pPD95.75 backbone) was digested with KpnI and EcoRI to release GFP and ligated with insert of lon-2 cDNA (yk1346g07) amplified with primers oCB1034 (CATGATGGTACCATGGTCTTCCGGTGGCTCATTC) and oCB1035 (ATCATGGAATTCTCAAAAAAGTTTAATAACTGC) to add on KpnI and EcoRI sites.Vector PXmaI and NheI and ligated with insert of lon-2 cDNA (yk1346g07) amplified by primers oCB963 (CATGATCCCGGGATGGTCTTCCGGTGGCTCATTC) and oCB964 (ATCATGGCTAGCTCAAAAAAGTTTAATAACTGC) to add on XmaI and NheI sites.Vector pPD96.41 was digested with dpy-7::lon-2 (pCB268) was digested with HindIII and XmaI to release Pdpy-7 and ligated with insert of Pelt-2 1-kb promoter (coordinates on cosmid C33D3: 2933–3875) amplified out of N2 genomic DNA (modeled after [HindIII and XmaI sites.Vector Ped after ) using pdpy-7::lon-2) was digested with HindIII and XbaI to release Pdpy-7 and was ligated with insert of Pmyo-3 digested out of vector pPD95.86 with HindIII and XbaI.Vector pCB268 (Pdpy-7::lon-2 (pCB268) was digested with HindIII and XmaI to release Pdpy-7 and ligated with insert of lon-2 3-kb promoter  amplified out of N2 genomic DNA (modeled after [HindIII and XmaI sites.Vector Ped after ) using plon-2::lon-2) was digested with XmaI and EcoRI to release the lon-2 cDNA and ligated with insert of lon-2 cDNA (yk1346g07) amplified with primers oCB963 (CATGATCCCGGGATGGTCTTCCGGTGGCTCATTC) and oCB1074 (ATCATGCTCGAGTCAATCCGGCTGAATTTCTTTTTCC) to add on XmaI and EcoRI sites and generate a truncated form of LON-2 after amino acid 488 to remove the GPI anchor (modeled after [Vector pCB246 (Ped after ).dpy-7::lon-2) was digested with XmaI and EcoRI to release the lon-2 cDNA and was ligated with insert of lon-2 cDNA (yk1346g07) amplified by primers oCB963 (CATGATCCCGGGATGGTCTTCCGGTGGCTCATTC) and oCB1075 (ATCATGGAATTCTCACCTTCCGAGTCGGTCCCACG), to add on XmaI and EcoRI sites and generate a truncated form of LON-2 after amino acid 368, i.e., containing amino acids 1–368 (modeled after [Vector pCB268 (Ped after ).lon-2::lon-2) was digested with XmaI and NheI to release lon-2 cDNA and was ligated with insert of lon-2 cDNA amplified by primers oCB964 (ATCATGGCTAGCTCAAAAAAGTTTAATAACTGC) and (1) oCB1253 (TCCGTCCTACCTGCAGAAGAAGTGAAAATCTGTGATCACTCG), (2) oCB1254 (ATTCTTTTTGTATTGCTCTACCGGTCCGTCCTACCTGCAGAAG), and (3) oCB1255 (CATGATCCCGGGATGGTCTTCCGGTGGCTCATTCTTTTTGTATTGCTCTACC) to add on XmaI and NheI sites, to add on the 22 amino acid lon-2 signal peptide sequence, and to truncate LON-2 to begin at amino acid 369. Thus, this construct codes for 22 amino acid residues of the signal peptide of LON-2, followed by residues 369–508.Vector pCB246 (PXmaI and XhoI and ligated with insert of sdn-1 cDNA (yk139f3) amplified with primers oCB903 (CATGATCCCGGGATGATTCTGAAACTCAATTTC) and oCB904 (ATCATGCTCGAGTTACGCGTAAAATTCTTTTG) to add on XmaI and XhoI sites.Vector pPD96.41 was digested with lon-2::lon-2) was digested with XmaI and EcoRI to release lon-2 and was ligated with an insert of sdn-1 cDNA digested out of pCB242 (Pmec-7::sdn-1) with XmaI and EcoRI.Vector pCB246 (Pttx-3::mCherry (50 ng/μL), Pceh-22::gfp (50–75 ng/μL), and/or Punc-122::rfp (50–75 ng/μL). pBSK+ (90–100 ng/μL) was used to increase total DNA concentration if needed. For details on specific coinjection marker(s) used for each rescued transgenic line, see Transgenic animals were generated by standard microinjection techniques . Each colqIs4), LON-2::GFP (TLG257), and LON-2ΔGAG::GFP (TLG199) worms were collected in buffer and protease inhibitors (Roche). Worm pellets were subjected to repeated freeze-thaw cycles. Protein concentration was measured using the Pierce 660 nm Protein Assay on a Nanodrop. 70 μg of samples mixed with 2x Laemmli sample buffer (Bio-Rad) were boiled, separated by SDS-PAGE on a 4%–20% Mini-Protean TGX gel (Bio-Rad), and transferred to PVDF membrane. Membranes were incubated in 1:3000 anti-GFP primary antibody (Millipore #AB3080) and 1:9000 goat anti-rabbit HRP secondary antibody (Bio-Rad #166-2408EDU). For the loading control, membranes were incubated in 1:5000 anti-HSP90 antibody (CST #4874) and 1:10000 goat anti-rabbit HRP secondary antibody (Bio-Rad #166-2408EDU). Signal was revealed using Clarity Western ECL Substrate (Bio-Rad) and imaged using film (LabScientific).Mixed-stage wild-type (N2), GFP control  was amplified by PCR with primers adding the EcoRI and ApaI restriction enzyme sites and cloned into pBlueScript II (Life Technologies). HA::LON-2 was made by synthesizing a DNA fragment, carrying the restriction sites EcoRI and HincII and containing the 5ʹ end of the lon-2 cDNA with the HA tag added after the signal peptide sequence (at cDNA position 61) and cloned into pBlueScript/lon-2. The HA::LON-2 EcoRI/ApaI fragment was then cloned into the pActin5.1/V5-His vector (Life Technologies).The lon-2 cDNA (bases 739–1527) with the myc tag DNA sequence before the GPI anchor domain sequence (at cDNA position 1375) and an ApaI restriction site at the 3ʹ end. The PmlI/ApaI synthesized fragment was then cloned into the pActin5.1/HA::LON-2 (pCB285) construct cut with the same restriction enzymes.The HA::LON-2::myc construct was made by synthesizing the 3ʹ end of the XhoI/BsmI fragment (bases 803–1340 of the lon-2 cDNA) in the pBlueScript/HA::lon-2 plasmid with synthesized fragment where the lon-2 sequence is mutated at the three predicted heparan sulfate chain attachment sites  modeled after [lon-2, the HA::LON-2ΔGAG EcoRI/ApaI fragment was then cloned into the pActin5.1/V5-His vector (Life Technologies).The LON-2ΔGAG construct was made by replacing the ed after . SimilarBsmI/ApaI fragment (bases 1340–1527 of the lon-2 cDNA) in the pBlueScript/HA::LON-2ΔGAG plasmid with a synthesized fragment containing the myc tag DNA sequence before the GPI anchor domain sequence (at cDNA position 1375) and an ApaI restriction site at the 3ʹ end. The HA::LON-2ΔGAG::myc EcoRI/ApaI fragment was then cloned into the pActin5.1/V5-His vector (Life Technologies).The double tagged HA::LON-2ΔGAG::myc construct was made by replacing the C. elegans unc-6 full-length cDNA (yk603d12) was amplified by PCR with primers adding the EcoRI and ApaI restriction enzyme sites and cloned into pBlueScript II. The superfolder-GFP::UNC-6 construct was made by synthesizing a DNA fragment, carrying the restriction sites EcoRI and HincII, and containing the 5ʹ end of the unc-6 cDNA with the superfolder-GFP sequence after the signal peptide sequence (cDNA position 70) and cloned into pBlueScript/unc-6. The superfolder-GFP::UNC-6 EcoRI/ApaI fragment was then cloned into the pActin5.1/V5-His vector.The C. elegans full length unc-40 cDNA (yk449d8) was amplified by two sequential PCR reactions that added the XhoI and ApaI restriction enzyme sites, as well as the FLAG tag at the 3ʹ end before the stop codon (adding the FLAG tag at the C-terminus of UNC-40 after the intracellular domain). This UNC-40::FLAG cDNA was then cloned into the pActin5.1/V5-His vector.The unc-40 intracellular fragment (cDNA bases 3022–4245 that include the coding sequence for the transmembrane and intracellular domains of UNC-40), along with the FLAG tag, was amplified by PCR using the UNC-40::FLAG cDNA as template and primers carrying the EcoRI and ApaI sites. A start codon (ATG) was added to the forward primer. The UNC-40ΔNt::FLAG fragment was then cloned into the pActin5.1/V5-His vector.The qyIs155, which contains a deletion of fibronectin III domains 4 and 5 of the unc-40 cDNA , was amplified by two sequential PCR reactions adding the EcoRI and ApaI restriction sites and the myc tag sequence at the 3ʹ end before the stop codon. The sdn-1::myc EcoRI/ApaI fragment was then cloned into the pActin5.1/V5-His vector (Life Technologies).The The pAc/Evi::EGFP  and pAc/S2 cells were transfected with HA::LON-2::myc (pCB313) and HA::LON-2ΔGAG::myc (pCB330) constructs. Cells were washed once with 1X Phosphate Buffered Saline and lysed for 30 min at 4°C in 1X Phosphate Buffered Saline, 0.5% Triton X-100, and 1X Protease Inhibitor Cocktail (Roche). Samples of supernatant and cell lysates were each mixed with 2X Laemmli sample buffer (BioRad). Proteins were separated by SDS-PAGE and transferred to PVDF membrane. Membranes were incubated with rabbit anti-HA (Life Technologies #715500) and rabbit anti-myc (Santa Cruz #sc-789) primary antibodies and HRP-linked goat anti-rabbit (Bio-Rad #166-2408EDU) secondary antibody. Signals were revealed by chemiluminescence with Clarity Western ECL Substrate (BioRad) and imaged using the ChemiDoc System (BioRad).S2 cells were independently transfected with HA::LON-2::myc (pCB313), UNC-40::FLAG (pCB301), or SfGFP::UNC-6 (pCB292) constructs. 48 h after transfection, old culture medium was removed, and new medium was added to resuspend the cells. Equal volumes of resuspended cells that had been transfected with individual constructs were mixed and cocultured overnight. Cells were harvested, centrifuged, and combined with their corresponding supernatant from each of these cell mixes. 100 μL of supernatant of each mixture was saved and kept on ice. Cell pellets were washed once with 1X Phosphate Buffered Saline and lysed for 30 min at 4°C in 100 μL of ice-cold RIPA buffer  supplemented with Protease Inhibitor Cocktail (Roche) and PMSF. Cell lysates were combined with their corresponding supernatant and mixed with 2X Laemmli sample buffer (BioRad). Each sample was split into three in order to run three protein gels in parallel. Proteins were separated by SDS-PAGE and transferred to PVDF membrane. Membranes were incubated with rabbit anti-myc (Santa Cruz #sc-789), mouse anti-FLAG (Sigma #F3165), and rabbit anti-GFP (Millipore AB3080) primary antibodies as well as HRP-linked goat anti-rabbit (Bio-Rad #166-2408EDU) and HRP-linked horse anti-mouse (Vector Labs PI-2000) secondary antibodies. Signals were revealed by chemiluminescence with Clarity Western ECL Substrate (BioRad) and imaged using the ChemiDoc System (BioRad).S2 cells were maintained in SFX Insect Media (HyClone) containing 10% Fetal Bovine Serum (HyClone) and Penicillin-Streptomycin (50 units-50 μg/mL) (Sigma). 70%–90% confluent S2 cells were transfected with 500 ng of each construct using Effectene (Qiagen) according to the manufacturer’s protocol. 48 h after transfection, old culture medium was removed and new medium was added to resuspend the cells. Equal volumes of resuspended cells that had been transfected with individual constructs were plated onto coverslips and cocultured overnight. Cells were then fixed with 4% paraformaldehyde and immunostained with rabbit anti-HA (Life Technologies #715500) and mouse anti-FLAG (Sigma #F3165) primary antibodies and Alexa594 donkey anti-rabbit (Life Technologies #R37119) and Alexa647 goat anti-mouse (Life Technologies #A21235) secondary antibodies. Confocal analysis was performed on a Zeiss LSM 5 Pascal confocal microscope. Confocal images were processed using ImageJ. Each experiment was repeated at least three times.For the experiment in which we use HA::LON-2-conditioned medium (supernatant) of cells expressing HA::LON-2, the culture medium was also changed 48 h after transfection, fresh medium was added, and the cells were incubated for another 48 h. This medium was collected and centrifuged at 1,500 rpm to remove cells and debris. This supernatant was added onto cells expressing UNC-40::FLAG, incubated overnight, and as above, fixed, stained, and imaged.Independent populations of S2 cells were transfected with (1) 450 ng of pActin5.1::mCherry alone, (2) 50 ng of the UNC-40::FLAG construct plus 450 ng of the cotransfection marker pActin5.1::mCherry, or (3) 500 ng of HA::LON-2. The medium was changed and cells were mixed 48 h after transfection. Control mCherry-expressing cells were mixed with untransfected cells or with HA::LON-2-expressing cells. Similarly, UNC-40::FLAG/mCherry-expressing cells were mixed with untransfected cells or with HA::LON-2-expressing cells. To maintain the total number of cells constant in our different mixes, one volume of UNC-40::FLAG/mCherry cells was mixed with either (a) one volume of control/untransfected cells or (b) one volume of LON-2-transfected cells. Cell mixes were cocultured overnight. Cells were then fixed with 4% paraformaldehyde and examined under a Zeiss LSM 5 Pascal confocal microscope. Control mCherry-expressing cells or UNC-40::FLAG/mCherry-expressing cells were identified by the cotransfection marker mCherry. 20 fields of ~300 cells each per mix per were photographed for each of three independent experiments. Cells were categorized as having the typical S2 cell round and smooth shape, irregular edges, and/or extensions protruding from the cell.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S1 Figunc-6/netrin and slt-1/slit pathways result in partially redundant defects, as previously established by the Culotti and Bargmann labs [p ≤ 0.001 . ns, not significant.Mutations in ann labs . Mutants(TIF)Click here for additional data file.S2 Figgpn-1(ok377) and gpn-1(tm595) are deletions (brackets) in the gpn-1 locus. (B) RT-PCR using primers oCB834 and oCB1321 yields truncated products in gpn-1(ok377) and gpn-1(tm595). Y45F10D.4 is a housekeeping gene used as an RT-PCR control [gpn-1 RT-PCR products for ok377 (blue) reveals that the transcript lacks most of exon 3 and has several in-frame stop codons (*), as intronic sequence (hatch pattern) gets incorporated into the mature transcript. Sequencing of the gpn-1 RT-PCR product for tm595 (red) reveals that the transcript lacks exons 2 and 3 and has several in-frame stop codons (*), as intronic sequence (hatch pattern) gets incorporated into the mature transcript. No alternatively spliced products were detected in the mutants ok377 and tm595. (C) gpn-1(ok377) and gpn-1(tm595) are strong loss-of-function mutations, likely nulls, in which, at most, small truncated proteins would get produced.(A)  control . Sequenc(TIF)Click here for additional data file.S3 Figsqv-5, the gene coding for the chondroitin sulfate polymerase [lon-2 function. Error bars are standard error of the proportion. Asterisks denote significance: *** p ≤ 0.001 . Click here for additional data file.S4 Figsdn-1::gfp (opIs171), we found that SDN-1::GFP is expressed in hypodermal cells and neurons, as previously reported [sdn-1/syndecan expression in AVM (Pmec-7::sdn-1) rescues the defects of sdn-1 mutants Click here for additional data file.S5 Figlon-2 slt-1 double mutants exhibit enhanced AVM guidance defects as compared to slt-1 single mutants. The defects of the double mutants can be rescued back down to slt-1 single mutant levels with expression of wild-type Plon-2::lon-2(+). In contrast, expression of sdn-1(+) in which lon-2/glypican is normally expressed (using the Plon-2 promoter) or in the AVM neuron (using the heterologous promoter Pmec-7) cannot rescue the axon guidance defects of lon-2 slt-1 double mutants. Data for wild type, lon-2, slt-1, lon-2 slt-1, and Plon-2::lon-2 in lon-2 slt-1 are as in p ≤ 0.001, * p ≤ 0.05.  (see ons) see  Tables.(TIF)Click here for additional data file.S6 Fig(A) Diagram of LON-2/glypican variants expressed in S2 cells, HA::LON-2::myc and HA::LON-2ΔGAG::myc, in which the three HS attachment sites were mutated from Serine to Alanine residues. The core protein of LON-2/glypican is red, and the heparan sulfate chains (HS) are yellow. (B) In the supernatant of cells expressing HA::LON-2::myc, high molecular weight species were detected with the anti-myc antibody, which likely corresponds to full-length HA::LON-2::myc with HS chains attached. In contrast, the species detected in the supernatant of HA::LON-2ΔGAG::myc-expressing cells are smaller and fainter, indicating that HA::LON-2ΔGAG::myc indeed affects the synthesis of HS chains onto the LON-2/glypican core protein. No signal was detected with the anti-HA antibody in the supernatants, likely due to technical limitations. In the cell extracts from HA::LON-2::myc-expressing cells, the main species runs at ~90 kDa, and it is detected with both the anti-myc and anti-HA antibodies, suggesting that it is full length. This signal likely corresponds to the LON-2/glypican core protein devoid of HS chains, as it runs as a tight band. The slight mobility shift in cell extracts of HA::LON-2ΔGAG::myc-expressing cells compared to HA::LON-2::myc might correspond to a difference of mass and isoelectric point between HA::LON-2::myc and HA::LON-2ΔGAG::myc. Anti-actin and anti-HSP90 antibodies were used as loading controls. Representative blots of more than four independent repeats.(TIF)Click here for additional data file.S7 Fig(A) Diagram of constructs used to express these proteins in S2 cells, showing the tags used to detect them. (B) Western blots for detection of HA::LON-2::myc, UNC-40::FLAG, and SfGFP::UNC-6. Constructs were individually and transiently transfected in S2 cells. Two d later, cells from single transfections were mixed and incubated overnight. Cells were harvested and combined with their corresponding supernatant from each of these cell mixes. Samples of each cell mix were split into three in order to run three parallel western blots and detect the proteins. As shown in (TIF)Click here for additional data file.S8 FigIn order to distinguish HA::LON-2-producing cells from HA::LON-2-acquiring cells, two separate populations of cells were transfected. One population of S2 cells was transfected with UNC-40::FLAG. A second population of S2 cells was simultaneously transfected with both GFP and HA::LON-2. Two d later, the two populations of cells were mixed, incubated overnight, and immunostained with anti-HA and anti-FLAG antibodies, as described for (TIF)Click here for additional data file.S9 FigDrosophila type I transmembrane receptor Evi. Evi-expressing cells were mixed with cells expressing LON-2/glypican and/or UNC-40/DCC. As shown in experiment 2 and in p ≤ 0.001 (t-test versus untransfected cells). ns, not significant. (C) Quantification of the association of SDN-1::myc with cells expressing UNC-40::FLAG, HA::LON-2, and untransfected cells. Ten different optical fields containing ~300 cells from three independent experiments were quantified.(A) Experiments 1 and 2 show that HA::LON-2 does not associate with cells expressing the unrelated (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.