Picornaviridae family is poorly understood. Human parechovirus 1 is an example of such viruses that contains 60 short regions of ordered RNA density making identical contacts with the protein shell. We show here via a combination of RNA-based systematic evolution of ligands by exponential enrichment, bioinformatics analysis and reverse genetics that these RNA segments are bound to the coat proteins in a sequence-specific manner. Disruption of either the RNA coat protein recognition motif or its contact amino acid residues is deleterious for viral assembly. The data are consistent with RNA packaging signals playing essential roles in virion assembly. Their binding sites on the coat proteins are evolutionarily conserved across the Parechovirus genus, suggesting that they represent potential broad-spectrum anti-viral targets.Assembly of the major viral pathogens of the  The mechanism underlying packaging of genomic RNA into viral particles is not well understood for human parechoviruses. Here the authors identify short RNA motifs in the parechovirus genome that bind capsid proteins, providing approximately 60 specific interactions for virion assembly. Multiple X-ray crystallographic and electron microscopic three-dimensional picornavirus structures show very little ordered density for the encapsidated nucleic acid, which would be consistent with the RNA playing no role in virion assembly.Picornaviruses are one of the largest groups of positive-sense single-stranded (ss) RNA viruses. They are major pathogens in all kingdoms of life, including human and animal viruses such as polio, human rhinovirus, and foot and mouth disease virus. Their virion assembly mechanism(s) has been extensively studied. Previous results suggest that they are an exception to the general findings in other examples of RNA viruses, that typically appear to exploit at least one high affinity coat protein (CP) binding site within their genomes to initiate virion assembly. In contrast, many studies including extensive nucleotide recoding of large sections of the polio genome have failed to identify any RNA determinants involved in picornavirus assembly8. All these virions contain 60 such RNA fragments that make identical contacts with their CPs around the five-fold vertices. The occupancy levels of the density are high, suggesting that they reflect important aspects of the virion structure. All of these viruses differs from the majority of picornaviruses in that they do not cleave their VP0 polyproteins into VP2 and VP4. This may be related to the presence of ordered RNA density. The role(s) of this RNA density, however, requires a functional explanation, especially as these structures may represent an earlier evolutionary state of the wider group of picornaviruses. The assembly and positioning of defined RNA fragments at precise locations within these virions suggests that their genomes may play an important role(s) in virus assembly.Recently, starting with the medium resolution EM reconstruction of human parechovirus 1 (HPeV1), several parechovirus structures have been reported that do contain extensive density for RNA in close contact with the overlying protein capsid, amounting in some cases to over 10% of the genome13. The classical PS hypothesis assumes the presence of a single PS site (a specific RNA fragment) with affinity for cognate CP that results in formation of an RNA-CP assembly initiation complex. PS-mediated assembly differs from this by recognising that multiple sites across ssRNA viral genomes can act as PSs, although these sites vary around a consensus sequence and thus have widely varying intrinsic affinities for CP15. Indeed, mathematical modelling of such assembly mechanisms suggests that modulation of CP-PS affinity is an essential feature of efficient assembly in order to avoid kinetic trapping16. Sequence variability in the PSs are therefore expected. Due to this, PS sites cannot be identified by a simple bioinformatics search for a repeating sequence motif.Previously we identified, in plant and bacterial viruses, an assembly mechanism we have termed packaging signal (PS)-mediated assemblyParechovirus genus. The sequence-specific PS-CP contacts that we identified could be potential targets for a novel broad-spectrum anti-viral therapy. Our insights into the processes of genome encapsidation and virion assembly may also be useful in the development of stable vaccine candidates.Here, we identified multiple PSs dispersed throughout the HPeV1 RNA genome using RNA-based systematic evolution of ligands by exponential enrichment (RNA SELEX), bioinformatics analysis and mutational analysis of an infectious HPeV1 clone. These PSs are conserved throughout the 7  under which we could reproducibly generate CP pentamers from purified HPeV1 virions (Harris strain), i.e. pentamers that are assembly competent  library. This outcome, together with the change in nucleotide composition of the starting and final aptamer pools  and naïve library (red curve). Roughly 6% of the selected unique aptamer sequences match the genome with this stringency. The frequency of matches within the selected aptamer pool is much larger than for the naïve library, with one exception, a polyU tract towards the 3′ end of the RNA. For aptamers corresponding to peaks below the level of this highest background peak, SELEX has not resulted in a sufficient enrichment to conclude a priori that the matching sites in the genome sequence are more significant than peaks resulting from an alignment of the naïve library. We therefore initially excluded these sites from analysis, leaving 21 statistically significant peaks dispersed across the genome. This result suggests that there are sequences within the HPeV1 genome that are likely to show specific affinity for the overlying CP, and these in turn are candidates to correspond to the ordered segments seen in the virion structure.If the genomic segments in contact with the CPs in the virions were bound sequence-specifically, we would expect these aptamers to share some primary/secondary structure motifs with them. Since the minimal CP recognition motifs within a PS may be discontinuous and/or structurally degenerated, e.g. by differing in the sequences of non-contacted base pairs, the number of identical nucleotide positions might be quite low. Therefore, in order to identify putative genomic sites with affinity for CP, we aligned all the aptamer sequences against every position in the Harris genome using a 1 nt sliding window. We quantified the goodness of any match between an aptamer and the genomic sequence with reference to the probability equivalent of finding a contiguous match. For example, if a non-contiguous match across a length of N nucleotides has the same probability as a contiguous match across M < N nucleotides, then we used M, known as the Bernoulli score (see Methods), to quantify the goodness of fit of the alignment. Since alignments of 12 nucleotides in a row are rare , we identified all alignments with a Bernoulli score of at least 12. For each such aptamer, we increased a counter by one at every matching nucleotide position, resulting in the histogram plot in Fig. 19 using the polyprotein gene region as a constraint. Whenever peak positions were located within at most 10 nt from each other in this alignment, we grouped them. Such peak positions are represented by columns in Fig. The variation in PS-CP affinities in other viral systems is manifest in subtle variations in the PS sequences/structures across the genome, making identification of PS recognition motifs based on data from a single virus genome challenging. However, due to their functional importance in assembly, PS sites are potentially conserved across strain variants. We therefore used all 21 complete HPeV1 genome sequences available from GenBank . Such PSs would be expected to encompass a common CP recognition motif. To explore this possibility, we extracted 41 nt long fragments around each of the highly conserved peaks from the Harris strain , two higher peaks but with low conservation , a lower peak close to the statistical cut-off (PS9), and the highest peak in the non-coding regions PS21), each containing 20 nt of the genomic sequence on either side of each peak nucleotide in the histogram plot , each marked by an asterisk in Fig. 4. High-resolution icosahedral averaging often results in the loss of RNA density, because the genome is an inherently asymmetric object with a unique nucleotide sequence. The extent of ordered RNA in the X-ray map is thus significantly less than that in the EM map . The model is an excellent fit  of a number of the individual PS sites in the context of an infectious HPeV1 cDNA clone  under the control of a T7 promoter. We introduced the minimal number of silent mutations required to disrupt the potential secondary structure of each chosen PS separately. In order to assess whether the peak heights in the Bernoulli plot Fig.  reflect 1 being unambiguously detected. In order to exclude the possibility that the outcomes are the result of effects on replication, we confirmed that the wild-type and mutants produced similar amounts of viral RNA using real-time PCR. Further, immunofluorescence with an anti-dsRNA antibody showed that there are also similar numbers of viral replication centres for all mutants in all the cells used. All the mutant genomes tested (except PS9-M) also produce similar in vitro translation products. Hence, in PS21, PS14 and PS18, the decreases in viral titre are most likely the result of alterations in assembly efficiency  cell line transiently expressing T7 RNA polymerase that then transcribes the viral genome. The viral titres in each case were determined by preparing cell lysates, treating them with RNase and DNase and then assaying their titres on HT29 cells using the 50% tissue culture infectious dose (TCID50) method. HT29 cells were chosen as they give a clear cytopathic effect with TCID50/ml as low as 1016 with the differing impacts of mutating individual PSs on the overall assembly efficiency being expected in the context of a co-operative process. Given the number of individual putative PS sites that appear to contribute to viral assembly, this is strong evidence in favour of PS-mediated assembly in which multiple PS sites act collectively.PS9-M and PS21-M each result in 6-log drops in TCID50 with no cytopathic effect , hinting at the spectrum of effects PSs may have on assembly. Here, stabilisation of the HPeV1 virion is very likely to be one such PS function.The detailed molecular mechanisms of PS action can vary from virus to virus. In at least one case, they overcome repulsive electrostatic contacts between protomers in a capsomerTo assess other possible effects we carried out in vitro binding assays to characterize the interactions of oligonucleotides encompassing some of the individual PS sites and their mutated versions, with CP pentamers. This is challenging because of the small amounts of the CP readily available (<200 µg). In order to conserve the CP, we carried out microscale thermophoresis (MST) binding assays with fluorescent dye end-labelled RNA PSs (Methods). MST is a non-destructive technique that can be carried out in a volume of ~10 µl, allowing a CP titration to be performed into a fixed amount of RNA. All the PSs showed binding that can be roughly rank ordered by affinities as PS14 > PS7/21 > PS6/9 . In contrast, all the associated variant sites did not bind under these conditions, suggesting that the mutational changes introduced had ablated CP recognition both in this assay and presumably in vivo in the context of the intact genome. Transmission electron microscopy of the highest MST titration points suggested that the cognate PS oligos, but not their mutational variants, trigger a partial self-association reaction  at 56 °C for 30 min followed by incubation at 4 °C for 10 days. Whole capsids were removed by centrifugation . Biotinylated pentamers (180 µg) were immobilised on 300 µl 10 mg/ml streptavidin-coated paramagnetic beads  by rolling overnight at 4 °C. These beads (referred to as positive beads) were then washed with 1× selection buffer three times to remove excess protein. The beads were resuspended in 300 µl of 1× selection buffer to a final bead concentration of 10 mg/ml.Purified HPeV1 Harris strain was biotinylated in phosphate buffered saline (PBS), at a molar ratio of 1:20 of capsid lysines:biotin , according to the manufacturer’s protocol. Pentamers were isolated by heating the biotinylated virus in 1× selection buffer  double-stranded DNA library using HiScribe T7 High Yield RNA Synthesis kit (NEB). The DNA template was then removed by digestion with TURBO DNase (Thermofisher Scientific) and the RNA purified using RNAClean XP resin (Agencourt).The naïve RNA library was transcribed from a synthetic combinatorial N40 . Samples were incubated at 52 °C for 1.5 h, followed by the addition of KAPA2G robust PCR mix  and the enriched library PCR amplified for the next SELEX round. Stringency was increased across the ten rounds in three ways: 1. By decreasing the target bead incubation time over the first five rounds from 25 to 5 min; 2. For rounds 6–10, the amount of target beads was halved to 10 µl; 3. The number of washes was increased from 10 to 15 over rounds 6–10.Ten rounds of SELEX were performed using a Biomek2000 liquid-handling robot (Beckman Coulter). 50 µl of the purified RNA library (10 µM) was added to 50 µl 2× selection buffer and incubated with 20 µl of 10 mg/ml MyOne T1 Dynabeads with no immobilised protein for 15 min at 37 °C. The beads were partitioned via magnetic separation (2 min) and the unbound fraction removed and incubated with 20 µl target beads for 25 min at 37 °C. Beads were partitioned again (2 min) and the unbound fraction removed. Beads were washed ten times (1 min incubations) with 125 µl of selection buffer to remove unbound/weakly bound RNA species before being finally resuspended in 35 µl DEPC-treated HFor rounds 5 and 10, a competition was carried out to remove RNA sequences that had a higher affinity for the capsid than for the inner pentamer surface. The SELEX round was performed as above but after the washes, the positive beads were incubated with either 0.1 mg/ml of biotinylated capsid (round 5) or capsid (round 10). A further three washes were carried out to remove the capsids and any capsid bound RNA aptamers. Elution, etc, was then performed as above. The amplified DNA of round 10 was then subjected to Next-Generation sequencing using Illumina MiSeq  = L−log4x where P = (1/4)B that a random sequence of B letters would align precisely with the genome. Since typically the fragment contributing to the score was smaller than the length of the aptamer and contained some mismatches, we identified the largest fragment of the aptamer that had the highest Bernoulli score, and therefore, the lowest probability of having aligned to the genome fragment by chance. For each comparison frame, the fragment of the aptamer that aligned to the genome with the maximum Bernoulli score was identified via computation of the contributions of different mismatches to the scores of any contiguous subset of the frame. If this maximum score was larger or equal to 12, we logged it into the data file that was subsequently used to compute the histogram in Fig. Sliding the aptamer sequence along the genome in increments of 1 nt generated genome frames for comparison with the aptamer sequence. In order to treat information at the 5′ and 3′-end on an equal footing to the middle sections in the histogram plot, we also considered partial overlaps, obtained from alignments of the 3′ region of the aptamers to the 5′ regions of the genome and vice versa, gradually increasing in steps of 1 nt until full overlap was reached. For each such alignment, we calculated the maximum Bernoulli score, defined as *3i ref. . This is31. The primers are listed in Supplementary Table Mutations in the PSs and in the capsid protein regions were made in an infectious HPeV1 cDNA clone, pHPeV1 , using the Geneart site-directed mutagenesis system (Invitrogen) according to the manufacturer’s instructionsInfectious genomic RNA was produced after transfection and translation in GMK cells. For this, the plasmids containing the cDNA clones of mutants or wild-type, were used as the templates to produce linear cDNA by PCR amplification using the T7 promoter-containing forward primer, 5′-TAATACGACTCACTATAGGGTTTGAAAGGGGTCTCCTAGAGAG-3′ and polyT-containing reverse primer 5′-TTTTTTTTTTTGTCATGTCCAATGTTCC-3′. The PCR running conditions were 98 °C (5 min), [98 °C (30 s) + 55 °C (30 s) + 72 °C (5 min)] × 25, 72 °C (10 min), 4 °C. The resulting linear cDNA contained the T7 promoter and the appropriate full-length HPeV1 cDNA genome.32 was transfected into GMK cells (kind gifts of Petri Susi) using Lipofectamine 2000 . Next day, these GMK cells were transfected with the linear cDNA, to generate full-length genomic RNA, which in the control experiments with the wild type cDNA, generates virions. Cell lysate was prepared from the transfected cells on the 3rd day post the second transfection by two cycles of freeze-thawing at −180 °C and 37 °C. The cell lysate was treated with RNase I  and DNase I  for 30 min at 37 °C to degrade any residual free RNA or DNA and subsequently added to HT29 cells  and scored for CPE for 4 days. The viral titres were expressed as 50% tissue culture infectious dose (TCID50) per ml using an end-point titration method described earlier33. The HT29 cells are an indicator cell line which shows clear CPE for HPeV1 compared to GMK. The HT29 and GMK cells were tested for mycoplasma prior to use.To test these mutants in vivo, a plasmid containing the T7 RNA polymerase under the CMV promoterIn order to gauge the efficiency of viral RNA and protein production in the GMK cells, the transfected cells were produced as for the above end point assay. In addition, cells were infected with HPeV1 virions (MOI 0.1) to serve as a positive control, and untransfected cells were used as a negative control.In order to detect the formation of double-stranded viral RNA, an immunofluorescence assay was performed. The cells were fixed at 6 h post second transfection with 4% paraformaldehyde for 15 min at room temperature (RT). The cells were washed twice with 1× PBS and permeabilised with 0.1% Triton X-100 for 20 min at RT, followed by blocking  at RT for 1 h. The primary antibody incubation was done at 4 °C overnight with mouse J2 mAb against dsRNA  at a 1:1500 dilution. The cells were washed three times with 1× PBS and incubated with a fluorescein isothiocyanate (FITC) labelled anti-mouse secondary antibody (Sigma) at 1:200 dilution. The cells were washed three times with 1× PBS. The 4′,6-diamidino-2-phenylindole (DAPI) stain (Sigma) at a working concentration of 0.1 µg/ml was added to the cells. The images were obtained with a Floid Cell Imaging Station (Life Technologies) in the Biocenter Finland Light Microscopy Unit, University of Helsinki.On the 2nd day post the second transfection, the wild type, mutants, positive virus infection and negative controls from the GMK cells were used for both total RNA extraction with Trizol (Invitrogen) and the production of cell lysates by freeze-thawing.34.The total RNA was used for cDNA synthesis with random hexamers utilising a Phusion RT kit (Thermo Scientific). The cDNA was subjected to quantitative real-time PCR (Roche) using primers and a probe designed for a 142bp-long conserved 5′ UTR region as described earlier35S-methionine. The signal was obtained on a phosphoimager after overnight exposure.Plasmids carrying the wild type genome (pHPeV1) or its mutants were transcribed and translated using the TNT Quick Couple Transcription/Translation system (Promega) as per the manufacturer’s protocol. A plasmid carrying the luciferase gene (provided in the kit) was used as the positive control. The proteins were labelled with 35. Each pentamer dilution was mixed with an equal volume of 50 nM of RNA , incubated for 1 h at 37 °C, and subjected to thermophoresis using the standard, untreated, capillaries. Conditions for MST: 5 s LED on (Blue LED), 35 s laser on, 5 s recovery. A control was also performed using the MS2 CP and its highest affinity PS, the translational operator, TR RNA. The MST signal is a composite of positive and negative thermophoretic effects that are acutely sensitive to the nature and solution environment of the dye-labelled species. If multiple self-association occurs, as was observed here, the signal can reverse preventing accurate Kd determination. The binding data were therefore assessed semi-quantitatively (+/−) noting the amplitude of signal change over the range of pentamer concentrations and where significant binding began to occur. These were compared to a known PS-CP interaction, that of bacteriophage MS2 TR and its CP , which was designated as +++++. The highest protein concentration samples from these assays were recovered, negatively-stained and examined by electron microscopy.We generated 5′-Alexa-488-labelled RNA oligomers based on the genome sequences for the PSs  and their sequence variants and measured their affinities for HPeV1 pentamers, ranging in concentration from ~0.5–900 nM pentamer, using a microscale thermophoresis Monolith machine (NanoTemper)4. The un-averaged 2mFo−DFc map was of sufficient quality for structural interpretation, without further real-space averaging being required. Close inspection of the deposited model revealed that portions of the RNA molecule and some of the surrounding amino acid side-chains were not optimally modelled into the electron density. These errors were corrected by manual rebuilding in COOT36 and 10 cycles of refinement in REFMAC37 with the X-ray weight set to 0.008 and strict ten-fold non-crystallographic symmetry applied. Structures were refined in parallel with either A or G in position 1 of the RNA molecule, to Rwork/Rfree values of 0.258/0.261 and 0.259/0.262 respectively, and root-mean-square deviations in bond lengths/angles of 0.007 Å/1.25° , with 93.8%/5.8%/0.4% of residues lying in favoured/allowed/forbidden regions of the Ramachandran plot. For the model with A in position 1 of the RNA molecule, the MOLPROBITY score was 1.86 (100% percentile), with 93.7%/5.9%/0.4% of residues lying in favoured/allowed/forbidden regions of the Ramachandran plot.The coordinates and structure factors for the X-ray crystal structure of human parechovirus were obtained from the PDB (accession code 4Z92)Computer code for sorting the next generation sequencing data and producing the sequence alignment plots is available upon request.The refined model for HPeV1 has been deposited in the Protein Data Bank in Europe under accession code 5MJV. The authors declare that all other data are available from the authors upon request.Supplementary InformationSupplementary Figures, Supplementary Tables and Supplementary ReferencesPeer Review FileReviewer reports and authors' response from the peer review of this Article at Nature Communications


Semliki Forest virus (SFV) is an arthropod-borne alphavirus that induces membrane invaginations (spherules) in host cells. These harbor the viral replication complexes (RC) that synthesize viral RNA. Alphaviruses have four replicase or nonstructural proteins (nsPs), nsP1–4, expressed as polyprotein P1234. An early RC, which synthesizes minus-strand RNA, is formed by the polyprotein P123 and the polymerase nsP4. Further proteolytic cleavage results in a late RC consisting of nsP1–4 and synthesizing plus strands. Here, we show that only the late RCs are highly active in RNA synthesis in vitro. Furthermore, we demonstrate that active RCs can be isolated from both virus-infected cells and cells transfected with the wild-type replicase in combination with a plasmid expressing a template RNA. When an uncleavable polyprotein P123 and polymerase nsP4 were expressed together with a template, high levels of minus-strand RNA were produced in cells, but RCs isolated from these cells were hardly active in vitro. Furthermore, we observed that the uncleavable polyprotein P123 and polymerase nsP4, which have previously been shown to form spherules even in the absence of the template, did not replicate an exogenous template. Consequently, we hypothesize that the replicase proteins were sequestered in spherules and were no longer able to recruit a template. Togaviridae together with the rubella virus, and they are mainly arthropod-borne viruses that infect vertebrates. The alphavirus genome of about 11–12 kb contains a 5′ cap and encodes two polyproteins. The P1234 polyprotein is processed into four nonstructural proteins (nsPs), nsP1–4, and the structural polyprotein, translated from a subgenomic RNA, is cleaved to yield the capsid and envelope proteins [Positive-strand RNA (+RNA) viruses are the most common type of RNA viruses, and include numerous human pathogens . The recproteins ,7,8, andproteins ,14,15,16Membrane-associated RCs are typical of all eukaryotic +RNA viruses. Membrane connection is indispensable, and two types of replication-associated membrane modifications have been recognized: membrane invaginations called spherules, as e.g., induced by alphaviruses; and double-membrane vesicles (DMVs), as e.g., induced by coronaviruses . AlphaviThe early RCs of alphaviruses are formed when the polymerase nsP4 is first cleaved from the nonstructural polyprotein. The resulting complex of P123 and nsP4 mainly synthesizes minus-strand RNA using the genomic RNA as a template ,21. NextGAA) with its active site Gly-Asp-Asp (GDD) mutated to Gly-Ala-Ala (GAA) causes spherule formation when co-expressed with P123 or nsP1 and P23. Spherule morphology in the absence of the template is, however, more irregular compared to those formed during normal replication, confirming the idea that the template plays a central role in size determination.Plasmid-based trans-replication systems have been developed to study the formation and activity of alphaviral RCs and the functions of the nsPs outside the context of viral infection ,27,28,29The RNA-synthesizing activity of alphavirus RCs has also been studied using in vitro replication assays (IVRAs). CHIKV, SINV, and SFV synthesize single-stranded genomic and subgenomic RNA as well as double-stranded RNA in vitro ,31,32, aHere, we applied the IVRA developed for CHIKV  to studyv/v) fetal bovine serum , 2 mM l-glutamine (Gibco), 100 U/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco). BSR T7/5 cells, a derivative of BHK-21 cells stably expressing T7 RNA polymerase [v/v) Difco tryptose phosphate broth , 2 mM l-glutamine, 1% (v/v) non-essential amino acids (Gibco), 1 mg/mL G418 , 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). Both cell lines were grown at 37 °C and in 5% CO2. Virus-infected or transfected cells were grown in the same conditions.BHK (baby hamster kidney)-21 cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10%  according to the manufacturer’s instructions. After 16–18 h incubation in minimum essential medium  containing 0.2% (v/v) bovine serum albumin (BSA) and 2 mM l-glutamine, the supernatant was collected. This primary stock was used to infect BHK-21 cells at a multiplicity of infection (MOI) of 0.01 in MEM containing 0.2% BSA and 2 mM l-glutamine. After 1 h, cells were washed twice and new medium was added. The supernatant designated as secondary stock was collected after 16–18 h post infection (p.i.). Titers were quantified by plaque assay titration [All virus and plasmid constructs used in this study contain a hemagglutinin (HA) peptide (YPYDVPDYA) in a frame within nsP3. For generation of SFV4-HA virus, the oligos encoding the HA-tag and flanking glycines were 5′-TCGAGGGCTACCCATACGATGTTCCAGATTACGCTGGTG-3′ and 5′-TCGACACCAGCGTAATCTGGAACATCGTATGGGTAGCCC-3′. Oligos were annealed, phosphorylated, and cloned into the XhoI site of replicon pSFV1-ZsG, removing the ZsGreen gene . A largeitration .HA4GAA expression construct, a NotI-BamHI fragment of nsP4 containing GAA substitution was transferred from P1234GAA [HA4. For the construction of P12CA3HA, Bsu36I and AgeI were used to replace ZsGreen in P12CA3Z [To generate HA-tagged replicase polyprotein constructs, the ZsGreen gene in constructs P123Z4 and P1^2^3Z4  was replP1234GAA  to P123H P12CA3Z  with the P12CA3Z .v/v) fetal calf serum . After the adsorption, cells were washed and DMEM containing 2% FCS was added. Mock-infected cells served as a control. Thirty minutes before labeling, cellular transcription was inhibited by adding Actinomycin D . Metabolic pulse-labeling of approximately 6 × 105 mock- or SFV-infected cells was performed by incubating cells with 40 µCi of 3H-uridine  in medium for 1 h. After the labeling, cells were lysed in LiDS/LET , 100 mM LiCl, 2 mM EDTA, and 5 mM DTT) and treated with proteinase K  for 15 min at 42 °C. Total RNA was isolated using the acid phenol method and analysed by denaturing agarose gel electrophoresis as described below. Fluorographic detection of 3H-labeled RNA was performed as described in [3H-uridine was also quantified from 2-µL samples of isolated total RNA with a liquid scintillation counter .BHK-21 cells were infected using the secondary stock of SFV4-HA at an MOI of 50 for 1 h in PBS containing 2%  (35 mM HEPES (pH 7.4), 250 mM sucrose, 2.5 mM DTT, 7 mM KCl) containing 2 μg/mL Actinomycin D, 200 U/mL RiboLock (Thermo Fisher Scientific), and Pierce EDTA-Free Protease Inhibitor . After a 20-min incubation on ice, cells were disrupted using a Dounce homogenizer with 40 strokes and unlysed cells and nuclei were removed by centrifugation  yielding a post-nuclear supernatant (PNS). A membrane pellet (P15) and cytosolic fraction (S15) were prepared by further centrifugation of PNS . The P15 fraction was washed once with DB containing Actinomycin D, RiboLock, and protease inhibitor and resuspended in the same buffer.For virus infection, the secondary stock of SFV4-HA was used to infect approximately 1 × 106 cells, 5.5 µg of the replicase, and 6.9 µg of the template plasmid were co-transfected. Mock-transfected cells without LTX were used as a control. After a 16-h incubation in MEM containing 0.2% BSA and 2 mM l-glutamine, cells were harvested by trypsinization. PNS, P15, and S15 were prepared as described for virus infection.For the trans-replication system, BSR cells were co-transfected with the replicase and Tmed template plasmids containing the T7 promoter . LTX wasHA4 or P123HA4 plasmid DNA per 10-cm dish, cells were harvested at 6 or 16 h post transfection, and PNS was prepared as described above. HA-affinity capture was performed as described in [g, 18 h, 4 °C) in a discontinuous iodixanol (Sigma-Aldrich) gradient consisting of 1 mL DB, 9 mL 30% (w/v) iodixanol in DB, and 2 mL 35% iodixanol in DB. The final layer contained 0.5 mL PNS. Protease inhibitor was used throughout the purification. After the centrifugation, the light-scattering zone between DB and 30% iodixanol layers was collected and used for the Western blotting and replication assay described below.BSR cells were transfected with 12.4 µg of P1^2^3ribed in , except 32P-CTP (Perkin Elmer). For IVRA with exogenous template RNA, an in vitro transcript of Tmed was prepared using a SacI-linearized template plasmid and a mMESSAGE mMACHINE® T7 Transcription Kit  according to the manufacturer’s instructions, and 1 µg of RNA was added per reaction. After 1-h incubation at 30 °C, IVRA reactions were terminated by the addition of LiDS/LET containing 80 µg/mL proteinase K and incubating for 15 min at 37 °C. Unicorporated label was removed using RNase-free Micro Bio-Spin™ P-30 Gel Columns , and RNA was isolated using the acid phenol method.IVRA was designed based on that described in . Typical® Reagent (Invitrogen) was used to isolate total RNA from PNS, P15, and S15 fractions prepared from transfected BSR cells according to the manufacturer’s instructions. In both methods, GlycoBlue (Ambion) was used as a coprecipitant and isolated RNA was dissolved in 1 mM sodium citrate . Isolated RNA was analysed by denaturing formaldehyde agarose gel electrophoresis as described previously [w/v) agarose gels were used.The acid phenol method was used to isolate total RNA from IVRA samples as well as cell lysates, PNS, P15, and S15 prepared from SFV-infected BHK-21 cells. RNA was extracted with acid phenol:chloroform, premixed with isoamyl alcohol , precipitated with isopropyl alcohol, and washed with 70% ethanol. TRIzoleviously , except HA4 and Tmed were treated with RNase Cocktail™ Enzyme Mix (Ambion) containing RNase A and T1 . Incubation was in DB  for 15 min at 37 °C, and RNA was isolated using TRIzol® Reagent and analysed as above.PNS, P15, and S15 fractions prepared from BSR cells co-transfected with P1^2^332P-labeled oligonucleotides was performed as described in [In-gel hybridization to detect RNA molecules from dried gels with ribed in . All hyb32P-labeled RNA was detected by exposing Phosphor Imager screens to dried gels and scanning the screens with a Typhoon 9410 imager . Incorporated label was quantified by Quantity One software (Bio-rad).All ® T7 or SP6 Transcription Kit (Ambion) according to the manufacturer’s instructions. SacI-linearized Tmed or XhoI-linearized pEAV221Δ plasmids were used as templates for Tmed and EAV in vitro plus transcripts, respectively. pEAV221Δ was obtained by cleaving pEAV221 [+ membrane . The membranes were prehybridized in a hybridization mix [2PO4, 5 mM EDTA, pH 7.4) and 0.05% SDS, and exposed to Phosphor Imager screens. To test the specificity of the capture probes, the membranes were also hybridized with 32P-labeled minus and plus transcripts of the SFV4-HA and Tmed described above. The labeled transcripts were prepared using a T7 or SP6 Transcription Kit according to the manufacturer’s instructions, except for that a nucleotide mix containing 20 µM ATP, UTP, CTP, and GTP and 0.12 µM (10 µCi) of α-32P-CTP (Perkin Elmer) were used.In vitro transcripts containing nucleotides 8390–11,468 of SFV4-HA genomic RNA (capture probe for minus strands) and its complementary sequence (capture probe for plus strands), nucleotides 1–2963 of Tmed RNA (capture probe for minus strands), nucleotides 1–2878 of Tmed negative-sense RNA (capture probe for plus strands), and nucleotides 1–2042 of equine arteritis virus (EAV) genome were prepared using mMESSAGE mMACHINEtion mix  for 24 h35S-l-methionine and 35S-l-cysteine labeling was performed using PNS prepared from mock BSR cells or cells co-transfected with P123HA4 and Tmed. The 35S-labeling was performed in the same buffer conditions as IVRA by incubating the samples with 22 µCi of EasyTag™ EXPRESS35S Protein Labeling Mix (Perkin Elmer) for 1 h at 30 °C. Unicorporated label was removed using RNase-free Micro Bio-Spin™ P-30 Gel Columns (Bio-rad), and proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with 4% and 12% (w/v) acrylamide concentrations in the stacking and separation gels, respectively. SDS-polyacrylamide gels were stained with Coomassie blue to show total protein composition. All 35S-labeled proteins were detected by drying the gels and exposing them to Phosphor Imager screens. If samples were heat inactivated, they were incubated for 5 min at 96 °C before the labeling. If translation inhibitors were used, samples were incubated with puromycin  or cycloheximide  for 60 min at 4 °C before the labeling. The same pre-incubation was also used when translation inhibitors were tested in IVRA.Protein concentrations were measured by the Coomassie blue method  using BSw/v) acrylamide concentrations in the stacking and separation gels, respectively, and transferred to AmershamTM ProtanTM Nitrocellulose Blotting Membrane . After blocking with 5% (w/v) non-fat dry milk powder in Tris-buffered saline (TBS), membranes were incubated with rabbit polyclonal antibodies against SFV nsP1, nsP2, nsP3, or nsP4 [v/v) Tween-20. Mouse monoclonal antibody against β-actin (Sigma-Aldrich) was used as a cytosolic marker. Secondary antibodies IRDye® 800CW donkey anti-rabbit IgG  and Alexa Fluor 680 donkey anti-mouse IgG (Invitrogen) were used for fluorescent detection of the primary antibodies with an Odyssey system (LI-COR). Quantification was performed using Image Studio Software (LI-COR).For Western blotting, proteins were separated by SDS-PAGE with 4% and 10%  and subgenomic (26S) RNA species became detectable after 1.5 h.p.i.  and the subgenomic promoter was provided as a template plasmid [BSR cells were co-transfected with the SFV replicase and template plasmids to determine if the RCs isolated from the trans-replication system are active in RNA synthesis in vitro. PNS was prepared from the transfected cells at 16 h post transfection using the same method as optimized for SFV-infected cells. The wild-type replicase polyprotein, P1234, as well as the polymerase inactive mutant, P1234 plasmid .GAA, where the majority of nsP4 was found in S15 (~80%). The P15 fraction of the cleavable constructs, P1234 and P1234GAA, contained the majority of nsP1 . Approximately 30% and 20% of nsP2 and ~50 and 40% of nsP3 were found in P15 of P1234 and P1234GAA, respectively. The polyprotein P123 was detected when cells were transfected with the uncleavable constructs, P1^2^34 or P12CA3, and the majority was distributed in P15 . Besides the polyprotein P123, bands recognized by anti-nsP1 and anti-nsP2 antibodies were detected in these samples. Their size was slightly smaller compared to nsP1 and nsP2 in the cleavable constructs, and thus they most likely represent unspecific cleavage products. Approximately 90% of the band detected by the anti-nsP1 antibody was found in P15. However, the unspecific cleavage product of nsP2 was also mainly found in the membrane fraction (~80%) in contrast to nsP2 in cells infected with SFV or transfected with the wild-type replicase, in which the majority of nsP2 was recovered in the cytosolic fraction  gave some background, most likely from the T7 polymerase that is expressed in BSR cells  activity. In addition, an unspecific product was observed in all constructs with a size below that of the subgenomic RNA. The higher expression levels of nsP4 most likely explain why P12CA3 + nsP4 produced more labeled RNA than P1^2^34  control for binding specificity. The IVRA products synthesized by SFV RCs strongly hybridized with the capture probe for the plus-strand RNA, indicating that SFV RCs mainly produced RNA of positive polarity in vitro  we studied the effect of translation inhibitors. We first established whether translation occurred when PNS samples were incubated under IVRA conditions by metabolic labeling of protein synthesis with  protein A. A syntsing PNS B, despitPNS , without success. To give the complex more time to form, we also pre-incubated P1^2^34 replicase-containing membrane preparations with an exogenous template for 1 h at 4 °C before the reaction was performed, and we tested different reaction times from 30 min to 2 h. None of these experimental variations resulted in the replication of the exogenous template.Herein, we analysed SFV replication using a combination of in vitro activity and trans-replication systems. The results described in this study revealed the main requirements for RNA synthesis in vitro and are summarized in Highly active RNA synthesis was observed in vitro only when RCs were isolated from cells containing the fully processed SFV replicase, either resulting from virus infection or transfection with the wild-type replicase and template plasmids . Highly Infection and the trans-replication system only yielded isolated RCs that synthesized exclusively plus-strand RNA . These cGAA it did not become membrane-associated but mainly remained free in the cytosol  has been used to purify hepatitis C virus (HCV) RCs . NS4B isEscherichia coli synthesizes RNA in vitro if combined with a P123-membrane fraction and template RNA [It is intriguing that the uncleavable polyprotein P123 and polymerase nsP4 of SINV are able to start the minus-strand synthesis from an exogenous template in a recombinant vaccinia virus expression system  in contrlate RNA , and thuIn this study, we showed that SFV polyprotein P123 and polymerase nsP4 were active in RNA replication only in cells, and after isolation these early RCs were unable to effectively synthesize RNA in vitro. In contrast, the polymerase nsP4 together with nsP1, nsP2, and nsP3 was highly active in vitro and these late RCs could be isolated for in vitro analyses. The next step to shed light on the mechanisms and molecular details that underlie the differences between the early and late RCs would be to analyse their formation from template recruitment to RNA synthesis and membrane deformation. This will, however, require that we freeze RC assembly at different stages. It has been shown that P123 alone does not form spherule structures . Thus, a


Caenorhabditis elegans is a key model system for the study of human aging, and strains were generated with modified C. elegans ryanodine receptors corresponding to human myopathic variants linked with malignant hyperthermia and related conditions. The altered response of these strains to pharmacological agents reflected results of human diagnostic tests for individuals with these pathogenic variants. Involvement of nerve cells in the C. elegans responses may relate to rare medical symptoms concerning the central nervous system that have been associated with ryanodine receptor variants. These single amino acid modifications in C. elegans also conferred a reduction in lifespan and an accelerated decline in muscle integrity with age, supporting the significance of ryanodine receptor function for human aging.Delaying the decline in skeletal muscle function will be critical to better maintenance of an active lifestyle in old age. The skeletal muscle ryanodine receptor, the major intracellular membrane channel through which calcium ions pass to elicit muscle contraction, is central to calcium ion balance and is hypothesized to be a significant factor for age-related decline in muscle function. The nematode  During RYR1 gene, with its many introns across such a large coding region, is awkward to manipulate. The nematode Caenorhabditis elegans, however, has a compact genome and the RYR1 ortholog, unc-68, is only 30 kb (WormBase). Nevertheless, UNC-68 has ∼40% amino acid identity with the human RyR1, distributed along the entire length of the proteins, suggesting that the mammalian and nematode ryanodine receptor operate and are controlled in similar fashion   by bacterial transformation, using positive selection for the cassette. The cassette was then replaced, with incorporation of the desired point mutation in a second bacterial transformation with a second PCR product bearing the desired sequence alteration, using negative selection against the cassette. A dicistronic cassette was used containing the positive marker tetA(C) conferring tetracycline resistance (TcR), and the negative marker rpsL+ conferring streptomycin sensitivity (StrS) in the rpsL− (thus StrR) EL350 host, a recombineering competent Escherichia coli strain  worms by microinjection (unc-68(e540) carries a point mutation toward the center of the gene and behaves genetically as a null , which causes an obvious dominant roller phenotype  hermaphrodites to generate male progeny heterozygous for unc-68. These males were mated with RW1596 {]}. The resulting hermaphrodite progeny were then allowed to self-fertilize to generate uncoordinated rollers, homozygous for unc-68(e540) and bearing the extrachromosomal array containing myo-3::gfp, rol-6(su1006). These worms were subjected to UV mutagenesis and screened for uncoordinated rollers with progeny that are all rollers due to the extrachromosomal array having been stably integrated into a chromosome  transgenes. Self-fertilization and selection for well-coordinated rollers yielded myo-3::gfp, rol-6(su1006) homozygous strains bearing the unc-68 transgenes.GFP-myosin strains were developed by mating C. elegans strains were routinely maintained in culture at 20° on 50 mm plates of Nematode Growth Medium (NGM)   . NGM pla2PO4, 42 mM Na2HPO4, 86 mM NaCl, 1 mM MgSO4) (Eggs were prepared by bleaching to synchronize worm populations for assay. Mixed stage population of nematodes were washed from the NGM plates in 500 μl M9 buffer (20 mM KHM MgSO4) . A total2HPO4, 6 g KH2PO4, 1 ml cholesterol (5 mg/ml in ethanol), H2O to 1 liter and autoclaved], 10 ml 1 M potassium citrate pH 6, 10 ml trace metals solution , 3 ml 1 M CaCl2, 3 ml 1 M MgSO4}  producing dsRNA for cbd-1, osm-3, or che-3. cbd-1 RNAi was used in the longevity assays to exclude progeny from the assay  yielded progeny with apparently wild-type locomotion, suggesting WRM069cA02 contains the entire unc-68 gene and all that is required for its expression. Each rescued line moved with ∼200 body bends per min in liquid medium, considerably faster than the 60 mean body bends per min of the unc-68 null mutant. Transmission of this rescued phenotype to subsequent generations established that this fosmid, in an extrachromosomal transgenic array, provides appropriate levels of expression of the ryanodine receptor and yielded the reference strain, UL4140, used in subsequent comparisons examining ryanodine receptor variants.backward . While munc-68 transgene in UL4140 fully rescue for locomotion but the strain responded to increasing concentrations of halothane and caffeine, the drugs used in the MH IVCT, in the same manner as the standard wild-type strain, N2    . Previoucaffeine , yet loco 80 mM) .RYR1 variants were selected for study in C. elegans   as other MH- and CCD-associated versions of UNC-68, like the corresponding human condition. This could result from the nonfunctional variant being stably expressed and, in combination with the wild-type protein, generating a malfunctioning heteromeric ryanodine receptor.Genetic dominance, a striking property of many pathogenic human unc-68 variant strains were examined for age-related phenotypes in C. elegans. First, a dramatic age-related change in response to caffeine was found for the LOAM (K3452Q) variant strain . Hence although the ryanodine receptor appeared to remain functional, these single amino acid changes did shorten C. elegans lifespan.Second, the lifespan . All theN2, although the difference is not as marked . Presumably this small reduction in lifespan results from the difference in expression of unc-68 when present in multiple copies as an extrachromosomal transgene, rather than in the endogenous location in single copy, within a chromosome.It is also noted that the lifespan of UL4140 is significantly shorter than the lifespan of the nontransgenic wild-type control, unc-68 variants on C. elegans body-wall muscle. The progressive disorganization of the sarcomeric structure with age can be followed in live C. elegans by fluorescence microscopy using GFP-tagged myosin . No significant difference in whole-body score on day 0 or day 2 of adulthood was found for any of the variant strains when compared to the UL4140-based control, transformed with just wild-type unc-68. The whole-body score was significantly increased compared to the control by day 4 for the R163C, A4940T, and K3452Q variant strains, by day 6 for R2454H and R2458H, by day 8 for G341R, and by day 10 for R2163H and R4861H, indicating that the single amino acid change in all unc-68 variants examined did induce faster muscle aging, although the age when this became statistically significant varied.Third, we found a specific aging effect of e of 1–5 , from 1 sequence . OverallC. elegansUNC-68 and this is likely to be a key intracellular Ca2+ channel in all cells, and critically in excitable cells. Furthermore, caffeine-resistant mutations have been localized to two C. elegans genes, osm-3 and che-3, specifically required for chemosensory nerve cell function . In young adults of the strain transgenic for only wild-type unc-68, the stimulation of locomotion at low concentrations of caffeine was completely lost and the level of inhibition of locomotion at high concentrations was reduced upon osm-3 and che-3 RNAi knockdown. Exactly the same result was observed for strains transgenic for the EHI-associated variant R163C, for the LOAM-associated variant K3452Q, and for the standard wild-type strain, N2. The inhibition of locomotion by caffeine, across the entire range of concentrations assayed, in the strains transgenic for the MH-associated variant R2163H and the CCD-associated variant A4940T was reduced by osm-3 and che-3 knockdown at each caffeine concentration. Furthermore, the dramatic progressive stimulation of locomotion in response to increasing concentrations of caffeine in old adults with the LOAM variant version of unc-68 was also eliminated with knockdown of these genes. In summary, for all strains examined, the locomotive response to each caffeine application was eliminated or markedly reduced by osm-3 and che-3 RNAi. These results could indicate that the focus of influence of the UNC-68 amino acid changes upon caffeine response is actually in these chemosensory nerve cells. UNC-68 does have a presynaptic function at neuromuscular junctions (C. elegans (UNC-68 in other cells, including or specific to muscle cells.While RyR1 is considered predominantly a skeletal muscle isoform and other isoforms are expressed in other cell types, there is only a single ryanodine isoform in unc-68 variant-specific changes in the body’s response to caffeine may be pertinent to human biology. Central nervous system damage has been reported occasionally after MH episodes. The skeletal muscle–specific effects of RYR1 variants may be simply a reflection of the predominant tissue-specific distribution of this gene’s expression. While RYR2 is expressed predominantly in cardiac muscle and RYR3 is more broadly and weakly expressed, RYR1 is also expressed in other tissues, including the central nervous system (RYR1 variants to central nervous system damage in response to triggering events (The fact that compromising chemosensory nerve cells eliminates the C. elegans to caffeine and halothane on directed modification of unc-68, as described here, emphasizes the conservation of functionality of the ryanodine receptor from humans to nematodes. Sequence changes in UNC-68, equivalent to human disease–causing variants, conferred increased sensitivity of the whole organism to pharmacological agents of direct relevance to medical conditions. The variants even induced this increased sensitivity in C. elegans in the presence of the wild-type protein, mirroring the genetic dominance of the human variant alleles. The distinction of increased caffeine sensitivity being seen for the unc-68 variants corresponding solely to MH or CCD, but not those implicated in EHI or LOAM, is based on analysis of a limited number of variants. However, this finding may also be relevant to muscle biopsies of EHI patients and some MH-susceptible individuals responding to halothane but not to caffeine in the diagnostic IVCT (The response of C. elegans longevity have been instrumental in our current understanding on this subject (C. elegans, adding further support to the evidence suggesting equivalent effects in mammals. The progressive increase in sensitivity to caffeine stimulation of locomotion with age in the strain with a unc-68 variant equivalent to LOAM may reflect changes that occur with age in humans and contribute to this condition.Processes determining animal lifespan appear remarkably conserved and studies on www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.040535/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.


Concentration is important and not only while driving; a new study indicates how an adjacent genomic element helps to increase the efficiency of a specific adenosine to inosine RNA editing reaction, by providing a means to increase the local concentration of the RNA editing enzyme ADAR. RNA editing by base deamination is an endogenous process of site-directed mutagenesis employed by organisms in all metazoa to modify genetic information as it passes through RNA. Thus, RNA deaminases such as APOBEC1 and the adenosine deaminase that acts on RNA (ADAR) family of enzymes can potentially become tools for manipulating genetic information by modifying the RNA sequence. This has the major advantage over other systems such as CRISPR/Cas9 in that the genetic information of the cell remains untouched and no introduction of a foreign protein (CAS9) into cells is required. However, the current limited knowledge about the factors that determine editing target selection hinders the ability to utilize them efficiently. The manuscript by Daniel et al. , 3.In mammals, ADAR1 and ADAR2 are two functional adenosine deaminases that act on RNA . These eOriginally, only the primary sequence and the structure adjacent to a specific edited site were thought to affect editing specificity and efficiency, such as the sequence preference of ADAR and an imperfect helical structure neighboring the edited adenosine . HoweverThe unique mechanism suggested by Daniel et al.  is reminSince the long dsRNA is also promiscuously edited by ADAR enzymes, a reasonable speculation may be that these elements also have a role in releasing the ADARs from the RNA molecule. In this way, a molecule that has attracted a very high concentration of enzyme will be eventually hyperedited at the long stem, lowering the ability to recruit further ADARs Fig. .Long dsRNAs are largely composed of reversely oriented non-coding retrotransposons, which constitute approximately half of the mammalian genome. The effect of these elements in the cell is mostly unclear and they are generally considered non-functional. Daniel et al. provide a new noteworthy regulatory meaning for some of these mobile elements by showing they induce RNA editing activity. This may be also valid for additional dsRNA-binding proteins, potentially extending the role of these elements.A-to-I editing has been studied extensively and shown to play a role in developmental processes and disease. Aberrant editing has been associated with autoimmune disorders, cancer, and neurological disorders . Here, D


Mitochondrial transcript maturation in African trypanosomes requires RNA editing to convert sequence-deficient pre-mRNAs into translatable mRNAs. The different pre-mRNAs have been shown to adopt highly stable 2D folds; however, it is not known whether these structures resemble the in vivo folds given the extreme “crowding” conditions within the mitochondrion. Here, we analyze the effects of macromolecular crowding on the structure of the mitochondrial RPS12 pre-mRNA. We use high molecular mass polyethylene glycol as a macromolecular cosolute and monitor the structure of the RNA globally and with nucleotide resolution. We demonstrate that crowding has no impact on the 2D fold and we conclude that the MFE structure in dilute solvent conditions represents a good proxy for the folding of the pre-mRNA in its mitochondrial solvent context. The folding of RNA molecules into compact, native structures or ensembles of structures is dictated by a set of first-principle physicochemical forces . One of RNA editing describes a posttranscriptional modification reaction of mitochondrial pre-mRNAs that is characterized by the site-specific insertion and deletion of exclusively U-nucleotides . The reHere, we analyze the effect(s) of macromolecular crowding on the structure of the mitochondrial RPS12 pre-mRNA as an archetypical example of a mitochondrial transcript in African trypanosomes. We use high molecular mass polyethylene glycol (PEG) as a neutral macromolecular cosolute to mimic intramitochondrial solvent conditions and we monitor the structure of the RPS12 transcript by temperature-dependent UV-spectroscopy and by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). T. brucei Lister 427 genomic DNA  and transcripts were generated by run off in vitro transcription from linearized plasmid templates using T7-RNA polymerase. RNAs were purified from nonincorporated NTPs by size exclusion chromatography followed by EtOH precipitation. RNA pellets were redissolved in 10 mM Tris/HCl pH 7.5, 1 mM EDTA (TE).The mitochondrial gene encoding ribosomal protein S12 (RPS12) was PCR-amplified fromDNA (see ) using tμL), heated to 95°C (2 min), and snap-cooled on ice before the addition of a concentrated folding buffer to yield a final volume of 0.5 mL. Final buffer concentrations were as follows: 5 mM Na-cacodylate, pH 6.8, 70 mM NaCl, and ±2 mM MgCl2. Volume-occupied conditions were generated by supplementing the folding buffer with PEG4000 at a final concentration of 6% (w/w). This exceeds the polymer crossover concentration (ϕ∗) of PEG4000 by a factor of 1.5 and thus represents a highly crowded solvent regime. RNA concentrations were adjusted to A260 = 0.5. Denaturation/renaturation profiles were measured at 260 nm between 20°C and 95°C at a heating/cooling rate of 1°C/min (data acquisition: 0.3 data points/°C). Melting temperatures (Tm) were obtained from the maximum of the first derivatives (δA260/δT) of the melting curves.RPS12 transcripts were dissolved in 0.5x TE pH 7.5 (50 μM) was denatured by heating to 95°C (2 min) followed by snap cooling on ice. RNA refolding was achieved by equilibration in 20 mM HEPES, pH 7.5, 30 mM KCl, and ±10 mM MgCl2 for 30 min at 27°C, which represents the optimal growth temperature of insect-stage trypanosomes. Crowding conditions were induced by either 6% (w/w) PEG4000 (1.5ϕ∗); 12% (w/w) PEG4000 (3ϕ∗); or 9% (w/w) PEG2000 (1.5ϕ∗). RNA samples were split and treated either with 3.5 mM 1 M7 in DMSO or the same volume of neat DMSO. Modification reactions were quenched after 70 sec by the addition of an equal volume of water. RNAs were recovered by EtOH precipitation and desalted by size exclusion chromatography. The different crowding conditions resulted in nearly identical modification results  with Pearson's (r) and Spearman's (ρ) correlation coefficients ≥ 0.87 and an average standard deviation of ±0.11 SU (SHAPE units).The modification reagent 1-methyl-7-nitroisatoic anhydride (1 M7) was synthesized as described . RPS12 p2, 2.5 mM DTT, 0.25 mM each dNTP, and 0.75 U/µL RiboLock RNase inhibitor (Invitrogen). The reaction was started by prewarming the samples for 1.5 min prior to the addition of 5 U/µL SuperScript III reverse transcriptase (Invitrogen). RPS12 pre-mRNA was reverse-transcribed for 20 min at 40°C. Sequencing reactions were carried out using unmodified RNA, fluorescently labelled DNA-oligonucleotide primer, and ddCTP or ddGTP at a final concentration of 0.125 mM each. Reverse transcription was stopped by snap cooling and the addition of 0.1 volume of 4 M NaOH followed by heating to 95°C (5 min). Samples were pooled, EtOH precipitated, and redissolved in Hi-Di® formamide (ABI/Life technologies) for capillary electrophoresis. Raw electrophoretic traces were analyzed using SHAPEfinder ).., 2013, , 24. Theelements . This cae effect , 41–45.  freedom . Based ol., 2015 , 47. How2+ is present. This suggests a highly stable and compact structure that obviously is not affected by volume exclusion effects. Thus, we conclude that the in vitro experiments properly reproduce the in vivo situation, at least on the level of RNA secondary structure. Furthermore, we conclude that the described sensitivity of the trypanosomal in vitro RNA editing system [The RPS12 transcript displays nonvarying local nucleotide flexibilities as well as identical melting profiles at dilute and crowded conditions given that Mgg system  cannot bg system ).Mitochondrial transcript maturation in African trypanosomes and other kinetoplastid organisms requires RNA editing in order to generate functional mRNAs. The reaction takes place within the single mitochondrion of trypanosomes, which represents the most crowded environment within eukaryotic cells. RNA substrates of the editing reaction are sequence-deficient preedited mRNAs. Next to the lack of sequence information, they are characterized by the unusual feature of adopting extraordinarily stable secondary structures in dilute solvent conditions. Here we analyzed the effect(s) of macromolecular crowding on the structure of the mitochondrial RPS12 pre-mRNA as an archetypical example of a trypanosome mitochondrial transcript. We demonstrated that macromolecular crowding has no impact on the 2D fold of the RPS12 transcript and we conclude that the minimum free energy (MFE) structure identified in dilute solvent conditions represents a good proxy for the folding of the pre-mRNA in its crowded mitochondrial solvent context. We hypothesize that the data can be extrapolated to all other mitochondrial transcripts in African trypanosomes.Supplementary Figure S1: SHAPE-reactivity profiles of the RPS12 pre-mRNA in dilute and crowded solvent conditions.Supplementary Figure S2: Difference (dilute - crowded) SHAPE-reactivity plot of the RPS12 pre-mRNA in the absence of magnesium ions.Supplementary Figure S3: Summary of the basepairing patterns of the RPS12-transcript at different solvent conditions.Supplementary Table S1: Statistical comparison of the SHAPE-modification data.


C. elegans GLR glial cells regulate axon specification of their nearby GABAergic RME neurons through GLR-RME gap junctions. Disruption of GLR-RME gap junctions causes misaccumulation of axonal markers in non-axonal neurites of RME neurons and converts microtubules in those neurites to form an axon-like assembly. We further uncover that GLR-RME gap junctions regulate RME axon specification through activation of the CDK-5 pathway in a calcium-dependent manner, involving a calpain clp-4. Therefore, our study reveals the function of glia-neuron gap junctions in neuronal axon specification and shows that calcium originated from glial cells can regulate neuronal intracellular pathways through gap junctions.Axon specification is a critical step in neuronal development, and the function of glial cells in this process is not fully understood. Here, we show that DOI:http://dx.doi.org/10.7554/eLife.19510.001 Drosophila have identified important roles of glial cells in neurogenesis, axon development and pruning, and synapse formation and elimination  mutants, supporting the conclusion that unc-1 regulates RME axon specification by modulating GLR-RME gap junctions. Using a combination of in vivo imaging, biochemistry, and genetic studies, we demonstrate that GLR-RME gap junctions can regulate microtubule assembly in RME neurites through activation of the CDK-5 pathway in a calcium-dependent manner. As the function of the CDK-5 pathway in regulating microtubules is highly conserved from nematodes to mammals, a similar mechanism may also be used in other organisms.Here, we show that gap junctions between unctions . We showC. elegans RME circuit as a model  than in axonal boutons (40%), while in control animals 8% of SNB-1::GFP signals were observed in D/V neurites and 67% of them at axonal boutons  phenotypes. Based on SNB-1::GFP phenotypes, ju1057 was mapped to the left arm of the chromosome X, a region containing the classic kinker unc gene unc-1. ju1057 failed to complement two unc-1 loss-of-function alleles, e580 and e2522; and e580 and e2522 animals displayed SNB-1::GFP mislocalization defects in both axonal processes and D/V neurites resembling those in ju1057 animals  in the C-terminal domain of UNC-1  mutant background to eliminate the expression of Punc-25::SNB-1::GFP in D-type motor neurons in the dorsal and ventral cords. To verify that the defects we observed did not depend on unc-30(ju32), we examined unc-1 animals without unc-30(ju32). As shown in unc-1(lf) animals we examined (105/105), similar to those seen in unc-30(ju32) background. RME D/V neurites grow along the dorsal and ventral cords together with D-type motor neurons, making it difficult to observe RME D/V phenotypes. However, in about 2–3% of animals, RME D neurites grow out of the dorsal cord. After screening over 500 unc-1(lf) animals, we were able to recover 14 animals with RME D neurites outside of the dorsal cord, and all of them (14/14) displayed SNB-1::GFP puncta in RME D neurites (Our genetic screen was carried out in a neurites .unc-1(lf) alleles had a similar number of SNB-1::GFP puncta  animals (unc-1(lf) animals are likely associated with presynaptic vesicles. In our analyses of SNB-1::GFP phenotypes, we noticed that the defects in RME axonal processes and D/V neurites displayed an all-or-none pattern such that any animals with the D/V neurite defects had SNB-1::GFP puncta along axonal processes, while animals without SNB-1::GFP puncta in D/V neurites did not show defects in axonal processes. This observation suggests that the defects in RME axonal processes and in D/V neurites are likely two aspects of one phenotype. Therefore, we decided to focus the rest of our analyses on RME D/V neurites. To further confirm unc-1(lf) phenotypes, we tested the distribution of other axonal markers, the presynaptic active zone protein SYD-2 (unc-1(lf) caused misaccumulation of SYD-2::GFP and mCherry::RAB-3, and SYD-2::GFP was also co-localized with mCherry::RAB-3 in RME D/V neurites  phenotypes could be due to defects in either the establishment of axon specification or the maintenance of neuronal polarity. To distinguish between these two possibilities, we took advantage of a cold-sensitive unc-1 allele, that has strong unc-1(lf) locomotion phenotypes at 11°C and reverse to almost wild-type at 25°C (unc-1(hs1) animals had RME defects, while more than 85% of animals displayed RME defects at 11°C (unc-1(hs1) phenotypes. As shown in unc-1 functions in the larval stages, the same time period of RME D/V neurite development  in all tissues (Pdpy-30:: unc-1(cDNA)) rescued unc-1(lf) phenotypes, expression of unc-1 cDNA in all neurons (Punc-33:: unc-1(cDNA)) or in RME neurons (Punc-25:: unc-1(cDNA)) did not rescue the RME defects in unc-1(lf) animals  blocked the electrical current mediated by gap junctions in muscle (unc-1 cDNA did not rescue unc-1(lf) phenotypes, we questioned whether the expression of unc-1 in GLR cells was required for RME axon specification. We expressed unc-1 cDNA in GLR cells using four different promoters . In addition to GLR cells, Pgly-18, Plet-2, and Pegl-6 promoters could drive expression in head muscle and some neurons as well, but Pnep-2 showed restricted expression in GLR cells (unc-1 cDNA in GLR cells did not restore the distribution of SNB-1::GFP in unc-1(lf) animals, but co-expression of unc-1 cDNA in both RME neurons and GLR cells using any one of the four promoters significantly rescued unc-1(lf) phenotypes (unc-1(cDNA) in muscle (Pmyo-3:: unc-1(cDNA)), in both muscle and RME neurons (Pmyo-3:: unc-1(cDNA)+ Punc-25:: unc-1(cDNA)), or in both muscle and GLR cells (Pmyo-3:: unc-1(cDNA)+ Pgly-18:: unc-1(cDNA)) did not show any rescue ability  expressing unc-1(cDNA) in all tissues (Pdpy-30:: unc-1(cDNA)), a nuclear marker sur-5::mCherry in all tissues (Psur-5:: sur-5::mCherry), and mCherry in RME neurons (Punc-25::mCherry) and in GLR cells (Pgly-18::mCherry). yadEx644 had a similar rescue ability as other transgenes expressing unc-1(cDNA) under the Pdpy-30 promoter (unc-1(+) in RME neurons, 13 of them lacking unc-1(+) expression in GLR cells, and one animal without unc-1(+) expression in both RME neurons and GLR cells. None of these mosaic animals showed rescue of unc-1(lf) RME phenotypes  animals, and unc-7(lf) phenotype was stronger than that of unc-9(lf)  animals  phenotype is due to its roles in both cells. Indeed, expression of unc-7 cDNA in GLR cells or RME neurons only partially rescued unc-7(lf) phenotypes, and expression of unc-7 cDNA in both GLR cells and RME neurons completely restored SNB-1::GFP distribution in unc-7(lf) animals (unc-7 has been reported to regulate presynaptic development in a gap-junction-independent manner (unc-7(lf) animals were due to its non-gap-junction functions, we tested the rescue ability of a C191A mutant form of UNC-7. The C191A mutation has been shown to specifically interrupt UNC-7 gap junction functions, but not its ability to regulate presynaptic development (unc-7(C191A) (Pdpy-30:: unc-7(C191A)) failed to rescue unc-7(lf) phenotypes, while expression of wild-type unc-7 cDNA (Pdpy-30:: unc-7) completely restored SNB-1::GFP distribution in unc-7(lf) animals. In conclusion, these data suggest that GLR-RME gap junctions play an important role in regulating RME axon specification.One gap junction channel is composed of two hemichannels connected across the intercellular space between two cells. Eliminating the expression of gap junction channels in one of the two cells is likely to be sufficient to block gap junction formation. The innexin family of gap junction proteins has 25 members in LR cells . To furtnc-9(lf) . We furt animals . Since u animals . In addit manner . To exclelopment . As showunc-1 and unc-7 unc-9 mutants, almost all microtubules were plus-end-out , which mirrors the microtubule assembly found in axons (unc-1(lf) mutants do not seem to affect the distribution or the number of microtubules in RME D/V neurites. To visualize polymerized microtubules in RME neurons, we used EMTB::GFP (MT-binding domain of ensconsin fused to GFP), which binds along the side of polymerized microtubules and has been previously used to label microtubules in C. elegans neurons (unc-1(lf) did not change the overall distribution or intensity of EMTB::GFP in RME D/V neurites.Assembly and polarity of microtubule cytoskeleton display different properties in axons and dendrites. Axonal microtubules are all orientated with plus-end-out, while dendritic microtubules are either a mixture of plus-end-out and minus-end-out or minus-end-out only . The obsin axons . While G neurons . As showC. elegans neurons, the CDK-5 pathway, consisted of cdka-1/p35 and cdk-5, has been shown to regulate microtubule polarity (cdk-5(lf) animals had similar microtubule polarity defects in RME D/V neurites as those in unc-1(lf) animals (cdk-5 or its upstream regulator cdka-1/p35 induced misaccumulation of SNB-1::GFP in RME D/V neurites (cdk-5 cDNA in RME neurons rescued cdk-5(lf) phenotypes, supporting the conclusion that the CDK-5 pathway cell-autonomously regulates RME axon specification (cdk-5 mutants led us to test their genetic interactions. Similar to unc-1(lf) animals, almost all unc-1;cdka-1 and unc-1;cdk-5 animals had defects in RME axon specification  single mutants, further supporting the conclusion that the CDK-5 pathway and GLR-RME gap junctions function in the same genetic pathway . In Western blot analyses, we detected a cleaved protein fragment of CDKA-1 in control animals  phenotypes in a dose-dependent manner, further supporting that the cdka-1/p35-cdk-5 pathway acts downstream of GLR-RME gap junctions  and cdk-5(lf) animals  caused the same extent of defects as animals expressing calbindin D28K in RME neurons or in both GLR cells and RME neurons . We found that clp-4(lf) displayed a similar degree of RME defects as seen in cdk-5(lf) mutants, while mutations or RNAi of others did not produce obvious phenotypes  partially suppressed RME phenotypes in unc-1(lf) and transgenic animals expressing calbindin D28K in GLR cells (slo-1(lf) suppressed both the microtubule polarity defects and the blockage of CDKA-1 cleavage in unc-1(lf) animals , ced-9(gf), ced-3(lf) and ced-4(lf)) or gsnl-1(lf) prevent the elimination of transient synapses in D/V neurites, and as a result SNB-1::GFP puncta are observed at the end of D/V neurites in adult animals. The phenotypes of gap junction mutants  are different from those of CED and gsnl-1 mutants. Loss of function in CED or gsnl-1 animals do not show any difference with control animals at the L1 stage in that they have similar numbers of SNB-1::GFP puncta at the end of D/V neurites. In contrast, gap junction mutant animals reported here already exhibit differences with control animals in the L1 stage, as they have SNB-1::GFP puncta along the D/V neurites in addition to those at the end of D/V neurites. When animals reach the adult stage, CED or gsnl-1(lf) animals only have SNB-1::GFP at the end of D/V neurites, but gap junction mutants have SNB-1::GFP along the entire D/V neurites. While gap junction mutants have defects of SNB-1::GFP distribution in axonal processes, no such phenotypes were observed in CED or gsnl-1 mutants. Different from gap junction mutants, CED or gsnl-1 mutant animals have similar microtubule polarity in D/V neurites as that in control animals (data no shown). With these differences and other evidence presented in this study, we conclude that GLR-RME gap junctions are involved in regulating RME axon specification rather than synapse elimination.In our previous studies, we show that RME neurons form transient synapses at the end of D/V neurites at the L1 stage, and the cell death (CED) pathway can regulate the elimination of those transient synapses through cleavage of a F-actin severing protein GSNL-1/gelsolin . CED mutC. elegans, the presence of gap junctions between RME neurons and GLR cells has been well documented  in unc-30 (ju32) background to visualize RME neuron presynaptic terminals (hpIs3 (Punc-25::SYD-2::GFP) (unc-1(ju1057) was isolated from the strain CZ15720 unc-30(ju32) (IV) juIs1(Punc-25::SNB-1::GFP) (IV) after ethyl methane sulfonate mutagenesis. In experiments examining the localization of SYD-2::GFP, RAB-3::mCherry and EBP-2::GFP dynamics, we used another unc-30 allele ok613.We maintained erminals . hpIs3 (-2::GFP)  was usedEscherichia coli OP50) until they reached the adult stage (36–48 hr). Control animals were incubated in M9-containing DMSO solvent to the equivalent concentration as that used in the specific experiment.BAPTA-AM and PD150606 were obtained from Sigma and were dissolved in DMSO before dilution in M9. We treated animals with drugs as previously described . For Wesunc-1(hs1) phenotypes at 11°C and 25°C, animals were grown at 11°C/25°C for at least two generation before examining their phenotypes. 11°C->(L1) 25°C: L4 animals were cultured in 11°C, and in the next generation, adult animals were collected for synchronized eggs. Eggs were hatched at 11°C and then transferred to 25°C until examination of the phenotype at the day 1 adult stage. 25°C->(L1) 11°C->(L4) 25°C, animals were grown at 25°C for two generations, and then L1 animals were transferred to 11°C. When animals reached middle L4 stages at 11°C, those animals were transferred back to 25°C until examination of the phenotype at the day 1 adult stage. 25°C->(L4) 11°C: similar as described in the '11°C->(L1) 25°C' section, but using 25°C as the starting temperature and 11°C as the final temperature, and animals were transferred at L4 stage.For quantification of unc-1 and unc-9 cDNA were amplified from yk364C8 and yk1324e06. unc-7, ebp-2, cdka-1, and clp-4 cDNA were amplified from a homemade N2 cDNA pool. DNA construct information is listed in Pttx-3::RFP at 40 ng/µl.All DNA expression constructs were made using Gateway cloning technology . Sequences of the final clones were confirmed. Representative images were acquired with a Zeiss LSM700 confocal microscope using a Plan-Apochromat 40x/1.4 objective. Worms were immobilized in 1% 1-phenoxy-2-propanol  in M9 buffer. For quantification of polarity defects in RME neurons, we used a Zeiss Axio Imager 2 microscope equipped with Chroma HQ filters. RME axon specification defects were scored for the percentage of animals with evenly distributed SNB-1::GFP puncta (more than 25) in both RME D/V dendrites. Each condition consisted of at least three independent experiments and 200–300 1-day-old adults. For quantification of the number of SNB-1::GFP puncta in RME D neurites, we used the Zeiss Axio Imager 2 microscope with 63X objective. We selected animals with the dorsal side up and manually counted the number of all visible SNB-1::GFP puncta. For analysis of SNB-1::GFP intensity, as shown in yadIs13) expressing FLAG-CDKA-1 under the control of Pan-neuronal promoter Prgef-1. Proteins from mixed-stage animals were first extracted using sample buffer by freezing and thawing about 20 times, which consists of freezing in ethanol with dry ice followed by incubation at 95°C for 5 min. Protein samples were separated using SDS-PAGE Gradient Gels (4–20%), and then transferred to nitrocellulose. Blots were probed with rabbit anti-Flag antibodies , and visualized with Amerisham HRP-conjugated anti-rabbit secondary antibodies at 1:5000 using the SuperSignal West Femto kit .For the CDKA-1 cleavage study, we generated an integrated transgene (EBP-2::GFP dynamic experiments were performed using an Andor revolution microscope with a 60 x/1.46 Plan-Apochromat objective controlled by the MetaMorph software. All videos were acquired by an Andor EM-CCD camera (DU897). Young adult animals were immobilized in 5 mM levamisole and on 5% agar pads for imaging. Videos for EBP-2::GFP dynamic analysis were roughly 30 s with 10 frames per second. Kymographs were generated using ImageJ.We analyzed our data using one-tailed Student’s t-test, one-way ANOVA, and Fisher’s exact test in Graphpad Prism . In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.C. elegans" for consideration by eLife. Your article has been favorably evaluated by Marianne Bronner (Senior Editor) and two reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.Thank you for submitting your article "Regulation of Neuronal Axon Specification by Glia-Neuron Gap Junctions in The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Specifically, the reviewers expressed enthusiasm about the problems addressed in this paper and find the results of potential interest. However, the reviewers agreed that there are two issues that need to be fixed in order to support the author's conclusions:http://dev.biologists.org/content/139/22/4191.short).1) The authors have not provided any evidence of the key claim of the paper, namely that GLR cells provide calcium via gap junction to control RME axon specification. Their argument rests entirely on showing that a calcium-dependent pathway is required for axon specification, but whether GLR neurons provides calcium is not tested. One way to do this would be to titrate out calcium in GLR by expressing a calcium binding protein in the GLR neurons  and hence the rescue experiments performed do not proof GLR involvement. This is disappointing because there appear to be a number of more suitable drivers in the literature  that the authors should have used. The authors need to use drivers with greater specificity.2) There are some issues with the On a purely editorial level, the reviewers would also appreciate if the authors could integrate – in the Discussion section – their present findings with their previous finding which demonstrated (a) that RME neurites initially do accumulate presynaptic clusters; (b) the existence of a CED-3-based mechanism to remove these clusters. What are the potential link between these mechanisms? Along these lines, it appears confusing that the author use the word "axon specification" in this paper, given that the process that the study is really a pruning process. Are the authors thinking that there is no polarity initially, the CDK pathway establishes this polarity and CED-3 then cleans up the "mess" that existed previously?The authors will also need to have the manuscript undergo a substantial grammar check. […] Specifically, the reviewers expressed enthusiasm about the problems addressed in this paper and find the results of potential interest. However, the reviewers agreed that there are two issues that need to be fixed in order to support the author's conclusions:http://dev.biologists.org/content/139/22/4191.short).1) The authors have not provided any evidence of the key claim of the paper, namely that GLR cells provide calcium via gap junction to control RME axon specification. Their argument rests entirely on showing that a calcium-dependent pathway is required for axon specification, but whether GLR neurons provides calcium is not tested. One way to do this would be to titrate out calcium in GLR by expressing a calcium binding protein in the GLR neurons  or RME neurons. As shown in cdk-5(lf) animals and observed a similar extent of defects as those in cdk-5(lf) or in GLR calbindin D28K transgene animals in wild type .In those experiments, we also noticed that expression of calbindin D28K in GLR cells caused lethality in ~10-20% of animals (multiple lines), and abnormal GLR morphology in ~25-40% of animals (multiple lines). In animals with abnormal GLR morphology, all showed defects in RME axon specification. To quantify the effects of calbindin D28K expression on RME axon specification, we only considered animals with normal GLR morphology, and we have described how we quantified the phenotypes in the The difficulty in maintaining the integrated transgene animals precluded us to test the effect of titrating out calcium from GLR cells on CDKA-1 cleavage.Nevertheless, our genetic analyses placed the CDK-5 pathway downstream of calcium coming from GLR cells.2) There are some issues with the gly-18 and nep-2 promoter specificity that the authors regrettably fail to mention: both promoters are also active in muscle cells  and hence the rescue experiments performed do not proof GLR involvement. This is disappointing because there appear to be a number of more suitable drivers in the literature  that the authors should have used. The authors need to use drivers with greater specificity.Pegl-6 and Plet-2 to express unc-1 in GLR cells, and expression of unc-1 under these two promoters showed similar effects as those using Pgly-18 and Pnep-2  that RME neurites initially do accumulate presynaptic clusters; (b) the existence of a CED-3-based mechanism to remove these clusters. What are the potential link between these mechanisms? Along these lines, it appears confusing that the author use the word "axon specification" in this paper, given that the process that the study is really a pruning process. Are the authors thinking that there is no polarity initially, the CDK pathway establishes this polarity and CED-3 then cleans up the "mess" that existed previously?unc-1, unc-7, unc-9, unc-7 unc-9) are different from those in CED or gsnl-1 mutants. Both CED/gsnl-1(lf) animals and control) animals at the L1 stage have similar number of SNB-1::GFP puncta at the end of D/V neurites. In contrast, gap junction mutant animals reported here already show differences of SNB-1::GFP from control animals in the L1 stage, that they have SNB-1::GFP puncta along the D/V neurites in addition to at the end of D/V neurites. When animals reach adult stages, CED or gsnl-1(lf) animals only have SNB-1::GFP at the end of D/V neurites, but gap junction mutants have SNB-1::GFP along the entire D/V neurites. While gap junction mutants have SNB-1::GFP distribution defects in axonal processes, no such phenotypes are observed in CED or gsnl-1 mutants. Different from gap junction mutants, CED or gsnl-1(lf) animals have microtubule polarity in D/V neurites similar as that in control animals. With those differences and other evidence presented in this study, we conclude that GLR-RME gap junctions are involved in regulating RME axon specification rather than synapse elimination.We added a Discussion section in the revised manuscript. In our previous studies, we show that RME neurons form transient synapses at the end of D/V neurites at the L1 stage, and the cell death pathway can regulate the elimination of those transient synapses through cleavage of a F-actin severing protein GSNL-1/gelsolin . The phenotypes of gap junction mutants (The authors will also need to have the manuscript undergo a substantial grammar check.Thanks. We revised our manuscript to correct grammar and article usage.