Vibrio cholerae O1 El Tor strain. The results showed that RNAs originating from intergenic regions were the most abundant. Our findings reveal a hitherto unrecognised feature of OMVs mimicking eukaryotic exosomes and highlight a need to evaluate the potential role of RNA-containing bacterial membrane vesicles in bacteria-host interactions.Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the host by delivering virulence factors. Here, we report for the first time that RNA is among the wide variety of bacterial components that are associated with OMVs. To characterize the RNA profiles of bacterial OMVs, we performed RNA deep sequencing analysis using OMV samples isolated from a wild type  Outer membrane vesicles (OMVs) are constantly being discharged from the surface of Gram-negative bacteria during growth. OMVs typically range between 50–200 nm in diameter and contain outer membrane proteins, LPS, phospholipids, and some periplasmic constituentsStudies of bacterial OMVs have revealed some similarities with vesicles known as exosomes that are secreted by most mammalian cell types. OMVs and exosomes have similar size ranges and both carry payloads of proteins, lipids, and genetic material enclosed in membrane-bound spherical structures. Both types can deliver functional molecules to distant extracellular compartments and tissues. Exosomes are involved in: horizontal transfer of genetic materialThe function of bacterial OMVs to aid in transport of a variety of bacterial products- such as proteins, enzymes, and DNA is well described. Extensive studies, reviewed elsewhere1689Their similarity to eukaryotic exosomes and the fact that OMVs act as transporters of various bacterial constituents prompted us to investigate if an “exosome-like” RNA delivery system by bacterial OMVs exists.V. cholerae strain A1552 were isolated as described earlier11V. cholerae strain used has no aberration of the normal process of cell division. Furthermore, the OMVs are distinctly smaller in size in comparison with minicells.OMVs from the wild type V. cholerae strain A1552 were subjected to hot phenol RNA extraction and purification . After two DNase treatments, the sample was dissolved in DEPC-H2O. Two 10 μg-samples were prepared where one sample was subjected to RNase treatment. Both samples were run on a 12% denaturing PAGE gel, GelRed stained and visualized by UV light  could visually be detected in the centrifuged tube  software is shown in As a mean to directly identify the RNA detected in vesicles, we performed sequencing using MiSeq (Illumina) of rRNA-depleted RNA preparations obtained from the vesicles released by strain A1552. The RNA isolation kit was used to acquire ultrapure RNA from which most of the rRNA was removed by the procedure of the Ribo-Zero Magnetic Kit (Epicentre) before RNA library preparation as described in the Materials and Methods. After paired-end sequencing, the resulting 80 bp sequences were aligned to the sequenced chromosomal DNA of et al. describes a sub-population of vesicles—outer-inner membrane vesicles (O-IMV) in which not only the outer but also the inner membrane is involved in the formation of the surfaceV. choleraeE. coli vesiclesExtensive studies of vesicles from Gram-negative bacteria over the last decades suggest that formation of vesicles occurs as the bacterial outer membrane creates a bleb with outer membrane proteins and lipids that surrounds the contents of the periplasm. However, a recent report by Pérez-Cruz vc2479 (uncharacterized protein) from chromosome I , the intergenic region between vc0190 (DNA helicase II) and vc0191 (uncharacterized protein) from chromosome I , and the intergenic region between vca0526 (H+/Cl- exchange transporter ClcA) and vca0527 (uncharacterized protein) from chromosome II  . The Northern blot analysis clearly revealed that these high-hit regions were transcribed as the transcripts were detected in the whole cell RNA preparations and they were present at detectable levels in the vesicles . These HTRRs are: the upstream region of 00 hits) . Ultrapuvesicles .vc2478,5 on extractions from the density gradient purified vesicle fractions also showed that fractions with RNA contained HTRRs  in which the chromosomal DNA corresponding to each of these HTRRs was deleted and replaced by Km-cassettes. RNA from vesicle preparations and whole cell extracts of wild type A1552 and the three mutants were subjected to a 12% polyacrylamide denaturing gel electrophoresis and stained with GelRed (*) in the AES207 and AES208 mutant lanes show where bands are missing in the samples from mutants. The gel-separated RNA transcripts were transferred to a membrane for Northern blotting and hybridized with probes vc2478,5  of RNA-containing bacterial membrane vesicles in bacteria-host and bacteria-bacteria interactions.In conclusion, we report for the first time the detection and successful isolation of bacterial RNA released via OMVs from −1; Kanamycin (Km), 25 μgml−1; and Rifampicin (Rif), 100 μgml−1.The strains and plasmids used in the present work are described in XhoI and XbaI and the desired fragments were gel purified. The Km-cassette was PCR amplified from plasmid pKD4 ) and gel-purified. Fifty to 90 ng of the flanking region DNA and 200 ng of the Km-cassette DNA were used for cross-over PCR  with the following conditions: anneal 43 °C–20 s, elongation 69 °C–1.45 min). The reaction was purified on a gel where the resulting 2500-bp fragments were excised and cloned into pJET1.2 for sequencing and amplification purposes. Correct constructs were digested with NruI and the gel-purified fragments were cloned into SmaI-digested pCVD442 yielding plasmids pCVD-190,5::Km, pCVD-2478,5::Km, and pCVD-526,5::Km.Mutant plasmid constructs were designed with a 1500 bp Km-cassette flanked by 500-bp sequences corresponding to upstream and downstream regions of the target gene. The Km-cassette and flanking regions co-amplified as one unit by PCR. The upstream (A and B primers) and downstream (C and D primers) flanking regions were PCR amplified  from A1552 chromosomal DNA and cloned into pJET1.2 for sequencing and amplification purposes. Correct pJET1.2 constructs were digested with V. cholerae A1552 (recipient) was cross streaked with SM10λ/pCVD-190,5::Km, SM10λ/pCVD-2478,5::Km, and SM10λ/pCVD-526,5::Km (donors), respectively, on non-selective LB plates and incubated at 37 °C for 6 h. A1552/pCVD-construct integrants (cross sections) were then selected for on LB + Km + Rif plates. To cure the integrants of transfer plasmids and to select for homologous recombination of Km-cassette replacement mutations, KmR + RifR-colonies were suspended in LB, serially diluted, and spread on LB + Km + Suc plates. Successful mutagenesis of plasmid-free (CbS) Km-cassette replacement clones was verified by PCR using AA and DD primers. Mutagenesis gave rise to strains AES206 (A1552Δvc0190 - vc0191), AES207 (A1552Δvc02478 - vc2479), and AES208 (A1552Δvca0527 - vca527).Wild type g for 15 min at 4 °C. Pellets were discarded and supernatant re-centrifuged twice. This supernatant was filtered twice through vacuum driven 0.2 μm pore size stericup filters (Millipore Express™ Plus) and ultra-centrifuged at 100,000 × g for 2 h at 4 °C in a 45 Ti rotor (Beckman). The vesicle pellets were re-suspended in 600 μl TE buffer and kept at −80 °C until further use.OMVs were isolated from bacterial culture supernatants essentially as described previouslyg, 3 h, 4 °C) in a SW60Ti rotor (Beckman). After centrifugation visual fraction bands were extracted (100–300 μl), washed in 60 ml TE buffer, and ultra-centrifuged at 100,000 × g for 2 h at 4 °C in a 45 Ti rotor (Beckman). The resulting fraction pellets were re-suspended in 20–80 μl TE buffer. 1 μl of the suspended fractions were taken for immunoblot analysis with anti OmpU and the rest kept at −80 °C until further use.Isolated vesicles suspended in TE buffer were fractionated by the density gradient centrifugation method slightly modified from previously describedg for 2 h at 4 °C in a 45 Ti rotor (Beckman). The resulting vesicle pellet was re-suspended in 60 μl SUPERase• In™ RNase Inhibitor buffer  and 100U inhibitor (Life Technologies) was added before analysis.For tests with RNase treatment, vesicles obtained from an overnight culture were suspended in 300 μl TE buffer and 50 μg RNase A (QIAgen) was added. The sample was kept at RT for 15 min, washed in 70 ml TE buffer, and ultra-centrifuged at 100,000 × V. cholerae O1 El Tor strain A1552 cultures grown to OD600 = 0.7 (log-phase) or OD600 = 4.6 (overnight culture) were harvested by centrifugation and suspended in 0.85% NaCl. After additional centrifugation and washing, bacterial suspensions of 106 bacteria/ml were subjected to staining as described by the manufacturer (Molecular Probes). Treatment of a sample with isopropanol was done to obtain a population of only dead bacteria for comparison. The staining was examined and visualized in a Nikon fluorescence microscope with bandpass filters for Texas Red and Fluorescein, respectively.Live/dead staining was performed using the LIVE/DEAD® BacLightTM Bacterial Viability Kit L13152 according to the manual. In short, 2O and immediately negatively stained in 50 μl of 1.5% uranyl acetate solution for 30 s. Negative-stained samples were examined on a JEOL JEM1230 TEM operating at 80 kV. Micrographs were recorded with a Gatan MSC 600CW CCD camera using Digital Micrograph software. For cryo-electron tomography 4 μl sample was vitrified on a holey carbon film using FEI Vitrobot. The tomography data was collected on a JEOL 2200FS TEM with SerialEM software and a pixel size of 0.57 nm at the Caesar institute in Bonn, Germany. The 3D reconstruction was performed with SIRT and visualized using the IMOD package from University of Colorado at BoulderFor negative staining 3.5 μl of sample was adsorbed for 2 min onto glow-discharged formvar and carbon-coated copper grids, washed in Het al.RNA was extracted from whole cells (1 ml culture) or vesicles isolated from 2.5–5 litre cultures. Extraction was initially made by modified hot phenol extraction methods described by von Gabain Less yield but cleaner RNA was obtained using the Total RNA Purification Plus Kit (Norgen Biotek corp) according to the manufacturer’s manual followed by treatment with DNase I. RNA was kept at −80 °C until further use.To check if the DNase I treatment efficiently removed any contaminating DNA, the RNA preparations were subjected to PCR amplification using DreamTaq Green PCR Master Mix (2×) (Fermenta) and two primer pairs, ompU1up + ompU3do and vc2478,5up + vc2478,5do, respectively. The templates for each set were 100ng vesicle RNA isolated with the Norgen kit and samples without or with DNase I treatment were tested.Isolated vesicle RNA was treated with the Ribo-Zero Magnetic Kit for Gram-Negative Bacteria (Epicentre) according to the manufacturer’s instructions to reduce the amount of rRNA. This rRNA-depleted RNA was used for library construction using the TruSeq RNA sample prep kit v2 (Illumina) according to the manufacturer’s protocol (#15026495 revD).Briefly, the rRNA-depleted RNA was fragmented and the RNA was reverse transcribed into cDNA using random-sequence primers and SuperScript III reverse transcriptase. The cDNA was purified using AMPure XP beads (Beckman Coulter) and amplified for 15 cycles of PCR with barcoded primers, followed by purification using AMPure beads.An 18-pM solution of RNA was subjected to cluster generation and paired-end sequencing with 80-bp read length on the MiSeq system (Illumina Inc.) using the v3 chemistry according to the manufacturer’s protocols.www.sequencing.se.Base calling was done on the instrument by RTA 1.18.42 and the resulting .bcl files were demultiplexed and converted to fastq format with tools provided by CASAVA 1.8.2 (Illumina Inc.), allowing for one mismatch in the index sequence. Additional statistics on sequence quality were compiled with an in-house script from the fastq-files, RTA and CASAVA output files. Sequencing was performed by the SNP&SEQ Technology Platform in Uppsala, Sweden Vibrio cholerae chromosomes using Integrative Genomics Viewer (IGV) software2526RNA sequences were aligned to the N16961 32P]ATP-labelled using the Prime-a-Gene® Labeling System (Promega) according to the manufacturer’s instructions. Unincorporated nucleotides were removed with MicroSpin™ G-25 Columns . Prehybridization (4 h) and hybridization (16 h) was performed in Roti-Hybri Solution (Roth) at 58 °C. Bands were visualized using a Phospho-Imager, Storm 860 (Molecular Dynamics).Whole cell RNA and vesicle RNA from the strains were separated on a 12% acrylamide:bis-acrylamide 19:1/8 M urea gel. The gel was stained with GelRed dye (Biotium) for visualization and then the RNA was transferred onto an Amersham Hybond™-XL membrane  for 1 h at 120 mA, 15 V on a semi-dry transfer unit . The blotted membrane was cross-linked with UV. 30 ng of specific probes generated by PCR were [α-Stripping of the membrane was done by 2 × 15 min washing on shaker at RT in Wash 1  followed by 2 × 15 min washing on shaker at RT in Wash 2 (0.02 × SSC).TM Western ECL substrate as described by the manufacturer (Bio-Rad). Bands were detected using the ChemiDoc XRS system (Bio-Rad).Protein extracts were separated by SDS-polyacrylamide gel electrophoresis and thereafter transferred to a polyvinylidene fluoride (PVDF) micro-porus membrane. OmpU polyclonal antibodies were used to detect outer membrane constituents (vesicles) in the Optiprep fractions. RpoS was used as possible cytoplasmic hallmark for lysed bacteria in the supernatants recognizable by anti-RpoS polyclonal antibodies. Anti-rabbit HRP-conjugated antibody (Agrisera) was used as secondary antiserum at a final dilution of 1:25,000. Further visualization was carried out using ClarityHow to cite this article: Sjöström, A. E. et al. Membrane vesicle-mediated release of bacterial RNA. Sci. Rep.5, 15329; doi: 10.1038/srep15329 (2015).


We recently developed an approach to profile transcriptome-wide RBP contacts on polyadenylated transcripts by next-generation sequencing. A comparison of such profiles from different biological conditions has the power to unravel dynamic changes in protein-contacted We compared protein occupancy profiles of polyadenylated transcripts in MCF7 and HEK293 cells. Briefly, we developed a bioinformatics workflow to identify differential crosslinking sites in cDNA reads of 4-thiouridine crosslinked polyadenylated RNA samples. We identified 30,000 differential crosslinking sites between MCF7 and HEK293 cells at an estimated false discovery rate of 10%. 73% of all reported differential protein-RNA contact sites cannot be explained by local changes in exon usage as indicated by complementary RNA-seq data. The majority of differentially crosslinked positions are located in 3′ UTRs, show distinct secondary-structure characteristics and overlap with binding sites of known RBPs, such as ELAVL1. Importantly, mRNA transcripts with the most significant occupancy changes show elongated mRNA half-lives in MCF7 cells.cis-regulatory RNA sequence space and its dynamics in growth, development and disease.We present a global comparison of protein occupancy profiles from different cell types, and provide evidence for altered mRNA metabolism as a result of differential protein-RNA contacts. Additionally, we introduce POPPI, a bioinformatics workflow for the analysis of protein occupancy profiling experiments. Our work demonstrates the value of protein occupancy profiling for assessing  Posttranscriptional regulation has emerged as a key factor in controlling eukaryotic gene expression by affecting virtually every aspect of RNA metabolism. RNA-binding proteins (RBPs) associate with their target mRNAs and form messenger ribonucleoprotein (mRNP) complexes that guide the processing of pre-mRNA into mature transcripts, control their nuclear export and finally regulate translation rates and decay . Importacis-regulatory mRNA regions [In this context we recently developed a method to display transcriptome-wide the contacts of the mRNA-bound proteome on polyadenylated RNA by next-generation sequencing ,14. Brie regions .cis-regulatory elements. To globally map regions of local differences in protein occupancy, we adapted count-based methods that are frequently used in differential gene expression analysis for comparison of T-C transitions  reference genome with TopHat2  . However, the profiles from different cell types were clearly distinguishable  and 3′ UTRs were occupied to almost the same extent in MCF7 cells  + RNA (mRNA-seq) from MCF7 cells in triplicates. Similarly, two replicate mRNA-seq data sets were generated for HEK293 cells. As expected, replicates from the same cell type showed higher correlation , with Ls being the total number of T-C transition event counts per sample s  normalization), Φp being the normalization factor (termed dispersion) and apc being the relative abundance of T-C transitions at position p in replicates of condition c, which sample s belongs to. Importantly, instead of performing the initial per-sample normalization and computing dispersion factors over all tested genomic positions at once , we compute the normalization as well as the sample- and tag-wise dispersion for each transcript individually. As a result, we normalize for global shifts in T-C transition count base levels that might result from technical variation such as different sequencing depth. In addition, a transcript-wise normalization adjusts for expected changes in T-C transition counts that result from changes in overall mRNA expression, which would otherwise be interpreted as differential occupancy  by repeating the same analysis, yet switching replicate assignment of the two conditions (one MCF7 replicate was assigned as HEK293 replicate and vice versa), thereby generating a null model distribution of ekutieli ,29 as thIn addition to identifying regions of significantly altered protein occupancy based on T-C transitions, we performed a similar analysis based on read coverage. Using a previously described peak calling approach ,31, we fIt appears easy to reconcile that binding of a single protein or a protein complex does not only affect a single T-C position but rather influences multiple locally clustered positions. To test this hypothesis, we computed the distance to the closest significantly altered T-C transition of significant or non-significant positions. In line with the premise of clustering, we found that significant positions are closer to each other than to non-significant positions Figure D. IntrigNext, we investigated the distribution of differential T-C transitions over different transcript regions and found a difference between sites with increased and decreased crosslinking signal in MCF7 compared to HEK293 Figure F. While post hoc if they fall into a differentially expressed exon, transcript, gene or any combination of these. For this study, we filtered out positions in exons with a significant change in expression across cell types . With these parameters, we retained 72.7% of all reported positions, which could not simply be explained by differential exon usage. This leaves 21,823 out of 30,006 positions with differential RBP occupancy in MCF7 versus HEK293 cells.Finally, we assessed the impact of differentially expressed exons as a possible source of differential T-C transitions. We would like to emphasize that our approach is not responsive to overall changes in T-C transition levels resulting from differential gene expression. However, a fraction of differential T-C transition positions might be a result of differential exon usage. In this scenario, skipping of a complete exon might lead to a local absence of transition events in one condition. To resolve this problem, we have implemented an additional filtering approach that optionally removes exons or transcripts based on differential expression analysis of mRNA-seq data. Significant T-C transitions can be removed All of the aforementioned analysis steps are implemented in the POPPI workflow , which mAs a next step we investigated the properties of mRNA regions with differential protein contacts. We selected the top 300 non-overlapping MCF7 positions with increased and reduced T-C transition events relative to HEK293 cells and excluded sites in differential exons  was observed. For the set of the top 300 positions with reduced protein occupancy in MCF7, we did not observe a significant overlap with any of the experimentally derived RBP binding sites. To further investigate our observation that MCF7 cells show a comparably higher occupancy on ELAVL1 PAR-CLIP sites, we performed a motif analysis in the surrounding areas ±25 nucleotides . We repeated the 7-mer analysis on regions with decreased occupancy. Compared to regions of elevated occupancy, we found a different set of enriched 7-mers , indicating that these transcripts represent ELAVL1-bound mRNAs that are differentially occupied in MCF7 cells compared to HEK293 cells.To further confirm binding of ELAVL1 to regions with increased protein-RNA contacts in MCF7, we compared our data to a previous study carried out in MCF7 cells that used RNA-immunoprecipitation experiments in combination with microarray analysis (RIP-Chip) to identify transcripts bound by ELAVL1 . We seleHaving analyzed properties of RNA regions differentially contacted by proteins, we were interested in the functional association of, and possible consequence for, the respective genes. We therefore defined the set of the top 300 target genes as those genes that harbor the most significantly increased or decreased T-C transition events in their respective mRNAs. While these two groups could overlap , their actual overlap was minor (36 out of 300 tested target genes). To gain insight into the associated gene functions, we performed a Gene Ontology (GO) term and pathway enrichment analysis of these targets using the R package g:Profiler , which iP-value <0.1 and at least five associated genes). For target mRNA transcripts with decreased positional occupancy in MCF7, we found an association to the regulation of cell cycle and gene expression as well as regulation of translation . Intriguingly, a more careful examination revealed elevated mRNA half-lives of many growth-promoting proto-oncogenic factors like CCNA2, CCNB2 and CDKN1A that are well-established targets of ELAVL1 [We have observed a significant enrichment of sequence motifs and experimentally determined binding sites for ELAVL1 and other regulators that affect RNA stability in our top differentially occupied target regions. Consequently, we tested whether the corresponding target genes exhibit changes in mRNA half-lives. We generated two replicate measurements of mRNA half-lives in both cell types by 4SU labeling and purification of labeled and unlabeled mRNA populations after 1 h of labeling and under steady state assumption as described by Dölken et al.  and Schw [et al. . Since tf ELAVL1  and showin silico analyses , we found 1) a significant increase of occupancy at putative ELAVL1 binding sites, 2) top differentially occupied genes to show a functional association to cell growth, cell proliferation as well as mRNA processing, and 3) increased half-lives of mRNA targets with differential local protein occupancy. These findings couple our predictions of local differential protein occupancy to a global regulatory outcome on the level of posttranscriptional gene regulation.Summarizing our results on the analysis of differential occupancy profiling experiments, gene expression measurements, estimation of mRNA half-lives and extensive cis-regulatory RNA regions involved in a posttranscriptional gene-regulation.Posttranscriptional gene regulation is elicited through a complex and highly interdependent network of RNA-binding proteins and non-coding RNAs that form dynamic ribonucleoprotein complexes to orchestrate specific regulation of RNA transcripts throughout their lifecycle . While tHere, we have established a bioinformatics workflow to compare protein occupancy profiles of polyadenylated RNA. Protein occupancy profiling data from HEK293 cells has been obtained previously [Utilizing established statistical methods that are frequently used in differential gene expression analysis, we identify differentially occupied positions based on a statistical test as implemented in the edgeR package . Insteadbona fide protein binding sites. Comparing a set of 300 top differentially contacted positions to known RBP-binding sites, we observed significantly increased protein occupancy on ELAVL1 binding sites in MCF7 cells. Interestingly, ELAVL1 was found to be up-regulated and preferentially localized to the cytoplasm in multiple cancer cell lines , which ing MCF7 -57. Giveing MCF7 -60. Furting MCF7 ,38,61. Aing MCF7 ,63, we dFinally, we set out to investigate the functional consequence of altered protein occupancy on the transcript level. Driven by the intriguing observation that regions with elevated protein occupancy in MCF7 cells showed significant enrichment of binding motifs and PAR-CLIP binding sites of ELAVL1, we reasoned that ELAVL1 might play a key role in explaining differences in protein occupancy between MCF7 and HEK293 cells. By analyzing ELAVL1 RIP-ChIP data we observed that transcripts with regions of elevated protein occupancy are significantly enriched in ELAVL1-RIPs in MCF7 cells. Considering the established function of ELAVL1 to increase the mRNA stability of important cellular transcripts with diverse roles in cell proliferation and carcinogenesis, we accessed differences in mRNA half-life between MCF7 and HEK293, possibly attributed to differential ELAVL1 binding. Thus, we can correlate differential protein-RNA contacts with a direct regulatory outcome, indicated by altered RNA metabolism. Strikingly, we observed an overall shift towards elevated mRNA half-lives of the top 300 differentially occupied transcripts. Importantly, we detected increased mRNA half-lives for cancer-related transcripts such as CCNA2, CCNB2 and CDKN1A that were previously shown to be stabilized by ELAVL1 .In addition, we introduced POPPI, a fully automated computational analysis pipeline specifically tailored to the analysis of protein occupancy profiling experiments. POPPI provides a highly flexible framework that streamlines the analysis steps and produces comparable statistics as well as intuitive figures to determine experimental quality, replicate correlation as well as functional analysis. Most importantly, we have added routines that identify local dynamic changes in occupancy profiles across different conditions .cis-regulatory RNA regions to uncover differences in posttranscriptional regulatory interactions.In conclusion, protein occupancy profiling is a powerful approach to study dynamics in protein-RNA interactions for coding transcripts as well as lincRNAs. Global mapping of protein-RNA contact sites on lincRNAs holds the potential to provide valuable insights into the modular design of these non-coding RNAs and determine the individual lincRNA-protein interaction domains. Using our approach, researchers gain an unbiased view of differentially protein-bound Binding of microRNA and RBPs to a large number of mRNA targets weaves a complex network of posttranscriptional gene regulation. Their combinatorial assembly, dynamic in time and space, determines the fate of protein-coding transcripts. Protein occupancy profiling provides an unbiased and system-wide insight into protein-contacted mRNA regions. We implemented a computational framework to streamline analysis steps and to detect differential protein occupancy on RNA across replicate experiments from different biological conditions. Importantly, our comparison of occupancy profiles in HEK293 and MCF7 cells is a first step in gaining a deeper understanding of the underlying posttranscriptional regulatory dependencies, which determine the fate of individual RNAs between cell types.HEK293 and MCF7 cells were grown in medium (DMEM high glucose with 10% (v/v) fetal bovine serum, 1% (v/v) 2 mM L-glutamine, 1% (v/v) 10,000 U/ml penicillin/10,000 μg/ml streptomycin) supplemented with 200 μM 4SU 16 h prior to harvest. For UV crosslinking, culture medium was removed and cells were irradiated on ice with 365 nm UV light (0.2 J/cm2) in a Stratalinker 2400 , equipped with light bulbs for the appropriate wavelength. Following crosslinking, cells were harvested from tissue culture plates by scraping them off with a rubber policeman, washed with ice-cold phosphate-buffered saline and collected by centrifugation . Resulting cell pellets were resuspended in five cell pellet volumes of lysis/binding buffer ) and incubated on ice for 10 minutes. Lysates were passed through a 21 gauge needle to shear genomic DNA and reduce viscosity. Oligo(dT) beads  were briefly washed in lysis/binding buffer, resuspended in the appropriate volume of lysate and incubated 1 h at room temperature on a rotating wheel. Following incubation, supernatant was removed and placed on ice for multiple rounds of mRNA hybridization. Beads were washed three times in one lysate volume lysis/binding buffer, followed by three washes in one lysate volume NP40 washing buffer . Following the washes, beads were resuspended in the desired volume of elution buffer  and transferred to a new 1.5 ml microfuge tube. Hybridized polyadenylated mRNAs were eluted at 80 degrees for 2 minutes and eluate was placed on ice immediately. Beads were re-incubated with lysate for a total number of three depletions by repeating the described procedure. Following RNAse treatment  protein-RNA complexes were precipitated by ammonium sulfate. After centrifugation , resulting protein pellets were resuspended in SDS loading buffer and separated on a NuPAGE 4-12% Bis-Tris gel ). Separated protein-RNA complexes were transferred to a nitrocellulose membrane, desired bands migrating between 10 kDa and 250 kDa were cut out and crushed membrane pieces were Proteinase K ) digested . Following Proteinase K treatment, RNA was phenol/chloroform extracted and ethanol precipitated. Recovered RNA was dephosphorylated using calf intestinal alkaline phosphatase ; 50 U, 1 h, 37°C). After dephosphorylation RNA was phenol/chloroform extracted, ethanol precipitated and subjected to radiolabeling using polynucleotide kinase  and 0.2 μCi/μl γ-32P-ATP ). Radiolabeled RNA was again phenol/chloroform extracted and recovered by ethanol precipitation. Subsequent small RNA cloning and adapter ligations were performed as described previously .Protein occupancy profiling sequencing data have been deposited under Gene Expression Omnibus (GEO) accession number GSE49831.MCF7 cells were maintained at 37°C in RPMI supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin. For RNA isolation, 5 × 105 cells were grown in triplicates under normal conditions, and harvested two days later. Cells were lysed and RNA was isolated using the InviTrap Spin Cell RNA Mini Kit ). RNA quality was analyzed with the Agilent RNA 6000 Nano Kit, and the concentration was measured with the Qubit RNA Assay Kit (Invitrogen). Library preparation was carried out with the TruSeq™ RNA Sample Preparation Kit ) using barcoded primers. Libraries were sequenced on Illumina HiSeq using a paired-end protocol (2 × 100 nucleotides).MCF7 mRNA-seq sequencing data have been deposited under GEO accession number GSE49831.HEK293 total RNA was extracted using the miRNeasy kit ) following the instructions of the manufacturer. RNA (4 μg) was used for poly(A) + mRNA library preparation following the TruSeq RNA sample Prep v2 LS protocol (Illumina). The libraries were sequenced on an Illumina Genome Analyzer GAII or Illumina HiSeq for 100 cycles (multiplexed 1 × 101 + 7 index).HEK293 mRNA-seq sequencing data have been deposited under GEO accession number GSE49831.For global mRNA half-life measurements, MCF7 and HEK293 cells were labeled with 700 μM 4SU for 60 minutes. Total RNA was extracted using the miRNeasy kit (QIAGEN). 4SU residues were biotinylated using EZ-Link biotin-HPDP ). Biotinylated 4SU-labled RNA was separated from non-labeled RNA using μMACS Streptavidin MicroBeads ) and 4SU-labeled RNA was eluted from μColumns by addition of 100 mM DTT. RNA was recovered from the flow-though and 4SU-labeled fractions using MinElute Spin columns (QIAGEN). Input , flow-though (non-labeled RNA) and eluted (4SU-labled RNA) samples were used for poly(A) + mRNA library preparation following the TruSeq RNA sample Prep v2 LS protocol (Illumina). The libraries were sequenced on an Illumina Hiseq 2500 for 100 cycles (multiplexed 1 × 101 + 7 index). mRNA half-lives were computed from gene-wise FPKM (fragments per kilobase of exonic sequence per million fragments mapped) as previously described . To acceMCF7 and HEK293 half-life measurement sequencing data have been deposited under GEO accession number GSE49831.BAM files were processed with the Samtool program . BED filTo streamline the analysis of protein occupancy profiling data and thereby leveraging its accessibility, we have bundled scripts used in this study into the protein occupancy profiling pipeline (POPPI). POPPI performs the following analysis steps: read processing, read mapping, transcriptome-wide read coverage and position-specific T-C transition event profiling as well as global comparison to genomic features and across different experiments  were filtered out.We additionally implemented the possibility to filter reported positions based on gene expression. To this end, we counted the number of reads assigned to each gene, transcript and exon and used these tables in a subsequent edgeR test for differential expression, again estimating both sample- and feature-wise dispersions. POPPI can be readily used for different species as it provides automatic data retrieval for species-specific annotation data like reference genome sequence and gene models. A detailed description of all implemented functionalities, output files and quality assessment plots is given in Additional file For functional analysis, we focused on the 300 most significant differential positions both increased and decreased in the comparison of MCF7 and HEK293 cell occupancies. To prevent any bias based on clustered positions, we required top positions to be more than 20 nucleotides apart. The respective random position set was produced by randomly shifting the initially defined top positions upstream or downstream by 100, 50 or 30 nucleotides while ensuring that the resulting position remained inside of a gene. To further reduce any potential sequence-dependent bias, we additionally ensured that each random position reflected a uridine in the transcript by shifting the resulting position to the closest genomic thymine or adenine dependent on transcriptional direction.To define the set of top targets associated to differentially increased and decreased occupancy, we sorted all target genes by the significance of their most significant differential position in both categories. Subsequently, the top 300 target genes from both groups were analyzed.k (with k being the length of the PWM) a score by multiplying the probabilities of the respective positions in the PWM. To prevent probabilities of zero, we added a small pseudo-count (0.01) to each column of the PWM initially and divided by the total count for each column. Second, we summed the resulting scores over the entire region. Third, we compared the PWM scores of the top and random regions using a one-sided Wilcoxon rank sum test and a significance threshold of 0.01. To associate PWMs to their respective RBPs, we used the 'RBP_information.txt' table that comes with the PWM annotation from the cisBP-RNA website.To define if certain RBP motifs were enriched around our top differential T-C transition positions, we downloaded all available PWMs and sequence logos annotated to human RBPs from the cisBP-RNA website . Followi4SU: 4-thiouridine; ARE: AU-rich element; CDS: coding sequence; CLIP: UV crosslinking and immunoprecipitation; DTT: dithiothreitol; FDR: false discovery rate; GEO: Gene Expression Omnibus; GO: Gene Ontology; HEK: human embryonic kidney; lincRNA: long intervening non-coding RNA; mRNP: messenger ribonucleoprotein; PAR-CLIP: photoactivatable ribonucleoside-enhanced CLIP; POPPI: protein occupancy profiling pipeline; PWM: position weight matrix; RBP: RNA-binding protein; SRSF: serine/arginine-rich splicing factor; TMM: trimmed mean of M-values; UTR: untranslated region.The authors declare that they have no competing interests.MS performed the computational data analysis. MM performed all protein occupancy profiling experiments as well as mRNA half-life measurements. LHG and AF contributed the HEK293 and MCF7 mRNA-seq experiments, respectively. MS and CD set up the POPPI pipeline. AL contributed to the design of the study. WC contributed computational analysis to estimate mRNA half-lives. ML designed the study and contributed analytic ideas. MS, MM, ML and CD wrote the manuscript. All authors read and approved the final manuscript.Read and mapping statistics.Click here for fileSupplementary Figures S1 to S11.Click here for fileAll positions with significantly changing occupancy (FDR <0.10) in MCF7 versus HEK293 cells. Positions additionally passing the differential exon filter are indicated in the last column.Click here for fileTop 300 positions with significantly enhanced occupancy in MCF7 versus HEK293 cells.Click here for fileTop 300 positions with significantly decreased occupancy in MCF7 versus HEK293 cells.Click here for fileP < 0.01) higher scores in the region ±25 nucleotides around top versus random positions with significantly enhanced occupancy in MCF7 versus HEK293 cells.Human RBP RNAcompete PWMs with significantly (Click here for fileP < 0.01) higher scores in the region ±25 nucleotides around top versus random positions with significantly decreased occupancy in MCF7 versus HEK293 cells.Human RBP RNAcompete PWMs with significantly  associated to top 300 target genes associated to positions with significantly enhanced occupancy in MCF7 versus HEK293 cells.Significant GO terms and pathways  associated to top 300 target genes associated to positions with significantly decreased occupancy in MCF7 versus HEK293 cells.Significant GO terms and pathways (adjusted Click here for fileHTML output of the POPPI pipeline run for the MCF7 and HEK293 protein occupancy profiling experiments.Click here for fileDetailed description of POPPI functionality and output files.Click here for file


S. pombe. Meiotic gene silencing occurs post-transcriptionally through recruitment of the exosome complex to promote RNA degradation. Extensive interaction analysis and functional characterizations link the NURS complex to specific RNA-binding and processing proteins and also chromatin modification machinery.The authors define a multiprotein nuclear RNA silencing (NURS) complex that mediates silencing of meiotic genes during vegetative growth in the fission yeast  Schizosaccharomyces pombe, an RNA-based mechanism represses meiotic gene expression during vegetative growth. This pathway depends on the zinc finger protein Red1, which is required to degrade meiotic mRNAs as well as to target histone H3 lysine 9 (H3K9) methylation, a repressive chromatin mark, to a subset of meiotic genes. However, the mechanism of Red1 function is unknown. Here we use affinity purification and mass spectrometry to identify a Red1-containing nuclear RNA silencing (NURS) complex. In addition to Red1, this complex includes the Mtl1, Red5, Ars2, Rmn1, and Iss10 proteins and associates with several other complexes that are involved in either signaling or mediating RNA silencing. By analyzing the effects of gene knockouts and inducible knockdown alleles, we show that NURS subunits regulate RNA degradation and H3K9 methylation at meiotic genes. We also identify roles for individual NURS subunits in interactions with Mmi1, an RNA-binding protein that marks meiotic RNAs for destruction, and the nuclear exosome RNA degradation complex. Finally, we show that the levels of H3K9 methylation at meiotic genes are not sufficient to restrict RNA polymerase II access or repress gene expression during vegetative growth. Our results demonstrate that Red1 partners with other proteins to silence meiotic gene expression at the post-transcriptional level. Conservation of a NURS-like complex in human cells suggests that this pathway plays an ancient and fundamental role in RNA silencing.RNA is a central component of gene-silencing pathways that regulate diverse cellular processes. In the fission yeast  Schizosaccharomyces pombe, small interfering RNAs (siRNAs) derived from noncoding centromeric transcripts are loaded onto the Argonaute (Ago1) subunit of the RNA-induced transcriptional silencing (RITS) complex. RITS then recruits the Clr4–Rik1–Cul4 (CLRC) methyltransferase complex to catalyze the di- and trimethylation of histone H3 lysine 9 (H3K9) to initiate heterochromatin formation at specific chromosome regions  and post-transcriptional (PTGS) levels in a broad range of organisms. These mechanisms are often regulated by specific RNA features that determine whether a nascent transcript becomes a functional RNA, is degraded, or feeds back on chromatin to silence transcription. In some cases, the RNA is processed into various classes of small RNAs that are associated with RNA interference (RNAi) pathways. In the fission yeast,  regions . Another regions . The TRA by RNAi . Long no by RNAi .S. pombe and involves the induction of hundreds of genes  complex. Consistent with the purification results, all members of the NURS complex colocalize in nuclear foci. However, these nuclear bodies do not colocalize with the mei4+ locus, suggesting that their formation occurs post-transcriptionally. By generating gene knockouts of iss10+ and rmn1+ and inducible knockdown alleles of the essential genes mtl1+, red5+, and ars2+, we show that Mtl1, Iss10, and Red5 are required for meiotic mRNA elimination and H3K9 methylation at meiotic genes. In addition, purification of Red1 after knockdown of Mtl1 or deletion of iss10+ revealed that Mtl1 is required for exosome recruitment, while Iss10 helps bridge the NURS complex to Mmi1. We also find that the expression of meiotic genes is not affected by the absence of heterochromatic silencing machinery and that the H3K9 methylation at meiotic genes does not restrict RNA polymerase (Pol) II access, suggesting that the presence of this histone mark at meiotic gene loci is not associated with the assembly of functional heterochromatin. Our results suggest that the NURS complex links various RNA processing signals to exosome-mediated nuclear RNA degradation. Very recently, an independent report (Here we use affinity purification and mass spectrometry to identify a multiprotein Red1-containing complex. We find that Red1 associates with an Mtr4-like helicase Mtl1 (SPAC17H9.02), two zinc finger proteins, Red5 (SPBC337.12) and Ars2 (SPBC725.08), the RNA recognition motif (RRM)- containing protein Rmn1 (SPBC902.04), and a serine- and proline-rich protein Iss10 (SPAC7D4.14c). These Red1-interacting proteins co-purify each other, indicating that they form a complex, which we name the t report  describeDrosophila Ars2 and Arabidopsis SERRATE. In these systems, Ars2/SERRATE interacts with the RNA cap-binding complex and regulates siRNA and micro (mi)RNA biogenesis  complex. Several members of this complex contain nucleic acid-binding domains, which may mediate interactions with nascent RNA or DNA to enable RNA degradation and H3K9 methylation.Red1 was originally identified in a screen for proteins that form nuclear bodies and was shown to localize in one to four nuclear foci in vegetative cells . To determn1+ did not affect the localization of Mtl1-GFP, Red5-GFP, Ars2-GFP, Iss10-GFP, or Red1-mCherry . Thus, Red1 and Iss10 are required for the NURS complex to concentrate in nuclear bodies. We note that +iss10 deletion only modestly increases meiotic RNA levels  array and expressed a GFP-tagged Tet repressor (TetR-GFP). The array was inserted upstream of +mei4, but because the 3′ UTR of the +cdk9 gene overlaps the 5′ UTR of +mei4, we duplicated the 567-bp region between the stop codon of +cdk9 and start codon of +mei4so that it appears both upstream of and downstream from the array (+mei4 and the levels of H3K9 methylation at this gene were unaffected by the presence of the tetO array and remained responsive to red1+ deletion (data not shown). We found that Red1-mCherry foci did not colocalize with the mei4+ locus  fused to the S. pombe Skp1, enabling the degradation of S. pombe proteins tagged with IAA degrons  in order to combine protein depletion and transcriptional shut-off, a strategy successfully used previously  system. In plants, the hormone auxin binds to the TIR1 F-box protein component of a Skp1–Cullin–F-box (SCF) E3 ubiquitin ligase complex and promotes its association with a degron present in the AUX/IAA family of transcriptional repressors . The ubi degrons . As in tetection . We fuseeviously . Howeverdg repeats. The addition of thiamine and auxin had no effect on RNA levels in the TIR1-expressing parent strain  to exponentially growing cultures and observed a drastic reduction in AID-tagged protein expression C. We thet strain D,E. As edeletion A,D. KnocA levels D,E. Simi5 mutant , but didA levels D,E. FusiA levels D. Finall and NAA D,E. It i and NAA C. Our reiss10+ had no effect on the composition of the NURS complex but resulted in a near complete loss of Mmi1 association . In agreement with previous results , we obsend crs1+ A. This wnd crs1+ A. Iss10, repeats A. Ars2 d repeats A. This liss10+ reduced H3K9 methylation at mei4+ and ssm4+ to near background levels but had no effect on H3K9 methylation at the centromeric dg repeats . However, red1Δ and red5-AID cells, as well as previously described temperature-sensitive red5 mutant cells  or deletion of the RITS subunit chp1+ (1.5–2.5-fold) depend on the presence of clr4+  tail  tail . Additioth nab2+ . Togethenuclear exosome targeting (NEXT) complex  and the  complex . CBCN anh ZFC3H1 . S. pomband Cbc2 C. Red1 iand Cbc2 . In humaand Cbc2 . CBCN aland Cbc2 . The hMTand Cbc2 . It willS. pombe , or whether the low levels of H3K9 methylation regulate the timing of meiosis by delaying the full-scale induction of meiotic genes. Alternatively, H3K9 methylation at meiotic genes may result from vestigial association of the CLRC and NURS complexes, which may have been critical at some point during evolution to silence these genes or other chromosome regions such as the pericentromeric repeats.The H3K9 methylation at meiotic genes in vegetatively growing rganisms . Howeverrganisms . We founrganisms A and thaic genes D,E. Furtic genes  and can eriments , but the4+ locus C. Togeth4+ locus . It remanmt41-GFP-3×FLAG plasmid was generated by first replacing the kanMX6 marker of pFA6a-kanMX6-Pnmt1-GFP with hphMX6 then performing DpnI mutagenesis to convert the nmt1 to the nmt41 promoter (nmt41-GFP-3×FLAG cassette was inserted into the S. pombe genome 1 kB upstream of the ura4+ start codon. The pFA6a-mCherry-kanMX6 plasmid was made by amplifying the mCherry coding sequence with flanking PacI and AscI sites and inserting the fragment into pFA6a-kanMX6. To generate pFA6a-2×HA-AID(IAA17)-kanMX6, the degron coding sequence was PCR-amplified with flanking BamHI and AscI sites and inserted into pFA6a-3×HA-kanMX6. The pFA6a-hphMX6-PCMV-TetR-GFP plasmid was created by cutting pFA6a-hphMX6-Pnmt1-GFP with BglII and PacI to remove the nmt1 promoter and inserting a PCR fragment containing the CMV promoter, tet repressor and λcI linker with flanking BamHI and PacI sites amplified from the genomic DNA of strain NBY3771 (CMV-TetR-GFP cassette was inserted into the S. pombe genome at the BamHI site downstream from the ura4-D18 locus (813 bp after the stop codon in ura4+ strains).All strains were constructed using a PCR-based gene targeting approach  and are promoter . DpnI mu NBY3771 . The hphtetO repeat array upstream of mei4+ without disrupting mei4+ expression, a pFA6a-ura4+-Pmei4 plasmid was constructed in two steps. First, a 567-bp fragment containing the region between the cdk9+ stop codon and mei4+ start codon was PCR-amplified from genomic DNA with flanking PciI and NcoI sites and inserted into the PciI site of pFA6a-kanMX6. The kanMX6 cassette was then replaced with the 1.8-kB ura4+ fragment from KS-ura4+ , and transformants were selected on medium lacking uracil. The pSR14 plasmid (tet operator (tetO), was modified to replace the LEU2 marker with the natMX6 cassette  and then screened for uracil auxotrophy.To integrate the KS-ura4+ . A PCR pKS-ura4+ , ura4+ c plasmid , which ccassette . A linea7 cells/mL at 32°C in yeast extract medium with supplements (YES) were harvested by centrifugation. Cell pellets were resuspended in a volume of lysis buffer  equal to one-fifth of the pellet volume then added dropwise to liquid nitrogen. Cells were lysed by three cycles of cryo-milling at 30 Hz for 3 min each in a Retsch MM301 mixer mill. Yeast powder was resuspended in a volume of IP buffer  equal to 1.2× the pellet volume and centrifuged at 16,000g for 5 min followed by filtration as described  were incubated with 38 μg of α-FLAG  or α-myc  antibody overnight at 4°C. Antibodies were cross-linked to the beads using 15 mM dimethyl pimelimidate (Pierce) in 0.2 M sodium borate, pH 9. Crosslinking was allowed to proceed for 30 min at room temperature and was quenched by the addition of 0.2 M ethanolamine, pH 8. Beads were washed twice with 10 volumes of IP buffer and resuspended in 1 volume of IP buffer before adding to cell extracts and incubating at 4°C for 3 h. Beads were washed with 1 mL ice cold IP buffer three times for 1 min each followed by one 5-min wash. Proteins were eluted in 687.5 μL of 500 mM ammonium hydroxide at 37°C for 20 min and samples were dried overnight using a SpeedVac concentrator (Savant). Immunoprecipitated proteins were resuspended in 75 μL of buffer containing 100 mM Tris, pH 8.2, and 2 M urea. DTT was added to a final concentration of 5 mM, and samples were incubated at 56°C for 30 min. After cooling to room temperature, iodoacetamide was added to a final concentration of 15 mM, and samples were incubated in the dark for 45 min. Unreacted iodoacetamide was quenched with addition of DTT to a final concentration of 10 mM. Samples were diluted with 100 mM Tris, pH 8.2 to bring the urea concentration to 1 M, and sequencing-grade trypsin (Promega) was added at a concentration of 15 ng/μL followed by incubation at 37°C overnight. Digested samples were then loaded onto stage tips and washed as described previously  and harvested by centrifugation. Pellets from ∼1.25 × 1010 of cells were resuspended in 600 μL of lysis buffer and lysed by six rounds of agitation with glass beads at 4500 rpm for 30 sec each in a MagNA Lyser instrument (Roche). Cell lysates were subjected to two rounds of centrifugation at 13,000g for 5 and 15 min. Approximately 20 mg of total protein was combined with 3 μg α-myc antibody  prebound to 20 μL Protein G Dynabeads (Life Technologies) in a total volume of 700 μL and incubated at 4°C for 2–3 h. Beads were washed three times in 1 mL lysis buffer for 5–10 min each, and bound proteins were eluted by incubation at 65°C for 8 min in loading buffer . RNase A was added to lysis buffer at a final concentration of 100 μg/mL and was present during both the binding and washing steps. For protein knockdown experiments, denatured whole cell extracts were prepared as described previously . Cells were pipetted onto slides and covered with a coverslip immediately prior to imaging using a Nikon Ti-E inverted epi-fluorescence microscope equipped with a Plan Apo 100× 1.4 NA objective lens. A Prior LumenPro light source and 490/20 and 572/35 excitation filters (Chroma) were used to excite GFP and mCherry fluorescence, respectively. Emission was collected using a 535/30 filter for GFP and a 630/60 filter for mCherry (Chroma). Images were acquired using a Hamamatsu ORCA-R2 cooled CCD camera and the Nikon Ti focus motor, controlled with MetaMorph v7.7 software (Molecular Devices). Single plane images are displayed, with brightness and contrast adjusted using MetaMorph.S. pombe Skp1 to the A. thaliana and O. sativa TIR1 F-box proteins  to final concentrations of 15 μM and 500 μM, respectively. Control cultures were treated with the same volume of DMSO (NAA vehicle). Cells were returned to 25°C for 6 h before processing for ChIP, Western blotting, co-immunoprecipitation, and RNA analysis and for 14 h for purification/mass spectrometry.Strains expressing target proteins tagged with 2×HA-AID(IAA17) and fusions of proteins  were groS. pombe cells using the hot phenol method  and lysed by three rounds of agitation with glass beads at 4500 rpm for 45 sec each in a MagNA Lyser instrument (Roche). Cell lysates were sonicated and clarified by centrifugation as described (Cells grown to a density of 2.5 × 10escribed . Pelletsescribed . After rescribed . Input a8 cells/mL. Three microliters of serial 10-fold dilutions were spotted onto nonselective YES medium and YES supplemented with 17 μg/mL TBZ. Plates were incubated at 32°C for 3–4 d, then photographed.Cells grown overnight at 30°C were resuspended in water at a density of 1 × 10Supplemental material is available for this article.


Bovine coronavirus is a primary cause of neonatal calf diarrhea worldwide, and is also associated with acute diarrhea in adult cattle during the winter season. There are no reports on molecular characterization of bovine coronavirus in Ireland, and little data exists apart from serological studies.In this study, 11 neonatal (mean age 9 days) calf BCoV strains from the south of Ireland were collected over a one year period and characterized using molecular methods. The spike gene which encodes a protein involved in viral entry, infectivity and immune response shows the most variability amongst the isolates and was subsequently selected for in depth analysis. Phylogenetic analysis of the spike gene revealed that the Irish strains clustered with novel BCoV strains from Europe in a unique clade, possibly indicating lineage partitioning. Direct analysis of alignments identified amino acid changes in the spike protein unique to the Irish clade.Thus, monitoring of bovine coronavirus in Ireland is important as the current isolates in circulation in the south of Ireland may be diverging from the available vaccine strain, which may have implications regarding future BCoV vaccine efficacy. Coronaviridae) are large enveloped viral particles containing a positive sense single stranded RNA genome (26–30 kb), coding for several structural proteins, including polymerase (Pol), nucleocapsid (N), membrane (M), hemagglutinin-esterase (HE), spike (S) proteins and several non-structural proteins (NSPs). Coronaviruses have been associated with respiratory and enteric infections in humans and ruminants [Coronaviruses  replicates in the epithelial cells of the gut, destroying villi, resulting in severe, often bloody diarrhea in calves, which can be life threatening, due to loss of electrolytes and malnutrition . DiseaseEscherichia coli, confering passive immunity to calves, the coronavirus aspect of the vaccine is based on an inactivated Mebus strain. [BCoV has been detected in Ireland using molecular or immunological techniques –6, but i strain.  In this Salmonella. A total of 11 coronavirus positive samples were collected from neonatal calves, mean age 9 days, presenting with diarrhea between 2010 and 2011. Samples were stored at −80 °C prior to analysis.Faecal samples were collected from the Cork Regional Veterinary Laboratory (CRVL) after they had tested positive for coronavirus using an immunochromatographic commercial kit (ICK), Corona Vet . Faecal samples were also screened for rotavirus, cryptosporidium and Prior to extraction, faecal samples were homogenized in an equal volume of 0.89 % NaCl, centrifuged and filtered using a 0.20 μm pore size. The RNA was then extracted from the cell free fluid using Qiagen Viral RNA mini kit (Qiagen), following the manufacturers’ instructions. Extracted RNA was stored at −20 °C prior to analysis. Extracted RNA was tested for the presence of Coronavirus using degenerate oligonucleotide primers described previously , specifiFollowing analysis of this region, the most variable isolate was selected for complete characterization of the S gene using primers previously described , 13. Reahttp://blast.ncbi.nlm.nih.gov/Blast.cgi), to identify homologous strains. Sequence alignment was carried out using Clustal W in Bioedit [Resulting sequence data was then analysed and edited using Bioedit v7.0.9.0  and onli Bioedit , and a s Bioedit  with a GPartial nucleotide sequences are available for Polymerase gene , Spike . The complete gene sequence of the spike protein of Irish BCoV strain RVLC7 was registered in Genbank under the accession number-KF272919.Eleven coronavirus ICK positive specimens were collected from the CRVL and then subjected to further analysis at the Virology Unit, Department of Biological Sciences, Cork Institute of Technology (CIT). Of the 11 positive faecal samples tested using an ICK, 8/11 (72.7 %) tested positive for coronavirus using molecular techniques  had 7 single nucleotide polymorphisms (SNPs) in a 464 bp sequence, resulting in 3 amino acid changes Fig. ; the amiFrom alignment analysis of the HVR sequences, one isolate was selected for complete sequencing of the whole S gene, using primers previously described , 13. IsoIn 2012, BCoV was the most commonly isolated virus from live clinical cases of pneumonia and is also an important cause of enteric disease in young Irish calves . In thisMonitoring of bovine coronavirus in Ireland is important as the data suggests that the current isolates in circulation maybe diverging. In addition, phylogenetic analysis demonstrated clear differences between European and global BCoV strains, therefore, continuous monitoring of BCoV is essential in order to detect new BCoV strains which may emerge and provide data which can be used to inform future BCoV vaccine design.


Cellular stressors and apoptosis-inducing agents have been shown to induce ribosomal RNA (rRNA) degradation in eukaryotic cells. Recently, RNA degradation in vivo was observed in patients with locally advanced breast cancer, where mid-treatment tumor RNA degradation was associated with complete tumor destruction and enhanced patient survival. However, it is not clear how widespread chemotherapy induced “RNA disruption” is, the extent to which it is associated with drug response or what the underlying mechanisms are.Ovarian  and breast  cancer cell lines were treated with several cytotoxic chemotherapy drugs and total RNA was isolated. RNA was also prepared from docetaxel resistant A2780DXL and carboplatin resistant A2780CBN cells following drug exposure. Disruption of RNA was analyzed by capillary electrophoresis. Northern blotting was performed using probes complementary to the 28S and 18S rRNA to determine the origins of degradation bands. Apoptosis activation was assessed by flow cytometric monitoring of annexin-V and propidium iodide (PI) binding to cells and by measuring caspase-3 activation. The link between apoptosis and RNA degradation (disruption) was investigated using a caspase-3 inhibitor.All chemotherapy drugs tested were capable of inducing similar RNA disruption patterns. Docetaxel treatment of the resistant A2780DXL cells and carboplatin treatment of the A2780CBN cells did not result in RNA disruption. Northern blotting indicated that two RNA disruption bands were derived from the 3’-end of the 28S rRNA. Annexin-V and PI staining of docetaxel treated cells, along with assessment of caspase-3 activation, showed concurrent initiation of apoptosis and RNA disruption, while inhibition of caspase-3 activity significantly reduced RNA disruption.Supporting the in vivo evidence, our results demonstrate that RNA disruption is induced by multiple chemotherapy agents in cell lines from different tissues and is associated with drug response. Although present, the link between apoptosis and RNA disruption is not completely understood. Evaluation of RNA disruption is thus proposed as a novel and effective biomarker to assess response to chemotherapy drugs in vitro and in vivo.The online version of this article (doi:10.1186/s12885-016-2197-1) contains supplementary material, which is available to authorized users. RNA molecules play critical roles in cells, including the facilitation of protein synthesis and regulation of gene expression. To prevent errors in the biosynthesis and function of cellular RNAs, quality control surveillance mechanisms have evolved to identify and preferentially degrade aberrant or nonfunctional RNAs. Degradation of nonfunctional RNAs occurs in regulated stages and involves specialized mechanisms . MechaniThe degradation of DNA into internucleosomal fragments is a well-known hallmark of apoptosis . AlthougAlthough apoptosis-inducing agents have been shown to induce rRNA degradation in mammalian cells, other cytotoxic agents, such as chemotherapy drugs, have not yet been investigated in this respect. Recently, image-guided tumor core biopsies were taken from patients with locally advanced or inflammatory breast cancer enrolled in the CAN-NCIC-MA.22 clinical trial . SamplesIn the current study, we describe for the first time an in vitro cell model system for the study of chemotherapy-dependent RNA disruption. This included an investigation into the relationship between RNA disruption, drug type, drug dose, and drug incubation time. We also examined whether RNA disruption reflected the sensitivity of cells to various drugs (previously measured using the clonogenic assay). In addition, we explored the origins of the rRNA fragments and the temporal relationship between the induction of apoptosis by docetaxel, as measured by enhanced caspase activity and annexin V staining, and RNA disruption. Finally, we show that inhibition of caspase-3 activity reduces, but does not eliminate, RNA disruption in response to docetaxel.This study did not require ethics approval from an ethics review committee or board because the study did not involve animals, humans, human data or material directly collected from humans or animals.The A2780 parental cell line was acquired from the European Collection of Cell Cultures. The development and characterization of the docetaxel-resistant A2780DXL and carboplatin-resistant A2780CBN cell lines used in this study were described previously . Cell liTo assess the effect of drug dose on RNA disruption, cells were seeded into six-well plates for 24 h, following which each well was exposed to increasing doses of paclitaxel, docetaxel or carboplatin. After determining the most effective doses to induce RNA disruption, time exposure experiments were performed where cell cultures were exposed to specific drug doses for varying amounts of time . Replicate experiments were performed at least three times.Cells were harvested by scraping the adherent cells in lysis buffer and collecting them, along with any floating cells by centrifugation. Total RNA was isolated from harvested cells using miRNeasy kits . The quantity and integrity of isolated RNA was determined by capillary electrophoresis on an Agilent 2100 Bioanalyzer  with known reference RNA standards of various masses. The RNA Disruption Index (RDI) was calculated for each sample using a proprietary algorithm , which computes the ratio of abnormal to normal rRNAs .. [. [32P-ATP and the DNA 5’ End Labeling System by Promega . Hybridization was performed according to Brown and Mackey [Total RNA was isolated from A2780 cells treated with or without 0.2 μM docetaxel for 48 h. The component RNAs (5 μg per lane) were resolved by denaturing formaldehyde 1 % agarose gel electrophoresis, transferred to BioTrace PVDF membranes , and UV cross-linked. The membranes were then incubated with various radiolabeled probes hybridizing to sequences within the 28S and 18S rRNAs. These probes   and the percentage of apoptotic cells  was determined by flow cytometry on a BD FACS Canto II flow cytometer . The effect of docetaxel on cell cycle progression was also assessed by flow cytometry after cells were fixed and stained with PI alone as described previously .Caspase-3 activity in extracts of control and docetaxel-treated cells was assayed by monitoring cleavage of a DEVD substrate using the CPP32 Colorimetric Assay Kit from BioVision Inc. . The effects of caspase-3 inhibition on docetaxel-induced caspase activity and docetaxel-dependent RNA disruption were determined by treating cells with and without docetaxel and/or the caspase-3 inhibitor, Q-DEVD-Oph , and then assaying extracts of these cells for caspase-3 activity and RNA disruption as described above.p < 0.05 were considered statistically significant. Significance was determined using a two-way ANOVA with Bonferroni post-hoc test when comparing two variables. A one-tailed t-test was performed to determine the impact of increasing carboplatin concentration on cellular RDI values. For all other data, a two-tailed t-test was performed after application of the F-test to determine equality of variance.Statistical analyses were performed using Microsoft Excel or GraphPad Prism 5 software and differences with RNA disruption in A2780 cell cultures became evident at a dose of 0.005 μM for both docetaxel and paclitaxel, but peaked at 0.2 and 1 μM for these drugs, respectively. RNA disruption was evident in the taxane-treated cells by the presence of abnormal bands on the electropherogram, distinct from the normal RNA banding pattern seen in untreated cells Fig.  and c. FThe CaOV3 ovarian carcinoma cell line was also treated with docetaxel to determine if other ovarian cancer cell lines could exhibit taxane-induced RNA disruption. Using the same docetaxel doses as for A2780 cells, RNA disruption was observed in CaOV3 cells Fig.  after 48Carboplatin, a structurally distinct drug with a very different mechanism of action from taxanes, required a longer exposure time to induce RNA disruption in A2780 and CaOV3 cells. Therefore, A2780 and CaOV3 cells were treated for 72 h with increasing doses of carboplatin. Figure To investigate if RNA disruption can be induced by multiple cytotoxic chemotherapy agents, with differing mechanisms of action, A2780 ovarian and MDA-MB-231 breast cancer cells were treated with a panel of chemotherapy drugs. RNA disruption was induced in A2780 cells by treatment with paclitaxel (TAX), docetaxel (DXL), carboplatin (CBN), cisplatin (CIS), etoposide (ETOP), vincristine (VIN), irinotecan (IRN) and doxorubicin (DOX), as shown in the electropherogram in Fig. The abnormal RNA disruption bands that occur upon chemotherapy drug exposure are smaller in molecular weight than the 28S and/or 18S rRNAs. To determine whether the abnormal bands originate from the 28S and/or 18S rRNAs, Northern blotting experiments were performed on total RNA prepared from A2780 cells after incubation in the absence or presence of docetaxel for up to 48 h Fig. . Figure . demonstrated lack of cross resistance, using a clonogenic assay, which showed that A2780DXL cells are sensitive to killing by carboplatin and that A2780CBN cells are sensitive to killing by docetaxel [To investigate the relationship between drug sensitivity and drug-induced RNA disruption, total RNA was isolated from docetaxel-sensitive (A2780) and docetaxel-resistant (A2780DXL) cells after treatment with 0.005 and 0.2 μM docetaxel for 48 or 72 h. Consistent with their known drug sensitivity using clonogenic assays , A2780 cocetaxel . Using R1 peak, often associated with apoptotic bodies, was evident in the docetaxel treated cells after 24 h, and by 72 h, almost all the PI signal was in the sub G1 peak. This shows that extended docetaxel treatment generated cell fragments with less than a diploid amount of DNA content, representing apoptotic bodies or micronuclei. Finally, to see if DNA laddering (a late apoptosis biomarker) also occurred, A2780 and Jurkat cells were treated with or without docetaxel (A2780 cells) or etoposide (Jurkat cells) for similar lengths of time. Genomic DNA was prepared from the cells and resolved by agarose gel electrophoresis  and propidium iodide (PI) and analyzed by flow cytometry Fig. . At 24 hsis Fig. . Interessis Fig. , it appeTo further support the induction of apoptosis in A2780 cells in response to docetaxel, we examined the effect of docetaxel treatment on caspase-3 activity at 24, 48, and 72 h following treatment. Figure Previous studies have shown that a variety of apoptosis-inducing agents with distinct mechanisms of action , dexamethasone, a calcium ionophore and a tricothecene mycotoxin) were able to induce an ordered, apoptosis-associated rRNA degradation in several different cell types, including plant cells and the unicellular organism yeast –17, 24. We further observed that RNA disruption is both dose- and time-dependent Figs.  and 2. M. depending upon the apoptosis-inducing agent used and the cell line being investigated. They observed a unique cleavage site in the 28S rRNA in bovine endothelial cells, although the RNA cleavage patterns in different cell types were remarkably similar overall [. in murine macrophages, rRNA cleavage was induced by the tricothecene mycotoxin deoxynivalenol. The pattern observed by He et al. contains a similar fragment just below the 18S rRNA and contains three fragments in the region between the 28S and 18S rRNA bands – similar to patterns observed by Houge et al. [., two different apoptosis-inducing agents were used in Jurkat and U937 cells to initiate rRNA fragmentation (Fas ligand and TNFα), producing a pattern with a band just below the 18S rRNA but with only one clearly discernable band in the region between the 28S and 18S rRNAs [. in S49 cells treated with several different apoptotic stimuli. No degradation band below the 18S rRNA was observed and all 5 degradation bands were derived from the 5’-end of the 28S rRNA [Significant RNA disruption occurred after 24 h exposure to paclitaxel and docetaxel in A2780 cells and after 48 h in CaOV3 ovarian tumor cell lines treated with docetaxel. RNA disruption was always maximal at the longest incubation time (72 h) Fig. . Carbopl overall . In yeas overall . In anote et al. . In a st8S rRNAs . A very 28S rRNA . In our In this study, we have documented that the ability of specific chemotherapy agents to induce cell death/growth arrest in a clonogenic assay is also reflected in their ability to induce RNA disruption in tumor cells in vitro Fig. . Our dat. [. [Northern blots of total RNA isolated from A2780 cells treated with docetaxel were used to identify the approximate origins of the RNA disruption bands. As described above, the 28S-5 probe hybridized to the full-length 28S rRNA and to two 28S rRNA fragments – one migrating faster than the full-length 18S rRNA  and one migrating between the intact 28S 5,025 nt) and 18S rRNA bands Fig. . Their s nt and 1.  and Houg. [.  which ar. [. , 24, 25.1 DNA content . These RNases could be homologs of RNases recently identified to play a role in stress-induced rRNA degradation in yeast . These iIn this study, we show that RNA disruption can be induced in various cell types by structurally distinct chemotherapy agents with contrasting mechanisms of action in a dose- and time-dependent manner. The disruption fragments (at least for docetaxel) initially stem from cleavages within the expansion segments (variable regions) of the 28S rRNA. The extent of RNA disruption observed in cells reflects their sensitivity (or lack thereof) to chemotherapy drugs and cells can only tolerate a specific level of RNA disruption, above which they become nonviable. Moreover, while the induction of RNA disruption is temporally correlated with the induction of apoptosis, RNA fragmentation products accumulate over time, while apoptotic biomarkers wane. RNA disruption is associated with caspase activation and a known caspase-3 inhibitor can substantially reduce RNA disruption by docetaxel. The link between drug sensitivity in cell lines (previously assessed by clonogenic assay) and drug-induced RNA disruption strongly support our recent clinical findings that high tumor RNA disruption is associated with tumor sensitivity to drug, expressed as a pathologic complete response and enhanced disease-free survival after neoadjuvant chemotherapy in patients with locally advanced or inflammatory breast cancer . RNA dis


C. elegans PLM neurons as a model, we show that UNC-44/ankyrin acts upstream of UNC-33/CRMP in regulation of a potential kinesin VAB-8 to control gap junction dynamics, and loss-of-function in the UNC-44/UNC-33/VAB-8 pathway suppresses the turnover of gap junction channels. Therefore, we first show a signal pathway including ankyrin, CRMP, and kinesin in regulating gap junctions.Gap junctions are present in both vertebrates and invertebrates from nematodes to mammals. Although the importance of gap junctions has been documented in many biological processes, the molecular mechanisms underlying gap junction dynamics remain unclear. Here, using the  C. elegans PLM neurons as a model, we carried out an unbiased genetic screen and isolated mutants altering gap junctions. In this study, we focus on two of them, unc-44/ankyrin and unc-33/CRMP. Through genetic analysis in combination with live imaging, we find that UNC-44/ankyrin and UNC-33/CRMP play important roles in gap junction turnover and demonstrate that UNC-44/ankyrin acts upstream of UNC-33/CRMP and VAB-8/ kinesin to regulate the removal of gap junction channels from gap junctions.The nervous system is made up of individual neurons connected by junction structures called synapses. There are two fundamentally different types of synapses: chemical synapses and electrical synapses . Through studies in different model organisms, we have gained rich knowledge about the development and dynamics of chemical synapses. However, we still know little about how gap junctions are formed during development and what regulates the dynamic of gap junctions in functional circuits. Using  Gap junctions were first discovered in the myocardium and nerves for their properties of electrical transmission between two adjacent cells ,2, and tRegulation of gap junctions is observed at two levels: fast regulation involving change of channel conduction and open probability, and slow regulation including alternation of composition and turnover of channels . StudiesC. elegans . El. ElC. el of tail . Three i neurons . To visuterminus . Taggingt zone 2 , and thet zone 2 . Using at zone 2 . We furtle cells . Consist neurons . The con neurons . We foununc-1(lf) background and found that loss of function of unc-1 did not affect UNC-9 puncta, suggesting UNC-1 was not required for the formation of UNC-9 puncta in PLM neurons  mutant animals lost UNC-9 puncta  as a starting strain and isolated mutants with three types of phenotypes: 1, type one mutants lost UNC-9 puncta; 2, type two mutants had more UNC-9 puncta close to the original gap junctions; 3, type three mutants had more UNC-9 puncta along the axon  phenotypes. After testing some unc genes on chromosome IV, we found yad21 failed to complement the loss-of-function allele of unc-44(e362), and yad26 failed to complement the loss-of-function allele of unc-33(mn407). unc-44(e362) had the same UNC-9 punctate defects as that seen in yad21, and unc-33(mn407) and unc-33(e204) had identical phenotypes with yad26 (yad21 introduced a point mutation (P4813S) and a premature stop codon (Q6827*) in the neuronal specific long isoform of unc-44, and yad26 had two point mutations (E168K and G328D) in unc-33  and yad26  animals, PLM axons grew straight from cell bodies to the middle section of the body similar to wild type animals, but about 70% of yad21(lf) and yad26(lf) animals had either shorter or longer PLM axons  was same as those seen in all animals. Loss of function of unc-34/Enabled/VASP affected PLM development. Loss-of-function mutation in rpm-1 caused overextension of PLM axons, but we did not observe any defects of UNC-9 distribution in these mutants (unc-44(lf) and unc-33(lf) were not results of axon growth defects.elopment ,39, raisLM axons . However mutants  41]. Th. Thunc-4unc-33 in C. elegans, named L (long), M (middle) and S (short) isoforms based on the length of cDNA, and only the long isoform could rescue unc-33(lf) polarity defects [unc-33(lf) animals, supporting the phenotypes we observed were not results of mis-regulation of neuronal polarity (unc-33(lf) phenotypes by expressing unc-33 cDNA in PLM neurons also showed that unc-33 cell autonomously regulated UNC-9 puncta  and unc-44(lf) mutants  contained transient gap junctions, and most of these gap junctions are eliminated in adults [unc-33(lf) and unc-44(lf) mutants, we observed significantly more NSY-5 puncta  in all examined animals . Using an antibody specifically recognizing the neuronal specific long isoform of UNC-44 [unc-33 loss-of-function mutants did not affect UNC-44 distribution (unc-33(S) on unc-44(lf) phenotypes. As shown in unc-33(S) at high level suppressed unc-44(lf) phenotypes. In conclusion, by analyzing mutants with abnormal accumulation of UNC-9 in PLM neurons, we uncovered an important role of the UNC-44/UNC-33 pathway in the regulation of gap junctions.To determine whether f UNC-44 , we founribution . These runc-44 and unc-33 suggested that this pathway might regulate gap junction dynamics. To test this possibility, we analyzed GFP::UNC-9 movement at the gap junction zone one. In control animals, we observed bidirectional movement of UNC-9 both anterior and posterior to gap junctions, and each animal had almost equivalent numbers of UNC-9 particles moving toward and away from gap junctions  and unc-33(lf) phenotypes might be due to suppression of gap junction turnover. To further examine this hypothesis, we used a transgene expressing photoactivatable GFP (PAGFP) tagged UNC-9 [unc-44 and unc-33 suppressed the decrease of fluorescence intensity (unc-44 and unc-33 were involved in the assembly of UNC-9 puncta using the Fluorescence Recovery After Photobleaching (FRAP) assay. Briefly, we photobleached the GFP signal in zone 1 and measured the recovery of GFP signal after 3 hours. As shown in unc-44 nor unc-33 affected the recovery of GFP signal, supporting the UNC-44/UNC-33 pathway did not regulate the assembly of UNC-9 puncta. We believed that the UNC-44/UNC-33 pathway regulated gap junction turnover through suppression of transport of UNC-9 out of gap junctions.The striking phenotypes of unctions , implicadynamics  Movies. ed UNC-9 . In thisntensity . We alsounc-44/unc-33 on UNC-9 dynamics suggested that they might regulate some motor proteins. We first tested three classic motor proteins, UNC-104, UNC-116 and DHC-1, that are known to be important for neuronal development. UNC-104 is the kinesin transporting synaptic vesicles from cell bodies to chemical synapses [C. elegans neurons [vab-8 induced similar phenotypes as those seen in unc-44(lf) and unc-33(lf)  and vab-8(S), and the major difference between these two isoforms is that vab-8(S) lacks the kinesin domain present in vab-8(L) [vab-8(L) in PLM neurons was able to rescue vab-8(lf) phenotypes, suggesting the potential kinesin function of VAB-8 was essential for gap junction regulation  and unc-44(lf) phenotypes  . Furtherturnover . Indeed,turnover . vab-8 hvab-8(L) . We foungulation . Since Uenotypes . These rC. elegans PLM neurons as a model, we investigated the molecular mechanisms underlying gap junction regulation. In adult animals, PLM neurons form gap junctions at two regions, at zone 1 with PVC, LUA and PVR neurons and at zone 2 with BDU neurons [C. elegans ankyrin/unc-44 and CRMP/unc-33 in regulating gap junctions. By imaging GFP::UNC-9 dynamics and PAGFP::UNC-9 decay, we further demonstrated that the UNC-44/UNC-33 pathway regulated UNC-9 dynamics and turnover. In searching for motor proteins contributing to unc-33/unc-44 phenotypes, we uncovered a role for a potential kinesin VAB-8 in UNC-9 turnover. VAB-8(L) directly bind to UNC-33(S), and overexpression of VAB-8(L) suppressed both unc-33(lf) and unc-44(lf) phenotypes, supporting the hypothesis that VAB-8 is the downstream target of the UNC-44 pathway in gap junction regulation. Double mutants of vab-8 and unc-33 had stronger phenotypes than each of single mutant and showed similar phenotypes as unc-44(lf), suggesting that UNC-33 and VAB-8L function in parallel downstream of UNC-44. The suppression of unc-33(lf) phenotypes by VAB-8L overexpression was likely due to the enhanced contribution from VAB-8L  phenotypes by overexpressing unc-33(s), we concluded that unc-44 acted upstream of unc-33 in gap junction regulation. However, the localization of UNC-33(S) and UNC-44 appeared to be different, that UNC-33(S) was concentrated at the nerve ring, but UNC-44 had a broader distribution in neurons. One concern is that the transgene used to label UNC-33(S) (Prgef-1::FLAG:: unc-33(S)) may not reflect its in vivo localization. We compared our results of UNC-33(S) with the previous description of UNC-33(L) localization [in vivo localization of UNC-33(S). Since UNC-44 plays important roles in many aspects of neuronal development, the broad distribution of UNC-44 seems consistent with its multiple functions in neurons.In our experiments, we found that the localization of UNC-33(S) was regulated by lization  and founThe UNC-44 homolog ankyrins have the ability to bind to channels and to integrate them to cytoskeleton, and the interactions between ankyrins and channels are dynamically regulated by intracellular signals . It is pC. elegans strains on NGM plates at 20–22.5°C. All transgenes and strains are described in the yadIs12 (Pmec-4::GFP::UNC-9) to visualize gap junctions in PLM neurons. Using yadIs12 as a starting strain, we performed a clonal recessive screen following standard ethyl methane sulfonate (EMS) mutagenesis protocol. In 1000 mutagenized haploid genomes we examined, 12 mutants were isolated with three type phenotypes as mentioned in the manuscript.We maintained unc-33 (L) cDNA was obtained from Dr. Cori. Bargmann, and unc-33(M) and unc-33(S) was amplified from unc-33(L) cDNA. vab-8(L) and vab-8(S) cDNA was amplified from yk clones from Dr. Yuji Kohara lab. Photoactivatable GFP (PAGFP) plasmid was obtained from Addgene. PAGFP coding sequence was inserted in an ASCI enzyme site before the start codon of UNC-9. All primer sequences are available upon request. Transgenic animals were generated following standard procedures. In general, plasmid DNAs of interest were used at 1–50 ng/ml with the co-injection marker Pttx-3::rfp/Pttx-3::gfp at 50 ng/μl.All DNA expression constructs were made using Gateway cloning technology (Invitrogen). Sequences of the final clones were confirmed. The Representative images and immunostaining results were acquired with a Zeiss LSM700 confocal microscope using a Plan-Apochroma 40x/1.4 objective. Worms were immobilized in 1% 1-phenoxy-2-propanol  in M9 buffer. For quantification of the percentage of animals with gap junction defects in PLM neurons, we used a Zeiss Axion Imager 2 microscope equipped with Chroma HQ filters. Each analyzed data performed takes at least three independent experiments and total 200–300 1-day old adults. For quantification of the length of gap junction zone, Images were acquired with LSM700 confocal microscope using a Plan-Apochroma 40x/1.4 objective, and the length of gap junction zone was measured using Zeiss Zen Black software.The photoactive experiments were carried out with a Zeiss LSM700 confocal microscope using a Plan-Apochroma 40x/1.4 objective. 1-day young adult transgenes expressing mCherry and PAGFP::UNC-9 in PLM neurons were immobilized in 1% 1-phenoxy-2-propanol. We first use mCherry signal to localize PLM neurons and use 405 laser to globally or locally photoactivate PAGFP::UNC-9. After photoactive animals were recovered on the NGM plates for 3 hours before the second images were taken. The fluorescent intensity was analyzed using Image J software.GFP::UNC-9 dynamic experiments were preformed using an Andor revolution microscopy with a 60 x /1.46 Plan-Apochromat objective controlled by MetaMorph software. All videos were acquired by an Andor EM-CCD camera (DU897). 1-day adult animals were immobilized in 5mM levamisole and on 5% agar pads for imaging. Videos for GFP::UNC-9 dynamic analysis were roughly 30–40s with 8 frames per second. Kymographs were generated using ImageJ, and the direction of GFP::UNC-9 movement was judged by the direction of the black lines in the kymograph pictures. In general, dynamic puncta were defined as their velocities >0.1 μm/s for last at least 5s. For puncta that change directions during experiments, we trace them for the overall direction.3h-Ibleach)/(Ipreblach -Ibleach)x100. I3h: the intensity at the region of interest (ROI) three hours after photobleaching; Ibleach: the intensity at the ROI after photobleaching; Iprebleach: the intensity in the ROI before photobleaching. Background (the intensity in the non-bleached part of ROI) was subtracted, respectively.1-day adult animals were immobilized in 5mM levamisole and on 5% agar pads. FRAP was performed using Zeiss LSM700 confocal microscope using a Plan-Apochromat 40x/1.4 objective. A high-powered laser  was used to photobleach the region of interest. Worms were recovered on NGM plates with food for 3 hours before taking the second image. Quantification of GFP::UNC-9 was carried out using Image J. Percentage Recovery = (IAll immunostaining experiments were carried out following the standard protocol using 1-day young adults. The Rabbit anti-UNC-9 antibody was a gift from Dr. Zhao-Wen Wang. The Rabbit anti-UNC-44(L) antibody was a gift from Dr. Anthony Otsuka. Mouse anti-FLAG M2 antibody (Cat# F1804), mouse anti- acetylated tubulin antibody (Cat# T7451) and rabbit anti-GFP (Cat# G1544) were purchased from Sigma. The dilutions for each antibodies are: anti-UNC-9(1:100), anti-UNC-44(1:100); anti-FLAG (1:300), anti-acetylated tubulin(1:300) and anti-GFP (1:150). Alexa Fluor 488 Donkey-anti-rabbit IgG (H+L) antibody (Cat# A-11008) and Alexa Fluor 594 Goat Anti-Mouse IgG (H+L) Antibody (Cat# A-11005) from Molecular Probes were used as secondary antibody in 1:500 dilution.yadEx421) expressing FLAG-VAB-8(L) and HA-UNC-33(S) under the control of Pan-neuronal promoter Prgef-1. Proteins from mixed stages animals were first extracted using RIPA buffer by frozen-throw about 50 times in ethanol with dry ice, and protein lysis was incubated with mouse anti-FLAG M2 antibody (Cat# F1804) in room temperature for 5 hours and then precipitated using Protein A/G PLUS-Agarose (Santa Cruz Bio. esc-2003). Heated Protein samples were separated using SDS-PAGE Gradient Gels (4–20%), and then transferred to nitrocellulose. Blots were probed with mouse anti-Flag antibodies  and rabbit anti-HA(Sigma H6908), and then visualized with Amerisham HRP-conjugated anti-rabbit secondary antibodied at 1:5000 using the SuperSignal West Femto kit .For the immunoprecipitation experiment, we generated an transgene .S1 Table(DOCX)Click here for additional data file.S1 FigA) A cartoon shows the predicted structure of UNC-9, that UNC-9 has four transmembrane domains, two extracellular and one intracellular loop, and intracellular N- and C- termini. (B) GFP::UNC-9 puncta localized to PLM-PVC junctions. PVC axons was visualized by expressing Pglr-1::mCherry. An enlarged view of PLM-PVC gap junctions is displayed at the right side. (C) Mutating four conserved Cys required for UNC-9 gap junction functions alters UNC-9 localization. (D) Loss of function of Netrin (unc-6) or its receptor unc-5 did not affect UNC-9 plaques, but loss of function of DCC(unc-40) suppressed the formation of UNC-9 plaques in about 3–5% animals. (E) Loss of function of unc-34 did not change UNC-9 puncta. (F) Loss of function of unc-33 or unc-44 did not change GFP-UNC-9 expression level. GFP signal from expression of co-injection marker Pttx-3::GFP was used as the loading control. The number displayed at each images shows the number of animals with wild type GFP::UNC-9 plaques/ total animals. Scale bar: 10 μm.((PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(A) Representative images shows axons morphology of PLM neurons in control, unc-44(yad21) and unc-33(yad26). For unc-44 and unc-33, the examples of “wild type”, shorter and longer axons were displayed from top to bottom. The “length” of axons was defined by the relative positions between PLM axon terminals (red arrowheads) and ALM cell bodies (yellow stars). The number displayed at each images shows the number of animals with according phenotypes/ total animals. (B) Representative images of immunostaining results for UNC-9(green) and acetylated tubulin (Pink) in unc-44(lf) and unc-33(lf) animals. CB: cell bodies. Scale bar: 10 μm.(PDF)Click here for additional data file.S4 Fig(A) and quantification(D) show that the fluorescence intensity at zone 1 recover 20% 3 hours after photobleaching. The thermal bar shows the color code for fluorescence intensity from purple (low) to red (high). Images and quantification(D) show that loss of function of unc-33 or unc-44 doesn’t affect the recovery after photobleaching. N>10 animals for each experiments. Data are shown as mean ± SD. ns: no significant difference. Scale bar: 10 μm.Image(PDF)Click here for additional data file.S5 Figunc-104 and unc-116, and dynein heavy chain dhc-1 does not affect UNC-9 plaques. The number displayed at each images shows the number of animals with wild type GFP::UNC-9 plaques/ total animals. Scale bar: 10 μm.Loss of function of neuronal kinesin (PDF)Click here for additional data file.S1 Movie(MP4)Click here for additional data file.S2 Movie(MP4)Click here for additional data file.S3 Movie(MP4)Click here for additional data file.S4 Movie(AVI)Click here for additional data file.S5 Movie(AVI)Click here for additional data file.S6 Movie(AVI)Click here for additional data file.