Here, we explored the mode of in vivo recruitment process of Rho. We show that sequence specific recognition of the rut site, in majority of the Rho-dependent terminators, can be compromised to a great extent without seriously affecting the genome-wide termination function as well as the viability of Escherichia coli. These terminators function optimally only through a NusG-assisted recruitment and activation of Rho. Our data also indicate that at these terminators, Rho-EC-bound NusG interaction facilitates the isomerization of Rho into a translocase-competent form by stabilizing the interactions of mRNA with the secondary RNA binding site, thereby overcoming the defects of the primary RNA binding functions.The bacterial transcription terminator, Rho, terminates transcription at half of the operons. According to the classical model derived from  The well-conserved bacterial transcription terminator, Rho, is a hexameric RNA helicase that terminates transcription at about half of the operons ,2. Rho-dIn vitro, NusG induces early termination and increases the speed of RNA release, and thereby is thought to function in the RNA release step of Rho-dependent termination , and facilitates the transcription termination process . Exact mmination .rut sites (Rho utilization), using its primary RNA binding sites (PBSs). Following that, it isomerizes into a translocase-competent form by threading the mRNA through its secondary RNA binding sites (SBSs), undergoes ATP-hydrolysis-dependent translocation along the mRNA and, finally, dislodges the elongation complex  RNAs comprising of rut sites (in vivo regulations involved in the recruitment of Rho to the EC by comparing the behaviors of Rho mutants defective for the PBS (PBS mutants) and the SBS functions (SBS mutants). The effect of these mutants on the genome-wide transcription pattern revealed that primary RNA binding function of Rho, unlike ATPase and translocase activities, can be compromised in majority of the terminators including those in the rac  and other prophages, whose suppression by Rho is essential for the cell viability  and slow RNA-induced isomerization of Rho at the rut sites of trac strongly indicated that Rho-EC bound NusG interactions act during the recruitment of Rho to the EC and provide additional stability to the Rho–mRNA complex at the weak rut sites of many terminators.In our previous work, using direct ut sites . Here, wiability . Howeverin vitro transcription reactions were purchased from GE healthcare. [γ-32P]ATP (3000 Ci/mmol) and [α-32P]CTP (3000 Ci/mmol) were from Jonaki, BRIT, India. Antibiotics, isopropyl β-D-1-thiogalactopyranoside (IPTG), lysozyme, dithiothreitol (DTT) and bovine serum albumin (BSA) were from USB. Primers for polymerase chain reaction (PCR) were obtained either from Sigma or MWG. Restriction endonucleases, polynucleotide kinase and T4 DNA ligase were from New England Biolabs. WT Escherichia coli RNAP holoenzyme was purchased from Epicentre Biotechnologies. Taq DNA polymerase was from Roche Applied Science. Ni-NTA agarose beads were from Qiagen. Streptavidin-coated magnetic beads were from Promega. Bacterial RNA purification kit was from NEB. RNAlaterTM used for storing RNA samples used in microarray experiments were from Ambion. All the bacterial growth media were from Difco.Nucleoside triphosphates (NTPs) for Rkan cassette from rho; rho::FRT) of chromosomal rho (strains RS1458 and RS1490) have been made by expressing flip recombinase from a temperature-sensitive (‘ts’) plasmid, pRS766. Subsequently, the plasmid was removed by growing the strains at 42°C. nusG mutations, G146D and L158Q, were inserted into the chromosome by linear DNA transformation. In brief, the strain RS1428 was transformed with PCR-amplified DNA fragments containing the mutations. RS1428 expresses λ red recombinase from a ‘ts’ plasmid and has a chromosomal insertion of a trac-LacZYA reporter cassette (as a λRS45 lysogen). The recombinants were screened on minimal media containing lactose. Colonies with nusG mutants was able to read through the trac terminator and thereby ensured the cells to utilize lactose. Presence of nusG mutants was further confirmed by suppression of the phenotype by expressing WT nusG (pRS695) and sequencing.Scar-less deletions  were at first transformed with WT and different Rho mutants expressed from a low copy number plasmid, pCL1920 (pHYD567), and, subsequently, the chromosomal rho was removed by inserting a Rkan cassette (Rrho::kan) via P1 transduction . The transductants were re-streaked on the LB plates supplemented with appropriate antibiotics .To study the differential growth behavior between the WT and different s Figure , MG1655rs Figure . In this−rac) during the propagation, and deletion of this prophage allowed the strain to survive in the presence of all the rho and nusG mutants. This MC4100 also has the trac terminator cassette inserted into the genome as a λRS45 lysogen (RS1428). nusG mutations were then inserted into the genome of RS1428 by linear DNA transformations to produce RS1514 (G146D) and RS1523 (L158Q). These two resultant strains were transformed with WT and different Rho mutants (Y80C and N340S) cloned in a low copy number plasmid, pCL1920 (pHYD567), and subsequently the chromosomal rho was removed by transducing a Rrho::kan cassette via P1 transduction. Two transductants of each mutants were re-streaked to measure the synthetic lethality  so that they are less dependent on Rho-dependent termination for their survival , Plac-H-19B tR1-lacZYA (RS1490) and PRM-racR/trac-lacZYA (RS1430) reporter cassettes as λRS45 lysogens. In all these reporters, expression of lacZ occurs only in the absence of Rho-dependent termination, and hence was used for measuring the termination defects . Total RNA was isolated from MC4100 (RS734) strain transformed with pHYD3011 plasmid expressing either WT or the nusG mutants, L158Q and G146D. RT-PCR reactions were performed on two genes; lacZ, fused downstream of tR1 terminator, and ybcK, which follows an untranslated region likely to have Rho-binding site(s) . The primer pairs RS401/402 and RS848/849 (see Supplementary Table S1) were used to amplify the cDNA products made from the RNA transcripts of lacZ and ybcK genes, respectively. PCR products were visualized in a 1.5% agarose gel stained with EtBr , RS1428 was transformed with either WT or N340S Rho mutants cloned in pCL1920 plasmid and the genomic copy of rho was deleted by P1 (RΔrho:kan) transduction. In all the RT-PCR assays, total RNA was isolated from these strains using RNeasy RNA isolation kit (Qiagen). cDNA was synthesized from 2 μg of isolated RNA using SuperScript III First Strand Synthesis System (Invitrogen Inc.) as per the manufacturer's protocol. cDNA was used as a template in RT-PCR with different oligos designed inside the gene of interest . To identify the in vivo Rho-dependent termination events in the trac terminator region, we compared the amounts of RT-PCR products obtained from the strains expressing WT and N340S Rho mutant. Higher amount of PCR products in N340S lanes  is due to impaired termination events.To measure the MG1655 strain was at first transformed with pCL1920 plasmids containing WT and different Rho mutants, and the chromosomal rho was deleted (Rrho::kan) by P1 transduction. For the PBS mutants, +MG1655rac, and for the SBS mutants, −MG1655rac strains were used. Deletion of rac prophage (rac−) allows the strains to grow in the presence of SBS mutants. Overnight cultures of these strains were sub-cultured in 10 ml LB with appropriate antibiotics, and were allowed to grow until OD600 ∼ 0.3–0.4. Culture was spun down and the cell pellet was re-suspended in 1 ml of RNAlater TM.The E. coli K12 microarray array slides in a 8 × 15k format were used. Agilent's two-color microarray-based gene expression analysis uses cyanine 3- and cyanine 5-labeled targets to measure gene expression in experimental and control samples. The total number of coding regions available on the chip was 4294. The average number of probes per coding region sequence was 3, and the total number of probes designed was 10 828. The total number of probes used for non-coding region was 4380. Two independent biological replicates for each strain were taken for the analyses. Fold change in gene expression for each strain was calculated with respect to WT. We have considered the genes whose expressions were changed 5-fold (log 2) or more with respect to the WT.RNA isolation and the microarray experiments were performed by Genotypic Technology, Bangalore, India. In brief, RNA was extracted from the cell pellets suspended in RNAlater solution using Qiagen's RNeasy kit. Agilent t, the number of threshold cycle , was used to quantitate the amount of cDNA. We have used the following mathematical equations to express the amount of the qRT-PCR products. 2−ΔΔCt method was used to calculate the fold change (2−ΔΔCt) in the mRNA level for the mutants with respect to the WT, where Ct = number of threshold cycle; ΔCt = Ct of target gene – Ct of internal control; ΔΔCt = ΔCt of the mutant – ΔCt of the WT. The level of rpoC mRNA was independent of the Rho mutants and, therefore, it was used as an internal control. Primer pair for each gene was chosen based on the following criteria: (i) comparable TM between the primers, (ii) located at the central part of the gene and (iii) the length of the cDNA product is ∼200–300 nt.The total RNA used in the quantitative RT-PCR (qRT-PCR) experiments was prepared in the same way as for microarray experiments. The amount of cDNA produced during the PCR cycles was monitored using SYBR green dye in an Applied Biosystem 7500 PCR system. Cin vitro transcription assays were made by PCR amplification from the plasmids, pRS22 , pRS106 , pRS604  and pRS1352 . When required, a lac operator sequence was inserted either after tR1 or trac terminators using a downstream primer having the operator sequence. In pRS385, the lac operator sequence is cloned after the H-19B tR1 terminator  of yagNM operon, multiple-rounds transcription was employed by omitting rifampicin from the reaction mixture  to make a 23-mer EC (EC23). The EC23 was then chased with 250 μM each of ATP, GTP, CTP and UTP. For multiple-round transcription, the RNAP–promoter complex was chased with 250 μM each of ATP, GTP and UTP. Concentration of CTP was 50 μM and the RNA was labeled with [α-32P]CTP (3000Ci/mmol). The reactions were stopped by extraction with phenol followed by ethanol precipitation. Samples were loaded onto a 6% sequencing gel and analyzed using FLA 9000 phosphorimager (Fuji).For the transcription using strong T7A1 promoter, we followed the regime of single-round transcription. However, due to the poor yield from PlacO site present downstream of the terminator region. The DNA templates were immobilized on streptavidin-coated magnetic beads (Promega) through streptavidin–biotin interaction. The transcription reactions were continued for 3 min to form the stalled EC, and the excess NTPs were removed by thoroughly washing the beads, following which 50 nM Rho plus 1 mM ATP was added. Ten microliters of samples were removed at different time points, and separated into supernatant (S) and pellet (P) on magnetic stands. Half of the supernatant (S) was directly mixed with equal volume of formaldehyde loading buffer. Rest of the sample (S+P) was phenol extracted and mixed with dye. Fraction of released RNA [(2S)/(S) + (S+P)] was plotted against time and the curves were fitted to exponential rise equations either of the form y = a(1−expbx−) or y = y0 +a(1−expbx)−, where ‘b’ denotes the rate, ‘a’ is amplitude of the RNA release process and ‘y0’ is minimum value of y at 0 time point. RNA release kinetics on trac terminator had a distinct lag period and was fitted to a sigmoidal equation of the form: y = y0 + (a)/[1 + (x/x0)b], where ‘x0’ is the inflection point of the curve and ‘b’ is the slope factor.For measuring the RNA release kinetics from the stalled EC, transcription reactions were performed in presence of 100 nM lac repressor so that the EC gets road-blocked at the tR1 or a trac sequence, respectively. To get adequate amount of RNA for the ATPase assays, the concentrations of DNA template and RNAP were increased to 4-fold compared to that used in the in vitro transcription assays. On these templates, road-blocked (RB) complexes were formed in a similar way as in RNA release kinetics assays. Due to the weaker transcription initiation efficiency of PRM promoter fused to the trac, compared to that of the T7A1 promoter fused to tR1, two reactions using the former fusion were pooled together and concentrated through Amicon-100 columns to get the desired volume. Rho + 1 mM ATP duped with [γ-32P]-ATP (3000Ci/mmole) was added to the RBs, and the time course of inorganic phosphate (Pi) release (ATP hydrolysis) was monitored on polyethyleneimine (PEI) TLC sheets (Merck) with 0.75 M KH2PO4 (pH 3.5) as the mobile phase. In all the ATPase assays, the composition of the reaction mixtures was 25 mM Tris-HCl (pH 8.0), 50 mM KCl and 5 mM MgCl2. The reactions at indicated time points were stopped with 1.5 M formic acid. TLC sheets were analyzed by Fuji FLA-9000. The amounts of [γ-32P] ATP and the released Pi were calculated from the intensities of the spots of these products using the Image-Quant software.We used the nascent RNA attached to the stalled ECs to activate the ATPase function of Rho . The stain vivo, we have chosen specific Rho mutants having changes in these two sites . PBS mutants are defective for rut site recognition , while SBS mutants can interact with RNA at the PBSs but have impaired RNA-dependent ATPase activity and, consequently, are defective in translocation along the mRNA . Upon re-streaking the transductants, we observed severe growth defect in the presence of SBS mutants, whereas the PBS mutants did not affect the cell viability significantly .MG1655y Figure . It was trac and at the well-known tR1 terminators  compared to tR1. This result is consistent with the observation that PBS mutants do not affect the viability of the cells that primarily arises due to severe termination defect at trac . The PBS mutants, on the other hand, were severely defective only on c Figure .These results indicate that defects of PBS mutants are terminator-specific. To check whether this differential effect of PBS mutants is general in nature, we decided to monitor the defects in genome-wide termination patterns of these Rho mutants.Δrho strain expressing WT and mutant Rho genes from a low copy pCL1920 plasmid and plotted the fold-change in gene expression of different mutants relative to that of the WT  were used. We observed the following.Among the genes expressed in the mid-log phase, ∼500–600 genes are affected due to the presence of the SBS mutants, and this is comparable to that observed in the presence of the Rho inhibitor, bicyclomycin , as well as some of the intragenic non-coding regions. Up-regulation of rho is consistent with the fact that its expression is terminated by a Rho-dependent terminator located upstream of this gene that directly binds rho when the latter's concentration is elevated in the cell  with the primary RNA-binding domain of Rho.The aforementioned data strongly indicated that the tolerance of mutations in the primary RNA binding domain of Rho is not confined to one specific terminator, in vitro transcription reactions using purified components. We used a DNA template having the trac terminator embedded inside the racR region , where transcription was initiated from its natural terminator PRM. We used trac as a prototype of the subset of terminators that are not affected by PBS Rho mutants for further in vitro and in vivo studies. Transcription termination at these terminators was compared with the classical Rho-dependent terminator tR1, where all the Rho mutants are defective  and SBS (N340S) Rho mutants in efective . Assays s Figure .in vitro transcription assays showed in Figure trac terminator . The partial restoration of the termination activity of Y80C by NusG on the trac terminator and the strong effect of NusG on this particular terminator led us to hypothesize that in vivo NusG might suppress the defects of primary RNA binding function on the trac-like terminators , which displayed differential responses to the PBS and SBS mutants  or N340S (SBS mutant) mutants of rho. Finally, the chromosomal rho was deleted by P1 transduction and the growth of the transductants upon re-streaking on LB plates was monitored. Figure If in vivo, and further reinforced the proposition that Rho recruitment steps of the trac-like terminators are NusG-assisted.These results strongly suggest that NusG specifically helps the PBS mutants tR1-like and trac-like terminators. We compared the sequence properties of the rut regions of these terminators  and also calculated the distances of their respective termination zones from the rut sites from in vitro as well as in vivo termination assays . As the termination zones of the trac terminator are inside the racR , we assumed that the Rho-loading site(s) would be in the preceding untranslated region (+1 to +210). From the sequence analyses , we observed that the segments of region +60 to +120 have the lowest propensity of forming secondary structures  and high C/G ratio. Sequence composition of the adjacent region, +1 to +60, also has a higher C/G ratio like other classical terminators . Hence, according to the definition of a Rho-binding site (trac terminator match the properties of bonafide rut site(s), and these sites are expected to offer optimal interactions to Rho, in a similar way as the tR1-like terminators. This assumption is also consistent with the fact that, in vitro, Rho terminates efficiently on trac in the absence of NusG . We observed that 61–120 nt and 41–87 nt of the untranslated regions of ybcK and yagNM operons, respectively, have low free energy of secondary structure formation and high C/G ratio.In a similar manner, assuming the untranslated regions of in vitro transcription assays), we noticed that the mean distances of the termination zones for trac and tyag (trac-like terminators) are unusually long compared to the tR1-like terminators . Using in vivo termination assays, we have also confirmed that for trac the termination region is indeed located inside the racR . These observations can be interpreted as much longer time requirements of Rho on these two terminators to dislodge the EC. Assuming that the translocation speed of Rho remains comparable on different RNA sequences, the following two parameters should determine the aforementioned distance as well as the time taken to dissociate the EC: the time taken to form a translocase-competent Rho at the rut site and the time taken to dislodge the EC. The latter parameter depends on the nature of the EC  in the supernatant (S) was followed over a time period . NusG did not have appreciable effect on the rate of RNA release, but improved the overall efficiency of the process modestly  were formed at the d Figure . We obsetrac terminator also exhibited a significant delay in starting the hydrolysis process, whereas the initiation of the process at the tR1 terminator was fairly instantaneous.Next we followed the nascent RNA induced ATP-hydrolysis rate of Rho using the same RBs described in Figure trac-specific lag observed in both the events is consistent with the slow isomerization process at the Rho–mRNA complex formation steps that we predicted from the unusually long distances between the rut site and the termination zone of this terminator . These results also indicated that NusG accelerates the isomerization process on the rac-tlike terminators.This In vitro, rut site-dependent Rho recruitment to the EC was studied using a few strong terminators, such as λtR1, trpt′, etc.  Growth assays and genome-wide expression analyses showed that Rho PBS mutants, unlike SBS mutants, neither induced growth defects of the cells nor induced termination defects in a large number of genes. These unaffected terminators include those present in rac and other prophages, where suppression of their induction is one of the essential functions of Rho  Th Figures  and 5. FRho binding site(s) on the mRNA are ambiguous in nature, defined by its C-richness and lack of secondary structures . And hentR1-like versus trac-like) and is likely to be dictated by the RNA sequences in and around the rut sites. Stability of Rho–RNA interaction will depend mostly on isomerization rate constant (2k). As the formation of the translocase-competent form is irreversible in nature, a higher value of 2k will drive the equilibrium toward forming this state by overcoming the reversible Rho–RNA binding step(s). We suggest that the terminator strength will be dependent on the values of this rate constant. The value of 2k will be higher in case of strong terminators. We also propose that Rho–NusG interaction increases the 2k at the weak terminators and thereby overcomes the defects in primary RNA binding function. This could be a general strategy utilized by Rho when it encounters weaker sites on the mRNA. It should be noted that the putative NusG binding region on Rho is quite distal to the secondary RNA binding channel of Rho  and closed hexamer (CH) ,26, and l of Rho , so NusGin vitro transcription reactions NusG induces early termination and increases the speed of RNA release, it was thought to function at the RNA release step of Rho-dependent termination , the unstable interactions between SBS and the sub-optimal rut sequences are likely to slow down the isomerization steps (having lower 2k). NusG suppresses this defect and these terminators exhibit enhanced dependence on NusG.Because in the mination . If NusGmination  concludetrac-like terminators will also depend on the availability of free NusG-CTD to Rho. It is likely that the ribosome-bound S10-NusG interaction (trac-like terminators, cannot be ignored. However, this Rho–RNAP interaction is not likely to dislodge the EC because Rho translocation along the mRNA a priori to the RNAP dissociation is mandatory (Figure in vivo cross-linking experiments (Optimal function of the eraction  will regy Figure ,14 and veriments .Supplementary Data are available at NAR Online.


Adenosine-to-inosine RNA editing is a highly conserved process that post-transcriptionally modifies mRNA, generating proteomic diversity, particularly within the nervous system of metazoans. Transcripts encoding proteins involved in neurotransmission predominate as targets of such modifications. Previous reports suggest that RNA editing is responsive to environmental inputs in the form of temperature alterations. However, the molecular determinants underlying temperature-dependent RNA editing responses are not well understood.Drosophila, we show that acute temperature alterations within a normal physiological range result in substantial changes in RNA editing levels. Our examination of particular sites reveals diversity in the patterns with which editing responds to temperature, and these patterns are conserved across five species of Drosophilidae representing over 10 million years of divergence. In addition, we show that expression of the editing enzyme, ADAR (adenosine deaminase acting on RNA), is dramatically decreased at elevated temperatures, partially, but not fully, explaining some target responses to temperature. Interestingly, this reduction in editing enzyme levels at elevated temperature is only partially reversed by a return to lower temperatures. Lastly, we show that engineered structural variants of the most temperature-sensitive editing site, in a sodium channel transcript, perturb thermal responsiveness in RNA editing profile for a particular RNA structure.Using the poikilotherm Drosophila transcriptome through RNA editing, presumably resulting in altered proteomic ratios of edited and unedited proteins.Our results suggest that the RNA editing process responds to temperature alterations via two distinct molecular mechanisms: through intrinsic thermo-sensitivity of the RNA structures that direct editing, and due to temperature sensitive expression or stability of the RNA editing enzyme. Environmental cues, in this case temperature, rapidly reprogram the The online version of this article (doi:10.1186/s12915-014-0111-3) contains supplementary material, which is available to authorized users. Natural DNAs are usually limited to double-stranded helical shapes; however, RNA is different — the repertoire of possible RNA secondary and tertiary structures appears limitless. RNA secondary structure is strongly correlated with function, and both the structure and corresponding thermodynamic stability of an RNA molecule contribute to functional regulation . DynamicRNA structure is fundamentally sensitive to abiotic factors, such as temperature and metal ion concentration. Bacterial RNA thermometers, riboswitches sensitive to temperature, are responsive regulatory elements that control translation of heat-shock, cold-shock, and virulence genes ,4. Yet, Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional modification known to be directed by secondary  and tertDrosophila ADAR (dADAR) appear to function primarily in the neuronal nucleus [Drosophila) and chemotaxis [Caenorhabditis elegans). Such phenotypic consequences of ADAR deficiency indicate that editing plays an integral role in organismal behavior and viability.ADARs (adenosine deaminases acting on RNA), the highly conserved proteins responsible for A-to-I editing in all metazoa, are found localized to both cytoplasm and nucleus. While there are multiple ADAR proteins in mammals, the mammalian ADAR2 and single  nucleus . This is nucleus  and seiz nucleus  (mouse)  nucleus  (Drosophemotaxis  (CaenorhDrosophila adar (dadar) transcript decreases as temperature increases. Given that the ratio of edited to unedited dADAR isoforms fine-tunes the global ‘editosome’ [Some evidence suggests that environmental factors, specifically temperature, affect editing of select transcripts -17, and itosome’  as well itosome’ .Drosophila, a poikilotherm with robust RNA editing machinery and a well-characterized editosome.However, little research has delved into the widespread effect of temperature on editing, especially in a genetically tractable organism. Because ADARs recognize duplex RNA structures, the information directing editing is carried in RNA secondary and tertiary structures, and changes in editing should partially reflect thermal alteration in RNA shapes . To obseDrosophila to rapidly adapt to varying environmental temperatures. Approximately 28% of ADAR-mediated RNA modifications occur in coding regions in the Drosophila transcriptome [Although poikilotherms experience environmental temperature undiminished, they have evolved physiological processes that allow them to thrive within the natural temperature ranges of their habitats . It is pcriptome . More spcriptome ,25. As ncriptome , this suDrosophila editing sites across a 20°C biologically relevant temperature range [Drosophila strains in which stabilizing and destabilizing structural mutations have been introduced into the endogenous locus of a natural editing substrate [The above observations and hypotheses led us to undertake a comprehensive survey of 54 re range . We creare range . Certainubstrate , revealiDrosophila melanogaster, in which the editosome has been well characterized [Drosophila editosome [To investigate the effect of temperature on editing, we limited our scope to wild type Canton-S cterized . Insteadcterized ,9,28, weditosome . The resD. melanogaster at 25°C before transferring newly-ecclosed male animals to equivalent incubators held at 10°C, 20°C and 30°C. We used only males to control for possible sex-specific differences. Drosophila eggs are most viable at 20°C [We grew engineered  at 20°C ; therefo at 20°C .synaptotagmin-1 transcript is universally insensitive to temperature  epitope sequence using homologous recombination (HR) [mini-white gene, inserted within the same intron in the dadar locus, which produces a viable dADAR hypomorph with approximately 20% of the protein found in the loxP ADAR+ control [Changes in editing levels may manifest due to effects at the RNA level  and/or directly via changes in dADAR protein concentration. We assayed dADAR protein in extracts from the heads of male flies reared at the experimental temperatures, as described above. We used engineered animals in which Jepson ion (HR) . The appion (HR) . The int control .Drosophila ADAR protein levels are stable between 10°C and 20°C, but decrease significantly at 30°C , while edited transcripts produce the glycine isoform (dADARG). We previously reported that the dADARS and dADARG isoforms target the same editing sites, but edit them to slightly different levels, with the dADARG isoform displaying less activity in vivo [dadar editing decreases to 37% at 30°C   editing , we collD. erecta, D. sechellia, D. simulans and D. yakuba in the same manner as D. melanogaster above. We did the same with mutant D. melanogaster in which the endogenous dadar gene had been HA-tagged using homologous recombination, as well as the dADAR hypomorph [S and dADARG engineered animals [Drosophila with mutations in the paralytic locus [Additionally, we raised and collected ypomorph , the dAD animals  and engiic locus .For each unique species/genotype/temperature sample, we extracted RNA from male fly heads (N = 15 to 20) using TriReagent . We chose to use only males to control for sex-related editing changes. For each sample, three separately extracted RNA biological replicates were used. We amplified cDNAs via RT-PCR using random primers, performed PCR using target-specific primers  and electrophoresed samples on agarose gels. We cleaned the PCR products using Wizard Gel and PCR Cleanup Kits  and detected editing by Sanger sequencing . For species-specific editing, we designed primers to regions of high sequence conservation.We wrote a program to calculate the ratio of mixed peak areas in chromatograms generated by Sanger sequencing. This program replicates and replaces the procedure that was previously done by hand , generatWe prepared whole-head lysates from 30 male heads per 100 μl radioimmunoprecipitation assay (RIPA) buffer , including 1 mM dithiothreitol (DTT), 1 mM phenylmethanesulfonylfluoride (PMSF) and protease inhibitor mixed tablet (Roche). We mixed approximately 25 μg protein per sample with 6 X sample buffer containing SDS and β-mercaptoethanol and loaded samples on 12.5% SDS-PAGE gels, which we then transferred to a 0.2 μm pore size polyvinylidene difluoride membrane (Bio-Rad). Primary antibodies included anti-actin , and anti-HA . The secondary antibody used was horseradish peroxidase (HRP)-conjugated goat anti-mouse . Both technical replicates and biological replicates were performed for each analysis and are presented as supplemental figures. We performed densitometry using ImageJ analysis software .post-hoc tests in which editing at 10°C and 30°C was compared to editing at 20°C. Exact P-values are noted in supplemental tables  followed by Dunnett


Paramphistomum cervi on the basis of its morphology and histology to be the common cause of paramphistomosis in infected sheep and its differentiation from other similar Paramphistomes in Gujarat.This study was undertaken to identify Adult rumen flukes were recovered from the rumen of naturally infected sheep slaughtered in various abattoirs in Gujarat. Some adult flukes were flattened and stained in Borax carmine, and some were sectioned in the median sagittal plane and histological slides of the flukes were prepared for detailed morphological and histological studies.P. cervi.Microscopic pictures of the parasite used in identification define the similarity in the morphology and histology of the anterior sucker, pharynx, esophagus, genital atrium, posterior sucker (acetabulum) and testes to the It can be concluded that the most common species found in sheep infected with Paramphistomosis is P. cervi on the basis of its histo-morphological appearance in Gujarat. Paramphistomum spp. are Platyhelminth (flatworm) parasites (Platyhelminthes: Trematoda: Digenea) responsible for Paramphistomosis i.e. gastrointestinal parasitic disease in domesticated animals, which causes heavy economic losses [c losses  to the lc losses . It has c losses -7. Distrc losses , but thec losses ,9.Paramphistomum cervi is considered to be one of the most important species of Paramphistomes [P. cervi has di-heteroxenous life cycle, which involve snail  as intermediate host and mammalian as definitive host [It is caused by specific species of the parasite depending on the regions . The comhistomes . Adult fhistomes ,10. P. cive host ,12.P. cervi are plug feeders [ feeders  and caus feeders . Mature  feeders .The species identification is still neglected as the various species of the family Paramphistomatidae are difficult to be detected through a systematic point of view, and most of the reports do not quote the main one .P. cervi has economic importance in ruminants but presently no reliable methods are available to identify and differentiate this parasite from other Paramphistomes. The present study represents the morphological and histological identification of P. cervi using Borax carmine and H and E staining methods, in order to provide a base for future molecular studies.The rumen fluke, P. cervi was designed to fulfill the need for molecular understanding of this economically important parasite.The present study on morphological and histological identification of The rumen of 350 sheep between 1 and 2 years of age was inspected for the presence of rumen flukes during slaughter at various local abattoirs of Anand, Ahmedabad and Vadodara District in Gujarat state, of which 17 sheep were found positive for Paramphistomes. Infected part of the rumen was brought to the laboratory of Department of Veterinary Parasitology, where it was washed thoroughly. After washing, adult flukes were carefully picked up with the help of forceps from the mucosa of the rumen of naturally infected sheep and then washed 3-4 times with phosphate buffer saline. After that, samples were preserved in 10% buffered formalin until being processed for morphological and histological studies. The samples were carefully labeled with proper details.Paramphistomum spp. was done based on morphology of flukes; its shape, anterior sucker, posterior sucker (acetabulum), terminal genitalium and tegumental papillae, following the standard guidelines given by Urquhart et al. [Rumen flukes were preliminarily identified under microscope using low power magnification and then slides were prepared for detailed morphological studies and identification. The collected flukes were placed on petridish and observed through stereo microscope to appreciate the morphology. Final identification of t et al. . Parasitt et al. .Out of all recovered adult flukes, 10 flukes were randomly picked from each infected sheep and were washed in water. After washing, flukes were flattened between two glass slides and fixed in Bouin’s fluid at room temperature for 24 h. After that, these specimens were washed in water and then stained for 24 h in 0.5% Borax carmine and subsequently destained in 1% hydrochloric acid until the pink color was observed. The acid was thoroughly washed out from all specimens with water. Thereafter, the specimens were washed with tap water and subsequently dehydrated through 50-100% alcohol for 1 h each, and cleared by xylene for 30 min. The cleared specimens were mounted using DPX and covered with a coverslip. The mounted slides were allowed to the air dry and observed under the light microscope.The species thus identified was further confirmed by histological identification of 10 formalin preserved flukes from each sample, which was processed by paraffin embedding method and stained by H and E stain as per Luna . 10 longIt is very difficult to identify and differentiate the species of amphistomes on the basis of its morphology due to significantly less variation in size and shape of parasite and its internal organs, which varies even in mature and immature worms of same spp. Morphology cannot be very well appreciated in the stereo microscope, but can be confirmed well by the histological examination of the parasite.P. cervi is reported in several studies conducted on ruminants [P. cervi was carried out on the basis of size and shape of fluke and position of anterior and posterior sucker (acetabulum). In the present study, most of the species were of P. cervi, which were found mainly in the rumen and were light pink in color with a sucker at the tip of the cone  and convex dorsally. The worm measures about 3-8 mm in length and 1.5-3.0 mm in width at anterior end of posterior third or at its junction with middle third. Mouth was terminal, funnel-shaped, widened posteriorly. Caeca were wide, pursued a serpentine course and reached anterior level of acetabulum with blind ends more dorsal than lateral , terminal genitalium, testes, ovary and on the basis of histological peculiarities of the muscular organs such as pharynx, genital opening and posterior sucker (acetabulum).The histological identification of P. cervi had a Liorchis type of pharynx, Gracile type of genital opening and Paramphistomum type of acetabulum in median sagittal section , Gracile  and Paramphistomum (acetabulum) type, which is similar to this study. Coskun et al. [P. cervi from P. gotoi on the basis of papillae found in the pharynx and by the position of the blind caeca. Liorchis type of pharynx in P. cervi in comparison to calicophoron type of pharynx of Paramphistomum gracile [P. cervi from other paramphistomes such as P. gracile, P. gotoi, Calicophoron calicophorum, Cotylophoron cotylophorum.The described parasites belong to the genus Paramphistomum  (Platyhe Eduardo , Sey [20 Eduardo  and Cosk [et al. . Sey [20n et al.  collecte gracile , Gracile gracile  differenP. cervi was also reported in Nigeria [Calicophoron daubneyi has been reported in Europe, Asia, Africa Oceania and France [Gastrothylax cruminefer was found in Nigeria [Carmyerius synethes and Calicophoron microbothrium were also found in Nigeria from sheep [P. cervi was recorded in Kashmir from sheep [P. cervi was also reported from buffalo [Many scientists studied the Prevalence of various species of amphistomes in sheep in various countries.  Nigeria ,25, Egyp Nigeria , Mexico  Nigeria , Thailan Nigeria ,28, Iran Nigeria  and Paki Nigeria . Calicopd France . Gastrot Nigeria , Iran [3 Nigeria  and Kash Nigeria . Carmyerom sheep . P. cervom sheep , and in om sheep . In Paki buffalo  and goat buffalo ,37.P. cervi mainly in adult stage in the rumen of sheep infected with paramphistomosis as the most common species and etiology behind the economic impact of this disease and provides a solid foundation for studying the reproductive biology of Paramphistomes and other related trematodes using molecular techniques. This disease and its impact can be controlled by interrupting the lifecycle of the parasite with prevention of grazing nearby water logged areas and the proper use of effective dewormer.Morphological and histological studies of all parasites revealed presence of This study is the major component of the work towards the M.V.Sc thesis of Vijayata, under the guidance of JJH. MK and SP helped in histopathology, drafted and thoroughly revised the manuscript. VDC and SP helped in sample collection from various abattoirs. PVP was the member of the advisory committee for the research work and provided guidance. All authors read and approved the final version of the manuscript.


Our framework constructs a unified representation that characterizes the structural specificities of RBP targets in all three dimensions, which can be further used to predict novel candidate binding sites and discover potential binding motifs. Through testing on the real CLIP-seq datasets, we have demonstrated that our deep learning framework can automatically extract effective hidden structural features from the encoded raw sequence and structural profiles, and predict accurate RBP binding sites. In addition, we have conducted the first study to show that integrating the additional RNA tertiary structural features can improve the model performance in predicting RBP binding sites, especially for the polypyrimidine tract-binding protein (PTB), which also provides a new evidence to support the view that RBPs may own specific tertiary structural binding preferences. In particular, the tests on the internal ribosome entry site (IRES) segments yield satisfiable results with experimental support from the literature and further demonstrate the necessity of incorporating RNA tertiary structural information into the prediction model. The source code of our approach can be found in https://github.com/thucombio/deepnet-rbp.RNA-binding proteins (RBPs) play important roles in the post-transcriptional control of RNAs. Identifying RBP binding sites and characterizing RBP binding preferences are key steps toward understanding the basic mechanisms of the post-transcriptional gene regulation. Though numerous computational methods have been developed for modeling RBP binding preferences, discovering a complete structural representation of the RBP targets by integrating their available structural features in all three dimensions is still a challenging task. In this paper, we develop a general and flexible deep learning framework for modeling structural binding preferences and predicting binding sites of RBPs, which takes (predicted) RNA tertiary structural information into account  RNA-binding proteins (RBPs) play important roles in various cellular processes, such as alternative splicing, RNA editing, mRNA localization and translational regulation . RBPs coCLIP-seq) protocols, has greatly advanced the genome-wide studies of RNA–protein interactions  novel 3D structural binding motifs of RBPs derived by our model, which can provide important clues for understanding the mechanisms of RBPs’ action in the post-transcriptional control of RNAs; (vi) test results on predicting PTB binding sites for the internal ribosome entry site (IRES) segments that agree with the previous experimental studies.In this study, we develop a novel deep learning framework to predict RBP target sites and model their sequence and structural specificities by systematically integrating the RNA primary sequence, (predicted) secondary and tertiary structural features. We have made the following contributions: (i) introduction of a novel method to encode RNA base sequence and secondary structure; (ii) introduction of a new method to construct the RNA tertiary structural profiles; (iii) development of a deep learning model to integrate the RNA sequence, secondary and tertiary structural profiles, and construct a unified representation to extract the hidden structural features of RBP targets; (iv) the CLIP-based experiments and use the tool RNAshapes (JAR3D (R3DMA)  . The encMEMERIS  , which aij denote the real-valued weight associated with the edge between visible variable Vi and hidden variable Hj, let bi and cj be the bias terms associated with visible variable Vi and hidden variable Hj, respectively. We use lowercase letters i and hj to represent the values of the corresponding variables Vi and Hj, respectively. We use m visible variables and n hidden variables, its joint probability density function is defined bycontrastive divergence (CD) algorithm . In our context, we treat the RNA sequences (in which each letter stands for the nucleotide type or secondary structure type at the corresponding base position) as ‘documents’, RNA subsequences as ‘words’ and a collection of distinct RNA subsequences as a ‘dictionary’. The ‘latent topics’ in the replicated softmax represent unknown factors that determine the sequence or structural features of RBP binding sites. Hereinafter, we will use RNA documents, RNA words, RNA dictionary and RNA topics to denote the corresponding concepts that are similar to those in the natural language processing field.The replicated softmax model  is a topD with an RNA dictionary K, there exists a matrix representation K × D, where ith RNA word takes the kth value of N stands for the number of hidden variables representing the latent RNA topics, j and k as we treat the RNA words in the RNA document kth RNA word of M RNA documents ij, bi and cj  are first used to construct the RNA dictionary, which includes all possible subsequences (i.e. RNA words) of fixed length k that are composed of bases in different orders. These subsequences of length k are also referred to as k-mers. In principle, an RNA dictionary with longer RNA words can provide a more accurate description of the RNA sequence. On the other hand, it is usually time- and space-consuming to process relatively long RNA words, since both time and space complexities grow exponentially as the length of RNA words increases. In our framework, the length of RNA words is set to be six, i.e. k = 6. Our additional tests (see Supplementary Section S5) have shown that slightly different RNA word length does not impact much on the model performance. We then use a sliding window of size 6 and step size 1 to scan the viewpoint region for each RNA document. After that, we construct a vector, called count vector, to record the occurrence of each RNA word in the scanning process. In particular, the ith element in the count vector records the number of times that the ith word in the RNA dictionary appears during the scanning process. This count vector is then used as the input fed into the replicated softmax model, as described in the previous section, to extract the corresponding latent RNA topics of the base sequence  of the RNA secondary structure predicted by RNAshapes. In addition, as it has been shown in the literature that the length of 150 nucleotides has the best performance in predicting the secondary structure of mRNAs  to construct the RNA secondary structural profiles using the same procedure as described in Figure JAR3D   of 529 dimensions, corresponding to 253 hairpin loop motifs and 276 internal loop motifs in R3DMA v1.13. Each element in the indicating vector defines whether the nucleotide sites can fold into the corresponding tertiary structural motif.The procedure of constructing the RNA tertiary structural profiles is illustrated in Figure ff score  to selecIn the previous sections, we have introduced the procedures of encoding RNA primary sequence, predicted secondary and tertiary structures to construct individual types of structural profiles. Next, we describe how to integrate them into a unified representation using a multimodal DBN model.r and l mean that the variables are in the right and left wings (i.e. the bottom RBMs) of the DBN, respectively, and the superscript i  means that the variable is in the ith layer of the DBN, and The multimodal DBN is a specific form of the deep learning architecture that is used to capture common latent features from the multimodal input data. For a dual-wing multimodal  DBN shown in Figure d1,d2) –  – 3000 – 3000, where d1 and d2 stand for the dimensions of the count vectors for the primary sequence and secondary structural profiles, respectively. Note that except the dimensions d1 and d2, we use the same network architecture for all RBPs in our tests. The training and implementation details of our deep learning framework are provided in Supplementary Section S4.In our multimodal DBN model, we roughly halve the dimension of each input modality for the dimension reduction purpose and use 2000 and 1000 RNA topics to encode the base sequence and secondary structure, respectively. Then these 3000 latent RNA topics are concatenated with the predicted tertiary structural profiles , which constitute the hybrid layer in the network. Furthermore, we stack another two RBMs, each of which has a 3000-dimensional hidden layer. For each RBP, the overall architecture of the multimodal DBN is . In the sampling phase, we first clamp the label vertex to be one, and conduct the mean-field based Gibbs sampling in the top-layer RBM. Then the ancestral sampling method . In particular, we follow the same principle of  and pretg method  is appliP = P − P, is calculated, which can be treated as the likelihood of the RBP binding motifs modeled by our framework. Based on ΔP, we sample from the expected RNA word distribution when clamping label = 1 in a rejection-sampling-liked manner, i.e. at each sampling step, the RNA word with the probability difference larger than a threshold δ is kept; otherwise, it is dropped. More implementation details of generating RBP binding motifs can be found in Supplementary Section S4.To generate the specific RBP binding motifs, we also need to eliminate the background motifs that appear universally in both bound and unbound scenarios. To achieve so, we also compute the expected sequence and structural profiles of the unbound RNA sites by clamping the label vertex to be zero. After that, the difference of these two probabilities, i.e. ΔHITS-CLIP-, PAR-CLIP- and iCLIP-derived RBP binding sites, in which 23 datasets were derived from doRiNA (HITS-CLIP was derived from (CLIP-based experiments) and negative samples (i.e. the unbound RNA sites created by shuffling the positions of bound sites). The descriptive statistics of these 24 RBP datasets is provided in Supplementary Table S1.We validated the performance of our deep learning framework on 24 datasets of the m doRiNA , and theved from . The lisved from , which iarea under receiver operator characteristic (AUROC) to evaluate the performance of our method on predicting RBP binding sites. We compared the prediction performance of our deep learning framework with that of the state-of-the-art method GraphProt (relative error reduction that has been commonly used for comparing the prediction performance of two different approaches (c′ − c)/(1 − c), where c denotes the baseline performance and c′ is the performance of the new method. Table GraphProt and our framework, which indicates that our model yielded comparable or superior performance to that of GraphProt. In particular, among 24 RBPs, our method outperformed GraphProt for 19 RBPs, with an average relative error reduction of 22%, the median relative error reduction of 21% and the largest relative error reduction of 73% (achieved for the PTB dataset). Based on the prediction performance provided in n motif in both 3′ UTR and long intronic sequences, regulating pre-mRNA splicing or RNA transport and translation  was chosen only if it is different from that accompanying with the training samples we used. The enriched motifs of AGO1–4 identified in previous studies agreed with our results in Table e middle . The binelements ,42. EWSRelements ,44, conf studies . IGF2BP1patterns , which dnslation , and ourribed in  to bind ribed in , which aAU-rich stem loop in non-neuronal cells while this result may not be so reliable in the CHIP studies in neural cells  during the motif generating process. The test result showed that by inactivating the structural profiles for a fraction (20%) of training samples, our framework lost certain ability to fully discover the structural binding preferences of PTB, but still grasped some secondary structural specificities. More details of this test can be found in Supplementary Section S7.P of the RNA tertiary structural profiles for each RBP, we selected the top five potential tertiary structural binding motifs with the largest difference values. Detailed case studies on these three RBPs are provided in the next section.We also identified the highest scored tertiary structural binding motifs for three RBPs, i.e. ALKBH5, MOV10 and PTB, which were shown to own tertiary structural specificities for their binding targets based on the tests performed previously. Based on the probability difference ΔC and U bases in the top five motifs was high (≥60%), which was in line with the fact that PTB has CU-rich sequence binding motifs , which is referred to the methylation of the adenosine at the nitrogen-6 position  incorporated in the prediction model.ALKBH5 has been recognized as an important demethylase of Nposition ,54–55. Tunctions . The strded RNAs ,56, and 6 proton . In addiractions ,59. ProbMOV10 has been known as a putative RNA helicase, which involves various RNA-mediated functions through the RNA-induced silencing complex (RISC). The recent study in  has indiCU-rich sites of a U1 snRNA form a special spatial arrangement to prevent U1 from interacting with the downstream spliceosomal elements , PTB prefers binding to polypyrimidine sites, and plays important roles in many functions of mRNA metabolism, such as splicing regulation, internal ribosomal entry site (IRES) mediated translation and RNA localization . The facelements . In thisCUCUCU oligonuleotide which does not have an explicit tertiary structure. Thus, we used HADDOCK , giving us confidence to continue the docking experiments. Meanwhile, these docked complex structures for the domains RRM1, RRM2 and RRM3 were used as positive controls in the latter tests. On the contrary, the complex structure of the RRM4 domain with the docked RNA was distinct from the original NMR structure, whose binding might be influenced by its interaction with RRM3 , which indicated that our docked complex structures were probably reasonable. As presented in Supplementary Table S4, four of the top five motifs were successfully docked to at least one domain through the conserved binding residues without serious steric clash between atoms.The results showed that four of the top five motifs presented descent docking efficiency that were comparable to the positive controls (see Supplementary Table S4), suggesting that the score of the binding motif calculated by our framework may indicate the binding affinity to some extent. In addition, the enrichment pattern of pyrimidines was found when measuring the ratios of The docking results may provide useful hints for understanding the intermolecular recognition between PTB and RNA. An example of the docked structure between the derived RNA 3D motif and PTB is given in Figure In practice, it is generally expensive and time-consuming to experimentally study the atomic interactions between the derived RNA 3D structural motifs and PTB. Our docking procedure provided an alternative and cheap way to investigate the atomic details of their potential interactions. The motivation of our docking studies was mainly to offer a more detailed view about the RNA–PTB interactions predicted by our framework, rather than providing a direct validation of the interactions between the derived RNA 3D motifs and PTB. Thus, our docking results cannot rule out the possibility that PTB can also bind to other RNA 3D motifs that were not selected in the top list. Without experimental validation, our docking strategy can only provide a limited solution for investigating the atomic interactions between PTB and the derived RNA 3D structural motifs.GraphProt for the known IRES regions available from the database IRESite  segment is a nucleotide sequence that attracts the eukaryotic ribosomal translation initiation complex to facilitate the translation initiation without the usual 5′-terminal 7mG cap structure , and PTB IRESite , and evaHomo sapiens from . Figure GraphProt, DBN-3D  and DBN+3D , yielded low enrichment of the predicted binding sites on the negative control dataset; (ii) GraphProt yielded similar prediction results on both IRES segments and negative control dataset; (iii) our deep learning method incorporating RNA 3D structural information showed a significantly higher enrichment of the predicted binding sites in the IRES regions than the negative control dataset ; (iv) our DBN model integrating RNA 3D structural information achieved a much higher fraction of hits on the IRES segments than GraphProt and DBN-3D . Based on these observations, we concluded that: (i) the increased number of hits in the IRES regions produced by DBN+3D was not attributed to the false-positive issue, in other words, our deep learning method did not suffer from the false positive problem; (ii) compared to GraphProt and DBN-3D, which did not consider RNA 3D structural information, DBN+3D yielded a much higher enrichment of the predicted binding sites on the IRES segments, which was probably due to the PTB recognition preference in RNA 3D structure in these IRES regions. All these results further demonstrate the necessity of integrating RNA 3D structural information into the model of predicting RBP binding sites.We downloaded a set of 40 IRES segments in ens from  that havAPAF1 and UNR, in which the exact PTB binding sites have been verified experimentally (APAF1 as a case study. The prediction results of UNR (upstream of N-ras) can be found in Supplementary Section S9.We also examined individual prediction results of the 13 IRES segments, in which the PTB binding has been validated experimentally in the literature ), and provided the predicted PTB binding sites for the other 27 IRES regions ). More details about these individual results can be found in Supplementary Section S9. To further investigate the prediction performance of our method in more detail, we also investigated the details of the predicted binding sites in IRES segments mentally ,75–76. HAPAF1 is a well-studied IRES segment that is bound to PTB, and multiple functional assays on its mutations have been performed to identify the binding patterns  interactions, there may be some bias in our prediction. To demonstrate this point, we relaxed the ‘ground-truth’ bound sites to the entire hairpin loop covering the P2 sites and found that the consideration of such bias can yield a much better prediction result, as shown in Figure patterns . Importang sites . The expAPAF1 and UNR  provide excellent examples to explain PTB's role as a general RNA chaperone to mediate the translation of mRNAs. According to the previous experimental studies, the binding of PTB and other cofactors to these RNAs can generate a conformational change, usually the disturbance of the stem regions to release the structured regions into available loops, and make the RNA fold into a specific tertiary structure required for translation (The test results of nslation ,72,75,77CLIP-seq datasets showed that our framework can achieve the comparable or superior performance to the state-of-the-art method for predicting RBP binding sites. In addition, the structural motifs generated by our framework agreed well with the previous experimental studies in the literature, and may provide useful hints for further elucidating the molecular recognition mechanisms of RBPs.We have developed a deep learning framework to model the binding preferences of RNA-binding proteins by integrating the primary sequence, predicted secondary and tertiary structural profiles of the target sites. Our framework considered RNA tertiary structure for RBP binding site prediction, and provided strong evidence to support the view that the RNA tertiary structural features can contribute to RBP target recognition. Tests on real Supplementary Data are available at NAR online.


Synaptojanin and endophilin represent a classic pair of endocytic proteins that exhibit coordinated action during rapid synaptic vesicle endocytosis. Current models suggest that synaptojanin activity is tightly associated with endophilin through high-affinity binding between the synaptojanin proline-rich domain (PRD) and the endophilin SH3 domain. Surprisingly, we find that truncated synaptojanin lacking the PRD domain sustains normal synaptic transmission, indicating that synaptojanin's core function in vivo resides in the remaining two domains that contain phosphoinositide-phosphatase activities: an N-terminal Sac1 phosphatase domain and a 5-phosphatase domain. We further show that the Sac1 domain plays an unexpected role in targeting synaptojanin to synapses. The requirement for Sac1 is bypassed by tethering the synaptojanin 5-phophatase to the endophilin membrane-bending Bin–Amphiphysin–Rvs (BAR) domain. Together, our results uncover an unexpected role for the Sac1 domain in vivo in supporting coincident action between synaptojanin and endophilin at synapses.DOI:http://dx.doi.org/10.7554/eLife.05660.001 Nerve cells called neurons can rapidly carry information around the body. Each neuron forms connections called synapses with several other cells to build networks for information exchange. At most synapses, electrical activity in one neuron results in the release of chemicals called neurotransmitters from storage compartments called synaptic vesicles. The neurotransmitters leave the cell and cross the gap between the two neurons to activate the next cell.After the neurotransmitters have been released, the synaptic vesicles need to be regenerated via a recycling process called endocytosis. This recycling process is very important for synapses to work properly, but it is not clear exactly how it occurs. Two of the proteins involved are called synaptojanin and endophilin. Synaptojanin is made up of three structural units (or ‘domains’), including the proline-rich domain and the Sac1 domain. It has been proposed that interactions between endophilin and the proline-rich domain of synaptojanin are essential for vesicle recycling.Here, Dong et al. studied nematode worms that carry mutant forms of synaptojanin. The experiments show that the Sac1 domain, but not the proline-rich domain, is required for the synapses to work properly. However, the Sac1 domain is not required if synaptojanin is artificially linked to endophilin.Dong et al.'s findings suggest that synaptojanin uses its Sac1 domains to work with endophilin. A future challenge will be to understand the details of how this cooperative action occurs.DOI:http://dx.doi.org/10.7554/eLife.05660.002 Synaptic vesicle (SV) endocytosis occurs through rapid and coordinated actions of endocytic proteins . A classGenetic studies have shown that the function of synaptojanin is tightly linked to the endocytic protein endophilin . Mutant Synaptojanin harbors two phosphatase domains in addition to the PRD domain . The N-tHere, we show that the functional core of synaptojanin resides in its tandem phosphatase domains rather than the PRD domain. Our results reveal an unexpected mechanism whereby the Sac1 domain displays a non-catalytic function to support coordinated action between synaptojanin and endophilin at synapses.Caenorhabditis elegans, the unc-26 gene encodes a highly conserved synaptojanin homologue with identical domain structure to the mammalian synaptojanin (unc-26 synaptojanin have significantly decreased locomotion rates and largely diminished excitatory postsynaptic currents (EPSCs) at neuromuscular junctions  that lacks PRD in unc-26 null mutant worms. In transgenic animals, a single copy of the transgene (unc-26∆PRD::gfp) driven by a pan-neuronal promoter (Prab-3) was inserted into chromosome X to avoid confounding issues of overexpression  levels  levels , indicatunc-26 mutant worms was significantly lower: reduced by ∼40% compared to controls , consistent with defects in membrane recycling. About 32% of internalized dye  by the unc-26 mutants was released upon KCl challenge  and the KCl-dependent dye release   , indicatPunc-17::ChR2(H134R)::YFP were activated by 2-Hz photostimulation, and evoked EPSCs were recorded at neuromuscular junctions  to measure changes in surface synaptobrevin . In SVs, defects , demonstWe next investigated whether synaptojanin's unique configuration of tandem phosphoinositide phosphatase domains  mediatesTo ask whether the entire Sac1 domain plays any role in synaptojanin function, we generated a truncated UNC-26 that lacks the Sac1 domain . Surprisingly, we found that removal of the Sac1 domain severely disrupted the rescuing activity of UNC-26 , suggestNostoc punctiforme (NpuDnaE)  , as this elegans . Co-expr mutants , suggestovements . These dovements .To gain insights into the non-enzymatic function of Sac1, we investigated the possibility that Sac1 guides 5-phosphatase for synaptic localization. We quantified synaptic abundance of GFP-tagged UNC-26 variants. Full-length UNC-26 is enriched at synapses , but not the version with wt phosphatase domains, exhibits significant levels of dominant-negative inhibition, presumably by competing with wt UNC-26. When D716A UNC-26∆PRD was expressed in wt worms, the evoked EPSC amplitude was reduced by ∼50% . The domBecause our data suggest that Sac1 acts as a targeting domain rather than an enzyme, we speculated that it might be possible to bypass the Sac1 requirement by directly tethering the UNC-26∆Sac1 mutant with non-enzymatic proteins. We utilized three categories of proteins as candidate targeting tethers: (1) SV proteins SNB-1 (Synaptobrevin) and RAB-3; (2) lipid-binding domains that recognize specific phosphoinositides ; and (3)unc-26 mutants (unc-26 mutants (data not shown), even though the SH3 domain enhanced the synaptic enrichment of UNC-26∆Sac1  transgene significantly restored locomotion, endogenous EPSCs, double mutants P2 P2 . HoweverPhosphoinositide phosphatases often harbor multiple lipid-binding domains to detect coincident signals for restricted localization on membranes . SynaptoHow the synaptojanin Sac1 domain recognizes endophilin-membrane complexes is currently unknown. Biochemical studies have shown that the Sac1 domain does not directly bind endophilin in solution  de Heuv. Here, wC. elegans strains were maintained at 20°C on agar nematode growth media (NGM) plates seeded with OP50 bacteria. The N2 strain  was used as wt. Mutant unc-26(s1710) and unc-57(e406) strains were obtained from the Caenorhabditis Genetics Center and were subsequently outcrossed 10× times to the N2 strain. The following strains were used in this study:Strain maintenance and genetic manipulations were performed as described . All C. unc-57(e406); unc-26(s1710)BJH188 unc-26(s1710); pekSi8 BJH180 unc-26(s1710); pekSi7 BJH88 unc-26(s1710); pekEx15 [Psnb-1::mSyj1∆PRD]BJH40 unc-57(e406); pekSi19 ; unc-26(s1710); pekSi7 BJH298 unc-26(s1710); pekEx27 [Prab-3::gfp::unc-26(D716A)]BJH52 unc-26(s1710); pekEx30 [Prab-3::gfp::unc-26]BJH55 unc-26(s1710); pekEx66 [Prab-3::gfp::unc-26∆PRD]BJH312 unc-26(s1710); pekEx18 BJH43 unc-26(s1710); pekEx21 [Prab-3::unc-26∆Sac1]BJH46 unc-26(s1710); pekEx24 [Prab-3::unc-26Sac1::IntN]BJH49 unc-26(s1710); pekEx23 [Prab-3::IntC::unc-26∆Sac1]BJH48 unc-26(s1710); pekEx39 BJH145 NuIs269 [Punc-129::gfp::unc-26]KP5105 pekEx80 [Punc-129::gfp::unc-26∆Sac1]BJH360 pekEx28 [Punc-129::gfp::unc-26∆Sac1∆PRD]BJH53 pekEx92 [Prab-3::gfp::unc-26∆PRD(D716A)]BJH338 pekEx98 [Prab-3::gfp::unc-26∆Sac1∆PRD(D716A)]BJH344 unc-26(s1710); pekEx64 [Prab-3::plc∂ PH:unc-26∆Sac1]BJH310 unc-26(s1710); pekEx67 [Prab-3::bem1 PX::unc-26∆Sac1]BJH313 unc-26(s1710); pekEx68 [Prab-3::btk PH::unc-26∆Sac1]BJH314 unc-26(s1710); pekEx71 [Prab-3::aps-2::unc-26∆Sac1]BJH317 unc-26(s1710); pekEx73 [Prab-3::itsn-1::unc-26∆Sac1]BJH319 unc-26(s1710); pekEx74 [Prab-3::dyn-1::unc-26∆Sac1]BJH320 unc-26(s1710); pekEx75 [Prab-3::apm-2::unc-26∆Sac1]BJH321 unc-26(s1710); pekEx76 [Prab-3::apa-2::unc-26∆Sac1]BJH322 unc-26(s1710); pekEx84 [Prab-3::unc-57::unc-26∆Sac1]BJH330 unc-26(s1710); pekEx86 [Prab-3::unc-57::unc-26∆Sac1(D716A)]BJH332 unc-26(s1710); pekEx87 [Prab-3::apb-1::unc-26∆Sac1]BJH333 unc-26(s1710); pekEx89 [Prab-3::unc-57BAR::unc-26∆Sac1]BJH335 unc-26(s1710); pekEx90 [Prab-3::rEndoBAR::unc-26∆Sac1]BJH336 unc-26(s1710); pekEx91 [Prab-3::mAmphBAR::unc-26∆Sac1]BJH337 unc-26(s1710); pekEx94 [Prab-3::rEndoBAR ::unc-26∆Sac1]BJH340 unc-26(s1710); pekEx95 [Prab-3::rEndoBAR∆N::unc-26∆Sac1]BJH341 unc-26(s1710); pekEx97 [Prab-3::unc-26∆Sac1::snb-1]BJH343 unc-26(s1710); pekEx99 [Prab-3::mNadrin2BAR::unc-26∆Sac1]BJH345 unc-26(s1710); pekEx101 [Prab-3::unc-57::unc-26∆Sac1∆PRD]BJH347 unc-26(s1710); pekEx102 [Prab-3::unc-26∆Sac1::rab-3]BJH348 zxIs6 , acr-16(ok789)BJH396 zxIs6, acr-16(ok789); unc-26(s1710)BJH397 zxIs6, acr-16(ok789); unc-26(s1710); pekSi7 BJH398 pekEx122 [Prab-3::gfp::unc-26∆PRD]BJH399 pekEx123 [Punc-129::gfp::unc-26Sac1]BJH400 pekEx124 [Punc-129::gfp::unc-57SH3::unc-26∆Sac1]BJH401 pekEx126 [Punc-129::gfp]BJH403 unc-57(e406); unc-26(s1710); pekSi24 BJH405 pekEx127 [Punc-129::gfp::unc-26Sac1]BJH406 nuIs122 [Pacr-2::synaptopHluorin]BJH402 unc-57(e406); unc-26(s1710); nuIs122; pekEx125 BJH407 unc-57(e406); unc-26(s1710); nuIs122BJH408 Psnb-1 and Prab-3 promoters were used for neuronal rescue experiments, and Punc-129 for imaging analyses. cDNAs of unc-26, unc-57, snb-1, rab-3, dyn-1, apa-2, apb-1, apm-2, aps-2, and itsn-1 were amplified from total mRNA extracted from wt worms. cDNAs encoding rat endophilin A1, mouse Nadrin2, and mouse amphiphysin were amplified from a cDNA library from Clontech .−1) together with co-injection markers, including Pmyo-2::his11::gfp , Pvha-6::gfp , Pmyo2::NLS-MaxFP Green , and Pttx-3::DsRed . For dominate-negative inhibition experiments, plasmids  were injected at ∼60 ng µl−1. For imaging experiments, variants of Punc-129::gfp::unc-26 were injected at 15 ng µl−1, unless specified. Blank vector pBluescript was used as an injection filler to bring final DNA concentration to 100 ng µl−1. Integrated transgenes were obtained by UV irradiation of strains carrying extrachromosomal arrays. Transgenic worms were outcrossed at least 10 times.Transgenic strains for rescue experiments were generated by microinjection of various plasmids (2 ng µlunc-57 transgenes) and ttTi14024 . The following constructs were used to generate single-copy transgenes: BJP-B178 [Prab-3::unc-26::gfp for ttTi14024 (X)], BJP-B179 [Prab-3::unc-26∆PRD::gfp for ttTi14024 (X)], BJP-B384 [Psnb-1::unc-57∆SH3::mcherry for ttTi5605 (II)], and BJP-M208 [Prab-3::rEndoBAR::unc-26 5Pase for ttTi5605 (II)]. Transgenic worms carrying single copy insertion of transgenes were outcrossed at least 4 times.Mos1-mediated single-copy transgene insertion methods were used to generate transgenic animals carrying single-copy transgenes . The Moshttp://www.phage.dk/plugins/wrmtrck.html) in ImageJ . Average speed was determined for each animal. Statistical analysis was performed using Igor Pro 6 . Average values and standard error of the mean (SEM) were reported. p values were generated using one-way ANOVA followed by Dunnett's test.Worm movement on 10 cm agar plates without bacterial lawn was recorded for 30 s. Young adults (reared at 20°C) were transferred to room temperature 1 hr prior to behavior tests. Videos of individual animals were captured on a CCD camera  mounted on a stereomicroscope using 0.8× magnification. The center of mass was determined for each animal on each video frame using open-source object tracking scripts developed by Jesper S Pedersen . Animals were dissected in extracellular solution via a dorsolateral incision. Gonads and intestines were removed to reveal the underlying ventral nerve cord and body-wall-muscle quadrants as previously described . The worWhole-cell patch clamp recordings were carried out at 20°C. A body wall muscle cell was voltage clamped at −60 mV to record postsynaptic currents. Evoked EPSC responses were induced by applying a 0.4 ms, 30 μA pulse, generated by a stimulus isolator , through a borosilicate pipette (∼2 MΩ) placed in close apposition to the ventral nerve cord. Series resistance was compensated to 70% for the evoked EPSC recording. The currents were amplified using EPC-10 . The signals were sampled at 10 kHz using Patchmaster (HEKA) following low-pass filtering at 2 kHz. Patch pipettes (2–5 MΩ) were pulled using borosilicate glass and were fire polished. Extracellular solution contains (in mM) 150 NaCl, 5 KCl, 1 CaCl2, 5 MgCl2, 10 glucose, and 10 HEPES and was titrated to pH 7.3 with NaOH, 330 mOsm with sucrose. Internal solution contains 135 CH3O3SCs, 5 CsCl, 5 MgCl2, 5 EGTA, 0.25 CaCl2, 10 HEPES, and 5 Na2ATP and was adjusted to pH 7.2 using CsOH. All chemicals were purchased from Sigma .http://sulzerlab.org/Quanta_Analysis_8_20.ipf; Mosharov and Sulzer, 2005) in Igor Pro 6 (Wavemetrics). Average values and SEM were reported. Statistical analysis was performed using Igor Pro 6. p values were generated using one-way ANOVA followed by Dunnett's test. A p-value < 0.05 was considered to be significant.Electrophysiological data were analyzed with open-source scripts developed by Eugene Mosharov (NGM plates (60 mm) were seeded with 250 µl of OP50 bacteria and 4 µl of 100 mM all-trans retinal . Seeded retinal plates were kept in the dark at 4°C and were used within 7 days. Channelrhodopsin-2 transgenic worms (L4 hermaphrodites) were transferred from regular plates to retinal plates in the dark at room temperature and then grown for an additional 16 hr before electrophysiological experiments.2, quantified by an XR2100 power meter . Light pulses  were used to evoke post-synaptic currents at neuromuscular junctions.A TILL Oligochrome light source  was controlled by TTL signals from a HEKA EPC-10/2 amplifier. Blue light (460–500 nm) through a GFP filter set  was used to excite channelrhodopsin-2. The light intensity output from the 60× objective (1.0 NA) was 12 mW/mmApproximately, 5 adult hermaphrodites were loaded at room temperature into a 100 μm specimen chamber containing space-filling bacteria and M9 buffer. These worms were frozen instantaneously at ∼ −180°C in either a Leica EM PACT2  or a BAL-TEC HPM010  system. The frozen worms were fixed in a Leica EM AFS2 machine using 1% osmium in 0.1% UA in acetone fixative and then embedded in Eponate 12 from Ted Pella, Inc . Serial sections were cut at a thickness of 40 nm, collected on pioloform covered slotted grids  from Ted Pella, Inc., and counterstained in 6% aqueous uranyl acetate for 1.5 hr, followed by Reynolds lead citrate for 7 min. Images were obtained on a JEOL JEM 1400 transmission electron microscope  operating at 120 KV. Micrographs were collected using the Gatan Ultrascan 1000XP, 2k × 2k high-resolution camera . Synapse profiles were used to count the number of synaptic vesicles (∼30 nm in diameter). Each profile represents a single section that passes through the dense projection. Vesicle counting was performed blindly. p values were generated using one-way ANOVA followed by Dunnett's test.−1; Sigma–Aldrich) and were mounted on 2% agarose pads for imaging. Fluorescence images were collected on an inverted Olympus FV-1000 confocal microscope with an Olympus PlanApo 60× Oil 1.4 NA objective at 5× zoom. GFP was excited using a 488 nm argon laser (0.5% laser power). Images of fluorescent slides  were captured during each imaging session to provide a fluorescence standard for comparing fluorescence intensities between animals. Line scans were analyzed with custom-written scripts developed by Jeremy Dittman  is defined as (Fpeak − Faxon)/Faxon. All the values reported in the figures are mean ± SEM.Animals were immobilized with 2,3-Butanedione monoxamine (30 mg ml2+-free low-K+ buffer (0.5 mM EGTA and 1 mM ADVASEP-7 [Sigma]) for 5 min. Dye unloading from releasable vesicles was carried out by incubation with high-potassium medium without FM4-64 dye for 5 min. Imaging was done on a Zeiss LSM 780 confocal microscope  with a 40×/0.8 objective. FM4-64 was excited with a 561-nm laser (1% laser power), and fluorescence emission was collected between 643 nm and 751 nm. A set of Z-stack images  was obtained for each worm. Images were imported into ImageJ for data analysis (NIH). For all images in each Z-stack, an area of interest (AOI) was defined by drawing a circle of 45 µm in diameter around the neuron ganglion. Total fluorescence of each image was obtained by integrating fluorescence pixel intensity within the AOI. Background fluorescence was subtracted from all images. The fluorescence signal  with the highest value from each stack was used for comparison. Statistical analyses were performed using one-way ANOVA followed by Dunnett's test. All the values reported in the figures are mean ± SEM.Young adult worms were immobilized in standard medium  on Sylgard-coated coverslips with cyanoacrylate glue . The head neuron ganglion was exposed by a small incision using a sharp needle. After the ganglion was exposed, a second incision was made at the middle section of worm body to release internal pressure. Dissected worms were gently rinsed with standard medium. To stimulate FM4-64  loading, dissected worms were incubated with high-potassium buffer  in the presence of 10 µM FM4-64 dye for 1 min. Stimulated preparations were then incubated with standard medium containing 10 µM FM4-64 dye for 2 min to allow for vesicle recycling to proceed. To remove surface-bound dye, dissected worms were gently washed in a CaEscherichia coli strain BL21(DE3) as fusion proteins. DNA fragments encoding UNC-57 full-length, UNC-57 BAR (residues 1–283), and UNC-57 SH3 (residues 284–379) were inserted into pGEX4T-1 using BamHI and NotI sites. Recombinant GST::UNC-57 variants were immobilized onto glutathione beads . UNC-26 Sac1 (residues 1–493), 5-phosphatase (residues 494–986), and PRD (residues 987–1113) were fused to the C-terminus of the maltose-binding protein (MBP) using overlapping PCR. DNA fragments encoding MBP::UNC-26 variants were subcloned into PET28a using NdeI and XhoI sites to produce C-terminal His6-tagged fusion proteins. Recombinant MBP-UNC-26Sac1-his6 proteins were purified using Ni-NTA Agarose  and were eluted in HEPES buffer  plus 250 mM imidazole. For antibody development, DNA encoding UNC-57 BAR::mCherry was inserted into PET28 using BamHI and NotI sites, and DNA encoding SUMO::UNC-26 (residues 494–986) was inserted into PET28 using NcoI and NotI sites. Purification of GST- and His6-tagged proteins was performed essentially as previously described  were used as antigens. Mice  were immunized, and immune splenocytes were isolated from mice showing positive antisera titers . Isolated splenocytes were electrofused  to the NS-1/FOX-NY myeloma cell line. Antibody secreting hybridomas were identified using standard ELISA screens, and monoclonal hybridomas were isolated by limiting dilution subcloning. Monoclonal antibodies to UNC-57 and UNC-26 were further screened and characterized by Western blot analysis of recombinant proteins and C. elegans detergent extracts.Monoclonal antibodies to Worms were reared on 10–15 enriched peptone plates (15 cm) at 20°C. Adult animals were collected using the sucrose flotation method, frozen in liquid nitrogen, and homogenized for 20 s using a mini bead-beater16  in M9 buffer with 2 mM Ethylenediaminetetraacetic acid (EDTA), 1 mM DTT, and 1× protease inhibitor cocktail (Sigma). Worm samples were then cooled for 1 min on ice. The beat/cool cycle was repeated 6 times to completely lyse the worms. Triton-X100  was next added to extract total protein. After 15 min of incubation, crude protein extracts were centrifuged at 14,000 rpm in an Eppendorf centrifuge  for 10 min at 4°C to remove the beads, worm debris, and unbroken worms. The protein concentration of worm extracts was determined by the Pierce bicinchoninic acid (BCA) assay . Primary antibodies against UNC-57BAR and UNC-26 5-phophatase were used for Western blot. Immunoreactive bands were visualized using enhanced chemiluminescence and were quantified using a Bio-Rad ChemiDoc MP imaging system . review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a Reviewing editor, and three reviewers.Thank you for sending your work entitled “Synaptojanin cooperates in vivo with endophilin through an unexpected mechanism” for consideration at The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Graeme Davis (Reviewing editor) and Erik Jorgensen (peer reviewer). A further reviewer remains anonymous.The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.Summary:C. elegans. While these two proteins are well-established factors in the synaptic vesicle cycle (knockouts of either one severely impair synaptic transmission), how or whether they cooperate in this process is less well understood. There is published biochemical evidence for direct binding between endophilin and synaptojanin (SYNJ) via their SH3 and proline rich domains respectively, and this has led to a model where a direct interaction between these domains underlies their function in synaptic endocytosis and maintenance of the vesicle pool. This manuscript centers on the surprising result that the SYNJ PRD (and therefore its direct interaction with endophilin) is dispensible for its function in supporting normal synaptic transmission. Instead, SYNJ's Sac1 domain appears to play an important role (independent of its phosphatase activity) and is potentially required for enriching it at synapses. Overall, the experiments are solid, using a UNC-26 genetic deletion/transgene rescue approach with various mutants and truncations. The novel and non-catalytic role of the Sac1 domain and the surprising result that SYNJ's PRD is not essential for its role in synaptic transmission make this work conceptually interesting and challenges the existing model. The potential applicability of this mechanism to other forms of membrane trafficking make the work of broader interest beyond the synaptic vesicle trafficking field. While the role of the Sac1 domain remains mysterious, this domain is potentially interesting in light of recent evidence for a SYNJ Sac1 mutation  in a family with early onset Parkinsonism.The manuscript by Dong et al., investigates the involvement of synaptojanin/UNC-26 and endophilin/UNC-57 in synaptic vesicle endocytosis at the NMJ in Required modifications:1) For several experiments, the authors show only evoked EPSCs to document the function of chimeric proteins. While not a strict requirement for publication, it would be nice to also show mEPSCs and locomotion data whenever possible. unc-26 mutant, thus providing evidence that smaller EPSCs is not a consequence of fewer synapses.2) There are no direct measures of endocytosis presented in the manuscript. Two modifications would substantially strengthen the manuscript and make the train of logic more accessible to the general reader. First, it is important to show a direct measurement of endocytosis to establish that the defects in exocytosis can be ascribed to a defect in endocytosis. Second, the authors should be clear, in the text, that the defect in exocytosis is believed to be due to the cumulative effects of impaired endocytosis over time, resulting in diminished vesicle number and a diminished releasable vesicle pool. If there is another interpretation that can explain this association, this if fine, but should be explicitly stated. In dealing with this issue the text should also include reference to Ch'ng et al., PLoSG (2008) demonstrating that RIM puncta density is not impaired in the 3) Methods. Experimental procedures are not well described and will be hard for others to replicate the work. Please provide more details in each section.4) N-values are not apparent throughout. How many synapses were analyzed? How many animals were analyzed? Were the experiments blinded?Additional suggestions to improve the impact of this study:unc-57 unc-26 double mutant by a single-copy of the BAR-5-phosphatase chimera would be elegant if the authors can provide it.1) Rescue of 2) Is the isolated Sac1 domain  synaptically localized? This would be a nice way of documenting its function as a localizing domain. In other words, is the Sac1 sufficient to localize GFP to synapses in the wild-type?3) In 4) In the subsection headed “Endophilin BAR and synaptojanin Sac1 are functionally linked”, the authors state that fusing Endophilin's SH3 domain to UNC-26∆Sac did not rescue EPSC defects. Did the SH3 domain restore synaptic localization of ∆Sac? This would suggest that proper localization of the 5-Phosphatase domain is not sufficient to explain UNC-26 function in endocytosis.5) In the same section of the manuscript, the authors speculate as to why the SH3 and PRD domains are conserved despite the fact that they are not required for SV endocytosis. Another possibility the authors may want to suggest is that the PRD/SH3 interaction provides a backup mechanism for co-localizing Endophilin and SJN under conditions where Sac1 localization is disrupted. For example, if Sac1 binds PI3,4P lipids, its localization may be disrupted when the levels of these lipids are low. In this case, the SH3/PRD interaction may be required for endocytosis.6) In 7) In 8) In  1) For several experiments, the authors show only evoked EPSCs to document the function of chimeric proteins. While not a strict requirement for publication, it would be nice to also show mEPSCs and locomotion data whenever possible. We have added a table  and Figu2) There are no direct measures of endocytosis presented in the manuscript. Two modifications would substantially strengthen the manuscript and make the train of logic more accessible to the general reader. First, it is important to show a direct measurement of endocytosis to establish that the defects in exocytosis can be ascribed to a defect in endocytosis. Second, the authors should be clear, in the text, that the defect in exocytosis is believed to be due to the cumulative effects of impaired endocytosis over time, resulting in diminished vesicle number and a diminished releasable vesicle pool. If there is another interpretation that can explain this association, this if fine, but should be explicitly stated. In dealing with this issue the text should also include reference to Ch'ng et al., PLoSG (2008) demonstrating that RIM puncta density is not impaired in the unc-26 mutant, thus providing evidence that smaller EPSCs is not a consequence of fewer synapses.Several changes have been made to address these concerns.unc-26 mutants  to measure changes in surface synaptobrevin […] demonstrating that UNC-26∆PRD and UNC-57∆SH3 are functional to support SV endocytosis.”Third, to clarify the link between impaired endocytosis and diminished releasable vesicle pool, we have added the following text to the manuscript:“These defects are consistent with previous reports showing reduced SV pools and a corresponding decrease in synaptic transmission due to the cumulative effects of impaired endocytosis over time.”Fourth, to further strengthen the link between endocytosis and exocytosis, we have assayed for changes of synaptic transmission upon repetitive light stimuli, using worms carrying Channelrhodopsin-2 transgenes. We show that control synapses and synapses rescued by truncated UNC-26∆PRD sustain synaptic transmission to similar levels upon repetitive stimuli. We have added the following text to the paper:“Cholinergic neurons of transgenic animals carrying Punc-17::ChR2(H134R)::YFP were activated by 2Hz photostimulation […] supporting the notion that truncated UNC-26 functions sufficiently to supply SVs during sustained activity.”Finally, we have added the citation of Ch'ng et al., PLoSG (2008) to support the notion that smaller EPSCs is not a consequence of fewer synapses. It now reads:unc-26 mutants  remains unchanged in  mutants , reduced3) Methods. Experimental procedures are not well described and will be hard for others to replicate the work. Please provide more details in each section.We have expanded the Methods to provide more details.4) N-values are not apparent throughout. How many synapses were analyzed? How many animals were analyzed? Were the experiments blinded?All sample size values are now shown on the figures, as well as in the table and figure legends. Synaptic vesicle counting  and the Additional suggestions to improve the impact of this study:1) Rescue of unc-57 unc-26 double mutant by a single-copy of the BAR-5-phosphatase chimera would be elegant if the authors can provide it.Psnb-1::rEndoBAR::UNC-26 5Pase transgene. We found that rEndoBAR::UNC-26 5Pase chimera significantly rescues locomotion, endogenous EPSC frequency, and evoked EPSC amplitude in the unc-57; unc-26 double mutants. Interestingly, the single-copy transgene appears to rescue locomotion and endogenous EPSCs to a better degree than the high-copy simple arrays reported in the previous version of the manuscript. This difference may be due to the mosaicism in transgenic worms carrying simple high-copy extrachromosomal arrays.We have generated transgenic worms that carry a single-copy of the rEndoBAR::unc-26 5Pase (∆Sac1∆PRD) transgene significantly restored locomotion, endogenous EPSCs, double mutants  Is the isolated Sac1 domain  synaptically localized? This would be a nice way of documenting its function as a localizing domain. In other words, is the Sac1 sufficient to localize GFP to synapses in the wild-type?We show that isolated Sac1 domains  are sufficient to localize GFP to synapses in wild type worms .3) In, the authors show that the D716A 5-Phosphatase mutant has dominant negative activity, and that this inhibitory activity requires the Sac1 domain. This construct lacks the PRD. Have the authors tested if a ∆PRD construct containing wild type phosphatase domains also has dominant negative activity? This control would help conclude that the D716A mutation is required for the inhibitory action of this construct.We show that the UNC-26∆PRD with wild type phosphatase domains does not exhibit dominant-negative inhibition .4) In the subsection headed “Endophilin BAR and synaptojanin Sac1 are functionally linked”, the authors state that fusing Endophilin's SH3 domain to UNC-26∆Sac did not rescue EPSC defects. Did the SH3 domain restore synaptic localization of ∆Sac? This would suggest that proper localization of the 5-Phosphatase domain is not sufficient to explain UNC-26 function in endocytosis.We added the “The functional difference between the BAR domain and the SH3 domain is likely due to their distinct binding partners and the potential for differential targeting to sub-synaptic regions.”5) In the same section of the manuscript, the authors speculate as to why the SH3 and PRD domains are conserved despite the fact that they are not required for SV endocytosis. Another possibility the authors may want to suggest is that the PRD/SH3 interaction provides a backup mechanism for co-localizing Endophilin and SJN under conditions where Sac1 localization is disrupted. For example, if Sac1 binds PI3,4P lipids, its localization may be disrupted when the levels of these lipids are low. In this case, the SH3/PRD interaction may be required for endocytosis.The reviewers raised an interesting point. We agree that while the PRD domain alone is not sufficient, it is possible that it become required in some situations, e.g., when the Sac1-membrane interactions are reduced. We have added the following statement in the Discussion section of the manuscript:“Thus, it is possible that the PRD-SH3 interaction facilitates co-localization of endophilin and synaptojanin, helping the Sac1-dependent mechanism to sustain membrane recycling during persistent activity. In addition, while the PRD domain alone is not sufficient to promote SV endocytosis, it may become required in some situations, e.g., when the Sac1-membrane interactions are reduced.”6) In, what do the arrowheads indicate?We modified the legend of 7) In, panel C, it is unclear what the cyan domain indicates in the BAR domain containing protein.The cyan domain indicates the SH3 domain of endophilin. We have modified the figure now .8) In, was rescue done with high copy or single copy transgenes?The rescue experiments in


Trypanosoma brucei requires editing of mitochondrial mRNA by uridylate (U) insertion and deletion. Hundreds of small guide RNAs (gRNAs) direct the mRNA editing at over 3,000 sites. RNA editing is controlled during the life cycle but the regulation of substrate and stage specificity remains unknown. Editing progresses in the 3’ to 5’ direction along the pre-mRNA in blocks, each targeted by a unique gRNA. A critical editing factor is the mitochondrial RNA binding complex 1 (MRB1) that binds gRNA and transiently interacts with the catalytic RNA editing core complex (RECC). MRB1 is a large and dynamic complex that appears to be comprised of distinct but related subcomplexes (termed here MRBs). MRBs seem to share a ‘core’ complex of proteins but differ in the composition of the ‘variable’ proteins. Since some proteins associate transiently the MRBs remain imprecisely defined. MRB1 controls editing by unknown mechanisms, and the functional relevance of the different MRBs is unclear. We previously identified two distinct MRBs, and showed that they carry mRNAs that undergo editing. We proposed that editing takes place in the MRBs because MRBs stably associate with mRNA and gRNA but only transiently interact with RECC, which is RNA free. Here, we identify the first specialized functions in MRBs: 1) 3010-MRB is a major scaffold for RNA editing, and 2) REH2-MRB contains a critical trans-acting RNA helicase (REH2) that affects multiple steps of editing function in 3010-MRB. These trans effects of the REH2 include loading of unedited mRNA and editing in the first block and in subsequent blocks as editing progresses. REH2 binds its own MRB via RNA, and conserved domains in REH2 were critical for REH2 to associate with the RNA and protein components of its MRB. Importantly, REH2 associates with a ~30 kDa RNA-binding protein in a novel ~15S subcomplex in RNA-depleted mitochondria. We use these new results to update our model of MRB function and organization.Adaptation and survival of  Trypanosoma brucei and other kinetoplastid protozoa have a single mitochondrion with an unusual mitochondrial genome (kDNA) consisting of many copies of an identical “maxicircle DNA” (~23 kb) and several hundred different types of “minicircle DNAs” (~1 kb). Maxicircle DNA encode 18 mRNAs, most of which require maturation through a remarkable form of RNA editing that changes the length of the mRNA by insertion or deletion of uridylate (U) nucleotides. In contrast, each minicircle DNA encodes 3–4 genes for small guide RNAs (gRNAs) that bind the pre-mRNA in trans and direct editing at more than 3000 sites  identified in recent reports in procyclic strains Lister 427 and EATRO 164. A previously annotated encoded T in the ND7 gene (arrow) was missing in all 10 cDNA clones examined here from the Lister 427 strain.cDNA sequence of block 1 in mRNA ND7 3’ domain amplified from (A) 3010 and (B) REH2 IPs. PCR primers (arrows) flanking the first block target 5’ unedited and 3’ never-edited sequence. Precursor unedited or edited mRNA (boxed) with editing sites 1-to-8: unedited (gray) or with deletions ‘★’ and insertions ‘(TIF)Click here for additional data file.S2 FigThe mutated sites G1365A/K1366Q are shown with the carbons colored white. These mutations in the P loop or motif I (atoms of motif I are shown as sticks) remove one H-bond between beta phosphate of the ADP and REH2. Four H-bonds remain after the mutations.(TIF)Click here for additional data file.S1 TableWe designed the indicated primers to perform RT-qPCR of 3’ early editing sites, manual sequencing of block 1 sites, RT-PCR of the first ~200 bp of editing domain (A6 and ND7), RNA interference (RNAi), point mutations and generation of photo-reactive gA6 B1.alt. RT-PCR primers to amplify the entire RPS12 were as in [(TIF)Click here for additional data file.