C. elegans SMN ortholog, SMN-1, causes NMJ defects. We found that increased levels of the C. elegans Gemin3 ortholog, MEL-46, ameliorates these defects. Increased MEL-46 levels also restored perturbed microRNA (miR-2) function in smn-1(lf) animals. We determined that miR-2 regulates expression of the C. elegans M2 muscarinic receptor (m2R) ortholog, GAR-2. GAR-2 loss ameliorated smn-1(lf) and mel-46(lf) synaptic defects. In an SMA mouse model, m2R levels were increased and pharmacological inhibition of m2R rescued MN process defects. Collectively, these results suggest decreased SMN leads to defective microRNA function via MEL-46 misregulation, followed by increased m2R expression, and neuronal dysfunction in SMA.Spinal Muscular Atrophy (SMA) is caused by diminished Survival of Motor Neuron (SMN) protein, leading to neuromuscular junction (NMJ) dysfunction and spinal motor neuron (MN) loss. Here, we report that reduced SMN function impacts the action of a pertinent microRNA and its mRNA target in MNs. Loss of the DOI:http://dx.doi.org/10.7554/eLife.20752.001 Spinal muscular atrophy is a genetic disease that causes muscles to gradually weaken. In people with the disease, the nerve cells that control the movement of muscles – called motor neurons – deteriorate over time, hindering the person’s mobility and shortening their life expectancy. Spinal muscular atrophy is usually caused by genetic faults affecting a protein called SMN  and recent research suggested that disrupting this protein alters the function of short pieces of genetic material called microRNAs. However, the precise role that microRNAs play in the disease and their connection to the SMN protein was not clear.MicroRNAs interfere with the production of proteins by disrupting molecules called messenger RNAs, which are temporary strings of genetic code that carry the instructions for making protein. By disrupting messenger RNAs, microRNAs can delay or halt the production of specific proteins. This is an important part of the normal behavior of a cell, but disturbing the activity of microRNAs can lead to an unwanted rise or fall in crucial proteins.O’Hern et al. made use of engineered nematode worms and mice that share genetic features with spinal muscular atrophy patients, including disruption of the gene responsible for producing the SMN protein. These animal models of the disease were used to examine the relationship between decreased SMN levels and microRNAs in motor neurons. The experiments showed that reduced SMN activity affects a specific microRNA, which in turn causes motor neurons to produce more of a protein called m2R. This protein is a receptor for a molecule, called acetylcholine, which motor neurons use to send signals to muscle cells.Increased m2R may be detrimental to motor neurons. As such, O’Hern et al. decreased m2R protein activity to determine whether this could reverse the defects in motor neurons that arise in the animal models of the disease. Indeed, blocking this receptor rescued some of the defects seen in the animal models, supporting the link to spinal muscular atrophy.Several treatments that block m2R are already available to treat other conditions. As such, the next step is to determine whether these existing treatments are able to protect mice models of spinal muscular atrophy against muscle deterioration or increase their lifespan. If successful, this could open new avenues for the development of treatments in people.DOI:http://dx.doi.org/10.7554/eLife.20752.002 SMN1  gene, resulting in reduced Survival of Motor Neuron (SMN) protein levels  is an autosomal recessive neurodegenerative disease and the leading genetic cause of infant death in the US . SMA is n levels . SMN expn levels . Diseasen levels . Understn levels . Studiesn levels , messengn levels , and locn levels . SMN funmicroRNAs (miRNAs) are non-coding RNAs that often repress protein translation, by a mechanism that requires miRNA binding to the 3’UTR of mRNA targets. Disruption of the miRNA pathway in spinal MNs leads to severe degeneration . SMN losC. elegans SMA model to examine the connection between SMN, Gemin3, and miRNA function. SMN1, Gemin3, and multiple miRNA pathway components are conserved in C. elegans  animals also have neuromuscular junction (NMJ) defects, suggesting a functional role for SMN-1 in MNs (smn-1(lf) animals do not die, likely because of their short lifespan. However, smn-1(lf) neuromuscular defects may correspond to the early stages of SMA pathogenesis, characterized by NMJ dysfunction prior to MN degeneration  homozygous loss of function animals had severely decreased pharyngeal pumping rates. Pharyngeal pumping was restored to normal rates in mel-46(tm1739) animals using a previously described, broadly expressed mel-46 rescue array #1]), which utilizes the mel-46 promoter  or cholinergic neuron-specific mel-46(RNAi)  . We concC. elegans cholinergic MNs #1] rescue array restored aldicarb sensitivity in mel-46(tm1739) animals. Tissue-specific knock-down of mel-46 in cholinergic neurons resulted in aldicarb resistance, thus confirming that MEL-46 function is required in cholinergic neurons, as is SMN-1  animals? We evaluated localization of presynaptic proteins SNB-1 (synaptobrevin) and APT-4 (AP2 α-adaptin) in cholinergic dorsal A-type (DA) MNs of mel-46(tm1739) animals  animals also had increased APT-4 linear density, but no changes in puncta width or intensity compared to controls  animals. Because ok355 deletion in smn-1 leads to a complete loss of function, smn-1(ok355) animals were maintained over an hT2 balancer and sterile smn-1(ok355) homozygous progeny carry some maternally-loaded SMN-1 protein (smn-1(+)) and smn-1(ok355) animals. Our finding is consistent with previous work showing that Gemin3 localizes to granular structures in mammalian neurites; Gemin3 co-localizes with SMN in 50–60% of these granules, along with multiple mRNAs (smn-1(ok355) animals, we found that the density of MEL-46::GFP-positive granular structures was doubled compared to smn-1(+) controls (smn-1(ok355) animals (smn-1(ok355) synaptic defects (mel-46 gene dosage in smn-1(ok355) animals using the [mel-46(+)#1] rescue array and showed that this ameliorated smn-1(ok355) aldicarb resistance defects  promoter in an integrated array, referred to as [ACh::mel-46::GFP], rescued smn-1(ok355) aldicarb resistance  (mel-46::GFP#2])   and no d:GFP#2]) . It is p:GFP#2]) ,10.7554/smn-1(ok355) animals by increased mel-46 levels ([mel-46(+)#1] rescue array, mel-46 did rescue smn-1(ok355) synaptic protein localization defects. Using the [mel-46(+)#1] rescue array, we rescued smn-1(ok355) defective SNB-1 puncta width and intensity to normal levels, but did not ameliorate linear density defects (mel-46 in an smn-1(+) control background did not increase SNB-1 levels, suggesting that mel-46-induced up-regulation of SNB-1 is specifically beneficial in a smn-1(ok355) background. Increasing mel-46 with a second broadly-expressed mel-46 rescue array line, [mel-46(+)#2], also restored APT-4 puncta linear density to normal levels  animals by partially restoring levels and localization of synaptic proteins. Furthermore, these results suggest that mel-46 may act with or downstream of smn-1 in a pathway essential for NMJ function.The interdependency we report, between SMN-1 and MEL-46 levels, may be specific to particular tissues and/or neural circuits. For example, pharyngeal pumping rate defects were not ameliorated in  defects . Notablyl levels , withoutmel-46 stabilizes maternally-loaded SMN-1 protein and mRNA, a scenario which could also explain mel-46 rescue of smn-1(ok355) NMJ function. Since mammalian Gemin3 directly binds SMN #2] rescue array did not increase GFP::SMN-1 levels, but unexpectedly caused a modest overall decrease (smn-1(ok355) rescue by increased mel-46 is due to stabilization of maternally-loaded SMN-1. Using the same CRISPR-based method, we were unable to tag endogenous smn-1 on balancer chromosomes necessary for maintaining the smn-1(ok355) line. This approach would have allowed us to examine the effects of MEL-46 overexpression on the stability of maternally-loaded SMN-1 in smn-1(ok355) animals. Therefore, although we cannot rule out stability of maternally-loaded SMN-1 as a contributing factor, the large effect that decreased SMN-1 has on MEL-46 localization favors a mechanism in which MEL-46 overexpression rescues smn-1(ok355) defects, at least in part, by restoring MEL-46 functional deficits in this background.Alternatively, we considered the possibility that increasing inds SMN , increas protein  (Dickins protein . The GFPdecrease . It thermir-2(lf) animals. Two different deletion alleles, gk259 and n4108, caused resistance to aldicarb paralysis compared to wild type animals (unc-17 (ACh) cholinergic neuron-specific promoter (referred to as ([ACh::mir-2(+)]) ]) . Loss ofr-2(+)]) . Taken tmir-2(gk259) animals revealed increased SNB-1 puncta linear density, but no change in puncta width or intensity compared to wild type animals (mir-2(gk259) animals were indistinguishable from wild type control animals with respect to SYD-2 synaptic localization for all metrics analyzed (mir-2(gk259) and mir-2(n4108), animals compared to wild type controls (mir-2(gk259) and mir-2(n4108) had decreased APT-4 puncta width, intensity, and linear density (smn-1(ok355) animals , SYD-2 (α-liprin), ITSN-1 (DAP160/Intersectin), and APT-4 (AP2 α-adaptin). Analysis of tagged SNB-1 in  animals . Additioanalyzed , suggestanalyzed . ITSN-1 controls . Finally density . ITSN-1, density . Togethe density . Additio animals , a findigar-2, dbl-1, sek-1, and vab-2  background. Loss of any of these four genes suppressed mir-2(gk259) to some extent, but gar-2(ok520), which contains a large deletion removing gar-2 exons 6 and 7, resulted in the most complete suppression, thus suggesting that GAR-2 acts downstream of miR-2  carrying the disrupted PAM site mutation (C>T), we found that animals with the scrambled miR-2 binding site (gar-2 UTRscrC) were resistant to aldicarb, similar to miR-2 loss . A second control version of the construct contained the same scrambled UTR sequence as used in gar-2 UTRscrC animals (referred to as unc-17p-ACh::GFP::gar-2 3’UTRscr) (mir-2(gk259) animals expressing the intact 3’UTR construct as compared to control animals  loss of function animals  . Transge animals . Whereas control . To quanckground . By thisckground , indicat animals . These rA levels .smn-1 loss altered miR-2 regulation of the gar-2 3’UTR. Both GFP reporter transgenes were crossed into the smn-1(ok355) background. Using the same ratiometric strategy from smn-1(ok355) animals compared to smn-1(+) controls (smn-1(ok355) animals may have decreased miR-2 function and, as a consequence, an increase in GAR-2 translation. We showed above that increased mel-46 levels ameliorates smn-1(ok355) NMJ defects (mel-46 on miR-2 activity using the [mel-46(+)#2] rescue array. In control smn-1(+) animals, increasing mel-46 did not alter relative expression of the miR-2 GFP reporter. However, in smn-1(ok355) animals, increasing mel-46 caused decreased relative reporter expression by ~15%, compared to smn-1(ok355) animals lacking the mel-46 rescue array (smn-1(ok355) by increasing miR-2 activity.Next, we determined if controls . This fi defects , therefoue array . These dgar-2 transcript levels  MNs might contribute to the NMJ defects previously observed in these animals  animals  restored normal response to aldicarb in both smn-1(ok355) and smn-1(rt248) animals  restored normal response to aldicarb in mel-46(tm1739) animals (gar-2(ok520) also rescued numerous presynaptic protein localization defects caused by SMN-1 loss; SNB-1 puncta width, intensity and linear density defects were rescued in both smn-1(ok355) and smn-1(rt248) backgrounds (smn-1(+) control animals resulted in increased SNB-1 puncta width and intensity, but did not alter SNB-1 puncta linear density.If increased GAR-2 levels exacerbate NMJ dysfunction in animals lacking SMN-1, then decreasing GAR-2 function should ameliorate the NMJ defects caused by  animals , similar6 levels . However animals , consistto ok355 . Additio animals . Our reskgrounds . GAR-2 lsmn-1(ok355) motor neurons (smn-1(ok355) APT-4 puncta defects (smn-1(+) control animals. As loss of GAR-2 did not restore APT-4 synaptic defects, we conclude that there are additional pathways affected by smn-1 loss, beyond GAR-2 misregulation. Nevertheless, these results suggest that C. elegans GAR-2 levels are increased by smn-1 loss, which might contribute to NMJ defects in smn-1(lf) animals.Research in vertebrates has demonstrated m2R internalization normally occurs in response to chronic m2R stimulation, either by pharmacological agonist application or acetylcholinesterase inhibition . Since e neurons . Loss of defects , but decCHRM2 gene. GAR-2 and m2R are functionally conserved, as activation of these presynaptic receptors by acetylcholine in different species results in hyperpolarization and decreased NMJ acetylcholine release across species . A ~50% increase in m2R levels was observed, compared to wild type control MNs (The closest human ortholog of GAR-2 is the M2 muscarinic receptor (m2R), encoded by the  species . Previoud humans   (Decreased SMN levels results in axon outgrowth defects in MNs derived from a SMA mouse model  and incrMN2tg/0) . Wild tyMN2tg/0) . We concCHRM2 locus lacks introns  through miR-2 misregulation and that increasing MEL-46 (Gemin3) reduces GAR-2 protein levels in smn-1(ok355) animals via modulation of miR-2. Several lines of evidence support this conclusion, including miR-2 GFP reporter expression results, NMJ aldicarb sensitivity, synaptic protein localization changes, and qPCR measurements of mature miR-2 and gar-2 mRNA levels (The most direct molecular connection between SMN and the miRNA pathway is Gemin3. Results presented here indicate that decreases in SMN-1(SMN) or MEL-46(Gemin3) result in similar neuromuscular defects as well as decreased miR-2 levels in neurons. This analysis further suggests that decreases in A levels –5.Drosophila, Gemin3 is necessary for motor function, but the molecular mechanisms underlying this defect are unclear  of miRNAs . It was  binding . Both mi binding . More st binding .C. elegans, miR-2 loss does not cause overt defects and GAR-2 loss does not restore viability, fecundity, or normal development to animals lacking SMN-1 or MEL-46. This suggests miR-2 loss does not contribute to defects outside the NMJ caused by SMN-1 loss. And, at the NMJ, synaptic protein perturbations are more severe in animals with diminished SMN-1 or MEL-46, compared to those lacking either miR-2 or GAR-2, which is consistent with a broader range of defects caused by decreased SMN-1 levels. In mice, complete miR-128 knock-out results in decreased motor activity and premature death  and P/Q-type Ca2+ (Cav2.1) channels resulting in decreased SV release at the NMJ  animals, may act via SK channels  and smn-1(rt248), animals assayed were first generation progeny of hermaphrodites heterozygous for the hT2 balancer. To maintain a common genetic background, control smn-1(+) animals were also derived from +/hT2 parents. Similarly, for APT-4::GFP synaptic localization (mel-46(tm1739) animals were first generation progeny of parents heterozygous for the nT1 balancer. Control mel-46(+) animals were derived from +/nT1 animals. For all other assays involving mel-46(tm1739), animals were first generation progeny of parents carrying the ytEx211[mel-46(+)] rescue array; animals tested did not carry the array unless specified. For these experiments, N2 animals served as wild type controls. We attempted to generate smn-1(ok355);mel-46(tm1739) double mutant animals, but generation of heterozygous double mutant animals was not possible, using either balancer chromosomes or the ytEx211[mel-46(+)] rescue array.Strains listed in lization  and aldilization , mel-46(unc-17p::mir-2::unc-54 3’UTR) plasmid was generated by excising a 867 bp fragment from pHA#755 (aex-3p::mir-2::unc-54 3’UTR) using NheI and SpeI. This fragment, containing the genomic mir-2 pre-miRNA sequence along with unc-54 3’UTR sequence, was subcloned into pPD95.77  between NheI and SpeI sites, resulting in removal of the GFP sequence. Additionally, a 4466 bp fragment corresponding to the unc-17 promoter was inserted between pPD95.77 SphI and AscI sites. Information for all amplification primers can be found in unc-17p::GFP::unc-54 3’UTR) was generated by inserting the unc-17 promoter fragment between pPD95.77 SphI and AscI sites, without altering the GFP sequence. Plasmid pHA#758 (NLS::GFP::gar-2 3’UTRwt) contains a 269 bp fragment corresponding to the gar-2 3’UTR that was subcloned into pPD95.67  as a EcoRI and SpeI product. pHA#759 (unc-17p::NLS::GFP::gar-2 3’UTRwt) was generated by excising a 1286 bp fragment containing the NLS::GFP sequence and gar-2 3’UTR from pHA#758 using MscI and SpeI and ligating this fragment into pHA#756, thus removing the genomic mir-2 pre-miRNA and unc-54 3’UTR sequences. pHA#760 was generated by ligating the gar-2 3’UTR fragment into pBluescript KS+  using EcoRI and SpeI. To construct pHA#761, the last 85 bp of the gar-2 3’UTR were removed from pHA#760 using NcoI and SpeI. This fragment was replaced with an identical 85 bp sequence, but with 19 bp scrambled at the predicted miR-2 binding site sequence (gar-2 3’UTRscr). Primers were annealed to produce this 85 bp sequence (gar-2 3’UTRscr) was generated by subcloning the 269 bp gar-2 3’UTRscr fragment from pHA#761 into pPD95.67 with EcoRI and SpeI. pHA#763 (unc-17p::NLS::GFP::gar-2 3’UTRscr) was produced by subcloning the 1286 bp fragment containing NLS::GFP and gar-2 3’UTRscr sequences from pHA#762 into pHA#756 using MscI and SpeI, while removing the genomic mir-2 pre-miRNA and unc-54 3’UTR sequences. pHA#790 (unc-122p::mel-46::unc-54 3’UTR) was created by amplifying the MEL-46 coding region from the pRM8 plasmid . To create pHA#792 (unc-17p::mel-46::GFP::unc-54 3’UTR), a 906 bp GFP sequence was amplified from pHA#763 and subcloned by Gibson assembly into pHA#791 just before the MEL-46 stop codon (TGA). The small guide RNA (sgRNA) plasmids targeting the smn-1 gene (pHA#764 and pHA#765) and the sgRNA plasmid targeting the gar-2 3’UTR (pHA#793) for CRISPR/Cas9-mediated genome editing were produced by amplification of PU6::klp-12 ; myo-2p::RFP], wild type animals were injected with 133 ng/μl PRM8 plasmid  and 75 ng/μl pBluescript KS+. Animals injected with rtEx855 were crossed into a mel-46(tm1739) background to assure rescue of viability before further experiments were undertaken. Notably, expression of either rtEx855 or ytEx211 in smn-1(lf) animals did not rescue lethality or adult survival, further emphasizing a privileged relationship between SMN-1 and MEL-46 in cholinergic NMJ signaling. Lines for rtEx853 and rtEx854 were produced by injecting mir-2(gk259) animals with pHA#756 or pHA#757, respectively, at 40 ng/μl alongside 2.5 ng/μl myo-2p::mCherry  and gar-2(rt318) alleles were generated by injecting pha-1(e2123) animals with the pHA#793 sgRNA plasmid targeting the gar-2 3’UTR at 25 ng/μl with either 50 ng/μl of a mutant single-strand oligo DNA (ssODN) repair template (rt318) or a control ssODN repair template (rt317), alongside the injection cocktail as described in smn-1(rt280), which contains a GFP N-terminal insertion, wild type animals were injected with both pHA#764 and pHA#765 sgRNA plasmids targeting smn-1 at 50 ng/μl alongside 20 ng/μl of the GFP template plasmid pHA#766 and the standard injection cocktail described in Integrated arrays orgensen , and 75  plasmid , 5 ng/μlorgensen ; Addgenesmn-1(RNAi), C26C6.2/goa-1(RNAi), T06A10.1/mel-46(RNAi), or Y37D8A.23/unc-25(RNAi) (http://cmgm.stanford.edu/~kimlab/primers.12-22-99.html).RNAi studies involved animals from an RNAi-enhanced background (KP3948) , neuron-25(RNAi) . Primer Aldicarb resistance assay: 1 mM aldicarb assays were completed in at least three independent trials blinded to genotype  as described in previous work (smn-1(ok355), smn-1(rt248) or mel-46(tm1739) animals were completed at the early L4 stage. All other aldicarb experiments were done with young adult animals. Pharyngeal pumping: Assays were performed blinded to genotype as previously described (smn-1(ok355), smn-1(rt248) or mel-46(tm1739) animals were completed at day three post-hatching . Pumping experiments involving all other genotypes were done with young adult animals.ous work . Paralysescribed . PumpingDorsal cord protein localization: Images were obtained as Z-stacks of the dorsal cord above the posterior gonad reflex  AxioImager ApoTome and Axiovision software v4.8). For MEL-46::GFP analysis, a set area was defined for each image along the dorsal cord (25 µm x 5 µm). Using ImageJ (RRID:SCR_003070), a uniform threshold was used to eliminate background. The number (density), mean fluorescence (intensity) and area (size) for MEL-46::GFP granular structures were calculated using the ImageJ ‘particle analyzer’ program. For synaptic protein localization, mean puncta width (meanfixedwidth), intensity (meanfixedvolume) and linear density (fixedwidthlineardensity) were quantified with an in-house developed program called ‘Punctaanalyser’ using MatLab software  (smn-1(ok355), smn-1(rt248), or mel-46(tm1739) animals, all genotypes were examined at the early L4 stage, while other data sets were collected with young adult stage animals. GFP Fluorescence Quantification: GFP images of L4 animals were acquired . Mean GFP fluorescence was quantified using ImageJ (RRID:SCR_003070). A threshold was set to eliminate background fluorescence. For each data set, thresholds were kept constant. Average fluorescence values (±SEM) were combined from at least three independent trials for n > 25 animals/genotype; however certain backgrounds containing rtEx855[mel-46(+)] had a lower n (reported in legends) as these animals went sterile and/or did not throw many progeny carrying the mel-46 array. Ratios in rtIs56 background and divided by their respective average mean fluorescence in the control rtIs57 background. Ratio SEM was calculated by summing the SEM for each population  in M9 buffer. _001622) . At leastion see . All repact-1: 5’-acgccaacactgttctttcc-3’ and 5’-gatgatcttgatcttcatggttga-3’  incubation. 1 ng total RNA was used for reverse transcription with either the miScript II RT kit (Qiagen #218160) for miRNA or the SuperScript III First-Strand Synthesis Supermix kit (Invitrogen #11752050) for mRNA. Methodology followed manufacturer’s instructions. miRNA levels were determined in a 10 µl reaction using miScript SYBR Green PCR kit  and 300 nM of mature miR-2 primer/probe. miR-60 was used to normalize miR-2 expression as it is not expressed in the nervous system where SMN-1 or MEL-46 were knocked-down. Forward primer sequences for miR-2 and miR-60 were, respectively: 5’-TATCACAGCCAGCTTTGATGTGC-3’ and 5’-TATTTATGCACATTTTCTAGTTCA-3’. A universal reverse probe was provided by Qiagen. Primer sequences for gttga-3’ . Primer gttga-3’ . The stariation’ . mRNA leving SMN . Primer escribed .IMSR_JAX:001800) and SMA mice    as descr:005024) . IsolateAfter 10 days in vitro culture, total RNA was extracted from MNs using the mirVana total RNA isolation kit (Thermo Scientific). Nanodrop was used to measure RNA amount. Using 100 ng of total RNA, miR-128 expression levels were determined by real time PCR with mature miR-128a primer/probe . Actin-beta was used to normalize miR-128a expression. Primer sequences for actin-beta: 5'-agccatgtacgtagccatcc-3' and reverse 5'-ctctcagctgtggtggtgaa-3'. Methodology followed manufacturer’s instruction .AB_10858602; 1:1000) and β-actin  were used to detect proteins. Methoctramine  was treated 48 hr from DIV3 to DIV5 in various concentrations. After 5 days of in vitro culture, neuronal morphology was visualized with Tau  staining. Axon length was analyzed with ImageJ (RRID:SCR_003070).Proteins were extracted from motor neurons, after 10 days in vitro culture, using RIPA buffer and protease inhibitor cocktail . ExpressU-test, or t-test were used for C. elegans statistical analysis. The Mann Whitney U-test was chosen over t-test for experiments where homogeneity could not be assured . t-test was used to determine significance for spinal motor neuron Western blot quantification and qPCR quantification.Log-rank test, two-tailed Mann-Whitney  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Decreased SMN causes deleterious increases in neuronal M2 muscarinic receptors due to microRNA misregulation" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.mel-46), the miR-2 and the M2 muscarinic receptor. The study demonstrates the power of a genetically tractable system to understand fundamental biology. Based on their data and previously published work, the authors propose the following:The authors harness the power of C.elegans genetics to interrogate the defects incurred by loss of SMN on neuromuscular function. Using a series of approaches, the authors implicate the worm ortholog of Gemin3 (1) SMN-1 acts genetically through MEL-46/Gemin3. Molecularly, SMN-1 is necessary to achieve normal levels of MEL-46/Gemin3.mir-2.2) Normal levels of Gemin3 are necessary for correct miRNA processing/function, including mir-2 is necessary to repress gar-2. GAR-2 is an M2 receptor that is predicted to inhibit synaptic release at cholinergic NMJs and thus needs to be kept at relatively low levels for optimal NMJ function3) These conclusions are based on a number of relatively indirect measures, which are for the most part not sufficient to support all claims. In essence, a number of qPCR experiments are required to support a number of conclusions. Specifically:mel-46 overexpression rescues part of the defects observed in smn-1(lf) animals, most prominently, it suppresses the aldicarb resistance of smn-1(lf) animals. However, it does not rescue the pharyngeal defect of smn-1(lf), suggesting some kind of tissue specificity. Since the connection to miRNA processing/function is given by MEL-46/Gemin3, I think it's important to provide a more direct link and the authors need to measure mel-46 (RNA using qPCR and/or protein) in smn-1(lf).1) The authors propose that reduction of SMN-1 results in lower levels of MEL-46. This is based on one genetic interaction: the fact that smn-1(lf) background but not in the wt background, it is not clear if one would expect to see any changes in SMN-1 abundance in wt animals. A more direct test would have been to look at the remaining GFP in the smn-1(lf) progeny from heterozygous mothers and see whether this changes with overexpression of MEL-46. Also, given the tissue specificity observed phenotypically, it may be more revealing to focus e.g. on motorneurons. Although if levels are too low to see GFP, a western blot against GFP may already provide some information.This is important given that an alternative explanation is not satisfactorily rejected. Specifically, the authors discard an effect of MEL-46 in stabilizing the maternal contribution of SMN-1 by quantifying GFP fluorescence from a very nice SMN-1::GFP allele generated with Cas9, over whole worms, in wt animals with or without MEL-46 overexpression. However, given that in all phenotypic assays the effect of MEL-46 is only seen in the mir-2, the miRNA the authors find to be important for NMJ function. Based on the data provided, mir-2 could be acting in the NMJs in a parallel pathway that has no connection to SMN-1 or MEL-46. To strengthen this link, the authors need to measure mir-2 levels in smn-1(lf), mel-46 mutants or overexpression strains. However, one has to wonder whether there is a connection at all: The authors claim to provide a link  with an epistasis analysis shown in gar-2, the predicted target of mir-2. The authors compare a reporter with the wt 3'UTR and one without the mir-2 binding site in wt animals or smn-1(lf). In wt animals, the fluorescence ratio between the two reporters is ~1 and in smn-1(lf) animals it goes up by 5% (to 1.05). The authors suggest this is consistent with mir-2 levels being lower in smn-1(lf)  and that leads to gar-2 derepression. In addition to this being an extremely small effect that would need additional data to be convincing , there is a problem which is that the increase in ratio is not as one would expect from the reporter with the wt 3'UTR being derepressed (numerator going up) in smn-1(lf) but rather the reporter with the mutant 3'UTR is decreased in smn-1(lf) (denominator going down) (smn-1 and mel-46 may have on the NMJ and whatever function mir-2 might have there as well.2) The connection of SMN to miRNAs through Gemin3 is solely based on previous publications reporting the dysregulation of certain miRNAs when SMN or Gemin3 are decreased. There is no direct connection in this work between this pathway and gar-2 being a target of mir-2. Given the small magnitude of repression observed, this point could be made more convincingly, for example by mutating the mir-2 binding site in the 3'UTR of gar-2, which should not be too difficult given that the authors are able to generate alleles using Cas9.3) The phenotypic and reporter analyses are consistent with eLife. Your revised article has been favorably evaluated by a Senior Editor, a Reviewing Editor, and two reviewers.Thank you for resubmitting your work entitled "Decreased microRNA levels lead to deleterious increases in neuronal M2 muscarinic receptors in Spinal Muscular Atrophy models" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:mir2 measurements . One suggestion for improving reproducibility is to use a remarkably well-working protocol published by Kien Ly, Suzanne J. Reid, Russell G. Snell as "Rapid RNA analysis of individual Caenorhabditis elegans" in MethodsX 2 (2015) 59-63.The authors have done a highly appreciated effort to incorporate the reviewers’ comments and suggestions and I think this has paid off in strengthening their model. However, one concern remains about a critical link in the story: The changes in urements , an impo […] These conclusions are based on a number of relatively indirect measures, which are for the most part not sufficient to support all claims. In essence, a number of qPCR experiments are required to support a number of conclusions. Specifically:1) The authors propose that reduction of SMN-1 results in lower levels of MEL-46. This is based on one genetic interaction: the fact that mel-46 overexpression rescues part of the defects observed in smn-1(lf) animals, most prominently, it suppresses the aldicarb resistance of smn-1(lf) animals. However, it does not rescue the pharyngeal defect of smn-1(lf), suggesting some kind of tissue specificity. Since the connection to miRNA processing/function is given by MEL-46/Gemin3, I think it's important to provide a more direct link and the authors need to measure mel-46 (RNA using qPCR and/or protein) in smn-1(lf).This is important given that an alternative explanation is not satisfactorily rejected. Specifically, the authors discard an effect of MEL-46 in stabilizing the maternal contribution of SMN-1 by quantifying GFP fluorescence from a very nice SMN-1::GFP allele generated with Cas9, over whole worms, in wt animals with or without MEL-46 overexpression. However, given that in all phenotypic assays the effect of MEL-46 is only seen in the smn-1(lf) background but not in the wt background, it is not clear if one would expect to see any changes in SMN-1 abundance in wt animals. A more direct test would have been to look at the remaining GFP in the smn-1(lf) progeny from heterozygous mothers and see whether this changes with overexpression of MEL-46. Also, given the tissue specificity observed phenotypically, it may be more revealing to focus e.g. on motorneurons. Although if levels are too low to see GFP, a western blot against GFP may already provide some information.unc-17 promoter (smn-1(ok355) background, we found an obvious defect in MEL-46 localization in dorsal cord motor neurons processes. We find that MEL-46::GFP in wild type control animals is sparsely localized to punctate structures along the dorsal cord (see smn-1(ok355) animals, MEL-46::GFP is found in many more granules, with an average decrease in MEL-46::GFP fluorescence within granules (smn-1(ok355) cholinergic neurons support our model in which MEL-46 function is perturbed when SMN-1 levels decrease.We appreciate the reviewer’s criticism and the opportunity to show a more direct connection between SMN-1 and MEL-46. To confirm that loss of SMN-1 affects the function and/or levels of MEL-46, we have now generated two independent integrated lines expressing MEL-46 tagged with GFP behind the control of the cholinergic-specific promoter . Using tcord see . These sgranules -1D. MEL-smn-1(ok355) homozygous animals. The simplest strategy would be to tag the endogenous smn-1 gene on the balancer chromosome used to propagate smn-1(ok355). Using the CRISPR-based approach outlined in the manuscript and suggested by the reviewer, we attempted to GFP-tag endogenous smn-1 on balancer chromosomes hT2 and ccIs425. We were unsuccessful after multiple attempts. Therefore, we cannot unequivocally conclude that increased MEL-46 does not lead to increased stabilized maternally-loaded SMN-1. We make this caveat clear in the revised text (see “changes made to text” below). Given the severe MEL-46 localization defects we observe in smn-1(ok355) animals, however, it is more likely that increased MEL-46 proteins levels ameliorate smn-1(ok355) defectsby increasing MEL-46 within critical granules in motor neurons, like those in the dorsal cord. See Unfortunately, we were unable to directly evaluate levels of maternally-loaded SMN-1::GFP in Changes made to text for Editor Point 1:to smn-1(ok355) synaptic defects .”“MEL-46 might act together with or downstream of SMN-1 in pathways necessary for NMJ function. […] Our findings suggest that SMN-1 impairs MEL-46 function, which could contribute smn-1 on balancer chromosomes necessary for maintaining the smn-1(ok355) line. […] Therefore, although we cannot rule out stability of maternally-loaded SMN-1 as a contributing factor, the large effect that decreased SMN-1 has on MEL-46 localization favors a mechanism in which MEL-46 overexpression rescues smn-1(ok355) defects, at least in part, by restoring MEL-46 functional deficits in this background.”“Using the same CRISPR-based method, we were unable to tag endogenous 2) The connection of SMN to miRNAs through Gemin3 is solely based on previous publications reporting the dysregulation of certain miRNAs when SMN or Gemin3 are decreased. There is no direct connection in this work between this pathway and mir-2, the miRNA the authors find to be important for NMJ function. Based on the data provided, mir-2 could be acting in the NMJs in a parallel pathway that has no connection to SMN-1 or MEL-46. To strengthen this link, the authors need to measure mir-2 levels in smn-1(lf), mel-46 mutants or overexpression strains. However, one has to wonder whether there is a connection at all: The authors claim to provide a link  with an epistasis analysis shown in gar-2 transcripts greatly strengthens the manuscript. We focused specifically on how decreasing SMN-1 or MEL-46 within neurons impacts mature miR-2 levels and gar-2 mRNA levels. To do this, we undertook RNAi studies using animals sensitive to RNAi by feeding only in neurons . These animals were placed on bacteria expressing SMN-1 or MEL-46 RNAi constructs for at least two generations. We found that knock-down of either SMN-1 or MEL-46 resulted in a large (~80%) decrease in miR-2 levels. These results are consistent not only with a role for both SMN-1 and MEL-46 in miR-2 biogenesis, but further suggest a relationship between SMN-1 and MEL-46 in a pathway important for proper miRNA function. In measuring gar-2 transcript from these samples, we found that this decrease in miR-2 levels did not result in significantly increased gar-2 mRNA levels. This was unsurprising as gar-2 mRNA levels were also not significantly increased in mir-2(gk259) loss of function animals compared to N2 control animals  loss of function animals  The phenotypic and reporter analyses are consistent with gar-2 being a target of mir-2. Given the small magnitude of repression observed, this point could be made more convincingly, for example by mutating the mir-2 binding site in the 3'UTR of gar-2, which should not be too difficult given that the authors are able to generate alleles using Cas9.gar-2 3’UTR. Animals with a scrambled endogenous miR-2 binding site in the gar-2 3’UTR were resistant to aldicarb, when compared to control animals  animals did not have significantly increased gar-2 transcript levels. We note that other miR-2 family members likely bind this site, including miR-43, miR-250, and miR-797. Additionally, our qPCR results come from whole animal lysate. Thus, the increase in gar-2 messenger RNA likely results from the inability of all miR-2 family members to bind the gar-2 3’UTR throughout all tissues. These family members may act differently than miR-2, resulting in transcript destabilization when bound. Furthermore, since miR-2 appears to be limited to the nervous system, the effect it has on gar-2 transcript in neurons may be minimized by levels of the more broadly-expressed gar-2 transcript. This may explain why a significant difference in transcript levels is seen when the binding site is scrambled, but not in mir-2(gk259) loss of function animals.We agree. CRIPSR/Cas-9-based genome editing was used to scramble the microRNA binding site in the  animals . Furthernger RNA . These rSee Changes made to text for Editor Point 3:gar-2 3’UTR. […] Disruption of the 3’UTR site likely inhibits binding of other miR-2 family members, likely contributing to the effect we observe .”“To determine if miR-2 regulates GAR-2 expression directly, we examined the consequences of perturbing the putative miR-2 binding site in the The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:The authors have done a highly appreciated effort to incorporate the reviewers’ comments and suggestions and I think this has paid off in strengthening their model. However, one concern remains about a critical link in the story: The changes in mir2 measurements , an impo(act-1). After either mel-46 and smn-1 RNAi treatments, we observed decreased miR-2 levels, normalized to any of these 3 RNAs relative to empty RNAi . We utilized previously reported amplification primers for both 18S rRNA  and act-1 . This extended analysis makes clear that miR-2 levels decrease when smn-1 or mel-46 function decreases.We thank the reviewer for these suggestions. We undertook the additional experiments suggested by the reviewer. We increased the number of samples from 3 to 6 for each group and examined miR-2 expression levels relative to 3 different RNAs: a microRNA (miR-60), a ribosomal RNA (18S), and a messenger RNA pty RNAi . In the act-1 . As miR-60 is the most stable candidate, we use it as a control in the main text figures.Choosing an appropriate control RNA can be challenging; here we examined 3 different possibilities. We evaluated the stability of each of the putative “control RNAs” under the different experimental conditions using the “model-based approach to estimation of expression variation” . This mathematical model is used to describe the “stability value” of candidate genes- taking in account variability within a group of samples under the same treatment conditions and between groups of samples under different treatment conditions. Lower “stability values” for a given group of genes indicate more stable expression. We find that miR-60 is the most stable RNA , compared to 18S rRNA or See Changes made to text for Editor Point 1:gar-2 transcript levels . Primer sequences for 18S rRNA: 5’-TTGCTGCGGTTAAAAAGCTC’3’ and 5’-CCAACCTCAAACCAGCAAAT-3’ . The stability of miR-60, 18S rRNA, and act-1 housekeeping RNAswere evaluated using the ‘model-based approach to estimation of expression variation’ .”“Primer sequences for (RNAi), smn-1(RNAi), and mel-46(RNAi) young adult animals relative to housekeeping miRNA miR-60. t-test, two-tailed (n=6 for each condition).”“miR-2 levels were decreased in neurons when either SMN-1 or MEL-46 were decreased. Quantification of mature miR-2 for empty(RNAi), smn-1(RNAi), and mel-46(RNAi) young adult animals relative to housekeeping gene act-1. […] Quantification of mature miR-2 for empty(RNAi), smn-1(RNAi), and mel-46(RNAi) young adult animals relative to housekeeping rRNA 18s. t-test, two-tailed (n=6 for each condition).”“(C) miR-2 levels were decreased in neurons when either SMN-1 or MEL-46 were decreased. Quantification of mature miR-2 for empty


Based on these results, we suggest that SC79 protects RPE cells from UV damages possibly via activating Akt-Nrf2 signaling axis.Excessive Ultra-violet (UV) radiation causes oxidative damages and apoptosis in retinal pigment epithelium (RPE) cells. Here we tested the potential activity of SC79, a novel small molecule activator of Akt, against the process. We showed that SC79 activated Akt in primary and established (ARPE-19 line) RPE cells. It protected RPE cells from UV damages possibly via inhibiting cell apoptosis. Akt inhibition, via an Akt specific inhibitor (MK-2206) or Akt1 shRNA silence, almost abolished SC79-induced RPE cytoprotection. Further studies showed that SC79 activated Akt-dependent NF-E2-related factor 2 (Nrf2) signaling and inhibited UV-induced oxidative stress in RPE cells. Reversely, Nrf2 shRNA knockdown or S40T mutation attenuated SC79-induced anti-UV activity. For the  Excessive Ultra-violet (UV) radiation and reactive oxygen species (ROS) are known as the important contributors of age-related macular degeneration (AMD) and other retinal degenerative diseases –3. RetinAkt is a well-established pro-survival signaling molecule . Many stin vivo, it also protected against early brain injuries by subarachnoid hemorrhage [A recent study by Jo et al., has characterized a novel, selective, and cell-permeable small molecule Akt activator: SC79 . SC79 unmorrhage . Yet, Momorrhage . In the The transcriptional factor NF-E2-related factor 2 (Nrf2) dictates the transcription of key anti-oxidant genes , 15. ActThe structure along with the molecular/formula weight of SC79 were presented in Figure Our studies and others have shown that UV radiation induces RPE cell apoptosis , 17–20. Further studies showed that UV radiation also induced caspae-9 activation Figure  and mitoTo elucidate to link between Akt activation and SC79-induced RPE cytoprotection, we first applied an Akt specific inhibitor: MK-2206 [To rule out the possible off-target effect of MK-2206, we utilized targeted shRNA to selectively knockdown Akt1 in ARPE-19 cells   was intrWe also tested Nrf2 signaling in SC79-treated primary murine RPE cells. As shown in Figure in vivo. In line with our previous findings, twenty-four hours after the light exposure, retinal ERG a- and b-waves were markedly decreased  and protein kinase C (PKC) . More rein vivo study results showed that intravitreal injection of SC79 offered a significant protection of mice retina against light damages. Thus, SC79 might have therapeutic values for treatment of AMD and other retinal degenerative diseases.Based on these results, we propose that SC79 activates Akt to possibly phosphorylate Nrf2 at Ser-40, causing Nrf2 accumulation and activation. As a matter of fact, SC79-induced Nrf2 signaling activation was almost blocked by the Akt inhibitor MK-2206 or Akt1 shRNA silence. Significantly, Nrf2 shRNA knockdown or S40T mutation dramatically attenuated SC79-induced RPE cytoprotection against UV. Therefore, SC79 activated Akt downstream Nrf2 to attenuate UV-induced oxidative stresses and RPE cell apoptosis. Importantly, the All methods listed in the study were carried out in accordance with the approved ethics guidelines of all authors' institutions.MK-2206 S1078) and SC79 (S7863) were from Selleck . The anti-β-tubulin antibody (SAB4500088) and the heme oxygenase-1 (HO-1) inhibitor Zinc protoporphyrin  were obtained from Sigma . All other antibodies were from Cell Signaling Tech  and Santa Cruz Biotech  as described [78 and SCCulture of human retinal pigment epithelial cells (ARPE-19 line) and human lens epithelial cells (HLECs) were described previously , 27, 28.2). Afterwards, cells were returned for incubation in culture medium with indicated treatments.UV radiation (UVB and UVA2) to cultured cells was performed as reported –6. CellsThe cell viability was tested via the MTT assay, which was described in detail in our previous publications , 29. TheFollowing treatment of cells, trypan blue staining was performed to indentify the “dead” RPE cells (Trypan blue positive). The percentage (%) of trypan blue cells was calculated via an automated cell counter .+/+/ PI−/− cells were marked as early apoptosis cells [+/+/ PI+/+ cells were marked as late apoptosis cells [Apoptosis assays, including the Annexin V fluorescence-activated cell sorter (FACS) assay, Histone DNA apoptosis enzyme-linked immunosorbent assay (ELISA) assay and caspase-9 activity assay were described in detail in our previous studies , 20, 30.is cells , 30. Annis cells , 30.6) were tested via flow cytometry (BD bioscience). Relative H2DCFDA intensity (vs. untreated control cells) was recorded to reflect cellular ROS content [As previously reported , 17, fol content , 17.ΔΔCt method was applied to quantify mRNA expression Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was tested as an internal control. Primers were described in our previous studies [Total RNA was extracted using Trizol reagents . Five hundred ng of DNA-free total RNA was utilized to perform the reverse transcription with the 2-step RT-PCR kit  . The PCR studies  and in p studies .The detailed protocol of Western blot assay was described in our previous studies , 10, 20.As described in our previous study , the celNrf2 shRNA-1 (sc-37030-V), Akt1 shRNA (sc-29195-V) and scramble control shRNA lentiviral particles were purchased from Santa Cruz Biotech (Santa Cruz); Lentiviral Nrf2 shRNA-2, with non-overlapping sequence with Nrf2 shRNA-1, was designed and verified by Genechem . The lentiviral particles (10 μL/mL) were added directly to cultured ARPE-19 cells for 36 h. Cells were then subjected to puromycin (1.0 μg/mL) selection for 8–10 passages until resistant colonies can be identified. The expression of targeted protein (Nrf2 or Akt1) in stable cells was always tested by Western blot assay.The S40T dominant negative (“DN”) Nrf2 pSV2 puro Flag plasmid was described previously . The DN-in vivo activity of SC79, 30 min before light exposure, SC79  was injected intravitreally to the left eyes. For detecting ERG [2 for 10 ms) was applied [The weight-matched male BALB/c mice (6–8 week old) were utilized. The retinal light damage model was described in detail in our previous studies . To evalting ERG , twenty-f-test via SPSS 18.0 software . Significance was chosen as p < 0.05Quantitative results were normalized to the control values of each assay, and were presented as mean ± standard deviation (SD). Data were analyzed by one-way ANOVA followed by a Scheffe's


The mechanisms involved in malignant transformation of mature B and T lymphocytes are still poorly understood. In a previous study, we compared gene expression profiles of the tumor cells of Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) to their normal cellular counterparts and found the basic leucine zipper protein ATF-like 3 (BATF3) to be significantly upregulated in the tumor cells of both entities. To assess the oncogenic potential of BATF3 in lymphomagenesis and to dissect the molecular interactions of BATF3 in lymphoma cells, we retrovirally transduced murine mature T and B cells with a BATF3-encoding viral vector and transplanted each population into Rag1-deficient recipients. Intriguingly, BATF3-expressing B lymphocytes readily induced B-cell lymphomas after characteristic latencies, whereas T-cell transplanted animals remained healthy throughout the observation time. Further analyses revealed a germinal center B-cell-like phenotype of most BATF3-initiated lymphomas. In a multiple myeloma cell line, BATF3 inhibited BLIMP1 expression, potentially illuminating an oncogenic action of BATF3 in B-cell lymphomagenesis. In conclusion, BATF3 overexpression induces malignant transformation of mature B cells and might serve as a potential target in B-cell lymphoma treatment. Lymphomas are a diverse group of cancers derived from B or T lymphocytes, and rarely from natural killer cells. The development of B- and T-cell lymphomas is closely linked to lymphocyte-specific genetic alterations . EspeciaBesides lesions that induce proliferation or prevent apoptosis, another mechanism of B-cell transformation involves a block in the differentiation of GC B cells into resting memory B or plasma cells . During The basic leucine zipper protein ATF-like 3 (BATF3) is another member of the AP-1 family and favors heterodimerization with other family members like JUN . BATF3 wIn order to investigate the resemblance between tumor cells in classical Hodgkin lymphoma (HL) and their corresponding normal B-cell counterpart, we compared gene expression profiles (GEP) of isolated Hodgkin and Reed/Sternberg (HRS) tumor cells of HL, cases of diffuse large B-cell lymphoma (DLBCL), several HL cell lines and human GC B cells. These analyses revealed strong and specific overexpression of BATF3 in HL cell lines as well as primary HRS cells in comparision to normal GC B cells and DLBCL , 14. In In previous studies, we performed GEP of HL, DLBCL, ALCL, and normal B and T cells –16. WhenBATF3 mutation analysis was performed for all ALCL and DLBCL primary cases and three HL cell lines . In none of the samples we identified any mutations in the coding sequence of BATF3 (data not shown).in vitro stimulation of the isolated lymphocytes, we retrovirally transduced the cells with the human BATF3 gene, which has 80% aminoacid sequence identity with the murine counterpart. The BATF3 encoding gammaretroviral vector coexpressed enhanced green fluorescence protein (EGFP) as a marker gene via an internal ribosomal entry site (IRES) to enable detection of transduced cells -transgenic OT-II T cells. Intriguingly, after transplantation of BATF3-transduced B cells, all 14 transplanted animals developed malignancies with a latency of 56-213 days  cell lines served as positive control for upregulated BLIMP1 expression Figure . This prAs a next step, we wanted to functionally analyze the potency of BATF3-mediated inhibition of BLIMP1 expression in malignant B cells. Therefore, we analyzed BLIMP1-expressing MM cell lines, although in these cells BLIMP1 has an oncogenic and not a tumor-suppressive function as in mature B-cell lymphomas. We first tested BLIMP1-dependent survival of MM cell lines MOLP8 and L363. As expected and already reported , both ceNext, we transduced the BLIMP1-expressing cell lines with BATF3, to enforce inhibition of AP-1 and thereby BLIMP1 expression. As expected, in MOLP8 cells BATF3-overexpression resulted in downregulation of BLIMP1. Surprisingly, ectopic expression of BATF3 led to an unaffected BLIMP1 levels in L363 cells Figure . Consequ− DLBCL, in over 60% of CD30+ DLBCL, and in about 90% of primary mediastinal B cell lymphomas [BATF3 is a direct target of STAT factors and of the PI3K/AKT pathway [In a study of differential gene expression of HL cell lines, we observed increased BATF3-expression . This fiymphomas . Notablyymphomas , 21. Alt pathway , 21. As  pathway –25, STAT pathway , 21.BATF3 and transplanted the cells into immunocompromised recipients. T-cell transplanted animals did not show any sign of malignancy during the whole observation time of more than 250 days. Moreover, we overexpressed BATF3 in human T-cell lines, but did not observe any change in proliferation or enhanced cell survival (data not shown). Most likely, BATF3 is not a strong oncogene in T cells and needs further synergistic genetic events in order to induce transformation of mature T cells. Nevertheless, we recently showed that downregulation of BATF3 in ALCL cell lines is toxic for the cells, supporting oncogenic features of BATF3 also in this T-cell malignancy [To investigate the potential tumor-initiating capacity of BATF3 in lymphomagenesis of B and T cells, we retrovirally transduced murine mature T and B cells with human lignancy . In huma+ T cells expressed an irrelevant transgenic, ovalbumin-specific TCR, so that we can exclude a T-cell dependent GC formation in our mouse model. Moreover, lymphoma-affected tissues of diseased animals featured a low number of accompanying T cells (data not shown). Unexpectedly, the B-cell tumors that developed in the transplanted mice mostly had a GC B-cell phenotype, with expression of the GC B-cell markers Fas and GL7 by nearly all lymphomas, and Bcl6 and PNA each by about half of the cases , OT-II and Rag1-deficient (CD45.2+) mice were obtained from Charles River Laboratories  and from the commercial breading facility mfd Diagnostics GmbH . Animals were maintained under specific pathogen-free (SPF)-like conditions in the animal facilities of the Georg-Speyer-Haus . Cages were individually ventilated. Animals were killed after anesthesia by cervical dislocation. Diseased animals were examined for pathologic abnormalities, including histology, morphology, white blood cell counts and flow cytometry. The experiments were performed in compliance with the local animal experimentation guidelines. Animal experiments were approved by the regional council .Six- to eight-week-old C57BL/6J.Ly5.1 . Control vector MP91-EGFP was described elsewhere [The human lsewhere . The synBLIMP1-specific targeting oligonucleotides and non-specific sequences as a control (described elsewhere ) were us® (Life Technologies) supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin/streptomycin (Pen/Strep). Retroviral supernatant was produced in a split genome approach by calcium phosphate mediated transient transfection of HEK 293T human embryonic kidney producer cells. After 36 and 48 hours supernatant was collected, filtered (0.2 μm pores) and stored at 4°C. All supernatants were titrated on the embryonic murine fibroblast SC-1 cell line or HEK 293T cells.Vector supernatants were produced in DMEM GlutaMax®, Life Technologies). Stimulation and transduction of T cells was previously described [6 transduced mature T or B cells were injected intravenously into the tail vein of Rag1-deficient recipient mice.Murine B and T cells were isolated from spleen and lymph nodes  of C57BL/6J.Ly5.1 donor mice. After preparation of single cell suspensions untouched isolation of the two different cell types was performed with the respective isolation kits ; T cells: Dynabeadsescribed . B cells2). Cell lines were grown in RPMI 1640 GlutaMax™ (Life Technologies), with 1% Pen/Strep. L363 cells were cultured with 10% FCS, and MOLP8 were cultured with 20% FCS.Myeloma cell lines L363 and MOLP8  were transduced on retronectin-coated, vector supernatant-preloaded  non-tissue culture plates. Three days after transduction Cerulean- or EGFP-expressing cells were enriched via fluorescence activated cell sorting on an S3-Cell sorter  and cultured under standard culture conditions , supplemented with 10% FBS (Biochrom), 1% L-glutamine, 1% Pen/Strep, 1% sodium pyruvate, 1% nonessential amino acids, and 0.1% β-mercaptoethanol (Life Technologies). After two weeks in culture, the surviving, non-adhering cells were isolated and further cultivated. Three B-cell lines (T1-T3) were raised from primary mouse tumors.®450-conjugated anti-MHC-II (M5/114.152.2) phycoerythrin (PE) conjugated anti-IgG, APC conjugated anti-IgD (11-26c),  APC conjugated anti-CD45R/B220 (RA3-6B2), APC conjugated anti-CD83 (Michel-19), allophycocyanin cyanine7 (APC-Cy7)-conjugated anti-Igκ light chain (RMK-45), (Becton Dickinson (BD) Pharmingen, Heidelberg, Germany) PE-Cy7 conjugated anti-CXCR5 (2G8), APC conjugated anti-CD138 (281-2), PE conjugated anti-GL7 (Miltenyi Biotec), VioBlue conjugated anti-CD8a (53-6.7); hamster anti-mouse monoclonal antibody (BD Horizon), BV421 conjugated anti-Fas (Jo2); goat anti-mouse polyclonal antibody , PE conjugated anti-Igλ light chain. To prevent nonspecific binding of antibodies to Fc receptors, samples were incubated with mouse FcR Blocking Reagent (Miltenyi Biotec). Before staining, samples of blood, bone marrow and spleen were treated with Lysing Buffer (BD) to remove erythrocytes. Propidium iodide (Miltenyi Biotec) was used to exclude dead cells. Apoptotic cells were determined by Annexin V-APC staining (BD Pharmingen) and cell proliferation assay was performed by CellTrace Violet (Life Technologies) staining, according to the manufacturer´s protocols. All flow cytometric analyses were performed on a MACSQuant (Miltenyi Biotec) using the FlowJo 8.7 software .Flow cytometric analyses were performed on peripheral blood and single-cell suspensions of spleen, lymph nodes, bone marrow and solid tumors. The following anti-mouse antibodies were used for staining: rat anti-mouse monoclonal antibodies  phycoerythrin cyanine7 (PE-Cy7) conjugated anti-CD19 (1D3), PE-Cy7 conjugated anti-IgG1 (M1-14D12), PE-Cy7 conjugated anti-CD38 (90), PE-Cy7 conjugated anti-CD86 (GL1), allophycocyanin (APC)-conjugated anti-IgM (II/41), APC conjugated anti-CXCR4 (2B11), eFluor6 cells were lysed in 1x RIPA buffer , including protease inhibitor cocktail . Protein concentration was determined by DC protein assay (Bio-Rad Laboratories) and 40 μg per lane and sample were loaded. Western blotting was performed according to the operating instructions of the device manufacturer. Rabbit anti-mouse/−human antibodies were used in a 1:1000 dilution: , anti-Blimp1 (C14A4) , anti-HSP70 , anti-JUN , and mouse anti-human BATF3 antibody (3H1)  in a 1:5000 dilution and as loading control mouse anti-mouse/−human antibody anti-β-actin (AC-74)  in a 1:80,000 dilution. Secondary antibodies goat anti-rabbit and goat anti-mouse were conjugated with horseradish peroxidase and used in 1:5,000 dilutions .For Western blot analyses, 1-5 × 106 cells were lysed in 1x RIPA buffer , including protease inhibitor cocktail (Roche). 500 μg of each lysate were used according to the operating instructions of protein A/G PLUS-agarose protocol . 500 μg of cellular protein dosed with 2 μg of anti-BATF3 antibody (3H1) (Abnova) were incubated for 1 hour at 4°C. Afterwards 20 μl of protein A/G-agarose were added and again incubated at 4°C overnight. After overnight incubation the samples were prepared and used for Western blot analysis. Western blots were analyzed for BATF3, JUN and Hsp70.For Co-IP 5×10Gene expression profiling data were taken from data sets GSE 12453  and GSE Immunohistochemical stainings were performed on sections of formalin fixed and paraffin embedded HL cases (n = 12), DLBCL cases (n = 21), ALCL cases (n = 18) and murine tumor-infiltrated organs. Slides were stained with rabbit anti-mouse Bcl6 , PNA  and rabbit anti-human BATF3 antibody (Abcam). For detection, the EnVision System-HRP (DAKO) was used.BATF3 were amplified via nested polymerase chain reaction (PCR)  from whole tissues slices of DLBCL cases (n = 21), ALCL cases (n = 18) and three HL cell lines  and the exons of on (PCR) . AmplicoLM-PCR was performed as previously described , 18. The


C. elegans KLP-15 and KLP-16, two highly homologous members of the kinesin-14 family of minus-end-directed kinesins. These proteins localize to the acentriolar oocyte spindle and promote microtubule bundling during spindle assembly; following KLP-15/16 depletion, microtubule bundles form but then collapse into a disorganized array. Surprisingly, despite this defect we found that during anaphase, microtubules are able to reorganize into a bundled array that facilitates chromosome segregation. This phenotype therefore enabled us to identify factors promoting microtubule organization during anaphase, whose contributions are normally undetectable in wild-type worms; we found that SPD-1 (PRC1) bundles microtubules and KLP-18 (kinesin-12) likely sorts those bundles into a functional orientation capable of mediating chromosome segregation. Therefore, our studies have revealed an interplay between distinct mechanisms that together promote spindle formation and chromosome segregation in the absence of structural cues such as centrioles.In many species, oocyte meiosis is carried out in the absence of centrioles. As a result, microtubule organization, spindle assembly, and chromosome segregation proceed by unique mechanisms. Here, we report insights into the principles underlying this specialized form of cell division, through studies of  C. elegans as a model system, we set out to identify factors required for acentriolar spindle assembly in oocytes and found two microtubule motor proteins necessary for this process. When these motors are depleted in oocytes, microtubules fail to form stable bundles during spindle assembly, resulting in severely aberrant spindles. However, we were surprised to find that these disorganized microtubules were then able to reorganize into a spindle capable of segregating chromosomes during anaphase, revealing a second mechanism that can act to bundle and organize spindle microtubules. Studies of this unique anaphase reorganization phenotype then enabled us to uncover new proteins contributing to spindle organization, and to gain insights into the mechanisms driving chromosome segregation in this important cell type. The work presented here therefore deepens our understanding of the molecular mechanisms of acentriolar spindle assembly and chromosome segregation in oocytes.When cells divide, they must assemble a microtubule-based structure called a spindle on which the chromosomes are segregated. While in most cell types the microtubules that comprise the spindle are nucleated and organized by centriole-containing centrosomes, female reproductive cells (oocytes) of many species lack centrioles and therefore spindles in these cells assemble using unique mechanisms. Using  During mitosis, centriole-containing centrosomes duplicate and then move to opposite ends of the cell where they nucleate microtubules and form the spindle poles. However, oocytes of many species lack centrioles, and as a result, spindles in these cells assemble using a different pathway . We are C. elegans oocyte meiosis as a model, we recently found that acentriolar spindle assembly in this system proceeds by: 1) formation of a cage-like structure comprised of prominent bundles of microtubules that are constrained by the disassembling nuclear envelope, 2) reorganization of this structure such that the microtubule minus-ends are sorted away from the chromosomes to the periphery of the array where they are focused into multiple nascent poles, and then 3) coalescence of these poles until bipolarity is achieved . Aur. Aur4]. klp-15/16(RNAi) spindles, we found that the microtubule ball configuration observed in our imaging (klp-15/16(RNAi) anaphase spindles may not be organized like in wild-type spindles, where a high concentration of microtubule minus-ends are found at the spindle poles. To test this hypothesis, we assessed the localization of ASPM-1 and KLP-18 (a kinesin that is enriched at the poles of wild-type oocyte spindles  II (gift from Marie Delattre)ANA065: adeIs1 II; ltIs37 IV (gift from Marie Delattre)ANA072: zen-4(or153) IV (from the CGC). For experiments using zen-4(or153), plates were shifted to 25°C 16–18 hours before dissection and fixation.EU716: unc-119(ed3) ruIs32[unc-119(+) pie-1::GFP::H2B] III; ruIs57[unc-119(+) pie-1::GFP::tubulin] (gift from Bruce Bowerman)EU1067: unc-119(ed3) III; ltIs37 IV; ltIs25 IV (gift from Arshad Desai)OD57: klp-15(ok1958) I. ok1958 is a deletion allele of the last 391 amino acids of KLP-15 (from the CGC)RB1593: klp-18(tm2841)IV/nT1[qIs51]; unc-119(ed3) ruIs32[unc-119(+) pie-1::GFP::H2B] III; ruIs57[unc-119(+) pie-1::GFP::tubulin] SMW13: klp-16(wig1) I. This strain was generated using a CRISPR approach detailed below.SMW15: Pklp-16::klp-16::GFP (C1971>A–PAM site mutation) I. This strain was generated using a CRISPR approach detailed below.SMW16: Pklp-16::klp-16::GFP (C1971>A–PAM site mutation) I; ltIs37 [(pAA64) pie-1::mCherry::his-58 + unc-119(+)] IVSMW18: (SMW16 x OD56) dpy-10 crRNA (5’—GCUACCAUAGGCACCACGAG- 3’) (IDT), 1.34μM dpy-10 repair oligo , 9.6μM klp-16 crRNA (5’—UGUCUAGUUCAUAGACAUCU- 3’) (IDT); and 136ng/μL ssDNA klp-16 repair template into N2 worms, that were then allowed to produce progeny. Worms from plates containing rollers and dumpys were screened for GFP expression, and homozygous KLP-16::GFP worms were identified by PCR screening. To make the klp-16 repair template, we generated a C-terminal LAP tag using a GBlock (IDT) and Gibson Assembly to create an S-TEV-GFP construct. The tag was then amplified using PCR with primers that contained homology to the klp-16 gene with the final product containing 68 bp of homology upstream of the klp-16 stop codon and 100 bp of homology downstream of the stop codon. ssDNA was generated by asymmetric PCR. SMW16 (KLP-16::GFP) was also crossed with OD56 (mCherry::histone) to generate SMW18: Pklp-16::klp-16::GFP (C1971>A—PAM site mutation) I; ltIs37 [(pAA64) pie-1::mCherry::his-58 + unc-119(+)] IV.A CRISPR-based approach , 55 was klp-16 locus beginning ~100 bp upstream of the start codon. Essentially the same approach was used as above; the differences being two crRNAs (4.8μM) (5’- AGGCGGAGUUUAAGUUUGAG-3’ and 5’- CUCCUCAAGAAGCGUCACUU-3’) (IDT) , and a ssDNA oligo (4μM)  were used in the injection mix. The repair Ultramer sequence is homologous to the sequence just upstream and downstream to the two CRISPR cut sites thereby deleting the ~600 base pairs. Worms from plates containing rollers and dumpys were screened by PCR and homozygous mutants were isolated.A CRISPR-based approach similar to the one above was used to generate a worm strain with a ~600 bp deletion in the Protein domains were determined using PsiPred  and PairFrom a feeding library , 59, indYoung adult worms grown on control plates or plates containing RNAi-expressing bacteria were transferred to new plates containing either control or RNAi-expressing bacteria and allowed to lay eggs for 24 hours at 15°C before being moved to another fresh plate of either control or RNAi-expressing bacteria. The eggs were allowed to hatch for 24 hours and then the progeny (eggs and hatched worms) were counted. For each parent worm this process was repeated twice, resulting in three days of progeny being counted. For each condition, the progeny of at least 15 worms were scored.p-phenylenediamine, 20mM Tris-Cl, pH 8.8, 90% glycerol or ProLong Gold antifade Mountant (Molecular Probes), and sealed with nail polish; except for the overnight primary, the entire procedure was performed at room temperature. For experiments using the rabbit anti-KLP-16 antibody and staining of SPD-1::GFP with mouse anti-GFP, embryos were blocked in AbDil overnight at 4°C and incubated in primary antibody for 2 hours at room temperature. Primary antibodies used in this study: rabbit anti-ASPM-1 , rabbit anti-SEP-1 , rabbit anti-KLP-18 , rabbit anti-ZEN-4 , mouse anti-SUMO , mouse anti-GFP . Rat anti-AIR-2 was generated by Covance using the C-terminal peptide sequence KIRAEKQQKIEKEASLRNH (synthesized by the Peptide Synthesis Core Facility at Northwestern University), then affinity purified and used at 1:1000. Rabbit anti-KLP-16 was generated by Covance using the N-terminal peptide sequence CMNVARRRSGLFRSTIGAPPK (synthesized by the Peptide Synthesis Core Facility at Northwestern University), then affinity purified and used at 1:2000. Rabbit anti-SPD-1 was generated by Proteintech using the C-terminal peptide sequence CIASSTPSSAKKVLTRRNQFL, then affinity purified and used at 1:1000. Directly conjugated mouse anti-α-tubulin-FITC  and Alexa-fluor directly conjugated secondary antibodies (Invitrogen) were used at 1:500. All antibodies were diluted in AbDil.Immunofluorescence was performed by freeze cracking embryos and plunging into -20°C methanol as described . EmbryosAll fixed imaging and high resolution imaging of KLP-16::GFP and KLP-16::GFP;mCherry::histone was performed on a DeltaVision Core deconvolution microscope with a 100x objective (NA = 1.4) (Applied Precision). This microscope is housed in the Northwestern University Biological Imaging Facility supported by the NU Office for Research. Image stacks were obtained at 0.2μm z-steps and deconvolved using SoftWoRx (Applied Precision). All images in this study were deconvolved and displayed as full maximum intensity projections of data stacks encompassing the entire spindle structure, unless stated otherwise. For KLP-16::GFP and KLP-16::GFP;mCherry::histone imaging, live worms were mounted in anesthetic .Two-color live imaging was performed using a spinning disk confocal microscope with a 63x HC PL APO 1.40 NA objective lens. A spinning disk confocal unit  attached to an inverted microscope (Leica DMI6000 SD) and a Spectral Applied Imaging laser merge ILE3030 and a back-thinned electron-multiplying charge-coupled device (EMCCD) camera (Photometrics Evolve 521 Delta) were used for image acquisition. The microscope and attached devices were controlled using Metamorph Image Series Environment software (Molecular Devices). Typically, ten to twelve z-stacks at 1μm increments were taken every 20–45 seconds at room temperature. Image deconvolution was done using AutoQuant X3 (Media Cybernetics Inc.). Images were processed using ImageJ. Images are shown as maximum intensity projections of entire spindle structure. Live, intact worms were mounted on 5% agarose, M9 pads in 50% live imaging solution , 50% 0.1 micron polystyrene Microspheres (Polysciences Inc.), and covered with a coverslip. The spinning disk microscope is housed in the Northwestern University Biological Imaging Facility supported by the NU Office for Research.For  Slides made on the same day were imaged within an 8 hour window on a DeltaVision Core deconvolution microscope (see Microscopy section) using the same exposure conditions and times for all slides. In ImageJ, linescans of 154 x 75 pixels (L x W) were performed on 6 z-slice sum projections of representative spindles from control (n = 8) and klp-15/16(RNAi) (n = 9) embryos. In control spindles, the linescans were done along the pole-to-pole axis. In spindles from klp-15/16(RNAi) embryos, linescans were done straight along the x-axis of the image, since these spindles lack a discernable orientation. The average fluorescence intensity for each channel was graphed (solid line) along with the SEM (standard error of the mean) (shaded area) using the ggplot package in R Studio. The y-axes of the graphs are the same between control and experiment for a given channel. α-KLP-16 staining for each stage of spindle assembly in wild-type oocytes/embryos. Oocytes in prophase with the nuclear envelope intact were scored as “localized” if the KLP-15/16 signal was primarily cytoplasmic. During all other stages, oocytes were scored as “localized” if the KLP-15/16 signal was colocalized with spindle microtubules. The quantification is as follows: diakinesis 81.8% (n = 22), cage 0% (n = 13), multipolar 31.3% (n = 67), bipolar 64% (n = 114), anaphase 51.9% (n = 79), mitotic spindles 3.7% (n = 27). Although not every spindle is stained, we think that this represents variability with the immunofluorescence procedure and with the antibody  oocytes. The number of oocytes in which we could discern any spindle staining is as follows: microtubule ball stage 8% (n = 75), anaphase 2.9% (n = 35). α-KLP-16 staining was scored in klp-15(ok1958) oocytes. Oocytes in prophase with the nuclear envelope intact were scored as “localized” if the KLP-15/16 signal was primarily cytoplasmic. During all other stages, oocytes were scored as “localized” if the KLP-15/16 signal was colocalized with spindle microtubules. The quantification is as follows: diakinesis 100% (n = 3), cage 14% (n = 7), multipolar 44% (n = 25), bipolar 72.2% (n = 18), anaphase 38% (n = 21). α-KLP-16 staining was scored in klp-16(wig1) oocytes as above. The quantification is as follows: diakinesis 100% (n = 10), cage 42.9% (n = 7), multipolar 89% (n = 19), bipolar 81.8% (n = 11), anaphase 79.2% (n = 24). As with the control strain (see  Linescans of control anaphase spindles and anaphase spindles from klp-15/16(RNAi) oocytes stained for ASPM-1 were performed using the arbitrary profile tool in SoftWorx (Applied Precision). A spindle was scored as having staining at poles if the ASPM-1 signal was enriched at two ends of the spindle near the segregating chromosomes. ASPM-1 was enriched at the poles of 21/24 control spindles, but was largely diffuse along spindle microtubules in anaphase of klp-15/16(RNAi) oocytes . Linescans of control anaphase spindles and anaphase spindles from klp-15/16(RNAi) oocytes stained for KLP-18 were performed using the arbitrary profile tool in SoftWorx (Applied Precision). A spindle was scored as having staining at poles if the KLP-18 signal was enriched at two ends of the spindle near the segregating chromosomes. KLP-18 was enriched at poles in 11/12 control spindles but was diffuse in klp-15/16(RNAi) spindles (0/6 had KLP-18 concentrated into poles). Aneuploidy in MII embryos was quantified by counting the number of chromosomes in MII in immunofluorescence images of control and klp-15/16(RNAi) embryos. An embryo was scored as ‘aneuploid’ if the number of chromosomes was not 6. ZEN-4 and SPD-1 staining was scored in control spindles and spindles from klp-15/16(RNAi) oocytes. Staining of metaphase and anaphase spindles  was scored for each condition. ZEN-4: Control metaphase 1.6% (n = 63), Control anaphase 90% (n = 20); klp-15/16(RNAi) metaphase 2.9% (n = 134), klp-15/16(RNAi) anaphase 80.3% (n = 66). SPD-1: Control metaphase 9.5% (n = 42), Control anaphase 97% (n = 66); klp-15/16(RNAi) metaphase 1% (n = 100), klp-15/16(RNAi) anaphase 88.2% (n = 85). Quantification of microtubule bundling and chromosome segregation was done using immunofluorescence images of anaphase spindles with SEP-1 gone and AIR-2 relocalized to the microtubules (mid/late anaphase) for the conditions shown. We scored microtubule bundling by eye, looking through the entire z-stack in SoftWorx (Applied Precision). An anaphase spindle was scored as “bundled” if one or more microtubule bundles were discernable. Chromosomes were scored as “segregated” if two or more distinct masses of chromosomes were observed. The simple matching coefficient (SMC) for microtubule bundling and chromosome segregation = 0.82 (n = 251); in other words, 82% of the spindles were scored as microtubules bundled and chromosomes segregated or as no microtubule bundles and no chromosome segregation. To approximate anaphase microtubule lengths, we used the measure distances tool in SoftWorx (Applied Precision). Using this tool, a line was manually drawn (point by point) along the most prominent spindle microtubule bundle through the 3D stack of an image to measure its full length. Polar body number and maternal pronuclei number were quantified by scoring live EU1067 worms mounted in anesthetic  on a Leica DM5500B fluorescent microscope. Linescans of control anaphase spindles and anaphase spindles from klp-15/16(RNAi) oocytes stained for tubulin, SUMO, and SPD-1 were performed using the arbitrary profile tool in SoftWorx (Applied Precision). A spindle was scored as having oscillations if one or more instances of alternating MTs/SUMO and SUMO/SPD-1 signal was observed. This analysis was done by examining both single z-slices and max projections of spindles. Oscillations were observed in 9/11 control anaphase spindles and 12/18 klp-15/16(RNAi) anaphase spindles. Lateral microtubule associations to chromosomes were scored in control anaphase spindles and anaphase spindles from klp-15/16(RNAi) oocytes. A spindle was scored as having lateral microtubule/chromosome associations if a microtubule appeared to contact and run along the side of a chromosome. This analysis was done by examining both single z-slices and max projections of spindles. We observed clear lateral associations in 31/35 control anaphase spindles and in 22/31 klp-15/16(RNAi) anaphase spindles. Live, intact worms expressing GFP::tubulin, GFP::histone (EU1067) fed either control or klp-16(RNAi)-expressing bacteria were anesthetized in 0.2% tricaine, 0.02% levamisole in M9 and viewed on a Leica DM5500B widefield fluorescence microscope. Spindles in embryos in the -1, spermatheca, and +1 positions within the gonad were scored for microtubule organization by eye. A spindle was scored as “multipolar” if it had prominent microtubule bundles that formed more than two organized poles. A spindle was scored as an “array” if the microtubule structure lacked prominent bundles and organized poles. A spindle was scored as “MT ball” if the microtubule structure had collapsed around the chromosomes and lacked prominent bundles and organized poles. Spindle volumes were measured using the surfaces tool in Imaris (Bitplane). Using the full 3D image stack, this tool renders a 3D surface based on fluorescence signal . The volume of this 3D surface is then measured. The volumes of metaphase and early anaphase spindles  from klp-15/16(RNAi) oocytes were measured and compared to the volumes of the spindles used for the linescan measurements in  Live, intact worms expressing KLP-16::GFP, mCherry::histone (SMW18) were anesthetized in 0.2% tricaine, 0.02% levamisole in M9 and viewed on a Leica DM5500B widefield fluorescence microscope. The localization of KLP-16::GFP was scored in oocytes/embryos in the -1, spermatheca, and +1 positions within the gonad. KLP-16::GFP signal was scored as cytoplasmic if it was absent/reduced in the nucleus. Because the localization of KLP-16::GFP on the spindle looks identical to the organization of GFP::tubulin, we scored localization for the following categories: cage, multipolar, bipolar, and anaphase. The organization of the chromosomes visualized by mCherry::histone was used to identify and better stage the spindles. α-GFP staining for each stage of spindle assembly in SMW16 (KLP-16::GFP). Oocytes in prophase with the nuclear envelope intact were scored as “localized” if the α-GFP signal was primarily cytoplasmic. During all other stages, oocytes were scored as “localized” if the α-GFP signal was colocalized with spindle microtubules. The quantification is as follows: diakinesis 100% (n = 2), cage 0% (n = 4), multipolar 76.5% (n = 17), bipolar 96.2% (n = 26), anaphase 83.3% (n = 24), mitotic spindles 100% (n = 5). As with the KLP-15/16 antibody, we think that the lack of staining in all embryos represents variability with the immunofluorescence procedure, since we see robust localization of KLP-16::GFP to microtubules when we visualize this strain live.klp-16(RNAi) (EU1067 and SMW15), or klp-15(RNAi) (RB1593) plates onto new, empty (no bacteria) plates. The worms were washed off the plates with cold M9 and transferred to a 1.5ml microcentrifuge tube. Worms were pelleted by spinning at 2000 rpm for 1 minute, and the tube was put on ice for ~2 minutes to allow worms to slow down and form a tight pellet. The M9 was removed and the tube was filled with fresh, cold M9 and mixed. The worms were washed a total of 3 times. After the final wash, as much M9 was removed as possible and 2X SDS sample buffer was added to the remaining worm/M9 mixture and boiled for 10 minutes. Samples were run on a 10% SDS-PAGE gel and blotted. For western analysis, rabbit anti-KLP-16 antibody  and mouse anti-tubulin (1:5000)  were used.Seventy-five EU1067, RB1593, or SMW15 worms were picked off of control, S1 Figklp-15(RNAi) and the dashed line denotes the region of complementarity to klp-16(RNAi). The solid line under the N-terminal 20 amino acids marks the peptide sequence that was used to make our KLP-15/16 antibody; the red shaded residue is the single amino acid difference between KLP-15 and KLP-16 in the sequence used to make the antibody.Residues shaded in green are identical between the two proteins. Prolines in the proline-rich tail (1–149 aa) are denoted with asterisks. The dotted line denotes the region of complementarity to (TIF)Click here for additional data file.S2 Figklp-15(ok1958), and klp-16(wig1) worms. The wild-type and klp-16(wig1) worms were fed bacteria expressing the RNAi clone annotated as targeting KLP-16, and klp-15(ok1958) worms were fed bacteria expressing the RNAi clone annotated as targeting KLP-15. (B) Diagram of the C. elegans germline. The germline is organized in an assembly-line fashion where oocytes in prophase (-3 to -1 positions) are ovulated into the spermatheca where they are fertilized. These fertilized embryos begin the meiotic divisions and exit the spermatheca to the +1 position where they continue to progress through meiosis and subsequently mitosis. This organization enables staging of spindles based on the position of the oocyte/embryo in the germline. (C) Quantification of spindle phenotypes in control and klp-15/16(RNAi) worms. This analysis was done using live worms expressing GFP::tubulin, GFP::histone. n represents the number of oocytes/embryos analyzed for each condition.(A) Results of embryonic lethality assays from wild-type, (TIF)Click here for additional data file.S3 Figklp-15/16(RNAi) microtubule ball images used for the linescan analysis performed in klp-15(ok1598) and klp-16(wig1) oocytes, spindles are indistinguishable from wild-type spindles. n represents the number of spindles observed for each condition. Bars = 2.5 μm.(A) Shown are DNA (blue), microtubules (green), and ASPM-1 (red) for the nine (TIF)Click here for additional data file.S4 Fig(A) Quantification of KLP-16 localization in live worms expressing KLP-16::GFP and mCherry::histone (SMW18). KLP-16 localization was scored as “cytoplasmic” if the GFP signal was absent from inside the nucleus. At all other stages, the GFP signal was enriched on the spindle near the chromosomes. n represents the number of oocytes/embryos analyzed for each condition. (B and C) Examples of KLP-16 localization in live worms expressing KLP-16::GFP (SMW16). (B) KLP-16::GFP is absent from the nucleus prior to spindle assembly. During spindle assembly, KLP-16 localizes to the spindle microtubule bundles and remains associated through anaphase; the image of the cage stage is a sum projection of the spindle structure. (C) KLP-16 localizes to mitotic spindle microtubules and centrosomes in one-cell stage embryos. (D and E) DNA (blue), microtubules (green), GFP (red). Meiotic (D) and mitotic (E) spindles from worms expressing KLP-16::GFP (SMW16) were stained with an anti-GFP antibody. The staining pattern of KLP-16::GFP on meiotic spindles (D) is identical to the staining pattern using the anti-KLP-15/16 antibody. (E) KLP-16::GFP was detected on spindle microtubules and centrosomes . The mitotic image was not deconvolved. Bars = (B and D) 2.5 μm; (C and E) 10 μm.(TIF)Click here for additional data file.S5 Figklp-18(tm2841) mutant strain, early anaphase spindles  begin as a ball of microtubules surrounding the chromosomes, in both control and klp-15/16(RNAi) treated oocytes. n represents the number of spindles observed for each condition.DNA (blue), tubulin (green), AIR-2 (not in merge), and SEP-1 (red in merge). In the (TIF)Click here for additional data file.S1 MovieBar = 10 μm.(MOV)Click here for additional data file.S2 MovieBar = 10 μm.(MOV)Click here for additional data file.S3 MovieBar = 2 μm.(MOV)Click here for additional data file.S4 MovieBar = 2 μm.(MOV)Click here for additional data file.S5 MovieMitotically dividing cell is marked with an asterisk. Bar = 10 μm.(MOV)Click here for additional data file.S6 Movieklp-15/16(RNAi)).Bar = 5 μm (wild-type); 10 μm ((MOV)Click here for additional data file.S7 MovieBar = 5 μm.(MOV)Click here for additional data file.


Here we show the identification of tilivalline and analogs thereof in the entomopathogenic bacterium Xenorhabdus eapokensis as well as the identification of its biosynthesis gene cluster encoding a bimodular non-ribosomal peptide synthetase. Heterologous expression of both genes in E. coli resulted in the production of 1 and from mutasynthesis and precursor directed biosynthesis 11 new tilivalline analogs were identified in X. eapokensis. These results allowed the prediction of the tilivalline biosynthesis being similar to that in K. oxytoca.The pyrrolobenzodiazepine tilivalline ( Xenorhabdus live in symbiosis with nematodes of the genus Steinernema. Soil living infective juvenile nematodes are carrying Xenorhabdus, hunting and killing insect larvae as food source and reproductive space + , S1 Fig 0H19N3O2  characteN3O2 (1) . A subse oxytoca  and a syX. eapokensis extraction samples using the GNPS network analysis tool + (5)  and four derivatives thereof: 9-deoxy-TV (2) and the three hitherto undescribed analogs dihydroxy TV (3), dehydro TV (4) and dehydro-dihydroxy TV (5) , Figs , Table 1 missing  as was a oxytoca . Its typred to 1 . Its MS/red to 1  shows a ure data ,34,36, anome see    and m/z +H]+ (5)  are charanalysis . Albeit D C-ring . In summX. eapokensis cultivation extract that correlate to the first NRPS derived intermediate named tilimycin or kleboxymycin (16) + were plotted against concentration, where 1:10,000, 1:2000 and 1:1000 resulted in a linear function (R2 = 0.998) used for quantification. The calibration curve is shown in A 0.5 mg TV standard was dissolved in 1 mL methanol and dilutions were generated in a range from 1:1000 to 1:10,000 and measured in technical triplicates using UPLC-HR-MS. Signal area counts of EIC S1 Fig1) chemical structure. (B) Extracted ion chromatogram (EIC) of 1, m/z 334.1550 [M+H]+. MS/MS spectrum of natural (C) and synthetic 1 (D). Mass signals are annotated with predicted sum formulas, detection errors and putative molecule fragment structures. Red diamond indicates precursor ion.(A) Structure of tilivallin ((DOCX)Click here for additional data file.S2 Fig4). (B) MS/MS spectrum of dehydro-dihydroxy TV (5). Mass signals are annotated with predicted sum formulas, detection errors and putative molecule fragment structures. Red diamond indicates precursor ion.(A) MS/MS spectrum of dehydro-TV ((DOCX)Click here for additional data file.S3 Fig6), (B) 7-chloro-pyrrolobenzodiazepine (7), (C) 9-methyl-pyrrolobenzodiazepine (8), (D) 7-methyl-pyrrolobenzodiazepine (9), (E) 9-methoxy-pyrrolobenzodiazepine (10), (F) 1-methyl-9-hydroxy-pyrrolobenzodiazepine (11), (G) 9-hydroxy-5’-fluoro-pyrrolobenzodiazepine (12), (H) 9-hydroxy-5’phenyl-pyrrolobenzodiazepine (13), (I) 9-hydroxy-5’-methyl-pyrrolobenzodiazepine (14), and (J) 9-hydroxy-azaindole-pyrrolobenzodiazepine (15). Mass signals are annotated with predicted sum formulas, detection errors and putative molecule fragment structures. Red diamond indicates precursor ion.Shown are MS/MS spectra of (A) 9-chloro-pyrrolobenzodiazepine ((DOCX)Click here for additional data file.S4 Fig16 and 17 with corresponding fragment mass spectra (MS/MS) . Extracted ion chromatogram (EIC) of TV intermediates (DOCX)Click here for additional data file.S5 Figm/z 334.1550 [M+H]+ corresponding to the used dilution series . Each data point represents the average area count of a triplicate measurement . Graphical error indicators are below display limit. Regression equation and coefficient of determination are given in the diagram.Plotted are signal area counts of EIC (DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table“*” indicates no NCBI accession number available. “-”indicates no homolog was detected by pBLAST search.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Salmonella enterica subsp. enterica serovar Infantis strains were isolated from livestock for the first time in Germany. Here, we announce the complete genome sequence of the first German blaVIM-1-harboring Salmonella Infantis isolate (15-SA01028) originating from food.Carbapenems are considered last-resort antibiotics used to treat human infections caused by multidrug-resistant bacteria. In 2011, VIM-1 carbapenemase-producing  Salmonella enterica subsp. enterica serovar Infantis is an important zoonotic pathogen causing gastroenteritis in humans. This pathogen is commonly transferred via contaminated food products  showed microbiological resistance to meropenem , imipenem , and ertapenem  using the microdilution method (CLSI guideline CLSI M07-A9 ) and harbors 12 antibiotic resistance genes. The blaVIM-1 gene was located on a Tn21-like transposon harboring two class 1 integrons containing, apart from the sul1 gene, further resistance gene cassettes aadA1-ere(A) and aadA1-aacA4-blaVIM-1.Genome analysis using the ResFinder3.0 and PlasmidFinder1.3 tools provided by the Center for Genomic Epidemiology (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/). The results revealed the presence of 5,029 coding sequences , as well as 126 RNA genes  on the bacterial chromosome. With this sequence, we provide a high-quality reference for genome comparison studies.Genome annotation was performed using the automated Prokaryotic Genome Annotation Pipeline (CP026660 (chromosome) and CP026661 (pSE15-SA01028).Sequences were deposited in GenBank under the accession numbers