Protein synthesis is tightly regulated and alterations to translation are characteristic of many cancers. Translation regulation is largely exerted at initiation through the eukaryotic translation initiation factor 4 F (eIF4F). eIF4F is pivotal for oncogenic signaling as it integrates mitogenic signals to amplify production of pro-growth and pro-survival factors. Convergence of these signals on eIF4F positions this factor as a gatekeeper of malignant fate. While the oncogenic properties of eIF4F have been characterized, genome-wide evaluation of eIF4F translational output is incomplete yet critical for developing novel translation-targeted therapies.To understand the impact of eIF4F on malignancy, we utilized a genome-wide ribosome profiling approach to identify eIF4F-driven mRNAs in MDA-MB-231 breast cancer cells. Using Silvestrol, a selective eIF4A inhibitor, we identify 284 genes that rely on eIF4A for efficient translation. Our screen confirmed several known eIF4F-dependent genes and identified many unrecognized targets of translation regulation. We show that 5′UTR complexity determines Silvestrol-sensitivity and altering 5′UTR structure modifies translational output. We highlight physiological implications of eIF4A inhibition, providing mechanistic insight into eIF4F pro-oncogenic activity.Here we describe the transcriptome-wide consequence of eIF4A inhibition in malignant cells, define mRNA features that confer eIF4A dependence, and provide genetic support for Silvestrol’s anti-oncogenic properties. Importantly, our results show that eIF4A inhibition alters translation of an mRNA subset distinct from those affected by mTOR-mediated eIF4E inhibition. These results have significant implications for therapeutically targeting translation and underscore a dynamic role for eIF4F in remodeling the proteome toward malignancy.The online version of this article (doi:10.1186/s13059-014-0476-1) contains supplementary material, which is available to authorized users. Energetically, protein synthesis is the most costly step on the path toward gene expression and is thus a rigidly controlled process. In eukaryotes, protein synthesis occurs in three phases: translation initiation, elongation and termination. Although translation is controlled at multiple stages, regulation is primarily exerted at initiation, the phase in which 80S ribosomes assemble onto mRNA transcripts. Regulation of initiation is mediated by multiple factors, many of which converge on the assembly of the eukaryotic initiation factor 4F (eIF4F). This heterotrimeric complex is composed of eIF4E, the rate-limiting protein which binds the 5′-7-methylguanosine cap on cellular mRNA transcripts; eIF4A, a DEAD-box RNA helicase; and eIF4G, a scaffolding protein which bridges eIF4E and eIF4A, and recruits eIF3 and the 43S pre-initiation complex. Formation of eIF4F is tightly controlled by multiple mitogenic signaling pathways, namely mitogen-activated protein kinase (MAPK) and phosphoinositide-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR), and has been shown to stimulate translation of mRNAs involved in cell proliferation, growth, survival, cell cycle progression, and DNA damage repair –3. Moreoin vitro [Significant progress has been made toward understanding the machinery that drives protein synthesis. However, the underlying mechanisms by which individual eIF4F components contribute to translation regulation in the cell remain ambiguous. Emerging methods that allow for global dissection of translation have bolstered the long standing knowledge that translation is subject to considerable regulation and thus plays a key role in regulating gene expression –13. Studin vitro . These din vitro and in vivo, thereby impairing translation initiation [eIF4A is an integral part of the heterotrimeric eIF4F complex and the only component with known enzymatic activity. While several eIF4A-regulated genes have been identified, in-depth studies have yet to provide a genome-wide description of the eIF4A target gene landscape. We sought to comprehensively define the cellular mRNAs regulated by eIF4A and investigate the mRNA features that confer dependence on this helicase by directly blocking its activity. To achieve this, we employed the potent and specific eIF4A inhibitor Silvestrol. This compound has been shown to selectively target the RNA helicase activity of eIF4A both itiation ,22, and itiation ,24. Moreitiation . Here we35S-methionine incorporation into newly synthesized proteins. To mitigate off-target or secondary effects of drug treatment, we chose to treat cells with a low concentration of Silvestrol for a short time period. We selected a concentration of 25 nM Silvestrol, which had negligible effects on global translation after 2 hours of exposure  and a difference in distribution of ∆G values from genes with increased TE was also notable compared with the insensitive pool (P =4.25 × 10-7)  while those from genes with increased TE were comparatively shorter  and slightly higher in those from genes with increased TE (P =8.08 × 10-6)  and ROCK1 , were significantly more sensitive to Silvestrol than unstructured controls, CMV alone and PFN2   . The introduction of ARF6mut into our luc2CP reporter decreased the sensitivity of our reporter construct to Silvestrol  analysis of Silvestrol-sensitive genes revealed that a large proportion of eIF4A-dependent genes were involved in cell cycle progression 4 motif is present in both the ARF6 and ARF6mut 5′ UTR reporter constructs .Cells were seeded at a density of 1 × 105 cells/well in 6-well format and grown overnight. 24 hours after seeding, cells were washed in 1× phosphate-buffered saline  for 10 minutes prior to harvest. Cells were lysed in RIPA buffer  containing complete EDTA-free protease inhibitors , and phosphatase inhibitors . Total protein was trichloroacetic acid  and used to normalize radioactive counts in TCA precipitated protein.MDA-MB-231 cells were seeded at a density of 3 × 106 cells/plate in 15 cm plates and grown overnight. Cells were then treated with DMSO or Silvestrol (25 nM) for 2 hours prior to harvest. At time of harvest, 100 μg/ml cycloheximide  was added to each plate and incubated for 1 minute. Cells were washed twice in ice-cold 1× PBS +100 μg/ml cycloheximide and lysed in ice-cold polysome lysis buffer , 1% TritonX-100 ). Sucrose gradients (10 to 50%) were created by layering 10% and 50% sucrose solutions , 10 or 50% RNase-free sucrose) into Seton tubes  followed by mixing with the BioComp Gradient maker set at an 81.5° angle at 16 rpm . Lysates were layered on top of each gradient and subjected to ultracentrifugation in an SW41-Ti rotor at 35,000 rpm at 4°C for 3 hours. Polysome profiles were analyzed using a BioComp Gradient Station at constant speed with optical monitoring at a 260 nm wavelength. For polysome fractionation experiments, 1 ml fractions were collected and stored at -80°C for subsequent RNA isolation followed by quantitative RT-PCR.Cells were seeded at a density of 4 × 102, 1 mM DTT, 8% glycerol, 100 μg/ml cycloheximide (Sigma), 24 U/ml Turbo DNAse (Ambion)). Lysates were clarified, treated with RNAse I (Ambion) and overlaid on a 34% sucrose cushion. Monosomes were isolated by centrifugation at 69,000 rpm for 4 hours in a TLA110 rotor. Ribosome protected RNA fragments (RFs) were isolated from monosome fractions by acidic-phenol extraction and used to generate libraries for sequencing. The genome-aligned RF profiles in Additional file Ribosome profiling was performed as previously described ,41 with Total RNA was extracted from MDA-MB-231 cells using Trizol  following the manufacturer’s guidelines. Polyadenylated RNA was isolated from the total fraction using Oligotex mRNA kit . The resulting mRNA was partially fragmented by alkaline hydrolysis with sodium carbonate to generate approximately 150-nucleotide fragments on average. RNA fragments of 40 to 100 nucleotides were isolated by gel extraction and used to generate libraries for mRNA-Seq. The genome-aligned mRNA profiles in Additional file et al. [Strand-specific libraries were generated as described ,41 with et al. . SamplesSequence analysis was performed as described . Briefly5′ UTR sequences were retrieved from Ensemble using RefSeq mRNA numbers as query. Folding energies of the resulting sequences were analyzed using CONTRAfold  and McCaCyclinD1 and ROCK1 were reverse transcribed from total RNA isolated from MDA-MB-231 cells  and the resulting cDNA was amplified by high-fidelity PCR using 5′ UTR-specific oligonucleotides. The 5′ UTRs for ARF6, ARF6mut and PFN2 were synthesized by Blue Heron Biotechnology . 5′ UTRs were subcloned into the NcoI site of vector pMH2 to create vectors pCR300, pCR301, pCR302, pCR303, and pCR304 . Luciferase assays were performed by adding 100 μl/well Luciferase Assay Reagent (Promega E1501) and incubating for 10 minutes at room temperature before measuring luminescence. For luc2CP mRNA analysis, 4 × 105 cells/well were seeded in 6-well plates and grown overnight. Cells were then treated with the indicated amounts of Silvestrol for 40 minutes and lysed using Trizol reagent. RNA was isolated from Trizol lysates using standard methods. The abundance of luc2CP and β-actin mRNA was subsequently analyzed by quantitative RT-PCR.293 T cells were co-transfected with 5′ UTR-bearing Prior to RNA extraction from polysome fractions, 5 ng of Luciferase Control RNA (Promega) was added to each fraction. Samples were treated with 200 μg/ml Proteinase K for 1 hour at 50°C and RNA was extracted using the standard hot acid phenol method. RNAs were precipitated using isopropanol and resuspended in 10 mM Tris pH7.luc2CP mRNA) levels, transcript abundance was normalized to β-actin mRNA measurements.Total RNA was converted to cDNA using SuperScript III reverse transcriptase (Invitrogen) with oligo dT primers according to the manufacturer’s instructions. Transcript levels were measured by quantitative PCR using SYBR green FAST PCR mix  and oligo pairs specific to each transcript. In polysome fractionation experiments, transcript levels were normalized to luciferase control RNA abundance. To measure luciferase reporter mRNA  antibody  at room temperature for 30 minutes, washed, resuspended in PI/RNase solution (BD Biosciences #550825) and analyzed by fluorescence-activated cell sorting (FACS).MDA-MB-231 cells were seeded in 6-well dishes at 3.5 × 10MDA-MB-231 cells were treated with 25 nM Silvestrol and lysed in RIPA buffer  containing complete EDTA-free protease inhibitors (Roche #11836170001), and phosphatase inhibitors . Lysates were quantified using BCA reagent (Thermo Scientific #23228). Lysates were subjected to SDS-PAGE (NuPAGE) using 15 to 25 μg of cell lysate per well and transferred to PVDF (Invitrogen).The following antibodies were purchased from commercial suppliers: CyclinD1 , CyclinB1 , CyclinE1 , Bcl2 (BD Pharmingen #551097), Parp(Asp214) , Arf6 , GAPDH , β-Tubulin .Cell lines used in this study were obtained from ATCC. Cells were tested for mycoplasma by the Novartis Institutes for Biomedical Research and were mycoplasma-free._CATCTCCATGGGCAGAACTGGGAGGAGGAGT_3′; oARF6rev: 5′_GACCTCCATGGCGCGTCGGAGGAGCCGGGGCCG_3′; oCCND1fwd:_5′_GACCTCCATGGGCTTAACAACAGTAACGTCACACGG_3′; oCCND1rev:_5′_CAGCTCCATGGCTGGGGCTCTTCCTGGGCAG_3′; oROCK1fwd:_5′_GACCTCCATGGGCGCUGGUUCCCCTTCCGAGCGT_3′; oROCK1rev:_5′_CATCTCCATGGGTGTTGCTGCTGCTGTGACAATGCCCT_3′; oPFN2fwd:_5′_GAATCTCCATGGGCCGCTGGTTTGTCAGCC_3′; oPFN2rev:_5′_CTCGAGCCATGGCTTCGAGCCCTTCGC_3′; oARFmut_ampF:_5′_CCATGGTTGGGACGTGCACTGGCAGCCGGC_3′; oARFmut_ampR:_5′_GTGGGAGTTGCCTCCTAAGCTAATATGTGC_3′oARF6fwd: 5′_ATGTTGCAGGTGAGATGTGGT_3′,ARF6_qP_F8: 5′_TACCTGCTCCAGTCACCAATG_3′,ARF6_qP_R8: 5′_AGCCTCGCCTTTGCCGA_3′,ActB_qP_F1: 5′_GCGCGGCGATATCATCATC_3′,ActB_qP_R1: 5′_TTCTGCCCCTGCCAAATCTT_3′,BCL2_qP_F3: 5′_CATCTGAGAACCTCCTCGGC_3′,BCL2_qP_R3: 5′_TGAGGGACGCTTTGTCTGTC_3′,CCND1_qP_F2: 5′_CTTCTGCTGGAAACATGCCG_3′,CCND1_qP_R2: 5′_ACCTGCCCCTTACTCTGACT_3′,CDK6_qP_F2: 5′_AGCACCCAGTAAGACATCCAG_3′,CDK6_qP_R2: 5′_TGAAAGCCGCACTGATGGAT_3′,ROCK1_qP_F1: 5′_GCCATGAGAAAACACATTGCAG_3′,ROCK1_qP_R1: 5′_ATCCGGAAGCGACCAACGCC_3′,Luciferase_F: 5′_GTCGGGAAGACCTGCCACGC_3′,Luciferase_R: 5′_ATCCACCTTAACAGCCACGG_3′,Luc2CP_F: 5′_CAGGGTGTCTATCCATGCCG_3′.Luc2CP_R: 5′ARF6mut sequence. The (CGG)4 motif is shown in bold.Mutated residues are indicated by lower case lettering in the ARF6wt:_5′_AGAACUGGGAGGAGGAGUUGGAGGCCGGAGGGAGCCCGCGCUCGGGGCGGCGGCUGGAGGCAGCGCACCGAGUUCCCGCGAGGAUCCAUGACCUGACGGGGCCCCGGAGCCGCGCUGCCUCUCGGGUGUCCUGGGUCGGUGGGGAGCCCAGUGCUCGCAGGCCGGCGGGCGGGCCGGAGGGCUGCAGUCUCCCUCGCGGUGAGAGGAAGGCGGAGGAGCGGGAACCGCGGCGGCGCUCGCGCGGCGCCUGCGGGGGGAAGGGCAGUUCCGGGCCGGGCCGCGCCUCAGCAGGGCGGCGGCUCCCAGCGCAGUCUCAGGGCCCGGGUGGCGGCGGCGACUGGAGAAAUCAAGUUGUGCGGUCGGUGAUGCCCGAGUGAGCGGGGGGCCUGGGCCUCUGCCCUUAGGAGGCAACUCCCACGCAGGCCGCAAAGGCGCUCUCGCGGCCGAGAGGCUUCGUUUCGGUUUCGCGGCGGCGGCGGCGUUGUUGGCUGAGGGGACCCGGGACACCUGAAUGCCCCCGGCCCCGGCUCCUCCGACGCGCCAUG_3′.ARF6mut:_5′_CCAUGGuUGGGAcGuGcAcUgGcAGcCgGcAGaGAGCCCGCcCaCcGcuacGCGGCUuacccgucCcgAgCcAcUaggCcCGAGGAUCCAUGACCUGACGGGGCCCCGGAGCCGCGCUGCCUCUCGGGUGUCCUGGGUCGGUGGGGAGCCCAGUGCUCGCAGGCCGGCGGGCGGGCCGGAGGGCUGCAGUCUCCCUCGCGGUGAGAGGAAGGCGGAGGAGCGGGAACCGCGGCucuagaCGCGCGGCGCCUGCGGGGGGAAGGGCAGUUCCGGGCCGGGCCGCGCCUCAGCAGGGCGGCGGCUCCCAGCGCAGUCUCAGGGCCCGGGUGGCGGCGGCGACUGGAGAAAUCAAGUUGUGCGGUCGGUGAUGCCCGAGUGAGCGuauacagUcGaCaUaUuagCUUAGGAGGCAACUCCCACGCAGGCCGCAAAGGCGCUCUCGCGGCCGAGAGGCUUCGUUUCGGUUUCGCGGCGGCGGCGGCGUUGUUGGCUGAGGGGACCCGGGACACCUGAAUGCCCCCGGCCCCGGCUCCUCCGACGCGCCAUG_3′.CyclinD1:_5′_CCAUGGGCUUAACAACAGUAACGUCACACGGACUACAGGGGAGUUUUGUUGAAGUUGCAAAGUCCUGGAGCCUCCAGAGGGCUGUCGGCGCAGUAGCAGCGAGCAGCAGAGUCCGCACGCUCCGGCGAGGGGCAGAAGAGCGCGAGGGAGCGCGGGGCAGCAGAAGCGAGAGCCGAGCGCGGACCCAGCCAGGACCCACAGCCCUCCCCAGCUGCCCAGGAAGAGCCCCAGCCAUG_3′.ROCK1:_5′_GCUGGUUCCCCUUCCGAGCGUCCGCGCCCCGCAUGCGCAGUCUGCCCCGGCGGUCUCCGUUUGUUUGAACAGGAAGGCGGACAUAUUAGUCCCUCUCAGCCCCCCUCGCCCCACCCCCCAGGCAUUCGCCGCCGCGACUCGCCCUUUCCCCGGCUGGGACCGCAGCCCCUCCCAGAAGCUCCCCCAUCAGCAGCCGCCGGGACCCAACUAUCGUCUUCCUCUUCGCCCGCUCUCCAGCCUUUCCUCUGCUAAGUCUCCAUCGGGCAUCGACCUCGCCCUGCCCCACCGGACACCGUAGCAGCAGCCCCAGCAGCGACGGGACAAAAUGGGAGAGUGAGGCUGUCCUGCGUGGACCAGCUCGUGGCCGAGACUGAUCGGUGCGUCGGGCCGGGCCGAGUAGAGCCGGGGACGCGGGGCUAGACCGUCUACAGCGCCUCUGAGCGGAGCGGGCCCGGCCCGUGGCCCGAGCGGCGGCCGCAGCUGGCACAGCUCCUCACCCGCCCUUUGCUUUCGCCUUUCCUCUUCUCCCUCCCUUGUUGCCCGGAGGGAGUCUCCACCCUGCUUCUCUUUCUCUACCCGCUCCUGCCCAUCUCGGGACGGGGACCCCUCCAUGGCGACGGCGGCCGGGGCCCGCUAGACUGAAGCACCUCGCCGGAGCGACGAGGCUGGUGGCGACGGCGCUGUCGGCUGUCGUGAGGGGCUGCCGGGUGGGAUGCGACUUUGGGCGUCCGAGCGGCUGUGGGUCGCUGUUGCCCCCGGCCCGGGGUCUGGAGAGCGGAGGUCCCCUCAGUGAGGGGAAGACGGGGGAACCGGGCGCACCUGGUGACCCUGAGGUUCCGGCUCCUCCGCCCCGCGGCUGCGAACCCACCGCGGAGGAAGUUGGUUGAAAUUGCUUUCCGCUGCUGGUGCUGGUAAGAGGGCAUUGUCACAGCAGCAGCAACACCCAUG_3′PFN2:_5′_CGCUGCGGUAAGGAGCAGCCGCCACAGGCACAGCCGCUUCGCAGCCUCCCGCCGCUGGUUUGUCAGCCCCGCGGCUGCGGGCGGCCGGGCGGCCGAGCGCGCUCUGAGGUUCGUCCCUCAUCGCUGAACCCGCGUCCUCCCGCCGCAGCUCCUCGGGGAGGGGGGCGGUCGGUGCCUGCGCAGAGCCGCCUCCUCCCCGCCCCCGCCCCGCCUCCCCCCGCGCCGCCGCCGCCCGCUACCGCCGCCGCCGCCGCUGCGCCUGCUGCUCCUCGCCGUCCGCGCUGCAGUGCGAAGGGCUCGAGCCAUG_3′.Monosome preparations pelleted in ultracentrifuge tubes were washed with 1 ml ice-cold ethanol by addition of the ethanol followed by a 5 minute incubation on ice and aspiration of the ethanol via a vacuum trap. The pellet was allowed to air dry. Monosome pellet was dissolved in 100 μl of a solution of 20 mM ammonium bicarbonate, 8 M urea and 2 mM Tris(2-carboxyethyl)phosphine hydrochloride. MS-Grade Lys C (Thermo) was dissolved in water to a concentration of 0.5 μg/μl and 2 μl was added to the dissolved pellet. The Lys C digestion was incubated for 4 hours at room temperature. The digestion was further diluted with an additional 98 μl of water. MS-Grade Trypsin (Thermo) was also dissolved in water to a concentration of 0.5 μg/μl and 2 μl was added to the diluted Lys C digestion. This trypsin digestion was incubated overnight at room temperature.Digestion samples were prepared for liquid chromatography-tandem mass spectrometry (LC-MS/MS) by acidification. The digestion sample (20 μl) was acidified by the addition of 2 μl of 10% formic acid; 20 μl of this sample was injected onto a 2.1 × 50 mm Extend-C18 column  at 400 μl/minute flow rate. Elution of peptides was over a 51 minute gradient from 5 to 40% acetonitrile with 0.1% formic acid as the modifier, and a flow rate of 200 μl/minute. Data acquisition was on an Agilent 6530 Q-ToF instrument equipped with a dual electrospray source, and a reference mass of 922.009798 enabled. MS/MS spectra were acquired in Auto MS/MS mode with triggering of precursor ions of +2 charge state and higher and individual ions were excluded for 9 seconds after triggering an MS/MS scan. Up to five precursors were selected per MS scan, and MS/MS scans were for 50,000 counts or 333 mS, whichever came first. LC-MS/MS data were exported to a Mascot Generic Format file using MassHunter Qual 5.0 (Agilent) and searched against SwissProt 51.6 with taxonomy limited to human, without modifications other than variable pyroglutamate formation at Q and E, MS tolerance of 10 ppm and MS/MS tolerance of 0.1 Da.Digestion samples were prepared for LC-MS/MS by acidification. The digestion sample (10 μl) was acidified by the addition of 10 μl of 1% formic acid; 4 μl of this sample was injected onto a 4 mm 40 nL trap column at a flow rate of 3 μl/minute and analyzed on a 75 μm × 150 mm column containing Zorbax 300SB-C18 (5 μm beads) at a flow rate of 400 nl/minute. Elution of peptides was over a 100 minute gradient from 5 to 40% acetonitrile with 0.1% formic acid as the modifier. Data acquisition was on an Agilent 6550 Q-ToF instrument equipped with the ChipCube source, and a reference mass of 1221.990637 enabled. MS survey scans were acquired at a rate of 5 Hz, and MS/MS spectra were acquired in Auto MS/MS mode with triggering of precursor ions of +2 charge state and higher. Individual ions were excluded for 18 seconds after triggering an MS/MS scan. Up to five precursors were selected per MS scan, and MS/MS scans were for 25,000 counts or 200 mS, whichever came first. LC-MS/MS data were exported to a Mascot Generic Format file using MassHunter Qual 5.0 and searched against SwissProt 51.6 with taxonomy limited to human, without modifications other than variable pyroglutamate formation at Q and E, MS tolerance of 5 ppm and MS/MS tolerance of 0.1 Da.5′ UTR sequences were retrieved from Ensemble using RefSeq mRNA names as query. Sequences were broken into 20-nucleotide fragments in a stepwise manner beginning from the first nucleotide (n) and proceeding in 1 nucleotide steps  through the length of the UTR. The structure of each fragment was analyzed using the CONTRAfold algorithm and the resulting free energy values were plotted based on their position across the length of the UTR, as were the percentage GC values.5 cells in serum-free medium. Medium containing 5% fetal bovine serum was aliquoted to lower wells and cells were incubated for 22 hours. When measuring migration in the presence of Silvestrol, compound was included in the medium in both upper and lower chambers at the concentrations indicated. To assess cell migration after 22 hours, upper chambers were transferred to wells containing 1× PBS +8 μM Calcenein AM (Life Technologies) and incubated for 40 minutes. Fluorescence was evaluated by excitation at 485 nM wavelength followed by measuring emission at 520 nM.Migration assays were performed using trans-well migration chambers (BD Biosciences). MDA-MB-231 cells were seeded into upper chambers at a density of 0.5 × 105 cells/well in RPMI supplemented with 10% fetal bovine serum and grown for 24 hours to generate a monolayer. The following day cells were scratched to produce a wound in the monolayer and wound closure was monitored by microscopy using the Incucyte Live Cell Imaging system .Cells were seeded into 24 well plates at a density of 6 × 10The next generation sequencing data presented in this paper have been deposited in NCBI’s Gene Expression Omnibus  and are


Here we use transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technologies to interrogate the functional relevance of predicted miRNA response elements (MREs) to post-transcriptional silencing in zebrafish and Drosophila. We also demonstrate an effective strategy that uses CRISPR-mediated homology-directed repair with short oligonucleotide donors for the assessment of MRE activity in human cells. These methods facilitate analysis of the direct phenotypic consequences resulting from blocking specific miRNA–MRE interactions at any point during development.MicroRNA (miRNA) target recognition is largely dictated by short ‘seed’ sequences, and single miRNAs therefore have the potential to regulate a large number of genes. Understanding the contribution of specific miRNA–target interactions to the regulation of biological processes  Identifying miRNA response elements (MREs) within target mRNAs can be done computationally but the functional validation of putative MREs remains challenging. Here, Bassett et al. describe applications of genome engineering to target and assess the functional significance of MREs in different organisms and stages of development. To date, the standard strategy used to validate miRNA targets has relied on artificial sensor assays whereby the 3′-untranslated region (UTR) of the gene of interest (or a region flanking the predicted MRE) is coupled to a reporter gene. Although valuable, a critical limitation of this technology is its inability to infer the physiological relevance of a putative MRE in vivo, as these assays do not recapitulate the endogenous context and stoichiometry of miRNAs and their targets. An alternative strategy is provided by target protector (TP) oligonucleotides, which overlap with the seed region and a unique flanking sequence in the target 3′UTR, and can therefore block miRNA access to the MRE8910in vivo, few studies have been performed to address potential toxicity or off-target effects of TP oligonucleotides. Furthermore, TP assays are in most cases limited to transient effects in systems where oligonucleotides can be supplied by injection or transfectionMicroRNA (miRNA)-mediated post-transcriptional silencing represents an essential regulatory layer underlying cellular function, development and disease12ry high ~0%67, andin vivo. To generate targeted insertion and deletion (indel) mutations within MREs, we use both transcription activator-like effector nucleases (TALENs) in zebrafish and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas9) in Drosophila. In both systems, indels are created as a result of inefficient non-homologous end joining repair at the nuclease cleavage site. Using this approach, we have accurately defined bona fide MREs in these two species and have directly addressed their functional relevance in an endogenous context in vivo. Furthermore, we describe a novel approach for the identification of active MREs in cell culture systems, using CRISPR-mediated homology-directed repair (HDR) with short oligonucleotide donors. This now allows the effect of defined deletions or alterations within MREs to be rapidly assessed in heterogeneous cell populations. Finally, we present an online algorithm that computationally predicts CRISPR target sites neighbouring all predicted MREs in several species, and thereby show that these techniques may be generally applicable for investigation of the vast majority of MREs.To address these limitations, we have used genome engineering technologies to genetically ablate endogenous MREs within putative target genes lefty2 (lft2) 3′UTR, causing a midline development phenotype in the embryo as a consequence of impaired Nodal signallinglft2 DNA  of single experimental embryos showed that lft2 expression level at shield stage was significantly increased as compared with controls . miR-430 MRE disruption, as assayed by the resistance to Bsp1286I digest, was observed in DNA samples obtained from all tested embryos that exhibited mutant phenotypes , indicating highly efficient editing events at the targeted locus. These results establish the feasibility of this approach for analysis of miRNA–target regulatory interactions during zebrafish embryonic development, and validate the functional relevance of this MRE in the lft2 3′UTR. Notably, such mutants could be used to generate stable lines, allowing phenotypic analysis at all stages of development and during adult life.In zebrafish, morpholino TP oligonucleotides have been used to block a predicted miR-430 site in the lft2 DNA . Customilft2 DNA . The spaion site . In agreion site , with a bantam (ban) in the enabled (ena) gene in Drosophila. Previous work showed that overexpression of ban in the wing imaginal disc can reduce Ena levels in the context of a 3′UTR reporter assay, and that this effect was dependent on a conserved ban target siteTo realize the potential of our strategy in elucidating the functional significance of MREs throughout development, we next investigated the effect of removing a well-studied target site for the miRNA ban target site in the ena 3′UTR . The presence of the mutation in animals used for all subsequent steps was validated by PCR (ban using the decapentaplegic driver (dpp-GAL4) that expresses in a stripe of cells across the anterior/posterior boundary of the wing imaginal disc (ena-3′UTR-sensor construct (ena-3′UTR) and endogenous Ena protein levels (n>30). No change in Ena levels was observed in control discs expressing CD8-EGFP under the same GAL4 driver . This result indicates that overexpressed ban can directly target ena via this predicted MRE in its 3′UTR.To create a genomic deletion in the predicted MRE, we designed a synthetic guide RNA (sgRNA) that would target the Cas9 nuclease to the predicted na 3′UTR . The expquencing . We decid by PCR  and sequd by PCR . We overnal disc . Confirmn levels , n>30. N4 driver . When th protein , n>30. Tban regulates Ena expression on either side of the dorsal/ventral (D/V) boundary of the wing imaginal discsban-MREmut animals revealed no apparent changes to endogenous Ena expression pattern in the wing imaginal discs (compare n=10), or any detectable phenotypes in the adult wings (n=10). This indicates that although ban overexpression is able to downregulate Ena through this MRE, the endogenous expression pattern of Ena in the wing disc is not dependent on this target site. Thus, although it remains possible that ban participates in establishing the identity of the D/V boundary in wing discs, it is unlikely this activity is mediated by tuning Ena expression through this predicted MRE. These results emphasize the importance of directly establishing the in vivo physiological relevance of a MRE, and suggest that caution should be exerted when inferring its function from indirect assays.It was previously suggested that endogenous  compare . We also compare , n=10, olt wings , n=10. Tin vivo are critical for understanding the function of MREs in the context of the whole organism, cell culture systems offer a valuable tool for studying miRNA function in a well-defined context, and a means to directly investigate their role in human cells. We therefore developed a novel strategy to validate functional MREs in cultured cells, using the CRISPR/Cas9 system to enhance homologous recombination, and generate defined sequence alterations of a MRE of interest. The approach involves co-transfection of the Cas9/sgRNA targeting a MRE with two ~140 nt single-stranded DNA (ssDNA) oligonucleotide templates for HDR. One of these HDR templates inserts a T3 ‘barcode’ downstream of the target MRE locus, but maintains the intact MRE . Interestingly, C9orf7 is targeted by miR-92a via a non-canonical motif through complementary base pairing with its 3′ end, demonstrating that such interactions can be biologically relevant. The other putative MREs in PCMTD1 and MAPRE1 failed to show this effect, suggesting that they may not play a role in regulating the abundance of these transcripts, at least in this particular cell type. One consideration when designing this strategy is to determine whether integration of the barcodes creates or inadvertently removes endogenous MREs. To test this, the sequences generated upon integration of T7-MREmut and T3-MREWT barcodes were screened for miRNA target sites using the PITA algorithmmut barcode, as intended , and revealed that both of these MREs are functional in this cell type intended . All butell type .Based on these results, we propose that this strategy can be applied broadly to the identification of physiologically active MREs when the target mRNA is expressed at sufficient levels to allow detection by qPCR of homologous integration within the cDNA. In addition, our results suggest that care must be taken when extrapolating the functional relevance of a MRE from studies of miRNA binding to the target transcript or changes in gene expression resulting from miRNA inhibition.www.microrna.org, August 2010 release1921To establish the versatility of these technologies, and whether they can be generally applied to analysis of miRNA–MRE interactions, we performed computational analysis to identify CRISPR target sites in the vicinity of all predicted MREs in several organisms . CRISPR http://miR-CRISPR.molbiol.ox.ac.uk/fulga/miR-CRISPR.cgi). Information is provided for five species: human, mouse, rat, Drosophila and Caenorhabditis elegans. Upon entering a gene symbol and/or miRNA identifier, CRISPR sgRNA target sites are generated for each predicted MRE, along with the distance between the PAM and MRE, the forward and reverse oligonucleotides required for sgRNA production, and the number and details of putative off-target sites.CRISPR target sites for all predicted MREs are available via the miR-CRISPR online web interface lft2 3′UTR at the miR-430-binding site, separated by a 16-nt spacer. RVD arrays containing HD, NG, NI and NN monomers were assembled using the Golden Gate TALEN and TAL Effector Kit 2.0  as previously describedin vitro transcription of corresponding TALEN mRNAs. The RVD sequences of the left and right TALENs are:The TALE nuclease was designed using the TALEN Targeter algorithm , and capped TALEN mRNAs were in vitro transcribed using the mMESSAGEmMACHINE SP6 kit . RNAs were then polyadenylated with the polyA tailing kit  and purified with the RNeasy mini kit .Plasmid pMTB2-Goldy A measure of 25 pg of each TALEN mRNA was injected into single-cell stage embryos, using the Picospritzer II microinjector (Parker Instrumentation). Embryos were incubated in E3 mediumlft2 antisense probe was generated by PCR amplification of cDNA from 12 hours post fertilization wild-type zebrafish, using the following primers: SP6-lft2-F 5′-GATTTAGGTGACACTATAGgaccacagcgatctcactca-3′ and T7-lft2-R 5′-TAATACGACTCACTATAGGGgactggagggattttgtcc-3′. The PCR product was gel extracted and purified by ethanol precipitation. 1.5 μg of template DNA was transcribed using T7 RNA polymerase  in the presence of digoxygenin (DIG)-labelled dNTPs . Probes were purified using G-50 micro-columns . Embryos were permeabilized with 1% H2O2, equilibrated with hybridization buffer dimethylammonio]-1-propanesulfonate (CHAPS), 100 μg ml−1 Heparin) and incubated overnight with lft2 DIG-labelled probes. Embryos were blocked with 20% sheep serum+2% Boehringer Blocking Reagent . Probes were detected with an anti-DIG-alkaline phosphatase antibody  and the signal was developed using 30 μg ml−1 of each nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate .The template for Bsp1286I  for 4 h at 37 °C. Digested products were then analysed by gel electrophoresis (1% agarose). To identify the nature of indel mutations, PCR products were cloned into pGEM-T easy vector  and DNA was isolated from individual clones and sequenced using an SP6 primer.gDNA was extracted by homogenizing single zebrafish embryos in 50 μl of 50 mM NaOH, followed by incubation for 20 min at 95 °C, cooling to 4 °C and addition of 5 μl of 1 mM Tris-HCl pH=8 to neutralize the solutionFor coupled transcriptional/genomic analysis of single injected zebrafish embryos, total RNA and DNA were extracted using the RNAqueous Micro Kit . cDNA was synthesized using the Superscript III Reverse transcriptase kit . For quantitative PCR, the following gene-specific primers were used: Lft2-F (5′-CTGGCAGGAATACTCAGGGG-3′), Lft2-R (5′-TGGCCTCCATGTCGAACA-3′), ActB-F (5′-AATCCCAAAGCCAACAGAGA-3′), ActB-R (5′-ACATACATGGCAGGGGTGTT-3′). The levels of Lft2 transcript in individual embryo analysis were calculated using the standard curve method.20NGG). Potential off target sites within the Drosophila genome were identified by BLAST and using the CRISPR design tool ( http://crispr.mit.edu). The closest off-target site contained four mismatches to the 20 nt target sequence. Cas9 mRNA was in vitro transcribed from plasmid MLM3613 (ref. TAATACGACTCACTATAGGCGTATGAGATCGTGTGCTTGTTTTAGAGCTAGAAATAGC-3′) and a reverse oligonucleotide encoding the remainder of the sgRNA sequence (sgRNA-R=5′-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3′). Reaction solutions (100 μl) were cycled on a GStorm thermal cycler , 72 °C 10 min, 10 °C ∞) and purified with a PCR purification kit . In vitro transcription was performed with 300 ng purified DNA template for 4 h at 37 °C using the Megascript T7 kit , and sgRNA purified by phenol chloroform extraction and isopropanol precipitation. sgRNAs were diluted to 1 μg μl−1 in water and stored in aliquots at −80 °C  was used with an Injectman NI2 micromanipulator and Femtotip II needles . Embryos were incubated at 25 °C for the remainder of the development. Putative mosaic flies were crossed individually to yw; Sco/CyO flies. These flies were removed after 7 days, and analysed for mutant tissue by HRMA ; ena-3′UTR-sensor and UAS-bantam were a generous gift from Marco MilanDrosophila CRISPR injections are provided at the OxfCRISPR website ( http://www.oxfcrispr.org)A measure of 0.5 μg sgRNA and 10 μg Cas9 mRNA were precipitated with ethanol to purify and concentrate, and resuspended in water at 0.5–1 μg μl by HRMA . Only cr by HRMA . During −1 proteinase K , and heating to 37 °C for 30 min or 60 min, followed by inactivation at 95 °C for 2 min  and blocked in 5% normal goat serum in PBS-T. Discs were then incubated overnight at room temperature with primary antibodies mouse anti-Enabled , rabbit anti-GFP  and mouse anti-wingless , washed three times 20 min each in PBS-T and incubated for 2 h at room temperature with anti-mouse A568 , anti-rabbit A488  and DAPI . Samples were then mounted in Slowfade  and imaged on a Zeiss 780 confocal microscope with a × 25 oil immersion objective and × 0.7 digital zoom. Images were acquired using similar laser power, pinhole size and gain parameters, and adjusted post-acquisition for background intensity and contrast correction. For assessing Ena expression in the dpp-GAL4>UAS-ban background, a minimum of 30 third instar larval wing imaginal discs were analysed. The results displayed in ban-MREmut lines. To confirm the nature of ban-MREmut indels in animals processed for immunofluorescence and imaging, half of each larvae was snap frozen immediately after dissection and subjected to gDNA extraction followed by PCR validation and sequencing.20NGG were chosen as close as possible to the predicted MREs in the 3′UTRs of PCMTD1 (5′-GGCAATATATGAGTGCAATA-3′), MAPRE1 (5′-CCTTGGGATCTGCCAGGCTG-3′) and C9orf7 (5′-GTGGCATCTGAGGCCGGGAG-3′) in human cells, and Pck (5′-TCCATAGTGCCTTTAACAAT-3′) and CG13088 (5′-CATTTCTACCTCAATCCGTC-3′) in Drosophila cells. Target sequences were cloned into the pX330 vector Target sites of the form NPCMTD1 targetCCGGGCAATATATGA GTGCAATAACAATACAAGATATTGAATAATTTAGCTTTAAAAAATCCCACAAATTTTATG AAATTTT-3′5′-TTACTTTTGTTGCAATCACTGTTGTTGGGTTGCTGTATATATATTPCMTD1 ssOligoT7TAATACGACTCACTATAGGGAACAATACAAGATATTGAATAATTTAGCTTTAAA AAATCCCACAAATTTTATGAAATTTT-3′5′-TTACTTTTGTTGCAATCACTGTTGTTGGGTTGCTGTATATATATTGCGGGCAATATATGAPCMTD1 ssOligoT3GTGCAATAATTAACCCTCACTAAAGGGAAACAATACAAGATATTGAATAATTTAGCTTTAAAAAATCCCACAAATTTTATGAAATTTT-3′5′-TTACTTTTGTTGCAATCACTGTTGTTGGGTTGCTGTATATATATTGCGGGCAATATATGA MAPRE1 targetGGCAGGCTGGTGTTTTCGGTATCTGCTGTTCACAGCTCTCCA CTGTAATCCGAATACTTTGCCAGTGCA-3′5′-CTTTCTGGACCTCTGGCAAAGGGAGTGGTCAGTGAAGGCCATCGTTACCTTGGGATCTGCMAPRE1 ssOligoT7TAATACGACTCACTATAGGGGTGTTTTCGGTATCTGCTGTTCACAGCTCTCCACTGTAATCCGAA TACTTTGCCAGTGCA-3′5′-CTTTCTGGACCTCTGGCAAAGGGAGTGGTCAGTGAAGGCCATCGTTACCTTGGGATCTGCMAPRE1 ssOligoT3CAGGCTGGGAATTAACCCTCACTAAAGGGAGTGTTTTCGGTATCTGCTGTTCACAGCTCTCCACTGTAATCCGAATACTTTGCCAGTGCA-3′5′-CTTTCTGGACCTCTGGCAAAGGGAGTGGTCAGTGAAGGCCATCGTTACCTTGGGATCTGCC9orf7 targetAGGCCGGGAGTGGCATCTGAGGCCAGGAGTGGCAGGCTGGTGGGCTGGGCGTGGGGTT TTCTGGGCCCT-3′5′-AACTGTTTCCCAGGAACACCTCTCGGGCCCATCTGCGTCTGAGGCTGGGAGTGGCATCTGC9orf7 ssOligoT7TAATACGACTCACTATAGGGGTGGCATCTGAGGCCAGGAGTGGCAGGCTGGTGGGCTGGGCGTG GGGTTTTCTGGGCCCT-3′5′-AACTGTTTCCCAGGAACACCTCTCGGGCCCATCTGCGTCTGAGGCTGGGAGTGGCATCTGC9orf7 ssOligoT3AGGCCGGGAAATTAACCCTCACTAAAGGGAGTGGCATCTGAGGCCAG GAGTGGCAGGCTGGTGGGCTGGGCGTGGGGTTT TCTGGGCCCT-3′5′-AACTGTTTCCCAGGAACACCTCTCGGGCCCATCTGCGTCTGAGGCTGGGAGTGGCATCTGPCK targetCCGTCCATAGTGCCTTTAACAATCGACCATATGTATCTATATACACGCCGACTCAGCCGAG ATCAG-3′5′-ATTGGATCTTAGCTTAAGTTTTCAGACGCAATCTCGTGCCTCGAATCTGCGCAATCGACGPCK ssOligoT7TAATACGACTCACTATAGGGATAGTGCCTTTAACAATCGACCATATGTATCTATATACA CGCCGACTCAGCCGAGATCAG-3′5′-ATTGGATCTTAGCTTAAGTTTTCAGACGCAATCTCGTGCCTCGAATCTGCGCAATCGACGPCK ssOligoT3CCGTCCAATTAACCCTCACTAAAGGGAATAGTGCCTTTAACAATCGACCATATGTATCTATATACACGCCGACTCAGCCGAGATCAG-3′5′-ATTGGATCTTAGCTTAAGTTTTCAGACGCAATCTCGTGCCTCGAATCTGCGCAAT CGACGCG13088 targetC TCCGTCGGTATACGAATATATATGTAGATGGAGATCCAA ATGATATATCCTGAGTAAAATGTTGTA-3′5′-GAATTCAGTCCAGACTCGTAGTAGTCATTTGAAAAGACTTAAATGACATTTCTACCTCAACG13088 ssOligoT7 TAATACGACTCACTATAGGGGGTATACGAATATATATGTAGATGGAGATCCAAATGATATATCCTGAG TAAAATGTTGTA-3′5′-GAATTCAGTCCAGACTCGTAGTAGTCATTTGAAAAGACTTAAATGACATTTCTACCTCAACG13088 ssOligoT3TCCGTCCAATTAACCCTCACTAAAGGGAGGTATACGAATATATATGTAGATGGAGATCCAAATGATATATCCTGAGTAAAATGTTGTA-3′5′-GAATTCAGTCCAGACTCGTAGTAGTCATTTGAAAAGACTTAAATGACATTTCTACCTCAA 2 in DMEM with 25 mM glucose , 10% fetal bovine serum  and 1% penicillin-streptomycin . S2R+ cells were cultured at 25 °C in Schneider’s Drosophila medium , 10% fetal bovine serum  and 1% penicillin-streptomycin . Cas9/sgRNA expression vectors (1 μg) and homology oligonucleotides (0.5 μg each T3/T7 oligo) were co-transfected into HEK293T cells in a six-well dish using polyethylenimine (Sigma-Aldrich) as previously describedHEK293T cells were cultured at 37 °C and 5% CO−1 of proteinase K , and incubation at 55 °C for a minimum of 2 h. Proteins were removed by phenol/chloroform extraction, and DNA precipitated with 2.5 volumes ethanol. DNA was quantified with a Nanodrop spectrophotometer and diluted to a concentration of 100 ng μl−1 before analysis. RNA was extracted using a miRNeasy mini kit  including the additional DNAse treatment step. cDNA was synthesized from 1 μg RNA with the Quantitect Reverse Transcription kit  and diluted twofold before analysis.gDNA was extracted from zebrafish embryos by addition of 300 μl lysis buffer  containing 200 μg ml−ΔCt.Quantitative PCR was performed with gene-specific forward primers , and either T3as (5′-TCCCTTTAGTGAGGGTTAATT-3′) or T7as (5′-CCTATAGTGAGTCGTATTA-3′) reverse primers. Amplification was performed with SybrGreen JumpStart Taq Ready Mix  and detected on an ABI 7500 Fast thermal cycler. Results are shown as a ratio of the signal with the T7 primer to that with the T3 primer for gDNA and cDNA for three independent biological replicates, each analysed in technical triplicate. The final signal ratio was obtained by calculating the difference in Ct between T7 and T3 primers (ΔCt) during the exponential amplification phase, and subsequently transforming the resulting values using 2www.microrna.org, August 2010 Release) for four species, Drosophila, human, mouse and rat. A custom perl script was used to identify potential CRISPR target sites within a 200-bp window around each of the MREs from the appropriate genomic sequences . Potential off-targets for each of the CRISPR target sites were identified using the BatMis algorithmMREs for conserved miRNAs with a ‘good mirSVR score’ were predicted using the miRanda algorithm .Supplementary Figure 1 and Supplementary Table 1


The zinc finger proteins ZNF644 and WIZ regulate the G9a/GLP complex for gene repression. Published 19 March 2015TTCGATGATTCCCAGATACTAGTACGGTCAG-3′ (WIZ ‘WT DNA target’).There was an error in the WIZ binding sequence used for the EMSA experiments. The incorrect sequence was 5′-GAGTCTCACTCACGCGCCATTCCATTCCATTCAGATACTAGTACGGTCAG-3′ (WIZ ‘WT DNA target’).The correct sequence is 5′-GAGTCTCACTCACGCGCThe article has been corrected accordingly.


TMPRSS2:ERG gene fusion is common in androgen receptor (AR) positive prostate cancers, yet its function remains poorly understood. From a screen for functionally relevant ERG interactors, we identify the arginine methyltransferase PRMT5. ERG recruits PRMT5 to AR-target genes, where PRMT5 methylates AR on arginine 761. This attenuates AR recruitment and transcription of genes expressed in differentiated prostate epithelium. The AR-inhibitory function of PRMT5 is restricted to TMPRSS2:ERG-positive prostate cancer cells. Mutation of this methylation site on AR results in a transcriptionally hyperactive AR, suggesting that the proliferative effects of ERG and PRMT5 are mediated through attenuating AR’s ability to induce genes normally involved in lineage differentiation. This provides a rationale for targeting PRMT5 in TMPRSS2:ERG positive prostate cancers. Moreover, methylation of AR at arginine 761 highlights a mechanism for how the ERG oncogene may coax AR towards inducing proliferation versus differentiation.The DOI:http://dx.doi.org/10.7554/eLife.13964.001 Prostate cancers are among the most common types of cancer in men, which, like other cancers, are driven by genetic mutations. Roughly half of all prostate cancers contain a genetic change that incorrectly fuses two genes together, causing the cells to produce abnormally high levels of a protein called ERG.ERG is a transcription factor, a protein that binds to specific sequences of DNA to influence the activity of nearby genes. ERG represses genes that help to prevent prostate cancers from growing, and so promotes prostate cancer development. Like most other transcription factors, ERG is difficult to target with drugs and no therapies that directly prevent the activity of ERG currently exist.Mounir et al. wanted to find out whether ERG cooperates with other proteins to cause prostate cancer cells to grow, with the hope that these proteins could be more easily targeted with a drug. By using various biochemical techniques in human prostate cancer cell lines, Mounir et al. found that ERG interacts with an enzyme called PRMT5. This interaction enables PRMT5 to chemically modify other proteins to change their activity. In the case of prostate cancer cells, PRMT5 inappropriately modifies the androgen receptor, a protein that regulates the growth of normal prostate cells. This abnormal modification contributes to the excessive growth of the cancer cells.Although PRMT5 will be easier to target with drugs than ERG, it also has many other roles besides those described by Mounir et al. Much more work is therefore needed to investigate whether PRMT5 could be safely targeted to treat patients with prostate cancer.DOI:http://dx.doi.org/10.7554/eLife.13964.002 TMPRSS2:ERG fusion  is highly prevalent and lethal . Drugs tG fusion . TMPRSS2 in mice . ERG is  in mice , suggest in mice . We explTMPRSS2:ERG positive PC cells, we performed a pooled short hairpin RNA (shRNA) screen in TMPRSS2:ERG and AR-positive VCaP prostate cancer cells, using ERG-negative 22Rv1 cells as a control . The shRNA pool targets 648 genes involved in transcriptional and epigenetic regulation . Identified proteins  includedTMPRSS2:ERG positive NCI-H660) and one PRMT5 hairpin likely to have minimal off-target effects. This shRNA shows a trend of sensitivity in ERG-positive lines, in agreement with our findings -inducible shRNA vectors targeting PRMT5 and a non-targeting control shRNA (NTC). PRMT5 knockdown was robust in all cell lines . Robust findings .PRMT5 is a protein arginine methyltransferase that regulates multiple signaling pathways through the mono- and symmetric di-methylation of arginines on its target proteins .To deterPSA, NKX3-1 and SLC45A3  to investigate ERG, AR, and PRMT5 recruitment to the AR targets cterized  binding cterized . We alsocterized . In VCaPcterized . As expecterized , ERG expcterized , and ERGcterized . Like ERcterized .To extend these findings to a genome wide scale, we performed AR and ERG ChIP-seq experiments in androgen (DHT or R1881) stimulated VCaP cells upon PRMT5 knockdown . ERG and AR recruitment were robust in these experiments, as judged by recovery of the canonical DNA binding sites of these proteins . In agrePSA or NKX3-1 loci  and symmetric di-methylation (SDMA) levels compared to wild-type controls  , suggest3-1 loci . We ther3-1 loci . To confenotypes . Like VCcontrols . On the sus VCaP , suggestsus VCaP . In thessus VCaP , suggestTo further understand if the observed AR methylation was directly dependent on PRMT5, we performed biochemical assays using purified PRMT5 (complexed with its requisite binding partner MEP50/WDR77) and AR LBD. PRMT5 activity, as judged by production of SAH (the by-product of SAM-dependent substrate methylation), was observed in the presence of AR LBD as substrate, but not in the presence of ERG ETS DNA binding domain or pointed (PNT) domain. PRMT5 activity in the presence of AR LBD further increased with the addition of ERG ETS domain protein to the reaction, but not with PNT domain . Unlike To identify the arginine methylation site(s) on AR, we cloned AR cDNA and mutated all arginines in the LBD to lysine . We expressed each construct in AR-negative, ERG-negative RWPE-1 prostate cells. R1881 stimulates exogenous AR to induce target gene expression in these cells. ERG expression represses this effect  and induPSA locus as a model AR-regulated gene. Relative to WT AR, the AR R761K mutant showed enhanced recruitment to PSA and increased PSA expression. Moreover, R761K mutation prevented ERG-dependent attenuation of AR recruitment to PSA and PSA expression  and tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection kit (Lonza).Redundant siRNA Activity or RSA metric was used as described  were used for further analysis  targeting transcriptional and epigenetic regulators similar to a previously reported library . shRNA lescribed . Brieflyhttp://tools.proteomecenter.org/software.php]) using a false positive threshold of <1% for protein identifications (See Nuclear extracts of VCaP cells (~600 million cells) were pre-cleared using agarose beads  and used for pulldown with either an ERG antibody (Santa Cruz#353) or an anti-rabbit IgG antibody. All antibodies were pre-coupled to beads , washed then used for pulldown. Immunoprecipitations were eluted at pH2.5 followed by TCA protein precipitation, alkylated with iodoacetamide, and separated on a NuPage 4–12% Bis-Tris gradient gel (Invitrogen). Complete gel lanes were excised using a LEAP 2DiD robot and in-gel digested with trypsin (Tecan Freedom EVO 20). Peptide sequencing for the resulting resulting 16 digest samples was performed by liquid chromatography-tandem mass spectrometry using an Eksigent 1D+ high-pressure liquid chromatography system coupled to a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific). Peptide mass and fragmentation data were searched against a combined forward-reverse IPI database (v3.55) using Mascot 2.2 (Matrix Science). Peptide and protein validation were done using Transproteomic pipeline v3.3sqall  was previously described, as were the sequences of the nontargeting control and ERG shRNA inserts and stable cell line generation. PRMT5 sPRMT5 shRNA#1:AGGGACTGGAATACGCTAATTCTCGAGAATTAGCGTATTCCAGTCCCTPRMT5 shRNA#2:AGGGACTGGAATACGTTAATTGTTAATATTCATAGCAATTAGCGTATTCCAGTCCCTPRMT5 shRNA#3:GCGGATAAAGTTGTATGTTGTGTTAATATTCATAGCACAGCATACAGCTTTATCCGCAll shRNA were expressed from a puromycin resistant vector. Lentiviral production and cell transduction was as previously described .CGCGATTGGAACACGTTGATT .The Dox-inducible ERG constructs named ERG or ERG DNAx used for stable and inducible expression in 22Rv1 cells were generated as previously described . The cDNCAGAATCAAGCTCTACGCCGT .For shPRMT5-2: the GCGGATAAAGCTGTATGCTGT target sequence was converted to Full sequence of scPRMT5 below:scPRMT5 sequence: ATGTACCCCTATGACGTGCCAGATTACGCCATGGCGGCGATGGCGGTCGGGGGTGCTGGTGGGAGCCGCGTGTCCAGCGGGAGGGACCTGAATTGCGTCCCCGAAATAGCTGACACACTAGGGGCTGTGGCCAAGCAGGGGTTTGATTTCCTCTGCATGCCTGTCTTCCATCCCAGGTTCAAGCGCGAGTTTATTCAGGAACCTGCTAAGAATCGGCCCGGTCCCCAGACACGATCAGACCTACTGCTGTCAGGACGCGATTGGAACACGTTGATTGTGGGAAAGCTTTCTCCATGGATTCGTCCAGACTCAAAAGTGGAGAAGATTCGCAGGAACTCCGAGGCGGCCATGTTACAGGAGCTGAATTTTGGTGCATATTTGGGTCTTCCAGCTTTCCTGCTGCCCCTTAATCAGGAAGATAACACCAACCTGGCCAGAGTTTTGACCAACCACATCCACACTGGCCATCACTCTTCCATGTTCTGGATGCGGGTACCCTTGGTGGCACCAGAGGACCTGAGAGATGATATAATTGAGAATGCACCAACTACACACACAGAGGAGTACAGTGGGGAGGAGAAAACGTGGATGTGGTGGCACAACTTCCGGACTTTGTGTGACTATAGTAAGAGGATTGCAGTGGCTCTTGAAATTGGGGCTGATTTGCCCTCTAATCACGTCATTGATCGCTGGCTTGGGGAGCCCATCAAAGCAGCCATTCTCCCCACTAGCATTTTCCTGACCAATAAGAAGGGATTTCCTGTTCTTTCTAAGATGCACCAGAGGCTCATCTTCCGGCTCCTCAAGTTGGAGGTGCAGTTCATCATCACAGGCACCAACCACCACTCAGAGAAGGAGTTTTGTAGCTACCTGCAGTACCTGGAATACTTAAGCCAGAACCGTCCTCCACCTAATGCCTATGAACTCTTTGCCAAGGGCTATGAAGACTATCTGCAGTCCCCGCTTCAGCCACTGATGGACAATCTGGAATCTCAGACATATGAAGTGTTTGAAAAGGACCCCATCAAATACTCTCAGTACCAGCAGGCCATCTATAAATGTCTGCTAGACCGAGTACCAGAAGAGGAGAAGGATACCAATGTCCAGGTACTGATGGTGCTGGGAGCAGGACGGGGACCCCTGGTGAACGCTTCCCTGCGGGCAGCCAAGCAGGCCGACCGCAGAATCAAGCTCTACGCCGTGGAGAAAAACCCAAATGCCGTGGTGACGCTAGAGAACTGGCAGTTTGAAGAATGGGGATCCCAGGTCACGGTAGTCAGCTCAGACATGAGGGAATGGGTGGCTCCAGAGAAAGCAGACATCATTGTCAGTGAGCTTCTGGGCTCATTTGCTGACAATGAATTGTCGCCTGAGTGCCTGGATGGAGCCCAGCACTTCCTAAAAGATGATGGTGTGAGCATCCCCGGGGAGTACACTTCCTTTCTGGCTCCCATCTCTTCCTCCAAGCTGTACAATGAGGTCCGAGCCTGTAGGGAGAAGGACCGTGACCCTGAGGCCCAGTTTGAGATGCCTTATGTGGTACGGCTGCACAACTTCCACCAGCTCTCTGCACCCCAGCCCTGTTTCACCTTCAGTCACCCTAATCGCGACCCCATGATTGACAACAACCGCTATTGCACCTTGGAATTTCCTGTGGAGGTGAACACAGTACTACATGGCTTTGCCGGCTACTTTGAGACTGTGCTTTATCAGGACATCACTCTGAGTATCCGTCCAGAGACTCACTCTCCTGGGATGTTCTCATGGTTTCCTATTCTGTTTCCCATCAAGCAGCCCATAACGGTACGTGAAGGCCAAACCATCTGTGTGCGTTTCTGGCGATGCAGCAATTCCAAGAAGGTGTGGTATGAGTGGGCTGTGACAGCACCAGTCTGTTCTGCTATTCATAACCCCACAGGCCGCTCATATACCATTGGCCTCTGA.Generation of the PRMT5 catalytic dead mutant was performed by site-directed mutagenesis  through mutation of G365 to A and R368 to A .2 incubator at 37°C. Doxycycline  was used at 100 ng/ml. Cell proliferation was measured in 6-well plates (Corning) using automated confluence readings . R1881 (Sigma) and charcoal-stripped serum (Omega Scientific) were used where indicated.VCaP, 22Rv1, LNCaP and RWPE-1 cell lines (ATCC) were grown in vendor-recommended media and maintained in a humidified 5% CO2–5 ug of AR, ERG or IgG control antibody was coupled to 1mg of magnetic beads according to manufacturer’s protocol (Invitrogen Dynabeads Antibody Coupling kit#143.11D). After coupling, 1 mg of the antibody/bead mixture was incubated with 1–5 mg of protein lysate overnight under rotation at 4°C. IP samples were then washed with RIPA buffer containing protease/phosphatase inhibitor cocktail for 3–4 washes and resuspended in non-reducing loading buffer, boiled and loaded on a gel for western blot analysis. Immunoprecipitations were performed using the following antibodies: anti-ERG antibody (Epitomics# 2805–1), anti-HA antibody (Roche#11815016001), anti-AR antibody (Thermo Scientific# MA5-13426) or anti-IgG antibody .Procedures were previously described  and the Generation of labeled cDNA, hybridization to Affymetrix U133plus2 human arrays, and data normalization were performed as described . For theBlack line  represents expressed probe set position and is ranked by average fold-change. Blue, green, and red lines indicate where the probe sets mapping to genes in the androgen receptor activation signatures appear in our data set and show the cumulative sum of the probe sets in the androgen receptor activation signatures that overlap with our gene list (only the highest expressing probe set was used per gene). The dashed line represents the hypothetical cumulative sum for a random list of genes that are unenriched.PSA, NKX3-1 and SLC45A3 and VIC-labeled probe for Beta-2-macroglobulin (B2M) as a normalization control. Samples were run on a 7900HT Real-Time PCR machine (Applied Biosystems) and data was analyzed and normalized according to manufacturer’s instructions (2-ΔCt method).RNA isolation was performed as previously described . Taqman VCaP, 22Rv1 and RWPE-1 cells were treated as specified followed by cross-linking with 1% formaldehyde for 10 min. Cells were next lysed in 1% SDS and sonicated until DNA ladder is below 1 kb (Diagenode). Sheared chromatin was then used for IP with specific primary antibodies  pre-complexed with Protein A/G Dynabeads and incubated overnight under rotation at 4°C. The next day, ChIP samples were washed with RIPA buffer and TE followed by reverse crosslinking using 1%SDS and 30 ug/ml proteinase K (Invitrogen) at 65°C for 6 hr with beads. The eluates were then purified using the QIAquick PCR purification kit (Qiagen) and used for qPCR with the following primer sets:PSA -4100: acctgctcagcctttgtctc AND ttgtttactgtcaaggacaatcgPSA -3800: agaattgcctcccaacactg AND cagtcgatcgggacctagaaPSA -100: cttccacagctctgggtgt AND aaaccttcattccccaggacPSA +700: agccccagactcttcattca AND atgcagatttggggaatcagNKX3-1 -2800: gagagcagctgttcctccac AND acgagccttttccacctttcNKX3-1 -200: agggaggagagctggagaag AND tcctccctaggggattcctNKX3-1 +2150: accaggatgaggatgtcacc AND cagggacagagagagccttgNKX3-1 -+10800: tctctcgttggctcctgatt AND ccagcttttgttccttcctgNKX3-1 +62100: cggtttattgcccatgaaga AND aacagggctcacagtgctttVCaP cells harboring Dox inducible shRNAs targeting PRMT5 where grown to 80% confluency and re-seeded (day 0) into full media containing 100 ng/ml Doxycycline. On day 3 media was replaced with ‘hormone reduced’ media, containing 100 ng/ml Doxycycline. Cells were stimulated with adding indicated ligands (DHT (Sigma), R1881 [Sigma]) or vehicle on day 4 and harvesting of cells was performed on day 5. Cells were harvested by fixation using 1% methanol free formaldedyde  in PBS at room temperature. Fixation was stopped after 8 min by replacing fixation buffer with ice cold PBS containing 125 mM glycine and 5 mg/ml BSA. Cells were further washed once using ice-cold PBS and re-suspended into 500 ul of PBS containing Complete Protease Inhibitors (Roche). Cells where then pelleted and supernatant removed, and the resulting pellet either snap frozen in liquid Nitrogen or immediately re-suspended in lysis buffer for further processing.3 and 1% (w/v) SDS. Crosslink reversal was done at 65C°C for 6 hr and ChIP DNA were isolated using DNA purification beads (MagBio). ChIP-seq libraries were generated using the KAPA HTP library preparation kit (Kapa biosciences). All handling of samples after sonication was done on using a Sciclone NGS Workstation . Sequencing was performed on an Illumina NextSeq500 instrument.For ChIP the resulting cell lysate was sonicated using a Covaris E210 instrument according to manufacturers recommendations. Each ChIP reaction was performed using soluble fraction chromatin corresponding to 7.5 ug purifed DNA and 4 ug of antibodies. Antibodies were allowed to bind overnight before capture on protein A magnetic beads . Bound beads where washed 4 times in RIPA buffer containing 500 mM LiCl, and 2 times with TE buffer before being re-suspended in containing 100 mM NaHCOReads passing Illumina standard QC were mapped to genome version Hg19 using BWA, and binding sites (‘peaks’) were identified using MACS2, evolutionary conservation scores at peak locations was calculated using Phastcons and enriched DNA motifs using MDscan and Seqpos. These were performed using the ChiLin QC pipeline (liulab.dfci.harvard.edu/WEBSITE/software). Peaks from AR ChIP-sequencing samples with a MACS2 enrichment score higher than 10 were extended to a uniform 400 bp across all samples and overlapping peaks where collapsed to generate a union of all peaks. This resulted in a Cistrome of 25,593 peaks that were used in all genome wide ChIP analyses. Using the features of this Cistrome as GTF, read counts from BWA mapped Bam files were processed using the Qlucore 3.1.19 software. Heatmaps and statistical test (two-sided t-test using correction for multiple hypothesis testing) of differential binding scores on the 25,593 features were performed in Qlucore v3.1.19. The AR and ERG ChIPseq datasets are available at the NCBI Gene Expression Omnibus (Accession number GSE79128).RWPE-1 cells were treated as specified, fixed with 4% paraformaldehyde for 45 min at room temperature (Electron Microscopy Sciences), blocked with 5% goat serum, 0.5% Triton X-100 in PBS for 2 hr and incubated with 1:50 dilutions of AR (LSBIO #LS-C87494) and symmetric di-methyl arginine antibodies (CST#13222) in 5% goat serum and 0.05% Triton X-100. Fixed samples were incubated overnight at 4°C in primary antibody before incubations with proximity ligation assay (PLA) secondary antibodies .The secondary antibody incubation, ligation, amplification and final wash steps were performed according to the manufacturer’s specifications. Confocal microscopy was performed using an LSM 510 META  with a 40x C-Apochromat objective, NA 1.2. Images were collected and processed using Zen software .Homo sapiens androgen receptor (AR) sequence  was synthesized to include a 5’ NotI and 3’ BamHI sites and used as a template for the mutagenesis of each arginine in the ligand binding domain of AR into lysine . Following sequence verification to ensure mutation incorporation, each AR mutant sequence was cloned into the pLVX vector via the 5’ NotI and 3’ BamHI as previously described  and incubated with 1:50 dilution of AR antibody (LSBIO #LS-C87494). Confocal microscopy was performed using an LSM 510 META  with a 40x C-Apochromat objective, NA 1.2. Images were collected and processed using Zen software .The heterodimeric structure of PPARγ-RXRα (PDB Code: 3DZY) was used as a template to overlay individual domain structures of AR including the DBD in complex with DNA (PDB Code: 1R4I) and the LBD in complex with coactivator peptide TIF2(iii) and ligand R1881 (PDB Code: 2AO6). A superposition was achieved using secondary structure matching in COOT . The AR °C. Cells were resuspended in buffer A containing 50 mM Tris-HCl (pH 7.3), 150 mM NaCl, 10% glycerol, 0.25 mM TCEP, and 10 µM DHT, to which 50 µg/ml DNase I and protease inhibitor cocktail (Roche) were added. Cells were lysed using an M-110L Microfluidizer at 18,000 psi, followed by the addition of 0.5% CHAPS to the lysate prior to high speed centrifugation. For one-step batch purification, the soluble extract was incubated with 2 ml of glutathione sepharose 4 fast flow medium  for 1 hr at 4°C with rotational mixing. The sepharose medium was washed in buffer A with the addition of 0.5% CHAPS. Elution was accomplished by resuspending and incubating the media for 10 min in the wash buffer plus 10–20 mM reduced glutathione. The eluted fractions were then combined and concentrated to 0.3 mg/ml.The gene encoding human AR LBD (residues 663–919), was inserted into a pGEX-6P-1 vector and expressed as a GST-tagged fusion protein in BL21 Star (DE3) cells. Cells were grown in TB2 medium containing 10 µM dihydrotestosterone (DHT) and induced with 1 mM IPTG for 14 –16 hr at 16S-adenosyl-L-homocysteine (SAH) product formation utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). 0.5–1 uM of PRMT5/MEP50 recombinant enzyme  was incubated with 50 uM SAM, 2 uM GST-AR LBD and/or 5 uM ETS or PNT ERG protein for 2 hr at 37°C. Reactions were quenched to 0.1% HCOOH followed by addition of -SAH (SAH-D4) in 20% DMSO as an internal standard for MS quantification. Samples were sonicated with a Hendrix SM-100 sonicator (Microsonics Systems) and centrifuged. SAH was separated from the reaction mixture by reversed phase chromatography using polar endcapped C18 reversed phase columns  and detected using a 4000 QTRAP Hybrid Triple Quadrupole/Linear Ion Trap LC-MS/MS system (AB Sciex).PRMT5 enzymatic activity was assessed by monitoring  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 Kevin Struhl as the Senior Editor.Thank you for submitting your work entitled "ERG signaling in prostate cancer is driven through PRMT5-dependent methylation of the Androgen Receptor" 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. As you will see below, the reviewers have some concerns that will require quite an additional effort to address. Ordinarily, we allow just two months to return a revised version but we are concerned that this required experiments may take longer to complete. To help us assess the likelihood of a successful completion of the required additional work, please send us a note indicating how you plan to proceed and an estimate of the time it will take to complete the work.Summary:In this manuscript, the authors present two major findings: 1) PRMT5 methylates AR at R761 and these methylation results in decreased AR recruitment at target genes and decreased AR function and 2) ERG targets AR to be methylated. Overall, the findings address long-standing questions of ERG function in prostate tumorigenesis and possibly posit new therapies to ERG translocated prostate cancers. This would be certainly relevant to the prostate field. In addition, it is appropriate for a general audience for implications of a general enzyme that affects specific programs. The experiments are well done and logical. The manuscript is well written. However, at this point, there are several critical experiments that should be performed to solidify the conclusion.Essential revisions:1) Definition of the ERG and AR binding genome wide with and without PRMT5 and ERG knockdown and correlation with the gene expression is very important for the conclusion. Is AR binding specifically depleted in ERG co-bound sites? The changes genome wide may not reflect the selected sites, as highlighted by the effect of FOXA1 on AR. This does require time and effort. But AR and ERG ChIP-seq is well optimized and ChIP-seq today is cost effective so this is eminently doable.2) One weakness of the manuscript, intrinsic to prostate cancer research, is the use of a single ERG positive cell line. So any hit of the screen could be ERG specific, or even more likely, VCAP specific. Since there are no other available ERG dependent cell lines, the authors should note in the manuscript this major caveat. It would be very helpful if the authors could examine xenografts or perform some validation of the mechanisms in primary human tissue samples. For example, authors can IP AR from several ERG positive and ERG negative prostatectomy specimens to assess for methylation.3) Mass spec evidence of methylation at R761 would be helpful. Since you know what you're looking for, IP of AR in VCAP and another ERG negative line and looking for this should not be hard and would provide definitive evidence. The mutagenesis is highly suggestive, but could still change conformation in a way such that R761 is not the direct methylation site. Essential revisions:1) Definition of the ERG and AR binding genome wide with and without PRMT5 and ERG knockdown and correlation with the gene expression is very important for the conclusion. Is AR binding specifically depleted in ERG co-bound sites? The changes genome wide may not reflect the selected sites, as highlighted by the effect of FOXA1 on AR. This does require time and effort. But AR and ERG ChIP-seq is well optimized and ChIP-seq today is cost effective so this is eminently doable.KLK3 (aka PSA) and NKX3-1, the ChIPseq dataset shows a significant increase of AR recruitment upon PRMT5 knockdown at 6% of the total AR-bound cistrome. Genome-wide recruitment of ERG was not significantly affected upon PRMT5 knockdown, including to the sites where AR recruitment was increased . This data agrees with our model that ERG recruits PRMT5 to attenuate AR activity to a significant subset of its target genes.We thank the reviewers for raising this important point. To extend our findings beyond individual genes, we performed ChIPseq for both ERG and AR in the presence of the androgen molecules R1881 and DHT, and in the absence or presence of PRMT5 knockdown. This data is provided in 2) One weakness of the manuscript, intrinsic to prostate cancer research is the use of a single ERG positive cell line. So any hit of the screen could be ERG specific, or even more likely, VCAP specific. Since there are no other available ERG dependent cell lines, the authors should note in the manuscript this major caveat. It would be very helpful if the authors could examine xenografts or perform some validation of the mechanisms in primary human tissue samples. For example, authors can IP AR from several ERG positive and ERG negative prostatectomy specimens to assess for methylation.TMPRSS2:ERG positive models, we have explored the ERG/PRMT5 interaction using additional model systems. First, in TMPRSS2:ERG transgenic mice vs. control mice ; AR immunoprecipitates from the TMPRSS2:ERG mouse prostates show increased SDMA and MMA signals at the correct size for AR. Fifth, we show in We thank the reviewers for acknowledging the challenges of obtaining appropriate prostate models and patient specimens. To address the general lack of We further attempted to confirm our findings in human tissue samples. In the absence of an AR-R761me2s-specific antibody for immunoblotting or IHC, we planned to use a chromogenic in situ proximity ligation assay (PLA)  with primary antibodies recognizing AR and symmetric di-methyl arginine (SDMA) to detect AR-R761me2s in human prostate cancer samples. First, we focused on optimizing PLA for formalin-fixed paraffin-embedded (FFPE) tissues, using FFPE RWPE-1 prostate cells, FFPE human prostate cancer samples, and two primary antibodies against Ki67 . While numerous individual spots representing positive signal were detected in the nuclei of RWPE-1 prostate cells, few, if any, spots were seen in the human tissues see , suggest3) Mass spec evidence of methylation at R761 would be helpful. Since you know what you're looking for, IP of AR in VCAP and another ERG negative line and looking for this should not be hard and would provide definitive evidence. The mutagenesis is highly suggestive, but could still change conformation in a way such that R761 is not the direct methylation site.We agree that mass spectrometry data would be an important support of our findings. We made several attempts at mass spec identification of the arginine site using AR immunoprecipitated from VCaP cells. Unfortunately, we could not identify this site by mass spectrometry. In our hands, arginine methylation has been generally difficult to detect, including on histones, which are 'classic' PRMT5 targets. We suspect that R761me2s does not occur on the entire population of AR within the cell lysate, which 'masks' the detection of methylated AR by mass spec. This may be in line with the observation that PRMT5 knockdown does not affect AR recruitment to all possible sites in our ChIPseq experiments.While not directly addressing the reviewers’ question, we provide additional supporting data that PRMT5 can methylate purified AR ligand binding domain in vitro. These biochemical assays demonstrate PRMT5 activity  on purified AR but not ERG, and that addition of purified ERG DNA binding domain can facilitate greater PRMT5 activity on AR. This data is provided in


AbstractPhyllodonta latrata (Guenée) has been applied to what is a complex of three undescribed species in Costa Rica. They are very similar in maculation, but can be differentiated by genitalic characters and barcodes. P. alajuela Sullivan, sp. n. occurs at lower altitudes in the northwestern part of Costa Rica whereas P. intermediata Sullivan, sp. n. and P. esperanza Sullivan, sp. n. are found at partially overlapping altitudes in the central mountain ranges.Historically, the name  PageBreakPhyllodonta Warren currently consists of approximately 26 species of medium-sized geometrid moths that occur from the southern United States to Argentina (Phyllodonta latrata (Guenée) was described from Brazil and Colombia; in the British Museum the specimen from Nova Friburgo  is labeled as the type. It is unlikely that specimens from Colombia are conspecific. In Costa Rica, specimens that are very similar to Phyllodonta latrata occur from about 500–3000 m, but mtDNA barcode sequences of distinct clusters suggest a complex of species. Four additional species from Peru and Ecuador have been subject to barcode analysis but no haplotype clusters overlap those from Costa Rica. There are no specimens of Phyllodonta latrata from Brazil that have been barcoded nor have the types of Phyllodonta latrata nor Phyllodonta succedens   been dissected. Currently, Phyllodonta succedens is placed as a synonym of Phyllodonta latrata in the British Museum collections, although Phyllodonta nolckeniata (Snellen)  as a synonym.The genus rgentina . Currentrgentina . UnpubliPhotographic methods used herein are described in  Natural History Museum, London, UKBMNH Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa RicaINBio J. Bolling Sullivan, Beaufort, North Carolina, USAJBS National Museum of Natural History, Washington, District of Columbia, USAUSNMPageBreakPhyllodonta latrata (Gn.), Phyllodonta succedens (Wlk.) and Phyllodonta nolckeniata (Snellen) are distinguished by their rich brown color and will be referred to as the Phyllodonta latrata complex. The antemedial and postmedial lines are usually distinct and are wavy or undulating. On the underside, these lines are distinct and the characteristic whitish blotch  and Phyllodonta sarukhani Beutelspacher are similar in general color but the wing markings and genitalia are very different , Cartago Province, 1275 m, 7–9 July 2008, J. Bolling Sullivan (DNA voucher #11-CRBS-381) (INBio). Paratypes: 5♂, 4♀: 1♂, same data as holotype (DNA voucher #11-CRBS-288); 1♂, same data as holotype but 12–17 February 2005 (07-CRBS-1206), (dissection #JBS-3305); 1♂, Costa Rica, Parque National Volcan Poas , Alajuela Province, 2500 m, 7–8 August 2007, J. Bolling Sullivan (07-CRBS-359), (dissection #JBS-2006); 2♀, same data (07-CRBS-357), (JBS-2007); 3♂, Costa Rica, Villa Mills , Cartago Province, 2845 m, J. Bolling Sullivan , 2♀, same data . BMNH, INBio, JBS, USNM.Holotype male: Costa Rica, Tapanti Parque ; the vesica is unarmed with pouches emanating from the left side . In the female, the collar on the ductus bursae is narrow and the signum on the corpus bursae is crescent shaped, often with a medial kink and with the anterior and posterior sides narrowly separated. In the other two species the collar is wider and the signa are more oval shaped, never kinked.Maculation does not seem to be diagnostic for distinguishing this species. It is best characterized by barcode data and the genitalia. It has the highest altitude distribution but does overlap that of Male. . Wings–forewings warm brown with prominent antemedial and postmedial lines that undulate, in both cases forming two outward bulges. Antemedial bulges separated by prominent cleft. Both lines black edged with lighter grayish scales proximally. Black scaling distal to reniform at costa and forming a diffuse line paralleling antemedial line. Reniform spot small, black and forming center of a gray circle. Forewings long, outer margin truncated at costa with a distinct notch medially, forewing length 23.0 mm . Hindwing with prominent postmedial line, margin with submedial notch. Underside of wings warm brown, forewing with postmedial line prominent, medial line visible, antemedial line absent. White blotch subterminal at notch, larger black blotch proximal to it. Small discal spot present. Hindwing underside with prominent postmedial line, prominent discal spot, small yellow line of scales distal to postmedial line on both wings. Cream streak from wing base widening to anal angle. Male Genitalia  is:Twenty nine specimens have been barcoded and exhibit twelve haplotypes that differ from each other by a maximum of 0.8%. They differ from those of PageBreakGATGAACTGTTTATCCTCCTTTATCTTCTAATATTGCTCACGGTGGTAGTTCTGTTGACCTTGCTATTTTTTCATTACATTTAGCTGGTATTTCATCAATTTTAGGGGCTATTAATTTTATTACTACAATTATTAATATACGATTAAATAATTTATCTTTTGATCAAATACCTTTATTTGTATGAGCAGTAGGAATTACTGCATTTTTATTATTATTATCATTACCTGTTTTAGCTGGAGCTATTACTATATTATTAACAGATCGAAATTTAAATACATCTTTTTTTGATCCTGCTGGAGGAGGAGATCCAATTTTATACCAACATTTATTTAACATTATATTTTATTTTTGGGATTTGAGCTGGAATAGTAGGAACATCTTTAAGTTTATTAATTCGAGCTGAATTAGGAAATCCTGGATCTCTAATTGGAGATGATCAAATTTATAATACTATTGTAACTGCTCATGCTTTTATTATAATTTTCTTTATAGTAATACCTATTATAATCGGAGGATTTGGAAATTGATTAGTTCCTTTAATATTAGGAGCTCCTGATATGGCTTTCCCTCGAATAAATAATATAAGATTTTGATTACTTCCACCTTCTATTACATTATTAATTTCTAGAAGAATTGTGGAAAATGGAGCTGGGACAGKnown from above 1200 m in the Talamancas as well as the Central Volcanic and Tilaran ranges in Costa Rica. In flight throughout the year.Nothing is known about the biology of this species. Its range probably extends into the other mountain ranges in Costa Rican and perhaps in northern Panama.Taxon classificationAnimaliaLepidopteraGeometridaeSullivansp. n.http://zoobank.org/FEE6C9E5-A097-4088-983C-D0573F1F0E1C9.43°N, 83.46°W) Cartago Province, 1475 m, 7–8 July 2008, J. Bolling Sullivan (10-CRBS-283) (INBio), Paratypes. 5♂, 1♀: 2♂, 1♀, same data as holotype , 10-CRBS-282 (JBS-5409)); 2♂, Costa Rica, Tapanti Parque , Cartago Province, 1275 m, 7–9 July 2008, J. Bolling Sullivan, (11-CRBS-286 (JBS-5416); 1♂, Costa Rica, Vera Blanca, La Paz Waterfall Garden , Alajuela Province, J. Bolling Sullivan (07-CRBS-1202), (JBS-3306) .Costa Rica, tunnel road, Tapanti Parque , the vesica is unarmed and pouches emanate from the right side . In the female, the collar on the ductus bursae is narrow (broad in Phyllodonta esperanza)and the signum on the corpus bursae is peanut-shaped with moderate spacing between the anterior and posterior sides .Maculation is not diagnostic for identifying this species. It is best characterized by barcode data and the genitalia. In the male, the distal side of the socius is usually straight . Wings–forewings warm brown with prominent antemedial and postmedial lines that undulate, in both cases forming two outward bulges. Antemedial bulges separated by cleft. Both lines black edged with lighter grayish scales proximally. Black scaling distal to reniform at costa and forming a diffuse line paralleling antemedial line. Reniform spot small, black and forming center of a gray circle. Wings long, outer margin truncated at costa with a distinct notch medially, forewing length 23.1 mm . Hindwing with prominent postmedial line, margin with submedial notch. Underside of wings warm brown, forewing with postmedial line prominent, medial line visible, antemedial line absent. White blotch subterminal at notch, larger black blotch proximal to it. Small discal spot present. Hindwing underside with prominent PM line, prominent discal spot, small yellow line of scales distal to PM line on both wings. Cream streak from wing base widening to anal angle. Male Genitalia  is:Seven specimens have been barcoded and exhibit two haplotypes that differ from each other by a maximum of 0.5%. They differ from those of AACATTATATTTTATTTTTGGAATTTGAGCTAGAATAGTGGGAACGTCTTTAAGTTTATTAATTCGAGCAGAATTAGGGAATCCTGGGTCTTTAATTGGAGATGATCAAATTTATAATACTATTGTAACTGCACATGCTTTTATTATAATTTTCTTTATAGTAATACCTATTATAATTGGGGGATTTGGAAATTGATTAATTCCTTTAATACTAGGGGCTCCTGATATAGCTTTCCCTCGAATAAATAATATAAGATTTTGGTTACTTCCACCTTCCATTACATTATTAATTTTTAGAAGAATTGTAGAAAATGGAGCTGGAACAGGATGAACAGTTTACCCACCTTTATCTTCTAATATTGCTCATGGGGGTAGTTCTGTTGATCTTGCTATTTTTTCATTACATTTAGCTGGTATTTCATCAATTTTAGGAGCTATTAATTTCATCACCACAATTATTAATATACGATTAAATAATTTATCTTTTGATCAAATACCTTTATTTGTATGAGCGGTAGGAATTACTGCATTTTTATTATTATTATCATTACCTGTTTTAGCTGGAGCTATTACTATATTATTAACCGATCGAAATTTAAATACATCTTTTTTTGACCCTGCTGGTGGAGGAGATCCAATTTTATACCAACATTTATTTKnown from between 1275 to 2280 m in the Talamancas, the Central Volcanic range, and the Tilaran range in Costa Rica. The moths are probably in flight throughout the year.Nothing is known about the biology of this species. Its range may extend into the other mountain ranges in Costa Rican and perhaps into northern Panama.Taxon classificationAnimaliaLepidopteraGeometridaeSullivansp. n.http://zoobank.org/F193BB75-F3BF-42EC-9292-C10D65E1533110.22°N, 85.62°W), Alajuela Province, 850 m, 7–11 February PageBreak2005, J. Bolling Sullivan  (INBio). Paratypes: 4♂, 1♀: 2♂, same data as holotype , 1♂, 1♀, Costa Rica, Upata. Estacion San Gerardo , Alajuela Province, 550 m, 17–21 July 2006, J. Bolling Sullivan , JBS-3314). 1♂, Costa Rica, Upata Bijagua, Alberque Heliconias , 800 m, Alajuela Province, J. Bolling Sullivan (JBS-3310).Holotype male: Costa Rica, San Ramon Reserva Biol. Alberto M. Brenes Estacion Biol. . The female has a broadened collar on the ductus bursae (narrow in Phyllodonta esperanza)and the signum on the corpus bursae is almost round with equal-sized spines around it .Maculation does not seem to distinguish this species. It is best characterized by barcode data and the genitalia. To date it has been found below 1200 m and with no other member of the Male. . Wings–forewings warm brown with prominent antemedial and postmedial lines that undulate, in both cases forming two outward bulges. Antemedial bulges separated by cleft. Both lines black edged with lighter grayish scales proximally. Black scaling distal to reniform at costa and forming a diffuse line paralleling antemedial line. Reniform spot small, black, and forming center of a gray circle. Wings long, outer margin truncated at costa with a distinct notch medially, forewing length 24.1 mm . Hindwing with prominent postmedial line, margin with submedial notch. Underside of wings warm brown, forewing with postmedial line prominent, medial line visible, antemedial line absent. White blotch subterminal at notch, larger black blotch proximal to it. Small discal spot present. Hindwing underside with prominent PM line, prominent discal spot, small yellow line of scales distal to PM line on both wings. Cream streak from wing base widening to anal angle. Male Genitalia  is:Twenty eight specimens have been barcoded and exhibit 9 haplotypes that differ from each other by a maximum of 1%. They differ from those of PageBreakGAGATGATCAAATTTATAATACTATTGTAACTGCTCATGCTTTTATTATAATTTTTTTTATGGTAATACCTATTATAATTGGGGGATTTGGGAATTGATTAGTTCCTTTAATATTGGGGGCCCCAGATATAGCTTTCCCACGAATAAATAATATAAGATTTTGATTACTTCCGCCTTCTATTACACTTTTAATTTCTAGAAGAATTGTAGAAAATGGAGCCGGAACTGGATGAACTGTCTACCCTCCTTTATCTTCTAATATTGCCCACGGTGGTAGTTCTGTTGATCTTGCTATTTTTTCATTACATTTAGCTGGTATTTCATCAATTTTAGGGGCTATTAATTTTATTACAACAATTATTAATATACGATTAAATAACTTATCTTTTGATCAAATACCTTTATTTGTTTGAGCTGTAGGAATCACTGCATTTTTATTATTATTATCATTACCTGTTTTAGCTGGAGCTATTACTATATTATTAACTGATCGAAATTTAAATACATCTTTTTTTGACCCTGCTGGAGGAGGAGACCCAATTTTATATCAACATTTATTCAACATTATATTTTATTTTTGGAATTTGAGCTGGAATAGTAGGTACATCTTTAAGTTTATTAATTCGAGCGGAATTAGGAAACCCTGGGTCTTTAATTGKnown from 500 to 1150 m in the provinces of Alajuela and Guanacaste. Moths are probably in flight throughout the year.Witheringia solanacea L. Hér. in the Solanaceae. One record may represent a second species on Brugmansia × candida (Solanaceae). The present range of Phyllodonta alajuela seems to be limited to the Northwestern provinces of Costa Rica and may extend into Nicaragua. Southward and eastward its range is unknown. Specimens of the Phyllodonta latrata complex from Anchicaya and Calima Dam in Valle Province of Colombia (four dissections) have similar male genitalia, but the basal spines on the vesica are larger; no specimens of it have been barcoded. Species of non-migratory moths common to Colombia and Costa Rica are often distributed in the western lowlands of both countries.Phyllodonta latrata (Gn.) complex is represented in Costa Rica by three new species that can be differentiated by barcodes and genitalic features. Phyllodonta alajuela occurs at lower altitudes whereas Phyllodonta intermediata and Phyllodonta esperanza overlap at intermediate altitudes; but are often separate at the altitude extremes of their ranges. At the conclusion of this study, two male specimens in the Phyllodonta latrata complex from the states of Bahia and Rio De Janeiro in Brazil were borrowed from the USNM and dissected. Both dissections showed characters not seen in any other members of the complex  and are supported by other less obvious characters and geographic separation.The complex nature of species grouped under the name otropics . ConstanPhyllodonta latrata complex in South America. Specimens from lowland western Colombia may be conspecific with Phyllodonta alajuela but proof is lacking. Two male specimens taken above 2500 m in the central Andean range (Alto Rio Quindio) were dissected and differed from each other and all species from Costa Rica. Two specimens from coastal Brazil also were unique. Additional specimens barcoded from Ecuador and Peru are distinct. Thus, it is likely that the type locality for Phyllodonta latrata (Brazil) and high altitude locations throughout the Andes will provide a surprising number of additional species in the complex.Barcodes and genitalic analyses also support the differentiation of other populations of the PageBreak


AbstractDisphragis notabilis (Schaus) species-group are reviewed, including the types and their dissected genitalia. Disphragis hemicera (Schaus), stat. rev., is elevated to species rank, D. normula (Dognin) is retained as a synonym of D. notabilis, D. sobolis Miller is confirmed as distinct from D. hemicera, and D. bifurcatasp. n., is newly described. Both D. hemicera and D. bifurcata occur in Costa Rica. The known ranges of the other species are outlined. Defining characters of each species are presented and a key to species is provided. Unusual variation in the genitalia is noted.The four described taxa in the  Disphragis notabilis (Schaus), described from French Guiana, has been applied to prominent moths throughout Central and South America. Miller described Disphragis sobolis from Ecuador and indicated that genitalic characters reveal yet another member of the complex in Ecuador  from Costa Rica and Disphragis normula (Dognin) from Peru, so it is necessary to examine all named taxa in order to classify the species found in Costa Rica.The name  Ecuador . CollectPhotographic methods used herein are described in  Natural History Museum, London, UKBMNH Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa RicaINBio J. Bolling Sullivan collection, Beaufort, North Carolina, USAJBS National Museum of Natural History, Washington, District of Columbia, USAUSNMHeterocampa notabilis Schaus was named from French Guiana. Both Heterocampa hemicera Schaus  from Costa Rica and Heterocampa normula Dognin  from Peru are listed as synonyms of Disphragis notabilis by Disphragis sobolis from the mountains of eastern Ecuador and noted that it was easily separated from specimens of Disphragis notabilis by its darker color and more mottled appearance , Limon Province, 354’, 1–4 July 2008, J. Bolling Sullivan. INBio. Paratypes: 11♂, 3♀: 4♂, same data as holotype ; 1♀, 22 March 2003, Monty Wood (JBS-3030); 1♂, Costa Rica, Est. Biol. La Selva , Heredia Province, 50–150 m, 21–30 June 2003, Monty Volovsic (JBS-3040), 2♂, 29 Aug. –2 Sept. 2003, J. Bolling Sullivan (JBS-3038); 2♂, Costa Rica, Upata Estacion San Gerardo , Alajuela Province, 550 m, 17–21 July 2006, J. Bolling Sullivan, B. Espinosa (JBS-3035); 1♂, Costa Rica, Puriscal Chires, Mastatal , San Jose Province, 400 m, 16–18 Oct. 2011, J. Bolling Sullivan; 1♂, Costa Rica, Verugua Rainforest Campamento , Limon Province, 400–500 m, 12–16 March 2010, J. Bolling Sullivan (11-CRBS-2066), (JBS-5427); 1♀, Costa Rica, Tuis, 2500’, June, W. Schaus 1910-110. (BM-); 1♀, Costa Rica, Cashi, 8–10 1912 (Lankester), Rothschild Bequest, B. M. 1939-1. (BM-). Holotype male: Costa Rica, Reserva Hitoy Cerere . Longest rami 0.44 mm. Thorax a blend of fine brown and cream scales giving a tan appearance. Metathorax bearing a central white spot with row of darker brown scales anteriorly. Abdomen with appressed brown scaling. Forewing (17.5 mm N = 10) elongate, rounded apically and with broad tan subcostal streak from base of wing to apex. Streak encloses chocolate reniform spot and has several slightly darker brown lines crossing obliquely from costa. Basal dash below streak paralleling costa. White streak below basal dash; warm brown patch distal to white streak bordered by white; wavy antemedial (AM) and postmedial (PM) lines. Chocolate shading from middle of forewing below costal streak and forming a wedge to margin . Weak gray crescent on lower half of margin. Hind wing fuscous with darker margin and veins, weak darker brown anal markings almost a spot at anal angle. Underside of forewing fuscous, anal margin and cell yellowish. Basal 3/4 of hind wing yellowish, margin brown and well differentiated. Legs a mixture of brown and white scales, appearing almost yellowish, with white scales forming rings at distal end of tarsal segments. Tibial spines 0-2-4. Male genitalia  and with fasciculate antennae. Female genitalia  is:Five barcoded specimens exhibit two haplotypes that differ from each other by a maximum of 0.15%. They differ from AACCTTATATTTCATTTTTGGAATTTGAGCAGGAATAGTAGGAACCTCTTTAAGTCTTCTAATTCGTGCTGAATTAGGAACCCCCGGGACTTTAATTGGAGATGATCAAATTTATAATACTATTGTAACAGCTCATGCTTTCATTATAATTTTTTTTATAGTAATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTACCTTTAATATTAGGAGCCCCAGACATAGCTTTCCCACGAATAAATAATATAAGTTTTTGATTATTACCTCCTTCTTTAATACTTTTAATTTCGAGAAGTATTGTAGAAAATGGAGCAGGAACAGGATGAACAGTTTACCCACCACTGTCATCTAATATTGCTCATAGAGGAAGCTCTGTTGATTTAGCCATTTTTTCCCTTCACTTAGCTGGTATTTCATCAATTTTAGGGGCTATTAATTTTATCACAACAATTATTAATATACGATTAAATAATATATCTTTTGATCAAATACCTTTATTTGTGTGAGCTGTAGGAATTACTGCTTTTTTACTTTTACTTTCTCTCCCAGTTCTAGCTGGAGCTATTACTATACTTTTAACTGATCGTAATTTAAATACATCTTTTTTTGACCCTGCAGGGGGAGGAGATCCTATTTTATACCAACATTTATTTPageBreakKnown from Guatemala to Colombia , and probably extending south into northern Ecuador.Disphragis hemicera.This species occurs at lower altitudes and moderate elevations (1000 m) where it occurs with Taxon classificationAnimaliaLepidopteraNotodontidaestat. rev.Heterocampa hemicera Schaus, 1910, Annals and Magazine of Natural History 6: 582.Costa Rica.Disphragis hemicera and Disphragis sobolis from Disphragis notabilis and Disphragis bifurcata. Their appearance is mottled, grayish brown with a distinct dark band next to the PM line. Males may be distinguished by the shape of the phallus, which in Disphragis sobolis has a distinct dorsal projection. Females can be separated by the shape of the genital plate, which in Disphragis sobolis is bifurcate at the distal tip and in Disphragis hemicera has a middle phalanx with lateral “wings” from the base. Geographic distribution also separates Disphragis hemicera from Disphragis sobolis, with Disphragis hemicera in Central America and western Colombia, and Disphragis sobolis along the western slopes of the Andes.Maculation will usually separate Male. . Rami noticeably longer than in Disphragis bifurcata, longest rami 0.53 mm. Thorax a blend of brown and cream scales giving a tan appearance. Metathorax with a central white spot with row of darker brown scales anteriorly. Abdomen with appressed brown scaling. Forewing  elongate, rounded apically and with broad light brown subcostal streak from base of wing to apex. Streak encloses chocolate reniform spot and has several slightly darker brown lines crossing obliquely from costa. Basal dash below streak perpendicular to thorax. White streak below dash; warm brown patch distal to white streak bordered by white; AM and PM lines wavy. Distinct brown line bisecting warm brown patch. Chocolate shading from middle of forewing below costal streak and forming a wedge to margin . Gray crescent on lower half of margin with distinct brown band inward to PM line. Hind wing uniformly fuscous with brown anal markings forming something of a spot at anal margin. Light streak along anal edge. Underside of forewing fuscous with yellowish subapical crescent along costa. Basal half of hind wing yellowish, no well-differentiated margin. Legs a mixture of brown and white scales appearing somewhat yellowish with white scales forming rings at distal end of tarsal joints. Tibial spines 0-2-4. Male genitalia  is:Fifty eight barcoded specimens exhibit seven haplotypes that differ from each other by a maximum of 0.30%. They differ from those of AACTTTATATTTTATTTTTGGAATTTGAGCAGGAATAGTAGGAACTTCTTTAAGTCTTTTAATTCGTGCTGAATTAGGAACCCCCGGGACTTTAATTGGAGATGATCAAATTTATAATACTATCGTAACAGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTCCCTTTAATACTAGGAGCACCAGATATAGCTTTCCCACGAATAAATAATATAAGTTTTTGACTATTACCCCCTTCTTTAATACTTCTAATTTCAAGAAGTATTGTAGAAAATGGAGCTGGTACAGGATGAACAGTTTATCCCCCACTGTCATCAAATATTGCTCACGGAGGAAGCTCTGTTGATTTAGCTATTTTTTCCCTTCATTTAGCGGGTATTTCCTCAATTTTAGGGGCTATTAATTTTATTACAACAATTATTAATATACGATTAAATAATATATCTTTTGATCAAATACCTTTATTTGTATGAGCTGTAGGAATTACTGCTTTTCTACTTTTACTTTCACTCCCAGTATTAGCTGGAGCTATTACTATACTTTTAACCGATCGTAATTTAAATACATCTTTTTTCGACCCTGCTGGGGGAGGAGATCCTATTTTATACCAACATTTATTTPageBreakDisphragis hemicera occurs throughout Costa Rica at moderate altitudes. It is found south along the western coast of Colombia and may extend to the west coast of Ecuador. The northern limits are unknown but it probably occurs at least into Nicaragua.Disphragis hemicera is by far the most common member of the group in Costa Rica and appears to be absent below 500 m. At moderate altitudes both Disphragis hemicera and Disphragis bifurcata occur together.Taxon classificationAnimaliaLepidopteraNotodontidaeHeterocampa notabilis Schaus, 1906, Proceedings of the United States National Museum 29: 253.Heterocampa normula Dognin, 1909, Annales de la Société entomologique de Belgique 53: 81. Disphragis notabilis: French Guiana; Disphragis normula: PeruDisphragis notabilis and Disphragis bifurcata from Disphragis hemicera and Disphragis sobolis. Disphragis notabilis and Disphragis bifurcata are warm brown, not mottled or brownish gray like Disphragis hemicera and Disphragis sobolis. The male antennal pectinations are shorter in Disphragis notabilis than in Disphragis hemicera and Disphragis sobolis. Males of Disphragis notabilis are easily distinguished by their moderately wide socii, which taper to a single point with many ventral spines. In males of Disphragis bifurcata the socii are much broader and are bifurcate at the upturned apex. Females must be sorted by maculation and geography. Disphragis notabilis is Amazonian in distribution whereas Disphragis bifurcata occurs from central and western Colombia north into Central America.Maculation characters can usually be used to separate Male. . Longest rami 0.34 mm, shortest of all species. Thorax a blend of brown and cream scales giving a tan appearance. Metathorax bearing a central white spot with row of darker brown scales anteriorly. Abdomen with appressed brown scaling. Forewing  elongate, rounded apically and with broad tan subcostal streak from base of wing to apex. Streak encloses chocolate reniform spot and has several slightly darker brown lines crossing obliquely from costa. Basal dash below streak perpendicular to thorax, abbreviated relative to that of Disphragis bifurcata. White streak below dash; warm brown patch distal to white streak bordered by white; AM and PM lines wavy. Chocolate shading from middle of wing below costal streak and forming a wedge to margin . Weak gray crescent on lower half of margin. Warm brown from patch expanded almost to margin and reducing size of chocolate wedge seen in Disphragis bifurcata. Hind wing fuscous with darker margin, weak darker brown anal markings almost forming a spot. Underside of forewing fuscous, anal margin and cell yellowish. Basal 3/4 of hind wing yellowish, margin brown and moderately differentiated. Legs a mixture of brown and white scales appearing almost yellowish with white scales forming rings at distal end of tarsal joints. Tibial spines 0-2-4. Male genitalia  is:Two barcoded specimens exhibit 2 haplotypes that differ from each other by 0.30%. They differ from those of AACTTTATATTTCATTTTTGGAATTTGAGCAGGAATAGTAGGAACCTCTTTAAGTCTTCTAATTCGTGCTGAATTAGGAACCCCCGGGACTTTAATTGGAGATGACCAAATTTATAATACTATCGTAACAGCTCATGCTTTCATTATAATTTTTTTTATAGTAATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTACCTTTAATATTAGGAGCCCCAGACATAGCTTTCCCACGAATAAATAATATAAGTTTTTGATTATTACCTCCTTCTTTAATACTTTTAATTTCAAGAAGTATTGTAGAAAATGGAGCAGGAACAGGATGAACAGTTTACCCACCACTGTCATCTAATATTGCCCATAGAGGAAGCTCTGTTGATTTAGCCATTTTTTCCCTTCACTTAGCCGGTATTTCATCAATTTTAGGGGCTATTAATTTTATCACAACAATTATTAATATACGATTAAATAATATATCTTTTGATCAAATACCTTTATTTGTATGAGCTGTAGGAATTACTGCTTTTTTACTTTTACTTTCTCTTCCAGTTCTAGCTGGAGCTATTACTATACTTTTAACTGATCGTAATTTAAATACATCTTTTTTTGACCCTGCAGGGGGAGGAGATCCTATTTTATACCAACATTTATTTThis species occurs throughout the Amazon basin from western Venezuela eastward and southward to at least Bolivia.Disphragis notabilis is by far the most common member of the group in South America, however, earlier references to this species should be confirmed in light of the additional species described here.Taxon classificationAnimaliaLepidopteraNotodontidaeMiller, 2011Disphragis sobolis Miller, 2011. In Ecuador.Male. . Rami noticeably longer than in Disphragis hemicera, longest 0.59 mm. Thorax a blend of brown and cream scales giving a tan appearance. Metathorax bearing a central white spot with row of darker brown scales anteriorly. Abdomen with appressed brown scaling. Forewing  elongate, rounded apically and with broad light brown subcostal streak from base of wing to apex. Streak encloses chocolate reniform spot and has several slightly darker brown lines crossing obliquely from costa. Brown scaling throughout as well as several black streaks. Basal dash below streak perpendicular to thorax and greatly reduced in length. White streak below dash; warm brown patch distal to white streak bordered by white; AM and PM lines wavy. Distinct brown line bisecting warm brown patch. Chocolate shading from middle of forewing below costal streak and forming a wedge to margin  more extensive than in Disphragis hemicera. Prominent gray crescent on lower half of margin with distinct brown band inward to PM line. Hind wing uniformly fuscous with brown anal markings almost a spot. Light streak along anal edge. Underside of forewing fuscous with yellowish subapical crescent along costa. Basal half of hind wing yellowish, no well-differentiated margin. Legs a mixture of brown and white scales, appearing almost yellowish with white scales forming rings at distal end of tarsal joints. Tibial spines 0-2-4. Male genitalia  is:One specimen has been barcoded and differs from that of AACTTTATATTTTATTTTTGGAATTTGAGCAGGAATAGTAGGAACCTCTTTAAGTCTCCTAATTCGTGCTGAATTAGGAACCCCCGGGACTTTAATTGGAGATGATCAAATTTATAATACTATTGTAACAGCTCATGCTTTTATTATAATTTTTTTTATAGTAATACCCATTATAATTGGAGGATTTGGTAATTGATTAGTTCCTCTAATATTAGGAGCTCCAGATATAGCTTTCCCACGAATAAATAATATAAGTTTTTGATTATTACCCCCCTCTCTAATACTTTTAATTTCAAGAAGTATTGTAGAAAATGGAGCAGGAACAGGATGAACAGTTTACCCCCCACTGTCATCAAACATTGCTCATAGAGGAAGATCTGTTGATTTAGCTATTTTTTCCCTTCACTTAGCAGGTATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATTATTAATATACGATTAAATAACATATCTTTTGATCAAATACCTTTATTTGTTTGAGCTGTAGGAATTACTGCTTTTTTACTTTTACTCTCTCTTCCAGTATTAGCAGGAGCTATTACTATATTATTAACCGATCGTAATTTAAATACATCTTTTTTTGACCCCGCTGGGGGAGGAGATCCTATTTTATATCAACATTTATTTPageBreakThis species appears to be limited to the eastern slopes of the Andes from Bolivia to Villavicencio, Colombia.Disphragis sobolis was recently described from Ecuador; the species appears to have a much greater geographical range and occurs to almost 3000 m. The lower altitude limits of its range are undefined, as is the southern boundary.PageBreakDisphragis notabilis complex is typical of many neotropical species. When studied in detail, they frequently are found to consist of a number of very similar species that do have structural differences, can be separated by barcoding, and occupy different altitudes or geographic ranges. The correct generic placement of this complex is not in Disphragis  and makes the delineation of species and their defining characters with female specimens extremely difficult. Fortunately, such extreme variation has not been observed in males.A second difficulty encountered in this study was the hyper-variation of the female genital plate in hemicera . This inDisphragis notabilis complex found in Colombia (and probably Ecuador) remains to be elucidated but should highlight the individual habitat requirements of each species. Neither larvae nor foodplants are known. The geographical area where Disphragis bifurcata and Disphragis notabilis come into contact should be particularly interesting because the two species differ by 1.4% in their barcodes, a magnitude lower than between most congeneric species. However, there are a number of characters that separate the two species and characters do not seem to intergrade in individuals from central Colombia and western Venezuela.The exact distribution of the four species of the