The Malarial parasite resides in the host RBC during its erythrocytic cycle. Plasmodium meets its entire nutritional requirement fromRBC. It scavenges the hemoglobin of RBCs to meet its amino acid requirement. The host hemoglobin is made of different chains and itis dependent on age. Hemoglobin F (HbF), which has two-alpha and two gamma chain persists in children upto six years, andhemoglobin A (HbA) made of two alpha and two beta chains dominates. Therefore, it is of interest to compare the compositionalfeatures of HbA with HbF. Isoleucine is present in hemoglobin of children (gamma chain of HbF) while it is absent in adulthemoglobin (HbA). The presence of Isoleucine (I) makes HbF ideally suitable for the growth of parasite, as it does not have to dependupon the exogenous supply of the isoleucine, which might be responsible for making children more vulnerable to malaria as comparedto adults. Malaria is one of the most prevalent diseases in developingcountries . MalariaMalaria affects all age groups. However, the children are affectedthe most. According to WHO report there were an estimated 438000 malaria deaths around the world in 2015, 69% of total deathsdue to malaria are known to occur in children aged from 6months to 5 years .Though the parasite culture in RBC containing adult Hb isroutinely performed  The studIn this study, we show the abundance of different amino acids inPlasmodium falciparum 3D7 and distribution of different aminoacids according to its essential nature in host. We have alsocompared the amino acid composition of different chains ofhaemoglobin to determine the difference that leads to preferenceof RBC containing HbF, which might explain the disease severityin children.The FASTA format of all protein sequence, 5,369 proteins, ofPlasmodium was downloaded from PlasmoDB. Composition ofprotein sequences was completed using ProtParaman tool.ProtParaman tool is an online tool, which is freely available anddetermines the sequence composition and predicts other physicalparameters . Total aComplete protein sequences of 5369 were downloaded fromPlasmoDB for Plasmodium falciparum 3D7. The amino acid compositions of total 5369 proteins are represented by pie chart. As The The protein sequence of the haemoglobin chain was downloadedfrom NCBI and the composition of amino acid was determinedusing ProtParaman tool  as descrThe fasta formats for different chain of the haemoglobins wereobtained from NCBI. The assertion number of different chains ofare as follows:MGHFTEEDKATITSLWGKVNVEDAGGETLGRLLVVYPWTQRFFDSFGNLSSASAIMGNPKVKAHGKKVLTSLGDATKHLDDLKGTFAQLSELHCDKLHVDPENFKLLGNVLVTVLAIHFGKEFTPEVQASWQKMVTAVASALSSRYHMVHFTAEEKAAVTSLWSKMNVEEAGGEALGRLLVVYPWTQRFFDSFGNLSSPSAILGNPKVKAHGKKVLTSFGDAIKNMDNLKPAFAKLSELHCDKLHVDPENFKLLGNVMVIILATHFGKEFTPEVQAA WQKLVSAVAI ALAHKYHMVHLTPEEKTAVNALWGKVNVDAVGGEALGRLLVVYPWTQRFFESFGDLSSPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFSQLSELHCDKLHVDPENFRLLGNVLVCVLARNFGKEFTPQMQAAYQKVVAGVANALAHKYHMVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYHMVLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYMSLTKTERTIIVSMWAKISTQADTIGTETLERLFLSHPQTKTYFPHFDLHPGSAQLRAHGSKVVAAVGDAVKSIDDIGGALSKLSELHAYILRVDPVNFKLLSHCLLVTLAARFPADFTAEAHAAWDKFLSVVSSVLTEKYRMultiple sequence analysis of different chain of Hemoglobin wasdone using Clustal omega. The results show similarity betweendifferent chains of haemoglobin. Further, we looked at thedistribution of amino acid in different haemoglobin chain . We obsePlasmodium parasite, scavenges the haemoglobin to meets itsamino acid requirements while lipids are obtained from RBCsmembrane. The distribution of amino acids in the proteins ofPlasmodium shows incorporation of all the 20 amino acids.However, the amino acids, which are essential and conditionallyessential to the host, are present in higher percentage than nonessentialamino acids.Among the essential amino acids leucine is required mostfollowed by isoleucine. Among the conditionally essential aminoacids N is required the most. N is present in parasite as repeatwhich might have role in immune evasion by antigenic variation.Other amino acids like arginine, which is essential for polyaminesynthesis is important for robust growth of the parasite.I make up to 9% of total amino acids in Plasmodium falciparum hasto be obtained exogenously from blood, as it is absent in adulthaemoglobin. Istvan et al. has shown that the absence of I affectsparasite growth and I analogue inhibits the parasite growth .The gamAs Plasmodium solely depends upon the degradation ofhaemoglobin for its amino acid requirements it meets most of thenutritional requirements from haemoglobin. As HbF contains allthe amino acids including isoleucine (absent in HbA) required bythe parasite hence, this might be aid growth of parasite in HbFcontaining RBC. Besides at the age of 9 up to, 18% of RBCcontains HbF. Hence, we hypothesise that presence of HbFwould aid parasite growth leading to disease severity in children.Authors declare no conflict of Interest


AbstractMastigodiaptomus Light, 1939, named Mastigodiaptomuscuneatussp. n. was found in a freshwater system in the City of Mazatlán, in the northern region of Mexico. Morphologically, the females of this new species are distinguishable from those of its congeners by the following combination of features: the right distal corner of the genital double-somite and second urosomite have a wedge-shaped projection, the fourth urosomite has no dorsal projection and its integument is smooth. The males are distinct by the following features: the right caudal ramus has a wedge-shaped structure at the disto-ventral inner corner; the basis of the right fifth leg has one triangular and one rounded projection at the distal and proximal margins, respectively, plus one hyaline membrane on the caudal surface close to the inner margin; the aculeus length is almost the width of the right second exopod (Exp2); and the frontal and caudal surfaces of the right Exp2 are smooth. Furthermore, the analysis of the COI gene of Mastigodiaptomuscuneatus sp. n. has revealed that Mastigodiaptomusalbuquerquensis  is its nearest congener, with 18.64% of genetic distance. A key for the identification of the known species of the genus is provided.A new species of the genus  Diaptomidae G. O. Sars, 1903 is one of the most common families of freshwater copepods worldwide, some genera of this family have restricted distributional patterns and present endemic forms, as the genus Mastigodiaptomus Light, 1939. Recent studies of this genus in the Neotropical region have added new species of these diaptomids  , Mastigodiaptomustexensis , Mastigodiaptomusamatitlanensis , Mastigodiaptomusalbuquerquensis , Mastigodiaptomuspatzcuarensis , Mastigodiaptomusmontezumae , Mastigodiaptomusnesus Bowman, 1986, Mastigodiaptomusmaya Suárez-Morales & Elías-Gutiérrez, 2000, Mastigodiaptomusreidae Suárez-Morales & Elías-Gutiérrez, 2000, and Mastigodiaptomussuarezmoralesi Gutiérrez-Aguirre & Cervantes-Martínez, 2013.aptomids  or the mr, 1938) . CurrentCyclopidae genus Mesocyclops; and Cyclopidae genus Eucyclops, particularly the Eucyclopsserrulatus species complex. Mastigodiaptomus genus using differences in the integument of prosomal wings and fifth legs of both sexes. Empirical evidence gathered from several species of freshwater crustaceans has shown that these morphological differences are consistent with reproductive isolation  Chetumal, México and the Colección Nacional de Crustáceos (CNCR) del Instituto de Biología, Universidad Nacional Autónoma de México.The type material was deposited at the Colección de Referencia de El Colegio de la Frontera Sur  were preserved after capture in 96% ethanol and prepared for barcoding following standard methods. DNA was extracted using the HOTSHOT method . A segmeBOLD) produced sequences of Mastigodiaptomusalbuquerquensis, Mastigodiaptomuspatzcuarensis, Mastigodiaptomuscf.albuquerquensis , Microcrustacean from Mexico (MCM), and Zooplankton II (ZPII) and compared with our sequence of Mastigodiaptomuscuneatus sp. n., this latter sequence is into the project MCM. In these project files, the electropherograms, sequence data, photographs, primers data, and collection details are available (on the Barcode of Life Data System http://www.boldsystems.org). Fifty-two COI gene sequences > 500 bp were used for the analysis, and BOLD Aligner and the ID Tree using the model Kimura 2 parameter   were used as an outgroup.These sequences were downloaded from the ter K2P;  were utiPageBreakTaxon classificationAnimaliaCalanoidaDiaptomidaehttp://zoobank.org/FADC3B97-FB6F-4559-B71B-EB6F76A3246FECOCH-Z-09339. Collected 28.VIII.2014. Collectors: A. Cervantes-Martinez, N. Hernández López, M. Bastidas, and J. Aguilar Rubio.One adult female dissected on one slide: PageBreakECOCH-Z-09340. Collected 28.VIII.2014. Same collectors.One adult male dissected on one slide: ECOCH-Z-09341. Collected 28.VIII.2014. Same collectors.Four adult females and five adult males preserved in 90% ethanol with a drop of glycerine. PageBreakCNCR-31861. Collected 28.VIII.2014. Same collectors.Two adult females and two adult males preserved in 90% ethanol: 23°14'10"N; 106°26'18"W.A lagoon called Laguna El Camarón in Avenida Insurgentes, Mazatlán, Sinaloa City, México; The name of the species means “wedged” in Latin and refers to the chitinous protuberance present on the right disto-lateral corner of the first and second urosomites in females, and on the right caudal ramus on the ventral surface in males.Adult female: Cuticle surfaces of prosomal somites smooth dorsal and laterally Fig. . AntennuAdult male: The cuticle surfaces of prosomal somites are smooth dorsally and laterally Fig. . Right an = 6  with a movable seta at tip. Rectangular, nude coxa. Basis with 4 long setae. Enp two-segmented, Enp1 with 4 ely Fig. .PageBreakand 2-segmented endopodite: Enp1 and Enp2 with 5 and 4 setae, respectively. Basis rectangular with 4 setae; coxal endite quadrangular 4-setulated. Praecoxal arthrite with 15 spiniform setae, 11 anterior, 4 posterior.Maxillule Fig. : Coxal eMaxilla Fig. : First pMastigodiaptomusalbuquerquensis and Mastigodiaptomuspatzcuarensis (see Maxilliped (not figured): Same structure as described and illustrated for nsis see .Diaptomidae family  with a smooth hyaline membrane.PageBreakAntennule, antenna, mandible, maxillule, maxilla, maxilliped, and P1-P4 as described for female.Right wing of fifth pediger with 1 tiny dorsal spinule and 1 ventral spine Fig. ; left wiPageBreakUrosome: Urosomites nude dorsally and ventrally. First urosomite with thin spine on right side and fold on left side Fig. . Fourth PageBreakP5: Coxal segments with strong spines on caudal view; left and right basis with a lateral seta Fig. .Left basis with a triangular protuberance on distal margin of frontal surface Fig. . Both leRight basis basally and distally projected: basal projection rounded whereas distal projection triangle-shaped; one semi-triangular sclerotization on caudal surface of right basis Fig. . Right EMastigodiaptomuscuneatus sp. n. is shown below:The nucleotide sequence (607 bp) obtained for specimen MAGA-0156 , identified as GGAGCCTGGTCAGGCATAGTAGGAACAGGCCTTAGAATGATTATTCGGATGGAGTTAGGACAAGCCGGGTCTTTAATTGGAGATGACCAAATTTATAATGTAGTAGTTACTGCTCATGCTTTTGTTATAATTTTTTTTATGGTGATACCTATTTTAATTGGGGGGTTTGGTAATTGGCTTGTTCCGTTAATATTAGGTGCAGCGGATATAGCTTTCCCTCGAATAAATAATATAAGATTTTGATTTTTATTGCCAGCTTTAGTCATATTGTTATCTAGGTCGCTTGTTGAAAGAGGGGCGGGAACAGGGTGAACTGTGTATCCCCCCCTGTCTAGCAACATTGCCCATGCTGGCAGGTCCGTTGATTTTGCTATTTTTTCGCTTCATTTAGCTGGGGTTAGGTCTATTTTGGGCGCAGTAAATTTTATTAGCACATTAGGAAATTTGCGGGCGTTTGGAATAATTTTAGATCGAATACCACTTTTTGCTTGAGCCGTTTTAATCACGGCTATCTTGTTATTGCTTTCTCTTCCTGTTTTAGCCGGGGCGATTACAATGCTTCTTACAGATCGGAACCTCAACTCAAGATTTTATGAT.K2P maximum distance between the surveyed species reaching 5.52%  appeared to be morphologically close to Mastigodiaptomusamatitlanensis . Similarities between these species in females include the presence of one protrusion on the second urosomite and the bulbose lateral margins of genital double-somite. The similarities in males are the short aculeus and the lack of hyaline membrane on the right Exp2 of the fifth leg.Mastigodiaptomuscuneatus sp. n. can be separated from Mastigodiaptomusamatitlanensis by the following features: the dorsal projection on the last pediger absent vs. present; the genital double-somite with vs. without a protrusion on the distal right side; on the genital double-somite, the right spine located at a higher level than the left spine vs. both left and right spines placed at same level; and the endopod of fifth leg long and 2-segmented vs. short and 1-segmented.However, Mastigodiaptomuscuneatus sp. n. in comparison to the rectangular basis with a transversal, distal, cuneiform lamella in Mastigodiaptomusamatitlanensis. In addition, the distal margins of the left and right endopods bear short setules in Mastigodiaptomuscuneatus sp. n. whereas in Mastigodiaptomusamatitlanensis these distal margins bear one slender seta. The aculeus on the right Exp2 is clearly straight in Mastigodiaptomuscuneatus sp. n. but short, distal and curved in Mastigodiaptomusamatitlanensis. The Exp2 is smooth in Mastigodiaptomuscuneatus sp. n. but in Mastigodiaptomusamatitlanensis an oblique ridge on the caudal surface is present. Finally, we assume that the wedge on the right caudal ramus present in Mastigodiaptomuscuneatus sp. n. is absent in Mastigodiaptomusamatitlanensis because there is no mention of a similar feature in The males of these species show more morphological differences in the fifth leg: the caudal surface of the right basis is bulbose with the oblique, medial, angulose and curved cuticular process in Mastigodiaptomuscuneatus sp. n. is genetically closest to Mastigodiaptomusalbuquerquensis s. str. and the species recorded in Mexico with one sclerotization  on the right basis of fifth leg of males such as Mastigodiaptomuspatzcuarensis. As previously discussed , which probably are cryptic species , Mastigodiaptomuspatzcuarensis, Mastigodiaptomusmontezumae , Mastigodiaptomusnesus (Caribbean and south eastern Mexico), and Mastigodiaptomustexensis (Texas and south eastern Mexico), whereas the species that are assumed to have restricted distribution or endemics are Mastigodiaptomusreidae, Mastigodiaptomusmaya, Mastigodiaptomuspurpureus, Mastigodiaptomusamatitlanensis, Mastigodiaptomussuarezmoralesi (see Mastigodiaptomuscuneatus sp. n.lesi see  and, proMastigodiaptomuscuneatus sp. n. was compared with the ten known Mastigodiaptomus species, particularly in the female and male urosomes, the male right antennule and fifth legs, and in the COI gene sequence. This report increases the number of recognized species of Mastigodiaptomus to eleven. Mastigodiaptomuscuneatus sp. n. appears to be part of the Mastigodiaptomusalbuquerquensis complex.Morphological and genetic differences were found when MalesFemales


Molecular characterization showed that lncR492 interacts with the mRNA binding protein HuR and facilitates its inhibitory function by activation of Wnt signaling. Thus, lncRNAs modulate the fate decision of pluripotent stem cells.RNA interference (RNAi) screens have been shown to be valuable to study embryonic stem cell (ESC) self-renewal and they have been successfully applied to identify coding as well as noncoding genes required for maintaining pluripotency. Here, we used an RNAi library targeting >640 long noncoding RNAs (lncRNA) to probe for their role in early cell differentiation. Utilizing a Sox1-GFP ESC reporter cell line, we identified the lncRNA  Embryonic stem cells (ESC) are characterized by their ability of long-term self-renewal as well as their potential to differentiate into each cell type of the embryo proper. After the first isolation of embryonic stem cells from the mouse blastocyst , 2 the rRecent sequencing approaches have shown that the majority of the genome is transcribed . Among tTUNA was identified as sustainer of pluripotency but was also required for neural differentiation [Large-scale functional studies have identified numerous lncRNAs that play a regulatory role in the maintenance of pluripotency –10. It hlncR492 as an inhibitor of neural differentiation, which exerts its function by interacting with the mRNA binding protein HuR and activating Wnt signaling, thereby blocking ectodermal differentiation in a lineage specific manner.To identify specific neural differentiation regulating lncRNAs, we performed a large-scale loss-of-function RNAi screen in a Sox1-GFP ESC reporter line. We discovered the lncRNA Austin Smith and Konstantinos Anastassiadis kindly provided the Sox1-GFP and Oct4-GFP cell lines. The Foxa2-GFP and T-GFP cell lines were generated in the BAC TransgeneOmics project  and kind™) supplemented with 1x N2, 1x B27, 1x NEAA, 1x Penicillin/Streptomycin, 50 μM 2-mercaptoethanol , 3 μM CHIR99021, 1 μM PD0325901  and LIF (generated in house). For differentiation towards the ectoderm cells were cultured in N2B27 containing medium without the two inhibitors and LIF. For differentiation into endoderm (Foxa2-GFP ESC) and mesoderm (T-GFP ESC), the two inhibitors and LIF were replaced by 30 μg/ml ActivinA (MPI protein facility) or 10 μg/ml BMP4 (R&D Systems), respectively. The cells were seeded on gelatin-coated dishes with a density of 15000 cells/cm2 and grown for 4 to 5 days before they were harvested for experiments.Sox1-GFP, Foxa2-GFP, T-GFP ESC and R1/E ESC lines were cultured on gelatin-coated plates in DMEM  supplemented with 10% Pansera ES FBS (PAN Biotech), 1x NEAA, 1x Penicillin/Streptomycin, 50μM 2-mercaptoethanol  and LIF (MPI-CBG protein facility).Oct4-GFP ESC were additionally cultured on gelatin-coated plates in DMEM  or the corresponding non-labeled amino acids, respectively. All other supplements were maintained as described. Cells were grown for five passages, lysed and checked for successful label incorporation by MS.For SILAC-labeling the standard DMEM was replaced with SILAC-DMEM medium  supplemented with 40 mg/mL EsiRNAs were synthesized as described previously . For theNheI and XhoI restriction sites in the flanking regions. After sequencing, the PCR products were cloned into the pCGIT destination vector under the CAG promoter . pCGIT without insert was used as control (EV—empty vector).The cDNAs for lncR492 and HuR were amplified by PCR introducing ESCs were transiently transfected with the overexpression or control plasmids using Lipofectamine 2000 (ThermoFisher Scientific). 50 ng of plasmid DNA were combined with 0.2 μl Lipfectamine 2000 and incubated in 50 μl OptiMEM for 10 minutes. The transfection mix was added to one well of a 96-well plate. 4500 cells per well in 200 μl N2B27 medium supplemented with 2i+LIF were seeded on top. After 24h differentiation was initiated as described above.18 primer. Standard RT-PCR reactions were performed with a MyTaq™ Red DNA Polymerase (Bioline). Quantitative RT-RCRs (qRT-PCR) were run with the SYBR Green qPCR kit (Abgene) on an CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad). Measured transcript levels were normalized to Gapdh. Samples were run in duplicates. Primers used are listed below.Total RNA was isolated by using the RNeasy Mini kit (QIAGEN). For each RT reaction 1 μg of RNA was reverse transcribed with SuperScript III Reverse Transcriptase (ThermoFisher Scientific) utilizing oligo(dT)™ Eukaryotic mRNA Isolation Kit, EpiCentre). Subsequent 500 ng of RNA were reverse transcribed as described above.For endonuclease treatment 5 μg RNA were treated with a 5’-phosphate-dependent exonuclease according to manufactor’s instructions (mRNA-ONLYThe forward and reverse primer sequences (written 5’ to 3’) were as follows:Gapdh, ACTCCACTCACGGCAAATTC, GGATGCAGGGATGATGTTCTlncR492, GCTGCTGTTTCACACCCAAG, TGACTAGGCGATCCTGACCASox1, CCTTGCTAGAAGTTGCGGTC, TCACTCAGGGCTGAACTGTGSrrm4, TCTCGTCGAAGTCCCAGCTA, CACTGGTTATCCTCCGAGCCOct4, AGAGGGAACCTCCTCTGAGC, TGATTGGCGATGTGAGTGATBrachyury (T), GAACAGCTCTCCAACCTATG, AGACTGGGATACTGGCTAGAGFoxa2, GCTGCAGACACTTCCTACTAC, GGACACAGACAGGTGAGACTPax6, CACCAGACTCACCTGACACC, TCACTCCGCTGTGACTGTTCNestin, GCAGGAGAAGCAGGGTCT, AGGTGCTGGTCCTCTGGTNanog, GGAAGCAGAAGATGCGGACT, ATGCGTTCACCAGATAGCCCMalat1, GTTTGTGATTGGAGCCGAG, AAGGGAGGGGAGAGAGAACAU1 snRNA, GCGCGATTTGGCAAGATGA, TTCTTCCCGGGTTTCTGCTCStar solution, exposed to Amersham Hyperfilm , which was developed using an OPTIMAX (PROTEC).The sequences of the gene-specific Northern blot probes are listed below (written 5’ to 3’). Two probes per gene were mixed together. Northern blot was performed according to the DIG Northern Starter Kit’s (Roche) manual. Briefly, RNA was separated on a pre-cast 1% agarose gel at 35V for 3.5h in 1x MOPS buffer. The separated RNA was blotted on a nitrocellulose membrane  and cross-linked by UV-light. The membrane was prehybridized with DIG easy Hyb for 30 minutes at 68°C, followed by an hybridization step with a DIG-labeled, gene-specific RNA probe mix diluted in DIG easy Hyb over night at 68°C. Unspecific binding was eliminated by several stringency washes. Thereafter the membrane was blocked, incubated with a DIG-specific antibody and washed. Finally, the membrane was incubated with CDP-lncR492 probe 1UCAGAACCCAGCACACUGUCAGCCUCAGAGCAUACAAUCUUUUGAGUGAGUGAGCUAGAUUUGACAACGAUGCUUGAUGCUUGCGAGCAUAUGAGGGGCUCGCAGCCUCUUCCUAGGCACUACUGUCUCCCUCCGGAGACGCCUCCUGGCCUCCUUGAUUAUGAACACCUUUGCUAAUGCUCACAGUCUUCUUGUUCUCAUGGCCCUUGGAGGAUAUGUGCAGUGGUGAACACAGCUlncR492 probe 2AUGGCUUUUUCCUCGCUACAGUUCAGAGUCGCCACAAUCACAGGGGGCCGGGGAAGAUCACCAGCAAUCAGUGUUCAAACGGCCCAAAGAGAUUGUUCUGAGUCUCCUUGCCACUCCGCUGAUGGAGUAGGGCCUUCUAAUUGGCCCUUCUGUCUCUCCUGCCUCGCCCUUCCAAUCUUUUGCCUUCUUGGCAGCUAGGUUCAUGCUUAUAGACCAUUUUUCGGUGAGUCAUUGCCUUGAUUUAAAACCCUUCGGCUUCCACUUGAGACUUGAGCCAUCCAGGCUCCCUGCCAUGGAGGGAGAGGGCAGUCAUGAUCUGGUUUUGGCCAUCCAUCUCUCUCCUCUAUCCUGUUCCCUGAGUCCUUGUUCUGAGCAUCUACCUGGGGCAUCCUUCCUUCCCACUAACUCCUGUAUCAUGUCCCUGGACCACUCCCCACCUGACUGGUCCUCUGUGUUUUCAGAGUAGCUGAUUUCUUUUGCACUGCCUAGCGCCACUGGGAACAGACAUCGUCUGUGAGCUUUGGGAAGGGapdh probe 1CAGCGAACUUUAUUGAUGGUAUUCAAGAGAGUAGGGAGGGCUCCCUAGGCCCCUCCUGUUAUUAUGGGGGUCUGGGAUGGAAAUUGUGAGGGAGAUGCUCAGUGUUGGGGGCCGAGUUGGGAUAGGGCCUCUCUUGCUCAGUGUCCUUGCUGGGGUGGGUGGUCCAGGGUUUCUUACUCCUUGGAGGCCAUGUAGGCCAUGAGGUCCACCACCCUGUUGCUGUAGCCGUAUUCAUUGUCAUACCAGGAAAUGAGCUUGACAAAGUUGUCAUUGAGAGCAAUGCCAGCCCCGGCAUCGAAGGUGGAAGAGUGGGAGUUGCUGUUGAAGUCGCAGGAGACAACCUGGUCCUCAGUGUAGCCCAAGAUGCCCUUCAGUGGGCCCUCAGAUGCCUGCUUCACCACCUUCUUGAGapdh probe 2AAGCAGUUGGUGGUGCAGGAUGCAUUGCUGACAAUCUUGAGUGAGUUGUCAUAUUUCUCGUGGUUCACACCCAUCACAAACAUGGGGGCAUCGGCAGAAGGGGCGGAGAUGAUGACCCUUUUGGCUCCACCCUUCAAGUGGGCCCCGGCCUUCUCCAUGGUGGUGAAGACACCAGUAGACUCCACGACAUACUCAGCACCGGCCUCACCCCAUUUGAUGUUAGUGGGGUCUCGCUCCUGGAAGAUGGUGAUGGGCUUCCCGUUGAUGACAAGCUUCCCAUUCUCGGCCUUGACUGUGCCGUUGAAUUUGCCGUGAGUGGAGUCAUACUGGAACAUGUAGACCAUGUAGUUGAGGUCAAUGAAGGGGUCGUUGAUGGCAACAAUCUCCACU6 cells were washed with ice-cold PBS. Cells were resuspended in ice-cold, protease inhibitor (Roche) containing HD-buffer  and incubated on ice for 10 minutes. IGEPAL was added to a final concentration of 0.5%. Quickly citric acid (100 mM) was added to a final concentration of 1 mM. The cells were vortexed vigorously. The nuclei were separated from the cytoplasmic fraction by gentle centrifugation. Total RNA was isolated with the PARIS Kit (Ambion). 1 μg of RNA was reverse transcribed with SuperScript III Reverse Transcriptase (ThermoFisher Scientific) utilizing oligo(dT)18 primer. qRT-RCR were run as described above.Approx. 10x10lncR492 probe 2 was used as FISH probe.Sox1-GFP ESC were transfected with esiRNA using Lipofectamine2000 (Thermo Fisher Scientific) and seeded on gelatin-coated chambered slides (Ibidi). After 48 h RNA-FISH was performed as described earlier . The seqFor immunofluorescent stainings cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. All following steps were performed at room temperature. After washing with PBS cells were blocked and premeabilized with 10% FCS in staining buffer (0.3%TritonX-100 in PBS) for 30 minutes. The primary antibody rabbit-anti-Tubb3  was diluted 1:1000 in staining buffer and incubated for 1 h. After washing the cells were incubated with donkey-anti-rabbit-Cy3 secondary antibody (Jackson ImmunoResearch Laboratories) and DAPI (Sigma) for 30 minutes. Finally the cells were imaged using a Zeiss Axiovert 200M microscope equipped with an ApoTome (Zeiss). Images are projections of the maximum intensity. Total numbers of nuclei as well as Tubb3+ cells were counted manually.To create the 450 bp RNA baits, forward primers containing the T7 promoter sequence and reverse primers with the S1 aptamer sequence were used in a PCR amplification reaction on the pCAGGS plasmid. A fragment of the human PTPN13 mRNA of equal length (450 bp) was used as a control bait. The primer sequences are written 5’ to 3’.CGTTAATACGACTCACTATAGGCTCGAGATCCAGCTGGGCACAGGC.AK016992 (1–450 bp) + T7 forward primer, CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCAACTCTGCTGATCGGATCTGTCCTC.AK016992 (1–450 bp) + BioApt reverse primer, CGTTAATACGACTCACTATAGGGGCTCCCGGACCTTCCCAAAG.AK016992 (451–900 bp) + T7 forward primer, CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCAGATTGTTCTGAGTCTCCTTGCCAC.AK016992 (451–900 bp) + BioApt reverse primer, CGTTAATACGACTCACTATAGGCTTTGGGCCGTTTGAACACTG.AK016992 (901–1350 bp) + T7 forward primer, CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCTCTAGACGGGTACAATGCCTTC.AK016992 (901–1350 bp) + BioApt reverse primer, CGTTAATACGACTCACTATAGGAGGGCCATGAGAACAAGAAG.AK016992 (220–670 bp) + T7 forward primer, CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCGTTTTGGCCATCCATCTCTCTC.AK016992 (220–670 bp) + BioApt reverse primer, CGTTAATACGACTCACTATAGGCAGATCATGACTGCCCTCTCC.AK016992 (671–1120 bp) + T7 forward primer, CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCCGCTTGGGTGTGAAACAGCA.AK016992 (671–1120 bp) + BioApt reverse primer, CGTTAATACGACTCACTATAGG CAATATATTTTCTGCTATCAAGTC.PTPN13 + T7 forward primer, CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCCTTTATTAAAATATTGGAAAACATTTTTG.PTPN13 + BioApt reverse primer, in vitro transcription according to the manufacturer’s protocol (Fermentas). Successful transcription was monitored by agarose gel electrophoresis and RNA concentration was quantified by A280 absorbance on a Nanodrop system (Peqlab). 25 μg of each S1-tagged RNA was coupled to paramagnetic streptavidin C1 beads  in RNA binding buffer , 0.5% IGEPAL CA-620) and incubated on a rotation wheel for 30 min at 4°C. RNA-bound beads were washed 3 times with RNA washing buffer , 0.5% IGEPAL CA-620), prior to incubation with 400 μg nuclear extract, with 20 μg competitor yeast tRNA (Invitrogen) added, for 30 min at 4°C with gentle agitation. After mild washing, SILAC heavy and light fractions were combined 1:1 and samples were boiled in 1x LDS buffer (Invitrogen) and separated on a 4–12% NuPAGE Novex Bis-Tris precast gel (Life Technologies) at 180 V in 1x MOPS.The PCR products were used for 17703201) using two layers of C18material (Empore).Coomassie stained gels were cut in one slice and destained with 50% EtOH/25 mM ammonium bicarbonate (ABC). The resulting gel pieces were dehydrated with 100% acetonitrile (ACN) and dried for 5 min in a concentrator (Eppendorf). Samples were incubated with reduction buffer (10 mM DTT/50 mM ABC) for 30 min at 56°C and further alkylated for 30 min in the dark with iodoacetamide (50 mM IAA/50 mM ABC). Gel pieces were completely dehydrated with ACN and covered in trypsin solution (1 μg trypsin per sample). Proteins were digested over night at 37°C and peptides were extracted twice by incubation with extraction buffer (3% TFA and 30% ACN) for 15 min. The gel pieces were dehydrated with 100% ACN and the extracted volume reduced to aproximately 150 μl in a concentrator (Eppendorf). Extracted peptides were desalted in StageTips (PMID:17721543). MS full scans were obtained in the orbitrap at 70,000 resolution with a maximal injection time of 20 ms, while MS/MS scan resolution was set to 17,500 resolution and maximal injection for 120 ms. Unassigned and charge state 1 were excluded from MS/MS selection and peptide match was preferred. Raw files were processed with MaxQuant (version 1.5.2.8.) (PMID:19029910) and searched against human UNIPROT annotated protein database provided with MaxQuant  using the Andromeda search engine (PMID:21254760). Carbamidomethylation was set as a fixed modification, while acetyl (N-term protein) and oxidation (Met) were considered as variable modifications. Trypsin (specific) was selected as enzyme specificity with maximal two miscleavages for MaxQuant analysis. Proteins were quantified with at least 2 ratio counts based on unmodified unique and razor peptides. Known contaminants and reverse hits were removed before plotting the protein ratios of the forward and reverse experiments in R (version 3.2.2).Eluted peptides were injected via an autosampler into an uHPLC  and loaded on a 25 cm capillary  packed in-house with Reprosil C18-AQ 1.9 μm resin (Dr. Maisch) for reverse-phase chromatography. The EASY-nLC 1000 HPLC system was directly mounted to a Q Exactive Plus mass spectrometer (Thermo). Peptides were eluted from the column with a 90 min optimized gradient from 2 to 40% ACN with 0.1% formic acid at a flow rate of 200 nL/min. Chromatography was stabilized with a column oven set-up operating at 40°C (Sonation). The heated capillary temperature was set to 250°C. Spray voltage ranged from 2.2–2.4 kV. The mass spectrometer was operated in data-dependent acquisition mode with one MS full scan and up to ten triggered MS/MS scans using HCD fragmentation (PMID:RNA pull-down experiments were performed 3 times—once for mass spectrometry and twice for western blot to confirm the enrichment of HuR.® Reporter Assay (Promega) according to the manufacture’s instruction and the EnVision Multilabel Reader (PerkinElmer).Cells were co-transfected with Super 8x TOPFlash or Super 8x FOPFlash luciferase plasmid together with either target-specific esiRNA or overexpression plasmid as well as pCMV renilla plasmid to correct for transfection efficiency. Cells were cultured in N2B27+2i+LIF. 48 h after transfection cells were lysed and bioluminescence was analysed using the Dual-LuciferaseTotal cell lysates were harvest 72h after transfection using RIPA lysis buffer. 20 μg of each protein sample were loaded and analysed by western blot using a mouse anti-HuR monoclonal antibody , rabbit anti-Tubb3  and mouse anti-Gapdh  as primary antibodies. As secondary antibodies HRP-conjugated species-specific antibodies  were used. For quantitative analysis species-specific secondary antibodies from LI-COR Biosciences have been used. Blots were scanned and analysed with Odyssey.t-test.The results are shown as mean ± standard deviation (SD) as indicated in the figure legends. Statistical significance was calculated using the unpaired Student’s Sox1 is one of the earliest transcription factors marking the neural ectoderm  and Sox1lncR492 (accession no. AK016992) had the strongest effect (Z-score (1st esiRNA) = 4.0 and Z-score (2nd esiRNA) = 5.4; lncR492. Both esiRNAs achieved a knock-down efficiency of >60%  tail -tailed and capped , 6. cDNA(A) tail . Further capping . To furtock-down . Consistfraction .lncR492 as a non-coding nuclear transcript, which might play a role in regulating ectodermal cell fate commitment.Thus, the esiRNA-based screen in differentiating ESCs identified lncR492 acts as an inhibitor of ectodermal differentiation. To clarify its role and specificity in lineage commitment, we first tested its expression in pluripotent and differentiating ESCs. These results revealed that lncR492 has its highest expression in the pluripotent state. Interestingly, together with Oct4, lncR492 is down-regulated during neural differentiation , Brachyury-GFP+  and Foxa2-GFP+ (endoderm) cells after differentiation. In accordance with the reporter, the lineage specific genes were up-regulated when the cells were cultured under differentiation conditions in N2B27 alone (Sox1-GFP) or supplemented with 30 μg/ml Activin A (Foxa2-GFP) or 10 μg/ml BMP4 -positive cells in the lncR492 knock-down condition compared to cells treated with a control esiRNA as seen by immunofluorescent staining  and obtained similar results , which was most enriched in pull downs with fragment 1  was identified as well as an 11 bp long sequence in the 3’ region (HuR BS2) of the transcript  mRNAs  and RNA-anscript . The posern blot . ESC difock-down . Importantiation . Likewisntiation . In accopression . Howeverpression . Given tpression . Hence, lncR492 gene expression. Interestingly, knock-down and overexpression of HuR resulted in a respective decrease and increase of lncR492, whereas Srrm4 transcript levels were not affected Click here for additional data file.S2 FigLncR492 expression in differentiated ESCs. Therefore T-GFP and Foxa2-GFP reporter cells were differentiated N2B27-containing medium supplemented with 10 μg/ml BMP4 or 30μg/ml ActivinA for 4 days, respectively. Sox1-GFP ESCs were differentiated for 4 days in medium containing the serum replacement N2B27 only. GFP+ cells were sorted by FACS, RNA was isolated and lncR492 as well as lineage specific gene expression was analysed by qRT-PCR. Expression was normalized to undifferentiated ESC. Data presents mean ± SD of three independent experiments.(A) lncR492 and Srrm4 during the time course of differentiation. Data presents mean ± SD of three independent experiments.(B) QRT-PCR analysis of lncR492 knock-down or overexpression in T-GFP ESCs. Bar graph represents the quantification of three independent western blot experiments. Data presents the mean ± SD.(C) Western blot analysis of Tubb3 after lncR492 knock-down or overexpression. Cells were harvested after 4 days of differentiation in N2B27. Data presents the mean ± SD of three independent experiments.(D) Gene expression analysis of the T-GFP reporter ESC (R1/E) by qRT-PCR after lncR492 knock-down and overexpression in Oct4-GFP ESC cultured in N2B27+2i+LIF medium. Data represents mean ± SD of four independent experiments.(E) FACS analysis of GFP expression after lncR492 knock-down and overexpression in Oct4-GFP ESC cultured in medium supplemented with FCS+LIF. Knock-down of Rad21 was used as a positive control. Data represents mean ± SD of four independent experiments.(F) FACS analysis of GFP expression after * p<0.05; ** p<0.01; *** p<0.001; n.s.–not significant.(TIF)Click here for additional data file.S3 FigSrrm4 expression after lncR492 knock-down and overexpression measured by qRT-PCR. Data represents mean ± SD of three independent experiments.(A) (B) Summary table of proteins detected by mass spectrometry analysis. The lncRNA transcript was split into five overlapping fragments of 450 bp length each. The top ten putative interaction proteins for each lncRNA fragment are listed according to their abundance.lncR492. Putative binding sides for HuR are highlighted in red based on the consensus sequence NNUUNNUUU.(C) Nucleic acid sequence (mRNA) of (D) Western blot of HuR knock-down and overexpression. Gapdh was used as loading control. EV—empty vector.lncR492 and HuR knock-down or HuR overexpression. Cells were differentiated for 4 days in N2B27 supplemented with 30 ng/ml ActivinA. Data presents mean ± SD of three independent experiments.(E) FACS analysis of Foxa2-GFP expression after (F) FACS analysis of Oct4-GFP expression 48h after HuR knock-down and overexpression. Oct4-GFP cells were cultured in N2B27+2i+LIF medium. Data presents mean ± SD of three independent experiments.* p<0.05; ** p<0.01; *** p<0.001; n.s.–not significant.(TIF)Click here for additional data file.S1 TableZ-scores of the primary and the validation screen are shown for each replicate. Hits of the primary screen with an average Z-score >3 are highlighted in green (increasing the number of Sox1-GFP positive cells) and hits with an average Z-score < -3 are highlighted in orange (decreasing the number of Sox1-GFP positive cells). In the validation screen a Z-score > 2 or <-2 are considered as hit and highlighted in green.(XLSX)Click here for additional data file.S2 TableIdentified proteins for each fragment used in the pull-down experiment are shown.(XLSX)Click here for additional data file.


Such integrations rendered cells sensitive to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. We constructed dph3 and msh3 strains with mutated ATG start codons (ATGmut), which allowed investigating drug sensitivity without potential interference by marker insertions. The dph3-ATGmut and a dph3::loxP-ura4-loxM gene disruption strain, but not msh3-ATGmut, turned out to be sensitive to hydroxyurea and methyl methanesulfonate, likewise the strains with cassettes integrated at the msh3 locus. The fungicide sordarin, which inhibits diphthamide modified eEF2 of Saccharomyces cerevisiae, barely affected survival of wild type and msh3Δ S. pombe cells, while the dph3Δ mutant was sensitive. The msh3-ATG mutation, but not dph3Δ or the dph3-ATG mutation caused a defect in mating-type switching, indicating that the ura4 marker at the dph3 locus did not interfere with Msh3 function. We conclude that Dph3 is required for cellular resistance to the fungicide sordarin and to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. This is likely mediated by efficient translation of proteins in response to DNA damage and replication stress.Dph3 is involved in diphthamide modification of the eukaryotic translation elongation factor eEF2 and in Elongator-mediated modifications of tRNAs, where a 5-methoxycarbonyl-methyl moiety is added to wobble uridines. Lack of such modifications affects protein synthesis due to inaccurate translation of mRNAs at ribosomes. We have discovered that integration of markers at the The online version of this article (doi:10.1007/s00294-017-0711-x) contains supplementary material, which is available to authorized users. Saccharomyces cerevisiae    by Trm9–Trm112 and to 5-carbamoylmethyl-uridine (ncm5U34) by an unknown enzymatic activity  and methyl methanesulfonate (MMS). Msh3 is a eukaryotic homologue of bacterial MutS. MutS is a DNA mismatch binding protein that initiates removal of mismatched and unpaired nucleotides, which were incorporated into the nascent strand during replication  that drug sensitivity was indeed due to an impaired dph3 function. Thus, Dph3 plays a role in response to DNA damage and replication stress, likely through modifications of tRNA and/or eEF2, which allow efficient biosynthesis of DNA damage response proteins.In this study, we discovered that replacement of the open reading frame of S. pombe msh3 and dph3 genes share an intergenic region of only 268 base pairs (bp) between the two ATG start codons . Data obtained by functional genomics further revealed that the intergenic region constitutes the 5′ untranslated regions (5′ UTR) of the msh3 and dph3 mRNAs, with divergent and likely overlapping promoters  from here on  of base–base mismatches and loops of one to four nucleotides , demonstrating that the bands produced with the reverse-transcribed samples reflect amplification of cDNA derived from mRNA. In addition, PCR on a cDNA sample of a dph3::loxP-ura4-loxM strain yielded no bands , demonstrating that the amplified DNA was dph3 specific. We then used dph3q-F and seven reverse primers for PCR that prime at different positions, covering most of the dph3 mRNA. We could not detect any relevant differences between wild type and the msh3::kanMX mutant  colonies, indicative for a switching defect. The h90dph3::loxP-ura4-loxM and h90dph3-ATGmut mutants formed iodine positive colonies, although less homogeneously stained than wild type  colonies were not formed. Thus, replacement of dph3 by the ura4 marker did not disrupt the Msh3 function in mating-type switching. The phenotype of reduced sporulation of dph3 mutants is unrelated to Msh3 functions, as dph3-ATGmut does not have an integrated cassette that may interfere with msh3 expression.We noticed that colonies of the ies Fig. a. Both dres Fig. b. On thedph3 (kti11), like the other dph mutated genes in S. cerevisiae causes resistance to diphtheria toxin and sordarin due to the lack of the diphthamide modification of eEF2  mutants and unlike the other dph mutants  for lysine have low protein levels in elp3 mutants  after 3 days of incubation at 30 °C. Drugs used were HU (Formedium), MMS (ACROS Organics) and sordarin (Santa Cruz). Spot tests were carried out at least twice, with the exception of the experiments including the plates with 100–150 μg/mL sordarin. To determine spore formation efficiency 10 μL of 1:10 dilutions in H2O of stationary phase cultures were spotted on MEA and incubated for 2 days at 30 °C. Means and standard deviations were calculated from six independent experiments, where 200 units (asci and zygotes) were counted for each of them. S. pombe strains are listed in Supplemental Table S1. The S. cerevisiae strain RCY2459 (MATa ho::LYS2 ura3 lys2 leu2::hisG in SK1 background) was a kind gift of Rita Cha .Yeast media were YEA (yeast extract agar), YEL (yeast extract liquid), MEA  and MMA  as described  was obtained by transformation of the 972 wild type strain (h−) with a PCR product obtained with primers D3kf 5′-TTTTTAAAACGTCCGCCTATATTCATTTAGAAACTATAAATGCGTTACAAATCTATGTTGAGCAAATTTGATTGTACAGTTTTTATTTTCGCTTTATTACCGCCAGCTGAAGCTTCGTAC-3′ and Dk3r 5′-AGAACTTAAAACTGATGTATAGTTGCTTAAATTTATTTATACAGTAAAATCTTATGTGTTGAGTATCCTAATAAAGTAGTTAATTCAGTAATGCTCTCTCGGCCACTAGTGGATCTGATA-3′ using plasmid pFA6a-kanMX  was constructed by transformation of OL2137 (h−ura4-D18) with a PCR fragment obtained with primers D3kf and Dk3r (see above) and pFA6a-hphMX6  derived from transformation of EH238 (smt-0 leu1-32 ura4-D18) with a PCR fragment obtained with primers msh3_pAW1_For 5′-TTTTTAAAACGTCCGCCTATATTCATTTAGAAACTATAAATGCGTTACAAATCTATGTTGAGCAAATTTGATTGTACAGTTTTTATTTTCGCTTTATTACCGGATCCCCGGGTTAATTAA-3′ and msh3_pAW1_Rev 5′-AGAACTTAAAACTGATGTATAGTTGCTTAAATTTATTTATACAGTAAAATCTTATGTGTTGAGTATCCTAATAAAGTAGTTAATTCAGTAATGCTCTCTCGAATTCGAGCTCGTTTAAAC-3′ using pAW1  is a transformant of OL2137 with a PCR fragment obtained with primers dph3_pAW1_For2 5′-CTTAGCTTGTAGTTTTCATTATGGGCGGTTCCCACATATAAAACAAATTTTTGGTGGAGTGGCCACGCACCTTCTGCCAGTAGTGCATTGAAGCGGCAAAAGCTTAGCTACAAATCCCAC-3′ and dph3_pAW1_Rev2 5′-TATCACGATGTAAAGAGTAGCCCTCCTATCCTTCGTAATTTCAAGACATTTTGAGAATAAATAGAAGTAAAAAACCAAATAAGAAATTATAGGGAAAAAAGCTTGTGATATTGACGAAAC-3′ and pAW1 as template.DE4 , a derivative of pAW8 6 tag followed by three glycine-encoding codons, added between the natural ATG start codon and the second codon of msh3. Nucleotides underlined in msh3_pAW8_Rev 5′ changed the two last codons of msh3 from GAA ATC (encoding the amino acids glutamic acid and isoleucine) to GAG CTC (encoding glutamic acid and leucine), thereby introducing a SacI restriction site, allowing further manipulation if desired.Plasmid pAW8-msh3 was constructed by in vitro Cre recombination between the h−dph3-ATGmut ura4-D18) was constructed by transformation of DE4 with a PCR fragment obtained with primers dph3_ATGmut_For 5′-TAGCTTGTAGTTTTCATTATGGGCGGTTCCCACATATAAAACAAATTTTTGGTGGAGTGGCCACGCACCTTCTGCCAGTAGTGCATTGAAGCGGCAAA-TGATCATTTTACGACGAAATCG-3′ and dph3_Rev 5′-TCACGATGTAAAGAGTAGCCCTCCTATCCTTCGTAATTTCAAGACATTTTGAGAATAAATAGAAGTAAAAAACCAAATAAGAAATTATAGGGAAAAATTATGCTGCAATGATTATAGGTG-3′ and as template genomic DNA of strain RO144 (smt-0). “-”in primer dph3_ATGmut_For indicates deletion of an A of the wild type sequence, which causes an in frame TGA stop codon instead of the ATG start codon. By this procedure, the lox sites flanking dph3 in DE4 were replaced in DE5. Sequencing confirmed the correct mutation in DE5 and an additional T155A point mutation, which is in intron II of the dph3 gene.Strain DE5 (smt-0 msh3-ATGmut leu1-32 ura4-D18) originated from the transformation of KK83 with a PCR fragment obtained from a mutagenized pAW8-msh3 plasmid as template. This plasmid contains the msh3 gene, with the 31st codon mutagenized from ATG to ATC by site directed mutagenesis with a QuikChange lightning site-directed mutagenesis kit (Agilent Technologies) using primers msh3_M31I_S 5′-GGAGCAATATCAGAAGATATCGTTGCCCTCAGTGGTCCAG-3′ and msh3_M31I_AS 5′-CTGGACCACTGAGGGCAACGATATCTTCTGATATTGCTCC-3′. Nucleotides deviating from the wild-type sequence are underlined. Primers for amplifying the 5′ part of the open reading frame of msh3 with ATG mutations at codons 1 and 4 were msh3-ATGmut_For2 5′-TCCGCCTATATTCATTTAGAAACTATAAATGCGTTACAAATCTATGTTGAGCAAATTTGATTGTACAGTTTTTATTTTCGCTTTATTACTCGAGAGGATAGAGTTATAACATTACTCATG-3′ and msh3_Rev2 5′-CTTAAAACTGATGTATAGTTGCTTAAATTTATTTATACAGTAAAATCTTATGTGTTGAGTATCCTAATAAAGTAGTTAATTCAGTAATGCTCTCTCTCAGATTTCTTCGAAAGCGGTAAG-3′. Primer msh3-ATGmut_For2 contained base substitutions (underlined), which changed the ATG start codon of msh3 to TCG and the fourth codon from ATG to TAG. After integration into the genome, the mutated msh3 gene was amplified by PCR and sequencing confirmed the three desired ATG mutations. In addition, we found A555G, C696T and T1112C mutations. The primers used for PCR to construct an ATG mutated msh3 strain were designed in such a way that the (His)6 tag, the glycine linker and the lox sites of plasmid pAW8-msh3-M31I were not present in the genome of the resulting DE7 strain.DE7  including DNase I treatment. 2 µg of RNA were reverse transcribed with oligo (dT)18 primers using a Tetro cDNA Synthesis kit (Bioline). The resulting cDNA was subjected to 22–35 cycles of 30 s at 94 °C, 30 s at 57 °C and 30 s at 72 °C and a final extension for 10 min at 72 °C. For amplification of dph3 cDNA the forward primer dph3q-F 5′-AGATTTCACGTTTGACGCCG-3′  and the reverse primer dph3q-R 5′-CTTGGGCAACGAGCAACATC-3′ was used. In a pilot experiment , we used primers dph3_cPCR_For2 5′-CTATGATGAAGATGAATTCATGGAAGTTG-3′ and dph3_cPCR_Rev2 5′-GAACAACAAACGTACTATGCCAATACAAAACC-3′. Other reverse primers for amplification of dph3 cDNAs of various sizes were dph3q-R#2 5′-TCACCCGGACAATCAAGCTG-3′, dph3q-R#3 5′-AGGTGCTGTAGAAGCATCGT-3′, dph3q-R#4 5′-GTAGCCCTCCTATCCTTCGT-3′, dph3q-R#5 5′-TGGAAAAGTCGTACGCTCAA-3′, dph3q-R#6 5′-AGGTGGGCTTTTAGTTTCGAGT-3′, dph3q-R#7 5′-ACGTTGAGCACAAGTACGAA-3′ and dph3q-R#8 5′-CCACTGTGGTATGTCGCACT-3′. Primers for amplification of act1 cDNA were act1-For 5′-AAGTACCCCATTGAGCACGG-3′ and act1-Rev 5′-CAGTCAACAAGCAAGGGTGC-3′. PCR products were separated on 1.5% agarose gels in Tris–Borate-EDTA buffer and images taken with a Gel Doc 2000 system (Bio-Rad). Band intensities of PCR products were quantified with the ImageJ software (NIH). dph3-specific bands were normalised to blanks to subtract background and to act1 cDNA.10 mL cultures were grown in YEL at 30 °C to a density of approximately 10S. pombe cells Supplementary material 2 (DOCX 15 kb)Below is the link to the electronic supplementary material.


C. elegans VAP homolog VPR-1 is essential for gonad development. vpr-1 null mutants are maternal effect sterile due to arrested gonadogenesis following embryo hatching. Somatic gonadal precursor cells and germ cells fail to proliferate fully and complete their respective differentiation programs. Maternal or zygotic vpr-1 expression is sufficient to induce gonadogenesis and fertility. Genetic mosaic and cell type-specific expression studies indicate that vpr-1 activity is important in the nervous system, germ line and intestine. VPR-1 acts in parallel to Notch signaling, a key regulator of germline stem cell proliferation and differentiation. Neuronal vpr-1 expression is sufficient for gonadogenesis induction during a limited time period shortly after hatching. These results support the model that the secreted VPR-1 MSPd acts at least in part on gonadal sheath cell precursors in L1 to early L2 stage hermaphrodites to permit gonadogenesis.VAMP/synaptobrevin-associated proteins (VAPs) contain an N-terminal major sperm protein domain (MSPd) that is associated with amyotrophic lateral sclerosis. VAPs have an intracellular housekeeping function, as well as an extracellular signaling function mediated by the secreted MSPd. Here we show that the  Highlighted Article:vpr-1 null mutants are sterile upon hatching, a defect rescued by the expression of MSPd from almost any tissue except for the somatic gonad itself. See also the companion paper by Schultz et al. The major sperm protein domain (MSPd) is an evolutionarily conserved immunoglobulin-like structure found in unicellular and multicellular eukaryotes . The nam2+ dynamics and other processes. In addition, the VAP MSPd is cleaved from the transmembrane domain and secreted in a cell type-specific fashion  A Lev et. Althoug fashion . The sec fashion . Hence, C. elegans. The C. elegans genome encodes numerous proteins that contain an MSPd  and mouse Vapb mutants  null mutation eliminates the first two vpr-1 exons, which encode the MSPd and part of the coiled-coil motif (vpr-1(tm1411) mutants derived from P0 vpr-1(tm1411)/hT2 heterozygous hermaphrodites exhibit limited fertility, with an average brood size of ∼30 F2 progeny (vpr-1(tm1411) hermaphrodites lacking maternal (M−) and zygotic (Z−) vpr-1 expression (vpr-1(tm1411) mutant adults with maternal vpr-1 mRNA (M+ Z−) contain functional sperm and oocytes /+ progeny produce functional gametes, similar to wild-type and M+ Z− vpr-1 mutant hermaphrodites  null mutants transgenically expressing vpr-1 from fosmid DNA contain ∼10 sheath cells (vpr-1 M− Z− gonads contain severely reduced numbers of sheath (∼4) and spermathecal (∼6) cells  blast cells formed during L1 and early L2 C Hubbar. To inveransgene . In adulransgene E. We obscomplete E. Furtheskeleton . vpr-1 mth cells F. SS blath cells C. The adth cells C  cells H-J. The ntiation .vpr-1 is broadly expressed in most tissues (vpr-1 functions (i.e. in which cell type) to promote gonad development. Transgenic vpr-1(tm1411) null mutants were generated using a DNA fosmid containing the vpr-1 genomic locus and a plasmid containing the sur-5p::NLS-GFP lineage marker  worms rescued the gonad defects  or failed ovulation, two defects consistent with abnormal somatic gonad development or function  in the germ line using the pie-1 promoter (pie-1p::vpr-1g). Single-copy integrants on chromosome II were used to avoid germline silencing mechanisms. Two independently generated integrated transgenes completely rescued the vpr-1(tm1411) gonad and body wall muscle mitochondrial defects .Genetic mosaic worms lacking  fertile , consist defects A-D  gonad defects  background. A small percentage (<20%) of transgenic mutants exhibited the novel gonad phenotype described above. In addition to the neuronal promoters, overexpressing vpr-1g specifically in the intestine using the ges-1 promoter . These results show that vpr-1 overexpression in diverse cell types is sufficient to promote gonadogenesis.A caveat with integrated transgenes is that ectopic expression in unwanted tissues may result from surrounding regulatory sequences. To avoid this issue, we tested a panel of well-characterized neuron-specific promoters using high-copy extrachromosomal arrays . Overexppromoter  largely  defects B,F. Simirneurons , the unc neurons , and the neurons   null and clr-1(e1745ts) temperature-sensitive mutants have degenerating gonads , genetic mosaic and transgenic expression studies indicate that this degenerative defect is a consequence of hypodermal clr-1 loss, which causes massive fluid accumulation and larval lethality  (vpr-1(tm1411) clr-1 RNAi hermaphrodites and transgenic vpr-1(tm1411); clr-1(e1745ts) hermaphrodites expressing clr-1 in the hypodermis exhibit correct muscle mitochondrial localization, yet have arrested gonads . Therefore, the clr-1 muscle pathway is unlikely to influence gonadogenesis. The MSPd also antagonizes the VAB-1 Eph receptor, which is expressed throughout the nervous system (vab-1(dx31); vpr-1(tm1411) double-null mutants have arrested gonads , indicating that excess VAB-1 signaling does not cause arrested gonadogenesis. Collectively, the results are consistent with MSPd signaling being important for gonad development, but independent of excess CLR-1 or VAB-1 signaling individually.The VPR-1 MSPd has been shown to interact with two broadly expressed receptors, CLR-1 and VAB-1, as well as an unidentified receptor(s) . clr-1 esurvival . In adulsurvival . We consFig. S1) . Adult vs system . vab-1; glp-1(ar202ts) double mutants. glp-1(ar202ts) is a temperature-sensitive (ts) gain-of-function mutation (vpr-1(tm1411) single-mutant, glp-1(ar202ts) single-mutant, and vpr-1(tm1411); glp-1(ar202ts) double-mutant hermaphrodites grown at the restrictive temperature (25°C) from L1 (vpr-1(tm1411); glp-1(ar202ts) gonads (glp-1(ar202ts) gonads (vpr-1(tm1411) gonads  gonads D lack th) gonads C and ins) gonads B. This rglp-1(bn18ts) is a temperature-sensitive loss-of-function mutation (glp-1(bn18ts) and vpr-1(tm1411) single mutants are small, but with an important difference. Germ cells in glp-1(bn18ts) gonads differentiate as sperm, whereas germ cells in vpr-1(tm1411) gonads fail to differentiate. If GLP-1 signaling is independent of vpr-1, then vpr-1(tm1411); glp-1(bn18ts) double-mutant gonads should contain sperm. To test this prediction, we grew single- and double-mutant hermaphrodites at 25°C from L1, stained them with DAPI, and performed fluorescence deconvolution microscopy. Characteristic highly condensed sperm chromosomes are not observed in vpr-1(tm1411) gonads (glp-1(bn18ts) single-mutant (vpr-1(tm1411); glp-1(bn18ts) double-mutant (vpr-1 mutants. These genetic data support the model that vpr-1 and glp-1 act in independent genetic pathways to promote germ cell expansion and differentiation.mutation . Gonads ) gonads B, but are-mutant E and vpre-mutant F gonads.vpr-1 activity we used the Q system, a drug-inducible binary gene expression system , which is added to plates, inhibits QS repressor activity, thereby activating gene expression. We used the glr-5 promoter to drive QF and QS expression in ∼56 interneurons  mutants . Six lines grew robustly in the presence of QA. All seven lines exhibited minimal QA-independent vpr-1 expression, as indicated by gamete development in a small percentage of transgenic vpr-1(tm1411) hermaphrodites. The Q system was more tightly regulated than the heat shock promoter (data not shown). Providing QA produced functional VPR-1, as evidenced by rescue of the muscle mitochondrial defect . We selected line 3 for further characterization because it grew very slowly without QA but grew rapidly with QA. An advantage of this line is that sufficient numbers of transgenic vpr-1(tm1411) homozygotes could be generated for staging. Similar results were observed for lines that failed to grow without QA.SS blast cells form during L1 and early L2, but do not complete development until mid to late L4 . To deten system . The QF cription A. A tranrneurons A. Seven vpr-1 mutant gonads. Class 2 gonads contain visible oocytes or an expanded germ cell population, but are sterile with no fertilized eggs in the uterus. Class 3 gonads contain differentiated gametes and fertilized eggs. In the absence of QA, 57% of transgenic vpr-1 mutant gonads were class 1 and 19% were class 3  embryos is sufficient for gonad induction. As this maternal contribution is not renewable, we considered the possibility that vpr-1 is sufficient transiently, early in development. To investigate this idea further, we provided QA to transgenic vpr-1 mutant adults, let their progeny hatch on QA plates, and then moved the L2 worms to plates lacking QA. We found little difference in class 3 gonad percentage in these experiments compared with controls grown continuously on QA  or P4 (germ line) lineage caused gonads to arrest, although gonad development often appeared more advanced than that of gonads in M− Z− vpr-1 animals. Intestinal vpr-1 loss also caused sterility in some animals. Gonads from these mosaics produced oocytes but exhibited ovulation defects or other defects consistent with an abnormal somatic gonad. Our mosaics used fosmid DNA containing the entire vpr-1 genomic locus and likely most gene regulatory sequences. Although transgenes are typically silenced in the germ line due to their repetitive nature /hT2 [bli-4(e937) let-?(q782) qIs48] ; JK2868 qIs56 [lag-2p::GFP+ unc-119(+)]; WS2170 opIs110 [lim-7p::YFP::act-5+unc-119(+)]; OD58 unc-119(ed3) III; ltIs38 [pie-1p::GFP::PH(PLC1delta1)+unc-119(+)]; DG1575 tnIs6 [lim-7p::GFP+rol-6(su1006)]; CB4108 fog-2(q71); CZ337 vab-1(dx31); GC833 glp-1(ar202); DG2389 glp-1(bn18); XM1101 vpr-1(tm1411)/hT2 [bli-4(e937) let-?(q782) qIs48]; clr-1(e1745ts); XM1102 clr-1(e2530)/mIn1 [dpy-10(e128) mIs14]; XM1103 vpr-1(tm1411)/hT2 [bli-4(e937) let-?(q782) qIs48]; vab-1(dx31); XM1104 vpr-1(tm1411)/hT2 [bli-4(e937) let-?(q782) qIs48]; glp-1(ar202); Xm1105 vpr-1(tm1411)/hT2 [bli-4(e937) let-?(q782) qIs48]; glp-1(bn18). E. coli . The folclr-1(e1745), glp-1(ar202) and glp-1(bn18) temperature-sensitive alleles were conducted at permissive (16°C) and restrictive (25°C) temperatures, as indicated. Strain construction was performed using PCR, sequencing and phenotypic analyses. vpr-1(tm1411) mutants are maternal effect sterile. Phenotypes were evaluated in vpr-1(tm1411) homozygous F2 progeny from vpr-1(tm1411)/hT2 heterozygotes (P0), unless otherwise indicated. vpr-1(tm1411) homozygous F1 progeny contain maternal vpr-1 mRNA. To investigate zygotic vpr-1 activity, progeny of fertile F1 vpr-1 mutants mated to wild-type males were examined. RNAi was performed by the feeding method        . This vpr-1 genomic fragment enhanced rescue of the vpr-1 mutant gonadogenesis defect relative to the vpr-1 cDNA fused to the unc-54 3′ UTR. PCR was used to amplify sequences from genomic DNA. The unc-119p::vpr-1g, unc-25p::vpr-1g, unc-17p::vpr-1g, glr-5p::vpr-1g and ges-1p::vpr-1g constructs were generated using PCR and restriction enzymes in a TOPO vector backbone. The rol-6p::vpr-1g and myo-3p::vpr-1g constructs were generated using Gibson assembly (New England Biolabs) in a pGEM backbone. Primers are shown below. The pie-1p::vpr-1g construct was generated by Knudra Transgenics. The pie-1 promoter sequence included 1095 bp upstream of the translational start site. The vpr-1 DNA sequence included exons and introns, as well as 745 bp of the 3′ UTR.Pan-neuronal (2000 bp) , GABA mo1893 bp) , choline2003 bp) , head in0003 bp) , intesti2003 bp) , body wa2385 bp)  and hypo2000 bp)  promoterclr-1 genomic locus, Gibson assembly was used to construct a plasmid containing clr-1 2 kb left homology arm::tdTomato::clr-1 3′ UTR::C. briggsae unc-119::2 kb right homology arm. The single guide RNA (sgRNA) plasmid was derived from Addgene plasmid 46169. Cas9 targeting sequence was 5′-ACTATATCTCTAAGACATAT-3′. PCR was used to amplify the entire sgRNA backbone, except for 20 bp from unc-119. DNA fusions were constructed using Gibson assembly. The rol-6p::clr-1 construct was made with clr-1 genomic DNA. 2 kb upstream of the rol-6 start codon was amplified by PCR. All constructs were confirmed by sequencing (UAB Heflin Center for Genomics Sciences). Primers  were: vpr-1 F1 sacII, GGGGACAACTTTCCGCGGAAAAAAATGTCTGAAAAGCACAGTCTTCTG; vpr-1 R1 kpnI, GGGGACTGCTTTGGTACCCCGAGATAATACGGCGAAAA; glr-5 F1 BHI NtI, GGGGACAACTTTGGATCCGCGGCCGCGTCACAATTTTCGGGTGTCGTAG; glr-5 R1 NeI ScII, GGGGACTGCTTTCCGCGGGCTAGCGATGCTTATTATTCACATGTTTCAAACC; unc-17 F1 BHI NEI, GGGGACAACTTTGGATCCAGCGGCCGCTTCACACAATTAAGAATTTTAAGATTTGGG; unc-17 R1 NeI ScII, GGGGACTGCTTTCCGCGGGCTAGCCTCTCTCTCTCCCCCTGGAATATT; ges-1 F1 BHI NtI, GGGGACAACTTTGGATCCAGCGGCCGCAAACTCCGAACTATGATGACGAA; ges-1 R1 NeI ScII, GGGGACTGCTTTCCGCGGGCTAGCCTGAATTCAAAGATAAGATATGTAATAGATTTTT; unc-25 F1 BHI NtI, GGGGACAACTGGATCCAGCGGCCGCGAGAAATAAGAAATAATTGTATAATTTTTTTTTC; unc-25 R1 ScII NeI, GGGGACTGCTTTCCGCGGGCTAGCTTTTGGCGGTGAACTGAGCTTTT; topo F1 KpnI, GGGGACAACTTTGGTACCCCTGAATGGCGAATGGAC; topo pR1 BHI, GGGGACTGCTTTGGATCCAGCTCACTCAAAGGCGGTAA; vpr-1 F1 NheI, GGGGACAACTTTGCTAGCAAAAAAATGTCTGAAAAGCACAGTCTTCTG; glr-5 R1 ScII NeI, GGGGACTGCTTTGCTAGCCCGCGGGATGCTTATTATTCACATGTTTCAAACC; F1 rol-6 pgem5, AGGTCGACCATATGGGAGAGCTAGAAAAACGATGGATTGAGTTATCTGG; R1 vpr-1 rol-6, AGACTGTGCTTTTCAGACATCTGGAAATTTTCAGTTAGATCTAAAGATATATCC; F2 rol-6 vpr-1, GATCTAACTGAAAATTTCCAGATGTCTGAAAAGCACAGTCTTCTG; R2 pgem vpr-1, CTATGCATCCAACGCGTTGGGAACCATAAACATCAAATTTTATTGTACCATATAC; F1 pmyo-3 pgem5, GGTCGACCATATGGGAGAGCTGGCTATAATAAGTTCTTGAATAAAATAATTTTCCC; R1 vpr-1 pmyo-3, GCAGAAGACTGTGCTTTTCAGACATTTCTAGATGGATCTAGTGGTCGTGG; F2 pmyo-3 vpr-1, CCACGACCACTAGATCCATCTAGAAATGTCTGAAAAGCACAGTCTTCTGC; R2 pmyo-3 pgem5, GCTATGCATCCAACGCGTTGGGAACCATAAACATCAAATTTTATTGTACCATATAC.To create the Cas9 DNA template for tdTomato insertion into the Microscopy images were taken by a motorized Zeiss Axioskop equipped with epifluorescence and AxioVision software version 4.8. For DAPI staining, worms were fixed in 10% neutral buffered formalin (Sigma-Aldrich), mixed with 0.5 µg/ml DAPI, and incubated for 24 h at 4°C. Worms were then washed five to eight times with sterile water and mounted for microscopy. To image gonads, axial scans were performed and out-of-focus light was removed with deconvolution software (AxioVision).C. elegans, plasmids (5-60 ng/μl) were injected into wild-type or vpr-1(tm1411)/hT2 young adult hermaphrodite gonads. The myo-3p::mitoGFP or sur-5p::NLS-GFP constructs were used for selection  by Knudra Transgenics. Integrated transgenes were crossed into the vpr-1(tm1411) background and maintained as transgenic vpr-1 mutant homozygotes.To generate transgenic election . Multiplvpr-1 genomic locus together with 10 ng/µl pTG96 (sur-5p::NLS-GFP) plasmid into vpr-1(tm1411)/hT2 hermaphrodite gonads  null hermaphrodites (vpr-1(tm1411) worms were screened from three independent lines. +vpr-1 loss in the AB, P1, P2, P3, P4, E, EMS and other lineages was scored as previously described  worms. Progeny were screened for rescue of the unc-119 movement defect and loss of myo3p::mitoGFP. Individual worms were isolated repeatedly to ensure 100% segregation. PCR and sequencing were used to confirm tdTomato insertion. The clr-1::tdTomato Cas9 line did not exhibit the fluid accumulation phenotype caused by reduced clr-1 function . Primers were : XW08 F1, CGGTTTGAAACATGTGAATAATAAGCATCATGGGCGCGCCTCTAGAGGATC; XW08 R1, GTTCTACGACACCCGAAAATTGTGACGCATGCAAGCTTGGCGTAATC; XW08 glr-5 F1, GATTACGCCAAGCTTGCATGCGTCACAATTTTCGGGTGTCGTAGAAC; XW08 glr-5 R1, GATCCTCTAGAGGCGCGCCCATGATGCTTATTATTCACATGTTTCAAACCG; XW09 F1, GGTTTGAAACATGTGAATAATAAGCATCATGGGCGCGCCTCTAGAGGATCC; XW09 R1, CTACGACACCCGAAAATTGTGACGGCCGGCCCAGTCAGTGCG; XW09 glr-5 F1, CGCACTGACTGGGCCGGCCGTCACAATTTTCGGGTGTCGTAG; XW09 glr-5 R1, GGATCCTCTAGAGGCGCGCCCATGATGCTTATTATTCACATGTTTCAAACC; XW12 F1, GGGAAACTGCTTCAACGCATCATGAGTAAAGGAGAAGAACTTTTCACTG; XW12 R1, GCAGAAGACTGTGCTTTTCAGACATTTTTTCTACCGGTACCGTCGAC; VPR-1 F1, GTCGACGGTACCGGTAGAAAAAATGTCTGAAAAGCACAGTCTTCTGC; VPR-1 R1, AGGTGAAAGTAGGATGAGACAGCAACCATAAACATCAAATTTTATTGTACCATATACA; SL2 F1, TGTATATGGTACAATAAAATTTGATGTTTATGGTTGCTGTCTCATCCTACTTTCACCT; SL2 R1,CAGTGAAAAGTTCTTCTCCTTTACTCATGATGCGTTGAAGCAGTTTCCC.Q system plasmids ang Shen . Gibson C. elegans, glr-5p::QF-SL2::mCherry::3′UTR-unc-54 (10 ng/μl), glr-5p::QS-SL2::mCherry::3′UTR-unc-54 (50 ng/μl), QUASp::Δpes10::vpr-1-SL2::GFP::3′UTR-unc-54 (10 ng/μl) and myo-3p ::mito::GFP (30 ng/μl) plasmids were injected into vpr-1(tm1411)/hT2 young adult hermaphrodite gonads. Transgenic lines were selected based on mCherry and myo3p::mito::GFP expression. Seven independent lines were created. Six responded well to quinic acid (QA) treatment. For treatment, 300 µl 300 mg/ml pH 6.5 QA (Sigma-Aldrich) was mixed with 40 µl M9 buffer and added to NGM plates seeded with NA22 bacteria. All lines exhibited very low vpr-1 expression in the absence of QA, partially rescuing the vpr-1 mutant gonadogenesis defect in a small percentage of transgenic worms. Increasing the glr-5p::QS dosage in the transgenic arrays appeared to limit QA-independent vpr-1 expression.To generate transgenic t-tests were computed using Excel 2013 (Microsoft) without the assumption of equal variance.Two-tailed Student's


Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex. Published 21, April 2016D. melanogaster, sequence #13 in Supplementary file 1, was incorrect due to a mistaken copy-and-paste while preparing the manuscript. The correct D. melanogaster sequence is now entered in the corrected The protein sequence of EMRE from This error did not affect any results or conclusions of the paper.Incorrect sequence:MTSKTVFQNAFKTFLDFAINSLPSTQGGLNITATAPGGVGQRPFTNKAGVLKLIFVSASSLYIGGLIAHKGASYLEENEIFVPTDEDDDDCorrected sequence:MIVPRLALPISLALQRVSRRVAEHPHNLRILQRHMSSVYFRSGAIKPKPEEMPFGLLAIFCAVIPGLFVGATISKNVANFLEENDLFVPADDDDDEDThe article has been corrected accordingly.


The deubiquitylating enzyme USP15 plays significant roles in multiple cellular pathways including TGF-β signaling, RNA splicing, and innate immunity. Evolutionarily conserved skipping of exon 7 occurs during transcription of the mRNAs encoding USP15 and its paralogue USP4, yielding two major isoforms for each gene. Exon 7 of USP15 encodes a serine-rich stretch of 29 amino acid residues located in the inter-region linker that connects the N-terminal putative regulatory region and the C-terminal enzymatic region. Previous findings suggested that the variation in the linker region leads to functional differences between the isoforms of the two deubiquitylating enzymes, but to date no direct evidence regarding such functional divergence has been published. We found that the long isoform of USP15 predominantly recognizes and deubiquitylates mysterin, a large ubiquitin ligase associated with the onset of moyamoya disease. This observation represents the first experimental evidence that the conserved exon skipping alters the substrate specificity of this class of deubiquitylating enzymes. In addition, we found that the interactomes of the short and long isoforms of USP15 only partially overlapped. Thus, USP15, a key gene in multiple cellular processes, generates two functionally different isoforms via evolutionarily conserved exon skipping. In previous work, we isolated a key gene involved in the cryptogenic cerebrovascular disorder moyamoya diseasein vivo.Here, we report the first example of isoform-specific bias in substrate preference of USP15. Biochemical evaluation of the association between USP15 and mysterin revealed that mysterin is predominantly recognized by the long isoform of USP15. Moreover, the USP15 long isoform potently deubiquitylated mysterin and maintained its basal expression level, indicating that alternative exon skipping biases the deubiquitylation substrate preference of USP15. In addition, we performed an extensive LC-MS/MS analysis of USP15-binding proteins and identified multiple isoform-specific binding proteins, suggesting that the two isoforms of USP15 play roles in distinct proteome networks and have overlapping but independent cellular activities To elucidate the cellular function of the moyamoya disease-associated protein mysterin, we sought to identify mysterin-binding proteins, reasoning that such proteins would include cofactors and/or substrates of mysterin. To this end, we transiently overexpressed mysterin harboring a FLAG tag at its C-terminus (mysterin-FLAG) in HEK293T human embryonic kidney cells. We precipitated mysterin-FLAG from cell lysates using anti-FLAG antibody and performed LC-MS/MS analysis on total co-precipitates without an isolation process such as SDS-PAGE. Four positive hits, including USP15, LYAR, hnRNPK, and IP3 receptor isoform 3 (IP3R3) in addition to mysterin itself, were reproducibly identified in four trials. In a replication study, we transiently expressed each candidate mysterin-binding protein in HEK293T cells, immunoprecipitated using specific antibodies, and then immunoblotted for mysterin. In this analysis, USP15 and LYAR were detectably physically associated with mysterin, whereas the other two proteins were not . However23We obtained cDNAs encoding both isoforms of USP15 from HEK293 cells by reverse transcription–polymerase chain reaction (RT-PCR). To our surprise, the isoform of USP15 with longer linker region (hereafter USP15L) had significantly stronger affinity for mysterin than the other isoform with the shorter linker . Mysterin-3FLAG was precipitated using FLAG affinity beads, and its ubiquitylation state was examined by immunoblotting using anti-ubiquitin antibody. Overexpression of USP15L significantly decreased the polyubiquitin chain signal associated with mysterin-3FLAG, whereas overexpression of USP15S did not, suggesting that USP15L has the stronger deubiquitylation activity against mysterin . The pol0.5% SDS . Mutatio0.5% SDS , but did0.5% SDS , supportPrevious studies demonstrated that USP15 removes K48-linked polyubiquitin chains and thereby protects its substrates against proteasomal protein degradation triggered by K48-linked polyubiquitylation5How does the alternative variation in the linker region bias the affinity of USP15 for mysterin? Although the substrate-binding domain of USP15 has not been definitely identified, one simple model is that the linker region directly mediates mysterin binding. We evaluated the roles of structural regionss and domains in USP15, including the long linker region, in the mysterin-binding process using five truncated mutants of USP15, schematically represented in To further characterize the functional divergence of the two major isoforms of USP15, we performed an interactome analysis on each isoform, based on the expectation that each interactome will include substrates and/or cofactors specific for each isoform. We transiently overexpressed the long and short isoforms of USP15, C-terminally tagged with three tandem FLAG epitopes, in HEK293T cells; precipitated them from cell lysate using anti-FLAG antibody; and performed LC-MS/MS analysis on total co-precipitates without isolation. Positive hits reproducibly obtained in all four trials are shown in 927Alternative skipping of exon 7 in USP15 resulted in deletion of a short element consisting of 29 amino acids in the inter-region linker of USP15 , which s5The exon 7-encoded short element in the linker region of USP15 was necessary but not sufficient for the physical association with mysterin. Likewise, the DUSP domain and the C-terminal region were also essential but not sufficient. The C-region harbors the enzymatic core responsible for deubiquitylation activity12299279Sixma and colleagues recently reported that the DUSP domain of USP4 promotes the deubiquitylation cycle by discharging hydrolyzed free ubiquitin from the enzymatic core of USP4 through inter-domain interactionsVariation in the linker region is likely to have been maintained under natural selection. Phylogenic study suggested that USP4, 11, and 15 arose from a single ancestral USP, conserved between fungi and metazoans, following the emergence of vertebrates4in vivo. Despite the obvious phenotypes in mysterin knockdown/knockout zebrafish, the cellular and molecular functions of mysterin remain controversial. Recent studies proposed roles in insulin production in pancreatic β cellsWe initiated this study based on our interest in the function and/or regulation of the moyamoya disease susceptibility factor mysterin. The resultant data suggest that USP15L stabilizes mysterin via removal of K48-linked polyubiquitin chains. These observations were obtained under conventional cell culture conditions, suggesting that USP15L maintains the basal expression level of this ubiquitin ligase, which was previously shown to have autoubiquitylation  activity19NheI/NotI sites (Myc) or NheI/HindIII sites (3FLAG) into the mammalian expression vector pcDNA3.1+ . Restriction enzymes were purchased from New England Biolab  or Fermentas . DNA ligase was purchased from Takara . The enzymatic mutant of USP15L (15Lmt), which harbors alanines instead of the cysteines at positions 269 and 812, and that of USP15S (15Smt), which harbors alanine instead of the cysteine at positions 269, were generated by mutagenesis PCR using the QuikChange Site-Directed Mutagenesis kit  using primers USP15C298AF (GCCTCTGTGGCCTAAGTAACTTGGGAAATACGGCTTTCATGAACTCAGCTATTCAGTGTTTGAGCAACACACC), USP15C298AR (GGTGTGTTGCTCAAACACTGAATAGCTGAGTTCATGAAAGCCGTATTTCCCAAGTTACTTAGGCCACAGAGGC), USP15C812AF (GCTGAAGATCCCTGGTATTGTCCGAATGCTAAAGAACATCAGCAAGCCACAAAGAAATTGG), and USP15C812AR (CCAATTTCTTTGTGGCTTGCTGATGTTCTTTAGCATTCGGACAATACCAGGGATCTTCAGC). Deletion mutants were generated using seven primers USP15FNheI (described above), USP15F2NheI (ATGCGCTAGCATGCCATCATATACCGCTTATAAGAAC), USP15FdN2NheI (ATGCGCTAGCCGCCATGGGTCCTTCTACTCCTAAGTCC), USP15FdN3NheI (ATGCGCTAGCCGCCATGGTAAAGCACTGCAAAGTAGAA), USP15R13FLAGHindIII (ATGCAAGCTTCTACTTATCGTCGTCATCCTTGTAGTCGATGTCATGGTCTTTGTAGTCACCGTCATGATCCTTGTAATCAAGACAGTAATTTGAGTTTTTCACATT), USP15R23FLAGHindIII (ATGCAAGCTTCTACTTATCGTCGTCATCCTTGTAGTCGATGTCATGGTCTTTGTAGTCACCGTCATGATCCTTGTAATCAATAATCACGTGCTCTGTATCTTCTGTCC), and USP15R33FLAGHindIII (ATGCAAGCTTTTACTTATCGTCGTCATCCTTGTAGTCGATGTCATGGTCTTTGTAGTCACCGTCATGATCCTTGTAATCGTTAGTGTGCATACAGTTTTC). ΔC1, ΔC2, ΔN1, ΔN2, and ΔN3 deletion mutants were generated by PCR using USP15FNheI and USP15R13FLAGHindIII, USP15FNheI and USP15R23FLAGHindIII, USP15F2NheI and USP15R33FLAGHindIII, USP15FdN2NheI and USP15R33FLAGHindIII, and USP15FdN3NheI and USP15R33FLAGHindIII, respectively.Full-length cDNAs for USP15L (NM_001252078.1) and USP15S (NM_006313.2) were obtained simultaneously by RT–PCR using the OneStep RT-PCR kit  with template RNA extracted from HEK293 cells using the RNeasy Mini Kit (QIAGEN). USP15L or S with Myc or three tandem FLAG (3FLAG) epitope tag at the C-terminus was generated by PCR using forward primer USP15FNheI (ATGCGCTAGCACCATGGCGGAAGGCGGAGCGGC) and reverse primers USP15RMycNotI (ATGCGCGGCCGCCTACAGATCCTCTTCTGAGATGAGTTTTTGTTCGTTAGTGTGCATACAG) or USP15R3FLAGHindIII (ATGCAAGCTTTTACTTATCGTCGTCATCCTTGTAGTCGATGTCATGGTCTTTGTAGTCACCGTCATGATCCTTGTAATCGTTAGTGTGCATACAGTTTTC). These PCR products, harboring exogenous restriction sites and epitope tag at the 3′ terminus, were subcloned using KpnI, NotI, SalI, and BamHI. Exogenous NheI and XbaI sites and the Myc epitope sequence were introduced into the 5′ and 3′ termini by PCR using forward primer CloNheIIP3R3F1 (ATGCGCTAGCCGCCATGAGTGAAATGTCCAGCTTTCTTCACATCGG) and reverse primer CloXbaIIP3R3R53FLAG (TACGTCTAGATCAGATGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCCTTATCGTCGTCATCCTTGTAATCGCGGCTAATGCAGTTCTGG). The PCR product was subcloned into pcDNA3.1+ using the NheI/XbaI sites. Full-length cDNA of LYAR was obtained by RT-PCR using forward primer KpnILYARCloF (ATGCGGTACCGCCACCATGGTATTTTTTACATGC) and reverse primer NotI3FLAGLYARCloR (TACGGCGGCCGCTTAGATGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCCTTATCGTCGTCATCCTTGTAATCTTTCACAAGCTTGACTTTG), and then subcloned into pcDNA3.1+ using the KpnI/NotI sites.Mysterin-FLAG and -3FLAG were generated in our previous studies19HEK293 (used only for cloning of USP15) and HEK293T cells were maintained in DMEM  containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Plasmids were transfected into HEK293T cells using Lipofectamine LTX (Life Technologies) or PEI MAX . The amount of DNA was adjusted to obtain equivalent expression levels of the introduced proteins in each experiment. For detection of ubiquitylation, cells were treated with 1 μM epoxomicin  for 8 h (or other indicated period) before harvest.Antibodies used for immunoprecipitation (IP), immunoblotting (IB), or immunostaining (IS) in this study include anti-FLAG antibody , anti-Myc antibody , andi-HA antibody , anti-ubiquitin antibody , anti-K48 linked ubiquitin antibody , and anti-GAPDH antibody . Anti-human mysterin mouse monoclonal antibody (1C9) was generated with the method previously reportedMysterin-FLAG or USP15L/S-3FLAG was expressed in HEK293T cells, and associated proteins were recovered from cell extracts by immunoprecipitation with anti-FLAG antibody. The associated protein complexes with mysterin-FLAG were immediately digested with lysyl endopeptidase . Protein complexes associated with USP15L/S-FLAG were reduced with 5 mM TCEP, and then alkylated with 10 mM monoiodoacetoamide. The alkylated protein complexes were digested with Lys-C and trypsin . The resultant peptides were analyzed using a direct nano-flow LC-MS/MS system, as described previouslyg, and the supernatant was incubated for 1 hour with anti-FLAG M2 agarose beads (Sigma Aldrich) or anti-Myc antibody and protein G beads . At this point, aliquots of supernatant were resolved with Laemmli’s sample buffer containing 1 mM DTT as a control . The beads were washed three times with digitonin-containing HEPES buffer and resolved with Laemmli’s sample buffer containing 1 mM DTT.Protein-encoding or empty vector was transiently transfected into HEK293T cells. The cells were harvested 24 or 48 hours after the transfection and lysed with HEPES buffer  containing 1% NP40 (for ubiquitylation assay) or digitonin . The lysate was centrifuged at 13,000 Samples were separated with SDS-PAGE using 7.5% or 3–10% acrylamide gel  and transferred to nitrocellulose  or PVDF  membrane for 1.5 hours at 75 V at 4 °C. Membrane was blocked using Blocking One  for 1 hour at room temperature, and then incubated sequentially with primary and secondary antibodies diluted in Can Get Signal . Detection was performed via the chemiluminescence or the alkaline phosphatase method.2HPO4 and 1.47 mM KH2PO4, pH 7.4). After incubation with Blocking One  for 4 hours at room temperature, the cells were incubated sequentially with primary and fluorescence-conjugated second antibodies; each of the antibody incubations were followed by PBS washes. The nucleus was stained using Hoechst 33342. Fluorescence images were obtained by confocal microscopy .HEK293T cells expressing USP15L-Myc or S-Myc together with mysterin-3FLAG were immobilized with 4% paraformaldehyde  for 10 min at room temperature 24 hours after transfection. The cells were permeabilized with 100 ng/mL digitonin for 5 minutes, and then washed with phosphate-buffered saline .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Drosophila, we discovered a 29-bp consensus sequence that we designated as the Clock-Associated Transcriptional Activation Cassette or ‘CATAC’. To experimentally address the spatiotemporal expression information associated with this element, we generated constructs with four separate native CATAC elements upstream of a basal promoter driving expression of either the yeast Gal4 or firefly luciferase reporter genes. Reporter assays showed that presence of wild-type, but not mutated CATAC elements, imparted increased expression levels as well as rhythmic regulation. Part of the CATAC consensus sequence resembles the E-box binding site for the core circadian transcription factor CLOCK/CYCLE (CLK/CYC), and CATAC-mediated expression rhythms are lost in the presence of null mutations in either cyc or the gene encoding the CLK/CYC inhibitor, period (per). Nevertheless, our results indicate that CATAC's enhancer function persists in the absence of CLK/CYC. Thus, CATAC represents a novel cis-regulatory element encoding clock-controlled regulation.Circadian clocks are autonomous daily timekeeping mechanisms that allow organisms to adapt to environmental rhythms as well as temporally organize biological functions. Clock-controlled timekeeping involves extensive regulation of rhythmic gene expression. To date, relatively few clock-associated promoter elements have been identified and characterized. In an unbiased search of core clock gene promoters from 12 species of  Drosophila melanogaster, a heterodimer consisting of CLOCK (CLK) and CYCLE (CYC), acts as the core transcription factor , Per-Arnt-Sim (PAS) domain transcription factors (TFs) for transcriptional activation . In the n factor ,3. CLK/Cn factor –6. The dn factor ,8.Drosophila, studies of the promoter regions of known clock genes, period (per) and timeless (tim), found that E-box-dependent enhancers are necessary for circadian transcriptional modulation. The per promoter has, arguably, the best studied circadian enhancer motif to date. The enhancer is a 69-bp sequence upstream of the transcription start site (TSS) that activates circadian gene expression  boxes, which are a variant of the consensus E-box sequence  and transcription factor binding sites (TFBSs) have provided more tools to determine the elements responsible for transcriptional regulation . As a continuation of this work, we, hereby, identified a second novel and independent 29-bp motif. The present study describes spatiotemporal expression information contributed by the latter motif, which was named Clock-Associated Transcriptional Activation Cassette or ‘CATAC’.Increasing knowledge of the gulation ,11. In pgulation , we identim, vrille, Pdp1 and cwo (stich1) in 12 Drosophila species . This identified a conserved set of CATAC sequences (Drosophila melanogaster genome (BDGP R5/dm3) is included in the The bioinformatics model used to predict the ‘CATAC’ element is identical to that previously described for the discovery of the E1–E2 element . Brieflyequences . In ordeequences , we traiPdp1 or Slob promoters: Slob 4xCATAC enhancer, 5΄pGGCCGATAACGCGGCGTATGCGCAATGTCGAAGCATATTACGCATACGCCCCATCCGC; 5΄pGTTTGCTGTGGCGGATGGGGCGTATGCGTAATATGCTTCGACATTGCGCATACGCCGCGTTATC; 5΄pCACAGCAAACGCTGCGTATGCGTAATACTTTGTGCACACGTTGCGTATGAGTAATGTCCT; 5΄pAATTAGGACATTACTCATACGCAACGTGTGCACAAAGTATTACGCATACGCAGC, Slob mt4xCATAC enhancer, 5΄pGGCCGATATCCCGGCCTTTGCCCTATGTCGAAGCATATAAGGCAAGGCCCGAACCGC; 5΄pGATTGCTGTGGCGGTTCGGGCCTTTGCCTTATATGCTTCGACATAGGGCAAAGGCCGGGATATC; 5΄pCACAGCAATCCCTGCCTTTGCCT TATACTTTGTGCACTCCTTGCCTTTGACTTATGTCCT; 5΄pAATTAGGACATAAGTCAAAGGCAAGGAGTGCACAAAGTATAAGGCAAAGGCAGG, Pdp1 4xCATAC enhancer, 5΄pGGCCAGCACATTACGCATACGTCACGTGTTGCAAAAATAATACGTATACGACGCGTGTC; 5΄pTGGTACAGAAGACACGCGTCGTATACGTATTATTTTTGCAACACGTGACGTATGCGTAATGTGCT; 5΄pTTCTGTACCATGCGGCGTATGAGCAATCTGTTAATACGTTACCCATACGCCCCGTGGGC C; 5΄pAATTGGCCCACGGGGCGTATGGGTAACGTATTAACAGATTGCTCATACGCCGCA, Pdp1 mt4xCATAC enhancer, 5΄pGGCCAGCACATAAGGCAAAGGTCAGGAGTTGCAAAAATAAAAGGTAAAGGACGGGAGTC; 5΄pAGGTACAGAAGACTCCCGTCCTTTACCTTTTATTTTTGCAACTCCTGACCTTTGCCTTATGTGCT; 5΄pTTCTGTACCTTC CGGCCTTTGACCTATCTGTTAATACGTA AGCCAAAGGCCCGGAGGGCC; 5΄pA ATTGGCCCTCCGGGCCTTTGGCTTACGTATTAACAGATAGGTCAAAGGCCGGA, Pdp1 4xCATAC with Slob-like E-boxes enhancer, 5΄pGGCCAGCACATTACGCATACGTAACGTGTTGCAAAAATAATACGTATACGAAGCGTTTC; 5΄pTCGTACAGA AGAAACGCTTCGTATACGTATTATTTTTGCAACACGTTACGTATGCGTAATGT GCT; 5΄pTTCTGTACGATGGGGCGTATGAGCAATCTGTTAATACGTTACCCATACGCCGCGTTGGC C; 5΄pAATTGGCCAACGCGGCGTATGGGTAACGTATTAACAG ATTGCTCATACGCCCCA. Oligos were annealed to match their order and orientation in the native Pdp1 or Slob promoters. Flanking EagI and EcoRI sequences allowed for the 4xCATAC enhancers to be inserted upstream of the hsp70 basal promoter in the pPTGAL vector (luc) were created by blunt-end cloning to replace a 3193 bp fragment containing Gal4 flanked by PstI sites with a 1955 bp HindIII–BamHI fragment containing the luciferase gene from pGL3-Basic (Promega) (luc can be requested online (http://www.addgene.org/Herman_Wijnen/). For each reporter construct, several independent lines were generated.Synthetic oligonucleotides were designed to match four separate, native occurrences of the CATAC element from either the L vector  (obtainePromega) . Copies 1118w, UAS-CD8::GFP, and 2eya mutations were obtained from the Bloomington Drosophila Stock Center. The 01, per01cyc and tim-luc alleles have been described previously  and relative amplitude error (RAE) by an iterative, coupled fast Fourier transform-non-linear least squares (FFT-NLLS) multicomponent cosine analysis . RAE is Gal4 driven expression of membrane-tethered green fluorescent protein were dissected in Ringer's solution . Flies were harvested onto dry ice at time points CT0, CT6, CT12, CT18 and CT24, and adult heads were dissected on a chilled platform and transferred to guanidinium thiocyanate buffer. Four separate groups of flies (∼50 each) were used for each experimental condition. Total RNA was obtained from the heads using the RNAqueous4PCR kit (Ambion). Sample concentration and purity was analysed using a NanoDrop spectrophotometer. Samples exhibited OD 260/280 ratios between 1.8 and 2.1. Concentrations were adjusted to 25 ng/μl in 10 mM Tris–HCl 0.1 mM EDTA pH 8.0 buffer and samples were frozen at –80°C in aliquots until further use. The RNA samples were then analyzed with the SensiFAST SYBR No-ROX One-Step qPCR Kit (Bioline) using experimental primer pairs designed to specifically amplify fragments of the circadian Pdp1 or Slob transcripts, the transgenic luciferase transcript or the EF1β control transcript  relative to EF1β were determined using the comparative Cycle threshold (Ct) method . ript see . All pri,23. riptcis-acting elements associated with clock gene expression, we combined MEME  for 12 species of Drosophila. As a result, a 29-bp motif designated the Clock-Associated Transcriptional Activation Cassette or ‘CATAC,’ was identified as over-represented in these promoters  is provn = 97 genes) encoding transcripts predicted to exhibit circadian oscillations (Slob (Slowpoke binding protein)  predicted to be induced upon activation of the circadian regulator CLK  as well llations –28 (Suppllations . A promiprotein) . With th genomes .Based upon the sequence alignment, conservation, and subsequent CLK/CYC target gene enrichment analyses, CATAC was hypothesized to be a regulatory element involved in mediating spatiotemporal expression of clock-regulated genes.Pdp1 and Slob, that not only exhibit strong CLK/CYC-associated circadian oscillations, but also have an unusually high number of CATAC motifs. The Pdp1 gene encodes a clock component (PDP1-ε) that impacts molecular circadian oscillations, particularly in the clock neurons, as well as circadian behaviour  as well as the CRCGTG consensus for the E-box-like element within CATAC  which allowed measurement of the transcriptional reporter constructs at relatively high frequency in vivo. Native Pdp1 and Slob 4xCATAC both showed strong luciferase expression  and both mt4xCATAC constructs shared characteristically low level expression  in which the orientation of Slob 4xCATAC in the vector was inverted and ‘Pdp1 4xCATAC with Slob-like E-boxes’ (PSE) in which the four E-boxes of Pdp1 4xCATAC were mutated such that their sequence fidelity matched that of the four E-boxes in Slob 4xCATAC , was significantly stronger than those for mt4xCATAC and empty vector controls, respectively  and Slob (ZT 3–5). Heads showed luciferase expression in not only the compound eyes, but also the proboscis and antennae; each of which has been shown to possess a circadian oscillator  genetic background , by and large there was little effect of the 01per mutation on their expression levels  construct. PSE is a modified version of Pdp1 4xCATAC, which has E-box sequences that were mutated such that their deviation from the CACGTG consensus matched that of the four E-boxes in the Slob 4xCATAC enhancer , constructs. Thus, the considerable variation in quality of E-box sequences between these constructs did not obviously impact reporter gene rhythms. The sequence immediately flanking an E-box element has also been implicated in affecting regulation  is known to bind E-boxes in clock-controlled genes (For all the similarities that CATAC shares with E-box motifs, CATAC is not simply another E-box element. Normally, an E-box regulated transcriptional element exhibits low reporter expression in a in per01 ,41. CATAed genes ,43 and mPdp1 and Slob. Of course, this conclusion comes with the caveat that the multimerized constructs do not represent the complexity of the native genomic environment. Mutational analysis of CATAC sequences in their natural context will constitute an important next step in the functional analysis of this cis-regulatory element. Nevertheless, the identification of CATAC contributes to our knowledge of circadian transcriptional elements, and may be used to further characterize the regulatory regions of clock and clock-regulated genes.Thus, we have discovered a novel circadian regulatory element that, although it possesses an E-box-like motif, exhibits non-E-box-like responses. Through use of multimerized CATAC element, we concluded that CATAC is capable of contributing to the rhythmicity of clock genes, such as Supplementary DataClick here for additional data file.


Open Biol. 7, 170139. (Published online 21 November 2017). (doi:10.1098/rsob.170139).A correction is required to the sequence of sgRNA_FD2 in .The correct sequence is as follows:sgRNA_FD2:TTCCAGAAGCCGCCTTTGCCCGGGTTTTAGAGCTAGAAATAGCAAG-3′5′-TAATACGACTCACTATAAll the authors gave their consent to this correction.