S-adenosylmethionine-mediated hypermethylation of the PTEN promoter was important for the growth of tamoxifen-resistant MCF-7 (TAMR-MCF-7) cancer cells. Here, we found that the basal expression level of methionine adenosyltransferase 2A (MAT2A), a critical enzyme for the biosynthesis of S-adenosylmethionine, was up-regulated in TAMR-MCF-7 cells compared with control MCF-7 cells. Moreover, the basal expression level of MAT2A in T47D cells, a TAM-resistant estrogen receptor-positive cell line was higher compared to MCF-7 cells. Immunohistochemistry confirmed that MAT2A expression in TAM-resistant human breast cancer tissues was higher than that in TAM-responsive cases. The promoter region of human MAT2A contains binding sites for nuclear factor-κB, activator protein-1 (AP-1), and NF-E2-related factor 2 (Nrf2), and the activities of these three transcription factors were enhanced in TAMR-MCF-7 cells. Both the protein expression and transcriptional activity of MAT2A in TAMR-MCF-7 cells were potently suppressed by NF-κB inhibition but not by c-Jun/AP-1 or Nrf2 knock-down. Interestingly, the expression levels of microRNA (miR)-146a and -146b were diminished in TAMR-MCF-7 cells, and miR-146b transduction decreased NF-κB-mediated MAT2A expression. miR-146b restored PTEN expression via the suppression of PTEN promoter methylation in TAMR-MCF-7 cells. Additionally, miR-146b overexpression inhibited cell proliferation and reversed chemoresistance to 4-hydroxytamoxifen in TAMR-MCF-7 cells.We previously showed that  Breast cancer is one of the most common malignancies in Western countries. Because estrogen is essential for either mammary gland or breast cancer development, the administration of anti-estrogens to suppress breast tumor growth is an important therapeutic option against estrogen receptor-positive breast cancer . TamoxifMAT1A and MAT2A [S-adenosylmethionine (SAM), a principal biological methyl donor [PTEN promoter, leading to decreased PTEN expression and sustained activation of phosphoinositide 3-kinase (PI3K) [Methionine adenosyltransferase (MAT), encoded by the genes nd MAT2A , is an eyl donor –5. The pyl donor . Howeveryl donor . In hepayl donor , 6–9. Adyl donor . Outsideyl donor  and in hyl donor . Howeveryl donor . Those ce (PI3K) . Hence, MAT2A contains several functional binding sites for transcription factors, including nuclear factor-κB (NF–κB), activator protein-1 (AP–1), NF-E2 related factor 2 (Nrf 2), and specific protein1 (Sp1) [In liver cancer, the up-regulation of MAT2A occurs via transcriptional activation . The pron1 (Sp1) . In the a −570/+61-bp human MAT2A promoter showed that MAT2A-luc reporter activity was enhanced in TAMR-MCF-7 cells , respectively  data. The accession number was GSE32988, providing 62 pre-chemotherapy biopsies of HER2 normal breast cancer patients (ER-positive and ER-negative subtypes) with the results of the TAM-chemotherapy. Series matrix file was matched the probes on platform GLP96, excluded 5 normal samples, rearranged into groups according to existence of residual invasive cancer and normalized by a control gene. Interestingly, residual invasive cancer cases (TAM-resistant) showed the increasing tendency of MAT2A expression compared to no invasive cancer cases (P = 0.066)  .Although MCF-7 and T47D cells are the representative ER-positive breast cancer cell lines, T47D cells are relatively more resistant to TAM than MCF-7 clone . MoreoveMAT2A gene transcription in TAMR-MCF-7 cells.NF-κB and AP-1 are required for basal MAT2A expression and the tumor necrosis factor-α (TNF-α)-induced transactivation of MAT2A in human hepatoma cells . AdditioThe regulatory role of NF-κB in MAT2A expression was reported in several cancer types. p65 knockdown reduces the basal level of MAT2A expression and blocks the additive effect of IGF-1 on MAT2A in human colon cancer cells . NF-κB aAs a class of small non-coding RNAs, miRs control the expression of diverse genes through either the inhibition of translational initiation or induction of mRNA degradation . Among vWe previously revealed that increases in intracellular SAM and DNMT1 expression in TAMR-MCF-7 cells contribute to exaggerated methylation of the PTEN promoter, which causes reduced PTEN expression and enhanced Akt phosphorylation . BecauseOne of the most important causes of TAM resistance is dysregulation of ER activity, especially the loss of ERα expression . When weBesides miR-mediated post-translational control, MAT2A expression can also be controlled by diverse kinases. To determine the kinase(s) involved in MAT2A transcription, TAMR-MCF-7 cells were incubated for 24 h with mitogen-activated protein kinase family inhibitors, including extracellular signal-regulated kinase , p38 kinase , c-Jun N-terminal kinase , and a PI3K inhibitor , and then the MAT2A protein levels were determined. The results showed that only PI3K inhibition selectively decreased MAT2A expression in TAMR-MCF-7 Figure . To confPTEN promoter. When we determined the cellular levels of miR-146a and -146b in TAMR-MCF-7 cells incubated with LY294002 for 24 h, the decreased miR-146b expression was significantly recovered by PI3K inhibition . Antibodies specific for phosphorylated Akt and Akt were obtained from Cell Signaling Technology . Horseradish peroxidase-conjugated donkey anti-rabbit, anti-goat IgG, and alkaline phosphatase-conjugated donkey anti-mouse IgG were acquired from Jackson Immunoresearch Laboratories . Anti-actin antibody and most of the reagents used for molecular studies were obtained from Sigma . The MAT2A-luc reporter plasmid was kindly donated by Dr. SC Lu . An overexpression plasmid for inhibitor of κBα (IκBα) and a pGL-ARE minimal reporter (containing three copies of the ARE region of the quinone oxidoreductase promoter) were donated by Dr. KY Lee  and Dr. MK Kwak , respectively. pNF-κB-luc and pAP-1-luc reporter plasmids were purchased from Stratagene . Mimic hsa-miR-146b-3p , has-miR-146a  or mimic negative control  were supplied from Thermo Fisher Scientific .2/95% air in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin. TAMR-MCF-7 cells were established using methods previously reported [MCF-7 and MDA-MB-231 cells were cultured at 37°C in 5% COreported , 14, 15.Nuclear extracts preparation and sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblot analysis were performed according to previously published procedures .® reagent . The cells were then incubated in culture medium without serum. Firefly and hRenilla luciferase activities in the cell lysates were measured using a luminometer . Relative luciferase activities were calculated by normalizing the promoter-driven firefly luciferase activity to the hRenilla luciferase.A dual-luciferase reporter gene assay  was used to determine promoter activity. Briefly, cells were plated in 12-well plates and transiently transfected with 1 μg/ml reporter plasmids and phRL-SV plasmid  using HillymaxBlocks for all the samples were consecutively cut in 4 μm sections and mounted on poly-l-lysine coated glass slides. Xylene was used to remove the paraffin from the sections, and the samples were rehydrated. Antigen retrieval was performed by boiling sections for 5 min in 1 μM sodium citrate buffer (pH 6.0) in a microwave oven. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide in methanol for 10 min, followed by three times washing with PBS. Sections were then incubated overnight with anti-MAT2A antibody  at 4°C. After washing with PBS, sections were incubated with HRP-conjugated anti-rabbit IgG for 30 min and washed with PBS. The color was developed by incubation with DAB solution. Finally, sections were counterstained with hematoxylin, dehydrated, mounted, and observed.miR expression was determined by collecting from total RNA from 70% to 90% confluent cells. Total RNA was isolated by using Trizol  and was then converted to cDNA by using the miScript reverse transcription kit . miScript primers specific for mature miRNAs were hsa-miR-146a (5′-CCTCTGAAATTCAGTTCTTCAG-3′) and hsa-miR-146b (5′-TGCCCTGTGGACTCAGTTCTGG-3′). Real time PCR was performed using miScript PCR kit. All samples were normalized to small nRNA U6 to calculate the fold changes.Total RNA was isolated by using Trizol . qPCR was performed with MiniOpticon real time PCR detection system  and primers specific for MAT2A  and S16r .The methylation status of the PTEN, BRCA1 and ERα promoter was determined by methylation-specific PCR after bisulfite-modification. The methylation sites of PTEN gene promoter included three regions  , 17. CpGpmole/well mimic hsa-miR-146b-3p or mimic negative control and the transfected cells was then incubated in DMEM containing 5% charcoal-stripped FBS for additional 36 h. Media were then removed and the transfected cells were harvested with trypsin, stained with 0.4% trypan blue. Cell numbers were counted using automated cell counter .Cells were seeded into 12 well plates and 50% confluent cells were transiently transfected with 50 pmole/well mimic hsa-miR-146b-3p or mimic negative control. The transfected cells were incubated in the presence or absence of 3 μM 4-hydroxytamoxifen in serum free medium for 36 h. The cells were harvested with trypsin and stained with both annexin V-fluorescein isothiocyanate (FITC) and propidium iodide according to ApopNexin FITC apoptosis detection Kit , and analyzed by flow cytometry  set for FLH-1 (annexin V) and FLH-2 (propidium iodide). Total 104 cells were counted for each sample.TAMR-MCF-7 cells were plated in 6 well plates and transfected with 120 t-test or one-way analysis of variance (ANOVA) followed by Newman–Keuls multiple comparison test were used to examine between group differences. Statistical significance was accepted at either P < 0.05 or P < 0.01.Scanning densitometry was performed using an LAS-3000 mini . Student's


Transient gene expression assays showed that the promoter activity of PORC is indeed regulated by SCL27. Consistently, chromatin immunoprecipitation and quantitative PCR assays showed that SCL27 binds to the promoter region of PORC in vivo. An electrophoretic mobility shift assay revealed that SCL27 is directly interacted with G(A/G)(A/T)AA(A/T)GT cis-elements of the PORC promoter. Furthermore, genetic analysis showed that gibberellin (GA)-regulated chlorophyll biosynthesis is mediated, at least in part, by SCLs. We demonstrated that SCL27 interacts with DELLA proteins in vitro and in vivo by yeast-two-hybrid and coimmunoprecipitation analysis and found that their interaction reduces the binding activity of SCL27 to the PORC promoter. Additionally, we showed that SCL27 activates MIR171 gene expression, forming a feedback regulatory loop. Taken together, our data suggest that the miR171-SCL module is critical for mediating GA-DELLA signaling in the coordinate regulation of chlorophyll biosynthesis and leaf growth in light.An extraordinarily precise regulation of chlorophyll biosynthesis is essential for plant growth and development. However, our knowledge on the complex regulatory mechanisms of chlorophyll biosynthesis is very limited. Previous studies have demonstrated that miR171-targeted scarecrow-like proteins (SCL6/22/27) negatively regulate chlorophyll biosynthesis via an unknown mechanism. Here we showed that SCLs inhibit the expression of the key gene encoding protochlorophyllide oxidoreductase (POR) in light-grown plants, but have no significant effect on protochlorophyllide biosynthesis in etiolated seedlings. Histochemical analysis of β-glucuronidase (GUS) activity in transgenic plants expressing  PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR). SCL27 is highly expressed at the basal cell proliferation region of young leaves, suggesting an important role of SCLs in inhibiting chloroplast development before cell expansion. In addition, GT-cis elements were required for SCL27 directly binding to the PORC promoter. Furthermore, we showed that SCLs mediated GA-regulated chlorophyll biosynthesis through direct interaction with DELLA proteins. The interaction between SCLs and DELLAs reduced the DNA binding activity of SCL27. Our uncovered GA-DELLA-SCL module and its DNA binding targets provide new insights into molecular mechanisms by which chlorophyll biosynthesis and cell proliferation are coordinately regulated during leaf development in response to developmental and environmental cues.Chlorophyll biosynthesis is essential for plant growth and development. To date, the regulatory mechanisms of chlorophyll biosynthesis have been well understood only in dark conditions. Previous reports showed that miR171-targeted SCL6/22/27 proteins were involved in chlorophyll biosynthesis. However, the molecular mechanism of SCL action remains unclear. In this study, we found that SCLs negatively regulated chlorophyll biosynthesis though suppressing the expression of the key gene  Chlorophylls are complexed with their binding proteins and serve two primary functions in photosynthesis: they trap light energy and transfer it to the reaction centers of photosystems Arabidopsis genome contains three differentially regulated POR genes. It has been shown that PORA is expressed in etiolated seedlings and its mRNA level drops sharply in light; PORB is expressed in both etiolated seedlings and light-grown plants; PORC expression is activated by light in a fluence rate-dependent manner PORA and PORB is regulated by the transcription factors ethylene insensitive 3 (EIN3) and its homolog EIN3-like1 (EIL1) via directly binding to the EBS cis-elements in the promoter region PORC expression was reported to be directly induced by PIF1 PORC is regulated in light where PIF proteins are degraded.It has been well documented that chlorophyll biosynthesis is finely regulated at the multiple steps in the pathway and at both transcriptional and post-transcriptional levels Arabidopsisga1-3 mutant leads to increased accumulation of Pchlide and PORs in etiolated seedlings, which are substantially more resistant to photo-oxidative damage after transferred from darkness to light POR expression is not fully understood. Recently, the miR171-targeted scarecrow-like (SCL) transcription factors SCL6/SCL6-IV, SCL22/SCL6-III and SCL27/SCL6-II  have been demonstrated to play an important role in the proliferation of meristematic cells, polar organization and chlorophyll synthesis POR expression is, at least in part, mediated by miR171-targeted SCLs in light.Gibberellic acid (GA) is an important phytohormone that controls many aspects of plant development and growth via the GA-GID-DELLA signaling module in MIR171c over-expressors (MIR171c-OX) and scl6 scl22 scl27 triple mutants produce dark green leaves (LUC-rSCL27 (fused to the luciferase gene) results in leaf yellowing  gene driven by the SCL27 native promoter, designated pSCL27::rSCL27-GUS. We examined the pattern of GUS expression in the 3- to 11-day-old seedlings. The results of GUS staining clearly showed that the SCL27-GUS fusion protein started to accumulate in the newly developed leaves , which reflects the maximal photochemical efficiency of photosystem II (PSII) photochemistry (PSII activity). Compared to WT plants, PSII activity decreased more slowly in MIR171c-OX and scl6 scl22 scl27 plants but decreased more rapidly in LUC-rSCL27-OX plants . Quantitative PCR (qPCR) and northern blotting assays showed that among the inspected genes the levels of PORs and CAO transcripts were higher in MIR171c-OX and scl6 scl22 scl27 while were lower in LUC-rSCL27-OX, compared to those in the WT  with substantially reduced levels of POR expression were identified using qPCR  together with 6xMYC-rSCL27 in Nicotiana benthamiana leaves using a transient expression system. The expression of LUC was much lower in the leaves transformed with 6xMYC-rSCL27 than in leaves transformed with the empty vector and rSCL27-VP16  , whereas LUC expression driven by pPORC-455 did not significantly differ between WT and MIR171c-OX or between WT and scl6 scl22 scl27 (PORC promoter.The important role of PORs in SCL-regulated chlorophyll biosynthesis prompted us to investigate whether SCLs can directly control the promoter activity of tivator) , suggestL27-VP16 . Consist22 scl27 . These dPORC promoter (6xMYC-rSCL27 but not in those from WT plants (cis-elements. We next performed electrophoretic mobility shift assays (EMSAs) to confirm whether SCL27 can directly bind to fragments II and III of the PORC promoter. Consistent with the ChIP-qPCR results, shifted bands were observed when purified recombinant SCL27 protein (in vivo and in vitro data suggest that SCL27 inhibits PORC expression via directly binding to the PORC promoter.We then performed chromatin immuno-precipitation (ChIP) and qPCR assays to further define the SCL27-binding region within the promoter . Our resT plants , whereasT plants , indicat protein  was incu protein . However protein . Taken tcis-elements PORC promoter, we chose the 62-bp DNA fragment from −500 bp to −438 bp, which contains three G(A/G)(A/T)AA(A/T) GT element repeats PORC promoter.GT elements have been reported to be important for light-regulated gene expression, and DNA fragments II and III contain these PORs either in a PIF-dependent or PIF-independent manner POR expression. For this purpose, we generated LUC-rSCL27-OX/ga1-3 or pSCL27::rSCL27/pRGA::RGAd17 (the GA-insensitive form of RGA) plants via sexual crossing. Over-expressing LUC-rSCL27 in the WT or ga1-3 genetic background led to pale-green phenotypes and significantly decreased chlorophyll content  and della pentuple mutants. Indeed, over-expressing MIR171c in these plants resulted in dark green leaves and increased chlorophyll content (PORC expression was higher in MIR171c over-expressors than in the corresponding control WT (Ler) and della pentuple plants , which increases the levels of DELLA proteins. Our results showed that changes in SCL expression did not affect the greening ratio and Pchlide content in the absence or presence of PAC  and co-immunoprecipitation (Co-IP) assays using a transient expression system. A strong YFP signal was observed when either rSCL27-nYFP and RGA-cYFP or rSCL27-Nter-nYFP and RGA-cYFP were co-expressed in leaves  under the control of the 35S regulatory sequence in the scl triple mutant background; these plants were designated 35S::6xMYC-rSCL27-GR/scl6 scl22 scl27. Compared to mock -treated plants, transgenic plants treated with 10 µM dexamethasone (DEX) were pale green and accumulated less chlorophyll  was co-transformed with or without 6xMYC-rSCL27 into N. benthamiana leaves. As shown in pMIR171a::LUC activity was significantly increased by 6xMYC-rSCL27. These results indicate that SCLs can up-regulate MIR171 gene expression. To confirm that SCL27 directly regulates MIR171 gene expression, ChIP-qPCR was performed using three fragments: V , VI  and VII (the precursor of MIR171a). Indeed, only fragment VI was enriched in MYC antibody pulled-down precipitates obtained from the 6xMYC-rSCL27 over-expressing plants but not in those obtained from the WT plants  PORA and PORB expression directly via EIN3 and EIL1. DELLAs might also indirectly regulate chlorophyll biosynthesis through other interacting transcriptional factors, including brassinosteroid-resistant 1 (BZR1) and the jasmonic acid ZIM-domain proteins (JAZs) Furthermore, we observed another complex phenomenon: over-expression of SCLs did not completely rescue the dark-green phenotype of the MIR171c-OX, scl6 scl22 scl27 triple mutants, 35S::LUC-rSCL27, ga1-3, gai-2 (SAIL_587_CO2), rga rgl1 rgl2 rgl3 mutants, and pRGA::RGAd17 are in the Arabidopsis thaliana Columbia ecotype (Col) background ga1-3 mutant was backcrossed with the wild type (Col) for six generations; the por-amiR and pSCL27::SCL27-GUS were transformed in Col background; the por-amiR/MIR171c-OX, LUC-rSCL27-OX/ga1-3, and rSCL27/RGAd17 plants were generated by crossing; the della pentuple is in the Ler ecotype −2. s−1) in long days (16-h light/8-h dark).PORC and MIR171a genes in the Col genome, respectively, with primers listed in XhoΙ/BamHI sites of the pGREEN0800LUC vector pPORC-1685::LUC and pMIR171a::LUC vectors. The pPORC-861::LUC and pPORC-455::LUC vectors were constructed in a similar way. To make the POR amiRNA vector, the amiRNA target sequences for POR genes and primers including POR I miR-s, POR II miR-a, POR III miR*s and POR IV miR*a were designed using the WMD3 Web microRNA Designer (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi) and listed in POR target amiRNA foldback. DNA fragments were gel-purified and cloned into the Gateway cloning vector pENTR-SD/D/TOPO (Invitrogen) according to the manufacturer's instructions. After sequencing confirmation, the cloned DNA fragments were transferred to the 35S over-expression vector (pGWB2) (Invitrogen) using LR clonase (Invitrogen).About 1.7- and 1.2-kb promoter fragments at the upstream of the start codon were amplified from SCL22 cDNA was cloned into the pGBKT7 vector (Clontech). RGA, GAI, RGL1, RGL2 and RGL3 cDNAs were cloned into the pGADT7 vector (Clontech). SCL27 and RGA cDNAs were cloned into pDEST22 (Invitrogen); cDNAs encoding SCL27 and its N-terminal (1–267 amino acids) and GRAS domain (268–640 amino acids) were cloned into pDEST32 (Invitrogen). The primers used for these constructs are given in SCL27, SCL27 N-terminal, and SCL27-GRAS sequences were cloned into pCAMBIA1300 (nYFP), whereas RGA was cloned into pCAMBIA1300 (cYFP).For yeast two-hybrid analysis, in vitro protein-DNA binding analysis, SCL27 and RGA was cloned into the pET28b and pGEX6p-3 vectors, respectively. The constructs were transformed into the expression strain BL21 for protein expression. For co-IP analysis, RGAd17 and miR171-resistent SCL27 (rSCL27) were cloned into the binary vector with 3xHA or 6xMYC. Transgenic plants were generated by the floral dipping method For Fv/Fm parameter was measured using light-stressed leaf discs after 15-min adaptation to darkness Seedling greening was analyzed by exposing 5-day-old dark-grown seedlings to white light (16 h-light/8 h-dark) for 2 days. Chlorophyll autofluorescence was analyzed using a confocal laser scanning microscope . In the PAC-treated etiolated seedlings, 0.01 µM of PAC was used. Greening ratio was determined by counting the percentage of green cotyledons of each genotype. Pchlide was extracted from 5-day-old etiolated seedlings with 1 mL of ice-cold 80% acetone in the dark. The samples were centrifuged at 13000 rpm for 10 min, and fluorescence was excited by the wavelength of 440 nm and scanned from 600 nm to 700 nm using a fluorescence spectrophotometer (Hitachi) at room temperature Plasmids were transformed into yeast strain AH109 by the lithium chloride–polyethyleneglycol method according to the manufacturer's manual (Clontech). The transformants were selected on SD-Leu-Trp plates. The protein-protein interactions were tested on SD-Trp-Leu -His-Ade plates with or without 3-amino-1, 2, 4-triazole.A. tumefaciens strain GV3101 transformed with each of the two constructs for BiFC analysis was cultured in the solution containing 10 mM MES, 10 mM MgCl2, and 100 µM acetosyringone to an optical density (OD600) of 0.6 to 0.8. Then, two strains were mixed and incubated at the room temperature for at least 2 h. The YFP fluorescence was analyzed using a confocal laser scanning microscope  48 to 96 h after N. benthamiana leaves were infiltrated with the mixture.The Plant materials were submerged in 90% acetone for 15 min, and then transferred into 0.5 mg/mL X-Gluc solution . Plant materials were vacuumized, kept at 37°C and decolorized in 70% ethanol.N. benthamiana leaves and transgenic plants over-expressing 6xMYC-rSCL27 were used for Co-IP analyses. The soluble proteins were extracted with the extraction buffer . The beads were washed with the buffer . Immunoprecipitation was performed with the anti-MYC antibody using N. benthamiana leaves. For Arabidopsis samples, immunoprecipitation was performed with the anti-RGA antibody. RGAd17-3xHA and 6xMYC-SCL27 fusion proteins were detected by immunoblotting with anti-HA (Sigma) and anti-MYC antibodies (Santa Cruz). To analyze POR, LHCB1, LHCB2, LHCB5, LHCA1, PsaD, and AtpB protein levels in vivo, samples (0.1 g) were ground in liquid nitrogen and suspended with 200 µL extraction buffer . Total protein was extracted by incubating the samples in boiled water for 5 min, and then centrifuged at 13 000 rpm for 10 min. Proteins were detected with the anti-POR, LHCB1, LHCB2, LHCB5, LHCA1, PsaD, and AtpB antibodies (Agrisera) after total proteins were separated onto a SDS-PAGE gel and transferred to Hybond-ECL Nitrocellulose membrane (Amersham Biosciences).Agrobacteria-infiltrated N. benthamiana leaves were used for LUC/REN analyses. Leaf samples were collected for the transient expression assay using commercial Dual-LUC reaction (DLR) reagents, according to the manufacturer's instruction (Promega).The transient expression assay  was carried out as described previously ACTIN2 was used as an internal control for normalization. The primers are listed in One µg of total RNAs was used for reverse transcription in a 20 µL reaction system using the RNA PCR (AMV) kit (Promega). Quantitative PCR was performed with SYBR-Green PCR Mastermix (Takara), and amplification was real-time monitored on stepone and steponeplus real-time PCR system (Applied Biosystems). 6xMYC-rSCL27-OX transgenic plants were harvested. For GA3 or PAC treatment, samples were harvested from the plants treated with 10 µM GA3 and 0.1 µM PAC for 2 day. After fixation, the materials were resuspended in extraction buffer followed by sonification. One third of the solution was saved as input total DNA without precipitation; another one-third was mixed with the MYC-fused agarose (Sigma); and the remaining one-third was precipitated in parallel with HA-fused agarose as a negative control. The resulting DNA samples were purified using a PCR purification kit (Qiagen). The relative concentrations of the DNA fragments were analyzed by qPCR, using the β-TUBULIN2 gene promoter as the reference.ChIP experiments were performed according to published protocols The EMSA was performed as reported previously SCL27 (At2G45160), SCL22 (At3G60630), SCL6 (At4G00150), MIR171A (At3G51375), MIR171B (At1G11735), MIR171C (At1G62035), β-TUBULIN-2 (At5G62690), HEMA1 (AT1G58290), GUN4 (AT3G59400), GUN5 (AT5G13630), PORA (AT5G54190), PORB (AT4G27440), PORC (AT1G03630), GAI (AT1G14920), RGA (AT2G01570), RGL1 (AT1G66350), RGL2 (AT3G03450), and RGL3 (AT5G17490).Figure S1MIR171c-OX, scl6 scl22 scl27 triple mutant and LUC-rSCL27-OX plants grown in long-day conditions. Bar = 0.5 cm. (B) Northern blot analysis of the expression of the indicated genes in (A). Five micrograms of total RNA were loaded on each lane. The levels of rRNAs stained with ethidium bromide are shown as loading controls. (C) Immunodetection of POR levels in (A). (D and E) Relative expression levels of PORA, PORB, and PORC genes in Col and por-amiR (D), in scl6 scl22 scl27 and por-amiR/scl6 scl22 scl27 (E). Expression levels were normalized to that of ACTIN2. The expression levels in Col and scl6 scl22 scl27 were set as 1. Error bars indicate s.d. (n = 3). Two biological replicates were performed with similar results.Effect of miR171-targeted SCLs on chlorophyll biosynthesis in light. (A) Leaf phenotypes of WT, (TIF)Click here for additional data file.Figure S2pSCL27::rSCL27-GUS from 3-day to 11-day seedlings. Bars = 1 mm.GUS staining of transgenic plants (TIF)Click here for additional data file.Figure S3MIR171c-OX, scl6 scl22 scl27, LUC-rSCL27-OX etiolated seedlings. Error bars indicate s.d. (n = 30). Three biological repeats were performed. (C) Ultrastructure of plastids in 5-day-old etiolated seedlings. Bars = 1 µm.Effect of miR171-targeted SCLs on chlorophyll biosynthesis in the dark. (A) Greening ratio of 5-day-old etiolated seedlings transferred to white light for 2 days. Three biological repeats were performed. Error bars indicate s.d. (n = 30). (B) Pchlide levels of 5-day-old Col, (TIF)Click here for additional data file.Figure S4Effect of miR171-targeted SCLs on chloroplast development. (A) Ultrastructure of chloroplasts in mature leaves from 25-day-old plants. Bars = 1 µm. (B) Statistic analysis of stacked and stromal thylakoid membranes. Error bars indicate s.e. (n>110). (C) Immunoblot analysis of PsaD, LHCB1, LHCB2, LHCB5, LHCA1, and AtpB expression in 25-day-old plants.(PDF)Click here for additional data file.Figure S5PORC promoter in Arabidopsis. (A) The LUC reporter gene driven by pPORC-1685, pPORC-861 or pPORC-455 was transformed into Col, MIR171c-OX, and scl6 scl22 scl27 plants. The relative LUC activities were normalized to the 35S::REN internal control. Error bars indicate the s.d. (n = 4). Three biological replicates showed similar results. (B) The purified His-SCL27 protein used for EMSA in SCL27 binds to the (TIF)Click here for additional data file.Figure S6SCL27 and RGA. (A) Chlorophyll content of the genotypes shown in p values (Student's t-test) <0.01 relative to wild-type and ga1-3, respectively. Error bars indicate s.d. (n = 4). (B) Phenotypes of Col, pSCL27::rSCL27, pRGA::RGAd17, pSCL27::rSCL27/pRGA::RGAd17 plants grown in long-day conditions for 25 days. Bars = 1 cm. (C) Chlorophyll content of the genotypes shown in (B) based on the fresh weight (FW). ** represent p values (Student's t-test) <0.01 relative to wild-type and pRGA::RGAd17, respectively. Error bars indicate s.d. (n = 4).Genetic analysis of (TIF)Click here for additional data file.Figure S7MIR171c-OX, scl6 scl22 scl27, LUC-rSCL27-OX etiolated seedlings that were grown on the media with PAC or without PAC  and transferred to white light for 2 days. Three biological repeats were performed. Error bars indicate s.d. (n = 30). (B) Pchlide levels of 5-day-old Col, MIR171c-OX, scl6 scl22 scl27, LUC-rSCL27-OX etiolated seedlings grown in the media with PAC or without PAC (Mock). Error bars indicate s.d. (n = 3). Three biological repeats were performed.Effect of SCL in DELLA-regulated chlorophyll biosynthesis in the dark. (A) Greening ratio of 5-day-old Col, (TIF)Click here for additional data file.Figure S8SCL27-Nter-nYFP and RGA-cYFP, SCL27-Nter-nYFP and cYFP, SCL27-GRAS-nYFP and RGA-cYFP, and nYFP and cYFP, were co-transformed into N. benthamiana leaves. Bars = 50 µm.SCLs interact with DELLAs. (A) SCL22 interacts with DELLAs in yeast. (B) BiFC analysis of the interaction between the N-terminal of SCL27 (SCL27-Nter) and RGA. The following pairs of constructs, (TIF)Click here for additional data file.Figure S9SCLs and DELLAs is not affected mutually at the transcriptional level. (A and B) qPCR analysis of MIR171c, SCL27, SCL22, and SCL6 expression in seedlings treated with GA3 or Mock (ethanol) (A), and PAC or Mock (methanol) (B). (C) qPCR analysis of MIR171c, SCL27, SCL22, and SCL6 expression in GA mutants including ga1-3, gai-2 and rga rgl1 rgl2 rgl3. (D) qPCR analysis of RGA and GAI expression in Col, MIR171c-OX, scl6 scl22 scl27, LUC-rSCL27-OX seedlings. Expression was normalized to that of ACTIN2 and in WT treated with mock or in WT was set as 1 for each gene. Two biological replicates were performed with similar results. Error bars represent s.d. (n = 3).Expression of (TIF)Click here for additional data file.Figure S10PORC promoter. (A and B) The purified GST and GST-RGA proteins used for EMSA in β-TUBULIN-2 promoter was used as a reference. Error bars indicate the s.d. (n = 3). Two biological replicates were performed with similar results.RGA reduces the binding activity of SCL27 to the (TIF)Click here for additional data file.Table S1A list of primers used in this study.(DOCX)Click here for additional data file.


Recombination-based cloning is a quick and efficient way to generate expression vectors. Recent advancements have provided powerful recombinant DNA methods for molecular manipulations. Here, we describe a novel collection of three-fragment MultiSite Gateway cloning system-compatible vectors providing expanded molecular tools for vertebrate research. The components of this toolkit encompass a broad range of uses such as fluorescent imaging, dual gene expression, RNA interference, tandem affinity purification, chemically-inducible dimerization and lentiviral production. We demonstrate examples highlighting the utility of this toolkit for producing multi-component vertebrate expression vectors with diverse primary research applications. The vectors presented here are compatible with other Gateway toolkits and collections, facilitating the rapid generation of a broad range of innovative DNA constructs for biological research. Over time, the development of sophisticated techniques such as genetic knockdown and knockout allowed more intricate and detailed investigations. Today, the continued advancement of recombinant DNA technologies has provided the modern biologist with an arsenal of molecular tools. Use of these techniques, however, often requires the laborious construction and validation of complex, multi-component vectors.Most contemporary investigations of cellular and molecular processes necessitate the use of synthetic DNA vectors. Recombinant cloning of plasmid vectors is the most commonly used method for transgenic analyses. Shortly after the first successful demonstration of gene expression from exogenous DNA in mammalian cells , synthetTM cloning system (ThermoFisher) utilizes site-specific recombination to insert DNA elements into a vector [The effort associated with conventional cloning methods still prevents many researchers from exploiting recombinant DNA tools. Plasmids are often still constructed with traditional “cut-and-paste” restriction enzyme techniques that are difficult and time consuming. Moreover, restriction enzyme-based cloning methods are prohibitive for DNA sequences containing common endogenous restriction sites. This problem is exacerbated when insertion of more than one DNA sequence into a vector is desired. The MultiSite Gatewaya vector  and has a vector . Furthera vector –18. Addia vector –22, worma vector , 24, froa vector  and multa vector –29 have Here, we present a novel set of three-fragment MultiSite Gateway vectors which provide an expanded array of molecular tools. Our entry vectors cover a large variety of applications such as cell-specific expression, fluorophore-based imaging, bicistronic expression, inhibitory RNA (RNAi)-mediated gene knockdown, protein purification and inducible protein dimerization. Additionally, we created two destination vectors for lentiviral production and describe optimized entry vectors designed for enhanced viral titers.www.addgene.org). Together, the tools presented here will prove useful for cutting-edge molecular and genetic investigations.We illustrate the diverse uses of these vectors with examples from multiple primary research applications. Using an entry vector with a novel brainbow color palette, we label motoneuron circuitry in zebrafish. Next we demonstrate efficient artificial microRNA (amiRNA)-mediated knockdown of multiple genes in primary neuronal cultures using lentivirus produced from vectors containing our recently enhanced amiRNA expression scaffold . We thenStudies using rats or zebrafish were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the University of Oregon Institutional Animal Care and Use Committee (Permit Numbers: #13–19 and #11–20). Rats were anesthetized with isoflurane prior to cervical dislocation and culturing of neurons. Zebrafish embryos were anesthetized with MS-222 prior to embedding in agarose and imaging. All zebrafish embryos, larvae and adults were raised and maintained at 28.5°C with a 14/10 light/dark cycle according to standard protocols  in the Uelavl3 was made by inserting a XhoI-containing linker into the NotI site of a huC:Cam2.1 plasmid [gfap was made by cloning a 7437-nucleotide XhoI-BamHI fragment from a gfap:GFP plasmid [Unless otherwise stated, all 5’ entry vectors were generated by PCR amplification of the desired 5’ element using attB4/B1R-flanked oligonucleotide primers, followed by a BP reaction with pDONR P4-P1R (Invitrogen). p5E-CMVmin was created by amplification of the minimal cytomegalovirus immediate early enhancer/promoter (CMVmin) from pcDNA3 (Invitrogen). The human synapsin1 promoter alone or hybrid CMVmin/human synapsin1 promoter from pENTR-L1-ESYN-R5  were amp plasmid  and clon plasmid . p5E-gfa plasmid  into p5Ekrt5 [mnx1 [dusp6 [bactin2 [The generation of p5E-EF1α/β-globin and p5E-krt5 , p5E-mnxt5 [mnx1  and p5E-1 [dusp6  have beeloxP and lox2272 sites and 3 SV40 polyadenylation sequences was created by PCR. This 1024-nucleotide recombination scaffold was cloned into KpnI-SacI sites of pME-MCS. Then HA-tagged E2Crimson (Clontech), Myc-tagged tdTomato and EGFP were cloned in sequence into unique PacI, AscI and FseI sites within the recombination scaffold, respectively. pME-FlEx was created by annealing sets of oligonucleotides to produce antiparallel tandem loxP and lox2272 recombination sites which was then PCR amplified inserted into pDONR221.Unless otherwise stated, all middle entry vectors were generated by PCR amplification of the desired middle element using attB1/B2-flanked oligonucleotide primers, followed by a BP reaction with pDONR221 (Invitrogen). To create pME-mKate2 no-stop, the mKate2 coding sequence  was amplTo generate P2A middle entry vectors, the GFP, nlsGFP, and memGFP sequences were first subcloned into pcDNA3. Sequences for GFP or nlsGFP with Kozak sequences and without stop codons were amplified from pME-nlsEGFP  and inseThe generation of pME-eSIBR , pME-ERTR-FRT pA was cloned by amplifying the KanR cassette from pCRII (ThermoFisher) with FRT sites in the 5’ and 3’ primers and inserting into the BamHI site of p3E-polyA [Unless otherwise stated, all 3’ entry vectors were generated by PCR amplification of the desired 3’ element using attB2R/B3-flanked oligonucleotide primers, followed by a BP reaction with pDONR P2R-P3 (Invitrogen). p3E-GFP-HA, p3E-YFP-HA , p3E-CFP-HA , p3E-mCherry-HA (from p3E-mCherrypA ), p3E-mK3E-polyA . FRB var3E-polyA –47 to geGFP, nlsGFP, GFPmem and CMVmin-promoted 3’ entry vectors were generated by first subcloning GFP, nlsGFP, or GFPmem coding sequences into pcDNA3. GFP and nlsGFP were amplified from pME-nlsGFP and inserted between HindIII/NotI to make pcDNA3-GFP and pcDNA3-nlsGFP. GFPmem, which contains the palmitoylation domain of human H-Ras on the c-terminus, was amplified from pME-EGFPCAAX  and inseTo make P2A 3’ entry vectors, the MCS from pBluescript II SK(+) (Agilent) was amplified to make p3E-MCS. Sense and antisense oligonucleotides containing the P2A sequence were annealed and inserted into the KpnI site of p3E-MCS to create p3E-P2A-MCS, which has unique restriction enzyme sites downstream of the P2A sequence in the 5’ to 3’ order of: XhoI, SalI, AccI, ClaI, HindIII, XmaI, SmaI, SpeI, BstXI and SacII. Coding sequences for GFP, CFP, and mKate2 were then amplified and inserted between XhoI/HindIII of p3E-P2A-MCS to produce p3E-P2A-GFP, p3E-P2A-CFP and p3E-P2A-mKate2 no-pA.R cassette by AgeI/ApaI restriction enzyme digestion, filling in 5’ overhangs with DNA polymerase I Klenow fragment, and blunt-end ligation with T4 DNA ligase.pEpic was made by blunt-end cloning the XhoI/ClaI-defined attR4-attR3 cassette from pDestTol2pA2  into thebactin2, p5E-MCS, p3E-polyA, p3E-mCherrypA, p3E-MTpA, pME-EGFPCAAX, pME-MCS and pME-nlsEGFP [huC:Cam2.1 plasmid was a gift from Joe Fetcho. The gfap:GFP plasmid was generously provided by Pam Raymond. pDONR223-ErbB3 and pDONR223-ErbB2 [Tol2kit vectors pDestTol2CG2, pDestTol2pA2, p5E-UAS, p5E-CMV/SP6, p5E-Fse-Asc, p5E-23-ErbB2  were gifVectors were generated using recombination reactions with LR-clonase II (Invitrogen). UAS:BrainbowTEC was made by combining p5E-UAS, pME-BrainbowTEC, p3E-polyA, and pDestTol2CG2. pEpic_Lite mCMV:eSIBR-nlsGFP pA or no-pA vectors were created by combining pEpic_Lite, p5E-CMVmin, pME-eSIBR constructs with pre-inserted amiRNAs, and p3E-nlsGFP pA or no pA, respectively. pEpic_Lite UbiC:eSIBR-nlsGFP no-pA vectors were produced by combining pEpic_Lite, p5E-Ui4-eSIBR constructs with pre-inserted amiRNAs, pME-nlsGFP, and p3E-HA no-pA. pEpic_Lite mCMV:ErbB3-P2A-GFP was made by combining pEpic_Lite, p5E-CMVmin, pDONR223-ErbB3, and p3E-P2A-GFP no-pA. ErbB2-myc was created by combining pEpic_Lite, p5E-CMVmin, pDONR223-ErbB2, and p3E-MTpA. pEpic_Lite mCMV:memGFP-P2A-HA-Neuroligin1 was made by combining pEpic_Lite, p5E-CMVmin, pME-memGFP-P2A, and p3E-HA-Neuroligin1. pEpic hPGK:Baf57c-SGTAP was constructed by combining pEpic, p5E-hPGK, pME-Baf57c, and p3E-SGTAP no-pA. pEpic CMV:OGT1-mCherry-FRB-HA was made by combining pEpic, p5E-CMV/SP6 [cmlc:GFP-positive) produced from these founders were indistinguishable from siblings lacking transgenic insertions (cmlc:GFP-negative).Transgenic UAS:BrainbowTEC lines were generated by co-injecting plasmid DNA and Tol2 transposase RNA  into themnx1:GAL4)b1222; Tg(hsp70l:cre)zdf13 were crossed to Tg(UAS:BrainbowTEC) founders. To induce Cre expression, embryos at 6–7 hpf were heat-shocked for 30 min by transfer into embryo medium pre-heated to 38.5°C as optimized previously [To label neurons in the ventral spinal cord of zebrafish embryos, Tg culture and transfection, and production and titration of lentivirus was previously described [COS7 and HEK293T cell (ATCCescribed . For nlsin vitro (DIV); for sub-saturating transduction 2,000 infectious lentiviral particles were added. Cells were used for western blotting, qRT-PCR, or immunocytochemistry at 14DIV.Primary rat hippocampal cell cultures were prepared and maintained as previously described . For sat® acoustic focusing cytometer (Applied Biosystems). GFP+ cells were determined as cells with >2x the maximum signal observed from non-transduced sister cultures for the BL1 channel . Mean BL1 values of all GFP+ cells in a culture were used for comparisons.Single-cell GFP intensity was measured by flow cytometry on an Attunewww.gimp.org).Standard SDS-PAGE western blotting procedures using nitrocellulose membranes were performed as previously described . Chemilut method. Primer sets used to measure mRNA levels were: nlgn1 , nlgn2 , nlgn3 .First-strand cDNAs were synthesized from total RNA isolated from cultured hippocampal neurons using oligo-dT primers and qRT-PCR was performed as described previously . Values For COS7 cells and primary hippocampal neurons, cells grown on circular glass coverslips (ThermoFisher) were fixed in 4% PFA and 4% sucrose in 1X PBS for 15 min at 4°C, rinsed 1X with PBS, and blocked for 1 hr at RT in 1X blocking buffer (10% BSA (Sigma Aldrich), 1X blocking reagent (Roche) and 1% normal donkey and goat serums (Jackson ImmunoResearch) in 1X PBS). Cells were then incubated with a primary antibody for HA.11  in 0.33X blocking buffer diluted in 1X PBS for 2 hr at RT. Cells were then washed 3 x 5 min with 1X PBS, permeabilized with 0.25% Triton X-100 in 1X PBS for 5 min at RT, then re-blocked with 1X blocking solution for 1 hr at RT. Cells were then incubated with primary antibodies overnight at 4°C; GFP  and neurons additionally used Synapsin1 . The following day cells were rinsed 3 x 5 min with 1X PBS and incubated for 1 hr at RT with secondary antibodies; anti-chicken Alexa Fluor 488  and anti-mouse Cy3 and anti-rabbit Cy5 . Cells were washed 3 x 5 min in 1X PBS and mounted on slides with Fluoromount-G (Southern Biotech). Images were taken using a 40X air or 100X oil-immersion objective on an inverted Nikon Eclipse C1 confocal microscope. The brightness and contrast of images was adjusted using GIMP. The binary GFP mask was produced by intensity thresholding of the GFP image in Image-Pro Plus 6.3 (Media Cybernetics).Nuclear extracts  were preHEK293T cells were co-transfected with 500 ng each of pEpic CMV:OGT1-mCherry-FRB-HA and pS-FKBPNES (which expresses human FKBP12 fused to the nuclear export sequence from HIV REV protein) per well of a 24-well plate using Xfect transfection reagent (Clontech). 24 hours later, the cells were treated with 50 nM rapamycin (LC Labs) and time-lapse imaged using a Nikon Eclipse Ti–E fluorescent inverted widefield microscope equipped with a LiveCell environmental control system (Pathology Devices).P-values obtained by statistical comparisons of two sample groups with normal distributions verified by Shapiro-Wilk tests for normality in R  used Student’s two-tailed, unpaired t-tests in Microsoft Excel. Sample group variances were compared using F-tests in R, and comparisons with equal variances used type 2 (homoscedastic) assumptions, whereas comparisons with unequal variances used type 3 (heteroscedastic) assumptions.R), which can contain additional vector-specific 5’ and 3’ flanking sequences. Next, using an “LR” reaction for site-specific recombination between pairs of unique attL and attR sites, DNA elements from a 5’, middle, and 3’ entry vector recombine to replace the destination vector ccdB/CmR cassette, allowing for the selection of recombinant clones  for clonal selection purposes. Because large inserts between the viral long terminal repeats (LTRs) can decrease lentiviral titers [R, which we call pEpic_Lite , middle (pME) and 3’ (p3E) entry vectors is provided in Tomato, EGFP, and the far-red fluorophore E2Crimson [loxP and lox2272 sites allows for either Cre-mediated recombination between different lox sites or failure to recombine dictating which fluorophore is expressed. tdTomato-myc is expressed as the default, no recombination configuration; whereas loxP recombination causes expression of GFP and lox2272 recombination causes the expression of E2Crimson-HA . This line also allowed temporal control of Cre expression by placing Cre downstream of the heat shock protein 70 promoter (hsp70l:Cre). The resulting embryos  were heat shocked at 7 hours post fertilization (hpf) to induce Cre expression and recombination . In conin vivo injections. Therefore, we investigated these factors in an attempt to optimize eSIBR-based gene knockdown and viral production from our pEpic destination vectors.Lentivirus provide a means for gene transfer into many cell types which are not amenable to transgenesis by other methods, including neurons, and are therefore a useful approach for introducing amiRNAs. Because transgenes carried by lentivirus are genomically integrated, they are beneficial for many research applications such as clonal selection and long-term expression. As with other expression constructs, promoter selection and inclusion of other factors such as polyadenylation signal (pA) sequences can influence the efficacy of gene expression from lentiviral vectors. Additionally, high viral titers are often necessary for efficient transgenesis in certain applications, such as Because previous reports have observed that internal pA sequences can impact lentiviral production , 66, we To compare the effect of promoter choice on knockdown potency, we cloned cadm1-3 and scrambled1-3 eSIBR amiRNAs into p5E-Ui4-eSIBR. We then performed LR reactions using p5E-Ui4-eSIBR constructs with a middle entry vector carrying nlsGFP with a stop codon (pME-nlsGFP ) and ourWe previously showed that chained eSIBR amiRNAs expressed from lentiviral vectors potently knocked down Cadm1-3 in cultured rat hippocampal neurons when cells were infected at saturating titers . To detePrior Gateway toolkits have provided entry vectors containing miR-30a scaffolds for amiRNA expression , 67. HowDespite the rapid advancement in recombinant DNA strategies, expression of more than one transgene from a single vector often remains challenging. A common method for dual-gene expression includes use of the internal ribosome entry site (IRES) between two protein coding sequences; however, IRES is notoriously inefficient and expression of the downstream transgene tends to be greatly diminished , 70. AnoRecently, viral 2A peptides have been extremely successful for stoichiometric expression of two proteins from a single open reading frame (ORF) in recombinant DNA vectors , 78–82. To validate the efficacy of p3E-P2A vectors for bicistronic fluorophore expression, we generated a plasmid driving expression of the receptor tyrosine kinase ErbB3 conjugated to P2A-GFP to make mCMV:ErbB3-P2A-GFP . ErbB3, Because “cleavage” of the 2A peptide leaves the majority of the P2A sequence attached to the C-terminus of the upstream protein, p3E-P2A constructs are not suitable for bicistronic expression of proteins where addition of a C-terminal peptide would compromise protein function. For example, many cell adhesion molecules, including the neuroligin family, present a PDZ-binding motif in their terminal 4 amino acids ; the addWe additionally determined if this vector worked for bicistronic expression in cultured rat hippocampal neurons. Again, surface labeling for HA in neurons transduced with lentivirus carrying mCMV:memGFP-P2A-HA-Neuroligin1 showed that Neuroligin1 was correctly inserted into the plasma membrane . Furtherstreptavidin binding protein/Protein G tandem affinity purification), based on an optimized tandem affinity purification (TAP) method that is amenable to much smaller amounts of starting material [Affinity purification has been instrumental in determining protein complex compositions; however, pull-downs from fusion proteins with a single affinity tag, such as Protein A or Protein G, are often impure and may lead to false-positive identification of protein complex members. Protein purification using sequential isolations from two different affinity tags considerably reduces non-specific protein pulldowns, but is notorious for low overall yield. We developed a 3’ entry vector, p3E-SGTAP  to enable their chemically-inducible dimerization (CID). CID can be widely applied, for example, for inducible membrane recruitment, nuclear import/export, and protein degradation . One exescopicus , forms ascopicus , 96. We scopicus . Furtherscopicus . Proteinscopicus .The FRB-rapamycin-FKBP system has been widely applied including to force protein subcellular localization and to modulate cellular signaling , 97. To elavl3 [elavl3). We offer four additional zebrafish gene promoters for cell/tissue-specific expression in glia [gfap), primary motoneurons [mnx1), epidermis [krt5), and FGF-responsive cells [dusp6).We generated additional 5’ entry vectors containing promoters for ubiquitous, semi-ubiquitous, or cell/tissue-specific gene expression. These include two promoters for ubiquitous or semi-ubiquitous expression: frog elongation factor 1α fused to rabbit β-globin intron (p5E-EF1α/β-globin) or EF1α/β-globin additionally enhanced with a zebrafish β-actin 2 promoter  , which allows the insertion of a protein coding sequence between two pairs of heterotypic, antiparallel on sites , 104. Cr[in vivo .lox site recombination. pME-ERT2-Cre-ERT2 differs from a previously described Cre-ERT2 middle entry vector [Finally, we offer vectors for tamoxifen-inducible gene expression by linking the estrogen receptor variant ERT2 onto both ends of Cre (pME-ERT2-Cre-ERT2) which can be used for y vector  in that y vector . ERT2 way vector , 38.E. coli DNA adenine methyltransferase (p3E-Dam-myc no-pA), which, when fused to a chromatin-associated protein of interest, can be used to identify DNA binding sequences [FRT-flanked kanamycin resistance cassette (p3E-FRT-KanR-FRT pA) for FLP-induced antibiotic resistance.We developed additional 3’ entry vectors for generating C-terminal fusion proteins. This list includes a no-pA vector with GFP (p3E-GFP no-pA) and pA and no-pA vectors with a GFP that targets to the plasma membrane (GFPmem) via the addition of an hRas palmitoylation domain on its C-terminus (p3E-GFPmem pA and no-pA). We stress the distinction of GFPmem in the 3’ entry vectors from memGFP in pME-memGFP-P2A because they use distinct mechanisms to localize at the plasma membrane. We also provide no-pA vectors containing mKate2 with myc or HA tags (p3E-mKate2-myc and p3E-mKate2-HA no-pA). Additionally, we generated five no-pA vectors for generating C-terminal HA-tagged fluorophore fusion proteins . We also cloned a myc-tagged equences , 108. LaWe also developed 3’ entry vectors for two-gene expression constructs from independent promoters. Using a dedicated CMVmin promoter, we made both pA and no-pA vectors for independent expression of GFP, nlsGFP or GFPmem . However, we warn against using these vectors to generate bicistronic constructs if robust expression of an upstream transgene is required because, as with other reports , 77, 109www.dnasu.org). Therefore, this toolkit should prove useful for rapidly generating constructs that will facilitate molecular and cellular research.Multisite Gateway cloning is a powerful method for generating multi-component vectors. Collectively, we have developed more than 50 novel entry vectors and two lentiviral destination vectors for vertebrate expression constructs. These vectors are available, either individually or as an entire kit, via Addgene. Our constructs complement existing vertebrate-compatible Gateway toolkits, including the widely-used Tol2kit . FurtherS1 Movie(AVI)Click here for additional data file.


MicroRNAs are key regulators associated with numerous diseases. In HEK293 cells, miR-153-3p and miR-205-5p down-regulate alpha-synuclein (SNCA) and Leucine-rich repeat kinase 2 (LRRK2), two key proteins involved in Parkinson’s disease (PD). We have used two-dimensional gel electrophoresis (2D-PAGE) coupled to mass spectrometry (MS) to identify a spectrum of miR-153-3p and miR-205-5p targets in neuronal SH-SY5Y cells. We overexpressed and inhibited both microRNAs in SH-SY5Y cells and through comparative proteomics profiling we quantified ~240 protein spots from each analysis. Combined, thirty-three protein spots were identified showing significant  changes in abundance. Modulation of miR-153-3p resulted in seven up-regulated proteins and eight down-regulated proteins. miR-205 modulation resulted in twelve up-regulated proteins and six down-regulated proteins. Several of the proteins are associated with neuronal processes, including peroxiredoxin-2 and -4, cofilin-1, prefoldin 2, alpha-enolase, human nucleoside diphosphate kinase B (Nm23) and 14-3-3 protein epsilon. Many of the differentially expressed proteins are involved in diverse pathways including metabolism, neurotrophin signaling, actin cytoskeletal regulation, HIF-1 signaling and the proteasome indicating that miR-153-3p and miR-205-5p are involved in the regulation of a wide variety of biological processes in neuroblastoma cells. Parkinson’s disease (PD) is the most common neurodegenerative movement disorder characterized by degeneration of dopaminergic neurons in the substantia nigra pars compacta . Most PDMicroRNAs have been used to decode different pathways associated with several diseases . HoweverIndividual microRNAs can regulate several mRNAs . Therefo2 atmosphere at 37°C. Transfections were performed, in triplicate, with scrambled control mimic, miR-153-3p mimic, miR-205-5p mimic, scrambled control hairpin inhibitor, miR-153-3p hairpin inhibitor and miR-205 hairpin inhibitor, all mirVanaTM (Life Technologies), at a final concentration of 20 nM. Cells were seeded in 6-well plates at 5x105 cells/well. 2μl μRNA (20 μM), diluted with 100 μl of Opti-MEM, and 7 μl Lipofectamine RNAiMax (Invitrogen) diluted with 100 μl of Opti-MEM was incubated for 5 minutes (min) at room temperature (RT). The two solutions were mixed and incubated for 15 min at RT. The transfection mix was diluted to 2 ml with Opti-MEM, added to the wells and incubated at 37°C for 4–6 hours before replacing with full media. Cells were harvested after 24 hours for quantitative PCR (qRT-PCR) analysis and after 48 hours for Western blotting and 2D-PAGE analysis.SH-SY5Y cells  were cultured in a base medium mixture (Full medium: 1:1 DMEM/Ham’s-F12) (Invitrogen) supplemented with 10% v/v fetal bovine serum (Atlanta biologics) and 2 mM GlutaMAX (Invitrogen) in 5% COTM microRNA cDNA Synthesis kit (Quanta Biosciences) and used for both semi-quantitative (25 cycles) and qRT-PCR. miR-153-3p forward primer (5’ GCCGGGCTTGCATAGTCACAA 3’), miR-205-5p forward primer (5’ GTTTCCTTCAT TCCACCGG 3’), U6 forward primer (5’ CGCTTCGGCAGCACATATAC 3’) and PerfeCTa®Universal PCR primer along with PerfeCTa® SYBR® GREEN SuperMix for IQTM were used for qRT-PCR in triplicates for each biological replicate.RNA was isolated, in triplicate, 24 hours post-transfection using the miRCURY RNA isolation kit (Exiqon) treated with 1 unit/μg of RNA of DNaseI (Thermo Scientific) for 30 minutes at 37°C followed by 10 min at 65°C with 50mM EDTA. cDNA was synthesized using the qScriptWhole cell lysates were prepared using RIPA buffer  48 hours post-transfection and used for western blot analysis following published protocol . PrimaryTotal protein lysates were prepared using Urea solublization buffer  CHAPS and 30mM Tris, 1X protease/phosphatase inhibitor cocktail) and sonication. The supernatant of centrifuged lysates was concentrated using Amicon® Ultra centrifugal filters . Protein concentrations were determined using the Bradford assay (Bio-Rad).TM DryStrip 7 cm, pH 3–10 NL  for overnight passive rehydration. Isoelectric focusing was conducted on a PROTEAN® IEF Cell, according to the manufacturer’s recommendation (Bio-Rad). Following rehydration, proteins were reduced with DTT and subsequently alkylated with iodoacetamide in equilibration buffer . Electrophoresis in the second dimension was performed on 12% SDS-PAGE at 100V. Gels were stained overnight with colloidal Coomassie Blue G-250 [Protein lysates were diluted with rehydration buffer  CHAPS, 40 mM DTT, 0.5% IPG buffer, pH 3–10 NL, and 0.4% Bromophenol Blue) and applied to Immobilineue G-250 .Gels were scanned using EPSON scan perfection V750 PRO software  at 600 dpi/16-bit grayscale. ImageMaster 2D platinum 7  was used for spot detection, background subtraction, matching, and to identify statistically significant (ANOVA) differences between protein spots i.e. fold change over control. The experiments were performed in triplicate.The differentially expressed protein spots were excised, cut into small pieces and placed in 0.6 ml Eppendorf tubes. The gel pieces were destained by incubating in 200 μl of 100 mM ammonium bicarbonate: acetonitrile (50:50 v/v) with shaking. When fully destained, the gel fragments were dehydrated with two washes of 100 μl of 100% acetonitrile (ACN) and were then dried in a vacuum centrifuge (Speed-Vac) for 5 min. The proteins were then cleaved enzymatically into peptides. For this, trypsin solution (2 μl of 0.02 μg/μl) was added to the wet the gel pieces and incubation was carried out for 4 hours at RT. Thirty μl of 50 mM ammonium bicarbonate was added to the gel pieces and left overnight at RT to allow for diffusion of the peptides from the gel. The digested proteins were stored at -80°C until further analysis.18 (Millipore). The ZipTips were conditioned with 10 μl of 0.1% TFA twice, 70% ACN/0.1% TFA twice, and 10 μl of 0.1% TFA twice. Samples containing the digest and bead mixture were transferred to the ZipTips and bound to the C18 resin. The loaded tips were then washed with 10 μl of 0.1% TFA. The peptide digests were eluted by placing 2 μl of 10 mg/mL CHCA matrix solution in 0.003% TFA, 13% ethanol, and 84% ACN onto the top of the ZipTips and slowly dispensing onto the MALDI plate. Mass spectrometric analysis was performed using a Thermo LTQ XL linear ion trap mass spectrometer (Thermo Scientific) equipped with a vacuum MALDI source, after the solvent evaporated at RT and the CHCA matrix was crystallized with peptides on the MALDI plate. A data-dependent acquisition was performed using Xcalibur software, in which the top 40 of the most abundant precursor ions from the survey scan (mass range 700–3500 Da) were chosen and MS/MS acquisition was triggered to fragment them by CID (collision-induced dissociation). The normalized collision energy was 50%, and the isolation width was 3 Da. The raw-files from the LTQ mass spectrometer were analyzed by using Mascot Distiller 2.3.2  for protein identification. Peptide masses were matched against the taxonomy Homo sapiens in the National Centre for Biotechnology Information non-redundant (NCBInr) database. One missed trypsin cleavage per peptide was allowed and an initial mass tolerance of 0.3 Da was used in all searches. Complete carboxyamidation of cysteine sulfhydryls and partial oxidation of methionine were assumed [After digestion, POROS 20 R2 resin (Applied Biosystems) was added to the digested gel samples with 5% formic acid and 0.2% trifluoroacetic acid for extraction at 4°C for 4 hours on a shaker. Prior to MALDI-MS analysis, the peptide digests were further desalted using ZipTip C assumed .Cell viability was measured using the neutral red uptake assay 48 hours post-transfection. Cells were washed with PBS, 100μl of neutral red working solution (40μg/ml) added to each well and plates were incubated for 2 hours. Cells were then washed with PBS, neutral red extracted using 150μl of destain solution  per well and the plates were subjected to shaking for 10 min. Absorbance was measured at 540nm using an Epoch microplate spectrophotometer (BioTeck).4 cells per well and after 48 hours incubated with 100μl of DCF-DA (25μM) for 45 min. Fluorescence was measured using a GLoMas®-Multi Detection System fluorescence plate reader (Promega) at 485nm (excitation) and 528nm (emission). The assays were performed in triplicate.Cellular reactive oxygen species (ROS) were measured using 2’, 7’- dichlorofluoresceine diacetate (DCF-DA) (Sigma-Aldrich). Cells were plated on solid black opaque plates at 5x10Western blot images were analyzed using IQTL software . Microsoft excel tools was used for two-tailed Student’s t-test. The standard error was used to display variation. The targets of miR-153-3p and miR-205-5p were used as input queries for the Partek Genomics Suite software, version 6.6 (Partek) to perform Gene ontology (GO) analysis and generate interactive maps and pathways.miR-153 overexpression in HEK293 cells downregulate SNCA whilst miR-205 overexpression in HEK293 cells has been shown to downregulate LRRK2 , 9. In tWe next sought to identify additional targets of miR-153-3p and miR-205-5p in SH-SY5Y cells using 2D-PAGE analysis. However, before performing 2D-PAGE analysis we showed that miR-153-3p and miR-205-5p transfections had no significant effect on SH-SY5Y cell viability ensuring that any observed protein changes were due to changes in miR-153-3p and miR-205-5p levels .We performed comparative 2D-PAGE analysis comparing control mimic and control antagomir transfected cells with cells transfected with the miR-153-3p mimic and the miR-153-3p antagomir, respectively . The samHomo sapiens) using Mascot Distiller revealing the identity of the differentially expressed proteins  between control transfected and miR-153-3p/miR-205-5p-transfected SH-SY5Y cells. In response to altered levels of miR-153-3p seven protein spots were up-regulated whilst eight protein spots were down-regulated , Table 1proteins .Overexpression of miR-153-3p resulted in up-regulation of proteasome subunit alpha type-1 isoform 2 (PSMA1) . miR-205miR-153-3p overexpression also resulted in increase abundance of Prefolding subunit 2 (PFDN2) , spot 7,We also found that cathepsin Z (CTSZ) , spot 13Inhibition of miR-153-3p also results in the up-regulation of the calcium activated chloride channel family member 4 (CLCA4) , spot 9.The stress-induced phosphoprotein 1 (STIP1) , spot 4 In response to miR-153-3p inhibition cofilin-1 was down-regulated , spot 32Our data indicates that both miR-205-5p and miR-153-3p influence direct and peripheral processes associated with neurodegenerative disorders, providing clues towards the possible regulation of key pathways , Fig 4.We found that miR-153-3p overexpression leads to an up-regulation of peroxiredoxin 2 (PRDX2) , spot 1 The regulation of PRDXs by miR-153-3p and miR-205-5p suggest that miR-153-3p and miR-205-5p may affect cellular ROS levels. Indeed, overexpression of miR-153-3p and miR-205-5p causes significant ROS reduction . Combinec-myc [c-myc promoter, but in contrast to Nm23, represses c-myc expression [Numerous microRNAs are involved in the cell cycle, cancer proliferation and metastasis . In respc-myc . In contpression .We also found that altered levels of miR-205-5p affect proteins associated with tumor proliferation and invasion . miR-205Galectin-1, a beta-galactoside binding protein associated with cell proliferation and differentiation is also down-regulated in response to miR-205-5p inhibition , spot 30Combined these results indicate that miR-153-3p and miR-205-5p may play a role in cell proliferation and migration involving various target proteins.Glucose stimulation increases miR-153 expression and miR-153 expression is reduced in PTPRN2 (Protein tyrosine phosphatase receptor type N polypeptide 2) mouse knockout models . We founmiR-153-3p overexpression also resulted in the up-regulation of High mobility group protein B1 (HMGB1) , spot 3,2 activity [Similar to miR-153-3p, miR-205-5p also down-regulates Serpin A12 , spot 26Collectively, miR-153-3p and miR-205-5p appears to regulate proteins involved in metabolic pathways and in particular carbohydrate metabolism .miR-205-5p appears to be affecting the abundance of proteins that influence mRNA expression and processing . The serAs microRNAs are most commonly involved in translational regulation, the up-regulation of eukaryotic translation initiation factor 5A-1 isoform B (EIF5A) , spot 17MicroRNA biology is complex and we have shown that miR-153-3p and miR-205-5p influences the abundance of numerous proteins integral to many biological processes in neuroblastoma cells . InteresSome of these processes associated with the identified proteins are fundamental in nature whilst others are specifically associated with cell survival, cell proliferation and neuroprotection. Although we acknowledge that the altered abundance of a small number of proteins in a pathway may not necessarily indicate that the entire pathway is affected, our study highlights that to fully understand microRNA-mediated processes a holistic approach is needed, which will pave the way for further insight into neuronal processes associated with normal development and disease.S1 FigShown are representative spectra for the peptide sequence shown at the top of each spectrum. The spectra are derived from Mascot search results.(TIF)Click here for additional data file.S2 FigProteasome subunit alpha type-1 isoform 2 (PSMA1) (regulated by miR-153-3p and miR-205-5p) and proteasome subunit p42 (PSMC6) (regulated by miR-205-5p) are integral parts of the 26S proteosome.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


C. elegans spread among cells, leading to RNAithroughout the body. During systemic spread of RNAi, membrane trafficking is thoughtto play important roles. Here, we show that RNAi Spreading Defective-3(rsd-3), which encodes a homolog of epsinR, a conserved ENTH  domain protein, generally participates in cellular uptake ofsilencing RNA. RSD-3 is previously thought to be involved in systemic RNAi only ingerm cells, but we isolated several deletion alleles of rsd-3, and found thatthese mutants are defective in the spread of silencing RNA not only into germ cellsbut also into somatic cells. RSD-3 is ubiquitously expressed, and intracellularlylocalized to the trans-Golgi network (TGN) and endosomes. Tissue-specificrescue experiments indicate that RSD-3 is required for importing silencing RNA intocells rather than exporting from cells. Structure/function analysis showed that theENTH domain alone is sufficient, and membrane association of the ENTH domain isrequired, for RSD-3 function in systemic RNAi. Our results suggest that endomembranetrafficking through the TGN and endosomes generally plays an important role incellular uptake of silencing RNA.RNA silencing signals in  In multicellular organisms, cells communicate with each other through various signalingmolecules, including proteins and lipids, to maintain tissue homeostasis and regulategrowth and differentiation. Growing evidence indicates that RNA also function as anintercellular signaling molecule14C. elegans, introduction of double-stranded RNA (dsRNA)into cells induces RNA interference (RNAi) not only in the cells, but also in cellsdistant from the cells where dsRNA is initially introduced6C. elegans,intercellular RNAi signal, which is assumed to dsRNA91112C.elegans11C. elegans, but little is known about how membranetraffic regulates the phenomenon and which trafficking pathways are involved.In some animals, including rsd (RNAi Spreading Defective) screen, rsd-3 alleles have beenisolatedrsd-3 encodes a homolog of epsinR (epsin-relatedprotein), an ENTH domain protein involved in endomembrane trafficking in mammaliancells17rsd-3 mutants showed defects in RNAi in the germline, but were not defectivein RNAi in the somarsd-3, andrevealed that RSD-3 is involved in systemic RNAi not only in germ cells but also insomatic cells, indicating that RSD-3 is a general component of systemic RNAi. Inaddition, we found that RSD-3 is required for importing dsRNA into cells rather thanexporting from cells. We also found that RSD-3 is associated with endomembranes such asthe trans-Golgi network (TGN) and endosomes, and membrane association of the ENTH domainof RSD-3 is required for function. From these results, we suggest that endomembranetrafficking through the TGN and endosomes generally plays an important role in cellularuptake of silencing RNA.Through a previous genetic screen to search for genes involved in systemic RNAi, namelyC. elegans, we searched for mutants of membranetrafficking-related genes exhibiting defects in systemic RNAi spreading. Weassayed various mutants of endocytosis, exocytosis or intracellular membranetrafficking-related genes for their sensitivity to RNAi induced by feedingtransformed bacteria expressing dsRNA (feeding RNAi). Among 61 strains examined(ok498) in the ctbp-1 gene, a homolog ofC-terminal binding protein 1 (CtBP1), which is involved in macropinocytosis inmammalspos-1 andsoma expressed gene bli-3, an epithelial gene (ctbp-1 mutants (tm6130 and tm6188) did not phenocopythe RB733 strain  in the RB733 strain. We performed whole-genomesequencing of the RB733 strain and identified a large deletion in thersd-3 gene. This mutation was confirmed by Sanger sequencing andrevealed a 7809 bp deletion and 7 bp insertion locatedin the genomic region of rsd-3. We named this mutation tm9006 mutation or only the rsd-3(tm9006) mutation.Mutants that contained only rsd-3(tm9006) were resistant to bothpos-1 and bli-3 feeding RNAi (ctbp-1(ok498) showed a normal response to feeding RNAi (rsd-3(tm9006) mutation is essentially responsible for the systemicRNAi defects observed in the RB733 strain.To understand the role of membrane trafficking in the intercellular transport ofsilencing RNA in examined, wefounial gene . However3 strain  and the n tm9006. rsd-3 aing RNAi , but muting RNAi , suggestrsd-3(tm9006) shows systemic RNAi defects in somatictissues other than epithelia, where bli-3 is expressed, we fedrsd-3(tm9006) bacteria that express dsRNA targeting themuscle-expressed gene (unc-22) and intestine-expressed gene(elt-2), and found that rsd-3(tm9006) was resistant tounc-22 and elt-2 feeding RNAi (rsd-3(tm9006) is resistant tofeeding RNAi throughout the organism. However, unlike sid-1 mutants, thatwere fully resistant to systemic RNAi, rsd-3 mutants were partiallyresistant to silencing soma-expressed genes  and endosomes17182223et al. performeda forward genetic screen named rsd (RNAi Spreading Defective) screen, andrsd-3 alleles such as pk2013 were isolatedrsd-3(pk2013) showed defectsin transmitting the RNAi effect to the germline, but show a normal RNAi responsein somatic tissues. We also confirmed the phenotypes of rsd-3(pk2013);i.e., rsd-3(pk2013) showed no RNAi phenotype when fed pos-1 dsRNA,but displayed RNAi phenotype when fed bli-3 dsRNA (rsd-3(tm9006) exhibits systemic RNAi defects inboth the germline and the soma, we examined whether RSD-3 actually plays a rolein systemic RNAi in somatic tissue. We generated a transgene containingrsd-3 genomic sequences including 4 kb of upstreampromoter sequences, the complete ORF and introns, fused in frame to GFP (seersd-3(tm9006). We foundthat expression of RSD-3::GFP completely rescued the silencing defects in thesoma of rsd-3(tm9006) . From these results, we conclude that RSD-3 is requiredfor systemic RNAi in somatic cells as well as in germ cells.-3 dsRNA . Because(tm9006) . To provd tm6420 , and foud tm6420  but alsod tm6420  as was trsd-3(tm9006)to RNAi triggered by a transgene. We generated an RNAi-inducing transgeneexpressing gfp hairpin RNA and mCherry under the control of the panneuronalsnb-1 promoter and introduced this transgene into let-858::gfptransgenic animals, which express nuclear-localized GFP in all somaticcellsrsd-3(tm9006) background  exhibitedno defects in systemic RNAi in somatic tissues. In pk2013, Tc1 transposon(a 1.6-kb repeated DNA sequence) is inserted within the first exon ofrsd-3, immediately after the start codonpk2013 mutation affects the expression or functionof RSD-3 in the soma, we constructed a pk2013 mutation-containingrsd-3, amplified from the rsd-3(pk2013) genome, with aC-terminal fusion to GFP under the control of the rsd-3 promoter(genomic rsd-3_pk2013::gfp) (see rsd-3(tm9006). We detected GFP expression in all somatic tissues, andfound that the subcellular localization of the GFP signal was indistinguishablefrom that of normal RSD-3::GFP (genomic rsd-3_pk2013::gfp completely rescued the systemicRNAi defects in the soma of rsd-3(tm9006) (pk2013 mutation did not strongly affect RSD-3expression and function in the soma. As reported for some Tc1 insertions27rsd-3(pk2013), Tc1 may beexcised from the genome, or may be removed from pre-mRNA by splicing, resultingin the production of an in-frame message to produce RSD-3 protein that isfunctional in vivo.We also investigated why the originally identified (tm9006) . These rrsd-3 cDNA derivedfrom the Tc1-containing allele of rsd-3 in somatic cells. We used anextrachromosomal array of genomic rsd-3_pk2013::gfp described above,because transgene expression from an extrachromosomal array is typicallyrestricted to somatic cellsrsd-3(tm9006) and rsd-3(tm9006);Ex[genomic rsd-3_pk2013::gfp]were reverse transcribed, and rsd-3 cDNAs around the Tc1 insertion sitewere PCR amplified (see rsd-3(tm9006) as expected. The size of the PCR product fromrsd-3(tm9006);Ex[genomic rsd-3_pk2013::gfp] was almost the same asthat from wild type, suggesting that most of Tc1 is spliced from mature mRNA ofgenomic rsd-3_pk2013::gfp or imprecisely excised from theextrachromosomal array (rsd-3(tm9006);Ex[genomicrsd-3_pk2013::gfp] and sequenced five independent cDNA clones(#1~5). As shown in rsd-3 translational reading frame. The predicted amino acid sequencestranslated from such mRNAs contain extra 16 amino acids at the site 11 aminoacids downstream from the N-terminus. This insertion site is located upstream ofthe functionally important region of the ENTH domain (see below), and therefore,such insertion may not significantly affect the function of RSD-3. Meanwhile,two clones (#4 and #5) have a small deletion and/or insertion, resulting in aframeshift with a premature stop codon . To identify earlyendosomes, we used phosphatidylinositol-3-phosphate (PI3P)-binding construct2xFYVE::Venus. Near the apical membrane of hypodermal cells, most 2xFYVE::Venusformed ring-like structures (trans-Golgi network(TGN), almost all RSD-3::ECFP-positive structures colocalized extensively withthe TGN marker, Venus::SYN-16To examine the subcellular localization of RSD-3, we performed a series ofcolocalization studies in the hypodermis of transgenic animals expressingECFP-tagged RSD-3 and a set of Venus-tagged organelle markers under the controlof the ructures , althougructures . More spructures , insets.The subcellular localization of RSD-3 in the coelomocytes was also checked withdouble labeling of RSD-3::mCherry see . Theser17rsd-3 mutants might be due todisruption of cellular RNAi machinery itself rather than to a systemic RNAidefect, we examined whether RSD-3 plays a role in cell-autonomous RNAi.Tijsterman et al. showed that, by injection of pos-1 dsRNA intoone gonad arm, cell-autonomous RNAi machinery is intact in germ cells ofrsd-3(pk2013)pos-1 dsRNA into both gonad arms ofrsd-3(tm9006) and found that RNAi efficiency in germ cells ofrsd-3(tm9006) is comparable to that in the wild type , whichexpress GFP in the muscle nuclei and mitochondria. In the wild type background,GFP was largely silenced in red muscle cells expressing the GFP-RNAi construct(rsd-3(tm9006)background (rsd-3(tm9006)muscle cells. These results indicate that RSD-3 is not involved in the RNAimachinery itself, but rather required for the systemic spread of silencing RNAin both somatic and germ cells.To examine the involvement of RSD-3 in the RNAi mechanism in somatic cells, wegenerated an extrachromosomal array expressing gfp hairpin RNA and mCherry underthe control of the muscle-specific onstruct. GFPexpckground ,suggestC. elegans is fed with dsRNA, the dsRNA is first taken up byintestinal cells from the intestinal lumen. The silencing RNA is then exportedfrom intestinal cells to the pseudocoelom and subsequently imported into thegerm and somatic cellsrsd-3(tm9006), we performed tissue-specific rescueexperiments. We used myo-3p::gfp transgenic animals (ccIs4251) andfed them with bacteria expressing GFP dsRNA. In the wild type background, GFPexpression in muscle cells was significantly decreased, but in muscle cells withan rsd-3(tm9006) background, GFP was not silenced prominently afterexposure to GFP dsRNA . We found thatexpression of RSD-3 and mCherry under the control of the muscle-specificmyo-3 promoter efficiently restored GFP feeding RNAi sensitivity inmuscle cells of rsd-3(tm9006);ccIs4251 animals (rsd-3(tm9006);ccIs4251expressing RSD-3 and DsRed under the control of the intestine-specificges-1 promoter (pos-1dsRNA (10 ng/μl) into the pseudocoelom. In wild typeanimals, the injection induced efficient RNAi in the germline, and resulted inalmost complete embryonic lethality in the F1 embryos (rsd-3(tm9006) animals were partiallyresistant to the injection , functioning at the TGN and endosomes  thatis mainly generated on the TGN membrane 1724. The17ndosomes 333435. Trsd-3(tm9006). We generated expression plasmids for ECFP-taggedfull-length RSD-3 and RSD-3 truncations, namely the ENTH domain  and the C-terminal region (rsd-3(tm9006) and examined the ability to restoresensitivity to bli-3 feeding RNAi. We also observed the distribution ofthese constructs in hypodermal cells. Expression of full-length RSD-3::ECFP inthe hypodermis completely rescued the bli-3 feeding RNAi defects ofrsd-3(tm9006) (bli-3 feeding RNAi in rsd-3(tm9006) (rsd-3(tm9006) at all . We expr(tm9006) , and RSD(tm9006) .Also, e(tm9006) . RSD-3_E(tm9006) . Incont) at all , and in) at all . These rC. elegans RSD-3.Meanwhile, mammalian ENTH domain proteins are thought to have other functionsnot directly related to membrane trafficking, such as transcriptional regulationin the nucleusbli-3 feeding RNAi sensitivity of rsd-3(tm9006) at all  upon binding to phosphoinositide17404140) at all . This reSaccharomyces cerevisiae, are reported to beinvolved in retrograde transport from endosomes to the TGN18rsd-3 mutants were partial, especially in the soma,indicating the existence of functionally redundant genes or pathways.Our data indicates that endomembrane-associated RSD-3 generally plays an importantrole in cellular uptake of silencing RNA. It is known that plasma membrane-localizedENTH domain proteins, such as epsin1/EPN-1, regulate clathrin-mediatedendocytosis21vti-1, a homolog ofVti1b/Vti1p, but vti-1 mutants did not show systemic RNAi defects (ourunpublished observation). Other SNAREs may function redundantly withvti-1.The ENTH domain, which lacks clathrin and clathrin adaptor protein-binding motifs, isnecessary and sufficient for RSD-3 function in systemic RNAi, suggesting that RSD-3function is not mediated by formation of clathrin-coated vesicles. Instead, becausethe ENTH domain of mammalian epsinR/Ent3p interacts with some SNAREs such asVti1b/Vti1p and functions as a cargo adaptor36373839The mechanism by which RSD-3 is involved in the uptake of silencing RNA into cellsremains unclear. At present, we suggest the following two possibilities. First,RSD-3 may regulate SID-1 localization. Several plasma membrane transporters, such asGLUT4 (glucose transporter) and Fet3p/Ftr1p (iron transporter), are known to cyclebetween the plasma membrane and the TGN, via endosomes4749Oligonucleotides hold outstanding promise as potential therapeutic agents, but amajor concern is their poor accessibility to the targets within the cells. Recently,some studies using mammalian cells suggested that the uptake and trafficking pathwayof oligonucleotide could affect the ultimate pharmacological effectiveness5253C. elegans methods were performed according to standardprotocolsabt-6(tm5404), aex-3(tm5659), arf-6(tm1447), arl-3(tm1703),arl-6(tm2622), ctbp-1(tm6130), ctbp-1(tm6188), mtcu-1(tm5041),rab-6.1(tm2124), rab-8(tm2526), rab-10(tm2992), rab-11.1(tm2287),rab-11.2(tm2081), rab-14(tm2095), rab-18(tm2121), rab-19(tm2629),rab-21(tm2999), rab-27(tm2270), rab-28(tm2636), rab-30(tm2653),rab-33(tm2641), rab-35(tm2058), rab-37(tm2089), rab-39(tm2466),rsd-3(tm6416), rsd-3(tm6420), scrm-2(tm650), sec-22(tm4552),sid-1(tm2700), snx-1(tm847), snx-3(tm1595), snx-6(tm3790),syx-17(tm3181), tat-1(tm3117), tat-2(tm2332), C52B11.5(tm3007),C56E6.2(tm3008), F11A5.3(tm2585), F11A5.4(tm2567), K02E10.1(tm2564),T04C9.1(tm5548), T28D6.6(tm5550), 4R79.2(tm2640). The following mutantsand transgenic animals were obtained from the Caenorhabditis GeneticsCenter : arf-1.2(ok796), cav-1(ok2089), cav-2(hc191),ctbp-1(ok498) (RB733), efsc-1(ok2572), evl-20(ok1819),exoc-7(ok2006), exoc-8(ok2523), max-2(ok1904), pak-1(ok448), pak-2(ok332),pkc-2(ok328), rab-7(ok511), rap-2(gk11), rsd-3(pk2013),sdpn-1(ok1667), sec-8(ok2187), src-2(ok819), ssr-2(ok1375),tufm-2(ok2850), vps-35(hu68), ccIs4251[myo-3p::gfp::LacZ::NLS &myo-3p::mitochondrial gfp], cdIs39[pcc1::gfp::rme-1],cdIs54[pcc1::mans::gfp], cdIs66[pcc1::gfp::rab-7],cdIs85[pcc1::2xFYVE::gfp], pkIs1582[let-858::gfp],pwIs50[lmp-1::gfp]. rsd-3(tm9006) was identified as describedin “Identification of rsd-3(tm9006)” section andisolated by crossing RB733 strain with wild type. Mutations were outcrossed atleast five times before further analysis except where otherwise indicated.Bristol N2 was used as wild type strain. Worm cultures, genetic crosses, andother ok498, we found twopotential deletions larger than 1 kb on chromosome X. One of the twogenomic regions contains no protein-coding gene, thus we focused on anotherregion, around chromosome X: +6.73 cM. This mutation candidate wasconfirmed by Sanger sequencing and revealed a 7809 bp deletion and7 bp insertion located in the genomic region of rsd-3.RB733 genomic DNA was purified by phenol and chloroform extraction includingRNase A treatment. 1 μg DNA was subjected tofragmentation and adaptor ligation using an Ion Xpress Plus Fragment Library Kit(Life Technologies), according to the manufacturer’s instructions.The library was subjected to emulsion PCR using the Ion OneTouch 200 TemplateKit v2 DL (Life Technologies), followed by bead enrichment. Then, whole-genomesequencing was performed with an Ion Torrent PGM system using the Ion PGM 200Sequencing Kit and Ion 318 chip (Life Technologies). Using IGV (IntegrativeGenomics Viewer), we inspected the genome region where no read was aligned asthe potential deletion candidates. Except for genomic rsd-3::gfp, the genomic region of rsd-3(3969 bp upstream the ATG initiation codon and the full-lengthrsd-3) was amplified by PCR from N2 genomic DNA and inserted intopPD95.75 (a gift from Dr. A. Fire) at the SalI and SmaI sites.snb-1p::gfp-hairpin was generated as described previouslypPD49.26_GST-loop. GFP sense sequence was amplifiedby PCR from pPD95.67 (a gift from Dr. A. Fire) and inserted intopPD49.26_GST-loop at the XbaI and SmaI sites, yieldingpPD49.26_GFPsense_GST-loop. GFP antisense sequence was amplified byPCR from pPD95.67 and inserted into pPD49.26_GFPsense_GST-loop at theNheI and SacI sites, yielding pPD49.26_GFPsense_GST-loop_GFPantisense.The promoter region of snb-1 (3015 bp) was amplified by PCRfrom N2 genomic DNA and inserted intopPD49.26_GFPsense_GST-loop_GFPantisense at the HindIII and PstIsites. To generate genomic rsd-3_pk2013::GFP, the genomic region ofrsd-3 (3969 bp upstream the ATG initiation codon and thefull-length rsd-3 containing Tc1 insertion) was amplified by PCR fromrsd-3(pk2013) genomic DNA and inserted into pPD95.75 at the SalI andSmaI sites. To generate dpy-7p::rsd-3::ECFP, rsd-3 cDNA wasamplified by PCR from N2 cDNA and inserted into pPD95.75 at the SalI and SmaIsites, yielding pPD95.75_rsd-3cDNA. The promoter region of dpy-7(431 bp) was amplified by PCR from N2 genomic DNA and inserted intopPD95.75_rsd-3cDNA at the HindIII and SalI sites, yieldingpPD95.75_dpy-7p_rsd-3cDNA. The ECFP sequence was amplified by PCRfrom pFX_ECFPTdpy-7p::aman-2::Venus, the Venus sequence wasamplified by PCR from pFX_VenusTpPD95.75_Venus. Theaman-2 cDNA fragment (1-84aa) was amplified by PCR from N2 cDNA andinserted into pPD95.75 at the SalI and SmaI sites, yieldingpPD95.75_aman-2_Venus. dpy-7 promoter was subcloned intopPD95.75_aman-2_Venus at the HindIII and SalI sites. To generatedpy-7p::2xFYVE::Venus, a dimeric FYVE domain from Hrs (2xFYVE)sequence was amplified by PCR from cdIs85 genomic DNA and inserted intopPD95.75_Venus at the SalI and SmaI sites. Then, dpy-7promoter was subcloned at the HindIII and SalI sites. To generatedpy-7p::Venus::rab-7, Venus::rab-7cDNA sequence was subclonedfrom pFX_vha-8p_VenusT/rab-7 into pPD95.75 at the SalI/SmaI sites. Then, dpy-7 promoter was subclonedat the HindIII and SalI sites. To generate dpy-7p::Venus::syn-16, genomicsyn-16 (1261 bp) was amplified by PCR from N2 genomic DNAand using dpy-7p::Venus::rab-7, substituted rab-7 cDNA withgenomic syn-16 at the BglII and SmaI sites. To generatedpy-7p::Venus::chc-1, genomic chc-1 (5535 bp) wasamplified by PCR from N2 genomic DNA and using dpy-7p::Venus::rab-7,substituted rab-7 cDNA with genomic chc-1 at the BglII and SmaIsites. To generate unc-122p::rsd-3::mCherry, the mCherry sequence wasamplified by PCR from pFX_mCherrypPD95.75_mCherry. Thepromoter region of unc-122 (685 bp) was amplified by PCR fromN2 genomic DNA and inserted into pPD95.75_mCherry at the HindIII and XbaIsites, yielding pPD95.75_unc-122p::mCherry. Genomic rsd-3(4743 bp) was amplified by PCR from N2 genomic DNA and inserted intopPD95.75_unc-122p::mCherry at the XbaI and SmaI sites. To generatemyo-3p::gfp-hairpin, the promoter region of myo-3(2599 bp) was amplified by PCR from N2 genomic DNA and usingsnb-1p::gfp-hairpin, substituted snb-1 promoter withmyo-3 promoter at the HindIII and PstI sites. To generatemyo-3p::rsd-3, rsd-3 cDNA was amplified by PCR from N2 cDNAand inserted into pPD95.75 at the SalI and SmaI sites, yieldingpPD95.75_rsd-3 cDNA. The myo-3 promoter was subcloned at theHindIII and SalI sites. To generate ges-1p::rsd-3, the promoter region ofges-1 (3275 bp) was amplified by PCR from N2 genomic DNAand inserted into pPD95.75_rsd-3cDNA at the HindIII and SalI sites. Togenerate dpy-7p::rsd-3_ENTH::ECFP, rsd-3_ENTH sequence (1-189 aa)was amplified by PCR and using dpy-7p::rsd-3::ECFP, substitutedrsd-3 with rsd-3_ENTH at the SalI and SmaI sites. To generatedpy-7p::rsd-3_Cterm::ECFP, rsd-3_Cterm sequence (190-483 aa)was amplified by PCR and using dpy-7p::rsd-3::ECFP, substitutedrsd-3 with rsd-3_ENTH at the SalI and SmaI sites. To generatedpy-7p::rsd-3_ ENTHΔα0::ECFP,rsd-3_ ENTHΔα0 sequence (37-189 aa)was amplified by PCR and using dpy-7p::rsd-3::ECFP, substitutedrsd-3 with rsd-3_ ENTHΔα0 atthe SalI and SmaI sites. The micro-injection of the DNA constructs describedabove was performed as previously reportedtmEx3441[snb-1p::gfp-hairpin & snb-1p::mCherry], tmEx3578[genomicrsd-3::GFP], tmEx3681[genomic rsd-3_pk2013::GFP],tmEx3713[dpy-7p::rsd-3cDNA::ECFP &dpy-7p::aman-2(1-84aa)::Venus], tmEx3717[dpy-7p::rsd-3cDNA::ECFP& dpy-7p::Venus::rab-7], tmEx3936[dpy-7p::rsd-3cDNA::ECFP &dpy-7p::Venus::syn-16], tmEx3938[dpy-7p::rsd-3cDNA::ECFP &dpy-7p::2xFYVE::Venus], xhEx3601[dpy-7p::rsd-3cDNA::ECFP &dpy-7p::Venus::chc-1], tmEx3845[unc-122p::rsd-3::mCherry] (recipient:cdIs66), tmEx3851[unc-122p::rsd-3::mCherry] (recipient:cdIs39), tmEx3853[unc-122p::rsd-3::mCherry] (recipient:cdIs54), tmEx3856[unc-122p::rsd-3::mCherry] (recipient:pwIs50), tmEx3859[unc-122p::rsd-3::mCherry] (recipient:cdIs85), tmEx3612[myo-3p::gfp-hairpin &myo-3p::mCherry], tmEx3933[myo-3p::rsd-3 &myo-3p::mCherry], tmEx3750[ges-1p::rsd-3 &ges-1p::DsRed], tmEx3723[dpy-7p::rsd-3_ENTH::ECFP],tmEx3725[dpy-7p::rsd-3_Cterm::ECFP],tmEx3615[dpy-7p::rsd-3_ENTHΔα0::ECFP].For each construct, at least three independent transgenic lines were analyzed. Togenerate f myo-329 bp was E. coli HT115(DE3), and the bacteria was grown on100 μg/mL ampicillin and 1 mMisopropyl-beta-D-thiogalactopyranoside (IPTG). RNAi clones for pos-1,mex-3, lin-31, bli-3 and unc-22 were taken fromthe Ahringer RNAi library (GeneService). RNAi clones for elt-2 and GFPwere generated as following: cDNA fragment of elt-2 was amplified from N2cDNA. cDNA fragment of GFP was cut from pPD79.44 using NotI/BglII sites. Then,cDNA fragments of elt-2 or GFP were cloned into the L4440 (pPD129.36)vector, and transformed E. coli HT115 (DE3) with each construct. Forpos-1 and mex-3 RNAi assay, more than twenty L1 larvae werefed with bacteria expressing pos-1 or mex-3 dsRNA, reared toadulthood and allowed to lay eggs for several hours. Then adult animals wereremoved and 24 hours later, the number of dead eggs and hatchedlarvae were counted. At least 300 eggs and larva were counted per strain. Forlin-31 RNAi assay, L1 larvae were placed on plates seeded withbacteria expressing lin-31 dsRNA and 54 hr later, the percentage ofanimals with Muv (multi-vulva) phenotype was scored. At least 50 animals werecounted per strain. For bli-3 RNAi assay, L1 larvae were placed on platesseeded with bacteria expressing bli-3 dsRNA and 48 hr later , the percentage of animals that reached adulthood wasscored. At least 100 animals were counted per strain. For unc-22 RNAiassay, L4 larvae were placed on plates seeded with bacteria expressingunc-22 dsRNA and 48 hr later, the percentage of twitching animals wasscored. At least 100 animals were counted per strain. For elt-2 RNAiassay, L4 larvae were placed on plates seeded with bacteria expressingelt-2 dsRNA, reared to adulthood and allowed to lay eggs for severalhours. Then adult animals were removed and 54 hours later, thepercentage of animals that reached adulthood was scored. At least 100 animalswere counted per strain. For GFP RNAi, L1 larvae were fed with bacteriaexpressing GFP dsRNA, reared to L4 larvae and photographed. For injection RNAiexperiments, pos-1 dsRNA was synthesized as following: pos-1 cDNAfragment attached with T7 promoter at both ends was amplified from N2 cDNA, andpos-1 dsRNA was transcribed using T7 polymerase (Ambion). Synthesizedpos-1 dsRNA was treated with DNase I, purified by phenol andchloroform extraction, and then injected into both gonad arms or thepseudocoelom of more than twenty day 1 adult hermaphrodites for each strain. Thehatching rate of embryos laid between 12 to 24 hr post injection was scored foreach injected animal. At least 300 progenies were counted per strain. Fortransgene-induced RNAi, gfp hairpin RNA was expressed from extrachromosomalarrays and GFP fluorescence was photographed at the L4 stage.Feeding RNAi was carried out as describedtm9006;tmEx3681[genomicrsd-3_pk2013::gfp] animals using TRIzol reagent according to theprotocol supplied by the manufacturer (Invitrogen). First-strand cDNA was reverstranscribed from the total RNA with an oligo-dT primer using SuperScript IV(Invitrogen). rsd-3 cDNAs in the vicinity of the Tc1 insertion site werePCR amplified using primers shown in Total RNA was extracted from mixed-stage Worms on NGM (nematode growth medium) plates were imaged with a Leica DFC280camera using Leica IM500 imaging software. For confocal images, animals in20 mM azide were mounted on a 5% agar pad on a glass slide.Micrographs were taken on a Zeiss LSM 710 inverted confocal microscope with 488and 543 lasers and images were processed using ZEN software (Carl Zeiss) exceptfor t-test. Data were consideredsignificantly different if P-values were lower than 0.05.The standard error of the mean (SEM) was used as the error bar for bar chartsplotted from the mean value of the data. All the data were compared usingtwo-tailed Student’s How to cite this article: Imae, R. et al. Endomembrane-associated RSD-3is important for RNAi induced by extracellular silencing RNA in both somatic andgerm cells of Caenorhabditis elegans. Sci. Rep. 6, 28198; doi:10.1038/srep28198 (2016).